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https://openalex.org/W3025607100	https://europepmc.org/articles/pmc7217902?pdf=render	English	null	Association between serum uric acid and metabolic syndrome: a cross-sectional study in Bangladeshi adults	Scientific reports	2,020	cc-by	6,081	Nurshad Ali ✉, Rakib Miah, Mahmudul Hasan, Zitu Barman, Ananya Dutta Mou, Jaasia Momtahena Hafsa, Aporajita Das Trisha, Akibul Hasan & Farjana Islam Elevated levels of serum uric acid (SUA) have been suggested to associate with cardiovascular disease, diabetes and metabolic syndrome (MetS). However, information is limited on the association between SUA and MetS in general adults. This study aimed to assess the relationship of SUA with MetS and its components in general adults in Bangladesh. A total of 420 participants were enrolled in this study and biochemical parameters including SUA, fasting blood glucose (FBG) and lipid profile were analyzed using standard methods. The NECP criteria were applied to define MetS. The association of SUA with MetS and its components were evaluated by multinomial logistic regression models. The overall prevalence of MetS was 22% with 21.9% in males and 22.1% in female participants. Male subjects had a high prevalence of elevated components of MetS than in the female subjects (p < 0.05 for all cases). The mean concentration of SUA was significantly higher in subjects of the MetS group compared to the non-MetS group (p < 0.05). The components of MetS were raised with the increasing concentrations of SUA across the quartiles. In regression analysis, SUA was significantly associated with the prevalence of MetS in Bangladeshi adults. In conclusion, elevated SUA was significantly associated with the prevalence of MetS and its components. Metabolic syndrome (MetS) consist of several risk factors including central obesity, elevated blood pressure, hyperglycemia, high triglycerides, and reduced high-density lipoprotein cholesterol1. MetS is associated with the increased risk of type 2 diabetes, cardiovascular disease (CVD) and mortality2,3. Several population-based studies showed an increased risk of CVD in individuals with MetS compared to those who do not have the syndrome4,5. Besides the traditional risk factors, other factors including microalbuminuria, inflammatory markers and hype- ruricemia have been suggested to be involved in the MetS6–8. Along with MetS, obesity has also been found as an important risk factor for CVD. Furthermore, a link has been found between obesity and hyperuricemia in various studies9–11. The prevalence of MetS is increasing at an alarming rate both in developed and developing countries. MetS is highly prevalent among Bangladeshi adults and has been increased rapidly in the last few decades. A recent review reported a high prevalence of MetS (30%) in the Bangladeshi population with 32% in females and 25% in males12.i Uric acid in serum is the final oxidation product of purine metabolism in human13. www.nature.com/scientificreports www.nature.com/scientificreports Department of Biochemistry and Molecular Biology, Shahjalal University of Science and Technology, Sylhet, 3114, Bangladesh. ✉e-mail: nur_rubd@yahoo.com Association between serum uric acid and metabolic syndrome: a cross-sectional study in Bangladeshi adults Nurshad Ali ✉, Rakib Miah, Mahmudul Hasan, Zitu Barman, Ananya Dutta Mou, Jaasia Momtahena Hafsa, Aporajita Das Trisha, Akibul Hasan & Farjana Islam Materials and methods Study population. This study was a cross-sectional design conducted between November 2017 and May 2019 at the Department of Biochemistry and Molecular Biology of Shahjalal University of Science and Technology, Sylhet, Bangladesh. A total of 420 general adults (aged ≥ 18 years) were enrolled from university students, academic and non-academic staff and local city people of the Sylhet and Dhaka region of Bangladesh. The inclusion criteria were: both gender, aged above 18 years, free from severe chronic illness and willing to participate. Exclusion criteria were: pregnant women, lactating mother and participants with a history of hepa- totoxic drug intake, kidney disease, alcohol intake and self-reported evidence of acute or chronic hepatitis. We also excluded participants with missing anthropometric data or blood samples. This study was approved by the Internal Ethics Committee at the Department of Biochemistry and Molecular Biology of Shahjalal University of Science and Technology, Bangladesh. All participants provided written informed consent before inclusion in the study. All steps in the methods section were performed in accordance with the relevant guidelines and regulations. General data collection. A standard questionnaire was used to collect demographic and lifestyle informa- tion from the participants. Individual anthropometric data such as age, gender, weight and height were recorded in the questionnaire form followed a standard procedure described elsewhere21–26. Briefly, systolic and diastolic blood pressure (SBP and DBP) were measured twice in the left arm of the participants with an automated sphyg- momanometer (Omron M10, Omron Corporation, Tokyo, Japan) in the seated position after at least 10 minutes of rest. Body mass index (BMI) was calculated as weight in kilograms divided by height in square meters (kg/m2). Waist circumference (WC) was measured using general tape that was placed midway between the lowest border of the ribs and iliac crest. Hip circumference (HC) was measured at the largest circumference of the buttocks to the nearest 0.5 cm. Waist-hip ratio (WHR) was measured as waist circumference divided by hip circumference. Blood sample collection and laboratory measurements. Venous blood samples were collected after an overnight fast from each subject. The blood samples were centrifuged and stored the isolated serum at −20 °C until laboratory analysis. Serum uric acid (SUA), fasting blood glucose (FBG), total cholesterol (TC), triglycerides (TG), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) were measured by colorimetric methods with a semi-auto biochemistry analyzer (Humalyzer 3000, USA) described elsewhere21,22,26. Materials and methods The diagnostic kits were purchased from Human Diagnostic (Germany) for analysis of the above clinical parame- ters. The measurements were carried out according to the standard manufacturer’s protocols provided within the kit. The precision of the measurements was maintained regularly by method standard calibration. Diagnostic criteria. In present study, hyperuricemia was defined as SUA concentration >7.0 mg/dL (416.4 µmol/L) in men or >6.0 mg/dL (356.9 µmol/L) in women27,28. All volunteers were stratified into four quartiles based on SUA concentrations (Q1: ≤ 243.9, 244–309.3, 309.4–380.7 and> 380.7 µmol/L). Metabolic syndrome (MetS) was diagnosed according to the National Cholesterol Education Program – Adult Treatment Panel III (NCEP-ATP III) criteria29. The components of the MetS were defined as following: i) Elevated BP (SBP ≥ 130 mmHg and/or DBP ≥ 85 mmHg or intake of an antihypertensive medication); ii) raised WC (> 102 cm for males and> 88 cm for females); iii) hyperglycemia (FBG ≥ 100 mg/dL). iv) hypertriglyceridemia (TG ≥ 150 mg/dL); v) low HDL-C (< 40 mg/dL for males and <50 mg/dL for females) and subjects with at least three of the above components were identified as having MetS. Statistical analysis. Statistical data analyses were performed using IBM SPSS version 23. Data are presented as mean ± SD and quartile ranges. The tests applied during data analysis already described in our previous stud- ies21,26. In brief, the baseline characteristics of the volunteers in the SUA quartiles were compared by one-way ANOVA. A Chi-square test was applied to differentiate the proportions of the categorical variables. Differences in the anthropometric and baseline characteristics between the gender groups were done by an independent sample t-test. Pearson’s correlation coefficient test was performed to assess the relationships between baseline variables and SUA concentrations. The association between MetS and SUA levels was evaluated by multinomial logistic regression models. MetS was categorized as yes (presence) and no (absence). In regression analysis, MetS (yes) was considered as the dependent variable and SUA as the independent variable. SUA and other covariates were used as continuous variables in the regression models. We applied three models in the regression analysis. Model 1 was adjusted for age (years) and gender (male and female). Model 2 was adjusted for age, gender and BMI (kg/ m2), Model 3 was further adjusted for variables used in model 1 and 2 and LDL-C (mg/dL). A p-value of <0.05 was considered statistically significant. Nurshad Ali ✉, Rakib Miah, Mahmudul Hasan, Zitu Barman, Ananya Dutta Mou, Jaasia Momtahena Hafsa, Aporajita Das Trisha, Akibul Hasan & Farjana Islam Recent epidemiological studies have demonstrated an association of serum uric acid (SUA) with MetS and its components in different populations14–19. Some other studies have also found that elevated SUA levels are an independent predictors of the components of MetS, such as high blood pressure and hyperglycemia20. However, information is limited regarding the relationship of SUA with MetS in general adults. Moreover, no study has been conducted to exam- ine the association between SUA and MetS in Bangladeshi adults. Given the increased prevalence of MetS in the Bangladeshi population, this cross-sectional study aimed to investigate the relationship of SUA with MetS and its components in general adults. This study also aimed to assess whether SUA is an additional component of MetS in this population. Scientific Reports \| (2020) 10:7841 \| https://doi.org/10.1038/s41598-020-64884-7 www.nature.com/scientificreports/ Results Total MetS Non-MetS p-value N 420 93 327 — Gender, m/f 420 57/36 200/127 — Age, year 30.5 ± 12.4 39.5 ± 14.1 27.8 ± 10.4 0.000 WC, cm 83.9 ± 10.6 90.5 ± 12.3 81.4 ± 8.6 0.000 HC, cm 92.8 ± 8.2 96.0 ± 8.6 91.5 ± 7.6 0.000 WHR 0.68 ± 0.39 0.91 ± 0.19 0.62 ± 0.41 0.000 BMI, kg/m2 23.9 ± 3.8 26.3 ± 3.8 23.3 ± 3.6 0.000 SBP, mmHg 118.2 ± 15.1 128.8 ± 18.1 115.1 ± 12.5 0.000 DBP, mmHg 73.6 ± 14.9 81.6 ± 8.4 71.4 ± 15.6 0.000 SUA, µmol/L 319.2 ± 107.8 332.0 ± 111.8 308.2 ± 106.6 0.026 FBG, mg/dL 98.8 ± 37.5 130.4 ± 66.2 89.9 ± 17.7 0.000 TG, mg/dL 135.1 ± 101.6 211.6 ± 107.2 113.5 ± 88.9 0.000 TC, mg/dL 157.7 ± 63.9 189.2 ± 75.7 148.8 ± 56.3 0.000 HDL-C, mg/dL 31.9 ± 17.1 29.6 ± 11.1 33.4 ± 18.5 0.046 LDL-C, mg/dL 93.3 ± 58.3 115.9 ± 73.2 85.9 ± 50.4 0.000 Table 1. Baseline characteristics of the participants based on the presence of MetS. M Table 1. Baseline characteristics of the participants based on the presence of MetS. MetS: Metabolic Syndrome. Values are presented as mean ± SD. P-values are obtained from independent sample t-test. Parameters Total Male Female P-value MetS, % 22.0 21.9 22.1 >0.05 Raised WC, % 13.3 4.6 30.9 <0.01 Elevated SBP, % 17.9 24.1 8.6 <0.01 Elevated DBP, % 17.4 18.4 15.9 <0.05 Hyperglycemia, % 25.7 23.4 29.2 <0.05 Elevated TG, % 40.4 52.2 22.9 <0.01 Reduced HDL-C, % 80.7 79.5 82.5 >0.05 Hyperuricemia 16.6% 21.3 8.3 <0.01 Table 2. Prevalence of MetS and its components in the male-female group. The difference between male and female is expressed as p-value. P-values are obtained from Chi-square test. Table 2. Prevalence of MetS and its components in the male-female group. The difference between male and female is expressed as p-value. P-values are obtained from Chi-square test. Table 2. Prevalence of MetS and its components in the male-female group. The difference between male and female is expressed as p-value. P-values are obtained from Chi-square test. Table 2. Prevalence of MetS and its components in the male-female group. The difference between male and female is expressed as p-value. P-values are obtained from Chi-square test. Figure 1. Prevalence of MetS and its components in the hyperuricemic and non-hyperuricemic group. Results Baseline characteristics of the participants in the MetS and non-MetS group. In total, 420 par- ticipants (257 male and 163 female) were enrolled in the present study. The baseline characteristics of the par- ticipants in the MetS and non-MetS groups are presented in Table 1. Among the participants, 93 subjects were diagnosed with MetS according to the diagnostic criteria. There were significant differences in the mean of age, WC, HC, WHR, BMI, SBP, DBP, FBG, TG, TC, LDL-C (p < 0.001 for all cases) between the MetS and non-MetS groups. The average level of SUA was also higher in the MetS group compared to the non-MetS group (p < 0.05). In contrast, subjects in the non-MetS group had a higher level of HDL-C level than the subjects in the MetS group (p < 0.05). Prevalence of MetS and its components in gender and hyperuricemic group. The prevalence of MetS and its components are presented in Table 2 and Fig. 1. Overall, the prevalence of MetS was 22% with 21.9% in males and 22.1% in female subjects. The MetS components such as raised WC, high blood pressure, Scientific Reports \| (2020) 10:7841 \| https://doi.org/10.1038/s41598-020-64884-7 www.nature.com/scientificreports/ Total MetS Non-MetS p-value N 420 93 327 — Gender, m/f 420 57/36 200/127 — Age, year 30.5 ± 12.4 39.5 ± 14.1 27.8 ± 10.4 0.000 WC, cm 83.9 ± 10.6 90.5 ± 12.3 81.4 ± 8.6 0.000 HC, cm 92.8 ± 8.2 96.0 ± 8.6 91.5 ± 7.6 0.000 WHR 0.68 ± 0.39 0.91 ± 0.19 0.62 ± 0.41 0.000 BMI, kg/m2 23.9 ± 3.8 26.3 ± 3.8 23.3 ± 3.6 0.000 SBP, mmHg 118.2 ± 15.1 128.8 ± 18.1 115.1 ± 12.5 0.000 DBP, mmHg 73.6 ± 14.9 81.6 ± 8.4 71.4 ± 15.6 0.000 SUA, µmol/L 319.2 ± 107.8 332.0 ± 111.8 308.2 ± 106.6 0.026 FBG, mg/dL 98.8 ± 37.5 130.4 ± 66.2 89.9 ± 17.7 0.000 TG, mg/dL 135.1 ± 101.6 211.6 ± 107.2 113.5 ± 88.9 0.000 TC, mg/dL 157.7 ± 63.9 189.2 ± 75.7 148.8 ± 56.3 0.000 HDL-C, mg/dL 31.9 ± 17.1 29.6 ± 11.1 33.4 ± 18.5 0.046 LDL-C, mg/dL 93.3 ± 58.3 115.9 ± 73.2 85.9 ± 50.4 0.000 Table 1. Baseline characteristics of the participants based on the presence of MetS. MetS: Metabolic Syndrome. Values are presented as mean ± SD. P-values are obtained from independent sample t-test. Results Dependent variable is MetS (yes) and independent variable is SUA (µmol/L). Reference category is normal (non-MetS). Model 1: adjusted for age (years) and gender (male and female). Model 2: model 1+ BMI (kg/m2) Model 3: model 2+ LDL (mg/dL). OR, odds ratio; CI, confidence interval; SE, Standard error. B SE Wald df OR (95% CI) P-value Model 1 0.011 0.003 10.570 1 1.011 (1.004–1.017) 0.001 Model 2 0.008 0.004 5.727 1 1.008 (1.002–1.016) 0.016 Model 3 0.007 0.004 3.872 1 1.006 (1.000–1.013) 0.042 Table 4. Multinomial logistic regression analysis to evaluate the association between SUA levels and MetS. Dependent variable is MetS (yes) and independent variable is SUA (µmol/L). Reference category is normal (non-MetS). Model 1: adjusted for age (years) and gender (male and female). Model 2: model 1+ BMI (kg/m2) Model 3: model 2+ LDL (mg/dL). OR, odds ratio; CI, confidence interval; SE, Standard error. Table 4. Multinomial logistic regression analysis to evaluate the association between SUA levels and MetS. Dependent variable is MetS (yes) and independent variable is SUA (µmol/L). Reference category is normal (non-MetS). Model 1: adjusted for age (years) and gender (male and female). Model 2: model 1+ BMI (kg/m2) Model 3: model 2+ LDL (mg/dL). OR, odds ratio; CI, confidence interval; SE, Standard error. Table 4. Multinomial logistic regression analysis to evaluate the association between SUA levels and MetS. Dependent variable is MetS (yes) and independent variable is SUA (µmol/L). Reference category is normal (non-MetS). Model 1: adjusted for age (years) and gender (male and female). Model 2: model 1+ BMI (kg/m2) Model 3: model 2+ LDL (mg/dL). OR, odds ratio; CI, confidence interval; SE, Standard error. hyperglycemia and high TG were significantly higher in male than in the female subjects (p < 0.01 for all cases). In contrast, there was no significant difference for low HDL-C between the male-female groups. The prevalence of hyperuricemia was 16.6% with significant differences between the male (21.3%) and female (8.3%) group (p < 0.01). All components of MetS were significantly higher in subjects in the hyperuricemic group compared to the subjects in the non-hyperuricemic group (p < 0.05 for all cases). Baseline characteristics of the study subjects according to SUA quartiles. The baseline charac- teristics of the study participants were also evaluated according to SUA quartiles (Table 3). Results The volunteers were divided into 4 groups based on SUA levels (Q1: ≤ 243.9; Q2: 244–309.3; Q3: 309.4–380.7 and >380.7 µmol/L). No significant difference was observed in the mean age across the SUA quartiles. Participants in the fourth quartile of SUA had a significantly higher WC, WHR, BMI, SBP, DBP, SUA, TG, TC LDL-C and lower FBG and HDL-C than the subjects in the other quartiles of SUA (p < 0.05 for all cases). Association of SUA with the prevalence of MetS and its components. Taking SUA as the inde- pendent variable and MetS as the dependent variable, multinomial logistic regression was performed to assess the relationship between SUA and MetS. The detailed results are presented in Table 4. In regression analysis, a positive association was observed between SUA and MetS. In all regression models, SUA showed a significant association with the prevalence of MetS. We further assessed the relationship of SUA with the individual components of MetS (Table 5). After adjustment for age, a positive association was observed between SUA and the components of MetS except for hyperglycemia and low HDL-C. Results P < 0.05 for all cases when the prevalence of MetS and its component are compared between the groups. P-values are obtained from the Chi-square test. Figure 1. Prevalence of MetS and its components in the hyperuricemic and non-hyperuricemic group. P < 0.05 for all cases when the prevalence of MetS and its component are compared between the groups. P-values are obtained from the Chi-square test. Scientific Reports \| (2020) 10:7841 \| https://doi.org/10.1038/s41598-020-64884-7 www.nature.com/scientificreports/ Q1 ≤ 243.9 µmol/L Q2 244–309.3 µmol/L Q3 309.4– 380.7 µmol/L Q4> 380.7 µmol/L p-value for trend N 104 110 105 101 — Sex, m/f 34/70 57/53 80/25 86/15 — Age, year 34.1 ± 13.9 31.2 ± 11.9 31.2 ± 12.0 31.3 ± 12.3 0.338 WC, cm 81.9 ± 9.9 82.6 ± 9.7 83.8 ± 8.0 87.3 ± 13.5 0.010 HC, cm 91.7 ± 8.4 92.0 ± 8.5 93.2 ± 6.8 94.1 ± 8.7 0.282 WHR 0.72 ± 0.36 0.75 ± 0.34 0.78 ± 0.31 0.88 ± 0.22 0.010 BMI, kg/m2 23.9 ± 3.7 24.2 ± 3.8 24.6 ± 3.5 25.4 ± 3.9 0.008 SBP, mmHg 114.7 ± 15.1 116. 7 ± 13.9 120.5 ± 13.0 124.9 ± 15.2 0.000 DBP, mmHg 69.2 ± 21.1 73.7 ± 12.0 76.0 ± 12.1 77.3 ± 11.9 0.003 SUA, µmol/L 196.2 ± 38.7 282.0 ± 19.7 347.1 ± 20.9 465.6 ± 85.1 0.000 FBG, mg/dL 108.7 ± 55.6 102.7 ± 44.4 90.7 ± 15.8 98.2 ± 28.6 0.025 TG, mg/dL 122.2 ± 73.9 145.9 ± 96.5 156.1 ± 92.4 171.6 ± 122.7 0.012 TC, mg/dL 139.4 ± 71.1 147.4 ± 47.3 164.1 ± 54.9 188.7 ± 77.6 0.000 HDL-C, mg/dL 35.9 ± 12.3 37.7 ± 14.6 31.9 ± 12.1 27.1 ± 13.9 0.000 LDL-C, mg/dL 80.6 ± 68.4 80.9 ± 42.1 98.9 ± 52.7 119.6 ± 66.6 0.000 Table 3. Baseline characteristics of the participants according to SUA quartiles. Values are presented as mean ± SD. P-values are obtained from one-way ANOVA. Table 3. Baseline characteristics of the participants according to SUA quartiles. Values are presented as mean ± SD. P-values are obtained from one-way ANOVA. B SE Wald df OR (95% CI) P-value Model 1 0.011 0.003 10.570 1 1.011 (1.004–1.017) 0.001 Model 2 0.008 0.004 5.727 1 1.008 (1.002–1.016) 0.016 Model 3 0.007 0.004 3.872 1 1.006 (1.000–1.013) 0.042 Table 4. Multinomial logistic regression analysis to evaluate the association between SUA levels and MetS. Discussionh A previous study reported that subjects with hyperuricemia have a higher chance of developing of MetS than in the non-hyperuricemic subjects40. This predictive role of SUA has been demonstrated in individuals who were free of all components of MetS at baseline27. Moreover, a recent clinical study reported that elevated levels of SUA can play a pathogenic role in MetS41. Due to controversial findings, NCEP could not include hyperuricemia as an individual component of MetS yet, and it seems that further longitudinal investiga- tions are required to elucidate whether hyperuricemia is another component of the MetS or not17.i q yp p In regression models, SUA showed a significant association with MetS and several components of MetS. However, after adjusting some confounding factors, we did not find the significant association of SUA with hyper- glycemia and low HDL-C. This might be happened because of the inverse relationship of SUA with the preva- lence of diabetes in the Bangladeshi population26. On the other hand, we observed a very high prevalence of low HDL-C among the participants that could be a reason for the non-significant association of SUA with HDL-C.hh i The underline mechanisms between SUA and MetS are not fully understood yet. The possible mechanisms of SUA in inducing MetS are as follows: First, hyperuricemia has been demonstrated to cause endothelial dysfunc- tion in human and animal models42,43. Second, SUA has been exhibited to hinder the production of NO44, which is considered as important for insulin function45,46. Deficiency of endothelial-formed NO is thought to reduce blood flow to cells leading to stop the insulin action and inducing hyperinsulinemia17. Thus, hyperuricemia may have a potential role in inducing or worsening insulin resistance by itself. In turn, the resistance of insulin is believed to play a vital role in the pathogenesis of MetS47. Although a positive association of hyperuricemia with MetS has been demonstrated through epidemiological and animal studies, the exact mechanisms by which SUA leads to this disorder are still at the beginning need to be explained. Thus, it is obvious that further prospective studies are required to determine the role of SUA in the development of MetS.h There were some limitations to the study. First, the sample size in the present study was relatively small. Second, the cross-sectional nature of the data did not allow us to draw a causal relationship between SUA and MetS. Discussionh The present study investigates the relationship between SUA and MetS in general adults. Although, the associ- ation of SUA with MetS has been studied in diabetic and hypertensive subjects, however, limited studies have documented the information regarding the link of SUA with MetS in general adults. In this study, we first report a positive association of SUA with MetS and its components in general adults in Bangladesh.if In the present study, no significant difference was observed in the prevalence of MetS between the gender groups. Among the MetS components, high TG and reduced-HDL-C were the more common abnormalities found in the participants. Anthropometric variables, lipid profile, FBG and SUA were significantly higher in subjects with MetS than in the subjects who do not have this syndrome. In this study, we observed an increasing Scientific Reports \| (2020) 10:7841 \| https://doi.org/10.1038/s41598-020-64884-7 www.nature.com/scientificreports/ B SE Wald df OR (95% CI) P-value Abdominal obesity 0.009 0.004 6.008 1 1.009 (1.002–1.017) 0.014 High blood pressure 0.008 0.004 4.445 1 1.008 (1.001–1.016) 0.035 Hyperglycemia −0.001 0.001 0.638 1 0.999 (0.997–1.001) 0.425 High TG 0.11 0.003 17.956 1 1.011 (1.006–1.016) 0.000 Low-HDL-C 0.002 0.002 1.024 1 1.002 (0.998–1.007) 0.312 Table 5. Age-adjusted logistic regression analysis to evaluate the association between SUA and the components of MetS. The dependent variable is MetS components (yes) and the independent variable is SUA (µmol/L). The reference category is normal. The model is adjusted for age (years). OR, odds ratio; CI, confidence interval; SE, Standard error. trend in the levels of individual components of MetS across the SUA quartiles. These results are consistent with the findings reported in Japanese30,31, Chinese19, Iranian17, the United States32,33, and European population34. trend in the levels of individual components of MetS across the SUA quartiles. These results are consistent with the findings reported in Japanese30,31, Chinese19, Iranian17, the United States32,33, and European population34. i g Several epidemiological studies demonstrated a positive association between SUA and the prevalence of MetS in human cohorts. However, it is still debated whether increased SUA concentration is a risk factor or only a biomarker in the progression and development of MetS19. Some studies reported that hyperuricemia may be an individual component of the MetS35,36, whereas, other works suggested to include hyperuricemia as an additional component of MetS37,38. In prospective studies, SUA at baseline level was associated with an increased risk of MetS in both genders27,39. Discussionh Moreover, we adjusted some probable confounding factors in determining the relationship between SUA and MetS; however, other confounding factors effects cannot be excluded. For example, we did not have informa- tion on renal function tests and diet habits that can affect SUA levels. Therefore, the present study findings should be generalized cautiously to other general populations. Conclusionsh The present study showed that the components of MetS are raised with the increasing concentrations of SUA across the quartiles. In this study, SUA was significantly associated with the prevalence of MetS and its compo- nents. Given the high prevalence of MetS among Bangladeshi adults, more studies are required to examine the role of SUA in the pathogenesis of MetS. Furthermore, attention should be paid to the increased risk of MetS and hyperuricemia for the general population. Data availability ll l d d All related data are included in this paper. The datasets produced and/or analyzed during the present study are available from the corresponding author upon reasonable request. Received: 10 December 2019; Accepted: 24 April 2020; Published: xx xx xxxx Received: 10 December 2019; Accepted: 24 April 2020; Published: xx xx xxxx References 1. Grundy, S. M. Metabolic syndrome: a multiplex cardiovascular risk factor. J. Clin. Endocrinol. Metab. 92, 399–404 (2007). 2. Thomas, G. N. et al. Metabolic syndrome increases all-cause and vascular mortality: the Hong Kong Cardiovascular Risk Factor Study. Clin. Endocrinol. (Oxf.) 66, 666–671 (2007). 3. Wilson, P. W. F., D’Agostino, R. 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Acta Diabetol. 48, 79–88 (2011). h and normal fasting glucose subjects. Acta Diabetol. 48, 79–88 (201 g g j 18. Nagahama, K. et al. Hyperuricemia predicts future metabolic syndrome: a 4-year follow-up study of a large screened cohort in Okinawa, Japan. Hypertens. Res. 37, 232–238 (2014). p yp 19. Wang, H.-J., Shi, L.-Z., Liu, C.-F., Liu, S.-M. & Shi, S.-T. Association between uric acid and metabolic syndrome in elderly women. Open Med. 13, 172–177 (2018).h p 0. Taniguchi, Y. et al. Serum uric acid and the risk for hypertension and Type 2 diabetes in Japanese men: The Osaka Health Survey. J Hypertens. 19, 1209–1215 (2001). yp 1. Ali, N. et al. Relationship between serum uric acid and hypertension: a cross-sectional study in Bangladeshi adults. Sci. Rep. 9, 9061 (2019).hi 2. Ali, N. et al. The relationship between serum uric acid and lipid profile in Bangladeshi adults. BMC Cardiovasc. Disord. 19, 42 (2019) h p p pi g ( ) 3. Islam, S. et al. 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Scientific Reports \| (2020) 10:7841 \| https://doi.org/10.1038/s41598-020-64884-7 Competing interestsh p g The authors declare no competing interests. Acknowledgementsh g The authors would like to thank all staff and volunteers for their active cooperation in the study. References JCMA 69, 104–109 (2006). 38 Sheu, W H H & Tseng, Y-H Uric acid: an additional component of metabolic syndrome? J Chin Med Assoc JCMA 69, 99–100 37. Liou, T.-L. et al. Is hyperuricemia another facet of the metaboli 37. Liou, T.-L. et al. Is hyperuricemia another facet of the metabolic syndrome? J. Chin. Med. Assoc. JCMA 69, 104–109 (2006). 38. Sheu, W. H. H. & Tseng, Y.-H. Uric acid: an additional component of metabolic syndrome? J. Chin. Med. Assoc. JCMA 69, (2006). , yp y J J , ( ) 38. Sheu, W. H. H. & Tseng, Y.-H. Uric acid: an additional component of metabolic syndrome? J. Chin. Med. Assoc. JCMA 69, (2006). 39. Ryu, S. et al. 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Insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo is NO dependent. Am. J. Physiol. 274, E692–699 (1998). p y 46. Wu, G. & Meininger, C. J. Nitric oxide and vascular insulin resistance. BioFactors Oxf. Engl. 35, 21–27 (2009). 47. Lann, D. & LeRoith, D. Insulin resistance as the underlying cause for the metabolic syndrome. Med. Clin. North Am. 91, 1063–1077 viii (2007) p y 46. Wu, G. & Meininger, C. J. Nitric oxide and vascular insulin resistance. BioFactors Oxf. Engl. 35, 21–27 (2009). h l h d l f h b l d d l h 46. Wu, G. & Meininger, C. J. Nitric oxide and vascular insulin resi Scientific Reports \| (2020) 10:7841 \| https://doi.org/10.1038/s41598-020-64884-7 www.nature.com/scientificreports/ Author contributions N.A. played a major role in the conception and design of the study, data interpretation and wrote the manuscript. R.M., M.H., Z.B. and A.D.M. experimented and analyzed the data. J.M.H., A.D.T., and A.H. helped in sample collection and contributed to results analysis. F.I. helped in result analysis and revision of the manuscript draft. All authors read the manuscript and approved the final version. Additional information Correspondence and requests for materials should be addressed to N.A. Reprints and permissions information is available at www.nature.com/reprints. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2020 Scientific Reports \| (2020) 10:7841 \| https://doi.org/10.1038/s41598-020-64884-7
https://openalex.org/W2158780262	https://europepmc.org/articles/pmc4680163?pdf=render	English	null	Performance characteristics of methods for quantifying spontaneous intracerebral haemorrhage: data from the Efficacy of Nitric Oxide in Stroke (ENOS) trial	Journal of neurology, neurosurgery and psychiatry	2,015	cc-by	7,493	Performance characteristics of methods for quantifying spontaneous intracerebral haemorrhage: data from the Efﬁcacy of Nitric Oxide in Stroke (ENOS) trial Kailash Krishnan,1 Siti F Mukhtar,1 James Lingard,1 Aimee Houlton,1 Elizabeth Walker,1 Tanya Jones,1 Nikola Sprigg,1 Lesley A Cala,2 Jennifer L Becker,3 Robert A Dineen,4 Panos Koumellis,5 Alessandro Adami,6 Ana M Casado,7 Philip M W Bath,1 Joanna M Wardlaw7 ▸Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jnnp-2014-309845). ABSTRACT Background Poor prognosis after intracerebral haemorrhage (ICH) is related to haemorrhage characteristics. Along with developing therapeutic interventions, we sought to understand the performance of haemorrhage descriptors in large clinical trials. Methods Clinical and neuroimaging data were obtained for 548 participants with ICH from the Efﬁcacy of Nitric Oxide in Stroke (ENOS) trial. Independent observers performed visual categorisation of the largest diameter, measured volume using ABC/2, modiﬁed ABC/2, semiautomated segmentation (SAS), fully automatic measurement methods; shape, density and intraventricular haemorrhage were also assessed. Intraobserver and interobserver reliability were determined for these measures. ▸Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jnnp-2014-309845). functional outcome is related independently to increased age, systolic blood pressure, stroke sever- ity and reduced level of consciousness. Radiologically, haemorrhage size, haemorrhage expansion, evidence of continuing bleeding (eg, ‘spot sign’ on CT angiography)2 3 and presence of intraventricular haemorrhage (IVH) are all also associated with poor outcome.4 5 The combination of ICH volume and initial Glasgow Coma scale was reported to be the strongest independent predictor of 30-day outcome.1 Hence, reliable measurement of haemorrhage size is an important component of early clinical management and for stratiﬁcation in clinical trials. For numbered afﬁliations see end of article. Correspondence to Professor Philip M W Bath, Stroke, Division of Clinical Neuroscience, University of Nottingham, Clinical Sciences Building, City Hospital campus, Hucknall Road, Nottingham NG5 1PB UK; philip.bath@ nottingham.ac.uk Although any effective intervention will ultim- ately need to show an effect on functional outcome (eg, using the modiﬁed Rankin Scale (mRS))6 in phase III trials, earlier developments will need to study the effect of treatment on a surrogate measure. Where the intervention aims to limit haemorrhage expansion, accurate, reproducible and validated measurement of haemorrhage size (thereby allowing measurement of the change in volume) will be critical. Performance characteristics of methods for quantifying spontaneous intracerebral haemorrhage: data from the Efﬁcacy of Nitric Oxide in Stroke (ENOS) trial Hence, phase II trials of blood pressure lowering and haemostatic interven- tions have utilised measurement of haemorrhage expansion as a key primary outcome.7 8 Received 3 November 2014 Accepted 8 December 2014 Published Online First 9 January 2015 Results ICH volume was signiﬁcantly different among standard ABC/2, modiﬁed ABC/2 and SAS: (mean) 12.8 (SD 16.3), 8.9 (9.2), 12.8 (13.1) cm3, respectively (p<0.0001). There was excellent agreement for haemorrhage volume (n=193): ABC/2 intraobserver intraclass correlation coefﬁcient (ICC) 0.96–0.97, interobserver ICC 0.88; modiﬁed ABC/2 intraobserver ICC 0.95–0.97, interobserver ICC 0.91; SAS intraobserver ICC 0.95–0.99, interobserver ICC 0.93; largest diameter: (visual) interadjudicator ICC 0.82, (visual vs measured) adjudicator vs observer ICC 0.71; shape intraobserver ICC 0.88 interobserver ICC 0.75; density intraobserver ICC 0.86, interobserver ICC 0.73. Graeb score (mean 3.53) and modiﬁed Graeb (5.22) scores were highly correlated. Using modiﬁed ABC/2, ICH volume was underestimated in regular (by 2.2-2.5 cm3, p<0.0001) and irregular- shaped haemorrhages (by 4.8-4.9 cm3, p<0.0001). Fully automated measurement of haemorrhage volume was possible in only 5% of cases. Open Access Scan to access more free content There are multiple methods for measuring haem- orrhage volume on a CT scan, ranging from quali- tative visual estimation to computerised automatic measurement. Visual approaches (ie, visual size cat- egorisation based on the largest diameter) are quick while computerised methods might provide a con- tinuous volume measure but require access to good quality electronic image ﬁles. A common approach is the ABC/2 method,9 which has been modiﬁed such that haemorrhage slices are excluded if the area is <25% of the largest slice and counted as half if it is 25–75% of the largest slice.10 Alternatively, semiautomatic or fully automatic computerised methods may be used, usually based on manually outlining or thresholding areas or volumes within regions of interest (ROI). The rela- tive advantages and disadvantages of these approaches reﬂect the balance between measure- ment time, type of data required, accuracy and Open Access Scan to access more free content Conclusions Formal measurement of haemorrhage characteristics and visual estimates are reproducible. The standard ABC/2 method is superior to the modiﬁed ABC/ 2 method for quantifying ICH volume. Clinical trial registration ISRCTN9941422. Cerebrovascular disease Cerebrovascular disease RESEARCH PAPER ▸Additional material is published online only. To view please visit the journal online (http://dx.doi.org/10.1136/ jnnp-2014-309845). METHODS Patients Data came from the prospective international multicentre ENOS trial, a study of blood pressure management in patients with acute ischaemic stroke or ICH, and high blood pressure.18 The trial had regulatory and ethics approvals in each participating country, and all patients gave written consent, or a relative pro- vided proxy consent if the patient lacked capacity. The main inclusion criteria were acute stroke with motor deﬁcit, systolic blood pressure of 140–220 mm Hg, and treatment could start within 48 h of onset. Patients were recruited from 173 sites in 23 countries across ﬁve continents. Following the entry of base- line demographic and clinical data into a secure internet-fronted database, patients were randomised to transdermal glyceryl tri- nitrate (5 mg) versus none for 7 days; those taking antihyperten- sive agents immediately prior to their stroke were also randomised to continue versus stop these, again for 7 days. For SAS and AVC, it was possible to use thresholds so that the haemorrhage ROI was measured within certain segmentation parameters. Upper and lower attenuation values (typically 40– 80 HU) of haemorrhage were established manually by sampling from the haemorrhage and normal brain. Where the threshold did not exclude non-haemorrhage areas (as with the pineal gland, calciﬁed choroid plexus, bone) in close proximity with the haemorrhage, the haemorrhage boundary was manually edited. The area of each slice that was included within these parameters was measured, added together and multiplied by scan thickness to obtain the SAS volume. AVC used an Osirix ‘3D growing region (entire series)’ method after selecting a threshold part of the haemorrhage near to its centre. With all methods, IVH blood was included in the ﬁnal ICH volume. Participants had a baseline CT or MRI brain scan as part of clinical care, usually before randomisation. Where possible, a second (research) CT or MRI scan was performed at day 7±1 (end-of-treatment). DICOM, JPEG, PNG or GIF ﬁles are sent to the coordinating centre. Any scans sent on ﬁlm were digitised using a VICOM digitiser (VIDAR Diagnostic PRO Advantage, USA). The following study assesses only those patients with a CT scan. For each patient, images were received with the thin- nest slices provided by CT scan machines using standardised protocols in the recruiting centres. However, during the course of the trial, more volumetric images were made available because of advances in imaging technology. METHODS Patients Additionally, four haemorrhage characteristics (area, perimeter, mean attenuation and SD of attenuation) were recorded for the haemorrhage from the slice with the largest area of haemorrhage. Haemorrhage shape and density indices were then calculated as: Shape index ½ref: 22] ¼ perimeter2=4 Y area Density index ½ref: 23] ¼ SD=meanðie; coefficient of variationÞ Shape index ½ref: 22] ¼ perimeter2=4 Y area Density index ½ref: 23] ¼ SD=meanðie; coefficient of variationÞ Shape index ½ref: 22] ¼ perimeter2=4 Y area Scan adjudication The divergence in visual appearance of haemorrhage shape and density were also assessed used an ordered categorical scale (1–5) where for an extra lesion edge or degree of density variation an additional point was given on the shape scale or density scale.15 Complete CT scan series were made available for assessment by a group of seven adjudicators comprising accredited neurora- diologists or neurologists trained in CT brain imaging assess- ment in stroke (http://www.neuroimage.co.uk/sirs, with coordination by JMW).19 Images were viewed over the web, blind to all clinical and treatment information except for patient age, time since onset of stroke and side of brain symptoms. Responses were entered directly into the trial database via a web-based response form. Collected information included whether the patient had an ICH and its location; an estimate of its size (sorted into ordered categories based on the longest diameter in any plane): <3, 3–4.9, 5–8, >8 cm; and the pres- ence of other qualitative ﬁndings (mass effect,19 atrophy,19 white matter disease,20 old infarct or haemorrhage) using vali- dated scoring methods. Similarly, intraventricular blood volume was assessed visually using the Graeb score and modiﬁed Graeb ordered categorical scales.16 17 The Graeb score is calculated by scoring the amount of blood in the lateral ventricles separately (score of 1=trace of blood to a maximum of 4=ventricle full of blood and expanded) and adding this to the scores for the third and fourth ventricles separately (1=blood present and 2=ﬁlled with blood and expanded) to derive a maximum score of 12. The modiﬁed Graeb score allocates scores for separate ventricular compart- ments (1≤25% ﬁlled with blood to 4≥75% ﬁlled with blood) to reﬂect selective regional accumulation of blood and an extra one point for expansion of each ventricle. The maximum score is 32. INTRODUCTION Spontaneous intracerebral haemorrhage (ICH) is usually a severe form of stroke with a high mortal- ity rate (∼50%) by the end of the ﬁrst year.1 Poor Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 1258 Cerebrovascular disease availability of computer workstations, and sample size, as studied in part.11–14 Assessment of additional properties of haemorrhage, such as shape and density15 and extension into the ventricles or subarachnoid space, may also be useful.16 17 slices). Both observers assessed scans blinded to each other’s data, and repeated a proportion of the scans (47 and 34 scans for KK and SFM, respectively) at a different time blinded to their original measurements. Thus, intraobserver and interobser- ver variation could be estimated. For the remaining 355 patients, one observer (KK) calculated ICH volumes using all four methods. slices). Both observers assessed scans blinded to each other’s data, and repeated a proportion of the scans (47 and 34 scans for KK and SFM, respectively) at a different time blinded to their original measurements. Thus, intraobserver and interobser- ver variation could be estimated. For the remaining 355 patients, one observer (KK) calculated ICH volumes using all four methods. In this study, we assessed and compared methods for measur- ing haemorrhage size, shape and density and IVH size. The data come from the ‘Efﬁcacy of Nitric Oxide in Stroke’ (ENOS) trial,18 which assessed the management of blood pressure in acute stroke and included patients with ICH. The ABC/2 method requires measurement of the longest diameter of the haemorrhage in the axial plane (‘A’), longest axis at 90° to ‘A’ in the axial plane (‘B’), and the number of slices (‘C’) showing the haemorrhage (noting that slice thickness may vary through the scan). ‘C’ is the product of slice thickness and number of slices showing any haemorrhage. Volume is calcu- lated as A×B×C/2.9 In the modiﬁed ABC/2 method, the area of haemorrhage seen on a slice has to be at least 25% of the haem- orrhage area seen on the largest slice if the slice is to be consid- ered in ‘C’; if the area is more than 75% of the largest area, the slice counts as 1, and 0.5 if the measured value of the area is between 25% and 75%.10 Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 Scan haemorrhage quantiﬁcation l d d l d Intraobserver and interobserver reliability was determined for visual categorisation of shape and density. In addition, intraobserver reliability was assessed for measured shape, density index, Graeb, modiﬁed Graeb and IVH volume over different reading sessions, separated by a minimum of 14 days. All adjudication and haemor- rhage measurements were made blind to baseline and follow-up clinical information, other imaging and treatment assignment. Scans were visualised and analysed using OsiriX for Mac (V.3, 32 bit, http://www.osirix-viewer.com21 on a 26 inch Apple iMac. Two trained observers (KK, SFM) measured baseline ICH volumes of 193 patients using ABC/2, modiﬁed ABC/2, semiautomatic seg- mentation (SAS) and automatic volume calculation (AVC, using three-dimensional (3D) rendering of a stack of two-dimensional 1259 Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 RESULTS Altogether, 629 patients with ICH were enrolled into ENOS.25 26 Of these, 548 patients had CT-conﬁrmed ICH and a baseline scan available for measurement; 81 other patients either had ICH diagnosed on MRI or had no CT scan available, and these were excluded. Patient demographic and clinical details are shown in table 1. The mean age of the 548 patients in the present analysis was 67 (SD 12) years, 66% of patients were male, mean baseline blood pressure was 171 (19)/92 (13) mm Hg, and the median time from onset of ictus to per- forming neuroimaging was 4.5 (IQR 5.6) hours. When adjudi- cated visually by experts, 63% of haematomas were located in the middle cerebral artery territory (table 1); most haemor- rhages caused a mass effect (86%) and many patients had leu- koaraiosis (66%) and/or a previous stroke lesion (49%) present on their scans. The most frequent visually assessed haemorrhage length category was 3–5 cm (224, 41.3%), closely followed by <3 cm (220, 40.6%) with much fewer larger haemorrhages. The mean measured haemorrhage length was 3.4 cm (longest diameter; table 1). Statistical analysis Statistical analysis y Data are shown as number (%), median (IQR) or mean (SD). Measurement of intraobserver and interobserver variability was assessed using the intraclass correlation coefﬁcient (ICC).24 A probability value of <0.05 was considered statistically signiﬁ- cant. Analyses were performed using SPSS (V.21) and checked with Medistat running on an Apple Mac. Cerebrovascular disease Table 1 Baseline demographic, clinical and neuroradiological factors in 548 patients with primary intracerebral haemorrhage in the ENOS trial Variable Data Demographics Age (years) 67.9 (12.1) Sex, male (%) 360 (65.7) Country, UK (%) Asia 117 (21.4) Europe 67 (21.4) Other (Africa, Australasia and North America) 42 (7.7) UK 322 (58.8) Clinical findings Premorbid modified Rankin Scale=0 (%) 418 (76.3) Previous stroke (%) 69 (12.6) Prior antihypertensive medication use (%) 227 (41.4) Prior history of high BP (%) 341 (62.2) Diabetes mellitus (%) 67 (12.2) Ischaemic heart disease (%) 56 (10.2) Atrial fibrillation (%) 30 (6.5) Total anterior circulation syndrome (%) 195 (35.6) SSS (/58) 30.1 (12.3) National Institutes of Health Stroke Scale (/42) 12.8 (5.3) Systolic BP (mm Hg) 171.6 (19.3) Diastolic BP (mm Hg) 92.2 (13.3) Heart rate (bpm) 77.7 (14.5) Time, stroke to neuroimaging (h) 4.5 [11.6] Adjudicated CT scan findings Location of haemorrhage (%) MCA 346 (63.1) ACA 22 (4.0) PCA 5 (0.9) MCA+ACA 2 (0.4) Borderzone 14 (2.6) Lacunar (ie, small subcortical) stroke 142 (25.9) Brainstem and/or cerebellum 16 (2.9) Leukoariosis 362 (66.1) Lesion mass effect (minimal to extreme swelling) 470 (85.9) Previous stroke lesion 270 (49.3) IVH 141 (25.7) Intracerebral haematoma size category (%) <3 cm 220 (40.6) 3 to <5 cm 224 (41.3) 5 to 8 cm 87 (16.1) >8 cm 11 (2.0) Measured CT scan findings Volume, ABC/2 (cm3) 12.77 (16.32) Longest diameter (cm) 3.38 (1.4) With IVH, n=141 Graeb score (/12)16 3.52 (2.4) Modified Graeb score (/32)17 5.19 (4.7) Without IVH, n=407 Shape (/5)15 3.00 (1.4) Shape index22 1.22 (1.1) Density (/5)15 2.54 (1.3) Density index23 0.19 (0.1) Data are number (%), median (semiquartile range), or mean (SD). NIHSS calculated from SSS scores range from 0 (comatose with quadriplegia) to 58 (normal neurological status). ACA, anterior cerebral artery territory; BP, blood pressure; ENOS, Efficacy of Nitric O id i St k IVH i t t i l h h MCA iddl b l t ICH volume l ICH volume was signiﬁcantly different between ABC/2, modi- ﬁed ABC/2 and SAS: 12.8 (mean) (SD 16.3), 8.9 (9.2), 12.8 (13.1) cm3, respectively (p<0.0001) (table 2). The ICC was ‘excellent’ at 0.84–0.96 when comparing the observers’ measure- ments for each of ABC/2, modiﬁed ABC/2 and SAS (table 2). The observers found haemorrhage volume to be larger, by an average of 2.7–4 cm3 with standard ABC/2, as compared to modiﬁed ABC/2, and 1.4–4 cm3 smaller with SAS as compared to modiﬁed ABC/2. As the mean ICH value increased, the differ- ence between ICH volume measured by modiﬁed ABC/2 versus the other two methods increased (ﬁgures 1 and 2), but no signiﬁ- cant difference was observed between standard ABC/2 and SAS (table 2; ﬁgure 3). The slope of the best-ﬁt regression line for the increasing difference between modiﬁed ABC/2 versus ABC/2 was −0.44, and −0.36 for modiﬁed ABC/2 versus SAS (both p<0.0001) (ﬁgures 1 and 2). There was good intraobserver agreement for measurements assessed on 34–47 scans (5–10% of the total of 548). For ABC/2, modiﬁed ABC/2 and SAS, the intraobserver ICC was ‘excellent’, ranging between 0.97–0.98, and 0.95–0.99, respectively (table 3). There was excellent interobserver agreement (ICC) based on 193 scans at 0.88 for ABC/2, 0.91 for modiﬁed ABC/2 and 0.93 for SAS (table 4). Both ABC/2 and modiﬁed ABC/2 showed excellent correlation with SAS (p<0.0001, see online supplementary ﬁgures S1–S3). Only 23 of 548 scans were amen- able to analysis using the fully AVC method as the software was unable to handle the scan image with varying slice thickness; this approach was therefore ignored in further analyses. Across the ICH visual size categories, ICH volumes calculated by modiﬁed ABC/2 were signiﬁcantly smaller compared to SAS (ﬁgure 4). As the ICH size category increased, the difference between modiﬁed ABC/2 and SAS also increased. By compari- son, there was no signiﬁcant difference between ICH volumes Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 1260 Cerebrovascular disease modiﬁed ABC/2, haemorrhage volume was signiﬁcantly lower for regular-shaped haemorrhages when compared with ABC/2 (by 2.2 cm3) and SAS (by 2.4 cm3) (table 5 and ﬁgure 5); the difference was greater with larger irregular-shaped haemor- rhages (between standard ABC/2 and modiﬁed ABC/2 by 4.8 cm3 and SAS and modiﬁed ABC/2 by 4.9 cm3). When com- pared by shape, ICH volume calculated by standard ABC/2 did not differ from that measured by SAS (ﬁgure 5). IVH volume and severity I b f Intraobserver agreement for assessment of IVH volume on base- line scans from 49 patients was excellent (ICC 0.98–0.99) whether using the Graeb score, modiﬁed Graeb score or SAS (see online supplementary table III). The Graeb and modiﬁed Graeb scores were highly correlated with each other (rs=0.88, p<0.01). Both were also highly correlated with measured IVH volume using the SAS method: Graeb, (rs=0.73, p<0.01); modiﬁed Graeb (rs=0.72, p<0.01). measured by standard ABC/2 and SAS when compared by ICH visual size categorisation (ﬁgure 4). There was good agreement between the adjudicators across ICH visual size categories (n=47, ICC 0.82, p<0.001) (see online supplementary table I) using the ordered categorical scale (<3, 3–4.9, 5–8, >8 cm)19 and strong agreement between the visual size category and the observer’s largest measured diameter in the axial plane ‘A’ (ICC 0.71, p<0.001) (see online supple- mentary table II). Comparison of ICH volume by ABC/2, modiﬁed ABC/2 to SAS We performed a non-systematic review to compare our study with previously published work comparing ABC/2, modiﬁed ABC/2 and SAS volume measures in ICH10–14 27–30 (see online supplementary ﬁgures S4–S7). Some studies used the standard ABC/2 formula while others used variations of it;10 11 Gebel et al11 used the central haemor- rhage slice to measure the largest diameter ‘A’ and found higher volumes computed by ABC/2 when compared to SAS. With regard to shape, three studies studied errors in warfarin-related haemorrhage due to higher frequencies of irregular shape.12–14 ICH volume l Table 2 Comparison by two observers of different methods for measuring haemorrhage volume (cm3) on CT scans: ABC/2 versus modified ABC/2 versus SAS Haematoma volume Difference p Value ICC Observer 1 n=548 ABC/2 12.77 (16.32) Modified ABC/2 8.90 (9.21) 3.96 <0.0001 0.84 ABC/2 12.77 (16.32) SAS 12.76 (13.06) −0.01 1.00 SAS 12.76 (13.06) Modified ABC/2 8.90 (9.21) 3.97 <0.0001 Observer 2 n=193 ABC/2 12.05 (12.40) Modified ABC/2 9.70 (10.05) 2.67 <0.0001 0.96 ABC/2 12.05 (12.40) SAS 11.08 (11.38) 0.05 0.89 SAS 11.08 (11.38) Modified ABC/2 9.70 (10.05) 1.38 <0.0001 Data are mean (SD), difference (Δ) in haemorrhage volume between ABC/2 and SAS and ICC. ICC, intraclass correlation coefficient; SAS, semiautomated segmentation. ICH shape and density in patients with no IVH Intra-agreements and interagreements were both ‘good’ for visual assessments of haemorrhage shape: intraobserver ICC 0.88 (see online supplementary table III), interobserver ICC 0.75 (n=47) (see online supplementary table IV); and density: intraobserver ICC 0.86, interobserver ICC 0.73. Intra-observer ICC was 0.53 for calculated shape index and 0.86 for density index (see online supplementary table III). DISCUSSION Huttner et al12 showed that volumes of regular shaped haema- tomas using ABC/2 were signiﬁcantly increased in regular and irregular shaped haemorrhages. However, the large slice thick- ness used during CT image acquisition in this study may have produced signiﬁcant errors during calculation.12 To the best of our knowledge, this is the ﬁrst study that com- pares several methods for assessing volume of spontaneous ICH and IVH volume on CT scanning and tests the effect of haema- toma shape and regularity on these measures. The major ﬁnd- ings are as follows: (1) Agreement within and between observers was excellent for measures of haemorrhage volume (ABC/2, modiﬁed ABC/2, SAS) and other haemorrhage parameters (visual categorisation by maximum length, description of shape, density and IVH); (2) haemorrhage volumes measured by modi- ﬁed ABC/2 were signiﬁcantly lower than those measured by ABC/2 and SAS planimetry; the discrepancy increased as the haematoma size became larger; (3) larger haemorrhages were signiﬁcantly more irregular in shape; (4) agreement was good between visual size categorisation and measured computerised To the best of our knowledge, this is the ﬁrst study that com- pares several methods for assessing volume of spontaneous ICH and IVH volume on CT scanning and tests the effect of haema- toma shape and regularity on these measures. The major ﬁnd- ings are as follows: (1) Agreement within and between observers was excellent for measures of haemorrhage volume (ABC/2, modiﬁed ABC/2, SAS) and other haemorrhage parameters (visual categorisation by maximum length, description of shape, density and IVH); (2) haemorrhage volumes measured by modi- ﬁed ABC/2 were signiﬁcantly lower than those measured by ABC/2 and SAS planimetry; the discrepancy increased as the haematoma size became larger; (3) larger haemorrhages were signiﬁcantly more irregular in shape; (4) agreement was good between visual size categorisation and measured computerised This review comparing all three methods showed that the ABC/2 formula tended to marginally overestimate the ICH volume, but there was no absolute signiﬁcant difference com- pared to SAS (see online supplementary ﬁgure S4); when assessed by variability in haemorrhage shape, the results follow the same sequence (see online supplementary ﬁgure S5). By comparison, ICH volumes computed using modiﬁed ABC/2 are signiﬁcantly smaller when compared with SAS; the difference is greater as the haemorrhage sizes became larger and more irregu- lar (see online supplementary ﬁgures S6 and S7). ICH volume and shape The most common shape was irregular (64%), followed by regular ICH. Small ICHs (5.5–8.06 cm3) were more regularly shaped than larger haemorrhages (p<0.0001) (table 5). Using Figure 1 The Bland–Altman plot for assessment of variation in estimating haematoma volume between modiﬁed ABC/2 and standard ABC/2 (n=548), r2=0.64, p<0.0001. The continuous and dotted lines represent the regression lines. The slope of the best-ﬁt regression line gradient was −0.44 (p<0.0001). Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 1261 Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 Figure 2 The Bland–Altman plot for assessment of variation in estimating haematoma volume between modiﬁed ABC/2 and semiautomatic segmentation (n=548), r2=0.45, p<0.0001. The continuous and dotted lines represent the regression lines. The slope of the best-ﬁt regression line was −0.36 (p<0.0001). Cerebrovascular disease DISCUSSION Figure 3 The Bland–Altman plot for assessment of variation in estimating haematoma volume (n=548) using ABC/2 and semiautomatic segmentation (n=548), r2=0.03, p<0.0001. The continuous and dotted lines represent the regression lines. The slope of the best-ﬁt regression line was 0.02 (p<0.0001). 1262 Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 1262 Cerebrovascular disease Table 3 Intraobserver comparison for two observers of haemorrhage size (n=47, 34 scans, respectively) on baseline CT scans from patients with ICH Difference (p) ICC Difference (p) ICC Observer 1 2 Measurement 1 2 1 2 Modified ABC/2 (cm3) 8.67 (8.34) 9.10 (8.34) 0.43 (0.80) 0.97 16.88 (14.60) 16.90 (14.63) 0.016 (0.97) 0.95 ABC/2 (cm3) 10.42 (10.28) 10.98 (10.79) −0.55 0.97 20.97 (19.32) 20.15 (17.98) 0.82 (0.39) 0.96 SAS (cm3) 11.11 (10.38) 11.69 (10.99) 0.58 (0.80) 0.98 19.41 (15.32) 19.42 (15.51) −0.11 (0.87) 0.99 Diameter (A) (cm) 3.41 (1.28) 3.37 (1.31) 0.04 (0.89) 0.98 3.89 (1.48) 4.01 (1.45) −0.12 (0.43) 0.96 Data are mean (SD), difference in means, and ICC. ICC, intraclass correlation coefficient; ICH, intracerebral haemorrhage; SAS, semiautomatic segmentation volume. instance, it was difﬁcult to set segmentation parameters to iden- tify a haemorrhage that was adjacent to the bone; and the threshold may miss an area of haemorrhage. Additionally, the threshold method will not account for oedema associated with the ICH, which increases the mass effect; thus, the space- occupying effect of the ICH may be much larger than its mea- sured hyperattenuated area. As segmentation is semiautomatic, it is feasible to adjust manually for errors if they are apparent, although any adjustment may be subjective. All images require visual checking and manual correction; failure to do this would result in erroneous measurements. The problem is compounded by more irregular spontaneous haemorrhages, for example, due to amyloid, in anticoagulant-associated bleeds and in traumatic haematomas, where typically lesions are irregular, of varied attenuation and often next to the bone. measurement of maximum haemorrhage diameter; (5) attempts to perform a fully automated volume measurement method failed in more than 95% of patients, as the software was unable to work with scans in which slice thicknesses varied. The sources of error that effect variation between ABC/2 and SAS merit consideration. First, the largest diameter of haemor- rhage was measured in the axial plane but this may not be the largest ICH diameter, which may also have contributed to dif- ferences between ‘A’ and the visual size categorisation. DISCUSSION Sucu et al31 suggested using the maximum length and width not necessarily on the same slice and found better correlation between ABC/2 and SAS. However, this adaptation applies only to chronic subdural haematomas which differ from spontaneous haemorrhages by extending to the cranial vault and being cres- centic.31 Second, when using ABC/2, the scan plane is measured through AB/2, the formula for the area of a triangle. However, a triangle is not necessarily the most appropriate description of a haematoma. Last, standard ABC/2 approximates the haematoma volume as an ellipsoid with all three axes extending in three per- pendicular directions9; to compensate for the varying slice thickness, ‘C’ is derived by multiplying the number of slices of which the haematoma is seen by the slice thickness in centi- metres. For the modiﬁed ABC/2, ‘C’ does not include slices if the area of the haemorrhage is less than 25% of the largest area,10 an approach that has no theoretical justiﬁcation. Hence, this method does not estimate all three Cartesian coordinates of the ellipsoid. Since the modiﬁed ABC/2 method underestimated the ICH volume (by 2–4 cm3), and small differences in volume equate to the variation in outcome,32 the modiﬁed ABC/2 method cannot be recommended. Further, modern CT scanners provide thin slices and the ability to directly measure ‘C’, thereby eliminating the need for approximating slice areas.33 34 In this series of patients with spontaneous ICH, a high pro- portion (64%) of irregular-shaped haemorrhages were found. The effectiveness of the ABC/2 method has been validated in regular, oval-shaped haemorrhages but researchers have doubted its accuracy in complex irregular haemorrhages (eg, as seen with warfarin or in large or amyloid-related haemorrhage) and those with intraventricular extension.12 35 It has been suggested that adjusting the denominator from 2 to 3 in ABC/2 in irregularly shaped haematomas may produce more accurate measures, but this concept is yet to be supported.12 14 Combining the present and published ﬁndings,29 it may be postulated that as the haem- orrhage became more irregular, the surface area and volume changes more than the largest diameter. Hence, the area of the largest haemorrhage slice may be more representative of the total haemorrhage size than its diameter. Cerebrovascular disease Figure 4 Box plots of intracerebral haemorrhage (ICH) volumes (n=548) by visually estimated size and corresponding volume measured by ABC/2, modiﬁed ABC/2 and semiautomatic segmentation (SAS). The difference between modiﬁed ABC/ 2 versus standard ABC/2 and modiﬁed ABC/2 versus SAS increases as the size category increases and is present in all four size categories (<3 (p<0.0001), 3–5 (p<0.001), 5–8 (p<0.0001) and >8 (p<0.0001)). There was no signiﬁcant difference between standard ABC/2 and SAS in all four size categories. increases the potential for errors. Furthermore, observers may have chosen slightly different window settings. Additionally, although the observers were all trained in using OsiriX, accuracy in measurement does depend on operator experience.33 36 volume of an irregular haemorrhage may be better estimated using SAS. This ﬁnding is important since irregular shape hae- morrhages are more likely to expand and affect morbidity and mortality.15 Our study is novel in design as it compares two commonly used methods ABC/2 and the modiﬁed formula (25%/75% distinction) with computer-assisted SAS and pragmatic visual scoring. Moreover, haemorrhage shape was incorporated in the analysis so that the source of error within each individual measurement method was assessed further. Our results show good compatibility between ABC/2 and computer-assisted SAS, irrespective of haema- toma shape and poor approximation using modiﬁed ABC/2. When comparing haemorrhage size on the basis of its longest measured diameter (‘A’ in ABC/2) with an adjudicated visual cat- egorisation, there was ‘strong’ agreement, within the adjudica- tors and between the observers and adjudicators, with ICC 0.72. Hence, it is unsurprising that the visual category versus the largest measured axial diameter has a slightly lower agree- ment than some of the other measured values. Note that the visual categorisation assigns an ordinal value based on the largest size in any plane, not just the axial plane, and thus is not the same as a linear measure of largest axial diameter. There are several potential explanations for variation in inter- observer performance. Thinner slices may produce more accur- ate volume measurement,33 34 although the scans were identical for each observer. DISCUSSION As a result, the true Table 5 Comparison of volumes (cm3) using ABC/2, modified ABC/2 and semiautomated segmentation (SAS) (n=548) by haematoma shape Haematoma volume Difference (Δ) p value Regular (1,2) n=198 ABC/2 8.06 (10.43) Modified ABC/2 5.56 (6.31) 2.51 <0.0001 ABC/2 8.06 (10.43) SAS 7.77 (8.75) 0.29 0.20 SAS 7.77 (8.75) Modified ABC/2 5.56 (6.31) 2.21 <0.0001 Irregular (3,5) n=350 ABC/2 15.48 (18.37) Modified ABC/2 10.66 (10.07) 4.81 <0.0001 ABC/2 15.48 (18.37) SAS 15.62 (14.22) −0.14 0.82 SAS 15.62 (14.22) Modified ABC/2 10.66 (10.07) 4.96 <0.0001 Data are mean (SD), difference (Δ) in volume and p value. Table 5 Comparison of volumes (cm3) using ABC/2, modified ABC/2 and semiautomated segmentation (SAS) (n=548) by haematoma shape Although computer-assisted methods are considered the gold standard for volume measure,1 11 12 35 the SAS method was i i d i h i ll h ll i F Although computer-assisted methods are considered the gold standard for volume measure,1 11 12 35 the SAS method was time consuming and at times technically challenging. For standard for volume measure, the SAS method was time consuming and at times technically challenging. For Table 4 Interobserver comparison for two (observer 1 and 2; n=193 scans) of haemorrhage volume (cm3) on CT scans from patients with intracerebral haemorrhage (ICH) Observer 1 2 Difference (Δ) ICC ABC/2 10.58 (10.20) 12.05 (12.40) −1.40 0.88 Modified ABC/2 8.42 (8.53) 9.70 (10.05) −1.3 0.91 SAS 12.02 (12.05) 11.08 (11.38) 0.95 0.93 Data are mean (SD), difference (Δ) in haemorrhage volumes and ICC. ICC, intraclass correlation coefficient; SAS, semiautomatic segmentation. Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jn 1263 Figure 4 Box plots of intracerebral haemorrhage (ICH) volumes (n=548) by visually estimated size and corresponding volume measured by ABC/2, modiﬁed ABC/2 and semiautomatic segmentation (SAS). The difference between modiﬁed ABC/ 2 versus standard ABC/2 and modiﬁed ABC/2 versus SAS increases as the size category increases and is present in all four size categories (<3 (p<0.0001), 3–5 (p<0.001), 5–8 (p<0.0001) and >8 (p<0.0001)). There was no signiﬁcant difference between standard ABC/2 and SAS in all four size categories. Cerebrovascular disease REFERENCES 1 Broderick JP, Brott TG, Duldner JE, et al. Volume of intracerebral hemorrhage. A powerful and easy-to-use predictor of 30-day mortality. Stroke 1993;24: 987–93. 2 Wada R, Aviv RI, Fox AJ, et al. CT angiography “spot sign” predicts hematoma expansion in acute intracerebral hemorrhage. Stroke 2007;38:1257–62. 3 Almandoz JED, Yoo AJ, Stone MJ, et al. Systematic characterization of the computed tomography angiography spot sign in primary intracerebral hemorrhage identiﬁes patients at highest risk for hematoma expansion the spot sign score. Stroke 2009;40:2994–3000. In conclusion, the modiﬁed ABC/2 formula signiﬁcantly under- estimates haemorrhage volume when compared to standard ABC/ 2 and computer assisted semiautomated segmentation (SAS) the difference increased as haematoma volume became larger and more irregular in shape. Most relevant for clinicians, our results show that the standard ABC/2 method offers more accurate quan- tiﬁcation of ICH volume by the bedside. Although SAS is consist- ent and reliable, the method is slow, reliant on manual correction, advanced software and likely to cause delay in clinical decision-making often in emergency situations. Further research is required to create a faster computerised volumetric model for accurate and reliable measurement of haematomas in the clinical setting. Visual assessment using an ordered categorical scale has the strength of providing an ‘instant’ marker of haemorrhage size in the acute situation and can be applied in the absence of meas- urement tools and historical cut ﬁlm data, and where very large study sizes or very moved images preclude computer based ABC/ 2 or SAS measurements. This study importantly shows that haem- orrhage characteristics can be reliably measured as accuracy is pivotal to clinical trials in which ICH volume change may be a surrogate end point. 4 Mendelow AD, Gregson BA, Fernandes HM, et al. Early surgery versus initial conservative treatment in patients with spontaneous supratentorial intracerebral haematomas in the International Surgical Trial in Intracerebral Haemorrhage (STICH): a randomised trial. 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See: http://creativecommons.org/ licenses/by/4.0/ REFERENCES Efﬁcacy and safety of recombinant activated factor VII for acute intracerebral hemorrhage. N Engl J Med 2008;358:2127–37. 9 Kwak R, Kadoya S, Suzuki T. Factors affecting the prognosis in thalamic hemorrhage. Stroke 1983;14:493–500. 10 Kothari RU, Brott T, Broderick JP, et al. The ABCs of measuring intracerebral hemorrhage volumes. Stroke 1996;27:1304–5. 11 Gebel JM, Sila CA, Sloan MA, et al. Comparison of the ABC/2 estimation technique to computer-assisted volumetric analysis of intraparenchymal and subdural hematomas complicating the GUSTO-1 trial. Stroke 1998;29: 1799–801. 11 Gebel JM, Sila CA, Sloan MA, et al. Comparison of the ABC/2 estimation technique to computer-assisted volumetric analysis of intraparenchymal and subdural hematomas complicating the GUSTO-1 trial. Stroke 1998;29: 1799–801. 12 Huttner HB, Steiner T, Hartmann M, et al. Comparison of ABC/2 estimation technique to computer-assisted planimetric analysis in warfarin-related intracerebra parenchymal hemorrhage. Stroke 2006;37:404–8. 12 Huttner HB, Steiner T, Hartmann M, et al. Comparison of ABC/2 estimation technique to computer-assisted planimetric analysis in warfarin-related intracerebral parenchymal hemorrhage. Stroke 2006;37:404–8. Acknowledgements The authors thank the investigators and patients who participated in ENOS. Acknowledgements The authors thank the investigators and patients who participated in ENOS. Funding ENOS was funded by the UK BUPA Foundation and Medical Research Council (G0501797). PMWB is The Stroke Association Professor of Stroke Medicine. JMW received funding from the Scottish Funding Council and Chief Scientist Ofﬁce SINAPSE Collaboration (Scottish Imaging Network, A Platform for Scientiﬁc Excellence, http://www.sinapse.ac.uk). This study has several strengths including the use of multiple assessors, multiple methods for assessing haemorrhage characteristics, and a large data set. To minimise measurement error, observers were trained to use the OsiriX software for measuring brain CT scan parameters and in recognition of hae- morrhages. Similarly, CT adjudicators were experienced neuror- adiologists. Multiple observers were used to allow measurement of intraobserver and interobserver variation. The data set involved patients from ﬁve continents and so the ﬁndings have excellent external validity. Contributors The study was conceived by KK and PMB with imaging guidance led by JMW. KK wrote and prepared the ﬁrst draft of the manuscript with input from the writing committee, all of whom approve for the ﬁnal version of the manuscript to submitted and agree to be accountable for all aspects of the work undertaken in this research project. Cerebrovascular disease Alternatively, each additional slice to measure g Our study also shows that qualitative descriptors of haemor- rhage characteristics of haemorrhage shape, density, presence of Figure 5 Box plots of intracerebral haemorrhage (ICH) volumes (n=548) by visually assessed shape and corresponding volume assessed by ABC/2, modiﬁed ABC/2 and semiautomatic segmentation (SAS) show: (1) larger ICHs are irregular in shape (p<0.0001); (2) the mean difference between modiﬁed ABC/2 versus standard ABC/2 and modiﬁed ABC/2 versus SAS increased as the haematoma shape became more irregular (p<0.0001). No signiﬁcant difference was observed between standard ABC/2 and SAS. 1264 Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 1264 Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 Competing interests None declared. Ethics approval UK and local research ethics committees in each participating country. Nevertheless, the study has three signiﬁcant limitations. First, the data come from a randomised controlled trial in acute stroke. Trial exclusion criteria can limit the type of patients (and therefore vari- ation in haemorrhage) so that the data set studied here did not include patients with normal/low blood pressure, GCS<8, or without motor signs. As a result, patients with very large haema- toma were under-represented. Second, the time from stroke onset to neuroimaging was relatively long (median 4.2 h), reﬂecting that the ENOS protocol allowed enrolment up to 48 h after stroke. Hence, ICH on later scans may already have developed some peri-ICH oedema. Finally, just over 25% of the patients had meas- urable IVH, and therefore the results do not represent a population where IVH is more frequent. The failure to get a fully automated volume measure in 95% of scans may be less of a problem with other software that can handle scans of variable slice thickness. Ethics approval UK and local research ethics committees in each participating country. Cerebrovascular disease Cerebrovascular disease IVH using the Graeb and modiﬁed Graeb scores can be reliably measured and are reproducible. IVH using the Graeb and modiﬁed Graeb scores can be reliably measured and are reproducible. 5Department of Neuroradiology, Nottingham University Hospitals, Queen’s Medical Centre, Nottingham, UK 6Stroke Centre, Ospedale Sacro Cuore Via Sempreboni, Verona, Italy 7Division of Neuroimaging Sciences, Centre for Clinical Brain Sciences, Western General Hospital, Edinburgh, UK Of note, when measuring density heterogeneity, we used only the coefﬁcient of variation (cv), whereas Barras used four add- itional measures (although these were apparently inferior to cv).23 Calculation of shape index using parameters obtained from the windowed haemorrhage had a low ICC of 0.53. When this result is held in context with similar computed volumes, it could be interpreted that the observer chose different threshold settings which explain the source of variability. Correction notice Since this paper was ﬁrst published online Lesley A Cala and Jennifer L Becker have been added as authors. Cerebrovascular disease 16 Graeb DA, Robertson WD, Lapointe JS, et al. Computed tomographic diagnosis of intraventricular hemorrhage- etiology and prognosis. Radiology 1982;143:91–6. 27 Wang CW, Juan CJ, Liu YJ, et al. Volume-dependent overestimation of spontaneous intracerebral hematoma volume by the ABC/2 formula. Acta Radiol 2009;50:306–11. g gy p g gy 17 Morgan T, Dawson J, Spengler D, et al. The modiﬁed Graeb score an enhanced tool for intraventricular hemorrhage measurement and prediction of functional outcome. Stroke 2013;44:635–41. y 28 Maeda AK, Aguiar LZ, Martins C, et al. Hematoma volumes of spontaneous intracerebral hemorrhage: the ellipse (ABC/2) method yielded volumes smaller than those measured using the planimetric method. Arquivod de Neuro-Psiquiatria 2013;71:540–4. 18 The ENOS Trial Investigators. Glyceryl trinitrate vs. control, and continuing vs. stopping temporarily prior antihypertensive therapy, in acute stroke: rationale and design of the Efﬁcacy of Nitric Oxide in Stroke (ENOS) trial (ISRCTN99414122). Int J Stroke 2006;1:245–9. 29 Yang W, Feng Y, Zhang Y, et al. Volume quantiﬁcation of acute infratentorial hemorrhage with computed tomography: validation of the formula 1/2ABC and 2/ 3SH. PLoS ONE 2013;8:e62286. 19 Wardlaw JM, Sellar R. A simple practical classiﬁcation of cerebral infarcts on CT and its interobserver reliability. Am J Neuroradiol 1994;15:1933–9. 30 Zhao KJ, Liu Y, Zhang RY, et al. A precise, simple, convenient and new method for estimation of intracranial hematoma volume-the formula 2/3sh. Neurol Res 2009;31:1031–6. 20 Sandercock P, Lindley R, Wardlaw J, et al. Third international stroke trial (ist-3) of thrombolysis for acute ischaemic stroke. Trials 2008;9:37. 31 Sucu HK, Gokmen M, Gelal F. The value of XYZ/2 technique compared with computer-assisted volumetric analysis to estimate the volume of chronic subdural hematoma. Stroke 2005;36:998–1000. y 21 Rosset A, Spadola L, Ratib O. OsiriX: an open-source software for navigating in multidimensional DICOM images. J Digit Imaging 2004;17:205–16. 32 Brott T, Broderick J, Kothari R, et al. Early hemorrhage growth in patients with intracerebral hemorrhage. Stroke 1997;28:1–5. 22 Pabst W, Gregerova E. Characterisation of particle and particle systems. Prague: ICT, 2007;27–29. 23 Barras CD, Tress BM, Christensen S, et al. Quantitative CT densitometry for predicting intracerebral hemorrhage growth. Am J Neuroradiol 2013;34:1138–44. 33 Divani AA, Majidi S, Luo X, et al. The ABCs of accurate volumetric measurement of cerebral hematoma. Stroke 2011;42:1569–74. 34 Prionas ND, Ray S, Boone JM. Volume assessment accuracy in computed tomography: a phantom study. J Appl Clin Med Phys 2010;11:3037. 24 Shrout P, Fleiss J. Author afﬁliations 1 k f 13 Freeman WD, Barrett KM, Bestic JM, et al. Computer-assisted volumetric analysis compared with ABC/2 method for assessing warfarin-related intracranial hemorrhage volumes. Neurocrit Care 2008;9:307–12. 13 Freeman WD, Barrett KM, Bestic JM, et al. Computer-assisted volumetric analysis compared with ABC/2 method for assessing warfarin-related intracranial hemorrhage volumes. Neurocrit Care 2008;9:307–12. 1Stroke, Division of Clinical Neuroscience, University of Nottingham, Nottingham, UK 2School of Pathology and Laboratory Medicine, The University of Western Australia, Nedlands, Australia 3 1Stroke, Division of Clinical Neuroscience, University of Nottingham, Nottingham, UK 2School of Pathology and Laboratory Medicine, The University of Western Australia, Nedlands, Australia g 14 Sheth KN, Cushing TA, Wendell L, et al. Comparison of hematoma shape and volume estimates in warfarin versus non-warfarin-related intracerebral hemorrhage. Neurocrit Care 2010;12:30–4. 14 Sheth KN, Cushing TA, Wendell L, et al. Comparison of hematoma shape and volume estimates in warfarin versus non-warfarin-related intracerebral hemorrhage. Neurocrit Care 2010;12:30–4. 3Department of Medical Imaging, College of Medicine, The University of Arizona, Arizona, USA 3Department of Medical Imaging, College of Medicine, The University of Arizona, Arizona, USA 4Radiological Sciences Research Group, Division of Clinical Neuroscience, University of Nottingham, Nottingham, UK 15 Barras CD, Tress BM, Christensen S, et al. Density and shape as CT predictors of intracerebral hemorrhage growth. Stroke 2009;40:1325–31. Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845 1265 Cerebrovascular disease Intraclass correlations: uses in assessing rater reliability. Psychol Bull 1979;86:420–8. 25 ENOS Investigators. Baseline characteristics of the 4011 patients recruited into the “efﬁcacy of Nitric Oxide in Stroke (ENOS) trial. Int J Stroke 2014;9:711–20. 35 Kosior J, Idris S, Dowlatshahi D, et al. Quantomo: validation of a computer-assisted methodology for the volumetric analysis of intracerebral haemorrhage. Int J Stroke 2011;6:302–5. 26 Bath PMW, Woodhouse L, Scutt P, et al. Management of high blood pressure in acute stroke: efﬁcacy of Nitric Oxide in Stroke (ENOS), a partial-factorial randomised controlled trial. Lancet 2014. Published online 22 Oct 2014 http://dx.doi.org/ 10.1016/ S0140-6736(14)61121-1 36 Hussein HM, Tariq NA, Palesch YY, et al. Reliability of hematoma volume measurement at local sites in a Multicenter Acute Intracerebral Hemorrhage Clinical Trial. Stroke 2013;44:237–9. 1266 Krishnan K, et al. J Neurol Neurosurg Psychiatry 2015;86:1258–1266. doi:10.1136/jnnp-2014-309845
W1581144881.txt	https://journals.iucr.org/e/issues/2009/08/00/hk2730/hk2730.pdf	en		2-[2-(Methylsulfonyl)ethyl]isoindoline-1,3-dione	Acta crystallographica. Section E	2,009	cc-by	2,046	organic compounds Acta Crystallographica Section E Mo K radiation = 0.29 mm1 T = 294 K 0.20 0.10 0.10 mm b = 17.766 (4) Å c = 8.9940 (18) Å = 112.31 (3) V = 1123.9 (5) Å3 Z=4 Structure Reports Online ISSN 1600-5368 Data collection 2-[2-(Methylsulfonyl)ethyl]isoindoline1,3-dione Qiong Tang, Qi Feng, Jian Xu and Cheng Yao* Department of Applied Chemistry, College of Science, Nanjing University of Technology, Xinmofan Road No. 5 Nanjing, Nanjing 210009, People’s Republic of China Correspondence e-mail: yaocheng@njut.edu.cn Received 28 June 2009; accepted 3 July 2009 Key indicators: single-crystal X-ray study; T = 294 K; mean (C–C) = 0.004 Å; R factor = 0.048; wR factor = 0.144; data-to-parameter ratio = 13.2. Enraf–Nonius CAD-4 diffractometer Absorption correction: scan (North et al., 1968) Tmin = 0.944, Tmax = 0.972 2182 measured reflections 2027 independent reflections 1567 reflections with I > 2(I) Rint = 0.030 3 standard reflections frequency: 120 min intensity decay: 1% Refinement R[F 2 > 2(F 2)] = 0.048 wR(F 2) = 0.144 S = 1.01 2027 reflections 154 parameters H-atom parameters constrained max = 0.23 e Å3 min = 0.35 e Å3 Table 1 Hydrogen-bond geometry (Å, ). In the molecule of the title compound, C11H11NO4S, the isoindoline ring system is almost planar with a maximum deviation of 0.008 (3)Å. In the crystal structure, intermolecular C—H O interactions link the molecules into a three-dimensional network. – contacts between the isoindoline rings [centroid–centroid distances = 3.592 (1) and 3.727 (1) Å] may further stabilize the structure. Related literature For a related structure, see: Kilburn et al. (2007). For bondlength data, see: Allen et al. (1987). D—H A i C1—H1A O3 C11—H11A O1ii D—H H A D A D—H A 0.93 0.96 2.34 2.51 3.189 (5) 3.463 (4) 152 175 Symmetry codes: (i) x þ 1; y þ 1; z þ 1; (ii) x 1; y þ 12; z 12. Data collection: CAD-4 Software (Enraf–Nonius, 1989); cell refinement: CAD-4 Software; data reduction: XCAD4 (Harms & Wocadlo, 1995); program(s) used to solve structure: SHELXS97 (Sheldrick, 2008); program(s) used to refine structure: SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997); software used to prepare material for publication: SHELXL97 and PLATON (Spek, 2009). Supplementary data and figures for this paper are available from the IUCr electronic archives (Reference: HK2730). References Experimental Crystal data Monoclinic, P21 =c a = 7.6030 (15) Å C11H11NO4S Mr = 253.27 o1932 Tang et al. Allen, F. H., Kennard, O., Watson, D. G., Brammer, L., Orpen, A. G. & Taylor, R. (1987). J. Chem. Soc. Perkin Trans. 2, pp. S1-19. Enraf–Nonius (1989). CAD-4 Software. Enraf–Nonius, Delft, The Netherlands. Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565. Harms, K. & Wocadlo, S. (1995). XCAD4. University of Marburg, Germany. Kilburn, J. P., Andersen, H. S., Kampen, G. C. T. & Ebdrup, S. (2007). PCT Int. Appl. WO 2007051811. North, A. C. T., Phillips, D. C. & Mathews, F. S. (1968). Acta Cryst. A24, 351– 359. Sheldrick, G. M. (2008). Acta Cryst. A64, 112–122. Spek, A. L. (2009). Acta Cryst. D65, 148–155. doi:10.1107/S160053680902580X Acta Cryst. (2009). E65, o1932 supporting information supporting information Acta Cryst. (2009). E65, o1932 [doi:10.1107/S160053680902580X] 2-[2-(Methylsulfonyl)ethyl]isoindoline-1,3-dione Qiong Tang, Qi Feng, Jian Xu and Cheng Yao S1. Comment The title compound is an important pharmaceutical intermediate, which is used in treatment of metabolic syndrome. As part of our studies in this area, we report herein the crystal structure of the title compound. In the molecule of the title compound (Fig 1), the bond lengths (Allen et al., 1987) and angles are within normal ranges. Rings A (N/C4-C7) and B (C1-C4/C7/C8) are, of course, planar and the dihedral angle between them is A/B = 0.61 (3)°. The isoindoline ring system is planar with a maximum deviation of -0.008 (3) Å for atom N. In the crystal structure, intermolecular C-H···O interactions (Table 1) link the molecules into a three-dimensional network, in which they may be effective in the stabilization of the structure. The π–π contacts between the isoindoline rings, Cg1—Cg2i and Cg2—Cg2ii [symmetry codes: (i) 2 - x, -y, 2 - z, (ii) 1 - x, -y, 2 - z, where Cg1 and Cg2 are centroids of the rings A (N/C4-C7) and B (C1-C4/C7/C8), respectively] may further stabilize the structure, with centroidcentroid distances of 3.592 (1) and 3.727 (1) Å, respectively. S2. Experimental The title compound was prepared according to the literature method (Kilburn et al., 2007). Crystals suitable for X-ray analysis were obtained by dissolving the title compound (0.1 g) in acetone (25 ml) and evaporating the solvent slowly at room temperature for about 7 d. S3. Refinement H atoms were positioned geometrically with C-H = 0.93, 0.97 and 0.96 Å for aromatic, methylene and methyl H atoms, respectively, and constrained to ride on their parent atoms, with Uiso(H) = xUeq(C), where x = 1.5 for methyl H and x = 1.2 for all other H atoms. Acta Cryst. (2009). E65, o1932 sup-1 supporting information Figure 1 The molecular structure of the title molecule, with the atom-numbering scheme. 2-[2-(Methylsulfonyl)ethyl]isoindoline-1,3-dione Crystal data C11H11NO4S Mr = 253.27 Monoclinic, P21/c Hall symbol: -P 2ybc a = 7.6030 (15) Å b = 17.766 (4) Å c = 8.9940 (18) Å β = 112.31 (3)° V = 1123.9 (5) Å3 Z=4 F(000) = 528 Dx = 1.497 Mg m−3 Mo Kα radiation, λ = 0.71073 Å Cell parameters from 25 reflections θ = 9–13° µ = 0.29 mm−1 T = 294 K Block, colorless 0.20 × 0.10 × 0.10 mm Data collection Enraf–Nonius CAD-4 diffractometer Radiation source: fine-focus sealed tube Graphite monochromator ω/2θ scans Absorption correction: ψ scan (North et al., 1968) Tmin = 0.944, Tmax = 0.972 2182 measured reflections 2027 independent reflections 1567 reflections with I > 2σ(I) Rint = 0.030 θmax = 25.3°, θmin = 2.3° h = 0→9 k = 0→21 l = −10→9 3 standard reflections every 120 min intensity decay: 1% Refinement Refinement on F2 Least-squares matrix: full R[F2 > 2σ(F2)] = 0.048 wR(F2) = 0.144 S = 1.01 2027 reflections 154 parameters Acta Cryst. (2009). E65, o1932 0 restraints Primary atom site location: structure-invariant direct methods Secondary atom site location: difference Fourier map Hydrogen site location: inferred from neighbouring sites sup-2 supporting information (Δ/σ)max < 0.001 Δρmax = 0.23 e Å−3 Δρmin = −0.35 e Å−3 H-atom parameters constrained w = 1/[σ2(Fo2) + (0.09P)2] where P = (Fo2 + 2Fc2)/3 Special details Geometry. All e.s.d.'s (except the e.s.d. in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell e.s.d.'s are taken into account individually in the estimation of e.s.d.'s in distances, angles and torsion angles; correlations between e.s.d.'s in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell e.s.d.'s is used for estimating e.s.d.'s involving l.s. planes. Refinement. Refinement of F2 against ALL reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > σ(F2) is used only for calculating R-factors(gt) etc. and is not relevant to the choice of reflections for refinement. R-factors based on F2 are statistically about twice as large as those based on F, and R- factors based on ALL data will be even larger. Fractional atomic coordinates and isotropic or equivalent isotropic displacement parameters (Å2) S O1 O2 O3 O4 N C1 H1A C2 H2A C3 H3A C4 C5 C6 C7 C8 H8A C9 H9A H9B C10 H10A H10B C11 H11A H11B H11C x y z Uiso/Ueq 0.44021 (11) 0.8805 (3) 0.7221 (3) 0.3992 (4) 0.5068 (4) 0.8004 (3) 0.7239 (5) 0.6981 0.7703 (5) 0.7765 0.8074 (4) 0.8368 0.7993 (4) 0.8318 (4) 0.7531 (4) 0.7534 (4) 0.7155 (4) 0.6854 0.8028 (4) 0.8480 0.8919 0.6092 (4) 0.6228 0.5626 0.2381 (5) 0.1381 0.2668 0.1978 0.29490 (4) 0.29528 (10) 0.50798 (11) 0.33791 (14) 0.21958 (12) 0.39124 (12) 0.55296 (19) 0.5972 0.4883 (2) 0.4901 0.42096 (18) 0.3774 0.42102 (15) 0.35948 (15) 0.46788 (15) 0.48617 (15) 0.55292 (16) 0.5963 0.34924 (16) 0.2986 0.3730 0.34476 (15) 0.3201 0.3954 0.29295 (18) 0.2665 0.2677 0.3435 0.03299 (8) 0.4407 (3) 0.1325 (3) 0.1503 (3) 0.0776 (3) 0.2541 (3) 0.6247 (4) 0.6679 0.7187 (4) 0.8239 0.6577 (4) 0.7197 0.5027 (3) 0.4035 (3) 0.2484 (3) 0.4094 (3) 0.4690 (4) 0.4066 0.1157 (3) 0.1496 0.0766 −0.0211 (3) −0.1123 −0.0538 −0.1438 (4) −0.1253 −0.2263 −0.1773 0.0425 (3) 0.0524 (6) 0.0555 (6) 0.0700 (7) 0.0662 (7) 0.0394 (6) 0.0582 (9) 0.070 0.0584 (9) 0.070* 0.0496 (8) 0.060* 0.0397 (7) 0.0390 (6) 0.0398 (7) 0.0399 (7) 0.0495 (8) 0.059* 0.0447 (7) 0.054* 0.054* 0.0399 (7) 0.048* 0.048* 0.0541 (8) 0.081* 0.081* 0.081* Acta Cryst. (2009). E65, o1932 sup-3 supporting information Atomic displacement parameters (Å2) S O1 O2 O3 O4 N C1 C2 C3 C4 C5 C6 C7 C8 C9 C10 C11 U11 U22 U33 U12 U13 U23 0.0537 (5) 0.0670 (15) 0.0708 (16) 0.0893 (18) 0.0747 (17) 0.0463 (14) 0.053 (2) 0.054 (2) 0.0470 (18) 0.0328 (15) 0.0365 (15) 0.0378 (15) 0.0336 (14) 0.0469 (18) 0.0505 (18) 0.0516 (17) 0.052 (2) 0.0383 (4) 0.0347 (12) 0.0379 (11) 0.0790 (17) 0.0441 (13) 0.0293 (12) 0.0501 (19) 0.072 (2) 0.0546 (19) 0.0393 (15) 0.0337 (15) 0.0293 (14) 0.0344 (15) 0.0367 (16) 0.0384 (16) 0.0323 (14) 0.057 (2) 0.0401 (4) 0.0607 (13) 0.0535 (13) 0.0613 (14) 0.0763 (16) 0.0421 (13) 0.077 (2) 0.057 (2) 0.0502 (18) 0.0475 (16) 0.0458 (16) 0.0483 (16) 0.0515 (17) 0.064 (2) 0.0490 (17) 0.0404 (15) 0.0540 (18) −0.0056 (3) 0.0102 (10) 0.0018 (10) −0.0205 (14) −0.0027 (11) 0.0008 (10) −0.0085 (15) −0.0098 (17) −0.0024 (14) −0.0028 (11) −0.0012 (12) −0.0010 (12) −0.0033 (12) −0.0022 (13) 0.0016 (13) −0.0016 (12) −0.0038 (15) 0.0229 (3) 0.0302 (11) 0.0189 (11) 0.0507 (14) 0.0248 (13) 0.0161 (11) 0.0302 (18) 0.0289 (16) 0.0219 (14) 0.0158 (13) 0.0147 (12) 0.0117 (13) 0.0159 (13) 0.0199 (15) 0.0231 (14) 0.0227 (13) 0.0208 (16) −0.0007 (3) 0.0087 (9) 0.0063 (10) −0.0248 (12) 0.0196 (11) −0.0030 (9) −0.0260 (18) −0.0198 (17) 0.0019 (14) −0.0039 (12) 0.0020 (12) −0.0003 (12) −0.0065 (12) −0.0097 (14) −0.0025 (12) 0.0002 (11) 0.0012 (15) Geometric parameters (Å, º) S—O3 S—O4 S—C11 S—C10 O1—C5 N—C5 N—C6 N—C9 C1—C8 C1—C2 C1—H1A O2—C6 C2—C3 C2—H2A C3—C4 1.430 (2) 1.433 (2) 1.743 (3) 1.774 (3) 1.207 (3) 1.392 (3) 1.404 (3) 1.457 (3) 1.377 (5) 1.391 (5) 0.9300 1.210 (3) 1.389 (4) 0.9300 1.371 (4) C3—H3A C4—C7 C4—C5 C6—C7 C7—C8 C8—H8A C9—C10 C9—H9A C9—H9B C10—H10A C10—H10B C11—H11A C11—H11B C11—H11C 0.9300 1.394 (4) 1.490 (4) 1.483 (4) 1.376 (4) 0.9300 1.520 (4) 0.9700 0.9700 0.9700 0.9700 0.9600 0.9600 0.9600 O3—S—O4 O3—S—C10 O3—S—C11 O4—S—C10 O4—S—C11 C11—S—C10 C5—N—C6 C5—N—C9 C6—N—C9 116.42 (16) 108.61 (14) 108.68 (16) 109.11 (14) 109.42 (15) 103.86 (15) 112.1 (2) 124.3 (2) 123.5 (2) N—C6—C7 C8—C7—C4 C8—C7—C6 C4—C7—C6 C7—C8—C1 C7—C8—H8A C1—C8—H8A N—C9—C10 N—C9—H9A 105.8 (2) 121.5 (3) 130.2 (3) 108.2 (2) 117.4 (3) 121.3 121.3 113.3 (2) 108.9 Acta Cryst. (2009). E65, o1932 sup-4 supporting information C8—C1—C2 C8—C1—H1A C2—C1—H1A C3—C2—C1 C3—C2—H2A C1—C2—H2A C4—C3—C2 C4—C3—H3A C2—C3—H3A C3—C4—C7 C3—C4—C5 C7—C4—C5 O1—C5—N O1—C5—C4 N—C5—C4 O2—C6—N O2—C6—C7 121.4 (3) 119.3 119.3 120.9 (3) 119.5 119.5 117.6 (3) 121.2 121.2 121.1 (3) 130.9 (3) 108.0 (2) 124.9 (3) 129.1 (3) 105.9 (2) 124.4 (3) 129.8 (3) C10—C9—H9A N—C9—H9B C10—C9—H9B H9A—C9—H9B C9—C10—S C9—C10—H10A S—C10—H10A C9—C10—H10B S—C10—H10B H10A—C10—H10B S—C11—H11A S—C11—H11B H11A—C11—H11B S—C11—H11C H11A—C11—H11C H11B—C11—H11C 108.9 108.9 108.9 107.7 112.58 (19) 109.1 109.1 109.1 109.1 107.8 109.5 109.5 109.5 109.5 109.5 109.5 C8—C1—C2—C3 C1—C2—C3—C4 C2—C3—C4—C7 C2—C3—C4—C5 C6—N—C5—O1 C9—N—C5—O1 C6—N—C5—C4 C9—N—C5—C4 C3—C4—C5—O1 C7—C4—C5—O1 C3—C4—C5—N C7—C4—C5—N C5—N—C6—O2 C9—N—C6—O2 C5—N—C6—C7 C9—N—C6—C7 C3—C4—C7—C8 −0.8 (5) 0.9 (5) −0.6 (4) 179.7 (3) −177.4 (3) 6.6 (4) 0.8 (3) −175.2 (2) −3.1 (5) 177.2 (3) 178.8 (3) −0.9 (3) 178.9 (3) −5.1 (4) −0.4 (3) 175.6 (2) 0.3 (4) C5—C4—C7—C8 C3—C4—C7—C6 C5—C4—C7—C6 O2—C6—C7—C8 N—C6—C7—C8 O2—C6—C7—C4 N—C6—C7—C4 C4—C7—C8—C1 C6—C7—C8—C1 C2—C1—C8—C7 C5—N—C9—C10 C6—N—C9—C10 N—C9—C10—S O3—S—C10—C9 O4—S—C10—C9 C11—S—C10—C9 −179.9 (3) −179.1 (3) 0.7 (3) 1.3 (5) −179.5 (3) −179.4 (3) −0.2 (3) −0.2 (4) 179.0 (3) 0.5 (5) 112.9 (3) −62.6 (3) −63.7 (3) 67.7 (2) −60.2 (2) −176.8 (2) Hydrogen-bond geometry (Å, º) D—H···A i C1—H1A···O3 C11—H11A···O1ii D—H H···A D···A D—H···A 0.93 0.96 2.34 2.51 3.189 (5) 3.463 (4) 152 175 Symmetry codes: (i) −x+1, −y+1, −z+1; (ii) x−1, −y+1/2, z−1/2. Acta Cryst. (2009). E65, o1932 sup-5
https://openalex.org/W3127722406	https://ejournal.almaata.ac.id/index.php/JNKI/article/download/1403/pdfikdw	English	null	Implementation of Second Step Learning (SSL) as Psychosocial Stimulation of 7-12 Years Old Children with Neglectful Parents	JNKI (Jurnal Ners & Kebidanan Indonesia)/Jurnal Ners dan Kebidanan Indonesia	2,021	cc-by-sa	4,929	Implementation of Second Step Learning (SSL) as Psychosocial Stimulation of 7-12 Years Old Children with Neglectful Parents Ika Arum Dewi Satiti1, Waifti Amalia2 Ika Arum Dewi Satiti1, Waifti Amalia2 1Departement of Nursing STIKES Widyagama Husada 2Study Program D3 Kebidanan, STIKES Widyagama Husada Jalan Taman Borobudur Indah 3A Malang Email : waiftiamalia@gmail.com Abstrak Pola asuh orang tua merupakan komponen penting dalam perkembangan soasial emosional anak . Anak yang memiliki orang tua dengan tipe pola asuh neglectful cenderung amat agresif atau amat pasif dan menyendiri sehingga beresiko tertinggi terlibat berbagai kesulitan. Stimulasi yang tepat perlu diberikan pada anak-anak dalam kondisi ini untuk memperbaiki emosi dan keterlibatan sosial. Second step Learning (SSL) merupakan metode stimulasi perkembangan social emosional yang digunakan untuk melatih keterampilan empati, manajemen emosi, pemecahan kasus dan bersosialisasi. Tujuan penelitian ini adalah menerapkan metode SSL sebagai stimulasi psikososial anak usia 7-12 tahun dengan neglectful parents di SDN Bendosari 2 Pujon,Kabupaten Malang. Metode penelitan yang digunakan adalah quasy eksperiment dengan analisa data uji dependent T Test untuk melihat perubahan perkembangan psikosial anak sebelum dan sesudah implementasi SSL. Implementasi dilakukan tehadap 50 anak dengan orang tua yang mempunyai pola asuh tipe neglectful. Metode SSL dilakukan dengan 4 langkah yakni pemaparan materi, diskusi kasus, aktivitas dengan kelompok dan aktivitas dengan orang tua. Hasil Penelitian ini menunjukkan adanya perubahan yang signifikan dari rata-rata nilai social emosional anak sebelum stimulasi SSL (80,5 ± 11,7) dan setelah stimulasi SSL (90,1± 11,) dengan P value 0.000. Dari hasil penelitian ini dapat disimpulkan bahwa stimulasi second step learning (SSL) efektif dalam memperbaiki social emosinal anak usia 7-12 tahun dengan neglectful parents di SDN Bendosari 2 Pujon, Kabupaten Malang. Kata Kunci : ssl; sosial emosional; anak usia 7-12 tahun INDONESIAN JOURNAL OF NURSING AND MIDWIFERY INDONESIAN JOURNAL OF NURSING AND MIDWIFERY Implementation of Second Step Learning (SSL) as Psychosocial Stimulation of 7-12 Years Old Children with Neglectful Parents Ik A D i S titi1 W ifti A li 2 Implementation of Second Step Learning (SSL) as Psychosocial Stimulation of 7-12 Years Old Children with Neglectful Parents Ika Arum Dewi Satiti1, Waifti Amalia2 Abstract Parenting style is an important component in children’s emotional social development. Children who have parents with the neglectful parenting type tend to be either very aggressive or very passive and aloof so they are at highest risk of getting into various difficulties. The appropriate stimulation needs to be given to children in this condition to improve emotional and social engagement. Second step learning (SSL) is a method of stimulating social emotional development that is used to train empathy skills, emotional management, case solving and socializing. The purpose of this study was to apply the SSL method as psychosocial stimulation for children aged 7-12 years with neglectful parents at SDN Bendosari 2 Pujon, Kabupaten Malang. The research method used is a quasy experiment with data analysis of the dependent T test to see changes in children’s psycho-social development before and after the implementation of SSL. Implementation was carried out on 50 children aged 7-12 years old with parents who have a neglectful type of parenting style. The SSL method is carried out in 4 steps, namely material presentation, case discussions, activities with groups and activities with parents. This study used a total sampling technique with an instrument in the form of a child psychosocial development test of 0.75 The results of this study indicate a significant change in the mean social emotional values of children before the stimulation of SSL (80.5 ± 11.7) and after stimulation of SSL (90.1 ± 11) with a P value of 0.000. From the results of this study it can be concluded that second step learning (SSL) stimulation is effective in improving the social emotionality of children aged 7-12 years with neglectful parents at SDN Bendosari 2 Pujon, Kabupaten Malang. The results of this study can be used as a new learning method and curricululum in the education line Keywords : ssl; emotional social; children aged 7-12 years Article info: Article submitted on November 2, 2020 Articles revised on November 30, 2020 Articles received on Desember 28, 2020 DOI: http://dx.doi.org/10.21927/jnki.2020.8(4).270-278 Articles received on Desember 28, 2020 DOI: http://dx.doi.org/10.21927/jnki.2020.8(4).270-278 parenting style does not involve parents in childcare and does not really care about children(3) mentation of Second Step Learning (SSL) as Psychosocial Stimulation of 7-12 Years Old Children with Neglectful Paren MATERIALS AND METHODS The research permit is submitted at the beginning of the research with the etical clearance letter number 086/S.Ket/ KEPK/STIkesKPJ/VI/2020. Sampling using a total sampling technique, where the sample was taken from the entire population with a total of 50 children. Initial data to identify children with neglectful parents were obtained by giving a parenting questionnaire. After that the group formation was assisted by a facilitator who was a teacher at SDN Bendosari 2. Social emotional data for children aged 7-12 years before and after SSL stimulation were identified through the provision of psychosocial questionnaires which uses the google form distribution mechanism because it is constrained by the COVID-19 pandemic. The research instrument is a social emotional development questionnaire with 48 questions with 7 question indicators, namely annoying friends, taking other people’s things without permission, doing activities that are not in accordance with the lesson; do not want to do the task; disobeying class hours; mock others and scold others.The proof of the validity of the psychosocial questionnaire is proven through expert judgment and the reliability value of 0.75 which means that the instrument in this study is reliable and this validity test is carried out before the questionnaire distribution method begins. The first step of this SSL is filling out a questionnaire related to the social emotional of children by filling in the question link that has been created and distributed via the WhatsApp (WA) group. Furthermore, Second Step Learning (SSL) is carried out in 4 stages, namely: the presentation of general material that is distributed through learning videos of <5 minutes duration The SSL method in children with parents who have neglectful parenting is expected to improve their emotional and social abilities so there are no cases of child abuse and bullying behavior. The formation of good social emotional development in children can form a competitive and quality human resource (6) (Upshur,2017). Hasil ini sesuai dengan penelitian Sabina (2015) yang menyatakan bahwa evaluasi anak sekolah dengan metode SSL menunjukkan sikap empati tinggi dan menurunnya perilaku antisosial(5) however, without the requisite social and emotional skills that facilitate learning, which has prompted schools nationwide to adopt specific curricula to teach students the social-emotional skills that enable them to maintain optimal engagement in the learning process. INTRODUCTION School age is a golden period in which children enter the education sector. School age is a golden period in which children enter the development of social industry vs inferiority. During this period, self-confidence, competitiveness and social feelings began to form. One of the factors that is very influential in developing children’s abilities is parents. Family is the first and foremost environment for child development. Family is very functional in instilling the basics of emotional experiences, because that is where the first experiences are obtained by children. An important component in the family that affects children’s emotional social development is the parenting style(1) . The phenomenon that occurs at this time, without realizing it, most parents have applied the neglectful type of parenting by reason of being busy and ignorant. Some of these neglected children tend to be very aggressive, others tend to be very passive and aloof and are at greatest risk of getting into difficulties. Based on data from KPAI (2018), there are 857 cases of child delinquency that occur due to poor parenting patterns. This data is in line with research conducted by Putri (2017) which reveals that 40% of children who have parents with indifferent parenting show bad social behavior (4).The appropriate stimulation needs to be given to children in this condition to improve emotional and social engagement. Parenting in the family means the habits of the parents, father and biological mother in leading, caring for and guiding children in the family (2). Parenting patterns are divided into four, namely authoritarian parenting, the type of parents who impose their will, democratic parenting, the type of parenting of parents that prioritizes common interests and permissive parenting, the type of parenting that educates children to become “spoiled” and patterns. Neglectful upbringing that combines low control and responsiveness. Relatively neglectful Second Step Learning (SSL) is a method of stimulating social emotional development used for elementary school aged children. This method trains skills of empathy, emotional management, case solving and socializing(5)however, without the requisite social and emotional skills that facilitate learning, which has prompted schools nationwide to adopt specific curricula to teach students the social-emotional skills that enable 271 272 Ika Arum Dewi Satiti, Waifti Amalia. JNKI, Vol. 8, Issue 4, 2020, 270-279 MATERIALS AND METHODS them to maintain optimal engagement in the learning process. Second Step® is one of the most widely disseminated social-emotional learning (SEL .Research conducted by Upshur in 2017 stated that the SSEL method was significant in increasing Emotional Skill (ES) and Social Skill (SS) in 31 schoolchildren children with parents working in America (6). Therefore, researchers are interested in applying this SSL method to children aged 7-12 years with neglectful parents. This research is a quasy experimental research with one group pre-post test design. The study population was children aged 7-12 years with neglectful parents at SDN Bendosari 2 Kabupaten Pujon. The research permit is submitted at the beginning of the research with the etical clearance letter number 086/S.Ket/ KEPK/STIkesKPJ/VI/2020. Sampling using a total sampling technique, where the sample was taken from the entire population with a total of 50 children. Initial data to identify children with neglectful parents were obtained by giving a parenting questionnaire. After that the group formation was assisted by a facilitator who was a teacher at SDN Bendosari 2. Social emotional data for children aged 7-12 years before and after SSL stimulation were identified through the provision of psychosocial questionnaires which uses the google form distribution mechanism because it is constrained by the COVID-19 pandemic. The research instrument is a social emotional development questionnaire with 48 questions with 7 question indicators, namely annoying friends, taking other people’s things without permission, doing activities that are not in accordance with the lesson; do not want to do the task; disobeying class hours; mock others and scold others.The proof of the validity of the psychosocial questionnaire is proven through expert judgment and the reliability value of 0.75 which means that the instrument in this study is reliable and this validity test is carried out before the questionnaire distribution method begins. The first step of this SSL is filling out a questionnaire related to the social emotional of children by filling in the question link that has been created and distributed via the WhatsApp (WA) group. Furthermore, Second Step Learning (SSL) is carried out in 4 stages, namely: the presentation of general material that is distributed This research is a quasy experimental research with one group pre-post test design. The study population was children aged 7-12 years with neglectful parents at SDN Bendosari 2 Kabupaten Pujon. MATERIALS AND METHODS Second Step® is one of the most widely disseminated social-emotional learning (SEL Based on a preliminary study, through the distribution of a questionnaire on parenting styles for 1 week in Dusun Cukal, Desa Bendosari, Kecamatan Pujon, Kabupaten Malang, out of 105 parents, 50 parents applied the neglectful type of parenting, and from the observations it was found that 7 children did not want to listen to what their parents said, 3 children rarely left the house and looked embarrassed when asked to talk. Based on the phenomenon that occurs, researchers are interested in applying this SSL method to children aged 7-12 years with neglectful parents. 272 Ika Arum Dewi Satiti, Waifti Amalia. JNKI, Vol. 8, Issue 4, 2020, 270-279 fourth SSL stage. This assessment is in line with research conducted by Satriawan (2020) that changes in behavior can be seen at least after 2 treatments (7). After all data collection steps are complete, the next process is tabulation of research results using exel which includes re- checking the completeness of the answers, giving respondent codes, and analyzing the results of the study using SPSS 21.0 for windows using statistical analysis of the paired T test, significant if P <0.05 . All data that has been collected will be kept confidential by researchers as a form of protecting the privacy of respondents. pan Penelitian which explains about SSL, then the exposure and discussion of the case phenomenon about bullying in children, the next day social activities with peers in the form of games based on cooperative methods such as puzzle games, stacking blocks and so on and the last one is activities with families in the form of studying with parents, reading stories or fairy tales. The first stage is carried out once with a duration of 30 minutes, the second stage to the fourth stage is carried out 2 sessions with a duration of 60 minutes. RESULTS AND DISCUSSION School age is a golden period where children enter the development of social industry vs inferiority. During this period, self-confidence, competitiveness and social feelings began to form. One of the factors that is very influential in developing children’s abilities is parents. Family is the first and foremost environment for children’s development, Schick, Andreas, Cierpka, & Manfred (2016) also argue that in middle or elementary school age children often engage in negative social emotional behavior, namely in the form of aggressive behavior (8). Aggressive behavior carried out by children can be in the form of physical aggression or verbal aggression, physical aggression such as hitting, pushing, pinching, kicking, biting and so on, then verbal aggression behavior such as insulting, threatening, scolding, shouting loudly, talking dirty and so on. Then according to the opinion of Oelfy, Basaria, and Ananta (2018) that aggressive behavior that causes physical (non-verbal) injuries, namely hitting, biting, kicking, pinching, stepping on, etc., then psychologically (verbally), including saying insulting or mocking words, cursing with profanity, harassing, threatening, yelling at older people, or even ordering others around casually. Negative social emotional development is influenced by several factors, namely age, gender, class, and parents (9). The results obtained in this study describe the characteristics of the respondents based on the age of the child, the class of the child, the order of the children in the family, the work status of the parents, the education of the parents and the class of the child. Specific data for the study were the social emotional abilities of children aged 7-12 years before and after Second Step Learning (SSL) stimulation. The research data was taken by distributing an online social emotional development questionnaire to 50 children with a summary table of the results as follows: Table 1. Data on Characteristics of Respondents Variable Amount Precentage (%) Children Age 7-8 TH 12 24 9-10 TH 21 42 11-12 TH 17 34 Gender P 33 60 L 17 34 Order in family 1 17 34 2 19 38 3 14 28 Parents Occupation Guru 1 2 IRT 18 36 Petani 31 62 Parental Education SD 29 58 SMP 17 34 ≥ SMA 4 8 class 1-2 12 24 3-4 21 42 5-6 17 34 Table 1. Data on Characteristics of Respondents Each person has different self-control. According to Hurlock (2008) in Retnowuri and Linda Yani (2019) states that the ability to control self develops with age. MATERIALS AND METHODS Data post test research was conducted 2 days after the respondent carried out the Flow Chart Ta Flow Chart Tahapan Penelitian RESULTS AND DISCUSSION Pre Test : Assessment of social emotional development scores Second Step Learning Stage 4: Activities with parents Stage 3: activity with friends Stage 2: Discussion of case phenomena Stage 1 (SSL Material): video lessons Case Discussion 2: bulliying Case Discussion 1: Physical abuse Game 2: teamwork Game 1: individual Activity 2: problem solving Activity 1: study Post Test: Score assessment of emotional social development Pre and post data analysis (paired T test) Pre Test : Assessment of social emotional development scores Second Step Learning Stage 2: Discussion of case phenomena Stage 3: activity with friends Stage 1 (SSL Material): video lessons Stage 4: Activities with parents Game 1: individual Case Discussion 1: Physical abuse Activity 2: problem solving Game 2: teamwork Case Discussion 2: bulliying Post Test: Score assessment of emotional social development Flow Chart Tahapan Penelitian mplementation of Second Step Learning (SSL) as Psychosocial Stimulation of 7-12 Years Old Children with Neglectful P The results obtained in this study describe the characteristics of t RESULTS AND DISCUSSION The theory states gender and the older a person is, the better his self- control, psychologically mature individuals will also be able to control their behavior because they have been able to consider which things are good and bad for them, the higher the level of education, the better the level(10). Parents’ knowledge, this is in accordance with Aulia (2019), namely the different levels of parental education make different ways how parents educate their children(11). The results of the above characteristics of the respondents, the data obtained a majority of respondents aged 9-10 years (42%), the majority of children to 2 in the family (38%), the majority of women as much as 66% and the majority of parents work as farmers (66%), education majority of parents are Elementary School (58%) (table 1). 274 4 Ika Arum Dewi Satiti, Waifti Amalia. JNKI, Vol. 8, Issue 4, 2020, 270-279 The development of children can grow optimally, if supported by a conducive one. Parents have a very important role in creating an environment to stimulate the potential that is owned by. Therefore, optimal parenting practices are needed. Parenting is the process of raising, providing protection, giving attention, and value to children’s development from birth to adulthood. This parenting task is generally left to the mother as the primary caregiver and the father. The care that is carried out by the mother is based on the knowledge that the mother has, however the knowledge and skills possessed by the mother are often inadequate. One of the influencing factors is the mother’s education level. Maternal education is a factor related to maternal knowledge (12). development causes children to be aggressive and withdraw, so it needs appropriate learning methods to improve self-confidence, empathy and socialization skills (13). The results of the social-emotional questionnaire before SSL showed that, on average, 26 children scored 86-97 (48%), 10 children scored ≥ 98 (20%), 12 children scored 75-85 (24%) and 1 child got a score of 63-74 (4%). Table 3. Score data after SSL stimulation Rentang nilai Frekuensi absolut Frekuensi relatif (5%) ≥ 98 10 20 86-97 26 52 75-85 12 24 63-74 2 4 ≤ 62 0 0 Table 3. Score data after SSL stimulation Table 2. RESULTS AND DISCUSSION shows The results of the social- emotional questionnaire before SSL showed that, on average, 24 children scored 75-85 (48%), 3 children scored ≥ 98 (6%), 11 children scored 86-97 (22%), 10 children got a score of 63-74 (20%) and 2 children got a score ≤ 62 (4%). At this time, children learn to get pleasure and satisfaction from completing tasks, especially academic tasks. Psychosocial stimulation is the embryo of the child’s learning process through education and training. Through psychosocial stimulation, children can control and coordinate their muscles and involve feelings of emotions and thoughts so that children get various life experiences. This is in line with Mulyanti (2017) who stated that good psychosocial stimulation is related to children’s health so that it can indirectly affect the child’s development status(14) . Table 2. Score data before SSL stimulation Table 2. Score data before SSL stimulation Range of score Absolut frequency Frekuensi relatif (%) ≥ 98 3 6 86-97 11 22 75-85 24 48 63-74 10 20 ≤ 62 2 4 Table 4. Data Analysis Results Before and after SSL Mean Std. Deviation Selisih P-value Before SSL 80,5400 11,69198 0.000 After SSL 90,1200 11,13120 9,58 Table 4. Data Analysis Results Before and after SSL The results of data analysis before the stimulation of SSL on 50 children obtained the highest social emotional ability score of 124, the lowest 59 and an average value of 80.5. While the data after the stimulation of SSL obtained the highest social emotional ability score of 132, the lowest 70, and an average value of 90.1. The score before and after SSL is 9.58 points with a P value of 0.000. From the results above, it can Successful completion at this stage will create children who can solve problems and are proud of their accomplishments. The acquired ego skills are competencies. On the other hand, children who are unable to find positive solutions and are unable to achieve what their peers have achieved will feel inferior. Psychosocial development affects the quality of the nation’s human resources. Poor psychosocial 275 mplementation of Second Step Learning (SSL) as Psychosocial Stimulation of 7-12 Years Old Children with Neglectful P be concluded that there are significant changes in the social emotional development of children before and after stimulation of SSL. skills, and responsible decision making. The adoption of the SSL learning method is very effective in improving children’s development. RESULTS AND DISCUSSION The phenomenon that occurs at this time, without realizing it, most parents have applied the neglectful type of parenting by reason of being busy and ignorant. Some of these neglected children tend to be very aggressive, others tend to be very passive and aloof and are at greatest risk of getting into difficulties(15). According to data from the Komnas Perlindungan Anak, the number of student brawls in 2018 was 4,885 cases of juvenile delinquency and 1,434 cases, followed by cases related to family and child care as many as 857 cases. The previous year, the number of cases of violence between students was 28. Some of the factors that influence this condition are peers, social environment, mass media and parenting styles(10). The phenomenon that occurs proves that school-age children need guidance to improve their social emotional behavior. This SSL method has been implemented as a curriculum in various schools abroad and shows good results (10). However, in Indonesia there is no specific curriculum on children’s psychosocial issues, the existing curriculum on psychosocial issues is only implicit in certain subjects, there has been no discussion of theories or cases.(16) In addition, the role of parents is also an important component in optimizing children’s development. Parenting in the family means the habits of the parents, father and biological mother in leading, caring for and guiding children in the family. Neglectful / Uninvolved Parenting is a parent who combines low control and responsiveness. Relatively not involved in child care and do not really care about children. Listiyaningsih (2017) argues that children from this parenting style tend not to have competence either socially or academically. They tend to be involved with juvenile delinquency and antisocial behavior when they are teenagers (18). Experts have found that parenting styles shown by parents have a correlation with children’s behavior. According to research conducted by Mirdat (2014), parenting contributes greatly to causing violent behavior against children (19). One parenting style that causes psychosocial barriers in children is called neglectful. This is in line with research conducted by Aulia in 2018 which stated that neglectful parenting can affect a child’s personality (7). In this study, stimulation of SSL was carried out on children aged 7-12 years with neglectful / indifferent parents, and this stimulation was said to be effective in changing the child’s social emotional development so that this method needs to be applied at school and at home. 276 Ika Arum Dewi Satiti, Waifti Amalia. JNKI, Vol. 8, Issue 4, 2020, 270-279 RESULTS AND DISCUSSION Children’s social emotional stimulation is related to children’s emotional and intellectual intelligence. Strong emotional control makes children confident academically and non-academically. The implementation of SSL can progressively improve physical and verbal violence behavior and tolerance of children. In the future, children can control their emotional social behavior so that the number of physical and verbal abuse among school children can decrease Research conducted by Upshur in 2017 stated that the SSEL method was significant in increasing Emotional Skill (ES) and Social Skill (SS) in 31 school children in America (2). This is in line with research conducted by Top and Leu in 2016, which stated that students in the treatment group (SSL curriculum) had the ability to solve problems and had higher academic scores than students in the control group (Non-SSL curriculum) (17). Second Step Learning (SSL) is a learning method developed for children’s social emotional improvement. SSL consists of 5 concepts, namely self-awareness, self- management, social awareness, socialization 276 Ika Arum Dewi Satiti, Waifti Amalia. JNKI, Vol. 8, Issue 4, 2020, 270-279 CONCLUSION AND RECOMMENDATION 8. Schick, Andreas, Cierpka & M. Risk Factor and Prevention of Aggresive behavior in childrens and adolescent. J Educ Res online. 2016;8:90–109. The results above can be concluded that there is a significant change in the social emotional development of children before and after the stimulation of SSL with a P value of 0.000. Thus, Second Step Learning stimulation is said to be effective in improving the psychosocial skills of children aged 7-12 years. For further research, it is hoped that the SSL method can be applied not only as a curriculum in schools module media but also by parents in educating the children. 9. Oelfy N, Basaria D, Nur Ananta S. Penerapan Asertive Behavior Therapy Dan Positive Reinforcement Untuk Mengurangi Kecenderungan Perilaku Agresivitas Verbal Pada Anak Usia Tengah. 2018;2(1):165–72. 10. Retnowuni A, Yani AL. Faktor-faktor yang Mempengaruhi Perilaku Agresif pada Remaja yang Tinggal di Pesantren. 2019;6(1):36–43. REFERENCES 11. Albab AU. 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Available from: http://doi.org/10.21009/JKKP
https://openalex.org/W2492139109	https://europepmc.org/articles/pmc4951010?pdf=render	English	null	Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant	PloS one	2,016	cc-by	8,489	RESEARCH ARTICLE Julie Thakur, Mayank D. Dwivedi, Pragya Sourabh, Prem L. Uniyal, Arun K. Pandey* Department of Botany, University of Delhi, Delhi, India a1111 OPEN ACCESS OPEN ACCESS Citation: Thakur J, Dwivedi MD, Sourabh P, Uniyal PL, Pandey AK (2016) Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant. PLoS ONE 11(7): e0159050. doi:10.1371/journal. pone.0159050 Editor: Randall P. Niedz, United States Department of Agriculture, UNITED STATES Received: December 26, 2015 Accepted: June 27, 2016 Published: July 19, 2016 Copyright: © 2016 Thakur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Citation: Thakur J, Dwivedi MD, Sourabh P, Uniyal PL, Pandey AK (2016) Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant. PLoS ONE 11(7): e0159050. doi:10.1371/journal. pone.0159050 Citation: Thakur J, Dwivedi MD, Sourabh P, Uniyal PL, Pandey AK (2016) Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant. PLoS ONE 11(7): e0159050. doi:10.1371/journal. pone.0159050 Received: December 26, 2015 Accepted: June 27, 2016 Published: July 19, 2016 Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Abstract Pittosporum eriocarpum Royle, a medicinally important taxon, is endemic to Uttarakhand region of Himalaya. It has become endangered due to over-collection and the loss of habi- tats. As raising plants through seeds in this plant is problematic, a reliable protocol for micro- propagation using nodal explants has been developed. High shoot regeneration (95%) occurred in MS medium augmented with BA 0.4mg/l in combination IBA 0.6mg/l. In vitro regenerated shoots were rooted in MS medium supplemented with three auxins, of which 0.6 mg/l indole butyric acid proved to be the best for rooting (90%) with maximum number of roots per shoot. Thereafter, rooted plants were hardened and nearly 73% of rooted shoots were successfully acclimatized and established in the field. Start codon targeted (SCoT), inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD) markers were used to validate the genetic homogeneity amongst nine in vitro raised plant- lets with mother plant. DNA fingerprints of in vitro regenerated plantlets displayed monomor- phic bands similar to mother plant, indicating homogeneity among the micropropagated plants with donor mother plant. The similarity values were calculated based on SCoT, ISSR and RAPD profiles which ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to 1.00 respec- tively. The dendrograms generated through Unweighted Pair Group Method with arithmetic mean (UPGMA) analysis revealed 97% similarity amongst micropropagated plants with donor mother plant, thus confirming genetic homogeneity of micropropagated clones. This is the first report on micropropagation and genetic homogeneity assessment of P. eriocar- pum. The protocol would be useful for the conservation and large scale production of P. eriocarpum to meet the demand for medicinal formulations and also for the re-introduction of in vitro grown plants in the suitable natural habitats to restore the populations. Citation: Thakur J, Dwivedi MD, Sourabh P, Uniyal PL, Pandey AK (2016) Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant. PLoS ONE 11(7): e0159050. doi:10.1371/journal. pone.0159050 Genetic Homogeneity Revealed Using SCoT, ISSR and RAPD Markers in Micropropagated Pittosporum eriocarpum Royle- An Endemic and Endangered Medicinal Plant Julie Thakur, Mayank D. Dwivedi, Pragya Sourabh, Prem L. Uniyal, Arun K. Pandey* Department of Botany, University of Delhi, Delhi, India Julie Thakur, Mayank D. Dwivedi, Pragya Sourabh, Prem L. Uniyal, Arun K. Pandey* Introduction Pittosporum eriocarpum (Pittosporaceae), commonly known as Agni [1], is an endangered [2] and endemic medicinal tree species of Uttarakhand region occurring at an altitudinal range of Competing Interests: The authors have declared that no competing interests exist. PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 1 / 17 Genetic Homogeneity Assessment of Pittosporum eriocarpum 1000-2200m [3]. It is represented by a few populations in lesser Himalayan range, including Mussoorie hills, Doon valley, Dehradun, Dhanlagiri, Chamba, Nagli in Tehri, Kurakhad, Jeulli- kot and Nainital [4,5]. It is reported to occur mostly on the calcium rich rocky slopes in exposed sites [6]. It is a small evergreen tree up to 5m height with alternate or whorled leaves. Flowers are pale yellow, which remain in many umbellate corymbs. Fruit is a globose capsule (10-12mm) with 8–10 blackish-red seeds. The bark is aromatic and used in bronchitis, and as expectorant and febrifuge. Application of root paste provides relief in rheumatic swelling [3]. It is also used as fodder. The tree is suitable for soil conservation and reclamation of degraded sites [4]. Micropropagation is an efficient and alternative method for large scale production of endangered plants in a short period of time. Effect of IBA on rooting in P. tobira var. variega- tum is reported to be insignificant [7]. Plant regeneration from nodal region of P. napaulensis (DC.) Reader & Wilson has been reported [8]. In vitro seed germination followed by hypocotyl sectioning to raise plants has been documented in P. tobira [9] P. eriocarpum can be regenerated asexually by layering or sowing [4], but such regenerates require periodic moist and dry conditions in moderate cool temperature. The seed coat is sur- rounded by thick gummy substance which delays seed germination. There is an urgent need to produce more plants through micropropagation which has advantage over conventional meth- ods [10]. Micropropagation is assumed to cause a change in genetic makeup of plant tissue and can lead to DNA sequence variation and generation of somaclonal variation [11]. Thus, it is a major issue of concern in micropropagation, where the regenerates are expected to be homoge- nous to donor mother plant (MP). To overcome this, it is a prerequisite to validate the genetic homogeneity to authenticate the quality of micropropagated plants for commercial use. Introduction The genetic homogeneity amongst the micropropagated plants with mother plant has been assessed by PCR based molecular markers, i.e., AFLP (Amplified Fragment Length Polymorphism), ISSR (Inter Simple Sequence Repeats), RAPD (Random amplified polymorphic DNA), SCoT (Start codon targeted) and SSR (Simple Sequence Repeats) [12–14]. The goal of this research was to provide an efficient and reproducible method for in vitro propagation of P. eriocarpum via nodal explants for large scale production and assessment of genetic homogeneity of micropropagated plantlets through SCoT, ISSR and RAPD markers. This objective was achieved by healthy material collection, in vitro nodal culture, greenhouse acclimatization and genetic homogeneity assessment using molecular markers. PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 Plant material and culture preparation Healthy shoots were collected from three different wild populations from sexually mature plants growing in Dehradun (300 35.05’N, 780 01.97’E), Nainital (290 22.627’N, 790 28.163’E) and Jeullikot (290 27.144’N, 790 24.795’E) in Uttarakhand region during March-May. The field studies did not involve specific permission for these locations. Nodal regions (2cm long) were used as explants after removing all the leaves and explants were washed with 2% (v/v) tween- 20 for 30 min under running tap water and then surface sterilized with 60% (v/v) ethyl alcohol for 10 minutes (Fig 1A). These explants were then soaked in 0.1% (w/v) mercuric chloride for 5 min followed by washing with autoclaved double distilled water under sterile conditions. Mur- ashige and Skoog medium [15] supplemented with different growth hormones and 2% (w/v) sucrose 20gm/l and one set without growth regulators as a control were prepared. The medium was solidified with 0.8% (w/v) agar and pH was adjusted to 5.8 with 1N NaOH or 1N HCl before autoclaving at 121°C and 108 K Pa for 20 minutes. Cultures were incubated at 25±2°C, PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 2 / 17 Genetic Homogeneity Assessment of Pittosporum eriocarpum Fig 1. Pittosporum eriocarpum. (A) Nodal explant. (B) Shoot induction in MS medium supplemented with BA (mg/l). (C) Root induction in MS medium supplemented with IBA (mg/l). (D) In vitro grown plantlets. (E) In vitro grown plantlets in soil rite. (F) In vitro raised plantlets in natural conditions. doi:10.1371/journal.pone.0159050.g001 Fig 1. Pittosporum eriocarpum. (A) Nodal explant. (B) Shoot induction in MS medium supplemented with BA (mg/l). (C) Root induction in MS medium supplemented with IBA (mg/l). (D) In vitro grown plantlets. (E) In vitro grown plantlets in soil rite. (F) In vitro raised plantlets in natural conditions. doi:10.1371/journal.pone.0159050.g001 doi:10.1371/journal.pone.0159050.g001 55–60% RH, under 16 hour photoperiod with light intensity of 2000 lux provided by cool white fluorescent lights. 55–60% RH, under 16 hour photoperiod with light intensity of 2000 lux provided by cool white fluorescent lights. Shoot induction and multiplication To determine the role of various combinations of growth hormones on shooting, freshly excised stem explants were cultured vertically in full strength MS medium. We used different levels ofBAP and levels of IBA as factors. We assessed three effects i.e. BAP, IBA and the inter- action between these two factors (BAPxIBA) on shoot length. There were 20 treatments to determine these three effects. Thus, the effect on shoot length were determined using 4x5 facto- rial design with BA varied at four levels (0.2, 0.4, 0.6, 0.8 mg/l) and IBA at five levels (0.0, 0.2, 0.4, 0.6, 0.8 mg/l), for a total of 20 treatments. Factorials were designed in a completely ran- domized manner. Each treatment combination, including the control had 25 replicates and the 3 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 Genetic Homogeneity Assessment of Pittosporum eriocarpum experiment was repeated twice. The average data from both experiments were combined for further statistical analysis. The data was analyzed by two-way ANOVA followed by Tukey’s multiple comparison test. Shoot length and number of shoots were recorded periodically. All cultures were maintained under the same growth conditions stated above and regenerated shoots were subcultured at least three times. After five months, regenerated shoots were sepa- rated and cut end of each was dipped for an hour in IBA and then subcultured in best suited combinations and change in the numbers of shoots, numbers of leaves per branch and callus diameter were recorded. One-way ANOVA was used to determine the effects of PGR combina- tions used for shoot multiplication on number of shoots, number of leaves per branch and cal- lus diameter Statistical analysis All experiments were carried out in a completely randomized manner. The results were statisti- cally analyzed using two-way Analysis of Variance (ANOVA) (Tables 1 and 2) and one-way ANOVA (Table 3) and through Statistical Packages for the Social Sciences (SPSS) version 16.0. Mean separation was doneusing Post-Hoc Tukey’s test for all the data (p<0.05) and presented in Tables 2 and 4. [16]. Rooting, acclimatization and field transfer To determine the influence of various root hormones on root induction, multiple shoots pro- duced in the shoot multiplication stage were separated carefully under sterile condition and subcultured in MS medium. We used three types of hormones (IAA, IBA and NAA) and differ- ent level of concentrations (0.2, 0.4, 0.6, 0.8, 1.0 mg/l) of each hormone as factors. The interac- tion between the types and concentrations of hormones was also analyzed. A 3x5 factorial design was used to determine the effects of PGRs (three) and their concentrations (Five) on number of roots per shoot, root length and basal callus formation separately. Two-way ANOVA was used followed by Tukey’s multiple comparison tests for statistical test. Replicates were handled in same manner as described in shoot and multiplication section. The cultures were maintained at 25±2°C, 55–60% RH under 16 hour photoperiod with light intensity of 2000 lux provided by cool white fluorescent lights. Rooted shoots with minimum 2–3 roots were carefully harvested from culture flasks and washed thoroughly under running tap water to remove traces of agar. These plantlets were then planted in plastic pots (9 cm in diameter and 11 cm in height) containing autoclaved mix- ture of soil and sand in the ratio of 1:1. We covered the plantlets with a polythene cap for one month and small slits were made at the edges to maintain high relative humidity (RH 80±5%) and irrigated them every alternate day with half strength MS liquid medium and incubated in a culture room. Micropropagated plantlets of minimum 5cm in height, after certain level of acclimatization were transferred on soil in culture room. Gradually rooted plantlets were transferred to green- house conditions (temperature 25°C and RH 45%) to allow them to acclimatize for 10 days, and gradual loosening of polythene caps was done and finally the caps were removed after 15 days. After 3 months of exposition under greenhouse conditions, the micropropagation-derived plants were transferred to the nursery. The number of surviving plants was recorded periodically. DNA isolation and genetic homogeneity assessment To evaluate the genetic homogeneity of micropropagated plantlets, nine hardened plants were randomly selected along with the mother plant for genetic homogeneity assessment. Total PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 4 / 17 Genetic Homogeneity Assessment of Pittosporum eriocarpum genomic DNA of all plants was extracted from leaves using DNeasy Plant Minikit (Qiagen, Netherlands). The leaf material (25mg) was powdered in liquid nitrogen and stored at -80°C until extraction. The quality and quantity of DNA samples were estimated on 1.5% agarose gel and spectrophotometer respectively. The extracted DNA was further preceded for genetic homogeneity assessment by PCR based SCoT, ISSR and RAPD markers. Table 1. Effect of various concentrations of BA and IBA (mg/l) on shoot length in P. eriocarpum. BAP (mg/l) IBA (mg/l) Shoot length (mm)(Mean± SD) 0.2 00 28.14±1.4 0.2 0.2 26.03±5.7 0.2 0.4 27.71±0.1 0.2 0.6 25.59±0.4 0.2 0.8 26.98±0.1 0.4 0.0 27.96±0.7 0.4 0.2 27.38±0.1 0.4 0.4 30.98±0.6 0.4 0.6 43.34±3.8 0.4 0.8 31.95±1.1 0.6 0.0 30.27±0.1 0.6 0.2 31.61±1.16 0.6 0.4 33.44±0.2 0.6 0.6 31.37±1.1 0.6 0.8 27.51±0.8 0.8 0.0 28.72±0.8 0.8 0.2 28.32±0.7 0.8 0.4 28.08±0.4 0.8 0.6 28.21±0.3 0.8 0.8 26.41±0.8 0.0 0.0 0 doi:10.1371/journal.pone.0159050.t001 Table 2. Effect of auxins (IAA, IBA or NAA) on in vitro rooting of P. eriocarpum shoots grown on MS medium. Auxin Concentration(mg/l) Number of rootsper shoot±SD Root length±SD(mm) Presence of basal callus±SD(mm) IAA 0.2 2.65±0.5 10.03±1.1 0 0.4 6.00±0.8 13.05±2.9 0 0.6 3.93±0.8 7.0±1.0 3.21±0.8 0.8 3.53±1.1 5.56±1.1 5.57±0.4 1.0 0 0 17.43±2.1 IBA 0.2 2.00±0.0 15.57±1.0 0 0.4 2.55±0.5 18.29±3.9 0 0.6 12.78±2.6 36.40±10.0 0 0 8 2 03 0 8 7 68 1 3 0 genomic DNA of all plants was extracted from leaves using DNeasy Plant Minikit (Qiagen, Netherlands). The leaf material (25mg) was powdered in liquid nitrogen and stored at -80°C until extraction. The quality and quantity of DNA samples were estimated on 1.5% agarose gel and spectrophotometer respectively. The extracted DNA was further preceded for genetic homogeneity assessment by PCR based SCoT, ISSR and RAPD markers. Table 1. Effect of various concentrations of BA and IBA (mg/l) on shoot length in P. eriocarpum. DNA isolation and genetic homogeneity assessment BAP (mg/l) IBA (mg/l) Shoot length (mm)(Mean± SD) 0.2 00 28.14±1.4 0.2 0.2 26.03±5.7 0.2 0.4 27.71±0.1 0.2 0.6 25.59±0.4 0.2 0.8 26.98±0.1 0.4 0.0 27.96±0.7 0.4 0.2 27.38±0.1 0.4 0.4 30.98±0.6 0.4 0.6 43.34±3.8 0.4 0.8 31.95±1.1 0.6 0.0 30.27±0.1 0.6 0.2 31.61±1.16 0.6 0.4 33.44±0.2 0.6 0.6 31.37±1.1 0.6 0.8 27.51±0.8 0.8 0.0 28.72±0.8 0.8 0.2 28.32±0.7 0.8 0.4 28.08±0.4 0.8 0.6 28.21±0.3 0.8 0.8 26.41±0.8 0.0 0.0 0 doi:10.1371/journal.pone.0159050.t001 genomic DNA of all plants was extracted from leaves using DNeasy Plant Minikit (Qiagen, Netherlands). The leaf material (25mg) was powdered in liquid nitrogen and stored at -80°C until extraction. The quality and quantity of DNA samples were estimated on 1.5% agarose gel and spectrophotometer respectively. The extracted DNA was further preceded for genetic homogeneity assessment by PCR based SCoT, ISSR and RAPD markers. Table 1. Effect of various concentrations of BA and IBA (mg/l) on shoot length in P. eriocarpum. BAP (mg/l) IBA (mg/l) Shoot length (mm)(Mean± SD) 0.2 00 28.14±1.4 0.2 0.2 26.03±5.7 0.2 0.4 27.71±0.1 0.2 0.6 25.59±0.4 0.2 0.8 26.98±0.1 0.4 0.0 27.96±0.7 0.4 0.2 27.38±0.1 0.4 0.4 30.98±0.6 0.4 0.6 43.34±3.8 0.4 0.8 31.95±1.1 0.6 0.0 30.27±0.1 0.6 0.2 31.61±1.16 0.6 0.4 33.44±0.2 0.6 0.6 31.37±1.1 0.6 0.8 27.51±0.8 0.8 0.0 28.72±0.8 0.8 0.2 28.32±0.7 0.8 0.4 28.08±0.4 0.8 0.6 28.21±0.3 0.8 0.8 26.41±0.8 0.0 0.0 0 doi:10.1371/journal.pone.0159050.t001 Table 1. Effect of various concentrations of BA and IBA (mg/l) on shoot length in P. eriocarpum. genomic DNA of all plants was extracted from leaves using DNeasy Plant Minikit (Qiagen, genomic DNA of all plants was extracted from leaves using DNeasy Plant Minikit (Qiagen, Netherlands). The leaf material (25mg) was powdered in liquid nitrogen and stored at -80°C until extraction. The quality and quantity of DNA samples were estimated on 1.5% agarose gel and spectrophotometer respectively. The extracted DNA was further preceded for genetic homogeneity assessment by PCR based SCoT, ISSR and RAPD markers. genomic DNA of all plants was extracted from leaves using DNeasy Plant Minikit (Qiagen, Netherlands). The leaf material (25mg) was powdered in liquid nitrogen and stored at -80°C until extraction. The quality and quantity of DNA samples were estimated on 1.5% agarose gel and spectrophotometer respectively. The extracted DNA was further preceded for genetic homogeneity assessment by PCR based SCoT, ISSR and RAPD markers. Netherlands). The leaf material (25mg) was powdered in liquid nitrogen and stored at -80°C until extraction. SCoT analysis Ten SCoT primers [17] were selected out of 20 primers screened for genetic homogeneity assessment. The PCR amplification was carried out in a total volume of 25 μl containing 2.5 μl of 10X PCR buffer containing 15 mM MgCl2, 0.2mM dNTPs, 1 unit Taq Polymerase (Sigma, USA), 20ng of genomic DNA and 20 ng of Primer (Integrated DNA technologies Inc., India). PCR was performed using following reaction conditions: one cycle of DNA denaturation at 94°C for 4 min, 38 cycles of 30 s denaturation at 94°C, annealing at Ta°C and 1 min of exten- sion at 72°C with a final extension at 72°C for 8 min. Genetic Homogeneity Assessment of Pittosporum eriocarpum SCoT analysis Ten SCoT primers [17] were selected out of 20 primers screened for genetic homogeneity assessment. The PCR amplification was carried out in a total volume of 25 μl containing 2.5 μl of 10X PCR buffer containing 15 mM MgCl2, 0.2mM dNTPs, 1 unit Taq Polymerase (Sigma, USA), 20ng of genomic DNA and 20 ng of Primer (Integrated DNA technologies Inc., India). PCR was performed using following reaction conditions: one cycle of DNA denaturation at 94°C for 4 min, 38 cycles of 30 s denaturation at 94°C, annealing at Ta°C and 1 min of exten- sion at 72°C with a final extension at 72°C for 8 min. ISSR analysis After preliminary screening of 15 ISSR primers, 10 primers were selected. DNA amplification reaction was performed in a total volume of 25 μl containing 2 5 μl of 10X PCR buffer 15 mM Table 3. ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length. Source Sum of squares df Mean Square F-Value p-Value BAP 380 3 126 44(3,60) <0.0001 IBA 172 4 43 15(4,60) <0.0001 BAPxIBA 603 12 50 17(12,60) <0.0001 Pure Error 173 60 Total (Corrected) 1327 79 R-Squared 0.87 Adj R-Squared 0.83 doi:10.1371/journal.pone.0159050.t003 Table 3. ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length. DNA isolation and genetic homogeneity assessment The quality and quantity of DNA samples were estimated on 1.5% agarose gel and spectrophotometer respectively. The extracted DNA was further preceded for genetic homogeneity assessment by PCR based SCoT, ISSR and RAPD markers. Table 2. Effect of auxins (IAA, IBA or NAA) on in vitro rooting of P. eriocarpum shoots grown on MS medium. Auxin Concentration(mg/l) Number of rootsper shoot±SD Root length±SD(mm) Presence of basal callus±SD(mm) IAA 0.2 2.65±0.5 10.03±1.1 0 0.4 6.00±0.8 13.05±2.9 0 0.6 3.93±0.8 7.0±1.0 3.21±0.8 0.8 3.53±1.1 5.56±1.1 5.57±0.4 1.0 0 0 17.43±2.1 IBA 0.2 2.00±0.0 15.57±1.0 0 0.4 2.55±0.5 18.29±3.9 0 0.6 12.78±2.6 36.40±10.0 0 0.8 2.03±0.8 7.68±1.3 0 1.0 1.75±0.5 10.43±2.3 7.06±1.5 NAA 0.2 1.63±0.5 18.83±8.6 0 0.4 7.63±0.5 22.47±3.1 7.18±1.5 0.6 1.80±1.0 8.25±1.7 10.18±1.7 0.8 1.5±0.6 13.16±1.4 12.06±0.7 1.0 0 0 21.90±0.4 Control 0 0 0 0 doi:10.1371/journal.pone.0159050.t002 . Effect of auxins (IAA, IBA or NAA) on in vitro rooting of P. eriocarpum shoots grown on MS medium. PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 5 / 17 RAPD Analysis RAPD analysis was done to support the results of SCoT and ISSR markers. A set of 10 decamer RAPD primers out of 15 primers (OPL 1–10) (Sigma Aldrich, USA) were used for amplifica- tion. The PCR amplification was carried out in a total volume of 25 μl containing 2.5 μl of 10X PCR buffer, 15 mM MgCl2, 0.2mM dNTPs, 1 unit Taq Polymerase (Sigma, USA), 20ng of genomic DNA and 20 ng of RAPD primer were subjected to one cycle of DNA denaturation at 94°C for 4 min, 45 cycles of 1 min denaturation at 94°C, 1 min annealing at Ta°C and 2 min of extension at 72°C with a final extension at 72°C for 7 min. The annealing temperature (Ta°C) was kept at 2°C below the melting temperature of all the three primers used. We used AB Verity Thermal Cycler (USA) for amplification and repeated all the reactions twice. Amplified fragments were run on a 2.0% (w/v) agarose gel in 1X Tris acetate EDTA (TAE) Buffer stained by ethidium bromide (0.5 μg/ml). A 1 Kb ladder was used to estimate the size of unknown DNA fragments. Gels were documented using an UVP Gel Documentation (Jena, Germany). DataAnalysis Only the consistent reproducible bands were scored and data matrix was prepared based on pres- ence (1) and absence (0). Intensity of the band was not considered while scoring. NTSYSpc (Numerical Taxonomy and Multivariate Analysis System) Version 2.1 software [18] was used to perform the similarity matrix and construction of dendrogram. Genetic association amongst the different individuals was measured by the Jaccard’s similarity coefficient [19] with the SIMQUAL (Similarity for qualitative data program in NTSYS) module of NTSYS-pc software. The similarity matrix was subjected to cluster analysis of UPGMA and a dendrogram was generated by using the SAHN (sequential, agglomerative hierarchical and nested clustering) module in NTSYSpc program. ISSR analysis After preliminary screening of 15 ISSR primers, 10 primers were selected. DNA amplification reaction was performed in a total volume of 25 μl containing 2.5 μl of 10X PCR buffer, 15 mM Table 4. ANOVA of the effect of growth hormones on number of roots/shoot, root length and basal callus formation. Factors Sum of squares df Mean Square F P-value No. of roots/shoot A. Hormone Type 30 2 15 18(2,45) <0.0001 B. Concentration 268 4 67 82(4,45) <0.0001 AxB 314 8 39 48(8,45) <0.0001 Error 37 45 0.9 Total (Corrected) 649 59 R-Squared 0.94 Adj R-Squared 0.92 Root Length A. Hormone Type 1112 2 556 36(2,45) <0.0001 B. Concentration 1826 4 456 29(4,45) <0.0001 AxB 1848 8 231 15(8,45) <0.0001 Error 697 45 15 Total (Corrected) 5484 59 R-Squared 0.87 Adj R-Squared 0.83 Basal Callus A. Hormone Type 788 2 394 460(2,45) <0.0001 B. Concentration 1683 4 421 491(4,45) <0.0001 AxB 321 8 40 47(8,45) <0.0001 Error 39 45 0.9 Total (Corrected) 2830 59 R-Squared 0.99 Adj R-Squared 0.98 doi:10.1371/journal.pone.0159050.t004 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 6 / 17 NOVA of the effect of growth hormones on number of roots/shoot, root length and basal callus formation. 6 / 17 Genetic Homogeneity Assessment of Pittosporum eriocarpum MgCl2, 0.2mM dNTPs, 1 unit Taq Polymerase (Sigma, USA), 20ng of genomic DNA and 20 ng of ISSR Primer (Sigma Aldrich, USA). PCR was performed using the following program: Initial denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 94°C for 40 s, anneal- ing at Ta°C for 60 s and extension at 72°C for 60 s with a final extension at 72°C for 8 min. PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 Shoot induction and multiplication Explants were inoculated on MS medium with various concentrations of BA alone and in com- bination with IBA. A two-way ANOVA was conducted to determine the effects of BA, IBA and BA+IBA on shoot length. First shoot initiation was observed in BA 0.4mg/l after 8 days of cul- ture. Among various concentrations of BA and IBA used, MS medium containing BAxIBA (0.4x0.6 mg/l) was found most suitable for shoot induction (93.3%), with 43.34±3.8 mm shoot length (Figs 1A–1B and 2) followed by BAxIBA (0.6x0.4 mg/l) which showed 86.7% shoot induction with 33.44±0.2 mm shoot length per explant (Table 1). Shoot induction frequency was significantly influenced by BA and IBA independently as well as their interaction was also found significant. The mean separation analysis by Tukey’s tests on shoot length showed that BAP has the largest effect on shoot length followed by interaction between BAPxIBA than IBA alone (Table 3). Out of 20 treatments at individuallevel, BAP in combination with IBA showed higher shoot regeneration frequency than BAP alone. BA is equivalent to 6-benzylaminopur- ine, BAP [20]. It is relevant to mention here that, the shoot induction is enhanced with the syn- ergistic effect of cytokinin and auxin [21]. In the highest tested concentrations of BA (0.8 mg/l) alone and in combination with IBA (BA+IBA 0.8x0.2 mg/l), the shootlength was decreased. PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 7 / 17 Genetic Homogeneity Assessment of Pittosporum eriocarpum Fig 2. Effect of various concentrations of BA and IBA on shoot induction from nodal explants of P. eriocarpum. doi:10 1371/journal pone 0159050 g002 Fig 2. Effect of various concentrations of BA and IBA on shoot induction from nodal explants of P. eriocarpum. Fig 2. Effect of various concentrations of BA and IBA on shoot induction from nodal explants of P. eriocarpum. doi:10 1371/journal pone 0159050 g002 doi:10.1371/journal.pone.0159050.g002 The explants of control set failed to develop shoots. The shoots obtained from BA 0.4 mg/l, BA 0.6 mg/l, BA+IBA 0.4+0.6 mg/l, BA+IBA 0.6+0.4 mg/l were further subcultured minimum of three times, and then after dipping in IBA for an hour were inoculated on MS medium in respective concentrations. Within five months of culture they developed multiple shoots. A one-way ANOVA was conducted to compare the effect of dipping of shoots obtained from four combinations on number of shoots per explant, leaves per branch and basal calls forma- tion. Rooting, acclimatization and field transfer Strength of MS medium, concentration and type of auxin and supplementation of additives are a few factors which affect the rate of in vitro rooting [25]. In vitro raised shoots obtained from cul- tures on MS medium supplemented with BA alone and BA in combination with IBA were rooted in different types and concentrations of auxins (IAA, IBA, NAA). A two-way ANOVA was con- ducted to determine the effects of type of hormones at varied concentrations and interaction between the two on number of roots per explant, root length and basal callus formation. First root- ing was observed in IBA 0.6 mg/l after 21 days of culture which showed 90% root induction with 12.78±2.6roots per shoot, 36.40±10.0 mm root length per root without any basal callus formation. It has been observed that with increase in the concentration of IBA, there was decrease in percent- age of root induction, number of roots per shoot but varied in root length per root (Fig 1C, Table 2). IAA (0.4 mg/l) showed 80% rooting response with6.00±0.8roots per explant, 13.05±2.9 mm root length per root without any basal callus formation. Decrease in percentage of root induc- tion, number of roots per explant and root length per root was observed with increase in IAA con- centration. Among different concentrations of NAA tested, NAA 0.4mg/l showed highest rooting response (80%) with 7.63±0.5 roots per explant, 22.47±3.1 mm root length per root with basal cal- lus formation. Increase in the concentration of NAA led to decrease in percentage of root induc- tion and number of roots per explant, however, increase in basal callus formation was observed. All three effects, type ofhormones, hormone concentrations and the interactions between the two factors (hormone type x concentration) showed significant influence on root induction, root length and basal callusformation (Table 4). No rooting was observed in IAA and NAA at concentration 1.00 mg/l. Among the three PGRs, IBA (0.6mg/l) stimulated early initiation of roots followed by IAA (0.8 mg/l) and NAA (0.4mg/l). Also, IBA 0.6 mg/l showed maximum root induction, root numbers per shoot and length among three auxins tested. Nearly 80–85% rooted explants devel- oped into plantlets (Fig 1D). Cultures raised in IBA 0.6 mg/l showed maximum survival rate (90%) followed by IAA and then NAA (Fig 4A–4C). Genetic Homogeneity Assessment of Pittosporum eriocarpum Table 5. Effect of one hour incubation in IBA on number of shoots, leaves and callus diameter per explants. PGR combinations used for shoot multiplication mg/l) Number of shoots per explant ±SD Number of leaves per branch ±SD Callus diameter (mm) ±SD BA 0.4 10.60±1.1 4.39±0.5 8.11±0.6 BA 0.6 9.85±1.1 7.58±1.3 7.97±0.3 BA 0.4+IBA0.6 14.40±3.3 14.85±1.7 11.27±1.7 BA0.6 +IBA0.4 9.10±1.2 10.33±1.6 9.43±1.9 doi:10 1371/journal pone 0159050 t005 e hour incubation in IBA on number of shoots, leaves and callus diameter per explants. Table 5. Effect of one hour incubation in IBA on number of shoots, leaves and callus diameter per expla doi:10.1371/journal.pone.0159050.t005 significant increment in shoot multiplication after subculturing the mother plant. This may be due to suppression of apical dominance due to repeated subcultures that in turn induce basal meristematic cells to produce new shoots [22]. This method of shoot multiplication has been employed in other plant species [23]. Treatment of low level of auxin and cytokinin in culture medium is known to stimulate number and elongation of shoots in several tree species [24]. Regenerated shoots remained viable for more than eight months when left in original culture medium. This is a low cost and short term storage method for germplasm of P. eriocarpum. Since it is a medicinally important and endangered plant, use of this method will provide pro- tocol for extraction of medicinal compound and release the pressure on natural populations. Shoot induction and multiplication BA+IBA 0.4+0.6 mg/l was found to be most suitable for multiplication of shoots with 14.40±3.3 shoots per explants and 14.85±1.7leaves per branch (Fig 3; Table 5). We observed Fig 3. The effect of BA and IBA on number of shoots produced per explant, number of leaves per shoot and basal callus. doi:10.1371/journal.pone.0159050.g003 Fig 3. The effect of BA and IBA on number of shoots produced per explant, number of leaves per shoot and basal callus. doi:10.1371/journal.pone.0159050.g003 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 8 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 Rooting, acclimatization and field transfer IBA stimulated rooting has also been reported in Pittosporum napaulensis, where direct root organogenesis is observed in the medium supple- mented with IBA and IAA, whereas higher concentration of NAA, led to the formation of callus [8]. IBA stimulated rooting is also reported in Pittosporum tobira variegatum[7]. Superiority of IBA as rooting hormone over other auxins is also reported in other genera [23, 26, 27]. PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 9 / 17 Genetic Homogeneity Assessment of Pittosporum eriocarpum Fig 4. Effect of types of auxins on root induction in P. eriocarpum. (A)Number of roots produced per regenerated shoot. (B)Root length. (C) Basal callus formation per explant. doi:10.1371/journal.pone.0159050.g004 Fig 4. Effect of types of auxins on root induction in P. eriocarpum. (A)Number of roots produced per regenerated shoot. (B)Root length. (C) Basal callus formation per explant. doi:10.1371/journal.pone.0159050.g004 doi:10.1371/journal.pone.0159050.g004 Transfer of in vitro raised plantlets to a similar environment that exists in nature is likely to be most suitable for the growth and survival of plantlets [28]. As P. eriocarpum grows on exposed sites with temperature range of 12−270 C,in vitro raised rooted plantlets were trans- ferred to the greenhouse (temperature 25°C) for three months. Nearly 73% plantlets survived and continued to grow (Fig 1E and 1F). In many other taxa, high mortality rate has been reported for in vitro raised plants when transferred to natural filed conditions, as they possess weak root system at this stage [29,30]. In the present study, no phenotypic variation was found amongst the in vitro raised plants. The reintroduction of native plants and especially of rare and endangered species has become important in conservation worldwide for restoration pur- poses, which has also become an important practice in biodiversity conservation [31–35]. Since P. eriocarpum is native to India and endangered species, the reintroduction in nature would save the species from becoming extinct in nature. Genetic Homogeneity Assessment of Pittosporum eriocarpum Table 6. Amplification products generated with SCoT, ISSR and RAPD markers among mother plant and in vitro raised plants. Markers Primerseries Primer sequence Number of scorable bandsper primer Monomorphic bands Polymorphic bands SCoT 1 5’CAACAATGGCTACCACCA3’ 5 5 0 2 5’CAACAATGGCTACCACCC3’ 3 3 0 3 5’CAACAATGGCTACCACCG3’ 6 6 0 4 5’CAACAATGGCTACCACCG3’ 4 4 0 5 5’CAACAATGGCTACCACCT3’ 4 4 0 10 5’CAACAATGGCTACCAGCC3’ 3 3 0 15 5’ACGACATGGCGACCGCGA3’ 6 3 3 18 5’ACCATGGCTACCACCGCC3’ 7 3 4 35 5’CATGGCTACCACCGGCCC3’ 5 5 0 36 5’GCAACAATGGCTACCACC3’ 4 4 0 ISSR 843 5’CTCTCTCTCTCTCTCTRA3’ 3 3 0 847 5’CACACACACACACACARC3’ 4 2 2 852 5’TCTCTCTCTCTCTCTCRA3’ 3 3 0 853 5’TCTCTCTCTCTCTCTCRT3’ 3 3 0 854 5’TCTCTCTCTCTCTCTCRG3’ 2 2 0 855 5’ACACACACACACACYT3’ 3 3 0 857 5’ACACACACACACACYG3’ 3 3 0 860 5’TGTGTGTGTGTGTGTGRA3’ 7 4 2 888 5’BDBCACACACACACACA3’ 4 4 0 891 5’HVHTGTGTGTGTGTGTG3’ 2 2 0 RAPD OPL-01 5’GGCATGACCT3’ 5 5 0 OPL-02 5’TGGGCGTCAA3’ 7 7 0 OPL-03 5’CCAGCAGCTT3’ 5 5 0 OPL-04 5’GACTGCACAC3’ 6 6 0 OPL-05 5’ACGCAGGCAC3’ 4 4 0 OPL-06 5’GAGGGAAGAG3’ 4 4 0 OPL-07 5’AGGCGGGAAC3’ 3 3 0 OPL-08 5’AGCAGGTGGA3’ 3 3 0 OPL-09 5’TGCGAGAGTC3’ 1 1 0 OPL-10 5’TGGGAGATGG3’ 6 4 2 doi:10.1371/journal.pone.0159050.t006 somaclonal variation in micropropagated plants [36]. The RAPD and ISSR analyses are based on the non-coding regions of DNA used for studying genetic diversity, variability or stability. However, SCoT is novel and gene targeted molecular marker technique derived from flanking ATG translation codon in plant gene and is considered to be more authentic in assessing genetic homogeneity [17,37]. All these markers are found to be simple, resolvable, and cost- effective in assessing the genetic homogeneity [13, 38–40]. Ten SCoT primers produced a total of 47 reproducible and scorable bands with an average of 4.7 bands per primer. Each primer produced amplification product in the range of 250–1500 bp. All banding profiles from micropropagated plants were monomophic and similar to mother plant except primers viz., primer 15 and 18, which showed polymorphic bands in two of the regenerates. Number of scorable band varied from 2 to 7 for each primer. In case of ISSR primers, 10 primers yielded reproducible bands out of 15 primers screened. They yielded 34 scorable bands with an average of 3.4 bands per primer ranging from 250–1500 bp. The band numbers varied from 2 to 7 in each primer. Genetic homogeneity assessment We evaluated the genetic homogeneity of the in vitro grown plants through SCoT, ISSR and RAPD markers. The use of more than one marker has always been recommended for a better analysis of genetic homogeneity of plants because micropropagation is known to provoke PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 10 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 All banding profiles from micropropagated plants were monomophic and similar to mother plant except the primers viz., primer 847 and 860 which showed polymorphic bands in two of the regenerates. Of the 15 RAPD decamer primers PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 11 / 17 Genetic Homogeneity Assessment of Pittosporum eriocarpum Fig 5. Assessment of genetic fidelity of in vitro raised plants of Pittospo fragments obtained with primer SCoT 35. (B) ISSR fragments obtained from fragments obtained with primer OPL10. L- 1Kb ladder; MP- mother plant (Co plants. doi:10.1371/journal.pone.0159050.g005 Genetic Homogeneity Assess :10.1371/journal.pone.0159050 July 19, 2016 Fig 5. Assessment of genetic fidelity of in vitro raised plants of Pittos fragments obtained with primer SCoT 35. (B) ISSR fragments obtained fro fragments obtained with primer OPL10. L- 1Kb ladder; MP- mother plant (C plants. doi:10.1371/journal.pone.0159050.g005 g y 050 July 19, 2016 Fig 5. Assessment of genetic fidelity of in vitro raised plants of Pittosporum eriocarpum. (A) Fig 5. Assessment of genetic fidelity of in vitro raised plants of Pitto fragments obtained with primer SCoT 35. (B) ISSR fragments obtained fr fragments obtained with primer OPL10. L- 1Kb ladder; MP- mother plant plants. doi:10.1371/journal.pone.0159050.g005 50 July 19, 2016 Fig 5. Assessment of genetic fidelity of in vitro raised plants of Pittosporum eriocarpum. (A) SCoT fragments obtained with primer SCoT 35. (B) ISSR fragments obtained from primer number 860. (C)RAPD fragments obtained with primer OPL10. L- 1Kb ladder; MP- mother plant (Control); A1-A9- in vitro raised plants. doi:10 1371/journal pone 0159050 g005 Fig 5. Assessment of genetic fidelity of in vitro raised plants of Pittosporum eriocarpum. (A) SCoT fragments obtained with primer SCoT 35. (B) ISSR fragments obtained from primer number 860. (C)RAPD fragments obtained with primer OPL10. L- 1Kb ladder; MP- mother plant (Control); A1-A9- in vitro raised plants. doi:10.1371/journal.pone.0159050.g005 12 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 Genetic Homogeneity Assessment of Pittosporum eriocarpum Fig 6. Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P. eriocarpum by UPGMA cluster analysis from the SCoT data set showing genetic relationship. MP- mother plant; A1-A9- in vitro raised plants. Fig 6 Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P eriocarpum by UPGMA cluster Fig 6. Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P. eriocarpum by UPGMA cluster analysis from the SCoT data set showing genetic relationship. MP- mother plant; A1-A9- in vitro raised plants. doi:10.1371/journal.pone.0159050.g006 doi:10.1371/journal.pone.0159050.g006 Fig 7. Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P. eriocarpum by UPGMA cluster analysis from the ISSR data set showing genetic relationship. MP- mother plant; A1-A9- in vitro raised plants. doi:10.1371/journal.pone.0159050.g007 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 13 / 17 Fig 7. Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P. eriocarpum by UPGMA cluster analysis from the ISSR data set showing genetic relationship. MP- mother plant; A1-A9- in vitro raised plants. Fig 7. Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P. eriocarpum by UPGMA cluster analysis from the ISSR data set showing genetic relationship. MP- mother plant; A1-A9- in vitro raised plants. doi:10.1371/journal.pone.0159050.g007 doi:10.1371/journal.pone.0159050.g007 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 13 / 17 Genetic Homogeneity Assessment of Pittosporum eriocarpum Fig 8. Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P. eriocarpum by UPGMA cluster analysis from the RAPD data set showing genetic relationship. MP- mother plant; A1-A9- in vitro raised plants. d i 10 1371/j l 0159050 008 Fig 8. Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P. eriocarpum by UPGMA cluster Fig 8. Dendrogram illustrating coefficient similarity among in vitro raised plants with mother plant of P. eriocarpum by UPGMA cluster analysis from the RAPD data set showing genetic relationship. MP- mother plant; A1-A9- in vitro raised plants. doi:10.1371/journal.pone.0159050.g008 screened initially, only 10 primers showed clear and reproducible bands. These primers gave rise to a total of 44 scorable bands. The number of bands for each primer varied from 1–7 with an average of 4.4 bands per primer ranging from 250 to 1000 bp. The highest number of bands were obtained in the primer 2 i.e., 7 and the lowest number of bands i.e., one, was obtained in the primer 9. All banding profiles from micropropagated plants were monomophic and similar to mother plant except the primer viz., primer OPL 10 which showed polymorphic bands in three of the regenerates. A comparative account of primer sequences used for SCoT, ISSR and RAPD analysis is given in Table 6. Monomorphic banding pattern was observed for all the amplified band classes from SCoT, ISSR and RAPD among the regenerates and mother plant, indicating the absence of variability (Fig 5A–5C). PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 Data analysis The similarity value on the basis of Jaccard’s similarity coefficient among nine micropropa- gated plants and mother plant was calculated for three markers viz., SCoT, ISSR and RAPD which showed similarity ranged from 0.89 to 1.00, 0.91 to 1.00 and 0.95 to1.00 respectively (Figs 6–8). The dendrograms generated through UPGMA analysis revealed 97% similarity among regenerated plants with mother plant. Dendrogram generated for SCoT marker showed that A1, A2, A3, A5, A7 and A8 are very identical to mother plant. A9 and A4 also clustered close to MP. Dendrogram generated for ISSR marker showed that except A8, rest of the micro- propagated plants were 100% identical to MP. Dendrogram generated for RAPD marker showed that A7 and A8 are almost identical to MP and A1-A6. It has been observed that, den- drograms generated for the three markers showed a slight change at DNA level of in vitro 14 / 17 PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 Genetic Homogeneity Assessment of Pittosporum eriocarpum raised A8 plant which probably is due to somaclonal variation [11]. The dendrogram generated through UPGMA analysis revealed 97% similarity amongst the regenerates and the donor mother plant. Acknowledgments Grant support (# BT/Env/BC/01/2010/23.3.2012) from Department of Biotechnology, Govern- ment of India, New Delhi, is thankfully acknowledged. We thank Prof. Mala Mazumdar, CPGS Jain University, Bangalore and an anonymous reviewer for their valuable comments. We are grateful to Prof Shailendra Goel, University of Delhi for help in molecular analysis and Dr GP Sharma andDr Pooja Jha Maity for help in statistical analysis. The authors warmly thank Dr. Randall P. Niedz for critically reading the manuscript. Conclusions Present study reports a holistic and reliable procedure for in vitro regeneration, greenhouse acclimatization and field transfer of P. eriocarpum. This is the first successful attempt to estab- lish a micropropagation protocol for this species by nodal explants, which will provide a alter- native method for its conservation as well as supply of the material for commercial use. It is estimated that a single explant can finally develop into 8–10 hardened plants within 8 months of culture period. We reported the genetic homogeneity and true-to-type nature of in vitro raised plants using SCoT, ISSR and RAPD analysis. Such protocol is useful for providing valu- able resource material and help in the delisting of species from red data list. Author Contributions Conceived and designed the experiments: AKP PLU JT PS. Performed the experiments: PS JT MDD. Analyzed the data: JT MDD. Wrote the paper: AKP PLU JT. Conceived and designed the experiments: AKP PLU JT PS. Performed the experiments: PS JT MDD. Analyzed the data: JT MDD. Wrote the paper: AKP PLU JT. S2 Table. Effect of auxins (IAA, IBA or NAA) on in vitro rooting of P. eriocarpum shoots grown on MS medium. (PDF) S2 Table. Effect of auxins (IAA, IBA or NAA) on in vitro rooting of P. eriocarpum shoots grown on MS medium. (PDF) S3 Table. ANOVA of the effect of BA and IBA and combined BA and IBA on shoot length (PDF) Supporting Information S1 Table. Effect of various concentrations of BA and IBA (mg/l) on shoot length in P. erio- carpum. (PDF) PLOS ONE \| DOI:10.1371/journal.pone.0159050 July 19, 2016 References 1. Semwal P, Kapoor T, Anthwal P, Thapliyal A. Pittopsoram eriocarpum Royle (Agni) endangered medic- inal plant species of Uttarakhand and its conservation. BTI 2013, 6: 25–30. 1. Semwal P, Kapoor T, Anthwal P, Thapliyal A. Pittopsoram eriocarpum Royle (Agni) endangered medic- inal plant species of Uttarakhand and its conservation. BTI 2013, 6: 25–30. 2. IUCN, The IUCN Red List of Threatened Species. 2014, Version 2014.3. Available: www.iucnredlist. org. Accessed 11 March 2015. Doi: http://dx.doi.org/10.2305/IUCN.UK.1998. RLTS.T31330A9627065. en. 2. IUCN, The IUCN Red List of Threatened Species. 2014, Version 2014.3. Available: www.iucnredlist. org. Accessed 11 March 2015. 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https://openalex.org/W2156923912	https://europepmc.org/articles/pmc3867857?pdf=render	English	null	Baseline Obesity Status Modifies Effectiveness of Adapted Diabetes Prevention Program Lifestyle Interventions for Weight Management in Primary Care	BioMed research international	2,013	cc-by	4,936	Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 191209, 7 pages http://dx.doi.org/10.1155/2013/191209 Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 191209, 7 pages http://dx.doi.org/10.1155/2013/191209 Hindawi Publishing Corporation BioMed Research International Volume 2013, Article ID 191209, 7 pages http://dx.doi.org/10.1155/2013/191209 Kristen M. J. Azar,1 Lan Xiao,1 and Jun Ma1,2 1 Palo Alto Medical Foundation Research Institute, 795 El Camino Real, Ames Building, Palo Alto, CA 94301, USA 2 Stanford Prevention Research Center, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA Kristen M. J. Azar,1 Lan Xiao,1 and Jun Ma1,2 1 Palo Alto Medical Foundation Research Institute, 795 El Camino Real, Ames Building, Palo Alto, CA 94301, USA 2 Stanford Prevention Research Center, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA 1 Palo Alto Medical Foundation Research Institute, 795 El Camino Real, Ames Building, Palo Alto, CA 94301, USA 2 Stanford Prevention Research Center, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA Correspondence should be addressed to Kristen M. J. Azar; azark@pamfri.org Received 30 September 2013; Accepted 13 November 2013 Academic Editor: Pierpaolo De Feo Copyright © 2013 Kristen M. J. Azar et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Objective. To examine whether baseline obesity severity modifies the effects of two different, primary care-based, technology- enhanced lifestyle interventions among overweight or obese adults with prediabetes and/or metabolic syndrome. Patients and Methods. We compared mean differences in changes from baseline to 15 months in clinical measures of general and central obesity among participants randomized to usual care alone (𝑛= 81) or usual care plus a coach-led group (𝑛= 79) or self-directed individual (𝑛= 81) intervention, stratified by baseline body mass index (BMI) category. Results. Participants with baseline BMI 35+ had greater reductions in mean BMI, body weight (as percentage change), and waist circumference in the coach-led group intervention, compared to usual care and the self-directed individual intervention (𝑃< 0.05 for all). In contrast, the self-directed intervention was more effective than usual care only among participants with baseline BMIs between 25 ≤35. Mean weight loss exceeded 5% in the coach-led intervention regardless of baseline BMI category, but this was achieved only among self-directed intervention participants with baseline BMIs < 35. Conclusions. Baseline BMI may influence behavioral weight-loss treatment effectiveness. Researchers and clinicians should take an individual’s baseline BMI into account when developing or recommending lifestyle focused treatment strategy. This trial is registered with ClinicalTrials.gov (NCT00842426). Clinical Study Baseline Obesity Status Modifies Effectiveness of Adapted Diabetes Prevention Program Lifestyle Interventions for Weight Management in Primary Care Kristen M. J. Azar,1 Lan Xiao,1 and Jun Ma1,2 1 Palo Alto Medical Foundation Research Institute, 795 El Camino Real, Ames Building, Palo Alto, CA 94301, USA 2 Stanford Prevention Research Center, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA 1. Introduction Coaches could view Heart360 patient self- monitoring records, which they reviewed regularly and used to tailor their ongoing progress feedback via secure messaging for participants in the coach-led intervention. Self-directed intervention intervention visits occurred at the clinic. Inclusion criteria included an age of at least 18 years, a BMI of at least 25, and the presence of prediabetes (defined by impaired fasting plasma glucose level of 100 to 125 mg/dL) or metabolic syndrome. Major exclusion criteria included serious medical or psychiatric conditions (e.g., stroke, psychotic disorder) or special life circumstances (e.g., pregnancy). Eligible and consenting overweight or obese adults with prediabetes and/or metabolic syndrome seen in primary care were randomized to receive usual care alone (𝑛= 81) or usual care plus a coach-led (𝑛= 79) or self-directed (𝑛= 81) behavioral weight-loss intervention. Height was measured at baseline only, and weight and waist circumference were measured at baseline and at 3, 6, and 15 months. Measurements were taken in duplicate per standardized protocols [17, 18]. Body mass index was calculated. Change in BMI from baseline to 15 months was the trial primary outcome [15]. interventions into real-world settings remains a challenge. Efforts have been made to facilitate this process while retain- ing essential components of efficacious interventions [13]. However, these same efforts have resulted in wide variation in intervention setting, structure, intensity and form of contact, and resources required—and (unsurprisingly) they have produced mixed results regarding clinical effectiveness. Improved ability to implement targeted interventions for readily defined subgroups of the intended population may result in more efficient and effective use of resources.h fif The “Evaluation of Lifestyle Interventions to Treat Ele- vated Cardiometabolic Risk in Primary Care” (E-LITE) study was one of few pragmatic randomized controlled trials that successfully translated the Diabetes Prevention Program (DPP) lifestyle intervention into a primary care setting in the US. Published E-LITE data have demonstrated that two adapted, technology-enhanced DPP interventions (further described in Section 2)—one using a self-directed approach and the other a coach-led approach—were both superior to usual care, whereas the coach-led intervention was superior to the self-directed one, in promoting weight loss among overweight or obese adults with prediabetes and/or metabolic syndrome [15]. The primary aim of the current study was to examine whether changes in clinical measures of general and abdominal obesity differed by baseline BMI category when comparing the two interventions to usual care and to each other. 1. Introduction medications have had limited effectiveness, some serious adverse effects, and limited uptake [7].i Obesity remains a pressing public health problem with adverse medical, psychological, social, and economical con- sequences. Nearly 70% of US adults are overweight (body mass index [BMI] in kg/m2 25 ≤30) or obese (BMI ≥30), with 36% obese [1]. More alarming still, the 6.3% prevalence of severely obesity (BMI ≥40) [1] is projected to increase by 130% over the next 2 decades [2]. Although bariatric surgery is the recommended treatment for severely obese individuals and/or moderately obese individuals (BMI 35 ≤ 40) with comorbidities [3], its implementation is limited by access, cost, recidivism, and complications [4, 5]. Only 1%- 2% of obese people eligible for insurance coverage of surgical treatment receive it, compelling an urgent need of alternative treatment strategies for this subpopulation [6]. Weight loss f Emerging data find intensive lifestyle interventions— focusing on calorie-reduced, healthful eating, increased physical activity, and self-management skills training—can lead to clinically significant weight loss in the short [8–10] and long term [10, 11] among individuals with a BMI ≥35, who also achieve improvements in cardiovascular disease (CVD) risk factors even despite persistent, albeit reduced, obesity after intervention. Despite a recent and renewed interest in examining the efficacy of intensive behavior therapy for obesity within higher BMI subcategories [12], very few studies have evaluated the comparative effectiveness of evidence-based, empirical lifestyle interventions in real- world settings by baseline obesity status. Implementation of efficacious but resource-intensive, research-based lifestyle 2 BioMed Research International Table 1: E-LITEa intervention: key components and features. Table 1: E-LITEa intervention: key components and features. y p Coach-led intervention Self-directed intervention (1) 12-week core curriculum sessionsb Clinic-based, small groups Home-based DVD (2) Online self-monitoring of weight and physical activityc Preferably daily but at least twice weekly; coach routinely reviewed records Preferably daily but at least twice weekly; coach did not routinely review records (3) Personalized lifestyle coachingd Proactive, coach-initiated As needed, patient-initiated aE-LITE: Evaluation of lifestyle interventions to treat elevated cardiometabolic risk in primary care. bDiabetes Prevention Program (DPP) investigators at the University of Pittsburgh developed the Group Lifestyle Balance (GLB) program following the DPP trial [13]. Its curriculum is publicly available online [14]. cVia the American Heart Association’s free, secure Heart360 Web portal (http://www.heart360.org/). dVia secure provider-patient online messaging embedded in a fully functional electronic health record system. 1. Introduction We hypothesized that baseline BMI modified participant response to treatment such that participants with baseline BMI 35+ would benefit from the more structured, coach-led intervention, whereas those with lower starting BMI would respond to either coach-led or self-directed intervention. 2.2. Intervention. The E-LITE study innovatively integrated the DPP-based Group Lifestyle Balance (GLB) core curricu- lum [13], which has been recognized by the Centers for Disease Prevention and Control’s National Diabetes Preven- tion Program, with lifestyle coaching and self-management support via high-reach, affordable technologies. Both E-LITE interventions adopted the DPP’s weight loss and physical activity goals [19] and delivered the GLB core curriculum for 12 weeks during the intensive treatment phase either through a self-directed, take-home DVD or coach-led, in- clinic small groups. The interventions also provided elec- tronically mediated lifestyle coach contact and online self- monitoring of weight and physical activity goal attainment during a 12-month maintenance phase. Table 1 summarizes the key components and features of the E-LITE self-directed and coach-led interventions. 2. Materials and Methods Complete E-LITE trial protocol and methods were published previously [16]. Data collection occurred in 2009–2011. Below we describe methodological details relevant to this study. 2.3. Statistical Analyses. Baseline characteristics of each study group by baseline BMI category (25 ≤30, 30 ≤35, or 35+) were examined using analysis of variance (ANOVA) for continuous variables and Chi-square test for categor- ical variables. Between-group differences by baseline BMI category at 15 months were evaluated by intention-to-treat using all available data and tests of group by baseline BMI category interactions in repeated-measures mixed models. A 2.3. Statistical Analyses. Baseline characteristics of each study group by baseline BMI category (25 ≤30, 30 ≤35, or 35+) were examined using analysis of variance (ANOVA) for continuous variables and Chi-square test for categor- ical variables. Between-group differences by baseline BMI category at 15 months were evaluated by intention-to-treat using all available data and tests of group by baseline BMI category interactions in repeated-measures mixed models. A 2.1. Study Population and Measures. Participants were recruited (July 2009–June 2010) from a single primary care clinic that is part of a large multispecialty group practice in the San Francisco Bay Area. All data collection and BioMed Research International 3 BMI 25 ≤30 BMI 30 ≤35 BMI ≥35 −4 −3 −2 −1 0 1 2 Body mass index (kg/m2) ∗ ∗ ∗ ∗ ∗ ∗∗ ∗∗ ∗∗ ∗∗ ∗∗† ∗∗† ∗† 95% CI (−2.8, −1.5) (−2.4, −1.1) (−1.1, 0.2) (−2.9, −1.6) (−2.4, −1.0) (−1.5, −0.1) (−2.9, −1.1) (−1.8, −0.04) (−0.9, 1.0) Body weight (%) −10 −5 0 5 95% CI (−8.8, −4.9) (−7.4, −3.6) (−2.6, 1.3) (−8.6, −4.6) (−7.4, −3.1) (−4.6, −0.4) (−8.4, −3.1) (−5.4, −0.4) (−3.7, 1.5) Waist circumference (cm) −25 −15 −5 5 15 95% CI (−23.2, −10.9) (−21.6, −9.9) (−12.8, −0.3) (−20.1, −8.1) (−20.0, −6.5) (−14.1, −0.1) (−17.9, −1.7) (−11.4, 4.9) (−7.3, 9.1) Coach-led Self-directed Usual care CI Confidence interval ∗P < 0.005 compared to usual care ∗∗P ≤0.01 compared to usual care †P < 0.005 compared to self-directed Figure 1: Outcomes by baseline body mass index category and intervention type. Waist circumference (cm) Figure 1: Outcomes by baseline body mass index category and intervention type. 2. Materials and Methods −1.1 kg/m2]), in percentage weight loss for the BMI 25 ≤30 (−5.5%, 95% CI [−7.4%, −3.6%]; 𝑃< 0.0001 versus usual care) and 30 ≤35 (−5.2%, 95% CI [−7.4%, −3.1%]; 𝑃= 0.02 versus usual care) categories, and in waist circumference for the BMI 30 ≤35 category (−13.2 cm, 95% CI [−20.0 cm, −8.1 cm]; 𝑃= 0.03). In the self-directed group mean weight loss reached 5% only among those with a baseline BMI of < 35. Moreover, reductions in BMI (𝑃= 0.01), weight as percentage change (𝑃= 0.04), and waist circumference (𝑃= 0.04) were significantly greater in the coach-led versus self- directed intervention within the BMI 35+ category, whereas the two interventions did not differ significantly for any of the three obesity measures in the two lower BMI categories. separate model examined change in each of the 3 obesity outcome variables: BMI and percent body weight change for general obesity and waist circumference for abdominal obesity. The group by baseline BMI category interaction terms were significant for all three outcomes (𝑃< 0.001). As in the main study [15], these models were adjusted for age, sex, race, and ethnicity, and missing data were handled directly through maximum likelihood estimation via mixed modeling. Model-based least-square mean changes and 95% confidence intervals (CIs) were obtained. All analyses were conducted using SAS, version 9.2 (SAS Institute Inc., Cary, North Carolina). 3. Results and Discussion Similarly, the coach-led intervention had no apparent The self-directed intervention led to greater improve- ments in BMI (𝑃= 0.03 versus usual care) only for the BMI 25 ≤30 category (−1.7 kg/m2, 95% CI [−2.4 kg/m2, BioMed Research International 4 Table 2: Baseline characteristics of the study participantsa. 3. Results and Discussion 3.2. Discussion. Efficacy research has unequivocally shown that intensive, highly structured, individual lifestyle interven- tion lowers cardiometabolic risk [20]. The unabated obesity epidemic and its associated health problems and rising societal and economical burdens compel the urgency of adapting proven, albeit expensive, interventions into increas- ingly resource-limited real-world settings while striving to retain the effectiveness of the original treatment. The current findings show that the effects of the successful E-LITE coach- led and self-directed interventions in primary care differed by starting obesity status, suggesting that one size may not fit all when it comes to lifestyle interventions. 3.1. Results. Table 2 shows baseline sample characteristics, stratified by baseline BMI. No signifıcant differences in baseline characteristics were detected between participants in each treatment group. The coach-led intervention resulted in significantly greater mean reductions from baseline to 15 months in BMI (ranging from −2.0 kg/m2, 95% CI [−2.9 kg/m2, −1.1 kg/m2] in the BMI 35+ category to −2.2 kg/m2, 95% CI [−2.9 kg/m2, −1.6 kg/m2] in the BMI 30 ≤ 35 category) and percent body weight change (ranging from −6.8%, 95% CI [−8.8%, −4.9%] in the BMI 25 ≤30 category to −5.7%, 95% CI [−8.4%, −3.1%] in the BMI 35+ category) for all three baseline BMI categories, and in waist circumference for the BMI 25 ≤30 (−17.1 cm, 95% CI [−23.2 cm, −10.95 cm]) and 35+ (−9.78 cm, 95% CI [−17.9 cm, −1.7 cm]) categories (𝑃< 0.05 versus usual care for all; Figure 1). The coach- led group achieved a mean percentage weight loss exceeding 5%, a commonly accepted threshold of clinically significant weight loss, and the upper 95% confidence limit was at least 3% weight loss, across the baseline BMI categories.h Notably participants with moderate or severe obesity (baseline BMI 35+) had greater reductions in all three obesity measures (BMI, percentage weight loss, and waist circumference) in the coach-led intervention, but not in the self-directed intervention, compared with usual care. They also responded more favorably to the coach-led intervention compared to the self-directed intervention. In contrast, over- weight participants (baseline BMI 25 ≤30) had similar mean BMI and percent body weight reductions in the two active interventions, both of which were more effective than usual care. 3. Results and Discussion Characteristic All Usual care Coach-led Self-directed 𝐹/𝜒2 (degree of freedom) 𝑃value Body mass index 25 ≤30 Age, year 53.8 ± 10.5 53.7 ± 10.3 54.7 ± 10.9 53.1 ± 10.6 0.22 (2, 108) 0.80 Female, % 32.4 32.4 33.3 31.6 0.03 (2) 0.99 Race/ethnicity, % 3.29 (6) 0.77 Non-Hispanic white 72.1 75.7 66.7 73.7 Asian/Pacific Islander 22.5 18.9 27.8 21.1 Latino/Hispanic 4.5 5.4 5.6 2.6 College level or above, % 97.2 97.2 94.3 100.0 2.22 (2) 0.33 Income, % 6.62 (6) 0.36 <$75,000 10.3 13.9 8.8 8.1 $75,000–$124,999 30.8 27.8 41.2 24.3 $125,000–$149,999 15.9 8.3 20.6 18.9 $150,000+ 43.0 50.0 29.4 48.6 Weight, kg 83.8 ± 9.9 85.4 ± 9.1 82.6 ± 10.2 83.5 ± 10.6 0.77 (2, 108) 0.46 Waist, cm 98.8 ± 6.4 98.3 ± 6.4 98.0 ± 6.4 100.1 ± 6.4 1.14 (2, 108) 0.32 Fasting plasma glucose, mg/dL 100.3 ± 9.3 99.9 ± 9.5 101.4 ± 9.3 99.6 ± 9.2 0.39 (2, 108) 0.68 Prediabetes, % 56.8 62.2 61.1 47.4 2.08 (2) 0.35 Metabolic syndrome, % 80.2 67.6 88.9 84.2 5.81 (2) 0.06 Prediabetes and metabolic syndrome, % 36.9 29.7 50.0 31.6 3.93 (2) 0.14 Body mass index 30 ≤35 Age, year 54.0 ± 10.8 54.5 ± 11.1 55.3 ± 12.9 52.3 ± 7.9 0.51 (2, 72) 0.60 Female, % 50.7 44.0 50.0 58.3 1.01 (2) 0.60 Race/ethnicity, % 5.25 (6) 0.51 Non-Hispanic white 80.0 80.0 80.8 79.2 Asian/Pacific Islander 14.7 16.0 7.7 20.8 Latino/Hispanic 4.0 4.0 7.7 0.0 College level or above, % 97.3 96.0 96.2 100.0 0.97 (2) 0.62 Income, % 5.16 (6) 0.52 <$75,000 12.5 8.7 15.4 13.0 $75,000–$124,999 23.6 30.4 23.1 17.4 $125,000–$149,999 9.7 0.0 15.4 13.0 $150,000+ 54.2 60.9 46.2 56.5 Weight, kg 94.2 ± 13.8 94.2 ± 12.9 97.2 ± 14.5 91.0 ± 13.8 1.26 (2, 72) 0.29 Waist, cm 106.7 ± 8.1 106.2 ± 8.9 109.2 ± 7.1 104.4 ± 7.9 2.35 (2, 72) 0.10 Fasting plasma glucose, mg/dL 100.3 ± 10.4 98.7 ± 8.6 100.4 ± 10.9 101.8 ± 11.6 0.52 (2, 72) 0.60 Prediabetes, % 50.7 44.0 50.0 58.3 1.01 (2) 0.60 Metabolic syndrome, % 88.0 92.0 80.8 91.7 1.97 (2) 0.37 Prediabetes and metabolic syndrome, % 38.7 36.0 30.8 50.0 2.06 (2) 0.36 Body mass index 35+ Age, year 49.7 ± 10.1 47.4 ± 10.6 53.4 ± 8.5 48.6 ± 10.4 1.79 (2, 52) 0.18 Female, % 69.1 73.7 76.5 57.9 1.74 (2) 0.42 Race/ethnicity, % 2.58 (4) 0.63 Non-Hispanic white 87.3 78.9 94.1 89.5 Asian/Pacific Islander 9.1 15.8 5.9 5.3 Latino/Hispanic 3.6 5.3 0.0 5.3 College level or above, % 98.2 94.7 100.0 100.0 1.93 (2) 0.38 Income, % 5.15 (6) 0.52 <$75,000 14.8 10.5 23.5 11.1 $75,000–$124,999 22.2 26.3 29.4 11.1 $125,000–$149,999 13.0 10.5 5.9 22.2 $150,000+ 50.0 52.6 41.2 55.6 Table 2: Baseline characteristics of the study participantsa. 3. Results and Discussion All Usual care Coach-led Self-directed 𝐹/𝜒2 (degree of freedom) 𝑃value BioMed Research International 5 Table 2: Continued. Table 2: Continued. Table 2: Continued. Characteristic All Usual care Coach-led Self-directed 𝐹/𝜒2 (degree of freedom) 𝑃value Weight, kg 113.4 ± 18.2 116.0 ± 20.0 111.6 ± 15.0 112.6 ± 19.4 0.29 (2, 52) 0.75 Waist, cm 120.1 ± 10.9 121.9 ± 11.7 117.3 ± 9.7 120.4 ± 11.4 0.78 (2, 52) 0.46 Fasting plasma glucose, mg/dL 98.7 ± 8.6 98.7 ± 8.7 98.7 ± 9.3 98.8 ± 8.3 0.00 (2, 52) 0.99 Prediabetes, % 54.5 52.6 58.8 52.6 0.18 (2) 0.91 Metabolic syndrome, % 98.2 100.0 94.1 100.0 2.28 (2) 0.32 Prediabetes and metabolic syndrome, % 52.7 52.6 52.9 52.6 0.0005 (2) 0.99 aPlus-minus values are means ± SD. suggests that the low cost, self-directed intervention can be a viable alternative to the coach-led intervention for high-risk adults with a BMI less than 35. The increased efficiency and reach of the self-directed intervention makes it an appealing public health intervention strategy. incremental benefit over the self-directed intervention for participants with a baseline BMI of 30 ≤35, although they led greater reductions in percentage weight loss and BMI (coach-led) or waist circumference (self-directed) compared with usual care. These results imply that the self-directed intervention can be an effective and efficient alternative to the coach-led intervention for overweight individuals with prediabetes and/or metabolic syndrome but that individuals with the same cardiometabolic risk factors who are moder- ately or severely obese may only benefit from the latter, more structured approach. These findings add to recent evidence that suggests a structured, intensive—and yet practical— lifestyle intervention is indicated for increased degree of obesity [10, 21], as opposed to a less structured, self-directed approach. Future studies are needed to explore factors that may modify or mediate the effectiveness of lifestyle interventions among persons with moderate or severe obesity. Poten- tial effect modifiers include sociodemographic character- istics (e.g., sex, race/ethnicity, and education), comorbid- ity (e.g., severity of coexisting chronic conditions), and community/societal resources (e.g., accessibility of grocery stores or farmers market, neighborhood walkability, and social norms). 3. Results and Discussion Possible effect mediators include outcome expectancy, self-efficacy, social support, and self-monitoring, which are theory-based variables that have been shown to predict weight loss in diverse populations [25, 26].hi Previously, in the absence of empirical evidence, lifestyle intervention was thought to be ineffective in severely obese individuals [22] but has recently been recognized as a promising approach among this subpopulation [3, 8–12], especially given the risk of postoperative complications, recidivism, and limited reach of surgical options [4, 5, 8]. Modest weight loss for individuals who are overweight or obese (5%–10% reduction in total body weight) has been shown to produce health benefits such as improvement in blood pressure, cholesterol and dysglycemia [19, 23, 24] and was achieved among all participants in the coach-led intervention, including those whose baseline BMI was ≥35. A randomized controlled trial by Goodpaster et al. showed that an intensive behavioral weight-loss intervention was effective for severely obese adults and that modest weight reduction, even despite persistent severe obesity, significantly improved cardiovascular risk factors in this population [9]. A secondary analysis of data from the Look AHEAD trial found that nearly 40% of severely obese participants in the intensive, DPP-like lifestyle intervention lost ≥10% of initial weight at 1 year [8], and 26% were able to maintain this weight loss at year 4 [11].hi The present findings should be interpreted with consid- eration of several study limitations. This was a secondary data analysis, and all findings warrant replication in future confirmatory research. The sample size for each BMI-by- treatment subgroup was small, and the trial duration was only 15 months. However, the effect size confidence intervals and data consistency across the three obesity outcome measures suggest that the E-LITE coach-led intervention may have clinically significant benefits beyond usual care for moder- ately and severely obese adults at high cardiometabolic risk that are worth further investigation in fully-powered, longer- term studies. Also, the generalizability of the current findings may be limited by a rather homogenous study sample in terms of race/ethnicity and socioeconomic status, and participants were recruited from a single primary care clinic. Future research is needed to confirm the generalizability of our findings to more ethnically and socioeconomically diverse populations. References [16] J. Ma, A. C. King, S. R. Wilson, L. Xiao, and R. S. Stafford, “Evaluation of lifestyle interventions to treat elevated car- diometabolic risk in primary care (E-LITE): a randomized controlled trial,” BMC Family Practice, vol. 10, article 71, 2009. [1] K. M. 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Nicholas, “One hundred- pound weight losses with an intensive behavioral program: changes in risk factors in 118 patients with long-term follow- up,” American Journal of Clinical Nutrition, vol. 86, no. 2, pp. 301–307, 2007. [6] M. Martin, A. Beekley, R. Kjorstad, and J. Sebesta, “Socioeco- nomic disparities in eligibility and access to bariatric surgery: a national population-based analysis,” Surgery for Obesity and Related Diseases, vol. 6, no. 1, pp. 8–15, 2010. Acknowledgments The content is solely the responsibility of the authors and does not necessarily represent the official views of any of the funding agencies. No sponsor or funding source had a role in the design or conduct of the study; collection, management, analysis, or interpretation of the data; or preparation, review, or approval of the paper. All authors comply with the editorial policies and have no conflict of interests. Kristen M. J. Azar and Jun Ma conceived the present study and interpreted data analysis. Lan Xiao analyzed data. All authors were involved in writing the paper and had final approval of the submitted and published versions. The E-LITE study was supported by Grant R34DK080878 from the National Institute of Diabetes and Digestive and Kidney Diseases, a Scientist Development Grant Award (0830362N) from the American Heart Asso- ciation, and internal funding from the Palo Alto Medical Foundation Research Institute. The authors wish to thank the participants and their families who made the E-LITE trial possible. They would also like to acknowledge the Diabetes Prevention Support Center (DPSC) of the University of Pittsburgh for training and supporting the Group Lifestyle Balance program; the E-LITE coach-led and self-directed interventions were derived from this material. [10] J. W. Anderson, L. Grant, L. Gotthelf, and L. T. P. Stifler, “Weight loss and long-term follow-up of severely obese individuals treated with an intense behavioral program,” International Journal of Obesity, vol. 31, no. 3, pp. 488–493, 2007. [11] J. L. Unick, D. Beavers, D. S. Bond et al., “The long-term effec- tiveness of a lifestyle intervention in severely obese individuals,” American Journal of Medicine, vol. 126, no. 3, pp. 236.e2–242.e2, 2013. [12] G. L. Blackburn, S. Wollner, and S. B. Heymsfield, “Lifestyle interventions for the treatment of class III obesity: a primary target for nutrition medicine in the obesity epidemic,” American Journal of Clinical Nutrition, vol. 91, no. 1, pp. 289S–292S, 2010. [13] M. K. Kramer, A. M. Kriska, E. M. Venditti et al., “Translating the diabetes prevention program. A comprehensive model for prevention training and program delivery,” American Journal of Preventive Medicine, vol. 37, no. 6, pp. 505–511, 2009. [14] Diabetes Prevention Support Center, “Group lifestyle bal- ance materials,” in A Modification of the Diabetes Prevention Program’s Lifestyle Change Program, University of Pittsburgh, Pittsburgh, Pa, USA, 2011. [15] J. Ma, V. Yank, L. 4. Conclusion This is the first study, to our knowledge, that suggests the potential effectiveness of a coach-led, technology-enhanced DPP translation intervention in reducing obesity among adults at high risk of Type 2 diabetes and CVD with a BMI of 35 or above. This is a growing segment of the overall population for which surgery is currently recommended; however, surgery cannot be the only solution to an epidemic. It is imperative that alternative strategies are developed that are effective, accessible, and affordable with potential for broad reach and impact. Equally important, our study In conclusion, baseline obesity status may influence behav- ioral weight loss treatment effectiveness. Less resource inten- sive approaches are perhaps adequate for individuals with lower baseline BMI in the overweight and obesity continuum, whereas the incremental benefit of more intensive, structured lifestyle change programs may not be evident except for those with higher BMI indicative of moderate or severe obesity. If confirmed in future definitive study, these findings would suggest that researchers and clinicians should take BioMed Research International 6 an individual’s baseline BMI into account when developing or recommending a weight-loss treatment strategy. Under- standing how to best allocate healthcare resources in weight- loss treatment may ultimately result in improved quality and affordability of obesity care. [8] J. L. Unick, D. Beavers, J. M. Jakicic et al., “Effectiveness of life- style interventions for individuals with severe obesity and type 2 diabetes: results from the Look AHEAD trial,” Diabetes Care, vol. 34, no. 10, pp. 2152–2157, 2011. [9] B. H. Goodpaster, J. P. DeLany, A. D. Otto et al., “Effects of diet and physical activity interventions on weight loss and car- diometabolic risk factors in severely obese adults: a randomized trial,” Journal of the American Medical Association, vol. 304, no. 16, pp. 1795–1802, 2010. Acknowledgments Xiao et al., “Translating the diabetes preven- tion program lifestyle intervention for weight loss into primary care: a randomized trial,” JAMA Internal Medicine, vol. 173, no. 2, pp. 113–121, 2013. References [22] “Clinical guidelines on the identification, evaluation, and treat- ment of overweight and obesity in adults—the evidence report. National Institutes of Health,” Obesity Research, vol. 6, supple- ment 2, pp. 51S–209S, 1998. [7] E. S. le Blanc, E. O’Connor, E. P. Whitlock, C. D. Patnode, and T. Kapka, “Effectiveness of primary care-relevant treatments for obesity in adults: a systematic evidence review for the U.S. preventive services task force,” Annals of Internal Medicine, vol. 155, no. 7, pp. 434–447, 2011. [23] G. Blackburn, “Effect of degree of weight loss on health bene- fits,” Obesity Research, vol. 3, supplement 2, pp. 211s–216s, 1995. BioMed Research International 7 7 [24] “The Diabetes Prevention Program. Design and methods for a clinical trial in the prevention of type 2 diabetes,” Diabetes Care, vol. 22, no. 4, pp. 623–634, 1999. [25] A. Bandura, Social Foundations of Thought and Action: A Social Cognitive Theory, Prentice Hall, Englewood Cliffs, NJ, USA, 1986. [26] A. L. Palmeira, P. J. Teixeira, T. L. Branco et al., “Predicting short-term weight loss using four leading health behavior change theories,” International Journal of Behavioral Nutrition and Physical Activity, vol. 4, article 14, 2007.
https://openalex.org/W3099651163	https://ogsh.abvpress.ru/jour/article/download/563/440	Russian	null	Oral mucositis in patients with oncological pathology of the oropharyngeal region: review	Opuholi golovy i šei	2,020	cc-by	6,904	Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Том 10 Vol. 10 Oral mucositis in patients with oncological pathology of the oropharyngeal region: review A. O. Eremina1, I. A. Zaderenko1, 2, S. Yu. Ivanov1, 3, S. B. Alieva2, A. B. Dymnikov1, A. V. Khromushina1, B. G. Pkheshkhova2 1RUDN University; 6 Miklukho-Maklaya St., Moscow 117198, Russia; 2N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia; 24 Kashirskoe Hwy, Moscow 115478, Russia; 3I.M. Sechenov First Moscow State Medical University, Ministry of Health of Russia; Bld. 4, 2 Bolshaya Pirogovskaya St., Moscow 119991, Russia A. O. Eremina1, I. A. Zaderenko1, 2, S. Yu. Ivanov1, 3, S. B. Alieva2, A. B. Dymnikov1, A. V. Khromushina1, B. G. Pkheshkhova2 1RUDN University; 6 Miklukho-Maklaya St., Moscow 117198, Russia; 2N.N. Blokhin National Medical Research Center of Oncology, Ministry of Health of Russia; 24 Kashirskoe Hwy, Moscow 115478, Russia; 3I.M. Sechenov First Moscow State Medical University, Ministry of Health of Russia; Bld. 4, 2 Bolshaya Pirogovskaya St., Moscow 119991, Russia Oral mucositis is one of the most common effects of chemoradiotherapy in patients with oropharyngeal cancer. The development of oral mu- cositis is the main cause of interruption of antitumor therapy, which significantly affects the results of treatment of the main disease. Despite the fact that the disease is well studied in the literature, today there is no universal treatment and prevention protocol. The aim of this review is to analyze scientific publications devoted to the problems of etiology, pathogenesis, clinic, diagnosis, treatment and prevention of oral mu- cositis. Key words: oral mucositis, oropharyngeal region, antitumor therapy, radiation exposure Key words: oral mucositis, oropharyngeal region, antitumor therapy, radiation exposure For citation: Eremina A. O., Zaderenko I. A., Ivanov S. Yu. et al. Oral mucositis in patients with oncological pathology of the oropharyngeal region: review. Opukholi golovy i shei = Head and Neck Tumors 2020;10(3):72–80. (In Russ.). Оральные мукозиты у пациентов с онкологической патологией орофарингеальной области: обзор литературы А. О. Ерёмина1, И. А. Задеренко1, 2, С. Ю. Иванов1, 3, С. Б. Алиева2, А. Б. Дымников1, А. О. Ерёмина1, И. А. Задеренко1, 2, С. Ю. Иванов1, 3, С. Б. Алиева2, А. Б А. В. Хромушина1, Б. Г. Пхешхова2 А. О. Ерёмина , И. А. Задеренко , С. Ю. Иванов , С. Б. Алиева , А. Б. А. В. Хромушина1, Б. Г. Пхешхова2 А. В. Хромушина1, Б. Г. Пхешхова2 1ФГАОУ ВО «Российский университет дружбы народов»; Россия, 117198 Москва, ул. Миклухо-Маклая, 6; 2ФГБУ «Национальный медицинский исследовательский центр онкологии им. Н. Н. Блохина» Минздрава России; Россия, 115478 Москва, Каширское шоссе, 24; 3ФГАОУ ВО Первый Московский государственный медицинский университет им. И. М. Сеченова Минздрава России; Россия, 119991 Москва, ул. Большая Пироговская, 2, стр. 4 Контакты: Александра Олеговна Ерёмина sas1.91@mail.ru Оральный мукозит – одно из наиболее частых последствий химиолучевой терапии у пациентов с раком орофарингеальной области. Именно развитие орального мукозита считается основной причиной прерывания противоопухолевой терапии, которое значи- тельно ухудшает результаты лечения онкологического заболевания. Несмотря на то что заболевание хорошо изучено, на сегод- няшний день не разработан универсальный общепринятый алгоритм его лечения и профилактики. В данном обзоре представлен анализ научных публикаций, посвященных проблемам этиологии, патогенеза, клинической картины, диагностики, лечения и про- филактики орального мукозита. Ключевые слова: оральный мукозит, орофарингеальная область, противоопухолевая терапия, лучевая нагрузка Для цитирования: Ерёмина А. О., Задеренко И. А., Иванов С. Ю. и др. Оральные мукозиты у пациентов с онкологической патоло- гией орофарингеальной области: обзор литературы. Опухоли головы и шеи 2020;10(3):72–80. DOI: 10.17650/2222-1468-2020-10-3-72-80 DOI: 10.17650/2222-1468-2020-10-3-72-80 Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Том 10 Vol. 10 Факторы риска К отдаленным клиническим проявлениям ОМ от- носятся повреждения сосудов и мягких тканей, которые могут вызывать вторичную позднюю атрофию с повы- шенной эпителиальной восприимчивостью к травми- рующим и инфекционным агентам [8]. Факторами риска развития ОМ являются вредные привычки (употребление алкоголя и курение), несо- стоятельность ортопедических и ортодонтических кон- струкций, наличие пломб, несанированное состояние полости рта, женский пол, большая площадь и интен- сивность облучения, кратность повторения и накоплен- ная доза, а также применение некоторых химиотера- певтических препаратов [22], например цисплатина и 5‑фторурацила, что увеличивает риск возникновения ОМ не только в комбинации с облучением, но и без не- го [23]. Ранние изменения эпителия при лучевой терапии регрессируют через несколько недель после завершения лечения. Однако они рассматриваются как фактор ри- ска развития поздних осложнений в ротовой полости (остеорадионекроза, хронических язв и др.) [13]. На сегодняшний день ОМ характеризуют 54 по- казателя, основными из которых являются 23, в том числе функциональное состояние (способность упо- треблять ту или иную пищу и жидкость), симптомы (дисгевзия, жжение, боль) и / или клинические при- знаки (гиперемия, язвы, отек, налет) в разнообразных сочетаниях. Патогенез Согласно основной патогенетической теории ОМ, он развивается вследствие нарушения микроциркуляции и имеет неинфекционную природу (вопреки представ- лениям, бытовавшим до середины 1990‑х). Доказано, что диффузия факторов роста из эндотелия сосудов под- слизистого слоя к базальной мембране определяет тол- щину эпителиального слоя. Данный путь регуляции нарушается из‑за гибели эндотелиоцитов [6]. Оценка тяжести Основываясь на этих данных, S. Sonis разработал классификацию ОМ, которая отражает 5 фаз его пато- генеза и сейчас является общепринятой [7]. Не существует общепризнанного стандарта диа- гностики и лечения ОМ, что затрудняет работу с данной группой пациентов и делает невозможным контроль эффективности проведенных манипуляций [19, 20]. Большинство специалистов используют классифика- цию Всемирной организации здравоохранения (ВОЗ). Данная модель патогенеза в настоящий момент яв- ляется базой для изучения ОМ, но остается во многом гипотетической, все еще требующей дальнейшего уточ- нения [8]. Разработаны 3 шкалы оценки токсичности у паци- ентов старше 18 лет: шкала оценки тяжести ОМ (Oral Mucositis Assessment Scale), индекс оральной токсич- ности ВОЗ (WHO Oral Toxicity Scale) и критерии общей токсичности Национального института рака США (National Cancer Institute Common Toxicity Criteria). Индекс оральной токсичности ВОЗ определяется с уче- том анатомических, симптоматических и функциональ- ных критериев. Осложнения Оральный мукозит – это состояние, при котором про- исходит воспаление слизистой оболочки рта, глотки, пи- щевода и других отделов желудочно-кишечного тракта как осложнение противоопухолевого лечения. Наиболее высок риск развития ОМ при проведении лучевой тера- пии в режиме ускоренного фракционирования (41–56 %) и сочетанной химиолучевой терапии (до 43 %). При стан- дартной лучевой терапии он составляет 25–34 % [5]. Наиболее тяжелое осложнение ОМ – присоедине- ние инфекции и развитие бактериемии, септицемии и фунгемии [14, 15]. Наиболее часто при этом в крови выявляют Streptococcus oralis, Streptococcus mitis. Инфек- ция, вызванная последним, на фоне терапии высокими дозами цитарабина может провоцировать развитие ре- спираторного дистресс-синдрома [16]. Кроме того, при ОМ возможно присоединение грибковой инфек- ции. Наиболее часто она вызвана Candida albicans, но выявляют и другие разновидности Candida, такие как C. glabrata, С. krusei, C. tropicalis, C. parapsilosis, а так- же Mucor и Aspergillus. Могут также развиваться ослож- нения со стороны пищеварительной системы, а именно гастроинтестинальные токсические реакции и венозно- окклюзионная болезнь печени [17, 18]. Классификация Исследователи уделяют основное внимание остро- му ОМ. Выделяют 4 степени тяжести острого ОМ, ко- торые имеют дозозависимый характер: от эритемы и незначительной боли до сливного эпителиита с вы- раженным болевым синдромом, отказом от приема пищи и др. [8–10]. Значительно реже у пациентов наблюдается хро- ническая форма ОМ, и, соответственно, она оказыва- ется менее изученной. Хроническая форма ОМ требует абсолютно другого подхода к лечению и профилактике, что подтверждается рядом исследований [11, 12]. При дифференциальной диагностике ОМ исклю- чают герпетический, афтозный, язвенно-некротиче- ский и гангренозный стоматит, хроническую травму, вызванную ношением съемного протеза, кандидоз [21]. Введение и лучевой терапии [2, 3]. Лучевая терапия как само- стоятельный вид лечения применяется в 17 % всех случаев рака полости рта и в 17,3 % всех случаев ЗНО глотки [1]. Одним из наиболее ранних и распростра- ненных осложнений химиолучевой терапии является оральный мукозит (ОМ), развивающийся более чем у 70 % пациентов и часто требующий прерывания противоопухолевой терапии [4]. и лучевой терапии [2, 3]. Лучевая терапия как само- стоятельный вид лечения применяется в 17 % всех случаев рака полости рта и в 17,3 % всех случаев ЗНО глотки [1]. Одним из наиболее ранних и распростра- ненных осложнений химиолучевой терапии является оральный мукозит (ОМ), развивающийся более чем у 70 % пациентов и часто требующий прерывания противоопухолевой терапии [4]. Заболеваемость злокачественными новообразова- ниями (ЗНО) полости рта в России в 2018 г. составила 28,5 случая на 100 тыс. человек, при этом смертность в течение 1‑го года после постановки диагноза дости- гает 34,2 % [1]. Максимальной эффективностью харак- теризуется комбинированное или комплексное лечение ЗНО полости рта и ротоглотки с включением химио- 72 72 Лечение и профилактика Исследователи уделяют большое внимание про- филактике и лечению ОМ. Более 300 исследований за последние 10 лет было проведено с целью оценки 73 73 Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Том 10 Vol. 10 8. Предполагается, что жидкость для полоскания рта с содержанием 0,5 % доксепина может быть эффективна в лечении боли, вызванной ОМ (УДД 4). 8. Предполагается, что жидкость для полоскания рта с содержанием 0,5 % доксепина может быть эффективна в лечении боли, вызванной ОМ (УДД 4). эффективности средств и методов профилактики и ле- чения ОМ. Всего несколько из них оцениваются учены- ми как эффективные, лишь десятки – как перспектив- ные, соответствующие современной теории патогенеза ОМ, но требующие дополнительных клинических ис- следований [10, 13]. 9. Предполагается, что пероральный прием препа ратов цинка может использоваться для предотвра- щения ОМ у пациентов с раком полости рта, про- ходящих лучевую терапию или химиолучевую терапию (УДД 3). На сегодняшний день вопрос о наиболее эффек- тивном методе профилактики и лечения ОМ остается открытым. Унифицированного алгоритма лечения и профилактики на сегодняшний день также не суще- ствует. Тем не менее исследовательская группа по муко- зитам Многонациональной ассоциации специалистов по поддерживающей терапии при раке и Междуна- родного общества по изучению онкологических забо- леваний полости рта (Multinational Association of Sup portive Care in Cancer and International Society of Oral Oncology, MASCC / ISOO) разработала практическое руководство по лечению вторичного ОМ, впервые опу- бликованное в 2004 г. [24]. Руководство было обновле- но в 2007 г. [25], а затем в 2014 г. [26]. ( ) В руководстве MASCC / ISOO перечислены и те ме- роприятия и средства, при исследовании которых полу- чены убедительные доказательства их неэффективно- сти. 1. Не рекомендуется использовать антимикробные пастилки PTA (полимиксин, тобрамицин, амфоте- рицин) и BCoG (бацитрацин, клотримазол, гента- мицин) и пасту PTA для предотвращения ОМ у па- циентов, проходящих лучевую терапию по поводу рака головы и шеи (УДД 2). 2. Не рекомендуется использовать противомикроб- ную жидкость для полоскания рта с изегананом для предотвращения ОМ у пациентов, проходящих высокодозную химиотерапию с полным облучени- ем тела или без него и трансплантацией гемопоэ- тических стволовых клеток (УДД 2), или у пациен- тов, проходящих лучевую терапию или сочетанную химиолучевую терапию при раке головы и шеи (УДД 2). Данное руководство включает рекомендации по профилактике ОМ, основанные на убедительных до- казательствах эффективности тех или иных лечебных мероприятий. 1. Лечение и профилактика Рекомендуется проводить 30‑минутную криотерапию полости рта для предотвращения ОМ у пациентов, проходящих болюсную 5‑фторурациловую химиоте рапию (уровень достоверности доказательств (УДД) 2). 3. Не рекомендуется применять сукральфат для по- лоскания рта с целью профилактики ОМ у пациен- тов, проходящих химиотерапию при раке (УДД 1), или у пациентов, проходящих лучевую терапию (УДД 1) или сочетанную химиолучевую терапию (УДД 2) при раке головы и шеи. 2. Рекомендуется использовать жидкость для поло- скания рта с бензидамином для предотвращения ОМ у пациентов с раком головы и шеи, получаю- щих умеренную дозу облучения (до 50 Гр) без со- путствующей химиотерапии (УДД 1). 3. В руководстве MASCC / ISOO также перечислены другие мероприятия, которые могут принести пользу, но их эффективность подтверждена менее убедительными доказательствами. 4. Не рекомендуется использовать сукральфат для по- лоскания рта с целью лечения пациентов, которые проходят химиотерапию при раке (УДД 1), или па- циентов, проходящих лучевую терапию (УДД 2) при раке головы и шеи. 4. Предлагается использовать протоколы по уходу за полостью рта для предотвращения ОМ во всех возрастных группах и при всех методиках лечения рака (УДД 3). В руководстве MASCC / ISOO также не рекомендо- вано применять некоторые средства, неэффективность которых не подтверждена достаточно убедительными доказательствами. 5. Предлагается использовать низкоинтенсивную ла- зерную терапию (с длиной волны 632,8 нм) для пре дотвращения ОМ у пациентов, проходящих луче- вую терапию без сопутствующей химиотерапии при раке головы и шеи (УДД 3). 1. Предлагается не использовать хлоргексидин для полоскания рта с целью профилактики ОМ у паци- ентов, проходящих лучевую терапию при раке го- ловы и шеи (УДД 3). 6. Трансдермальный фентанил может быть эффекти- вен в лечении боли, вызванной ОМ, у пациентов, проходящих традиционную или высокодозную хи миотерапию в сочетании с полным облучением тела или без него (УДД 3). 2. Предлагается не использовать мизопростол для по- лоскания рта с целью профилактики ОМ у пациен- тов, проходящих лучевую терапию при раке головы и шеи (УДД 3). 7. Предполагается, что 0,2 % раствор морфина для полоскания рта может быть эффективен в лечении боли при ОМ у пациентов, проходящих химиоте- рапию при раке головы и шеи (УДД 3). Со времени выхода последней редакции руковод- ства MASCC / ISOO в 2014 г. было опубликовано боль- шое количество работ по проблеме профилактики и лечения ОМ. Данные работы станут основанием 74 74 Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Том 10 Vol. 10 Лазерная и другая световая терапия для дальнейшего обновления практического руковод- ства. Природные и иные агенты В нескольких новых рандомизированных контро- лируемых исследованиях установлена неэффективность перорального приема препаратов цинка в профилак- тике ОМ у пациентов с раком полости рта, проходящих лучевую или химиолучевую терапию [61, 75, 76], но в 1 новом исследовании продемонстрирована его эф- фективность [77]. Из-за противоречивости данных при- менение цинка теперь не рекомендовано [78]. – – профессиональная гигиена полости рта [49, 51–55] и обучение пациентов правильной гигиене полости рта [56–58] (никаких руководящих указаний не раз работано). Противовоспалительные агенты Систематический обзор [59] подтвердил данную в руководстве [26] рекомендацию по применению бен- зидамина для полоскания рта с целью профилактики ОМ, вызванного лучевой терапией у пациентов с раком головы и шеи. Предложено также использовать бензи- дамин у пациентов с раком головы и шеи, проходящих химиолучевую терапию. Разработаны новых предложения. Во-первых, пред- полагается, что положительный эффект может оказы- вать комбинированное (местное и системное) исполь- зование меда для профилактики ОМ у больных раком головы и шеи, проходящих лучевую или химиолучевую терапию [79–84]. Во-вторых, не рекомендовано ис- пользование жевательной резинки для профилактики ОМ у детей с онкологическими заболеваниями систе- мы кроветворения или солидными ЗНО, проходящих химиотерапию [85]. Не дано рекомендаций по поводу применения ре- бамипида [60], целекоксиба [61], ирсогладина малеата [62], мизопростола [63–66]. Исследована эффективность витамина Е, селена, фолиевой кислоты и кальцитриола, но из‑за исполь- зования их в комбинации с другими средствами невоз- можно сделать какие‑либо точные выводы о каждом агенте в отдельности [86]. Лечение и профилактика Произошло повышение уровня убедительности рекомендаций по использованию низкоинтенсивной лазерной терапии (с длиной волны 632,8 нм) с целью предотвращения ОМ у пациентов, проходящих лучевую терапию без сопутствующей химиотерапии при раке головы и шеи [68, 69]. Основные методы лечения и профилактики, эф- фективность которых продолжает изучаться, можно разделить на 7 групп: 1) базовый уход за полостью рта; 2) факторы роста и цитокины; На основе новых публикаций была дана рекомен- дация по использованию низкоинтенсивной лазерной терапии с целью предотвращения ОМ у пациентов, проходящих лучевую терапию с сопутствующей химио терапией при раке головы и шеи [70–73]. 3) противовоспалительные агенты; 4) противомикробные препараты, покрывающие аген ты, анестетики и анальгетики; 4) противомикробные препараты, покрывающие аген ты, анестетики и анальгетики; ты, анестетики и анальгетики; 5) лазерная и другая световая терапия; 5) лазерная и другая световая терапия; 6) криотерапия; 6) криотерапия; 7) природные и разные другие агенты [26]. Базовый уход за полостью рта Данные исследований подтверждают обоснован- ность рекомендаций по применению криотерапии для профилактики ОМ у пациентов, которым болюсно вводится 5‑фторурацил. Сформулировать другие реко- мендации по применению криотерапии невозможно из‑за неубедительности доказательств [61, 74]. В данном разделе были рассмотрены: – – хлоргексидин (подтверждена рекомендация не ис- пользовать) [16, 27–46]; – – физиологический раствор и бикарбонат натрия для полоскания полости рта (рекомендовать или не рекомендовать невозможно, так как данные не поддаются оценке или противоречивы) [28, 47–50]; Л И Т Е Р А Т У Р А / R E F E R E N C E S 2008;113(10):2704–13. DOI: 10.1002/cncr.23898. 2008;113(10):2704–13. DOI: 10.1002/cncr.23898. 1. Злокачественные новообразования в России в 2018 году (заболеваемость и смертность). Под ред. А.Д. Каприна, В.В. Старинского, Г.В. Петровой. М.: МНИОИ им. П.А. Герцена, 2019. 250 с. Доступно по: https://glavonco.ru/ cancer_register/Забол_2018_Электр.pdf. [Malignant tumors in Russia in 2017 (morbidity and mortality). Ed. by A.D. Kaprin, V.V. Starinsky, G.V. Petrova. Moscow: MNIOI im. P.A. Gertzena, 2018. 250 p. 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Противомикробные препараты, покрывающие агенты, анестетики и анальгетики Актуальными остаются рекомендации, данные в ру- ководстве MASCC / ISOO по поводу нежелательности использования антимикробных пастилок PTA (поли- миксин, тобрамицин, амфотерицин), BCoG (бацитра- цин, клотримазол, гентамицин), пасты PTA, сукраль- фата и изеганана для предотвращения ОМ [61]. Рассмотрены результаты применения других средств, таких как прополис (также в сочетании с бикарбонатом натрия [87] и комплексом трав (алоэ вера, календулой и ромашкой [88])), траумель, куркумин, hangeshashinto (TJ-14) [89], ромашка, пикногенол [90], лист оливы [91], кангфуксин [92], листья чая, гидролизованный белок пшеницы [93], растительное соединение на основе гвоз- дики [94], лакрица [95], календула [96], тысячелистник [97], пыльца финиковой пальмы [98], смесь на основе лапачо (муравьиное дерево) и гиалуроновой кислоты, Проведены также исследования ацикловира, кла- ритромицина, нистатина, повидон-йода, флуконазо- ла, гиалуроната натрия местного действия, тетракаи- на, диклонин, MGI-209 (с бензокаином), кокаина, аметокаина, капсаицина, метадона, кетамина, нор- триптилина и габапентина. Какие‑либо рекомендации на данный момент отсутствуют [67]. 75 75 Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Том 10 Vol. 10 болевания, что является отрицательным социально- экономическим фактором. зеленого чая, эрисимо, прополиса, календулы и подо- рожника (OraSol Plus) [99] и смесь на основе черники, плодов маклейи сердцевидной и корня эхинацеи узко- листной [100]. Из-за ограниченности или противоре- чивости данных никаких рекомендаций по их приме- нению дать невозможно. Для системного лечения используют различные препараты, но ни один из них не улучшает микроцир- куляцию, нарушение которой рассматривается сегодня как основной патогенетический механизм развития ОМ. Поиск активных веществ и разработка препаратов для системной терапии при ОМ сложны, так как ми- кроциркуляция – многозвеньевый механизм. Прове- дено много исследований эффективности местных противовоспалительных, противомикробных средств, факторов роста и цитокинов, низкоинтенсивной ла- зерной терапии, криотерапии и препаратов на основе разных природных агентов, а также методов ухода за полостью рта. В доступной нам литературе имеются единичные упоминания об улучшении реологических свойств крови и микроциркуляции тканей в зоне пер- вичного воздействия. В 2 исследованиях продемонстрирован положи- тельный эффект пробиотиков на основе лактобацилл и лактококков, которые применялись внутрь и местно для профилактики ОМ у больных раком головы и шеи [101, 102]. Но из‑за ограниченности данных рекомен- довать этот метод нельзя. Изучено также влияние жидкости для полоскания рта с фенилбутиратным гелем [103], фенитоина [104], перорального бета-глюкана [105], супернатанта тромбо- цитарного геля [106] и анкаферда [107]. Ни для одного из этих средств невозможно было разработать ка- кие‑либо рекомендации. Дальнейшие исследования должны быть направле- ны на повышение доказательного уровня существующих методов лечения и профилактики, а также на разработ- ку новых эффективных лечебно-профилактических мероприятий. Заключение На сегодняшний день проблема лечения ОМ стоит достаточно остро. Во-первых, отсутствует общепри- нятый протокол лечения и профилактики данного за- Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Том 10 Vol. 10 Опухоли ГОЛОВЫ и ШЕИ 16. Ferretti G.A., Ash R.C., Brown A.T. et al. Control of oral mucositis and candidiasis in marrow transplantation: a prospective, double-blind trial of chlorhexidine digluconate oral rinse. Bone Marrow Transplant 1988;3(5):483–93. 38. Pitten F.A., Kiefer T., Buth C. et al. Do cancer patients with chemotherapy- induced leukopenia benefit from an antiseptic chlorhexidine-based oral rinse? A double-blind, block-randomized, controlled study. J Hosp Infect 2003;53(4): 283–91. 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Support Care Cancer / 68. Migliorati C., Hewson I., Lalla R.V. et al. Systematic review of laser and other light therapy for the management of oral mucositis in cancer patients. Support Care Cancer 2013;21(1):333–41. DOI: 10.1007/s00520-012-1605-6. 58. Yavuz B., Bal Yılmaz H. Investigation of the effects of planned mouth care 78 78 Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Том 10 Vol. 10 2017;39(9):1761–9. DOI: 10.1002/hed.24832. 2017;39(9):1761–9. DOI: 10.1002/hed.24832. Статья поступила: 19.05.2020. Принята к публикации: 22.09.2020. Article submitted: 19.05.2020. Accepted for publication: 22.09.2020. Вклад авторов Вклад авторов А.О. Ерёмина, И.А. Задеренко: написание текста статьи; С.Ю. Иванов, С.Б. Алиева, А.Б. Дымников: научное редактирование текста статьи; А.В. Хромушина, Б.Г. Пхешхова: обзор публикаций по теме статьи. Authors’ contributions A.O. Eremina, I.A. Zaderenko: article writing; S.Yu. Ivanov, S.B. Alieva, A.B. Dymnikov: commented on the manuscript; A.V. Khromushina, B.G. Pkheshkhova: reviewing of publications of the article’s theme. A.O. Eremina, I.A. Zaderenko: article writing; A.V. Khromushina, B.G. Pkheshkhova: reviewing of publications of the article’s theme. ORCID авторов / ORCID of authors А.О. Ерёмина / A.O. Eremina: https://orcid.org/0000-0002-7634-1384 И.А. Задеренко / I.A. Zaderenko: https://orcid.org/0000-0003-0183-4827 С.Ю. Иванов / S.Yu. Ivanov: https://orcid.org/0000-0001-5458-0192 С.Б. Алиева / S.B. Alieva: https://orcid.org/0000-0002-6835-5567 А.Б. Дымников / A.B.Dymnikov: https://orcid.org/0000-0001-8980-6235 А.В. Хромушина / A.V. Khromushina: https://orcid.org/0000-0003-1673-7261 Б.Г. Бхешхова / B.G. Pkheshkhova: https://orcid.org/0000-0003-1448-1733 Конфликт интересов. Авторы заявляют об отсутствии конфликта интересов. Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Опухоли ГОЛОВЫ и ШЕИ HEAD and NECK tumors 3’2020 Обзорная статья Том 10 Vol. 10 2017;39(9):1761–9. DOI: 10.1002/hed.24832. Antioxidant capacity of calendula officinalis flowers extract and prevention of radiation induced oropharyngeal mucositis in patients with head and neck cancers: A randomized controlled clinical study. Daru 2013;21(1):18. DOI: 10.1186/2008-2231-21-18. 106. Bonfili P., Gravina G.L., Marampon F. et al. Oral platelet gel supernatant plus supportive medical treatment versus supportive medical treatment in the management of radiation-induced oral mucositis. Am J Clin Oncol 2017;40(4):336–41. DOI: 10.1097/COC.0000000000000177. 87. Piredda M., Facchinetti G., Biagioli V. et al. Propolis in the prevention of oral mucositis in breast cancer patients receiving adjuvant chemotherapy: a pilot randomised controlled trial. Eur J Cancer Care (Engl) 2017;26(6). DOI: 10.1111/ecc.12757. 97. 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Работа выполнена без спонсорской поддержки. Финансирование. Работа выполнена без спонсорской подд Financing. The work was performed without external funding. Financing. The work was performed without external funding. 80 80
https://openalex.org/W2901896055	https://europepmc.org/articles/pmc6249377?pdf=render	English	null	Pax6 Lengthens G1 Phase and Decreases Oscillating Cdk6 Levels in Murine Embryonic Cortical Progenitors	Frontiers in cellular neuroscience	2,018	cc-by	7,661	ORIGINAL RESEARCH published: 15 November 2018 doi: 10.3389/fncel.2018.00419 Edited by: Tommaso Pizzorusso, Consiglio Nazionale delle Ricerche (CNR), Italy Reviewed by: Michele Studer, Institut National de la Santé et de la Recherche Médicale (INSERM), France Reviewed by: Michele Studer, Institut National de la Santé et de la Recherche Médicale (INSERM), France Federico Cremisi, Scuola Normale Superiore di Pisa, Italy Federico Cremisi, Scuola Normale Superiore di Pisa, Italy Correspondence: David J. Price David.Price@ed.ac.uk †Present address: Da Mi, Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology and Neuroscience and MRC Centre for Neurodevelopmental Disorders, King’s College London, London, United Kingdom Correspondence: David J. Price David.Price@ed.ac.uk Pax6 Lengthens G1 Phase and Decreases Oscillating Cdk6 Levels in Murine Embryonic Cortical Progenitors Da Mi†, Martine Manuel, Yu-Ting Huang, John O. Mason and David J. Price* Biomedical Sciences, The University of Edinburgh, Edinburgh, United Kingdom Pax6 is a key regulator of the rates of progenitor cell division in cerebral corticogenesis. Previous work has suggested that this action is mediated at least in part by regulation of the cell cycle gene Cdk6, which acts largely on the transition from G1 to S phase. We began the present study by investigating whether, in addition to Cdk6, other Pax6- regulated cell cycle genes are likely to be primary mediators of Pax6’s actions on cortical progenitor cell cycles. Following acute cortex-speciﬁc deletion of Pax6, Cdk6 showed changes in expression a day earlier than any other Pax6-regulated cell cycle gene suggesting that it is the primary mediator of Pax6’s actions. We then used ﬂow cytometry to show that progenitors lacking Pax6 have a shortened G1 phase and that their Cdk6 levels are increased in all phases. We found that Cdk6 levels oscillate during the cell cycle, increasing from G1 to M phase. We propose a model in which loss of Pax6 shortens G1 phase by raising overall Cdk6 levels, thereby shortening the time taken for Cdk6 levels to cross a threshold triggering transition to S phase. Keywords: Pax6, cortex, Cdk6, progenitor, cell cycle †Present address: Da Mi, Cerebral corticogenesis requires precise spatio-temporal control of the rates of progenitor cell division. Previous research on the developing mouse embryo established that the transcription factor Pax6 is a key regulator of this process (Götz et al., 1998; Warren et al., 1999; Estivill-Torrus et al., 2002; Heins et al., 2002; Haubst, 2004; Manuel et al., 2006, 2015; Quinn et al., 2007; Sansom et al., 2009; Asami et al., 2011; Georgala et al., 2011a,b; Mi et al., 2013). Pax6 is expressed by cortical radial glial progenitors, which are by far the most numerous cortical cell type present at the onset of corticogenesis at around embryonic day 12.5 (E12.5) in mouse. Previous functional studies have shown that Pax6 is an important cell autonomous repressor of radial glial cell cycle progression (Warren et al., 1999; Estivill-Torrus et al., 2002; Manuel et al., 2006; Georgala et al., 2011b; Mi et al., 2013). Loss of Pax6 shortens the overall length of cortical progenitor cell cycles and hence accelerates cell division (Mi et al., 2013). Previously, we identiﬁed a number of highly conserved cell cycle genes with actions fundamental to cell cycle progression across cell types and species whose levels of expression are aﬀected by constitutive loss of Pax6 function (Nurse, 2000, 2002). One of these was the cell cycle gene Cdk6 (Mi et al., 2013). Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology and Neuroscience and MRC Centre for Neurodevelopmental Disorders, King’s College London, London, United Kingdom Centre for Developmental Neurobiology, Institute of Psychiatry, Psychology and Neuroscience and MRC Centre for Neurodevelopmental Disorders, King’s College London, London, United Kingdom Received: 03 September 2018 Accepted: 26 October 2018 Published: 15 November 2018 Citation: Mi D, Manuel M, Huang Y-T, Mason JO and Price DJ (2018) Pax6 Lengthens G1 Phase and Decreases Oscillating Cdk6 Levels in Murine Embryonic Cortical Progenitors. Front. Cell. Neurosci. 12:419. doi: 10.3389/fncel.2018.00419 Received: 03 September 2018 Accepted: 26 October 2018 Published: 15 November 2018 Flow Cytometry Gene Forward primer Reverse primer Pax6 (exons 6–7) TATTACGAGACTGGCTCCAT TTGATGACACACTGGGTATG GFP CAGCCACAACGTCTATATCA GTGTTCTGCTGGTAGTGGTC Cdk6 GAGTGTCGGTTGCATCTTT GAGTCCAATGATGTCCAAGA Cdca7 GGAACGTCCATGCTTACTTG CACAACGTCGAGAACAAGAG Smad2 GCAGGAATTGAGCCACAGAG CGGAGAGCCTGTGTCCATAC Smad3 GATGACTACAGCCATTCCAT TCACTGGTTTCTCCATCTTC Smad4 TCCTGTGGCTTCCACAAGTC ATGGTAAGTAGCTGGCTGAG Smad7 GTGTTGCTGTGAATCTTACG CCATTGGGTATCTGGAGTAA Cdk2 GTGTACCCAGTACTGCCATC TCCATGAATTTCTTGAGGTC Cdk4 GAGGACATACCTGGACAAAG AGAATGTTCTCTGGCTTCAG Ccnd1 TTCATCGAACACTTCCTCTC GAGGGTGGGTTGGAAAT Ccnd2 AGTGTGCATGTTCCTAGCTT CAGGTTCCACTTCAGCTTAC Sesn1 TTGGCTGATTACCAAAGAAC GAGGCAAGAGAGTGGTAGTG Mcm3 GGACGATATAGCCAAGATCA GAGGATTGCCTTCTTGACAT Mcm6 GATTGTTGTGCCTGATGTCT GACCAGCCTGTATGACAGAT Smc2 GACCAGAACTGTAACCCTTG AGTTCATTCTCCTTGGTCCT Ccdc80 ATCTTTGGTCCTGTCAACAA CATTCCATACTCCTTCCTCA Ccdc90A GCACAGAAAAGAGAACTTGC CAGTGCAGACACAATGATTT TABLE 1 \| qRT-PCR primers. Gene Forward primer Reverse primer Pax6 (exons 6–7) TATTACGAGACTGGCTCCAT TTGATGACACACTGGGTATG GFP CAGCCACAACGTCTATATCA GTGTTCTGCTGGTAGTGGTC Cdk6 GAGTGTCGGTTGCATCTTT GAGTCCAATGATGTCCAAGA Cdca7 GGAACGTCCATGCTTACTTG CACAACGTCGAGAACAAGAG Smad2 GCAGGAATTGAGCCACAGAG CGGAGAGCCTGTGTCCATAC Smad3 GATGACTACAGCCATTCCAT TCACTGGTTTCTCCATCTTC Smad4 TCCTGTGGCTTCCACAAGTC ATGGTAAGTAGCTGGCTGAG Smad7 GTGTTGCTGTGAATCTTACG CCATTGGGTATCTGGAGTAA Cdk2 GTGTACCCAGTACTGCCATC TCCATGAATTTCTTGAGGTC Cdk4 GAGGACATACCTGGACAAAG AGAATGTTCTCTGGCTTCAG Ccnd1 TTCATCGAACACTTCCTCTC GAGGGTGGGTTGGAAAT Ccnd2 AGTGTGCATGTTCCTAGCTT CAGGTTCCACTTCAGCTTAC Sesn1 TTGGCTGATTACCAAAGAAC GAGGCAAGAGAGTGGTAGTG Mcm3 GGACGATATAGCCAAGATCA GAGGATTGCCTTCTTGACAT Mcm6 GATTGTTGTGCCTGATGTCT GACCAGCCTGTATGACAGAT Smc2 GACCAGAACTGTAACCCTTG AGTTCATTCTCCTTGGTCCT Ccdc80 ATCTTTGGTCCTGTCAACAA CATTCCATACTCCTTCCTCA Ccdc90A GCACAGAAAAGAGAACTTGC CAGTGCAGACACAATGATTT Citation: Mi D, Manuel M, Huang Y-T, Mason JO and Price DJ (2018) Pax6 Lengthens G1 Phase and Decreases Oscillating Cdk6 Levels in Murine Embryonic Cortical Progenitors. Front. Cell. Neurosci. 12:419. doi: 10.3389/fncel.2018.00419 Cdks drive progression through the eukaryotic cell cycle by forming complexes with cyclins (Morgan, 1997; Loog and Morgan, 2005; Hochegger et al., 2008; Coudreuse and Nurse, 2010; Uhlmann et al., 2011). In most natural systems studied so far, levels of Cdks remain relatively constant during the cell cycle whereas levels of the cyclins oscillate, causing Cdk activity to oscillate. November 2018 \| Volume 12 \| Article 419 1 Frontiers in Cellular Neuroscience \| www.frontiersin.org Pax6 and Cortical Progenitor Cell Cycle Mi et al. In experiments using engineered ﬁssion yeast, oscillations in Cdk activity alone, without the numerous additional regulatory mechanisms that normally aﬀect the cell cycle, can be suﬃcient to drive orderly progression through major cell cycle events (Coudreuse and Nurse, 2010). Cdk6 speciﬁcally partners a particular type of cyclin (the D-type cyclins) and primarily regulates the G1 to S phase transition (Hochegger et al., 2008). In cerebral cortical progenitors, Pax6 negatively regulates the expression of Cdk6 (Mi et al., 2013). In experiments using engineered ﬁssion yeast, oscillations in Cdk activity alone, without the numerous additional regulatory mechanisms that normally aﬀect the cell cycle, can be suﬃcient to drive orderly progression through major cell cycle events (Coudreuse and Nurse, 2010). Cdk6 speciﬁcally partners a particular type of cyclin (the D-type cyclins) and primarily regulates the G1 to S phase transition (Hochegger et al., 2008). In cerebral cortical progenitors, Pax6 negatively regulates the expression of Cdk6 (Mi et al., 2013). qRT-PCR q cDNA was synthesized from RNA with a Superscript reverse transcriptase reaction (Thermo Fisher Scientiﬁc, Perth, United Kingdom) and qRT-PCR was done with a Quantitect SYBR Green PCR kit (Qiagen, Hilden, Germany) and a DNA Engine Opticon Continuous Fluorescence Detector (MJ Research, QC, Canada). Primer pairs are listed in Table 1. For each sample, we normalized the abundance of each transcript to the GAPDH expression level and calculated the ratios of normalized expression levels in conditional knock outs (cKOs) to littermate controls. We used three biological replicates from three diﬀerent litters. For each biological replicate we ran three technical replicates. Here we addressed several questions. Are the eﬀects of Pax6 on the cell cycle likely to be mediated primarily through actions on Cdk6, or does Pax6 control multiple cell cycle regulators in parallel? Do the eﬀects of Pax6 vary between cell cycle phases? Are levels of Cdk6 normally constant throughout the cell cycle of cortical progenitors? Does Pax6 modulate Cdk6 levels equally or diﬀerentially across the phases? Flow Cytometry Aliquots of permeabilised and ﬁxed cells suspended in ﬂuorescence activated cell sorting (FACS) buﬀer were reacted with Hoechst 33342 (Thermo Fisher Scientiﬁc, Perth, United Kingdom; Cat# 1391095). Primary antibodies used were: anti-Pax6 (1:200, rabbit, Millipore, Livingston, United Kingdom; Cat# AB2237); anti-Tuj1 (1:800, mouse, Cambridge Bioscience, Cambridge, United Kingdom; Cat# MMS 435P); anti-PH3 (1:200, mouse, Cell Signaling Technology, Danvers, Massachusetts, United States; Cat# 9706); anti-Ki67 (1:500, rat, eBioscience, San Diego, CA, United States; Cat# 12-5698); anti-Cdk6 (1:500, rabbit, Santa Cruz Biotechnology, Dallas, TX, United States; Cat# SC-177). We used secondary- only and ﬂuorescence-minus-one controls to set thresholds for primary antibody-speciﬁc staining. Cells were analyzed using an LSRII ﬂow cytometer and FlowJo V10 software (BD Biosciences, Franklin Lakes, NJ, United States). In all of our experiments, we used the same gate for forward scatter for both wild-type and mutant progenitor cells. Since forward scatter values in ﬂow y To address the ﬁrst of these questions, we used quantitative real time polymerase chain reaction (qRT-PCR) to test for changes in the expression of a set of cell cycle regulators following acute cortical deletion of Pax6. The set we tested contained all cell cycle regulators that were shown in previous work to have altered cortical expression levels in constitutive Pax6−/−mutants (Mi et al., 2013). Here we studied embryos with tamoxifen- induced cortex-speciﬁc deletion of Pax6. This allowed us to focus on the eﬀects of Pax6 loss that are more likely to be direct by minimizing the possibility of secondary changes arising as a long- term consequence of cortical deletion or Pax6 removal from non-cortical tissues. We then used ﬂow cytometry to discover how Pax6 aﬀects the phases of the cell cycle and the levels of Cdk6 in each. We found that Pax6 loss increases Cdk6 levels similarly in all cell cycle phases and, particularly interestingly, that Cdk6 levels oscillate with cell cycle phase. This prompted us to extend our analysis to examine the relationship between Pax6 levels and Cdk6 levels across cell cycle phases. Our results indicated that Pax6 sets the level of expression around which Cdk6 oscillates but is unlikely to be responsible for controlling the oscillation itself. TABLE 1 \| qRT-PCR primers. MATERIALS AND METHODS Mice and Tissue Preparation Mice and Tissue Preparation Mice and Tissue Preparation All mice were bred in-house. The University of Edinburgh Animal Welfare and Ethics Board and a license from the Home Oﬃce UK issued under the UK Animals (Scientiﬁc Procedures) Act 1986 regulated all procedures. For constitutive inactivation of Pax6, we used the Pax6Sey allele (designated as Pax6−here; (Hill et al., 1991). For conditional inactivation of Pax6, we used the Pax6loxP allele (Simpson et al., 2009) and a green ﬂuorescent protein (GFP) reporter allele (Sousa et al., 2009) with BAC transgenic strain Emx1CreERT2 (Kessaris et al., 2006). Dams were killed by cervical dislocation, fetuses were removed and cortices were dissected and used for RNA extraction (RNeasy Plus micro kit; Qiagen, Hilden, Germany) or dissociated with papain (20 U ml−1; Dissociation kit; Worthington Biochemical Corporation, Lakewood, NJ, United States). Dissociated cell suspensions diluted to 1.8 × 106 ml−1 were ﬁxed and permeabilised in 100% ethanol and stored at −20◦C. November 2018 \| Volume 12 \| Article 419 Frontiers in Cellular Neuroscience \| www.frontiersin.org 2 Mi et al. Pax6 and Cortical Progenitor Cell Cycle Quantitative RT-PCR analysis of the effects of tamoxifen-induced cortex-speciﬁc Pax6 deletion on the expression of cell cycle genes. (A) Tamoxifen resulted in Pax6 mRNA loss, with a ∼60% reduction by E11.5 and a ∼100% reduction by E13.5. (B) This loss coincided with the production of els of GFP reporter gene mRNA. (C–R) Changes in the expression of 16 cell cycle genes. In all cases, the levels of cortical expression of each gene ed relative to levels of cortical expression of GAPDH in the same embryo. In panels (A) and (C–R), ratios of expression in conditional knock-outs (cKO) e controls were combined to give mean ± SEM at each age. All data are from three biological replicates at each age; ∗p < 0.05; ∗∗p < 0.01; Student’s ison of cKOs with paired controls at each age. FIGURE 1 \| Quantitative RT-PCR analysis of the effects of tamoxifen-induced cortex-speciﬁc Pax6 deletion on the expression of cell cycle genes. (A) Tamoxifen given at E9.5 resulted in Pax6 mRNA loss, with a ∼60% reduction by E11.5 and a ∼100% reduction by E13.5. (B) This loss coincided with the production of increasing levels of GFP reporter gene mRNA. (C–R) Changes in the expression of 16 cell cycle genes. Changes in the Expression of Cell Cycle Regulators Following Acute Cortical Deletion of Pax6 We gave tamoxifen to experimental Pax6ﬂ/ﬂ;Emx1CreER (designated cKO) and littermate control Pax6ﬂ/+;Emx1CreER embryos at E9.5. All embryos contained a ﬂoxed-stop GFP reporter allele (Sousa et al., 2009). We used qRT-PCR to measure levels of mRNA in cortical samples relative to levels of GAPDH. For all genes except GFP we then calculated ratios between the relative mRNA levels in cKO and control littermates at E11.5, 12.5, and 13.5 (Figure 1). We estimated the lengths of each phase in hours under diﬀerent conditions by multiplying the proportion of cells in that phase by the corresponding Tc derived from calculations above. These estimations gave values for G1 of 9.3 h in Pax6+/+ cortex and 7.7 h in Pax6−/−cortex at E12.5. At E14.5, values for G1 were 14.8 h in Pax6+/+ cortex and 11.5 h in Pax6−/−cortex. At E12.5, S phase was 1.7 h in Pax6+/+ cortex versus 1.8 h in Pax6−/− cortex, G2 phase was 0.9 h in Pax6+/+ cortex versus 1.0 h in Pax6−/−cortex and M phase was 0.2 h in Pax6+/+ cortex versus 0.25 h in Pax6−/−cortex. At E14.5, S phase was 1.9 h in Pax6+/+ Pax6 levels decayed exponentially and GFP levels rose steadily over the 4 days following tamoxifen administration (Figures 1A,B). Pax6 levels had declined to ∼40% of normal by E11.5, the earliest post-tamoxifen time-point examined. Cdk6 was the only cell cycle gene studied that showed a signiﬁcant change (upregulation) in gene expression at E12.5 and this was sustained at E13.5 (Figure 1C). Cdca7 showed a signiﬁcant upregulation but only later, at E13.5 (Figure 1D). Smad3, Smad7, and Sesn1 showed signiﬁcant downregulation but only at E13.5 (Figures 1F,H,M). These results suggest that no other Pax6- regulated cell cycle gene rivals Cdk6 as the top candidate among possible mediators of Pax6’s actions on cortical progenitor cell cycle lengths. Whereas genes such as Cdca7, Smad3, Smad7, and Sesn1 are almost certainly involved, it is possible that they alter their expression as a secondary consequence of changes in Cdk6 expression. FIGURE 2 \| Cell cycle lengths are shortened in Pax6−/−progenitors. Lengths of the cell cycles of progenitors in Pax6+/+ and Pax6−/−E12.5 and E14.5 cortex measured using double labeling with IdU and BrdU [n = 3 embryos of each genotype from three different litters for each age; mean ± SEMs are shown; in panel (A), p = 0.0099, Student’s t-test; in panel (B), p = 0.0004, Student’s t-test]. Immunohistochemistry for BrdU and IdU Immunohistochemistry for BrdU and IdU Pregnant females were injected intra-peritoneally with 200 µl of 100 µg ml−1 IdU and then 1.5 h later with the same dose of BrdU. They were sacriﬁced after 30 min and histological sections were cut through the cortex. Primary antibodies used were anti-BrdU/IddU (which recognizes both BrdU and IddU) (1:100, mouse, Becton Dickinson, Swindon, United Kingdom; clone B44) and anti-BrdU (1:100, rat, Abcam, Cambridge, United Kingdom; clone BU1/75). Nuclei were counterstained with TO-PRO-3 iodide (Molecular Probes). Analysis was as described in (Martynoga et al., 2005). We then used ﬂow cytometry to quantify the relative lengths of G1, S, G2, and M phases in cortical cells from E12.5 and E14.5 Pax6+/+ and Pax6−/−embryos (Figure 3). A combination of Hoechst and antibody staining identiﬁed cells in each of the phases (Figures 3A–F). Cells were labeled with Hoechst 33342 to measure DNA content and antibodies for Ki67 (a marker of proliferating cells), class III beta-tubulin (TuJ1; a marker of postmitotic neurons), phospho-Histone H3 (PH3; a marker of M phase), and Pax6. Cells were classiﬁed as G1 if they had 2n DNA content and were positive for Ki67 (Figure 3A). As expected, almost all of these cells (∼98%) were negative for TuJ1 (Figure 3C). Combining data from multiple embryos (four per genotype per age) showed signiﬁcant reductions by ∼8–10% in the proportions of cells in G1 at both ages in Pax6−/−embryos (Figures 3G,H). For phases other than G1, the only signiﬁcant diﬀerences were in S and G2 at E14.5 (Figures 3G,H), where mean values were higher in mutants in both cases. Mice and Tissue Preparation In all cases, the levels of cortical expression of each gene were measured relative to levels of cortical expression of GAPDH in the same embryo. In panels (A) and (C–R), ratios of expression in conditional knock-outs (cKO) over littermate controls were combined to give mean ± SEM at each age. All data are from three biological replicates at each age; ∗p < 0.05; ∗∗p < 0.01; Student’s t-test comparison of cKOs with paired controls at each age. November 2018 \| Volume 12 \| Article 419 Frontiers in Cellular Neuroscience \| www.frontiersin.org Frontiers in Cellular Neuroscience \| www.frontiersin.org 3 Pax6 and Cortical Progenitor Cell Cycle Mi et al. cytometry are directly linked to cell size, this indicates that the wild-type and mutant cells were in the same size range. At E12.5, Tc was ∼10% shorter in Pax6−/−cortex than in Pax6+/+ cortex (n = 3 embryos of each genotype from three diﬀerent litters; mean Tc ± SEM = 12.1 ± 0.15 h in Pax6+/+ and 10.75 ± 0.06 h in Pax6−/−; p = 0.0099, Student’s t-test). At E14.5, Tc was ∼15% shorter in Pax6−/−cortex than in Pax6+/+ cortex (n = 3 embryos of each genotype from three diﬀerent litters; mean Tc ± SEM = 18.0 ± 0.41 h in Pax6+/+ and 15.5 ± 0.42 h in Pax6−/−; p = 0.0004, Student’s t-test) (Figure 2). G1 Is Abnormally Short in Pax6−/− Mutants Given that Cdk6’s main action during the eukaryotic cell cycle is to promote the G1 to S phase transition (Hochegger et al., 2008), we hypothesized that elevation of Cdk6 in Pax6−/−mutants might shorten the cell cycle of cortical progenitors by shortening primarily G1 phase. We ﬁrst estimated the overall lengths of the cell cycles (Tc) of progenitors in Pax6+/+ and Pax6−/−E12.5 and E14.5 cortex using double labeling with iododeoxyuridine (IdU) and bromodeoxyuridine (BrdU) (Martynoga et al., 2005). FIGURE 2 \| Cell cycle lengths are shortened in Pax6−/−progenitors. Lengths of the cell cycles of progenitors in Pax6+/+ and Pax6−/−E12.5 and E14.5 cortex measured using double labeling with IdU and BrdU [n = 3 embryos of each genotype from three different litters for each age; mean ± SEMs are shown; in panel (A), p = 0.0099, Student’s t-test; in panel (B), p = 0.0004, Student’s t-test]. FIGURE 2 \| Cell cycle lengths are shortened in Pax6−/−progenitors. Lengths of the cell cycles of progenitors in Pax6+/+ and Pax6−/−E12.5 and E14.5 cortex measured using double labeling with IdU and BrdU [n = 3 embryos of each genotype from three different litters for each age; mean ± SEMs are shown; in panel (A), p = 0.0099, Student’s t-test; in panel (B), p = 0.0004, Student’s t-test]. November 2018 \| Volume 12 \| Article 419 Frontiers in Cellular Neuroscience \| www.frontiersin.org 4 Pax6 and Cortical Progenitor Cell Cycle Mi et al. \| Flow cytometric analysis of cell cycle phases of cortical progenitors in Pax6+/+ and Pax6−/−embryos at E12.5 and E14.5. (A–F) Representative of ﬂow cytometry showing how cells were assigned to speciﬁc stages of the cell cycle. Cells were labeled with Hoechst 33342 and antibodies for Ki67, FIGURE 3 \| Flow cytometric analysis of cell cycle phases of cortical progenitors in Pax6+/+ and Pax6−/−embryos at E12.5 and E14.5. (A–F) Representative examples of ﬂow cytometry showing how cells were assigned to speciﬁc stages of the cell cycle. Cells were labeled with Hoechst 33342 and antibodies for Ki67, class III beta-tubulin (TuJ1), phospho-Histone H3 (PH3) and Pax6. (A) Fluorescence intensity of Ki67 against Hoechst, shown as a heat-map with red areas containing most cells and blue areas fewest: Cells were classiﬁed as G1 if they had 2n DNA content and were positive for Ki67 (intensity values above the red line), which is a marker of proliferating cells. G1 Is Abnormally Short in Pax6−/− Mutants Signiﬁcant effects of genotype on each phase at each age are marked (Sidak’s multiple comparisons tests: ∗∗p = 0.0029; ∗∗∗p = 0.0001; ∗∗∗∗p < 0.0001). (I–L) Charts, drawn to scale, summarizing the relative lengths of the cell cycle phases across ages and genotypes. FIGURE 4 \| Flow cytometric analysis of Cdk6 levels at different phases of the cell cycle in Pax6+/+ and Pax6−/−cortex at E12.5. (A,B) Fluorescence intensity of Cdk6 against Ki67: Samples from a Pax6+/+ and a Pax6−/−embryo with cells classiﬁed according to their cell cycle phase by methods illustrated in Figure 3. G1 cells were distinguished from G0 cells by positivity for Ki67 in quadrants Q2-1 and Q4-1 (cells in quadrants 1-1 and 3-1 were negative for Ki67). (C) Relative levels of Cdk6 in the cortex of four Pax6+/+ and four Pax6−/−embryos at each phase of the cell cycle. Each data point is the average level in the cortex of one embryo. Regression analysis of levels of Cdk6 against cells cycle phase gave r2 values of 0.78 for Pax6+/+ data and 0.81 for Pax6−/−data. ANOVA showed a signiﬁcant effect of genotype (α = 0.05) and Sidak’s multiple comparisons tests showed signiﬁcantly higher levels of Cdk6 in Pax6-/−cortex in all phases (G1, p = 0.0075; S, p < 0.0001; G2, p = 0.0167; M, p = 0.0019). FIGURE 4 \| Flow cytometric analysis of Cdk6 levels at different phases of the cell cycle in Pax6+/+ and Pax6−/−cortex at E12.5. (A,B) Fluorescence intensity of Cdk6 against Ki67: Samples from a Pax6+/+ and a Pax6−/−embryo with cells classiﬁed according to their cell cycle phase by methods illustrated in Figure 3. G1 cells were distinguished from G0 cells by positivity for Ki67 in quadrants Q2-1 and Q4-1 (cells in quadrants 1-1 and 3-1 were negative for Ki67). (C) Relative levels of Cdk6 in the cortex of four Pax6+/+ and four Pax6−/−embryos at each phase of the cell cycle. Each data point is the average level in the cortex of one embryo. Regression analysis of levels of Cdk6 against cells cycle phase gave r2 values of 0.78 for Pax6+/+ data and 0.81 for Pax6−/−data. ANOVA showed a signiﬁcant effect of genotype (α = 0.05) and Sidak’s multiple comparisons tests showed signiﬁcantly higher levels of Cdk6 in Pax6-/−cortex in all phases (G1, p = 0.0075; S, p < 0.0001; G2, p = 0.0167; M, p = 0.0019). G1 Is Abnormally Short in Pax6−/− Mutants and whether the absence of Pax6 aﬀects Cdk6 levels in all phases of the cortical progenitor cell cycle. We used ﬂow cytometry to identify cells in each cell cycle phase (Figures 3A–F) and measure their Cdk6 levels. We found signiﬁcantly elevated levels of Cdk6 in all cell cycle phases of Pax6−/−progenitors (Figure 4C). In addition, Cdk6 levels varied across the phases, rising by ∼1.5–2- fold from G1 to M both in Pax6+/+ and Pax6−/−progenitors (Figure 4C). cortex versus 2.2 h in Pax6−/−cortex, G2 phase was 1.3 h in Pax6+/+ cortex versus 1.6 h in Pax6−/−cortex, and M phase was 0.1 h in Pax6+/+ cortex versus 0.2 h in Pax6−/−cortex. These estimations are summarized graphically in Figures 3I–L. These data suggest that in the absence of Pax6 G1 phase is shortened by ∼2–3 h while other phases are similar to or ∼15–20 min longer than normal. G1 Is Abnormally Short in Pax6−/− Mutants (B) Fluorescence intensity of TuJ1 against Hoechst: TuJ1 was used as a marker of differentiating neurons. (C) Fluorescence intensity of Ki67 against TuJ1, with different colors indicating cell cycle phase of each cell: In this sample, 98.0% (50.7/51.74) of Ki67+ cells were TuJ1– whereas 69.3% (33.5/48.3) of Ki67– cells were Tuj1+. (D) Fluorescence intensity of PH3 against Hoechst, plotted using the same method as in panel (A): Cells in M phase (Continued) FIGURE 3 \| Flow cytometric analysis of cell cycle phases of cortical progenitors in Pax6+/+ and Pax6−/−embryos at E12.5 and E14.5. (A–F) Representative examples of ﬂow cytometry showing how cells were assigned to speciﬁc stages of the cell cycle. Cells were labeled with Hoechst 33342 and antibodies for Ki67, class III beta-tubulin (TuJ1), phospho-Histone H3 (PH3) and Pax6. (A) Fluorescence intensity of Ki67 against Hoechst, shown as a heat-map with red areas containing most cells and blue areas fewest: Cells were classiﬁed as G1 if they had 2n DNA content and were positive for Ki67 (intensity values above the red line), which is a marker of proliferating cells. (B) Fluorescence intensity of TuJ1 against Hoechst: TuJ1 was used as a marker of differentiating neurons. (C) Fluorescence intensity of Ki67 against TuJ1, with different colors indicating cell cycle phase of each cell: In this sample, 98.0% (50.7/51.74) of Ki67+ cells were TuJ1– whereas 69.3% (33.5/48.3) of Ki67– cells were Tuj1+. (D) Fluorescence intensity of PH3 against Hoechst, plotted using the same method as in panel (A): Cells in M phase (Continued) Frontiers in Cellular Neuroscience \| www.frontiersin.org November 2018 \| Volume 12 \| Article 419 Pax6 and Cortical Progenitor Cell Cycle Mi et al. FIGURE 3 \| Continued were identiﬁed by their expression of PH3 (0.858%, above black line). (E) Fluorescence intensity of Ki67 against Hoechst: M phase cells expressed PH3 and contained the highest levels of Ki67. (F) Fluorescence intensity of Pax6 against Ki67, plotted using the same method as in panel (C): Very few Ki67– cells expressed Pax6 (11.5% [6.72/58.52] in this sample) whereas most Ki67+ cells co-expressed Pax6 (74.7% [31/40.5] in this sample). (G,H) Proportions of proliferating cells (i.e., G0 cells excluded) in each phase of the cell cycle in Pax6+/+ and Pax6-/- embryos at E12.5 and E14.5. Values are mean ± SEM (n = 4 embryos in all cases). ANOVA showed a signiﬁcant effect of genotype at both ages (α = 0.05). DISCUSSION Our new results support a central role for Cdk6 in Pax6’s regulation of cortical progenitor cell cycles. Figure 6 shows a model illustrating how elevated Cdk6 levels across the cell cycle could shorten G1 phase. We propose that the time of G1 and the G1 to S phase transition is determined by the time it takes Cdk6 activity to rise above a threshold; our model proposes that the threshold is similar in both Pax6+/+ and Pax6−/−progenitors (Figures 6B,C). Oscillation of Cdk6 activity at abnormally high levels would shorten the length of G1 since it would take less time to achieve the threshold required to exit G1. We also found evidence that other cell cycle phases were slightly longer than normal in Pax6−/−progenitors, particularly at the later age studied, E14.5. This is reﬂected in the way Figure 6 is drawn. These changes were not enough to compensate for the shortening of G1 and, overall, cell cycle lengths were reduced. We do not know how these other phases might be aﬀected. At least We identiﬁed cells in each phase using methods illustrated in Figures 3, 5A,B. We found that Pax6 levels varied signiﬁcantly with cell cycle phase at all ages studied (Figures 5C–E). In most phases at E12.5 and E13.5 (the one exception being M phase at E13.5), Pax6 levels were signiﬁcantly higher in rostral than in caudal cortex (p < 0.01 in all phases at both ages, Sidak’s multiple comparisons tests following ANOVA, α = 0.05). This was only the case in G1 at E14.5 (p < 0.01). These ﬁndings on rostral versus caudal diﬀerences are in excellent agreement with previous work showing a rostral [high] to caudal [low] gradient of Pax6 expression at E12.5 and E13.5 that ﬂattens by E14.5 (Mi et al., 2013). Pax6 levels changed by ∼1.5–2-fold from trough to peak during the cell cycle (Figures 5C–E). The oscillations in Pax6 FIGURE 5 \| Flow cytometric analysis of Pax6 levels at different phases of the cell cycle in Pax6+/+ cortex at E12.5, E13.5, and E14.5. Cells classiﬁed according to their cell cycle phase by methods illustrated in Figure 1. (A) Example showing how M phase cells were identiﬁed by their expression of PH3. (B) Example showing how G1 cells were distinguished from G0 cells by positivity for Ki67. Pax6 Levels Oscillate During the Cell Cycle in Cortical Progenitors In most systems studied to date, levels of Cdks remain relatively constant during the cell cycle (Hochegger et al., 2008). It was not known whether this is also the case for cortical progenitors A parsimonious explanation for the fact that Cdk6 levels oscillate not only in Pax6+/+ cortex but also in Pax6−/−cortex is that Pax6 inﬂuences the average level of Cdk6 expression rather than November 2018 \| Volume 12 \| Article 419 Frontiers in Cellular Neuroscience \| www.frontiersin.org 6 Mi et al. Pax6 and Cortical Progenitor Cell Cycle being directly responsible for the oscillations themselves, which would be caused by a diﬀerent mechanism. This would explain why Cdk6 levels still oscillate in the absence of Pax6. Nonetheless, a more complex scenario was conceivable. In normal cortex, Pax6 levels might oscillate during the cell cycle in such a way as to drive changes in Cdk6 levels; in Pax6−/−cortex, some other unknown mechanism might take over Pax6’s role. We addressed whether this second possibility might be feasible by measuring Pax6 levels in each phase of the cell cycle in normal embryos using ﬂow cytometry (Figure 5). levels were out of phase with the oscillations in Cdk6 levels shown in Figure 4. Levels rose to a peak in G2 and dropped in M, before Cdk6 levels fell. The rise of Pax6 from G1 to G2 paralleled that of Cdk6 through these three phases. DISCUSSION (C–E) Average relative levels (± SEM) of Pax6 in proliferating cortical cells (G0 cells excluded) in each cell cycle phase in rostral and caudal cortex (n = 4 embryos at each age). ANOVAs at each age showed signiﬁcant effects of cell cycle phase (α = 0.05). Sidak’s multiple comparisons tests showed signiﬁcant differences between phases, as marked (∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0001). FIGURE 5 \| Flow cytometric analysis of Pax6 levels at different phases of the cell cycle in Pax6+/+ cortex at E12.5, E13.5, and E14.5. Cells classiﬁed according to their cell cycle phase by methods illustrated in Figure 1. (A) Example showing how M phase cells were identiﬁed by their expression of PH3. (B) Example showing how G1 cells were distinguished from G0 cells by positivity for Ki67. (C–E) Average relative levels (± SEM) of Pax6 in proliferating cortical cells (G0 cells excluded) in each cell cycle phase in rostral and caudal cortex (n = 4 embryos at each age). ANOVAs at each age showed signiﬁcant effects of cell cycle phase (α = 0.05). Sidak’s multiple comparisons tests showed signiﬁcant differences between phases, as marked (∗p < 0.05; ∗∗p < 0.001; ∗∗∗p < 0.0001). November 2018 \| Volume 12 \| Article 419 Frontiers in Cellular Neuroscience \| www.frontiersin.org 7 Pax6 and Cortical Progenitor Cell Cycle Mi et al. FIGURE 6 \| A model. (A,B) Pax6 and Cdk6 oscillations are slightly out of phase. (C) A general elevation of Cdk6 activity throughout the cell cycle might allow levels critical for switching from G1 to S (horizontal line in B,C) to be achieved more rapidly than normal. scenario. Changes in Pax6 protein levels would need a certain time to inﬂuence transcription of the Cdk6 gene and the mRNA would then need to be translated to alter Cdk6 protein levels. Although we do not exclude the possibility that ﬂuctuations in Pax6 levels might contribute to the temporal pattern of change of Cdk6 levels, the simplest suggestion is that Pax6 reduces Cdk6 levels overall and that the oscillations in Cdk6 levels during the cell cycle are driven by other mechanisms. Our previous work showed that Pax6 overexpression signiﬁcantly reduced Cdk6 levels (Mi et al., 2013), and in the future it would be interesting to test how Cdk6 levels oscillate during the cell cycle in these mutants and how cell cycle phases change. DISCUSSION It is likely that post-translational processes are involved in the relatively rapid cyclical changes in Cdk6 and Pax6 levels identiﬁed here. Cortical progenitor cells might generate excess amounts of their mRNAs, which might be translated at levels that vary as the cell progresses through the cell cycle. A similar mechanism, termed “translation on demand,” was suggested in a recently published review paper (Liu et al., 2016). It suggests that quick generation of proteins in response to signals could be achieved purely by regulating the translation rate of pre-existing mRNA (Le Roch et al., 2004; Lee et al., 2011; Lackner et al., 2012; Eichelbaum and Krijgsveld, 2014; Jovanovic et al., 2015). Some studies suggested that transcription factors are more likely to be subject to translation on demand regulation, as some rapid cell state transitions require fast synthesis and reactions of transcription factors to modulate their downstream gene networks (Lee et al., 2013; Jovanovic et al., 2015; Liu et al., 2016). Rapid degradation via ubiquitination might also contribute to rapid adjustments of Pax6 and Cdk6 protein levels during the cell cycle. In eye development, for example, a post-translational ubiquitin-mediated proteasomal mechanism degrades Pax6 protein (Pﬁrrmann et al., 2016). It is possible that Pax6 regulates the overall levels of Cdk6 not only by direct repression of the Cdk6 gene itself (Mi et al., 2013) but also by aﬀecting the post-translational mechanisms that determine Cdk6 protein production and stability. FIGURE 6 \| A model. (A,B) Pax6 and Cdk6 oscillations are slightly out of phase. (C) A general elevation of Cdk6 activity throughout the cell cycle might allow levels critical for switching from G1 to S (horizontal line in B,C) to be achieved more rapidly than normal. one possibility is that they are lengthened as a consequence of accelerated progression through G1 phase rather than a direct action of Pax6 on regulators of these phases. Another possibility is that delayed changes in the expression of cell cycle genes other than Cdk6, such as those observed here (Figure 1: Cdca7, Smad3, Smad7, or Sesn1) directly aﬀect the lengths of these phases. This proposal has similarity to the mechanism proposed to regulate an engineered minimal control network in ﬁssion yeast (Coudreuse and Nurse, 2010). This control network is based on oscillating levels of CDK activity, which drive cell cycle progression as they cross thresholds triggering transition between cell cycle phases including G1 to S. Frontiers in Cellular Neuroscience \| www.frontiersin.org REFERENCES doi: 10.1038/nature09543 Lee, M. T., Bonneau, A. R., Takacs, C. M., Bazzini, A. A., Divito, K. R., Fleming, E. S., et al. (2013). Nanog, Pou5f1 and SoxB1 activate zygotic gene expression during the maternal-to-zygotic transition. Nature 503, 360–364. doi: 10.1038/ nature12632 Dehay, C., and Kennedy, H. (2007). Cell-cycle control and cortical development. Nat. Rev. Neurosci. 8, 438–450. doi: 10.1038/nrn2097 Eichelbaum, K., and Krijgsveld, J. (2014). Rapid temporal dynamics of transcription, protein synthesis, and secretion during macrophage activation. Mol. Cell. 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Neuron 21, 1031–1044. doi: 10.1016/ S0896-6273(00)80621-2 Manuel, M., Georgala, P. A., Carr, C. B., Chanas, S., Kleinjan, D. A., Martynoga, B., et al. (2006). REFERENCES Hochegger, H., Takeda, S., and Hunt, T. (2008). Cyclin-dependent kinases and cell-cycle transitions: does one ﬁt all? Nat. Rev. Mol. Cell Biol. 9, 910–916. doi: 10.1038/nrm2510 Arai, Y., Pulvers, J. N., Haﬀner, C., Schilling, B., Nüsslein, I., Calegari, F., et al. (2011). Neural stem and progenitor cells shorten S-phase on commitment to neuron production. Nat. Commun. 2:54. doi: 10.1038/ncomms1155 Jovanovic, M., Rooney, M. S., Mertins, P., Przybylski, D., Chevrier, N., Satija, R., et al. (2015). Dynamic proﬁling of the protein life cycle in response to pathogens. Science 347:1259038. doi: 10.1126/science.1259038 Asami, M., Pilz, G. A., Ninkovic, J., Godinho, L., Schroeder, T., Huttner, W. B., et al. (2011). The role of Pax6 in regulating the orientation and mode of cell division of progenitors in the mouse cerebral cortex. Development 138, 5067–5078. doi: 10.1242/dev.074591 doi: 10.1242/dev.074591 Kessaris, N., Fogarty, M., Iannarelli, P., Grist, M., Wegner, M., and Richardson, W. D. (2006). Competing waves of oligodendrocytes in the forebrain and postnatal elimination of an embryonic lineage. Nat. Neurosci. 9, 173–179. doi: 10.1038/nn1620 Calegari, F. (2003). An inhibition of cyclin-dependent kinases that lengthens, but does not arrest, neuroepithelial cell cycle induces premature neurogenesis. J. Cell Sci. 116(Pt 24), 4947–4955. doi: 10.1242/jcs.00825 Lackner, D. H., Schmidt, M. W., Wu, S., Wolf, D. A., and Bähler, J. (2012). Regulation of transcriptome, translation, and proteome in response to environmental stress in ﬁssion yeast. Genome Biol. 13:R25. doi: 10.1186/gb- 2012-13-4-r25 Calegari, F., Haubensak, W., Haﬀner, C., and Huttner, W. B. (2005). Selective lengthening of the cell cycle in the neurogenic subpopulation of neural progenitor cells during mouse brain development. J. Neurosci. 25, 6533–6538. doi: 10.1523/JNEUROSCI.0778-05.2005 Lange, C., Huttner, W. B., and Calegari, F. (2009). Cdk4/CyclinD1 overexpression in neural stem cells shortens G1, delays neurogenesis, and promotes the generation and expansion of basal progenitors. Cell Stem Cell. 5, 320–331. doi: 10.1016/j.stem.2009.05.026 Caviness, V. S., Goto, T., Tarui, T., Takahashi, T., Bhide, P. G., and Nowakowski, R. S. (2003). Cell output, cell cycle duration and neuronal speciﬁcation: a model of integrated mechanisms of the neocortical proliferative process. Cereb. Cortex 13, 592–598. doi: 10.1093/cercor/13.6.592 j Le Roch, K. G., Johnson, J. R., Florens, L., Zhou, Y., Santrosyan, A., Grainger, M., et al. (2004). Global analysis of transcript and protein levels across the Plasmodium falciparum life cycle. Genome Res. 14, 2308–2318. doi: 10.1101/gr.2523904 Coudreuse, D., and Nurse, P. (2010). Driving the cell cycle with a minimal CDK control network. Nature 13, 592–598. FUNDING expansion of the progenitor pool, and aﬀects laminar phenotypes later in development (Lange et al., 2009; Pilaz et al., 2009). This might be one mechanism by which Pax6 aﬀects the later stages of cortical formation despite being downregulated in postmitotic neurons. This research was funded by MRC grant N012291 and BBSRC grant N006542. AUTHOR CONTRIBUTIONS We thank Dr. Martin Waterfall at the Flow Cytometry Core Facility for his generous support, Dr. Thomas Tan at the School of Biological Sciences for technical assistance and Mr. Chenyi Wang at the Department of Clinical Neurosciences, University of Cambridge for the precious help of ﬂow cytometry analysis. DM, MM, and Y-TH performed the experiments. DP and JM helped to design the experiments, obtained the funding, and supervised the work. DP wrote the manuscript. DISCUSSION As in the ﬁssion yeast model, it is possible that the oscillations in Cdk6 in cortical progenitors result from phase-dependent changes in Cdk6 synthesis and degradation. A Pax6-loss-induced change in the length of G1 phase is likely to have consequences for the subsequent development of progenitors and the neurons that they generate. Previous studies have shown that cell cycle length has a direct impact on a cell’s mode of division, i.e., whether it is neurogenic, producing neurons, or proliferative, producing new progenitor(s). G1 length is increased in neurogenic progenitors compared with proliferative progenitors (Caviness et al., 2003; Lukaszewicz et al., 2005; Dehay and Kennedy, 2007; Salomoni and Calegari, 2010). This is in line with the ﬁnding that basal progenitors, which mostly generate neurons through neurogenic division, have a longer G1 phase than apical progenitors (Calegari et al., 2005; Salomoni and Calegari, 2010; Arai et al., 2011). The functional importance of G1 length was demonstrated by manipulating it. An increase in G1 length leads to neurogenic division and premature neurogenesis (Calegari, 2003; Calegari et al., 2005). Shortening G1 increases proliferative divisions, leading to an An alternative possibility is that cyclical changes in Pax6 levels might cause the oscillations in Cdk6 levels, given that Pax6 can directly repress Cdk6 expression (Mi et al., 2013). The oscillations in Pax6 were slightly out of phase with those in Cdk6 (Figures 6A,B) but it is very unlikely that ﬂuctuations in Pax6 protein levels alone cause the oscillations in Cdk6 protein levels. Additional factors would be required to explain the very diﬀerent rates of change of Pax6 during its rising phase and Cdk6 during its falling phase and vice versa. An additional complexity would be explaining the dynamics of this November 2018 \| Volume 12 \| Article 419 8 Pax6 and Cortical Progenitor Cell Cycle Mi et al. 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https://openalex.org/W2946411578	https://europepmc.org/articles/pmc6533644?pdf=render	English	null	Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients	Frontiers in immunology	2,019	cc-by	12,386	ORIGINAL RESEARCH published: 17 May 2019 doi: 10.3389/ﬁmmu.2019.01029 y, Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States Correspondence: Elisabet Svenungsson elisabet.svenungsson@ki.se †These authors share ﬁrst authorship ‡These authors share senior authorship Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identiﬁed Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). IRF5 was up-regulated while SPDEF was found to be Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks speciﬁc diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reﬂect differences in underlying pathogenesis. Our objective was to obtain protein proﬁles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratiﬁcation. Plasma samples from a cross-sectional study of well-characterized SLE patients (n = 379) and matched population controls (n = 316) were analyzed by antibody suspension bead array targeting 281 proteins. To investigate the differences between SLE and controls, Mann–Whitney U-test with Bonferroni correction, generalized linear modeling and receiver operating characteristics (ROC) analysis were performed. K-means clustering was used to identify molecular SLE subgroups. We identiﬁed Interferon regulating factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2) and S100 calcium binding protein A12 (S100A12) as the three proteins with the largest fold change between SLE patients and controls (SLE/Control = 1.4, 1.4, and 1.2 respectively). The lowest p-values comparing SLE patients and controls were obtained for S100A12, Matrix metalloproteinase-1 (MMP1) and SLC22A2 (padjusted = 3 × 10−9, 3 × 10−6, and 5 × 10−6 respectively). In a set of 15 potential biomarkers differentiating SLE patients and controls, two of the proteins were transcription factors, i.e., IRF5 and SAM pointed domain containing ETS transcription factor (SPDEF). y, Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States Correspondence: Elisabet Svenungsson elisabet.svenungsson@ki.se †These authors share ﬁrst authorship ‡These authors share senior authorship Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States Correspondence: Elisabet Svenungsson elisabet.svenungsson@ki.se †These authors share ﬁrst authorship ‡These authors share senior authorship Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States 1 Division of Rheumatology, Department of Medicine Solna, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden, 2 SciLifeLab, Division of Afﬁnity Proteomics, Department of Protein Science, KTH Royal Institute of Technology, Stockholm, Sweden, 3 Department of Medical Sciences, Akademiska Hospital, Uppsala University, Uppsala, Sweden, 4 Division of Physiological Chemistry 2, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden, 5 Department of Immunology, Genetics and Pathology, Uppsala University, Uppsala, Sweden, 6 Department of Medical Sciences, Rheumatology, Uppsala University, Uppsala, Sweden, 7 Department of Medical Sciences, Clinical Chemistry, Uppsala University, Uppsala, Sweden, 8 Department of Medical Sciences, Molecular Medicine and Science for Life Laboratory, Uppsala University, Uppsala, Sweden, 9 Science for Life Laboratory, Drug Discovery and Development & School of Engineering Sciences in Chemistry, Biotechnology and Health, KTH Royal Institute of Technology, Stockholm, Sweden Correspondence: Elisabet Svenungsson elisabet.svenungsson@ki.se Specialty section: This article was submitted to Autoimmune and Autoinﬂammatory Disorders, a section of the journal Frontiers in Immunology Received: 14 December 2018 Accepted: 23 April 2019 Published: 17 May 2019 Citation: Idborg H, Zandian A, Ossipova E, Wigren E, Preger C, Mobarrez F, Checa A, Sohrabian A, Pucholt P, Sandling JK, Fernandes-Cerqueira C, Rönnelid J, Oke V, Grosso G, Kvarnström M, Larsson A, Wheelock CE, Syvänen A-C, Rönnblom L, Kultima K, Persson H, Gräslund S, Gunnarsson I, Nilsson P, Svenungsson E and Jakobsson P-J (2019) Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients. Front. Immunol. 10:1029. doi: 10.3389/ﬁmmu.2019.01029 Specialty section: This article was submitted to Autoimmune and Autoinﬂammatory Disorders, a section of the journal Frontiers in Immunology Systemic Lupus Erythematosus (SLE) is a heterogeneous autoimmune disease, which currently lacks speciﬁc diagnostic biomarkers. The diversity within the patients obstructs clinical trials but may also reﬂect differences in underlying pathogenesis. Our objective was to obtain protein proﬁles to identify potential general biomarkers of SLE and to determine molecular subgroups within SLE for patient stratiﬁcation. y, Reviewed by: Carla Marie Cuda, Northwestern University, United States Betsy J. Barnes, Feinstein Institute for Medical Research, United States Correspondence: Elisabet Svenungsson elisabet.svenungsson@ki.se †These authors share ﬁrst authorship ‡These authors share senior authorship IRF5 was up-regulated while SPDEF was found to be Received: 14 December 2018 Accepted: 23 April 2019 Published: 17 May 2019 Helena Idborg 1†, Arash Zandian 2†, Elena Ossipova 1, Edvard Wigren 1, Charlotta Preger 1, Fariborz Mobarrez 1,3, Antonio Checa 4, Azita Sohrabian 5, Pascal Pucholt 6, Johanna K. Sandling 6, Cátia Fernandes-Cerqueira 1, Johan Rönnelid 5, Vilija Oke 1, Giorgia Grosso 1, Marika Kvarnström 1, Anders Larsson 7, Craig E. Wheelock 4, Ann-Christine Syvänen 8, Lars Rönnblom 6, Kim Kultima 7, Helena Persson 9, Susanne Gräslund 1, Iva Gunnarsson 1, Peter Nilsson 2, Elisabet Svenungsson 1‡ and Per-Johan Jakobsson 1‡ Edited by: Edited by: David Stephen Pisetsky, Duke University, United States INTRODUCTION division of SLE patients. In addition, experimental validation of biomarker candidates discriminating between SLE and control was performed. Our main focus was to identify molecular subgroups in SLE since these, despite similar clinical phenotypes, may beneﬁt from diﬀerent treatment perspectives. Systemic Lupus Erythematosus (SLE) is a heterogeneous systemic autoimmune disorder with a plethora of clinical manifestations. Clinical and immunological criteria, deﬁned by the American College of Rheumatology (ACR) (1), are used to classify the disease for research purposes, but reliable diagnostic biomarkers are lacking. The diversity of the disease is a great obstacle and might reﬂect diﬀerences in pathogenesis between diﬀerent subgroups. Several recent reviews highlight the importance of deﬁning subgroups of SLE to better treat patients with tailored medicine, and in order to increase eﬃcacy in clinical trials (2–5). Accordingly, there is a great need for exploring subgrouping and novel diagnostic biomarkers in SLE. Citation: Idborg H, Zandian A, Ossipova E, Wigren E, Preger C, Mobarrez F, Checa A, Sohrabian A, Pucholt P, Sandling JK, Fernandes-Cerqueira C, Rönnelid J, Oke V, Grosso G, Kvarnström M, Larsson A, Wheelock CE, Syvänen A-C, Rönnblom L, Kultima K, Persson H, Gräslund S, Gunnarsson I, Nilsson P, Svenungsson E and Jakobsson P-J (2019) Circulating Levels of Interferon Regulatory Factor-5 Associates With Subgroups of Systemic Lupus Erythematosus Patients. Front. Immunol. 10:1029. doi: 10.3389/ﬁmmu.2019.01029 May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 1 Idborg et al. SLE Subgroups Differ in IRF5 down-regulated in SLE patients. Unsupervised clustering of all investigated proteins identiﬁed three molecular subgroups among SLE patients, characterized by (1) high levels of rheumatoid factor-IgM, (2) low IRF5, and (3) high IRF5. IRF5 expressing microparticles were analyzed by ﬂow cytometry in a subset of patients to conﬁrm the presence of IRF5 in plasma and detection of extracellular IRF5 was further conﬁrmed by immunoprecipitation-mass spectrometry (IP-MS). Interestingly IRF5, a known genetic risk factor for SLE, was detected extracellularly and suggested by unsupervised clustering analysis to differentiate between SLE subgroups. Our results imply a set of circulating molecules as markers of possible pathogenic importance in SLE. We believe that these ﬁndings could be of relevance for understanding the pathogenesis and diversity of SLE, as well as for selection of patients in clinical trials. Keywords: Interferon regulating factor 5 (IRF5), antibody suspension bead arrays, subgroups, biomarker discovery, plasma proteomics, unsupervised clustering, hierarchical clustering, SLE - Systemic Lupus Erythematous Keywords: Interferon regulating factor 5 (IRF5), antibody suspension bead arrays, subgroups, biomarker discovery, plasma proteomics, unsupervised clustering, hierarchical clustering, SLE - Systemic Lupus Erythematous MATERIALS AND METHODS Plasma protein proﬁles were obtained for SLE patients and controls utilizing antibody suspension bead arrays for protein proﬁling. An overview of the study design can be found in Figure 1. Frontiers in Immunology \| www.frontiersin.org Patient Cohort and Controls Few biomarkers have been implemented in clinical routine reﬂecting the diﬃculties of biomarker research in lupus (6). Screening of a large number of proteins (>50) but in a limited number (<50) of SLE patients have been performed to identify biomarkers in SLE (7–10). In this study we analyzed 281 proteins using a suspension bead aﬃnity proteomics approach (11), in plasma samples from a total of 695 individuals comprising SLE and matched controls. Selection of proteins is crucial to obtain representative protein proﬁles. However, the current knowledge of protein functions is far from complete and transcription factors and other nuclear molecules could have unknown functions in the circulation or may, regardless of function, constitute novel biomarkers. The intra- and extracellular functions of a protein might be diﬀerent and unconventional secretion is also possible (12). Therefore, both nuclear and cytoplasmic molecules are relevant to study in the circulation with the aim to identify potential biomarkers and possible pathogenic pathways. Fasting plasma samples were obtained from patients in the Karolinska SLE cohort consisting of 379 SLE patients and 316 population-based controls with matching age, gender and residential area. All SLE patients included in this cross-sectional study, were adults and diagnosed according to the ACR SLE criteria (1). Both patients and controls underwent a structured interview and physical examination as previously described (14). Clinical and serological data for the SLE patients are summarized in Table 1 and in previous work (13). Medication is reported in Supplementary Table S-1, and demographic data for the controls are shown in Supplementary Table S-2. Protein Proﬁling by Antibody Suspension Bead Arrays y A number of 281 proteins were selected as previously described (13), i.e., based on published data on suggested biomarkers in inﬂammation/SLE/myositis, microarray data comparing SLE and controls and an untargeted mass spectrometry-based proteomic analysis suggesting additional biomarker candidates. A customized set of 367 antibodies (Supplementary Table S-3) was utilized to target unique epitopes of these proteins in a screening experiment (Figure 1B) (13). The antibodies were selected from the Human Protein Atlas (HPA, www.proteinatlas. org) project and are aﬃnity-puriﬁed polyclonal antibodies that In a previous study we presented protein proﬁles for two predeﬁned SLE subgroups, delineated based exclusively on the autoantibody proﬁles, but also corresponding to clinical observations and experience (13). In the present study we used a diﬀerent approach and performed unsupervised clustering of the obtained protein proﬁles to investigate an unprejudiced May 2019 \| Volume 10 \| Article 1029 2 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 1 \| Overview of the experimental workﬂow. Plasma samples (A) were randomized in set 1 and set 2 for screening phase (B) followed by validation phase (C). Data were analyzed to investigate SLE subgroups (D) as well as comparing SLE and control in a multivariate (E) and univariate (F) manner, respectively, and main results can be viewed in the referred ﬁgures. FIGURE 1 \| Overview of the experimental workﬂow. Plasma samples (A) were randomized in set 1 and set 2 for screening phase (B) followed by validation phase (C). Data were analyzed to investigate SLE subgroups (D) as well as comparing SLE and control in a multivariate (E) and univariate (F) manner, respectively, and main results can be viewed in the referred ﬁgures. have been extensively validated (17). Protein proﬁles were generated using antibody suspension bead array (18). In brief, the 367 HPA antibodies were attached to color-coded magnetic beads, then incubated with 45 µl diluted and biotinylated EDTA- plasma, followed by an addition of streptavidin-conjugated R-phycoerythrin (Invitrogen), and ﬁnally analyzed using a FlexMap3D instrument (Luminex Corp.). Data was evaluated as described below and 50 proteins (53 antibodies) were selected for further validation experiments (Figure 1C). In the validation experiment, additional HPA antibodies (n = 80) targeting other antigenic regions of these proteins were coupled to beads resulting in a validation assay of 133 antibodies toward the selected 50 proteins (Supplementary Table S-4). excluded from further analysis. Protein Proﬁling by Antibody Suspension Bead Arrays Subsequently, probabilistic quotient normalization (PQN) was performed on the MFIs to compensate for dilution errors and/or total amount of plasma proteins of the samples (21), followed by LOESS normalization on MA coordinates, per antibody, based on the MFIs to minimize the batch eﬀects (22). Data quality was assessed by comparing replicates per 96-well plate, in combined 384-well plates and inter 384-well plates. Thereafter the data was split into two separate but comparable datasets (Figure 1B) with similar age and gender distribution and equal number of SLE patients and controls (Supplementary Table S-2). Set 1 consisted of 190 SLE patients and 158 controls, and set 2 of 189 SLE patients and 158 controls. This data is referred to as the data from the screening phase. Proteins reaching signiﬁcance (after Bonferroni correction) comparing SLE and control, with the same direction in fold change between SLE/control, in both sample set 1 and set 2 in screening phase, were selected for validation (Figure 1C, n = 50). The validated proteins that were signiﬁcantly diﬀerent comparing SLE and controls (n = 15), were used for further interpretation. A generalized linear model with lasso regularization (R package: glmnet) Data Analysis of Antibody Suspension Bead Array Data The measured signals, reported as median ﬂuorescent intensities (MFI) from FlexMap3D were imported into R (19). As previously described (20), outliers were identiﬁed in the raw data by robust principal component analysis (R package: rrcov) and May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 3 SLE Subgroups Differ in IRF5 Idborg et al. TABLE 1 \| Clinical and serological data are reported for the three molecular subgroups as well as for the entire cohort of SLE patients. Entire SLE cohorta Molecular SLE subgroupsa Comparing SLE subgroupsb n = 357 RF- IgM/SSA/SSB subgroup N = 51 IRF5 low subgroup N = 129 IRF5 high subgroup N = 177 RF- IgM/SSA/SSB vs. IRF5 low RF- IgM/SSA/SSB vs. IRF5 high IRF5 low vs. Data Analysis of Antibody Suspension Bead Array Data IRF5 high Age (years) 47.2 (34.3–58.1) 45.4 (33.6–56.8) 41.4 (31.1–54.2) 51.0 (37.8–60.3) P = 0.50 P = 0.07 p = 0.0003 Gender %F 87% 90% 87% 86% 0.62 0.65 0.92 Disease duration (years) 11.5 (4.4–21.7) 6.9 (1.5–14.4) 11.6 (4.5–20.6) 12.9 (5.3–23.2) P = 0.06 P = 0.005 P = 0.19 SLE ACR criteria 6 (5–7) 6 (5–7) 6 (5–7) 6 (5–7) P = 0.81 P = 0.43 P = 0.18 SLAM 6 (4–10) 8 (5–12) 6 (3.5–9.5) 6 (3.5–9.5) P = 0.02 P = 0.02 P = 0.90 SLEDAI-2k 4 (0–7) 4 (1–7) 3 (0.5–7.5) 4 (0–7) P = 0.74 P = 0.70 P = 0.96 C3a Kruskal–Wallis test p < 0.0001 268.4 (192.7–537.1) 351.8 (243.2–991.4) 434.8 (181.7–3092) 250.9 (191.3–324.2) P = 0.83 P < 0.0001 P < 0.0001 RF IgA (IU/ml) Kruskal–Wallis test p < 0.0001 5.3 (3.4–12) 16.6 (5.6–66.9) 4.1 (2.8–7.3) 5.7 (3.7–11.2) P < 0.0001 P = 0.0001 P = 0.0005 RF IgG (µg/ml) Kruskal–Wallis test p = 0.0001 11 (6.9–23) 20 (8.9–54.6) 10 (6.5–17.5) 11 (6.8–19.2) P < 0.0001 P = 0.0004 P = 0.23 RF IgM (IU/ml) Kruskal–Wallis test p < 0.0001 1.3 (0.63–4.7) 28 (13.5–44.6) 1.1 (0.6–2.4) 1.1 (0.5–2.1) P < 0.0001 P < 0.0001 P = 0.67 IgA total (mg/ml) 2.8 (2–3.9) 3.1 (2.1–4.2) 2.7 (1.9–3.6) 2.9 (2–3.9) P = 0.08 0.38 P = 0.15 IgG total (mg/ml) 12.8 (10.4–16.6) 16.7 (12.7–20.6) 11.7 (9.5–14.7) 12.8 (10.3–16.1) P < 0.0001 P < 0.0001 P = 0.05 IgM total (mg/ml) 0.92 (0.58–1.4) 1.2 (0.92–2.1) 0.96 (0.62–1.40) 0.8 (0.49–1.3) P = 0.0017 P < 0.0001 P = 0.03 ESR (mm/hour) 19 (11–33) 30 (16.5–46) 14 (9–27) 21 (12–36) P < 0.0001 P = 0.04 P = 0.0004 hsCRP (mg/l) 1.7 (0.68–5.3) 1.4 (0.51–5.7) 1.1 (0.48–4.7) 2.2 (0.83–5.8) P = 0.28 P = 0.18 P = 0.0003 Fibrinogen (g/l) 4.1 (3.4–5.0) 3.9 (3.1–4.6) 3.8 (3.2–4.8) 4.4 (3.6–5.2) P = 0.95 P = 0.006 P = 0.0005 TNF-α (pg/ml) 4.5 (3.3–6.2) 4.8 (3.5–6.7) 4.0 (2.8–5.7) 5.1 (3.6–6.4) P = 0.015 P = 0.77 P = 0.0005 Fibronectin (mg/ml) Kruskal–Wallis test p = 0.0008 0.38 (0.25–0.46) 0.40 (0.29–0.50) N = 33 0.41 (0.32–0.48) N = 80 0.31 (0.19–0.44) N = 95 P = 0.78 P = 0.03 P = 0.0002 Leptin (mg/ml) Kruskal–Wallis test p = 0.0002 14294 (4776–27938) 13321 (5026–21162) 7878 (2240–20389) 19502 (8474–48617) P = 0.23 P = 0.05 P < 0.0001 SLE American College of Rheumatology (ACR) classiﬁcation criteria; SLAM, SLE Activity Measure (15); SLEDAI-2K, SLE Disease Activity Index (16). Data Analysis of Antibody Suspension Bead Array Data C3, Complement factor 3, RF IgA/G/M, Rheumatoid factor immunoglobulin A/G/M; ERS, erythrocyte sedimentation rate; hsCRP, high sensitivity C-reactive protein, aMedian (25% quantile - 75% quantile), NR, not reported. Serology data obtained as described in previous work. bMann–Whitney U-test for pairwise comparison of subgroups was used to characterize subgroups. P-values <0.001 without adjustment for multiple testing are highlighted in bold. Kruskal–Wallist test, i.e., comparing more than two groups and compensating for multiple testing, highlighted only RF-IgM, RF-IgG, RF-IgA, Leptin, Fibronectin and C3a as signiﬁcantly different (names highlighted in italic). LE 1 \| Clinical and serological data are reported for the three molecular subgroups as well as for the entire cohort of SLE patients. Comparing SLE subgroupsb SLE American College of Rheumatology (ACR) classiﬁcation criteria; SLAM, SLE Activity Measure (15); SLEDAI-2K, SLE Disease Activity Index (16). C3, Complement factor 3, RF IgA/G/M, Rheumatoid factor immunoglobulin A/G/M; ERS, erythrocyte sedimentation rate; hsCRP, high sensitivity C-reactive protein, aMedian (25% quantile - 75% quantile), NR, not reported. Serology data obtained as described in previous work. bMann–Whitney U-test for pairwise comparison of subgroups was used to characterize subgroups. P-values <0.001 without adjustment for multiple testing are highlighted in bold. Kruskal–Wallist test, i.e., comparing more than two groups and compensating for multiple testing, highlighted only RF-IgM, RF-IgG, RF-IgA, Leptin, Fibronectin and C3a as signiﬁcantly different (names highlighted in italic). clustering, emphasizing on the variables with greatest variance and the Calinski-Harabasz criterion was used to ﬁnd the number of clusters in the data. was used to ﬁnd panels of proteins to predict SLE patients and controls where the sample set 1 and set 2 corresponded to test set and training set, respectively. This was followed by analysis and visualization by performing receiver operating characteristic (ROC) analysis (R package: pROC) and conﬁdence intervals (CI) for the area under the curve (AUC) were calculated (23). Production of Recombinant IRF5 Protein Multiple constructs of IRF5 (Uniprot ID Q13568) were sub-cloned into the expression vectors pNIC28-Bsa4 and pNIC-Bio3 (Genbank acc. No EF198106, JN792439). After performing small-scale screening for soluble recombinant protein expression as previously described (24), clones corresponding to constructs covering the regions M1-V120 and E232-L434 were selected for generation of single-chain fragment variable (scFv) binders. Frontiers in Immunology \| www.frontiersin.org Generation of Antibody Fragments Against IRF5 5 Single-chain fragment variable (scFv) clone J-IRF5-5 was generated by phage display technology using a human synthetic library denoted SciLifeLib. The phage selection procedure was performed basically as described earlier (27), but the ﬁrst round of selection, including the steps of antigen-phage incubation to trypsin elution, was carried out in 1.5 ml tubes on a rotator with no automation. The number of washing steps was modiﬁed and increased with succeeding selection rounds; ﬁve in round one and seven in round four. Also, the recovered phages were propagated in XL1-Blue E. coli between the selection rounds. Re-cloning of the selected material in pool followed by transformation into TOP10 E. coli, small-scale expression of 94 randomly picked scFv and subsequent enzyme-linked immunosorbent assay (ELISA) for detection of recombinant full-length IRF5, i.e., verifying binding to target, and sequencing experiments were performed equivalent to previously reported (27). Aﬃnity measurements were performed using a Biacore T200 biosensor instrument (GE Healthcare) as described in Supplementary Figure S-1. The top candidate (J-IRF5-5), binding the construct region E232-L434 of IRF5, obtained a measured aﬃnity of 5 nM. Validation by ELISA and Biacore was extended by using IP-MS performed on cell lysate from HEK293 cells (300 µl) as previously described (27). The lysate was spiked with a small amount of recombinant IRF5 full-length protein (0.7 µg), as IRF5 is normally expressed at very low levels in HEK293 cells, and MS data was acquired in data dependent mode using a top 10 method. IRF5 was identiﬁed as the highest ranked protein in the obtained list of proteins (Supplementary Table S-5). This veriﬁes that the antibody can capture its target in a complex mixture. The top candidate, J-IRF5-5, was then used in IP-MS on plasma samples as described below. Peptides were separated using an Ultimate 3000 RSLCnano system. Samples were trapped on an Acclaim PepMap nanotrap column (C18, 3 µm, 100 Å, 75 µm × 20 mm), separated on an NanoEaseTM M/Z HSS column (C18, 1.8 µm, 100Å, 75 µm × 250 mm), (Thermo Scientiﬁc) and analyzed on a Q Exactive Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Fisher Scientiﬁc, San Jose, CA, USA). Peptides were separated using a gradient of A (3% ACN, 0.1% FA) and B (95% ACN, 0.1% FA), ranging from 3 to 40% B in 50 min with a ﬂow of 0.25 µl/min. Immunoprecipitation Followed by Mass Spectrometry (IP-MS) of IRF5 in Plasma Spectrometry (IP MS) of IRF5 in Plasma Heparin-plasma from a myositis patient and two SLE patients recruited at Karolinska University Hospital were analyzed by IP-MS as previously described (28) with a few adjustments. In brief, to an aliquot of 100 µl plasma, 400 µl of lysis buﬀer (1 mM Tris-HCl, 42 mM NaCl, and 0.01% NP-40 in water, pH 7.9) containing protease inhibitor cocktail (Roche) was added. Recombinant IRF5 protein (0.7 µg) was added to plasma and used as a positive control. An aliquot of 4 µg J-IRF5-5 was added to plasma samples and incubated overnight at 4◦C. As negative controls a scFv antibody, generated in the same way as J-IRF5-5 but targeting an unrelated antigen, was added to plasma and to another vial, J-IRF5-5 was added to a sample without plasma (lysis buﬀer only). Forty microliters of anti-FLAG M2 magnetic beads was added and incubated 2–5 h at 4◦C. The beads were washed three times (5–10 min in 4◦C) with low salt buﬀer (1 mM Tris-HCl, 10 mM NaCl, and 0.01% NP-40 in water, pH Data Analysis of Antibody Suspension Bead Array Data Expression and puriﬁcation of selected clones and full-length IRF5 was performed essentially as previously described (25, 26), and a detailed protocol can be In order to identify molecular SLE subgroups, unsupervised clustering was applied on the screening data. To prepare the data for principal component analysis (PCA), the data for each dataset (190 SLE patients in set 1 and 189 SLE patients in set 2) was log2-transformed and centered on the mean of each antibody. In set 1 PC1 and PC2 explained 14 and 12% respectively of the variance, and in set 2 the explained variances by PC1 and PC2 were 18 and 16% respectively. Clustering of samples was done on the ﬁrst two principal components by using K-means May 2019 \| Volume 10 \| Article 1029 4 SLE Subgroups Differ in IRF5 Idborg et al. found in Supplementary Methods and Results. Final protein batches were analyzed by SDS-PAGE and subsequently ﬂash frozen in liquid nitrogen and stored at −80◦C until use. 7.9) and two times with low salt buﬀer without NP-40. Elution was performed by 2 × 100 µl 0.5 M ammonium hydroxide and evaporated in Speedvac. Samples were reconstituted in 50 mM ammonium bicarbonate and subsequently reduced by 1 µl of 100 mM TCEP-HCl at 37◦C for 1 h, alkylated by 1 µl of 500 mM iodoacetamide in dark for 45 min and digested using 1 µg trypsin at 37◦C. Sample clean-up was performed in 50 mM ammonium bicarbonate using HiPPRTM Detergent Removal Spin Column Kit according to manufacturer’s instructions. Obtained peptide samples were desalted using ZipTip R⃝pipette tips or Pierce C18 Tips (Thermo Scientiﬁc) prior mass spectrometry analysis. A standard of IRF5 peptides were generated using full length IRF5 recombinant protein applying the same digestion protocol. Generation of Antibody Fragments Against IRF5 The Q Exactive was operated in a data dependent manner utilizing targeted SIM/ddMS2 method with an inclusion list containing masses corresponding to four unique IRF5 peptides. The survey scan was performed at 70.000 resolution from 400 to 1,200 m/z, with a max injection time of 100 ms and target of 1 × 106 ions. For generation of HCD fragmentation spectra, a max ion injection time of 250 ms and Automated Gain Control (AGC) of 3 × 106 were used before fragmentation at 30% normalized collision energy. Detection of IRF5 Positive Microparticles in Plasma in Plasma In another set of SLE patients (n = 63), citrate plasma was analyzed for detection of microparticles (MPs) expressing IRF5. Characteristics of these SLE patients and details about the sample collection can be found in Supplementary Methods and Results. Healthy controls (n = 20) matched for age and gender to the SLE patients were also included in this study. Platelet-poor plasma were centrifuged (2,000 g for 20 min followed by 13,000 g for 2 min) and the supernatants were then incubated with polyclonal anti-IRF5-Fluorescein isothiocyanate (FITC) (Biorbyt, UK) as described in Supplementary Methods and Results. MPs were measured by ﬂow cytometry on a Beckman Gallios instrument (Beckman Coulter, Bream CA, USA) and were deﬁned as particles between ∼0.3 µm and 0.9 µm in size. Frontiers in Immunology \| www.frontiersin.org General Biomarker Candidates of SLE General Biomarker Candidates of SLE Fifty-three antibodies, targeting 50 proteins, showed signiﬁcant diﬀerences between SLE patients and controls in both sample set 1 and set 2, i.e., in two separate experiments performed in parallel containing samples from diﬀerent patients/controls. In the following validation experiment, the plasma samples (n = 695) were analyzed using 133 antibodies targeting the 50 selected proteins. Protein proﬁles with low correlation (Spearman’s rho < 0.40) to the screening data were removed. The remaining 15 proteins, targeted by 16 antibodies (Table 2, Figure 1F), showed a median correlation to the screening data of rho = 0.78 with a minimum correlation of rho = 0.46. Antibody target sequence for all 15 proteins can be found in Supplementary Table S-6. Lipid Mediators and Cytokines Data Extracted From Related Projects In a previous study, sphingolipids were measured by LC-MS/MS in a selection of patients from our SLE cohort (29). Since one of the proteins characterizing the RF-IgM/SSA/SSB subgroup was ceramide synthase 5 (CERS5), which catalyzes the formation of C16 : 0-ceramide, we utilized data from this study where C16 : 0- ceramide was quantiﬁed. Among the analyzed patients (with data available from both C16 : 0-ceramide and antibody suspension bead array data), 16 patients were found to belong to the RF- IgM/SSA/SSB subgroup, 39 to IRF5 low subgroup and 44 patients belonged to the IRF5 high subgroup. p pp y The proteins yielding the largest fold change between SLE patients and controls (p < 0.05), were interferon regulatory factor 5 (IRF5), solute carrier family 22 member 2 (SLC22A2, organic cation transporter 2, OCT2) and S100 calcium binding protein A12 (S100A12, Calgranulin-C) (Figure 2). Of the 15 proteins in Table 2, three were found to be decreased, i.e., sterile alpha motif (SAM) pointed domain containing E26 transformation- speciﬁc (ETS) transcription factor (SPDEF), Apolipoprotein L6 (APOL6), and Cysteine-rich secretory protein 3 (CRISP3), and twelve were found to be increased in the SLE patients compared to controls. Seven of the proteins were classiﬁed as plasma proteins and two proteins, IRF5 and SPDEF, were transcription factors (Supplementary Table S-7). Levels of IRF5 were up- regulated and SPDEF were down-regulated in SLE compared to controls. Previously, 20 cytokines were analyzed in plasma from the entire Karolinska SLE cohort (14), and data from cytokines relevant in inﬂammation, i.e., TNF-α, IL-6, IL-8, Il-10, IL-16, and IP-10, were analyzed with respect to the identiﬁed molecular subgroups in this work. Interferon α (IFN-α) was measured by ELISA in the Karolinska SLE cohort in another study (30), and data was utilized in this work to study levels of IFN-α in IRF5 high and low subgroups. Data on IFN-α was obtained for 66% of the patients in the IRF5 low subgroup and in 70% of the patients in IRF5 high subgroup. Values below limit of quantiﬁcation (LOQ) was set to LOQ/2. Detection of IRF5 in Plasma by Sandwich ELISA Nunc immobilizer amino plates (Thermo scientiﬁc) were coated with commercial mouse anti-human IRF5, antibody targeting May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org Frontiers in Immunology \| www.frontiersin.org 5 SLE Subgroups Differ in IRF5 Idborg et al. aa 176–240 (Antibodies-online.com, ABIN121152). A standard curve was obtained using recombinant IRF5 protein at 0.156, 0.312, 0.625, 1.25, 2.5, 5.0, and 10 ng/ml (50 µl/well). Plasma samples from 25 SLE patients and 14 controls were diluted 1:2 in 0.1% BSA/PBS before adding 50 µl per well. As a secondary antibody, rabbit anti-human IRF5 (HPA046700, i.e., the antibody used in the antibody suspension bead array) was used. Donkey anti-rabbit IgG HRP-conjugated antibody was added for detection using TMB substrate and optical density was read at 450 nm. of antibody suspension bead array data, linear modeling and K-means clustering can be found in section Data Analysis of Antibody Suspension Bead Array Data. Calculations were performed using R (19), GraphPad Prism 7 and Excel 2016. IRF5 protein QTL analysis was performed on log10 transformed IRF5 protein levels using linear regression in R assuming an additive genetic model. Genetic Data on IRF5 The Karolinska SLE cohort had previously been genotyped using the Immunochip Illumina Inﬁnium Assay (31, 32). Two previously reported independent IRF5 SLE risk variants rs4728142 and rs10488631 (a proxy to rs35000415) were selected from this data for association with IRF5 protein levels in quantitative trait locus (QTL) analyses. Genotype data was available for 253 SLE patients and 280 controls. Proteins that showed signiﬁcant diﬀerences between SLE patients and controls (Table 2), were used to create a linear model (Figure 1E). Obtained model suggested a biomarker panel of 9 antibodies, targeting 8 proteins, i.e., GTP-binding protein Rhes (RASD2), S100A12, SLC22A2, Matrix metalloproteinase-1 (MMP1), CRISP3, complement component C6 (C6), Phospholipid phosphatase 1 (PPAP2A), SPDEF, achieving a ROC AUC of 0.78 (95% CI: 0.73–0.83) for prediction of SLE patients and controls (Figure 2). In comparison, the highest achieved AUC from a single protein was 0.73 (95% CI: 0.67–0.78) for MMP1 and 0.72 (95% CI: 0.66–0.77) for S100A12, and a panel of three proteins (S100A12, SLC22A2, and PPAP2A) yielding an AUC of 0.74 (95% CI: 0.69–0.80). This panel of 8 proteins is suggested as general biomarker candidates to diﬀerentiate between SLE and controls, independently of SLE subgroups. Frontiers in Immunology \| www.frontiersin.org Statistics For comparison between SLE and controls Mann-Whitney U-test was used. Bonferroni-corrected p-value at a threshold of 0.05 was used as a measure of signiﬁcance unless otherwise stated. When comparing three or more groups, i.e., when comparing the three molecular subgroups, Kruskal Wallis test or Fisher’s exact test (for categorical data) was used. In addition, in Table 1, Mann– Whitney U-test have been used for independent comparisons between molecular subgroups in favor for scientiﬁc reasoning of selected variables with cautious interpretation of p-values (33, 34). Spearman rank correlation was used to investigate correlations between variables. Additional details about analysis Applying strict statistical univariate analysis (Bonferroni correction) only two associations were identiﬁed between proteins and clinical data (i.e., serological data, clinical symptoms, disease activity scores). Lower levels of S100A12 were May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 6 SLE Subgroups Differ in IRF5 Idborg et al. TABLE 2 \| The 15 proteins (16 antibodies) differentially expressed comparing SLE and control. Protein name short Full protein name UniProta Correlation screening vs. validationb Fold change (SLE/Ctrl) p-value (SLE vs. Ctrl)c IRF5 Interferon regulatory factor 5 Q13568 0.96 0.48 4.5E-02 SLC22A2* Solute carrier family 22 (organic cation transporter), member 2 O15244 0.8 0.44 4.6E-06 S100A12* S100 calcium binding protein A12 P80511 0.77 0.28 3.3E-09 RASD2* GTP-binding protein Rhes Q96D21 0.86 0.26 1.7E-05 NOS3 Nitric oxide synthase 3 (endothelial) P29474 0.93 0.26 4.1E-02 MMP1* Matrix metallopeptidase 1 (or interstitial collagenase) P03956 0.63 0.17 3.2E-06 SPDEF* SAM pointed domain containing ETS transcription factor O95238 0.87 −0.14 1.3E-02 UBAC1 UBA domain containing 1 Q9BSL1 0.71 0.13 1.4E-04 TRIM33 Tripartite motif containing 33 Q9UPN9 0.84 0.13 3.0E-03 CFI Complement factor I P05156 0.65 0.13 2.9E-02 APOL6 Apolipoprotein L, 6 Q9BWW8 0.84 −0.13 4.5E-02 PPAP2A* Phosphatidic acid phosphatase type 2A (or Phospholipid phosphatase 1) O14494 0.82 0.12 9.9E-03 GRAP2 GRB2-related adaptor protein 2 O75791 0.69 0.11 3.4E-03 CRISP3* Cysteine-rich secretory protein 3 P54108 0.75 −0.10 5.5E-04 CRISP3* Cysteine-rich secretory protein 3 P54108 0.46 −0.10 1.8E-03 C6* Complement component 6 P13671 0.68 0.10 3.7E-03 Proteins are sorted based on log-fold change between SLE samples and controls. Proteins included in suggested biomarker panel are indicated by an asterisk (“”). aProtein ID in UniProt (35). bThe Speaman’s rho correlation coefﬁcients for screening and validation data are reported. cThe highest Bonferroni-corrected p-value among set 1 and set 2 comparing SLE and Controls is reported. Statistics NOS3, and interleukin-2 receptor subunit alpha (IL2RA) and is referred to as the IRF5 high subgroup. Levels of IRF5 in the three subgroups as well as in the two sample sets are shown in Figure 3D. associated in patients with a history of lupus nephritis, i.e., “nephritis ever” deﬁned by ACR criteria (median signal of 1591 vs. 1409, with IQR of 613 vs. 583, and Bonferroni-adjusted Mann-Whitney U-test p-value of 0.008) and IRF5 protein levels showed a weak negative correlation to C3a plasma concentration in SLE patients (Spearman’s rho = −0.32, p < 0.0001). Including all available clinical and serological data, considering associations comparing all three molecular subgroups (Kruskal–Wallis test), only rheumatoid factor (RF)-IgM, RF-IgG, RF-IgA, Leptin, Fibronectin, and C3a were found to be signiﬁcantly diﬀerent (p < 0.05) in at least one subgroup after correction for multiple testing (Supplementary Figure S-2). Molecular subgroup 1 was found to have high RF-IgM levels (Figure 4) as well as high levels of autoantibodies toward Sjögren’s Syndrom antigen A/B (SSA/SSB) (Supplementary Table S-8). This subgroup is further referred to as the RF-IgM/SSA/SSB subgroup. We also observed higher levels of RF-IgG and RF-IgA as well as higher levels of total IgG and IgM in this subgroup (Table 1, Supplementary Figure S-2). In addition, this subgroup showed higher frequency (45%) of patients with secondary Sjögren’s syndrome (sSS), as deﬁned according to the American-European Consensus criteria (36), compared to the IRF5 high and low subgroup (both 19%) (Supplementary Table S-9). Patients in the RF-IgM/SSA/SSB subgroup showed lower frequency of nephritis (20%) compared to IRF5 low (43%) and IRF5 high (48%) subgroups. Lower ESR were reported for the IRF5 low subgroup (Table 1). The IRF5 high subgroup was slightly older compared to other subgroups, showed lower levels of C3a and increased levels of inﬂammatory markers e.g., TNF-α, ﬁbrinogen and hsCRP (Table 1). Frontiers in Immunology \| www.frontiersin.org SLE Molecular Subgroups Unsupervised clustering of the 281 analyzed proteins (screening data, K-means clustering) was performed to ﬁnd potential molecular subgroups among the SLE patients (Figure 1D). Three distinct clusters were obtained in both experimental set 1 (Figure 3A) and set 2 (Figure 3B) and nine of the 10 proteins with the highest absolute PCA loadings were identical between the two sets. These 9 proteins were evaluated by analyzing the median protein levels and revealed concordant protein proﬁles between the sets (Figure 3C). This panel of biomarker candidates can be used to diﬀerentiate between suggested molecular subgroups. Molecular subgroup 1 (red, n = 51) showed higher levels of E-selectin (SELE), solute carrier family 22 (SLC22A2), Ceramide synthase 5 (CERS5) and Integrin subunit beta 1 (ITGB1, Glycoprotein IIA, CD29). Molecular subgroup 2 (green, n = 129) showed lower levels of IRF5, Ubiquitin-like protein ISG15 (ISG15), endothelial nitric oxide synthase (NOS3) and SLC22A2, and is further referred to as the IRF5 low subgroup. This subgroup was found to be similar to the control group (gray) as shown in Figure 3C. Molecular subgroup 3 (blue, n = 177) showed higher levels of IRF5, ISG15, May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 7 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 2 \| General biomarker candidates of SLE. Proteins showing the highest absolute fold change between SLE patients and controls (in both sample set 1 and 2) were (A) Interferon regulatory factor 5 (IRF5), (B) Solute carrier family 22 member 2 (SLC22A2) and (C) S100 calcium binding protein A12 (S100A12). A panel of 8 proteins, consisting of 9 antibodies proved to be the best panel for classifying SLE patients from controls. The panel of 8 proteins consist of RASD2, S100A12, SLC22A2, MMP1, CRISP3, C6, PPAP2A, and SPDEF and achieved an ROC AUC of 0.78 for the prediction of SLE patients and controls (D). FIGURE 2 \| General biomarker candidates of SLE. Proteins showing the highest absolute fold change between SLE patients and controls (in both sample set 1 and 2) were (A) Interferon regulatory factor 5 (IRF5), (B) Solute carrier family 22 member 2 (SLC22A2) and (C) S100 calcium binding protein A12 (S100A12). A panel of 8 proteins, consisting of 9 antibodies proved to be the best panel for classifying SLE patients from controls. SLE Molecular Subgroups The panel of 8 proteins consist of RASD2, S100A12, SLC22A2, MMP1, CRISP3, C6, PPAP2A, and SPDEF and achieved an ROC AUC of 0.78 for the prediction of SLE patients and controls (D). CERS5 was, as mentioned, increased in the RF-IgM/SSA/SSB subgroup, and is an enzyme catalyzing the formation of C16 : 0- ceramide. We have previously quantiﬁed levels of sphingolipids in SLE (29) and data was available for a selection of patients. C16 : 0-ceramide levels were 415 ± 143 nM (mean ± SD) in RF- IgM/SSA/SSB subgroup (n = 16), 305 ± 79 nM in IRF5 low subgroup (n = 39) and 331 ± 84 nM in IRF5 high subgroup 3 (n = 44) (Supplementary Figure S-3) (Kruskal–Wallis test p = 0.02) supporting our ﬁnding of higher levels of CERS5 in RF-IgM/SSA/SSB subgroup. CERS5 was, as mentioned, increased in the RF-IgM/SSA/SSB subgroup, and is an enzyme catalyzing the formation of C16 : 0- ceramide. We have previously quantiﬁed levels of sphingolipids in SLE (29) and data was available for a selection of patients. C16 : 0-ceramide levels were 415 ± 143 nM (mean ± SD) in RF- IgM/SSA/SSB subgroup (n = 16), 305 ± 79 nM in IRF5 low subgroup (n = 39) and 331 ± 84 nM in IRF5 high subgroup 3 (n = 44) (Supplementary Figure S-3) (Kruskal–Wallis test p = 0.02) supporting our ﬁnding of higher levels of CERS5 in RF-IgM/SSA/SSB subgroup. peaks corresponding to IRF5 were detected in the blank samples and no carry-over was observed between runs. MS/MS spectra of two of the peptides detected in plasma from a SLE patient is shown in Figure 5. IRF5 could repeatedly be detected in plasma aliquots from a myositis patient using IP-MS utilizing peptide exact mass (high-resolution m/z) and retention time. Levels were close to detection limit and fragment spectra of IRF5 peptides could not always be obtained although aliquots from the same sample were analyzed. Adding the criteria of reporting fragmentation spectra of the unique peptides, IRF5 was detected in two out of three separate experiments, not detected in one SLE patient and for the second SLE patient fragment spectra could be obtained in one out of two experiments. IP-MS, as used here, is not a quantitative method and the capture of IRF5 might slightly diﬀer between experiments and not reach detection limit. Therefore, this is not the method IRF5 in Plasma To conﬁrm the presence of IRF5 in plasma immunoprecipitation tandem mass spectrometry (IP-MS/MS) was used. The targeted MS/MS method was optimized for four unique tryptic IRF5 peptides using recombinant IRF5 protein as a standard. No May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 8 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 3 \| SLE molecular subgroups. K-means clustering, visualized on the two ﬁrst principal components (PC1 and PC2), identiﬁed three subgroups (1-red, 2-green and 3-blue) in sample set 1 (A) and set 2 (B) with a similar clustering pattern. The relative protein proﬁles (C) of the 9 proteins with the highest loadings in both sample sets for the RF-IgM/SSA/SSB (red, n = 51), the IRF5 low (green, n = 129) and the IRF5 high (blue, n = 177) molecular subgroups are shown and both sample set 1 (solid line) and set 2 (dashed line) shows concordant protein proﬁles. It is evident that the IRF5 high subgroup discriminate from the IRF5 low subgroup based on levels of IRF5, ISG15, and NOS3, while it is evident that the RF-IgM/SSA/SSB subgroup differentiate from the other two in levels of SELE, SLC22A2, CERS5, and ITGB1. Controls are included in gray for comparison but was not included in the clustering. Levels of IRF5 (D) are compared between the three molecular SLE subgroups RF-IgM/SSA/SSB (red), IRF5 low (green), and IRF5 high (blue) subgroup. FIGURE 3 \| SLE molecular subgroups. K-means clustering, visualized on the two ﬁrst principal components (PC1 and PC2), identiﬁed three subgroups (1-red, 2-green and 3-blue) in sample set 1 (A) and set 2 (B) with a similar clustering pattern. The relative protein proﬁles (C) of the 9 proteins with the highest loadings in both sample sets for the RF-IgM/SSA/SSB (red, n = 51), the IRF5 low (green, n = 129) and the IRF5 high (blue, n = 177) molecular subgroups are shown and both sample set 1 (solid line) and set 2 (dashed line) shows concordant protein proﬁles. It is evident that the IRF5 high subgroup discriminate from the IRF5 low subgroup based on levels of IRF5, ISG15, and NOS3, while it is evident that the RF-IgM/SSA/SSB subgroup differentiate from the other two in levels of SELE, SLC22A2, CERS5, and ITGB1. Controls are included in gray for comparison but was not included in the clustering. Frontiers in Immunology \| www.frontiersin.org IRF5 in Plasma The levels of RF-IgM are compared between the three molecular SLE subgroups RF-IgM/SSA/SSB (red), IRF5 low (green), and IRF5 high (blue) subgroup. We identiﬁed a weak positive association between IRF5 protein levels and the number of SLE risk alleles at one of two SNP representing the IRF5 SLE genetic association. However, this eﬀect was not apparent when separating the data from SLE patients and control individuals, thus it could be driven by the allele frequency and protein level diﬀerences between these two groups. This indicates that the IRF5 SLE risk variants are not the sole drivers for the diﬀerences in IRF5 plasma levels that we observe. As recently discussed elsewhere (41), the contribution of IRF5 genetic risk to disease susceptibility is not known, and it is possible that IRF5 may have both a genetic and non- genetic contribution. It is an intriguing and novel ﬁnding that the IRF5 protein occurs in the circulation and that it stands out as a potential biomarker for SLE. The high IRF5 levels in the circulation may reﬂect increased cell death in SLE patients. However, the IRF5 levels also vary to a large extent within the group of SLE patients. In addition, SPDEF, another transcription factor, showed the opposite regulation in SLE plasma (10% decrease) and unless SPDEF is strongly down-regulated in SLE, the diﬀerence in IRF5 cannot solely be explained by increased cell death/loss during apoptotic clearance in patients. Reports of transcription factors in circulation are sparse (42, 43) and by our approach using antibodies designed to target a short linear sequence of the protein, it is not possible to determine if the protein is full-length or represents a splice variant or other modiﬁed product. There is no information about extracellular function of IRF5. However, the fact that IRF5 may be found on microparticles, known to mediate cell-cell signaling, merits further investigations. In addition, further studies are needed to investigate if the IRF5 protein is actively secreted and to study possible extracellular functions of IRF5. FIGURE 4 \| Serological characteristics of SLE molecular subgroups. The levels of RF-IgM are compared between the three molecular SLE subgroups RF-IgM/SSA/SSB (red), IRF5 low (green), and IRF5 high (blue) subgroup. ELISA, showed low or medium levels of IRF5 as measured by the suspension bead array. IRF5 gene polymorphism is an established risk factor in SLE (32, 37). IRF5 in Plasma Levels of IRF5 (D) are compared between the three molecular SLE subgroups RF-IgM/SSA/SSB (red), IRF5 low (green), and IRF5 high (blue) subgroup. activity (p < 0.05) (SLE activity measure (SLAM) (15) equal or above 6) (Supplementary Figure S-4). of choice in a screening of the entire cohort comparing SLE and controls. Nevertheless, in cases where IRF5 was detected there is no doubt about the identity of IRF5 and that IRF5 is present in plasma. The IP, accurate retention times (RT) and high-resolution accurate-mass of unique IRF5 peptides and their fragment spectra, conﬁrm the presence of IRF5 in the circulation. In addition, we developed a sandwich ELISA for detection of IRF5 in plasma. IRF5 levels were signiﬁcantly higher (p = 0.014) in SLE (n = 25) compared to controls (n = 25) (Supplementary Figure S-5). However, the sensitivity of this assay was not suﬃcient for screening of the entire cohort since the majority of the SLE patients analyzed (56%, n = 14) report levels below quantiﬁcation limit. Within this data we aimed to correlate our results with the results obtained by the suspension bead array. Excluding data outside the quantitative range of the ELISA, Spearman’s rank correlation analysis was performed on data from 11 SLE patients. A strong correlation (Spearman’s rho = 0.63, p < 0.05) and a moderate R2 of 0.36 was obtained (Supplementary Figure S-6). However, the three samples resulting in levels above the quantiﬁcation range of the To further investigate the presence of IRF5 in plasma we analyzed IRF5 positive microparticles (MPs). The number of circulating MPs exposing IRF5 were signiﬁcantly higher in SLE (n = 63) compared to healthy controls (n = 20) (130.5 ± 88 vs. 36.5 ± 14 MPs/µl, p < 0.0001) (Figure 6A). IRF5 positive MPs were more frequently exposed on endothelial derived MPs (CD62E+ MPs) compared to platelet and leukocyte derived MPs (p < 0.0001) (Figure 6B). Furthermore, total IRF5+ MPs (regardless of origin) were signiﬁcantly higher in patients with higher disease May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 9 Idborg et al. SLE Subgroups Differ in IRF5 (detected by a diﬀerent antibody) are increased in SLE compared to controls. (detected by a diﬀerent antibody) are increased in SLE compared to controls. FIGURE 4 \| Serological characteristics of SLE molecular subgroups. IRF5 in Plasma To investigate whether IRF5 levels in plasma were regulated by known SLE genetic risk variants in IRF5 we performed a protein quantitative trait locus analysis for two previously reported SNPs (32). We identiﬁed a weak additive association between IRF5 protein levels and the IRF5 SLE risk variant rs4728142 (p = 0.003, beta = 0.07) in SLE patients and controls combined, but this eﬀect was not apparent in either group alone. There was no association between rs10488631 and IRF5 plasma protein levels (Supplementary Figures S-7, 8). Interestingly our unsupervised clustering of SLE patients demonstrate that IRF5 is characteristic for two diﬀerent SLE subgroups. The IRF5 low subgroup also showed lower levels of ISG15, an ubiquitin-like protein that is conjugated to intracellular target proteins upon activation by IFN-α and IFN-β (44, 45), suggesting that this subgroup might be described as a less interferon dependent subgroup. On the other hand, the IRF5 high subgroup seems to be an interferon-driven subgroup with higher levels of IRF5 and ISG15 and one might speculate that patients in these two subgroups could respond diﬀerently to IFN- α-inhibition. Serum levels of IFN-α did not diﬀer between IRF5 high and low subgroup and might be explained by that IFN-α is regulated by several genes and not only by IRF5. Building on these observations, we suggest stratiﬁcation of patients based on plasma levels of IRF5 prior to clinical trials targeting the IFN pathway. These subgroups need to be further investigated, e.g., in the light of type I IFN blockers (46) not reaching primary endpoint. Stratiﬁcation based on IRF5 levels may be more eﬃcient, deﬁnitely less expensive and more suitable to implement in clinical routine, than to measure interferon signature on a gene level. Serum levels of IFN-α in the IRF5 high subgroup was not signiﬁcantly diﬀerent compared to the IRF5 low subgroup. In both subgroups 40% were deﬁned as having detectable levels of IFN-α and the concentration (average ± SD) was 78 ± 122 pg/ml and 67 ± 149 pg/ml for the IRF5 low and IRF5 high subgroup, respectively. The number of IFN-α high patients, deﬁned as a concentration of >100 pg/ml) was 15 in both subgroups. Frontiers in Immunology \| www.frontiersin.org DISCUSSION IRF5, a transcription factor involved in regulation of interferon and cytokine production, showed the largest fold change among the diﬀerentially expressed proteins between SLE patients and controls. We also observed large variations in IRF5 levels between subgroups of SLE patients. IRF5 gene polymorphism is a well- known risk factor in SLE (38, 39) and in several other rheumatic diseases (40). IRF5 is an intracellular protein, nevertheless we detected IRF5 in plasma using aﬃnity-based proteomics and the extracellular location was conﬁrmed by IP-MS in a selection of plasma samples. To further illustrate the presence of IRF5 in the circulation we report that IRF5 expressing microparticles In the IRF5 high subgroup, we detected higher levels of NOS3 (endothelial (e)NOS) as compared to the IRF5 low subgroup. May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 10 Idborg et al. SLE Subgroups Differ in IRF5 E 5 \| Fragment spectra of endogenous IRF5 detected in plasma. The generated recombinant antibody (J-IRF5-5) was used to capture IRF5 by precipitation in a plasma sample from a SLE patient. We obtained fragment spectra of two unique peptides of IRF5, i.e., (A) LITVQVVPVAAR with [M+2H]2+ 633.4007 eluting at a retention time of 48.6 min and (B) FPSPEDIPSDK with [M+2H]2+ m/z of 616.29574 eluting at a retention time of 40.7 min. The retention he masses of the unique peptides and the fragment spectra of these peptides conﬁrms the presence of IRF5 in this plasma sample. an important regulator of nitric oxide (NO) production, is essential for cardiovascular and immune functions indicate an increased risk of cardiovascular events in the IRF5 low subgroup. It is also possible that the high levels of NOS3 in the FIGURE 5 \| Fragment spectra of endogenous IRF5 detected in plasma. The generated recombinant antibody (J-IRF5-5) was used to capture IRF5 by immunoprecipitation in a plasma sample from a SLE patient. We obtained fragment spectra of two unique peptides of IRF5, i.e., (A) LITVQVVPVAAR with [M+2H]2+ m/z of 633.4007 eluting at a retention time of 48.6 min and (B) FPSPEDIPSDK with [M+2H]2+ m/z of 616.29574 eluting at a retention time of 40.7 min. The retention times, the masses of the unique peptides and the fragment spectra of these peptides conﬁrms the presence of IRF5 in this plasma sample. FIGURE 5 \| Fragment spectra of endogenous IRF5 detected in plasma. DISCUSSION CCDC88A (girdin, APE), a protein important for angiogenesis (50), was also increased in the IRF5 high subgroup and decreased in the IRF5 low subgroup. Inﬂammatory markers were increased in this subgroup indicating that the IRF5 high subgroup is characterized by more pronounced inﬂammation and one may speculate that anti-inﬂammatory treatment is more likely to be beneﬁcial for this subgroup of SLE patients. Although validated HPA antibodies, targeting unique peptide sequences, were used, there is still a risk that these mono-speciﬁc polyclonal antibodies give rise to unspeciﬁc signals. Adding additional antibodies to the same protein enhance the probability of detecting the correct protein (56). However, the diﬀerent epitopes targeted by the additional antibodies might be subject to diﬀerences in post translational modiﬁcations or diﬀer in aﬃnity and might not conﬁrm the detection although the correct protein is present. In this work we conﬁrmed the identity of one protein (IRF5) in plasma by IP-MS using a recombinant monoclonal antibody. We were also able to conﬁrm the increased levels of IRF5 in SLE patients compared to controls in a subset of individuals utilizing a sandwich ELISA with a complementary capturing antibody. Although we did not validate the diﬀerences in IRF5 levels in subgroups of SLE in the entire cohort, we are conﬁdent of the detection of IRF5 in plasma. The RF-IgM/SSA/SSB subgroup is characterized by increased levels of SELE (endothelial cell adhesion molecule, E-selectin, CD62E, ICAM-1), ITGB1, SLC22A2, and CERS5. SELE is a cell adhesion glycoprotein on endothelium that can be stimulated by e.g., TNF-α (35, 51). ITGB1 is a cell surface receptor, which is part of the integrin family and it is important for cell adhesion (52). CERS5 synthesizes C16-ceramide and the increase in CERS5 was supported by the increase of C16- ceramide in this subgroup. Ceramides are signaling lipids involved in cell adhesion, inﬂammation as well as in a variety of other physiological functions (53, 54). This subgroup was also associated with higher levels of rheumatoid factor (RF) as well as higher levels of SSA/SSB antibodies. We previously reported high levels of RF-IgM (13), as well as higher levels of total IgG (55), in SLE patients with SSA/SSB antibodies. The RF-IgM/SSA/SSB subgroup share features with Sjögren’s syndrome. Diagnostic biomarkers and novel insight into possible pathogenic pathways in SLE are of great importance and we here report a panel of biomarker candidates that could diﬀerentiate between SLE and controls. DISCUSSION The generated recombinant antibody (J-IRF5-5) was used to capture IRF5 by immunoprecipitation in a plasma sample from a SLE patient. We obtained fragment spectra of two unique peptides of IRF5, i.e., (A) LITVQVVPVAAR with [M+2H]2+ m/z of 633.4007 eluting at a retention time of 48.6 min and (B) FPSPEDIPSDK with [M+2H]2+ m/z of 616.29574 eluting at a retention time of 40.7 min. The retention times, the masses of the unique peptides and the fragment spectra of these peptides conﬁrms the presence of IRF5 in this plasma sample. NOS3, an important regulator of nitric oxide (NO) production, which is essential for cardiovascular and immune functions through regulation of vascular tone, leucocyte adhesion and platelet aggregation (47, 48). NOS3 is vasoprotective and low levels of NOS3 are related to endothelial dysfunction (49). In this context, it is diﬃcult to dissect if the low levels of NOS3 indicate an increased risk of cardiovascular events in the IRF5 low subgroup. It is also possible that the high levels of NOS3 in the IRF5 high subgroup reﬂect damaged blood vessels since NOS3 is expressed in the endothelium and not expected to be increased in the circulation. In the microparticles, analyzed in another set of SLE patients, the IRF5 positive microparticles were mainly of May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 11 Idborg et al. SLE Subgroups Differ in IRF5 FIGURE 6 \| IRF5+ microparticles. Total IRF5+ MPs in SLE patients (n = 63) and healthy controls (n = 20) are shown (A). IRF5+ MPs in SLE patients were phenotyped based on cell origin (B). PMPs, platelet derived MPs; LMPs, leukocyte derived MPs; EMPs, Endothelial derived MPs. < 0.001 (Mann–Whitney). Data is presented as MPs/µl plasma. FIGURE 6 \| IRF5+ microparticles. Total IRF5+ MPs in SLE patients (n = 63) and healthy controls (n = 20) are shown (A). IRF5+ MPs in SLE patients were phenotyped based on cell origin (B). PMPs, platelet derived MPs; LMPs, leukocyte derived MPs; EMPs, Endothelial derived MPs. * < 0.001 (Mann–Whitney). Data is presented as MPs/µl plasma. Both the IRF5 high and the aPL+ subgroups were characterized by pronounced inﬂammation. Our conclusions are based on analysis of a large number of samples. However, validation in additional SLE cohorts and in other disease cohorts is needed. endothelial origin, suggestive of endothelial damage. Frontiers in Immunology \| www.frontiersin.org FUNDING Swedish Research Council (grant no: 2017-02577, grant no 2018-02399, grant no 2018-02535, grant no 2018-02000); Innovative Medicines Initiative (EU/EFPIA) (ULTRA-DD grant no: 115766); Stockholm County Council (ALF grant no: 20160378, grant no. 20170038), The Swedish Rheumatism Association (grant no: R-748261, grant no: R-755861, grant no: R-753741, grant no R-850611, grant no: R-739631); King Gustaf V’s 80-year foundation (grant no: n/a); Funds from Karolinska Institutet (grant no: n/a); Swedish Society of Medicine and the Ingegerd Johansson Donation (grant nr SLS-713911, grant nr SLS-708061); This study was supported by the AstraZeneca-Karolinska Institutet Joint Research Program in Translational Science. ACKNOWLEDGMENTS We would like to thank the patients and controls for participating in the study. We are grateful to Eva Jemseby for collecting all the blood-samples and coordinating nurses, and to Sonia Möller and Birgitta Mannerstedt Fogelfors for assistance in inclusion of patients. The IRF5 expression plasmid was prepared by Protein Science Facility (PSF) at Karolinska Institutet (http:// ki.se/psf). Professor Lennart Truedsson and coworkers at Lund University are acknowledged for measuring C2 and Professor Bo Nilsson and coworkers at Uppsala University for C3a and TCC measurements. We would like to acknowledge Susanna Eketjäll and AstraZeneca for performing the cytokine screening. Ju Li (Huai’an First People’s Hospital in China) is acknowledge for performing the IRF5 ELISA. DISCUSSION Utilizing unsupervised clustering of protein proﬁles, three molecular subgroups were revealed and could be characterized by another set of biomarker candidates. The RF-IgM/SSA/SSB subgroup essentially reﬂects the autoantibody deﬁned SSA/SSB+ subgroup, which has previously been described (13, 55). The novel ﬁnding of circulating IRF5 protein is of importance for the other two subgroups. We suggest that stratiﬁcation of patients based on circulating levels of IRF5 prior to e.g., IFN modulating treatments may be a valuable strategy. Furthermore, the IRF5 high subgroup expressed multiple signs of systemic inﬂammation, indicating that these patients may beneﬁt from anti-inﬂammatory treatment. This work adds new information to the emerging need to classify the heterogeneous sample groups within SLE. The extension of these observations indicate that subgroups might be subject to diﬀerent treatment perspectives, despite similar clinical proﬁle. g j g y In a parallel study the same proteins were investigated but the subgroups were predeﬁned by autoantibody proﬁle, building on previous studies and own clinical experiences (13). The SSA/SSB+ subgroup in that study consisted of 63 patients and the largest fraction (43%) was assigned to the RF-IgM/SSA/SSB subgroup in this study, while the second largest portion (32%) was found in the IRF5 high subgroup which is in line with a pronounced interferon signaling in the SSA/SSB+ subgroup. The frequency of nephritis was similar and relatively low in both RF-IgM/SSA/SSB and SSA/SSB+ subgroups (20 and 21% respectively) while higher in other subgroups (>40%). CERS5 and ITGB1 were proteins characteristic for both RF- IgM/SSA/SSB and SSA/SSB+ subgroups. The second subgroup in our previous work, i.e., an aPL+ subgroup (n = 66), was to the largest extent (58%) found in the IRF5 high subgroup in this work and only 5% overlapped with the RF-IgM/SSA/SSB subgroups. May 2019 \| Volume 10 \| Article 1029 Frontiers in Immunology \| www.frontiersin.org 12 SLE Subgroups Differ in IRF5 Idborg et al. SUPPLEMENTARY MATERIAL The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/ﬁmmu. 2019.01029/full#supplementary-material AUTHOR CONTRIBUTIONS HI substantial contributions to the design of the work, data interpretation, performed IP of IRF5, and drafted the manuscript. AZ substantial contributions to the design of the work, proteomic analysis, data interpretation, and drafted the manuscript. EO performed and analyzed IP-MS experiments, and wrote sections of the manuscript. EW produced recombinant IRF5 and wrote sections of the manuscript. CP performed phage display selections of recombinant antibodies and their subsequent validation and wrote sections of the manuscript. FM analysis and interpretation of IRF5 positive MPs and wrote sections of the manuscript. AC performed analysis of lipid mediators and critically revised the manuscript. AS performed analysis of RF and critically revised the manuscript. PP and JS performed genetic data analysis and wrote sections and critically revised the manuscript. CF-C developed and supervised the ELISA analysis and critically revised the manuscript. JR interpretation of RF data and design of RF experiments and critically revised the manuscript. VO performed analysis of IFN-α and critically revised the manuscript. GG clinical evaluation of sAPS patients and critically revised the manuscript. MK clinical evaluation of sSS patients and critically revised the manuscript. AL supervision of leptin and ﬁbronectin measurements and critically revised the manuscript. CW responsible for analysis of lipid mediators and critically revised the manuscript. AS and LR provided genetic data and critically revised the manuscript. KK responsible for MS-analysis of IRF5 and critically revised the manuscript. HP made the phage display library, supervised phage display selections and antibody validation, and wrote sections of the manuscript. SG supervision of antigen and antibody production and validation of recombinant antibodies and wrote sections of the manuscript. IG responsible for SLE cohort and clinical data and critically revised the manuscript. PN substantial contributions to the design of the work and responsible for ETHICS STATEMENT aﬃnity proteomic platform and drafting the manuscript. ES substantial contributions to the design of the work, responsible for SLE cohort and clinical data, and drafting the manuscript. PJ substantial contributions to the design of the work, responsible for biomarker study, and drafting the manuscript. All authors contributed to manuscript revision, read and approved the ﬁnal version. This study was carried out in accordance with the recommendations of The Local Ethics Committee of the Karolinska University Hospital/Karolinska Institutet in Stockholm. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by The Ethical Review Board in Stockholm (EPN Stockholm). This study was carried out in accordance with the recommendations of The Local Ethics Committee of the Karolinska University Hospital/Karolinska Institutet in Stockholm. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by The Ethical Review Board in Stockholm (EPN Stockholm). 3. Lisnevskaia L, Murphy G, Isenberg D. Systemic lupus erythematosus. Lancet. (2014) 384:1878–88. doi: 10.1016/S0140-6736(14) 60128-8 REFERENCES 3. Lisnevskaia L, Murphy G, Isenberg D. Systemic lupus erythematosus. Lancet. (2014) 384:1878–88. doi: 10.1016/S0140-6736(14) 60128-8 4. Yu C, Gershwin ME, Chang C. Diagnostic criteria for systemic lupus erythematosus: a critical review. J Autoimmun. (2014) 48–49:10–3. doi: 10.1016/j.jaut.2014.01.004 5. Sanz I. 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https://openalex.org/W3151015149	http://www.scielo.br/j/mioc/a/5tYQxw5PbLLrpR3jptmKJsb/?format=pdf&lang=en	English	null	Genomics and functional genomics in Leishmania and Trypanosoma cruzi: statuses, challenges and perspectives	Memórias do Instituto Oswaldo Cruz	2,021	cc-by	6,713	Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru 1Universidade Federal de Minas Gerais, Departamento de Parasitologia, Belo Horizonte, MG, Brasil 2Universidade Federal de Minas Gerais, Departamento de Bioquímica e Imunologia, Belo Horizonte, MG, Brasil 3Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular, Ribeirão Preto, SP, Brasil 1Universidade Federal de Minas Gerais, Departamento de Parasitologia, Belo Horizonte, MG, Brasil 2Universidade Federal de Minas Gerais, Departamento de Bioquímica e Imunologia, Belo Horizonte, MG, Brasil 3Universidade de São Paulo, Faculdade de Medicina de Ribeirão Preto, Departamento de Biologia Celular e Molecular, Ribeirão Preto, SP, Brasil The availability of Trypanosomatid genomic data in public databases has opened myriad experimental possibilities that have contributed to a more comprehensive understanding of the biology of these parasites and their interactions with hosts. In this review, after brief remarks on the history of the Trypanosoma cruzi and Leishmania genome initiatives, we present an overview of the relevant contributions of genomics, transcriptomics and functional genomics, discussing the primary obstacles, challenges, relevant achievements and future perspectives of these technologies. Key words: Leishmania - Trypanosoma cruzi - genomics - transcriptomics - functional genomics The Trypanosomatidae family comprises obligate protozoan parasites from the Kinetoplastida order, in cluding human and livestock disease agents. Trypano soma cruzi, Trypanosoma brucei and Leishmania spe cies are three parasites representative of this family and are the etiological agents of Chagas disease, African trypanosomiasis and leishmaniasis, respectively. Try panosomatid genomics was born at FIOCRUZ in 1994. The Parasite Genome Network Planning Meeting was held in April in Rio de Janeiro, and it was sponsored by FIOCRUZ and the World Bank/WHO (World Health Organization) Special Program for Research and Train ing in Tropical Diseases (TDR). The genome projects of T. cruzi, T. brucei, and Leishmania major were launched at this meeting. The Trypanosomatids Genome Initiative gathered several laboratories from different countries to sequence reference strains of T. brucei, T. cruzi, and L. major. Eleven years were needed to reach sequencing coverage and annotation status before the Tritryp ge nomes were considered complete.(1,2,3,4) Financial con straints, sequencing limitations of Sanger technology, the computational capacity of tools available at the time, and the limited number of groups with high-throughput sequencing capacity and computational expertise ex plain the length of time needed to complete the task. genome assembly is local (clones of tens of kilobases or chromosomes) and therefore not so complex, resulting in a high-quality assembled genome. Conversely, for T. REVIEW REVIEW 1\|7 Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 116: e200634, 2021 1\| Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru cruzi, the clone-by-clone approach was impractical due to the high repetitive content of the genome, and obtain ing chromosome-specific libraries was highly difficult to achieve due to the highly complex parasite karyotype. Therefore, a whole genome shotgun strategy was chosen to sequence the genome of T. cruzi.(2) In this strategy, the entire genome is fragmented, and no positional informa tion of the individual sequences is available to the as sembler algorithms to reconstruct the genome sequence. Compared to the T. brucei and L. major genomes, for T. cruzi, the assembly step was notably more complicated. Additionally, CL Brener, the reference strain for the T. cruzi genome project, is a hybrid between two divergent lineages (TcII and TcIII). This added another layer of complexity to the genome assembly because of the simi larity between the two parental haplotypes. The initial assembly attempt resulted in many pseudogenes, since the assembler merged sequences from the two parental haplotypes. More stringent assembly parameters were then applied in an attempt to assemble the two haplo types separately, but as a drawback, this strategy result ed in a more fragmented genome assembly. In contrast to L. major and T. brucei, no closure step to resolve gaps was applied in the CL Brener T. cruzi genomic assembly. All these aspects contribute to the difference in the qual ity of the first T. cruzi, T. brucei and Leishmania refer ence genomes available in public databases. In this review, we will focus on T. cruzi and Leishma nia omics, advances and challenges. The major achieve ments in these areas are depicted in Figure. However, a brief word on the first publications and the combined efforts on the Tritryps is warranted. L. major and T. brucei genomes were sequenced and assembled using a combination of a clone-by-clone approach and indi vidual chromosome shotgun sequencing,(3,4) in which the Although structural three-way genome comparisons among the Tritryps were somewhat limited due to the T. cruzi genome fragmentation, gene content comparisons among the three parasites revealed a core genome of ~6,200 genes distributed in syntenic polycistronic units containing a large number of genes transcribed from the same DNA strand. Despite evolutionary distance, the extent of syntenic blocks comparing T. doi: 10.1590/0074-02760200634 + Corresponding author: akcruz@fmrp.usp.br  https://orcid.org/0000-0002-4260-4713 Received 26 December 2020 Accepted 19 February 2021 online \| memorias.ioc.fiocruz.br Genomics and functional genomics in Leishmania and Trypanosoma cruzi: statuses, challenges and perspectives aniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cruz3/+ online \| memorias.ioc.fiocruz.br Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru brucei and Leish mania genomes is impressive; 68 and 75% of the anno tated genes are retained in the same genomic context, and almost all of these genes, 94%, are part of the core doi: 10.1590/0074-02760200634 + Corresponding author: akcruz@fmrp.usp.br  https://orcid.org/0000-0002-4260-4713 Received 26 December 2020 Accepted 19 February 2021 Daniella C Bartholomeu et al. Timeline showing some of the major achievements in the areas of genomics, transcriptomics and functional genomics in Leishmania and Try panosoma cruzi. Important genomic initiatives launched before 1994 were included in a comprehensive and recent review by Ramírez, 2020.(5) Timeline showing some of the major achievements in the areas of genomics, transcriptomics and functional genomics in Leishmania and Try panosoma cruzi. Important genomic initiatives launched before 1994 were included in a comprehensive and recent review by Ramírez, 2020.(5) meline showing some of the major achievements in the areas of genomics, transcriptomics and functional genomics in Le nosoma cruzi. Important genomic initiatives launched before 1994 were included in a comprehensive and recent review by proteome of the three trypanosomatids.(1) In terms of gene content, the total number of annotated protein-coding genes was similar for T. brucei and Leishmania, ~8,000, and significantly higher in T. cruzi, 12,000. Notably, only 32, 26 and 12% of species-specific genes were identified in T. brucei, T. cruzi and Leishmania, respectively; their predicted functions are clearly connected to these organ isms’ most clear biological differences.(1) 2007, Sanger sequencing technology was used. Since then, we have seen a notable improvement in new se quencing technologies, which provide considerably higher throughput and accurate short-read sequenc ing, as well as noisy long-read sequencing. The quality of long reads can be improved by sequencing a single template multiple times using hairpin adaptors or using high-quality short Illumina reads to correct long PacBio or Nanopore reads. The use of high-quality consensus long reads (10-100 kb) dramatically improves genome assembly, allowing the resolution of many repetitive regions, phasing alleles, distinguishing highly homolo gous sequences, and identifying structural variants, such as deletions, duplications, insertions, inversions, and translocations. Recently, Trypanosomatid genome projects have used both technologies, with long-read sequencing offering assembly continuity and short-read sequencing providing higher sequence accuracy. The genomes of several T. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru cruzi strains, such as Berenice, Dm28c, Bug2148, and TCC, were assembled using long reads or a combination of long and Illumina reads, en abling a reduction in the number of sequences generated and improving the contiguity of the assembled genome. (10,11,12) More recently, high-quality chromosomal level assemblies of Y and Brazil strains were obtained using a combination of long and Illumina reads and Hi-C and Chicago proximity-ligation libraries, improving genome contiguity and assembly accuracy.(13) The improvement of T. cruzi genome assemblies confirmed the compart mentalisation of the parasite genome’s organisation. Berná et al. proposed the occurrence of two compart ments named “core” and “disruptive” that differ in GC content and gene composition.(10) The core compartment comprises conserved and hypothetical genes and is syn tenic with the T. brucei and Leishmania genomes, while the disruptive core is composed of multigene families encoding MASP, TcMUC, and trans-sialidase variable surface proteins. Other multicopy gene families, such as DGF-1, GP63, and RHS, are evenly distributed through Shortly after the publications of the Tritryp genomes, the first comparative analysis of Leishmania genomes was conducted between L. (Leishmania) infantum and L. (Viannia) braziliensis with the available L. major ge nome in 2007.(6) These species are associated with dif ferent disease outcomes; typically, L. major leads to un complicated cutaneous disease (LCL), L. infantum leads to visceral disease (VL) and L. braziliensis is responsible for the morbid mucocutaneous forms (MCL and LCL). (6) As expected, the study demonstrated the genomes to be highly conserved at the content and synteny levels. (6) This comparative analysis contributed to unraveling similarities and differences due to the restricted number of species-specific genes detected; the highest number of L. braziliensis-specific genes was 49.(6) Notably, this study revealed that in contrast to the two Leishmania from the subgenus Leishmania, L. braziliensis from the Viannia subgenus contains an intact RNA-mediated in terference pathway. The RNAi pathway was subsequent ly shown to be functional and rapidly applied to gene function studies.(7,8) In 2009, a critical contribution to the CL Brener T. cruzi genome sequence was the generation of a chromo somal-level assembly by Weatherly et al.(9) A total of 41 pseudochromosomes for each CL Brener haplotype, TcII and TcIII, were assembled based on the scaffolds gener ated in 2005, BAC end sequences and synteny maps with the T. brucei genome. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru Advances and difficulties in data generation and analysis - For the first descriptions of the T. brucei, Leishmania and T. cruzi genomes generated in 2005 and Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 116, 2021 3\|7 out the genome.(10) Wang et al.(13) proposed the occurrence of three genomic compartments: (i) one corresponding to the disruptive compartment described by Berná encom passing rapidly evolving gene families associated with immune evasion; (ii) a second compartment containing gene families, such as TcSMUG, expressed in the vector stage, distributed as tandem arrays and displaying mini mal diversification; and (iii) a third compartment con taining all other core genes. Regardless of the number of compartments, it is clear that some T. cruzi genomic regions evolve under strong selective pressure for diver sification, while others exhibit low diversity.(10,13) These improvements in T. cruzi genome assembly have allowed a better understanding of the parasite genomic organisa tion and disclosed the complete repertoire of genes en coding surface proteins involved in several critical host- parasite interactions. the gain or loss of chromosomal copies, respectively. For a diploid genome, if the ratio chromosome median RDC/ genome median RDC is close to 1, the chromosome is disomic; if this ratio is 1.5, or 2, the chromosome is tri somic or tetrasomic, respectively, and so on. , p y, Allelic frequency analysis is another NGS-based ap proach to estimate ploidy. As in RDC analysis, the ini tial step consists of mapping NGS reads on a reference genome. Next, the frequency of heterozygous SNPs that have up to two and only two variants is computed. The chromosome somy can then be inferred by computing the proportion of reads that support each variant. In a disomic chromosome, the same number of reads are ex pected to support each heterozygous SNP, resulting in a peak allele frequency of 0.5 (50%). For trisomic chromo somes, the expected allele frequency is a combination of 0.33 and 0.66 frequencies, while in tetrasomic chromo somes, frequencies of 0.25, 0.5 and 0.75 are expected. For monosomic chromosomes, no SNP should be identi fied. Whenever possible, both RDC and allele frequency approaches should be used to estimate somy, so inde pendent evidence can be evaluated. However, the low frequency of allele polymorphisms in many Leishmania species/isolates may preclude allele frequency analysis, and the chromosome somy based on NGS data is mainly estimated by the RDC approach. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru p It is very important to keep in mind, however, that despite the many advances, the current sequencing and genome assembly approaches do not have enough resolution to generate the complete genome sequenc ing of diploid organisms. Only more recently have as sembler algorithms devoted to dealing with haplotype reconstruction, i.e., set of single nucleotide variations that distinguish chromosomal sequences from their ho mologous pairs, been developed.(14,15) Although the use of PacBio and Nanopore long reads by these assemblers improves the reconstruction accuracy, haplotype assem bly remains a complex computational task. Therefore, for most genomes, only a mosaic haploid representation of the genome sequence is available in public databases. This is especially relevant for T. cruzi and Leishmania genomes, as they are prone to extensive chromosome copy number variations.(16,17) These NGS-based methodologies and fluorescence in situ hybridisation (FISH) analyses have been recently used to infer ploidy in T. cruzi and Leishmania, reveal ing that aneuploidy seems to be the rule in these organ isms.(16,17,18) As many genomes of T. cruzi strains and Leishmania species/strains have been sequenced, it is now clear that these parasites display extreme chromo some copy number variations (CCNV), a phenomenon that affects many, if not all, chromosomes. Even within a given parasite population, FISH analysis of Leishma nia revealed variable chromosome ploidy among indi vidual cells generating intrastrain heterogeneity, which is a pattern known as mosaic aneuploidy.(18) The question that emerged from these studies is what is the biological meaning of these findings? Although complete haplotype reconstruction is still a challenge, computational methodologies based on next- generation sequencing (NGS) data have been developed to estimate genome ploidy and chromosome “somies”, such as allelic frequency analyses and read depth cover age (RDC) (the average number of times a base of a ge nome is sequenced). Both analyses require chromosom al-level assembly of a reference genome and high RDC provided by NGS data. Gene enrichment analysis of chromosomes consis tently supernumerary in T. cruzi(17) suggested a possi ble correlation between chromosome copy number and dosage of genes involved in crucial parasite biologi cal processes. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru brucei, sexual re production has been previously demonstrated to occur in their vectors.(24,25) The occurrence of recombination in Trypanosomatids implies that genetic traits associated with, for instance, virulence and drug resistance are not restricted to specific subdivisions but instead can be re combined and transferred, affecting parasite fitness and adaptation to new niches.(26,27) Whereas fast-evolving sequencing platforms and bio informatic tools are quickly overcoming most of the chal lenges faced by researchers during genome sequencing, assembly and annotation, as well as transcriptome analy ses, gene manipulation protocols are only now experienc ing significant improvements that will provide more sig nificant advances in functional genomic studies. Genome manipulation tools and protocols are es sential for any study aiming to determine gene function and identify the elements involved in gene expression. In the first report describing gene deletion in Leishmania, published in 1990, it was demonstrated that the single al lele of DHFR-TS can be replaced with high efficiency by homologous recombination after transfection of L. major promastigotes with linear dsDNA containing sequences homologous to the target gene.(35) In T. cruzi, studies based on gene manipulation began in 1991, when Lu and Buck transfected epimastigotes with a plasmid vector in which a sequence encoding the spliced leader (SL) repeat was inserted upstream from the bacterial chlorampheni col acetyltransferase (CAT) gene. Surprisingly, these au thors succeeded in detecting CAT activity in lysates from transfected parasites even in the absence of correct in formation regarding the sequences required for gene ex pression in T. cruzi. Similar to Leishmania, the first gene deletion experiment in T. cruzi described the disruption of only one allele of the gene encoding the flagellar ad hesion molecule GP72 by homologous recombination.(36) Genome and transcriptome information catalysing functional studies - A primary goal of any genome re search is the elucidation of gene function and the mecha nisms involved in the control of gene expression. The combination of genomics and transcriptomics data and analyses is greatly increasing our understanding of mo lecular mechanisms of parasite adaptation to distinct host environments. Due to recent advances in genomics, we are progressing toward a more complete understand ing of genome structure, gene function and, ultimately, how the information encoded in an organism’s DNA guides the synthesis of all RNA and protein molecules to determine the characteristics of a living cell. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru A plethora of data on differential gene expression and gene ontology analysis permit investigation of the host and parasite responses during infection; the available studies include a com parative analysis of different species of Leishmania in in vitro-infected human macrophages and a challenging analysis of pauci-parasitic L. braziliensis human lesions. Similar studies simultaneously capturing the transcrip tome dynamics in the parasites and host cells were re ported for an infection time course of human fibroblasts with two different T. cruzi strains, showing contrasting virulence phenotypes.(32,33,34) Such studies are invaluable for understanding parasite evasion, host response, and parasite-driven host cell subversion. ing transcript levels, although independent aneuploidy mechanisms of gene regulation were observed for chro mosomes 5, 8 and 31. As the control of gene expression in Leishmania operates at the posttranscriptional level, changes in aneuploidy have emerged as a major adapta tive strategy exploited by Leishmania to modulate ex pression by altering gene dosage, allowing rapid adapta tion to changing environments.(21) As more layers of novel information arise from com parison of Leishmania field strains and species from dif ferent geographical endemic areas and long-maintained laboratory strains or drug-resistant versus drug-sensi tive strains, an increasing number of questions emerge. Aneuploidy, chromosome-variable somy, loci-restricted DNA amplification, and expansion and deletions in gene arrays contribute together and to a different extent to gene copy number variation. Some paradigmatic studies have revealed the complexity of the genomic adaptation of these parasites to the field environment.(22,23) These studies and others indicate that the genomic plasticity of Leishmania plays a role in determining phenotypic variation and fitness gain in response to environmental changes and challenges. Whole-genome sequencing has also provided new insights into the population genetic structure of Try panosomatids, demonstrating that facultative sex is a common reproductive strategy in this group of parasites. Sequencing of 45 TcI T. cruzi field isolates from vec tors and mammalian hosts in southern Ecuador revealed that sexual meiotic reproduction occurs in some groups, while in others, long-term clonality remains after epi sodic hybridisation events. These distinct groups seem to be genetically segregated even though they may cooc cur in the same invertebrate and vertebrate hosts.(23) The site of T. cruzi genetic exchange, whether it occurs in the vector or mammalian host, remains unclear. On the other hand, in Leishmania and in T. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru As an example, chromosome 31, which was found to be supernumerary in the large majority of strains and isolates analysed, is enriched with genes involved in glycosylation and glycan biosynthesis, es pecially the enzyme UDP-GlcNAc-dependent glycosyl transferase, which is involved in mucin glycosylation and crucial for many parasite interactions with inverte brate and vertebrate hosts.(17,19) RDC analyses are based on the assumption that cov erage is directly related to copy number. Basically, the NGS reads are mapped on a reference chromosome se quence, and the number of reads covering each position or gene is computed. The number of copies of a given gene, genomic region, or chromosome can then be esti mated by dividing the median RDC in that region by the median RDC of the entire genome. In the case of very re petitive genomes, such as T. cruzi, instead of using RDC of the whole genome as a normaliser, it is more reliable to use the RDC of unique (single copy) genomic regions because the reads that map to repetitive regions have low mapping confidence.(17) It is also essential to control for RDC variations along each chromosome sequence to exclude segmental duplications/deletions that could bias the median RDC value. In summary, an increase or de crease in the median RDC of a chromosome compared to the whole genome (or single-copy regions) indicates Recent studies have disclosed direct evidence for the link between aneuploidy and gene expression in Leish mania. For instance, Dumetz et al.(20) examined aneu ploidy and gene expression in L. donovani strains under different in vitro and in vivo conditions. The results re vealed aneuploidy alterations among the different cul ture and infection conditions and a positive correlation between chromosome copy number and correspond 4\|7 Daniella C Bartholomeu et al. The ideal scenario for evaluating parasite gene ex pression within natural environments and/or concomi tantly analysing profiles of gene expression of hosts and parasites is currently possible. Of note, these analyses depend on robust computational tools and genome data of parasites and hosts. Quite informative studies on parasite-host interactions have been reported that high light the main differences between the transcriptomes of Leishmania sand fly stages(30,31) by comparing organisms maintained in vivo and in vitro. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru In addition to a poly-A tail at their 3’ end, ev ery trypanosomatid mature mRNA possesses a common sequence at its 5’ end, which is capped and contains the same 39 nucleotides (not 35 nucleotides as postulated in 1985) named the spliced leader (SL). Once polycistronic mRNAs are transcribed, coupled trans-splicing and poly adenylation machinery, driven by polypyrimidine-rich region sequences present within intergenic regions (IRs), promotes the cleavage between the coding sequences and the simultaneous insertion of the poly-A tail and SL on upstream and downstream genes, respectively. Another bizarre discovery is the enzyme that transcribes trypano some mRNAs. T. brucei possesses all three RNA poly merases and RNA polymerase II (RNA pol II) is respon sible for the transcription of most protein-coding genes, as in any eukaryote; however, two groups of T. brucei protein-coding genes, VSG and PARP, are transcribed by RNA polymerase I (RNA pol I), which, in most eukary otes, transcribes exclusively ribosomal RNA genes. Sur prisingly, in contrast to RNA polymerase I promoters, no consensus sequences for RNA pol II promoters have been identified in trypanosomatids, with the exception of genes encoding SL transcripts. Therefore, to express a foreign gene in T. cruzi or in Leishmania spp., IR con taining polypyrimidine-rich sequences must be inserted upstream and downstream from the exogenous gene in the expression vector to allow correct trans-splicing and polyadenylation. Moreover, as shown in early experi ments, it is possible to express, albeit at low levels, a for eign gene in T. cruzi or Leishmania without a transcrip tional promoter. However, even though transcription of endogenous protein-coding genes by RNA pol I has been described only in T. brucei, RNA pol I promoters derived from the ribosomal RNA locus have been successfully used to increase the expression levels of foreign genes in both T. cruzi and Leishmania.(40) Since the early 1990s, major advances in our under standing of the mechanisms controlling gene expression Gene knockout experiments are powerful tools used to determine gene function. In contrast to T. brucei, pro tocols that allowed reverse genetic manipulation of the T. cruzi and Leishmania genomes have been used in a limited number of studies that have revealed the function of parasite genes and their roles in complex interactions with parasite hosts. New protocols describing alterna tive methodologies to facilitate genetic manipulation in T. cruzi are indeed desirable. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru cruzi or in Leishmania spp., IR con taining polypyrimidine-rich sequences must be inserted upstream and downstream from the exogenous gene in the expression vector to allow correct trans-splicing and polyadenylation. Moreover, as shown in early experi ments, it is possible to express, albeit at low levels, a for eign gene in T. cruzi or Leishmania without a transcrip tional promoter. However, even though transcription of endogenous protein-coding genes by RNA pol I has been described only in T. brucei, RNA pol I promoters derived from the ribosomal RNA locus have been successfully used to increase the expression levels of foreign genes in both T. cruzi and Leishmania.(40) changes in the environment these parasites face during their life cycles. Regulatory sequences that are present mainly in the 3’ UTRs of mRNAs act as protein-binding sites, and the regulatory sequences in the 3’ UTR and RNA binding proteins (RBPs) are key factors that modu late individual mRNA levels during the parasite life cy cle.(37) Even before the availability of the whole genome sequence, a combination of experimental approaches, in cluding bioinformatics analyses and parasite transfection with reporter genes, were used to identify elements in the 3’ UTRs of several genes that control mRNA abundance, as well as translation. The identification of trans-acting elements that bind to mRNA regulatory motifs requires additional strategies, such as RNA affinity chromatog raphy with parasite protein lysates. The development of knockout or knockdown mutants is essential to directly test the role of an RBP on global gene expression. In con trast to T. brucei, in which the discovery of the RNAi machinery allowed the testing of several RBPs, the lack of RNAi machinery in T. cruzi(41) and all species of Leish mania(7) from the subgenus Leishmania delayed the stud ies in T. cruzi and Leishmania. revealed that this group of early branched eukaryotes has developed peculiar transcription and mRNA process ing mechanisms. Unlike most eukaryotes that transcribe their genes into monocistronic pre-mRNAs containing exons (coding sequences) and introns (mostly noncod ing sequences), almost all trypanosome protein-coding genes are intronless and transcribed into polycistronic pre-mRNAs, which require processing into mature mRNAs through “trans-splicing” and polyadenylation reactions. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru For Try panosomatids, such studies also allow us to understand species differences that help us to make rational choices in functional studies of pathways and gene networks rel evant to parasite survival.(20,28,29) The study of mechanisms involved in gene expres sion in T. cruzi and Leishmania yielded many surprises about the Trypanosomatid family (for a review, see(37)). Although studies on the variant surface glycoprotein (VSG) and tubulin genes showed identical 35 nucleotide sequences added on the 5’ ends of different mRNAs from T. brucei since 1985,(38) the coupled trans-splicing and polyadenylation mechanisms were elucidated only after gene manipulation protocols became well established in this parasite.(39) A remarkable series of experiments then Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol. 116, 2021 5\|7 revealed that this group of early branched eukaryotes has developed peculiar transcription and mRNA process ing mechanisms. Unlike most eukaryotes that transcribe their genes into monocistronic pre-mRNAs containing exons (coding sequences) and introns (mostly noncod ing sequences), almost all trypanosome protein-coding genes are intronless and transcribed into polycistronic pre-mRNAs, which require processing into mature mRNAs through “trans-splicing” and polyadenylation reactions. In addition to a poly-A tail at their 3’ end, ev ery trypanosomatid mature mRNA possesses a common sequence at its 5’ end, which is capped and contains the same 39 nucleotides (not 35 nucleotides as postulated in 1985) named the spliced leader (SL). Once polycistronic mRNAs are transcribed, coupled trans-splicing and poly adenylation machinery, driven by polypyrimidine-rich region sequences present within intergenic regions (IRs), promotes the cleavage between the coding sequences and the simultaneous insertion of the poly-A tail and SL on upstream and downstream genes, respectively. Another bizarre discovery is the enzyme that transcribes trypano some mRNAs. T. brucei possesses all three RNA poly merases and RNA polymerase II (RNA pol II) is respon sible for the transcription of most protein-coding genes, as in any eukaryote; however, two groups of T. brucei protein-coding genes, VSG and PARP, are transcribed by RNA polymerase I (RNA pol I), which, in most eukary otes, transcribes exclusively ribosomal RNA genes. Sur prisingly, in contrast to RNA polymerase I promoters, no consensus sequences for RNA pol II promoters have been identified in trypanosomatids, with the exception of genes encoding SL transcripts. Therefore, to express a foreign gene in T. REFERENCES 19. Buscaglia CA, Campo VA, Frasch ACC, Di Noia JM. Trypanoso ma cruzi surface mucins: host-dependent coat diversity. Nat Rev Microbiol. 2006; 4(3): 229-36. 1. El-Sayed NM, Myler PJ, Blandin G, Berriman M, Crabtree J, Ag garwal G, et al. Comparative genomics of trypanosomatid para sitic protozoa. Science. 2015; 309(5733): 404-9. 20. Dumetz F, Imamura H, Sanders M, Seblova V, Myskova J, Pescher P, et al. Modulation of aneuploidy in Leishmania donovani during adaptation to different in vitro and in vivo environments and its impact on gene expression. mBio. 2017; 8(3): e00599-17. 2. El-Sayed NM, Myler PJ, Bartholomeu DC, Nilsson D, Aggarwal G, Tran AN, et al. The genome sequence of Trypanosoma cruzi, etiologic agent of Chagas disease. Science. 2015; 309(5733): 409-15. 21. Iantorno SA, Durrant C, Khan A, Sanders MJ, Beverley SM, War ren WC, et al. Gene expression in Leishmania is regulated pre dominantly by gene dosage. mBio. 2017; 8(5): e01393-17. 3. Ivens AC, Peacock CS, Worthey EA, Murphy L, Aggarwal G, Berriman M, et al. The genome of the kinetoplastid parasite, Leishmania major. Science. 2015; 309(5733): 436-42. 22. Ubeda JM, Raymond F, Mukherjee A, Plourde M, Gingras H, Roy G, et al. Genome-wide stochastic adaptive DNA amplification at direct and inverted DNA repeats in the parasite Leishmania. PLoS Biol. 2014; 12(5): e1001868. 4. Berriman M, Ghedin E, Hertz-Fowler C, Blandin G, Renauld H, Bartholomeu DC, et al. The genome of the African trypanosome Trypanosoma brucei. Science. 2015; 309(5733): 416-22. 5. Ramirez JL. Trypanosoma cruzi genome 15 years later: what has been accomplished? Trop Med Infect Dis. 2020; 5(3): 129. 23. Schwabl P, Imamura H, Van den Broeck F, Costales JA, Mai guashca-Sánchez J, Miles MA, et al. Meiotic sex in Chagas dis ease parasite Trypanosoma cruzi. Nat Commun. 2019; 10(1): 3972. 6. Peacock CS, Seeger K, Harris D, Murphy L, Ruiz JC, Quail MA, et al. Comparative genomic analysis of three Leishmania species that cause diverse human disease. Nat Genet. 2007; 39(7): 839-47. 24. Akopyants NS, Kimblin N, Secundino N, Patrick R, Peters N, Lawyer P, et al. Demonstration of genetic exchange during cycli cal development of Leishmania in the sand fly vector. Science. 2009; 324(5924): 265-8. 7. Lye LF, Owens K, Shi H, Murta SM, Vieira AC, Turco SJ, et al. Retention and loss of RNA interference pathways in trypanoso matid protozoans. PLoS Pathog. 2010; 6(10): e1001161. 25. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru A major improvement that has allowed for better control of genetic manipulation in these parasites was the development of inducible expres sion of gene products using the tetracycline repressor. This system, initially developed for T. brucei, requires the generation of transgenic parasites that express the tetracycline repressor from Escherichia coli. Although tetracycline-dependent expression of a reporter gene cloned downstream from the tet operator(42) has been suc cessfully described in T. cruzi,(41) to date, this methodol ogy has not been widely used to control exogenous gene expression in either T. cruzi or Leishmania. We now have a much broader collection of vectors that can be used to perform genetic manipulation in T. cruzi and Leishma nia, including vectors containing sequences that allow the integration of the transfected gene into different loci of the parasite genome. The most recent improvement in our capacity to perform studies involving genome ma nipulation in these parasites came with CRISPR/Cas9 technology. Due to the efforts made by a somewhat lim ited number of research groups, highly efficient gene manipulation protocols based on CRISPR/Cas9 technol ogy have been developed for T. cruzi and Leishmania, enabling the generation of mutant parasite cell lines with targeted sequence modifications in their genomes in days instead of months.(43,44,45,46,47,48) Using CRISPR/Cas9, al terations in the genome can be created more precisely and more efficiently due to the induction of site-specific double-strand breaks (DSBs) caused by the RNA-guided Cas9 endonuclease. If a donor DNA template is provided, DSBs are efficiently repaired by the parasite homolo Since the early 1990s, major advances in our under standing of the mechanisms controlling gene expression in T. cruzi and Leishmania have been made due to ex periments involving transfection of exogenous DNA into parasites by conventional electroporation. More recently, a new transfection methodology named nucleofection using the Amaxa Nucleofector system resulted in con siderably improved transfection efficiency. These gene manipulation studies, performed mainly with T. cruzi epimastigotes and Leishmania promastigotes, also re vealed unique gene expression mechanisms. As a con sequence of polycistronic transcription of protein-coding genes, which are constitutively transcribed, as well as the absence of sequences controlling transcription initia tion, it became clear that T. cruzi and Leishmania genes must be posttranscriptionally regulated in response to the Daniella C Bartholomeu et al. 6\|7 13. Wang W, Peng D, Baptista RP, Li Y, Kissinger JC, Tarleton RL. ACKNOWLEDGEMENTS 17. Reis-Cunha JL, Rodrigues-Luiz GF, Valdivia HO, Baptista RP, Mendes TA, de Morais GL, et al. Chromosomal copy number vari ation reveals differential levels of genomic plasticity in distinct Trypanosoma cruzi strains. BMC Genomics. 2015; 16(1): 499. To our colleagues for many inspiring discussions and con tributions and apologise for those whose work was not cited here due to space limitations. 18. Sterkers Y, Lachaud L, Crobu L, Bastien P, Pagès M. FISH analysis reveals aneuploidy and continual generation of chromosomal mosa icism in Leishmania major. Cell Microbiol. 2011; 13(2): 274-83. Daniella C Bartholomeu1, Santuza Maria Ribeiro Teixeira2, Angela Kaysel Cru Strain-specific genome evolution in Trypanosoma cruzi, the agent of Chagas disease. PLoS Pathog. 2021; 17(1): e1009254. gous recombination DNA repair machinery, allowing not only target gene deletion but also sequence replacement that facilitates characterisation of functional domains in protein-coding genes as well as noncoding regulatory sequences.(49) Importantly, together with high-through put DNA/RNA sequencing technologies, CRISPR/Cas9 genome editing will allow genome-wide studies, which will produce a shift towards a global perspective on gene function studies with the examination of the expression and biological roles of gene networks in different stages of the parasite life cycle and under different contexts of host parasite interactions. 14. Sankararaman A, Vikalo H, Baccelli F. ComHapDet: a spatial community detection algorithm for haplotype assembly. BMC Genomics. 2020; 21(Suppl. 9): 586. 15. Majidian S, Kahaei MH, de Ridder D. Hap10: reconstructing ac curate and long polyploid haplotypes using linked reads. BMC Bioinformatics. 2020; 21(1): 253. 16. Rogers MB, Hilley JD, Dickens NJ, Wilkes J, Bates PA, Depledge DP, et al. Chromosome and gene copy number variation allow ma jor structural change between species and strains of Leishmania. Genome Res. 2011; 21(12): 2129-42. AUTHORS’ CONTRIBUTION All authors wrote and approved the final manuscript. REFERENCES Jenni L, Marti S, Schweizer J, Betschart B, Le Page RW, Wells JM, et al. Hybrid formation between African trypanosomes dur ing cyclical transmission. Nature. 1986; 322(6075): 173-5. 8. de Paiva RM, Grazielle-Silva V, Cardoso MS, Nakagaki BN, Mendonça-Neto RP, Canavaci AM, et al. Amastin knockdown in Leishmania braziliensis affects parasite-macrophage interac tion and results in impaired viability of intracellular amastigotes. PLoS Pathog. 2015; 11(12): e1005296. 26. 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First efficient CRISPR-Cas9-mediated genome editing in Leishmania parasites. Cell Microbiol. 2015; 17(10): 1405-12. 35. Cruz AK, Titus R, Beverley SM. Plasticity in chromosome num ber and testing of essential genes in Leishmania by targeting. Proc Natl Acad Sci USA. 1993; 90(4): 1599-603. 46. Lander N, Li ZH, Niyogi S, Docampo R. CRISPR/Cas9-induced disruption of paraflagellar rod protein 1 and 2 genes in Trypano soma cruzi reveals their role in flagellar attachment. mBio. 2015; 6(4): e01012. 36. Cooper R, de Jesus AR, Cross GA. Deletion of an immunodomi nant Trypanosoma cruzi surface glycoprotein disrupts flagellum- cell adhesion. J Cell Biol. 1993; 122(1): 149-56. 37. Teixeira SM, daRocha WD. Control of gene expression and genet ic manipulation in the Trypanosomatidae. Genet Mol Res. 2003; 2(1): 148-58. 47. Beneke T, Madden R, Makin L, Valli J, Sunter J, Gluenz E. A CRISPR Cas9 high-throughput genome editing toolkit for kineto plastids. R Soc Open Sci. 2017; 4(5): 170095. 38. Sather S, Agabian N. REFERENCES A 5’ spliced leader is added in trans to both alpha- and beta-tubulin transcripts in Trypanosoma brucei. Proc Natl Acad Sci USA. 1985; 82(17): 5695-9. 48. Burle-Caldas GA, Soares-Simões M, Lemos-Pechnicki L, Da Rocha WD, Teixeira SMR. Assessment of two CRISPR-Cas9 ge nome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants. Int J Parasitol. 2018; 48(8): 591-6. 39. Ullu E, Matthews KR, Tschudi C. Temporal order of RNA-pro cessing reactions in trypanosomes: rapid trans splicing precedes polyadenylation of newly synthesized tubulin transcripts. Mol Cell Biol. 1993; 13(1): 720-5. 49. Burle-Caldas GA, Grazielle-Silva V, Laibida LA, DaRocha WD, Teixeira SM. Expanding the tool box for genetic manipulation of Trypanosoma cruzi. Mol Biochem Parasitol. 2015; 203(1-2): 25-33.
https://openalex.org/W4383196698	https://www.qeios.com/read/V86ZGI/pdf	English	null	Review of: "Which sociocultural determinants of pre-drinking amongst undergraduate university students influence motivation"	null	2,023	cc-by	280	Qeios, CC-BY 4.0 · Review, July 6, 2023 Qeios ID: V86ZGI · https://doi.org/10.32388/V86ZGI Review of: "Which sociocultural determinants of pre-drinking amongst undergraduate university students influence motivation" Olanrewaju O.P. Ajakaiye1 Olanrewaju O.P. Ajakaiye1 1 Landmark University Potential competing interests: No potential competing interests to declare. Yes This is a well researched and articulately presented paper, however, I observed as follows: This is a well researched and articulately presented paper, however, I observed as follows: 1. The paper title can be framed as: sociocultural determinants of pre-drinking and drinking games amongst undergraduate university students 1. The paper title can be framed as: sociocultural determinants of pre-drinking and drinking games amongst undergraduate university students The influence and motivation should be proven in the paper. Since there is determinant in the title, no need to add influence and motivation. The influence and motivation should be proven in the paper. Since there is determinant in the title, no need to add influence and motivation. 2. Since the study is looking at Undergraduates, can university policies on alcohol consumption be included. This is to tell us if the University is predisposed to such social issue? 3. What is the country's law saying about alcohol consumption among young people. 4. What role does parent, guardian and care givers or anyone older than them, aside their peers that they had contact with before their university experience. 5. The social cultural aspect of the paper us not empirical enough. You can not base sociocultural on peers alone, without considering the influence of other social Institutions. 6. The limitations of the study was not reported. 6. The limitations of the study was not reported. Qeios ID: V86ZGI · https://doi.org/10.32388/V86ZGI 1/1
https://openalex.org/W4234237834	https://www.zora.uzh.ch/id/eprint/195481/1/2020_Schiedung_bg-17-6457-2020.pdf	English	null	Vertical mobility of pyrogenic organic matter in soils: A column experiment	null	2,020	cc-by	16,387	Zurich Open Repository and Archive Zurich Open Repository and Archive University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch University of Zurich University Library Strickhofstrasse 39 CH-8057 Zurich www.zora.uzh.ch Year: 2020 g y p Schiedung, Marcus; Bellè, Severin-Luca; Sigmund, Gabriel; Kalbitz, Karsten; Abiven, Samuel (2020). Vertical mobility of pyrogenic organic matter in soils: a column experiment. Biogeosciences, 17(24):6457-6474. DOI: https://doi.org/10.5194/bg-17-6457-2020 Correspondence: Samuel Abiven (abiven@biotite.ens.fr) Correspondence: Samuel Abiven (abiven@biotite.ens.fr) Received: 16 July 2020 – Discussion started: 3 August 2020 Received: 16 July 2020 – Discussion started: 3 August 2020 Received: 16 July 2020 – Discussion started: 3 August 2020 Revised: 11 November 2020 – Accepted: 11 November 2020 – Published: 22 December 2020 eceived: 16 July 2020 – Discussion started: 3 August 2020 evised: 11 November 2020 – Accepted: 11 November 2020 – Published: 22 December 2020 Revised: 11 November 2020 – Accepted: 11 November 2020 – Published: 22 December 2020 Abstract. Pyrogenic organic matter (PyOM) is a major and persistent component of soil organic matter, but its mobility and cycling in soils is largely unknown. We conducted a col- umn experiment with a topsoil and subsoil of a sand and a sandy loam to study the mobility of highly 13C labeled rye- grass PyOM (> 2.8 at. %), applied as a layer on a 7 cm long soil column, under saturated conditions. Further, we used fresh and oxidized PyOM (accelerated aging with H2O2) to identify changes in its migration through the soil with aging and associated surface oxidation. Due to the isotopic signa- ture, we were able to trace the PyOM carbon (PyOM-C) in the soil columns, including density fractions, its effect on na- tive soil organic carbon (nSOC) and its total export in perco- lates sequentially sampled after 1000–18 000 Lm−2. In total, 4 %–11 % of the added PyOM-C was mobilized and < 1 % leached from the columns. The majority of PyOM-C was mo- bilized with the ﬁrst ﬂush of 1000 Lm−2 (51 %–84 % of ex- ported PyOM-C), but its export was ongoing for the sandy soil and the loamy subsoil. Oxidized PyOM showed a 2–7 times higher mobility than fresh PyOM. In addition, 2-fold higher quantities of oxidized PyOM-C were leached from the sandy soil compared to the loamy soil. Besides the higher mobility of oxidized PyOM, its retention in both soils in- creased due to an increased reactivity of the oxidized PyOM surfaces and enhanced the interaction with the soil mineral phase. Density fractionation of the upper 0–2.3 cm, below the PyOM application layer, revealed that up to 40 % of the migrated PyOM was associated with the mineral phase in the loamy soil, highlighting the importance of mineral inter- action for the long-term fate of PyOM in soils. Correspondence: Samuel Abiven (abiven@biotite.ens.fr) The nSOC export from the sandy soil signiﬁcantly increased by 48 %– 270 % with addition of PyOM compared to the control, while no effect was found for the loamy soil after the whole perco- lation. Due to its high sorption afﬁnity towards the soil min- eral phase, PyOM can mobilize mineral-associated soil or- ganic matter in coarse-textured soils, where organo-mineral interactions are limited, while ﬁner-textured soils have the ability to re-adsorb the mobilized soil organic matter. Our results show that the vertical mobility of PyOM in soils is limited to a small fraction. Aging (oxidation) increases this fraction but also increases the PyOM surface reactivity and thus its long-term retention in soils. Moreover, the migration of PyOM affects the cycling of nSOC in coarse soils and thus inﬂuences the carbon cycle of ﬁre-affected soils. Vertical mobility of pyrogenic organic matter in soils: a column experiment Schiedung, Marcus ; Bellè, Severin-Luca ; Sigmund, Gabriel ; Kalbitz, Karsten ; Abiven, Samuel DOI: https://doi.org/10.5194/bg-17-6457-2020 DOI: https://doi.org/10.5194/bg-17-6457-2020 Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-195481 Journal Article Published Version The following work is licensed under a Creative Commons: Attribution 4. Posted at the Zurich Open Repository and Archive, University of Zurich ZORA URL: https://doi.org/10.5167/uzh-195481 Journal Article Published Version Originally published at: Schiedung, Marcus; Bellè, Severin-Luca; Sigmund, Gabriel; Kalbitz, Karsten; Abiven, Samuel (2020). Vertical mobility of pyrogenic organic matter in soils: a column experiment. Biogeosciences, 17(24):6457-6474. DOI: https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 https://doi.org/10.5194/bg-17-6457-2020 © Author(s) 2020. This work is distributed under the Creative Commons Attribution 4.0 License. 1 Introduction Pyrogenic organic matter (PyOM) is a product of artiﬁcial (biochar) or wildﬁre-induced incomplete combustion. It is one of the oldest global organic carbon (C) pools with res- idence times of several millennia (Coppola et al., 2018; Vertical mobility of pyrogenic organic matter in soils: a column experiment Marcus Schiedung1, Severin-Luca Bellè1, Gabriel Sigmund2, Karsten Kalbitz3, and Samuel Abiven1,4,5 1Department of Geography, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland 2Department of Environmental Geosciences, Centre for Microbiology and Environmental Systems Science, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria 3Institute of Soil Science and Site Ecology, Technische Universität Dresden, Pienner Straße 19, 01737 Tharandt, Germa 4L b t i d Gé l i CNRS É l l é i PSL U i it I tit t Pi Si L l 1Department of Geography, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland 2Department of Environmental Geosciences, Centre for Microbiology and Environmental Systems Science, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria 1Department of Geography, University of Zurich, Winterthurerstrasse 190, 8057 Zurich, Switzerland 2Department of Environmental Geosciences, Centre for Microbiology and Environmental Systems Science, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria 3Institute of Soil Science and Site Ecology, Technische Universität Dresden, Pienner Straße 19, 01737 Tharandt, Germ 4Laboratoire de Géologie, CNRS – École normale supérieure, PSL University, Institut Pierre Simon Laplace, Rue Lhomond 24, 75005 Paris, France 5 3Institute of Soil Science and Site Ecology, Technische Universität Dresden, Pienner Straße 19, 01737 Tharandt, Germany 4Laboratoire de Géologie, CNRS – École normale supérieure, PSL University, Institut Pierre Simon Laplace, Rue Lhomond 24, 75005 Paris, France 5CEREEP-Ecotron Ile De France, ENS, CNRS, PSL University, Chemin de busseau 11, 77140 Saint-Pierre-lès-Nemours, France 5CEREEP-Ecotron Ile De France, ENS, CNRS, PSL University, Chemin de busseau 11, 77140 Saint-Pierre-lès-Nemours, France M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experimen The quanti- ties and drivers controlling the vertical PyOM mobility in dissolved or particulate form in soils, its effect on non-ﬁre- derived SOC during its migration, and the inﬂuence of aging are not well understood so far. This is limiting our under- standing of the fate of PyOM in the terrestrial C cycle. Between the continuum of terrestrial and aquatic ecosys- tems, PyOM is continuously exported from soils to rivers with less seasonality compared to non-ﬁre-derived soil or- ganic matter (Dittmar et al., 2012a; Hockaday et al., 2007; Wagner et al., 2018). Globally, dissolved PyOM contributes to 0.1 %–15 % of the total riverine and marine dissolved or- ganic carbon (DOC) pool (Coppola and Druffel, 2016; Jones et al., 2020). Furthermore, the quantity of dissolved PyOM in rivers was found to be decoupled from the ﬁre history of the watershed (Ding et al., 2013; Santos et al., 2017), which indicates that soils are an important intermediate storage for PyOM in the terrestrial system prior to its export to aquatic ecosystems (Abiven and Santín, 2019; Bird et al., 2015; San- tín et al., 2016). In soils, PyOM contributes globally to 14 % (0 %–60 %) of the total soil organic carbon (SOC), which makes it one of the main components of organic matter (OM) in soils (Reisser et al., 2016). Pyrogenic organic matter is found with residence times in soils of several centuries to millennia, which is much more than the average age of SOC and is mainly attributed to its condensed and aromatic composition and thus increased stability against degradation (Kuzyakov et al., 2014; Santos et al., 2012; Schmidt et al., 2011; Singh et al., 2012). Within the soil proﬁle, PyOM contents can increase with depth, in- dicating that vertical transport determines its long-term fate in soils and terrestrial ecosystems (Hobley, 2019; Soucémar- ianadin et al., 2019). g y y In this study, we assessed the vertical PyOM mobility by conducting saturated soil column experiments (7 cm length) with ﬂow interruption. To trace mobilized PyOM, we applied highly 13C labeled (> 2.8 at. % in excess) ryegrass PyOM- carbon (PyOM-C) on a topsoil and subsoil of a sand and sandy loam and determined the effect of soil properties on the PyOM mobility. Moreover, we compared the vertical mo- bility of fresh and oxidized PyOM (accelerated aging with H2O2) to identify changes in its mobility with aging and associated surface oxidation. M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experimen 6458 Masiello and Druffel, 1998) and an annual global produc- tion from wildﬁres of 196–349 Tg of carbon (Jones et al., 2019). After a wildﬁre, around 12 %–27 % of the initially burned biomass can remain as PyOM on the site of burn- ing (Santín et al., 2015). It can directly enter the soils or can be transported laterally via mass transport prior to incor- poration. Mass transport of PyOM mainly occurs during the ﬁrst rain event after a ﬁre, resulting in a translocation and re- deposition within the landscape and eventually in a PyOM burial at depositional sites (Abney et al., 2019; Cotrufo et al., 2016; Rumpel et al., 2015). In addition to this natural- wildﬁre-derived PyOM, artiﬁcially produced biochar is used as agricultural soil amendment to improve soil properties and is recognized as a C sequestration strategy to mitigate climate change (Lehmann, 2007). interactions of PyOM with the soil mineral phase, which further depend on soil properties such as texture, pH and Fe/Al-(hydr-)oxides content (Hobley, 2019; Pignatello et al., 2015). In addition, PyOM is interacting with non-ﬁre-derived OM and inﬂuences its mobility by direct sorption and sta- bilization on PyOM surfaces (Jiang et al., 2019; Mukherjee and Zimmerman, 2013). However, due to its high molec- ular weight (rich in aromatic compounds), PyOM has a strong sorption afﬁnity to the soil mineral phase (Kaiser and Guggenberger, 2000). This can result in a mobilization of non-ﬁre-derived and less adsorbing OM that is already ad- sorbed on the soil mineral surfaces (Jiang et al., 2016; Oren and Chefetz, 2012; Zhang et al., 2017). Biotic and abiotic oxidation alter the surface reactiv- ity of PyOM by increasing the abundance of oxygen- and hydrogen-containing functional groups (e.g., carboxylic groups) and thus inﬂuencing its properties over time and with aging. Aging of PyOM is reported to enhance its water sol- ubility and thus its mobility in soils with vertical percolation (Abiven et al., 2011; Wagner et al., 2017). However, only a small fraction of the total PyOM (< 1%) was found to be solubilized and transported in soils (Abiven et al., 2011; Maestrini et al., 2014; Major et al., 2010). In addition, it is reported that PyOM found in ﬁre-affected watersheds under- went aging processes in soil prior to its export from the soils to the riverine system (Hockaday et al., 2006). https://doi.org/10.5194/bg-17-6457-2020 Published by Copernicus Publications on behalf of the European Geosciences Union. Published by Copernicus Publications on behalf of the European Geosciences Union. M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 2 Materials and methods resolution) by using diffuse reﬂectance infrared Fourier- transformed (DRIFT) spectroscopy (TENSOR 27 spec- trophotometer, Bruker, Fällanden, Switzerland). Figure 1 shows the differences in the spectra of oxidized and fresh PyOM for the three PyOM batches, indicating an overall in- crease in O- and H-containing functional groups after the PyOM oxidation (increases in absorbance at A, C, E and G; Chatterjee et al., 2012; Keiluweit et al., 2010; Wood, 1988), loss of aliphatic compounds (decrease in absorbance at B and H; Chatterjee et al., 2012; Keiluweit et al., 2010), a shift in aromaticity (increase at C and decrease at D) and decarbon- ation (decreases at D and H; Rechberger et al., 2017); see Fig. S2 in the Supplement for all spectra. 2.3 Soil column setup and percolation Columns of 11 cm length and 2.5 cm diameter were loaded with 49.0–57.5 g of soil to reach a bulk density of 1.4 ± 0.1 gcm−3. The columns were loaded (from bottom to top) with 1.5 cm of decarbonized and combusted quartz sand, 7 cm soil, 1 cm of a soil–PyOM mixture and 1 cm quartz sand. The soil–PyOM mixture contained 0.5 g of PyOM and 4.5 g of soil for each column, which is equivalent to 11 tPyOMha−1 input on the soil surface. Control columns without addition of PyOM were packed with 8 cm of soil. For each soil, four replicates with addition of fresh and oxidized PyOM and controls were used. The columns were packed dry and saturated from the bottom using a 0.01 M CaCl2 solution with a ﬂow of 0.1–0.2 mLmin−1. The percolation was conducted with 0.01 M CaCl2 from the top using Mar- iotte’s bottles for a constant pressure head and an adjusted ﬂow of 2 mLmin−1. In total, 8640 mL (on average 590 ± 7 soil pore volumes) was percolated through each column over 5 d. Subsamples of 200 mL were sequentially sampled after a percolation of 480, 1440, 3840, 6240 and 8640 mL. This is equivalent to 1000, 3000, 8000, 13 000 and 18 000 Lm−2. The percolation was stopped for 3–5 h (ﬂow interruption) during the sampling, but the columns were continuously sat- urated. The experiment was conducted at room temperature and the columns were protected from light to avoid photo- degradation. The percolated samples were not ﬁltered to re- duce the risk of sample cross contamination or losses. There- fore, the percolates contained the total mobile fraction of PyOM and nSOC including colloidal and dissolved forms. 2.1 Soils Topsoils (0–10 cm) and subsoils (40–60 cm) of a sand and a sandy loam, hereafter sandy and loamy soil, were used to obtain a range of texture and SOC con- tents between 2.64–21.91 gCkg−1 (Table 1). The sandy soil was sampled from an Entic Podzol near Gifhorn (55◦22′44.0′′ N; 10◦25′224.5′′ E), Germany, under pine for- est (Pinus sylvestris). The loamy soil was sampled from a Haplic Luvisol east of Eiken (47◦32′33.5′′ N; 8◦00′31.5′′ E), Switzerland, under mixed forest dominated by beech (Fagus sylvatica). Both sites are similar in terms of mean annual temperature (sandy soil = 8.8 ◦C and loamy soil = 10.0 ◦C) and mean annual precipitation (sandy soil = 620 mm and loamy soil = 780 mm). All soil samples were dried at 40 ◦C over night and sieved to < 2mm. The three batches of ryegrass PyOM varied in C content, δ13C, O : C, H : C ratios and DRIFT spectra (particularly batch 3). This can be attributed to growing differences even under similar and controlled conditions. To take into account the variability, all calculations are based on the individual C and δ13C values (Table 2). The pH was lower in the sandy topsoil (3.4 ± 0.1) com- pared to the loamy topsoil (5.3 ± 0.1) but similar in both subsoils with 4.1 ± 0.1 and 4.0 ± 0.1, respectively. Oxalate- extractable amorphous Fe and Al (hydr)oxides (Fe(o) and Al(o)) contents were higher in the loamy soil compared to the sandy soil (Table 1). The soils further differed in the SOC distribution between free particulate organic matter (fPOM) and mineral-associated organic matter (MAOM) obtained by density fractionation (see Sect. 2.4 for procedure). The MAOM fraction contained 82.8 ± 0.2 % and 85.7 ± 1.0 % of the SOC in the loamy topsoil and subsoil, respectively. The sandy topsoil contained 73.2 ± 0.6 % of the SOC as fPOM. In the sandy subsoil, the fPOM contributed to 41.1 ± 0.8 % of the SOC. M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experimen Using highly 13C labeled rye- grass PyOM under these controlled conditions allowed us, on the one hand, to trace even small proportions of mobi- lized PyOM-C in soils and PyOM-C potentially exported to aquatic ecosystems and, on the other hand, to detect changes in the native soil organic carbon (nSOC) mobility. We hy- pothesized that (i) PyOM is continuously exported from the soil but its rate decreases over time, (ii) a higher degree of oxidation (aging) increases the mobility of PyOM through soils, (iii) PyOM can be retained in soils during its migra- tion and (iv) PyOM migration through the soil inﬂuences the nSOC mobility. Pyrogenic organic matter and non-ﬁre-derived OM differ in biological (e.g., degradability), physical and chemical in- teractions in soils (Bird et al., 2015; Pingree and DeLuca, 2017). Pyrolysis affects the chemical and physical properties of the feedstock organic matter, which results in a high poros- ity and larger surface areas depending on the fuel biomass, duration and production temperature (Hammes and Abiven, 2013; Lehmann et al., 2015; Preston and Schmidt, 2006). The feedstock is one of the major precursors, and PyOM de- rived from grass, for example, is reported with smaller and lighter particles compared to wood-derived PyOM (Saiz et al., 2018). The chemical composition, physical properties and the particle size control the mobility in soils and the https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6459 https://doi.org/10.5194/bg-17-6457-2020 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6460 Table 1. Soil texture, total organic carbon (TOC), δ13C, pH, electrical conductivity (EC), oxalate-extractable Fe(o) and Al(o), and density ractions (free particulate organic matter, fPOM; and mineral-associated organic matter, MAOM) for the topsoil (0–10 cm depth) and subsoil 40–60 cm) of the loamy and sandy soil (±1 SE). See Sect. 2.4 for methods. Table 1. Soil texture, total organic carbon (TOC), δ13C, pH, electrical conductivity (EC), oxalate-extractable Fe(o) and Al(o), and density fractions (free particulate organic matter, fPOM; and mineral-associated organic matter, MAOM) for the topsoil (0–10 cm depth) and subsoil (40–60 cm) of the loamy and sandy soil (±1 SE). See Sect. 2.4 for methods. Table 1. Soil texture, total organic carbon (TOC), δ13C, pH, electrical conductivity (EC), oxalate-extractable Fe(o) and Al(o), and density fractions (free particulate organic matter, fPOM; and mineral-associated organic matter, MAOM) for the topsoil (0–10 cm depth) and subsoil (40–60 cm) of the loamy and sandy soil (±1 SE). See Sect. 2.4 for methods. Soil depth Texture∗ TOC δ13C pH (CaCl2) EC (CaCl2) Fe(o) Al(o) Density fractions [cm] [%] [gkg−1 soil] [‰] [−] [mScm−1] [gkg−1] [gkg−1] [% of total SOC] Sand Silt Clay fPOM MAOM Loamy soil 0–10 65 22 13 20.14 (0.67) −29.1 (0.2) 5.3 (0.1) 2.33 (0.01) 1.85 (0.17) 1.30 (0.17) 17.2 (0.2) 82.8 (0.2) 40–60 62 23 15 5.66 (0.18) −28.9 (0.2) 4.0 (0.1) 2.35 (0.03) 2.12 (0.10) 1.99 (0.15) 14.3 (1.0) 85.7 (1.0) Sandy soil 0–10 88 5 7 21.91 (0.01) −28.8 (0.1) 3.4 (0.1) 2.45 (0.01) 1.01 (0.12) 0.43 (0.03) 73.2 (0.6) 26.9 (0.6) 40–60 92 3 5 2.64 (0.08) −30.1 (0.2) 4.1 (0.1) 2.34 (0.01) 0.93 (0.04) 0.83 (0.10) 41.1 (0.8) 58.9 (0.8) ∗According to WRB texture classes. Table 2. Total C, δ13C, O : C and H : C ratios of PyOM used for the corresponding soil column experiment (±1 SE). See Sect. 2.4 for methods. Table 2. Total C, δ13C, O : C and H : C ratios of PyOM used for the corresponding soil column experim methods. Table 2. Total C, δ13C, O : C and H : C ratios of PyOM used for the corresponding soil column experiment (±1 SE). See Se methods. PyOM Soil Type Total C δ13C O : C H : C [%] [‰] [−] [−] 1. Loamy Fresh 40.6 (0.8) 4136 (5) 0.51 0.71 topsoil Oxidized 42.1 (1.5) 4202 (5) 0.50 0.75 2. M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment Loamy Fresh 35.7 (1.2) 3472 (8) 0.55 0.72 subsoil Oxidized 38.9 (2.1) 3458(19) 0.54 0.72 3. Sandy topsoil Fresh 48.5 (1.8) 2877 (6) 0.45 0.64 and subsoil Oxidized 49.4 (1.3) 2943 (3) 0.42 0.66 2.4 Soils and percolate sample preparation and analyses 2.2 13C labeled pyrogenic organic matter Highly 13C labeled ryegrass (Lolium Perenne L.) was pro- duced in the multi-isotope controlled environment facility at the University of Zurich (Studer et al., 2017). The ryegrass was oven-dried at 40 ◦C and pyrolyzed at 450 ◦C for 4 h un- der N2 atmosphere (Hammes et al., 2006). Three indepen- dent growing batches of the initial ryegrass were pyrolyzed separately and were used in our experiment as a proxy for PyOM (Table 2). Artiﬁcially altered PyOM was produced by chemical- and heat-accelerated oxidation presented by Cross and Sohi (2013); in brief, 1 g of C was oxidized with 0.1 mol of H2O2 at 80 ◦C for 2 d. The samples were gently shaken ﬁve to seven times a day to ensure a homogeneous reaction. The samples were dried at 105 ◦C over night. The oxidation was conducted in glass test tubes (20 cm length and 2.5 cm diameter) with 1 g of PyOM using a 5 % H2O2 solution. These test tubes ensured a continuous exposure of PyOM to the oxidant. The oxidized PyOM from ﬁve test tubes was ho- mogenized and stored in a desiccator. Mid-infrared spectra at wavelengths from 4000–400 cm−1 were recorded (average of 64 scans per sample at 4 cm−1 Biogeosciences, 17, 6457–6474, 2020 https://doi.org/10.5194/bg-17-6457-2020 6460 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment Table 1. Soil texture, total organic carbon (TOC), δ13C, pH, electrical conductivity (EC), oxalate-extractable Fe(o) and Al(o), and density fractions (free particulate organic matter, fPOM; and mineral-associated organic matter, MAOM) for the topsoil (0–10 cm depth) and subsoil (40–60 cm) of the loamy and sandy soil (±1 SE). See Sect. 2.4 for methods. Soil depth Texture∗ TOC δ13C pH (CaCl2) EC (CaCl2) Fe(o) Al(o) Density fractions [cm] [%] [gkg−1 soil] [‰] [−] [mScm−1] [gkg−1] [gkg−1] [% of total SOC] Sand Silt Clay fPOM MAOM Loamy soil 0–10 65 22 13 20.14 (0.67) −29.1 (0.2) 5.3 (0.1) 2.33 (0.01) 1.85 (0.17) 1.30 (0.17) 17.2 (0.2) 82.8 (0.2) 40–60 62 23 15 5.66 (0.18) −28.9 (0.2) 4.0 (0.1) 2.35 (0.03) 2.12 (0.10) 1.99 (0.15) 14.3 (1.0) 85.7 (1.0) Sandy soil 0–10 88 5 7 21.91 (0.01) −28.8 (0.1) 3.4 (0.1) 2.45 (0.01) 1.01 (0.12) 0.43 (0.03) 73.2 (0.6) 26.9 (0.6) 40–60 92 3 5 2.64 (0.08) −30.1 (0.2) 4.1 (0.1) 2.34 (0.01) 0.93 (0.04) 0.83 (0.10) 41.1 (0.8) 58.9 (0.8) ∗According to WRB texture classes. Table 2. 2.2 13C labeled pyrogenic organic matter Total C, δ13C, O : C and H : C ratios of PyOM used for the corresponding soil column experiment (±1 SE). See Sect. 2.4 for methods. PyOM Soil Type Total C δ13C O : C H : C [%] [‰] [−] [−] M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6461 Figure 1. Difference of oxidized and fresh PyOM DRIFT spectra for PyOM 1 (loamy topsoil), PyOM 2 (loamy subsoil) and PyOM 3 (sandy soil). Areas A–H indicate the main changes after oxidation, with the corresponding absorption bands indicating increases in A = 3500– 3200 cm−1, C−O bonds, hydroxyl groups and H2O; C = 1730–1680 cm−1, aromatic carbonyl/carboxyl C=O bonds and C=C bonds (1590– 1560 and 1620–1610 cm−1); E = 1375cm−1, O−H bonds and G = 1060–1020 cm−1, C−O bonds. Oxidation decreased the absorption at bands B = 2980–2820 cm−1, aliphatic C−H bonds; D = 1500cm−1, aromatic C=C bonds; F = 1280–1200 cm−1, C−O and H−O bonds and H = 880 and 805 cm−1, aliphatic C−H bonds. Decreases at 1480 and 864 cm−1 can be assigned to a decarbonation with oxidation. See Fig. S1 in the Supplement for full spectra. Figure 1. Difference of oxidized and fresh PyOM DRIFT spectra for PyOM 1 (loamy topsoil), PyOM 2 (loamy subsoil) and PyOM 3 (sandy soil). Areas A–H indicate the main changes after oxidation, with the corresponding absorption bands indicating increases in A = 3500– 3200 cm−1, C−O bonds, hydroxyl groups and H2O; C = 1730–1680 cm−1, aromatic carbonyl/carboxyl C=O bonds and C=C bonds (1590– 1560 and 1620–1610 cm−1); E = 1375cm−1, O−H bonds and G = 1060–1020 cm−1, C−O bonds. Oxidation decreased the absorption at bands B = 2980–2820 cm−1, aliphatic C−H bonds; D = 1500cm−1, aromatic C=C bonds; F = 1280–1200 cm−1, C−O and H−O bonds and H = 880 and 805 cm−1, aliphatic C−H bonds. Decreases at 1480 and 864 cm−1 can be assigned to a decarbonation with oxidation. See Fig. S1 in the Supplement for full spectra. between density fractions. All samples were dried at 40 ◦C over night. in 12 fractions each sampled after 6 min (see Fig. S2 for all BTCs). The percolated volume was added to the last perco- late fraction. Soil and density fraction samples were milled for further measurement. If the sample mass of the fPOM fraction was too little, the samples were ground manually with a mortar and pestle to reduce a potential loss of sample material. The freeze-dried percolate samples were homogenized by man- ual grinding. Bulk soil and bulk PyOM samples were dried at 40 ◦C over night and milled. 2.5 Hydraulic properties of soil columns Following the percolation experiment, breakthrough curves (BTCs) were conducted in order to evaluate similar hydraulic properties and ﬂow conditions. Afterwards the soils were sampled as described above (Sect. 2.4). The BTCs were per- formed using a NaCl (1 gL−1) solution as an inert tracer. The tracer solution was added for 25 min with a constant pressure head allowing an average ﬂow of 1 mLmin−1. Afterwards, the percolation was continued with 0.01 M CaCl2 for a fur- ther 47 min. The EC of the percolated solution was measured M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment The total C and δ13C, relative to the international Vienna Pee Dee Belemnite (VPDB) stan- dard, of all solid samples were measured using a dry com- bustion module cavity ring-down spectroscopy system (Pi- carro, Santa Clara, USA). Since our soils were carbonate-free (pH < 6), the total organic C (TOC) was equal to the mea- sured total C. To obtain O : C and H : C ratios of the PyOM, C and H were measured by dry combustion and O by high- temperature pyrolysis (TruSpec Macro analyzer, Leco, Saint Joseph, USA). The convective velocity v [cmmin−1] and the diffusion coefﬁcient D [cm2 min−1] were estimated by using STAN- MOD (version 2.08.1130; Šim˚unek et al., 2003) to solve the deterministic equilibrium convection–diffusion equation. Due to high ﬂow rates under saturated conditions, it can be assumed that diffusion of the tracer within the soil was negli- gible, and the dispersivity λ [cm] was calculated as the quo- tient of the D and v (Vanderborght and Vereecken, 2007). No signiﬁcant differences in dispersivity were found between the columns of the same soil: 0.29 ± 0.04 cm for the loamy topsoil, 0.19±0.04 cm for the loamy subsoil, 0.13±0.01 cm for the sandy topsoil and 0.14 ± 0.02 cm for the sandy sub- soil (see Table S1 in the Supplement for all values). There- fore, the packing and the addition of the soil–PyOM layer did not lead to any signiﬁcant trend for changes in the hy- draulic properties of the columns, and thus hydraulic param- eters were excluded as further explanatory variables in this experiment. 2.4 Soils and percolate sample preparation and analyses The soil columns were sampled after drainage from the bottom to the top to avoid any cross contamination with la- beled material from soil–PyOM layer. Three soil layers of 2.3 cm below the soil–PyOM layer were sampled and dried at 40 ◦C over night, corresponding to 0–2.3, 2.3–4.6 and 4.6– 7.0 cm depth layers. Amorphous Fe(o) and Al(o) were extracted by oxalate ex- traction according to McKeague et al. (1971) and measured using atomic absorption spectroscopy (ContrAA 700, Ana- lytik Jena, Jena, Germany). Soil texture was determined after oxidizing the organic material with H2O2, after which sam- ples were wet sieved < 63 mm and the silt / clay fraction fur- ther quantiﬁed by a Sedimat 4-12 (UGT, Müncheberg, Ger- many). Density fractionation was conducted with 5 g of the ﬁrst 0–2.3 cm below the soil–PyOM layer and bulk soil samples. A sodium polytungstate (SPT) solution adjusted to a density of 1.8 gcm−3, as recommended by Lavallee et al. (2020), was used to separate the fPOM after shaking and settling for 1 h. The ﬂoating fPOM was decanted after centrifugation (30 min at 4000 rpm) and vacuum ﬁltered using a glass ﬁber ﬁlter (< 0.7 µm). The fPOM on the ﬁlter was rinsed with deion- ized water to remove SPT. The remaining heavier MAOM was rinsed with deionized water and centrifuged three times to remove SPT. No further fractions were acquired, and the commonly used ultrasoniﬁcation, to separate occluded par- ticulate organic matter, was avoided to reduce the risk of physical breakdown of PyOM particles and potential shift At each sequential sampling time, the pH and EC of the percolates were measured (914 pH/Conductometer, Metrohm, Herisau, Switzerland). The pH and EC of bulk soils were measured using a soil–solution ratio of 1 : 2.5 in a 0.01 M CaCl2 solution after shaking and settling for 1 h. The percolate samples were stored at 4 ◦C for a maxi- mum of 3 weeks. If longer storage was required, the samples were stored frozen. All liquid samples were freeze-dried and weighed prior to further analysis. https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 2.6 Calculations and statistics The recovery of 13C derived from the labeled PyOM was calculated with the atomic 13C fractions following the rec- ommendations presented by Coplen (2011). The measured δ13C values were used to calculate the isotope-amount ratios R(13C/12C)sample of each sample, using an isotope-amount ratio of 0.01118 for the VPDB standard. The atomic frac- https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 6462 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment Figure 2. Cumulative leaching loss of percolated fresh and oxidized PyOM from sandy and loamy topsoil and subsoil. The p values indicate the signiﬁcance of differences between fresh and oxidized PyOM after a total percolation of 18 000 Lm−2. All values are shown with propagated SE. M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experimen 6462 Figure 2. Cumulative leaching loss of percolated fresh and oxidized PyOM from sandy and loamy topsoil and subsoil. The p values indicate the signiﬁcance of differences between fresh and oxidized PyOM after a total percolation of 18 000 Lm−2. All values are shown with propagated SE. tion of each sample x(13C)sample was calculated following Eq. (1): with less total C. The recovery of 13C in milligrams was cal- culated following Eq. (3): mrecovery13C = xE 13C sample xE 13C soil–PyOM × msample, (3) x 13C sample = R 13C/12C sample 1 −R 13C/12C sample . (1) (3) (1) where xE(13C)soil–PyOM is the excess-isotope-amount frac- tion of the corresponding soil–PyOM mixture, and msample is the total mass of C measured in the sample in milligrams. This recovery calculation allowed us to distinguish between three separate C pools: (1) TOC, (2) the labeled PyOM-C and (3) nSOC. On average, the total recovery of TOC in the soil and percolates was 91.6% ± 1.0 % and 91.4% ± 2.7 % of added PyOM-C. The recoveries were normalized to 100 % for further comparison. The excess-isotope-amount fraction of each sample xE(13C)sample was calculated following Eq. (2): xE 13C sample = x 13C sample −x 13C control, (2) (2) where x(13C)control is the atomic 13C fraction of the cor- responding soil. Here, the mean of the control columns was used to calculate the excess-isotope-amount fraction of the individual soil column depth after the percolation. 3.3 Percolated nSOC The percolation and export of oxidized PyOM-C from the columns were higher compared to fresh PyOM-C for all soils over the whole percolations (Fig. 2). After a total per- colation of 18 000 Lm−2, 0.70% ± 0.15 % of oxidized and 0.24%±0.03 % of fresh PyOM-C percolated from the sandy topsoil (p < 0.01). From the sandy subsoil, 0.64% ± 0.08 % of oxidized and 0.26% ± 0.07 % of fresh PyOM-C were leached (p < 0.01). The loamy topsoil showed a percola- tion of 0.40% ± 0.25 % of oxidized and 0.18% ± 0.03 % of fresh PyOM (p = 0.16). Signiﬁcantly more oxidized PyOM- C leached from the loamy subsoil, with 0.40% ± 0.06 % of oxidized PyOM-C compared to 0.17% ± 0.04 % of fresh PyOM-C (p < 0.01). The total amount of percolated oxi- dized and fresh PyOM-C did not differ signiﬁcantly between topsoil and subsoil for the loamy or the sandy soil. Between the two soils, the export of oxidized PyOM-C was signiﬁ- cantly increased (p < 0.01) for the sandy topsoil and subsoil compared to the loamy soil, while the export of fresh PyOM- C was not signiﬁcantly different (p = 0.38; Fig. 2). The addition of PyOM signiﬁcantly increased the total per- colated nSOC from the sandy topsoil and subsoil compared to the control but did not change the total nSOC export from the loamy soil (Fig. 3). The addition of fresh PyOM signiﬁ- cantly increased the total nSOC percolation compared to the oxidized PyOM in the sandy topsoil and subsoil (p = 0.01). In total, 3.5% ± 0.6% (0.73 ± 0.05 g of nSOC per kilogram of soil) of total initial nSOC leached with fresh PyOM and 2.5% ± 0.2 % (0.53 ± 0.02 g of nSOC per kilogram of soil) of total nSOC with addition of oxidized PyOM from the sandy topsoil. Thus, PyOM addition signiﬁcantly increased the nSOC leaching for the sandy topsoil compared to the control without PyOM addition, from which 1.7% ± 0.1% of the initial nSOC was leached (p < 0.01). From the sandy subsoil, 4.9%±0.4 % of the total nSOC percolated from the control while signiﬁcantly more nSOC percolated with fresh (9.9% ± 1.1 %; 0.26 ± 0.01 g of nSOC per kilogram of soil) and oxidized (7.8%±0.5 %; 0.21±0.01 g of nSOC per kilo- gram of soil) PyOM (p < 0.01). . Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experimen 6463 cant increase by 0.3–0.5 units. For the loamy topsoil, the ﬁrst ﬂush (1000 Lm−2) showed signiﬁcantly increased pH values by 0.5–0.8 units with addition of PyOM but thereafter ap- proached the pH of the percolates from the control. Changes in pH were less dominant in the loamy subsoil, but the pH increased continuously over the whole percolation. statistical analyses were performed using R version 4.0.0 (R Core Team, 2020). The standard error (error of the mean) of four replicates is presented for all data, and error propagation was applied for cumulative nSOC and PyOM-C ﬂuxes. Due to a potential sample contamination after the experiment, one control replicate of the sandy subsoil was excluded from the analysis. One replicate of the loamy subsoil with addition of oxidized PyOM was excluded from further analysis due to an unsteady ﬂow during the percolation. statistical analyses were performed using R version 4.0.0 (R Core Team, 2020). The standard error (error of the mean) of four replicates is presented for all data, and error propagation was applied for cumulative nSOC and PyOM-C ﬂuxes. Due to a potential sample contamination after the experiment, one control replicate of the sandy subsoil was excluded from the analysis. One replicate of the loamy subsoil with addition of oxidized PyOM was excluded from further analysis due to an unsteady ﬂow during the percolation. The EC increased signiﬁcantly with addition of PyOM (p < 0.01) in the ﬁrst percolates compared to the control for all soils (Table S2, Fig. S4). With further percolation, the EC equilibrated to the background value of the percolate solu- tion (2.20 mScm−1 of 0.01 M CaCl2) for all soils with and without addition of PyOM. 3.3 Percolated nSOC g y (p g ) The ﬁrst ﬂush of 1000 Lm−2 caused the highest export of PyOM-C from the soil columns (Table S2 in the Sup- plement) and contributed to 80.4 %–84.3 % of total perco- lated PyOM-C from the sandy soil and to 50.6 %–79.8 % of the total percolated PyOM-C from the loamy soil (Fig. 2). The ﬁrst ﬂush of PyOM-C was similar between the sandy topsoil and subsoil (2862.9–1114.7 µgPyOM-CL−1) and on- going over the whole percolation time. For the loamy top- soil, the ﬁrst ﬂush indicated higher PyOM-C concentra- tions (596.4–1411.4 µgPyOM-CL−1) compared to the sub- soil (347.5–724.2 µgPyOM-CL−1), but the concentrations decreased to non-detectable levels for the last percolation stage of 18 000 Lm2 from the loamy topsoil and were on- going for the subsoil. Between the loamy and sandy topsoil, the total relative nSOC percolation did not differ signiﬁcantly for the controls (p = 0.71) and with addition of oxidized PyOM (p = 0.86). But the addition of fresh PyOM resulted in a higher nSOC percolation in the sandy topsoil compared to the loamy top- soil (p = 0.01). For the subsoils, the total nSOC percola- tion was signiﬁcantly higher in sandy subsoil compared to the loamy subsoil with fresh PyOM (< 0.01) and oxidized PyOM (p < 0.01) but not for the controls (p = 0.28). The ﬁrst ﬂush (1000 Lm−2) showed the highest nSOC concentrations in the percolates (Table S2). From the sandy topsoil, 45.54±7.69 mgnSOCL−1 (p < 0.01) with fresh and 28.16 ± 2.09 mgnSOCL−1 (p = 0.05) with addition of oxi- dized PyOM were leached, whereas signiﬁcantly less nSOC was leached from the control (12.12 ± 0.24 mgnSOCL−1). The nSOC concentrations in the percolates were lower for the sandy subsoil (13.31–3.28 mgnSOCL−1) compared to the topsoil but higher for the last percolate, indicating an ongoing nSOC mobilization. The nSOC percolated from the loamy topsoil in the ﬁrst ﬂush was 9.65 ± 1.07 mgnSOCL−1 for the control and signiﬁcantly increased to 21.14±1.96 mgL−1 2.6 Calculations and statistics The mean atomic 13C fraction of all controls of the ﬁrst perco- late (1000 Lm−2) was used to calculate the excess-isotope- amount fraction of percolates from soil columns with addi- tion of PyOM. The ﬁrst percolates were observed to provide the most stable values for the atomic 13C fraction of the con- trol due to higher C contents compared to later percolates Signiﬁcant differences in PyOM-C between fresh and oxi- dized PyOM treatments were tested with a t test. To test sig- niﬁcant differences of nSOC in soils and percolates between controls and treatments with addition of PyOM, analysis of variance (ANOVA) was applied and p values were computed with the post hoc Tukey honest signiﬁcance difference of means method on a 95 % family-wise conﬁdence level. The https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 3.2 pH and electrical conductivity of percolates The initial pH of the percolates increased with addition of PyOM for all soils (Table S2, Fig. S3 in the Supplement). As an average of all percolates, the pH increased signiﬁ- cantly by 0.2±0.1 units in the percolates of the sandy topsoil. This effect was larger in the sandy subsoils with a signiﬁ- https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 6464 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment Figure 3. Cumulative leaching loss of native soil organic carbon (nSOC) from sandy and loamy topsoil and subsoil for controls and columns with addition of fresh and oxidized PyOM. The signiﬁcant differences (p < 0.05) of the total percolated nSOC after 18 000 Lm−2 are indicated by letters. All values are shown with propagated SE. M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6464 Figure 3. Cumulative leaching loss of native soil organic carbon (nSOC) from sandy and loamy topsoil and subsoil for controls and columns with addition of fresh and oxidized PyOM. The signiﬁcant differences (p < 0.05) of the total percolated nSOC after 18 000 Lm−2 are indicated by letters. All values are shown with propagated SE. (p = 0.01) and 15.89±2.08 mgL−1 (p = 0.16) with addition of fresh and oxidized PyOM, respectively. The loamy subsoil showed lower leached nSOC concentrations in the percolates with 7.86 ± 0.49 mgnSOCL−1 compared to the topsoil with addition of fresh (12.23±1.55 mgnSOCL−1; p = 0.07) and with oxidized PyOM (10.92±0.79 mgnSOCL−1; p = 0.07). recovered, respectively. The recovery of oxidized PyOM-C in these layers was mostly signiﬁcantly higher compared to the fresh PyOM-C (Fig. 4). Between topsoil and subsoil, the ﬁrst layer below the soil– PyOM layer (0–2.3 cm) showed no signiﬁcant differences in the recovery of PyOM-C for both sandy and loamy soil. The sandy subsoil indicated higher recoveries of oxidized PyOM- C (p = 0.12) compared to the topsoil in 2.3–4.6 cm depth but not for fresh PyOM-C (p = 0.68). In 4.6–7.0 cm depth be- low the PyOM layer, the recoveries of oxidized (p < 0.05) and fresh PyOM-C (p < 0.01) were signiﬁcantly higher in the sandy subsoils compared to the topsoil. The loamy sub- soil showed signiﬁcantly higher fresh (p = 0.05) and oxi- dized PyOM-C (p < 0.01) recoveries in 2.3–4.6 cm depth compared to the topsoil. 3.2 pH and electrical conductivity of percolates For the deeper layer (4.6–7.0 cm), the recoveries of oxidized (p < 0.05) and fresh PyOM-C (p < 0.01) were also higher in the subsoil than in the topsoil. The relative recovery of PyOM-C did not differ signiﬁcantly between the loamy and sandy soils for the same depths. 3.4 Changes in fresh and oxidized PyOM-C and nSOC in soil columns 3.4 Changes in fresh and oxidized PyOM-C and nSOC in soil columns After the percolation, 89 %–96 % of the fresh and oxidized PyOM-C remained at its initial location in the PyOM layer in both topsoils and subsoils. The ﬁrst 0–2.3 cm below the soil–PyOM layer contained the largest proportions of mo- bilized PyOM with no differences between oxidized and fresh PyOM (Fig. 4). The recovered PyOM-C from this layer ranged between 3.5 %–9.7 % (0.84–1.50 g of PyOM-C per kilogram of soil) and 3.6 %–10.1 % (0.52–1.06 g of PyOM- C per kilogram of soil) of added PyOM-C in the sandy and loamy soil, respectively. With greater soil depth, the recover- ies decreased to < 1 %. In the soil layers at 2.3–4.6 and 4.6– 7.0 cm below the soil–PyOM layer, only 0.01 %–0.13 % and 0.05 %–0.17 % of added fresh and oxidized PyOM-C were The total nSOC loss from the soil columns signiﬁcantly in- creased with PyOM for the sandy soil but not for the loamy soil (Fig. 5). The nSOC contents decreased by 1.8 ± 0.2 % https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 chiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6465 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6465 Figure 4. Recovery of PyOM-C in soil below the soil–PyOM layer of the sandy and loamy topsoil and subsoil (±1 SE). The p values indicate the signiﬁcance of differences between fresh and oxidized PyOM at each depth. Figure 4. Recovery of PyOM-C in soil below the soil–PyOM layer of the sandy and loamy topsoil and subsoil (±1 SE). The p values indicate the signiﬁcance of differences between fresh and oxidized PyOM at each depth. subsoil depth below the soil–PyOM layer (0–2.3 cm). In the loamy soil, 40.2±1.8 % of the PyOM-C was associated with the MAOM fraction in the topsoil and subsoil, given as an average of fresh and oxidized PyOM. In general, the propor- tions of PyOM-C found in the two fractions did not differ signiﬁcantly between the fresh and oxidized PyOM and be- tween the loamy topsoil and subsoil. The sandy subsoil in- dicated signiﬁcantly more oxidized PyOM-C in the MAOM fraction compared to the topsoil (p = 0.05). 3.4 Changes in fresh and oxidized PyOM-C and nSOC in soil columns (3.9 ± 2.6 g of nSOC per kilogram of soil) with fresh PyOM and by 0.8 ± 0.1 % (1.8 ± 1.3 g of nSOC per kilogram of soil) with addition of oxidized PyOM in the sandy top- soil (p < 0.01). In the sandy subsoil, the PyOM resulted in 4.8 ± 0.5 % (0.4 ± 0.2 g of nSOC per kilogram of soil) and 2.6 ± 0.3 % (0.3 ± 0.2 g of nSOC per kilogram of soil) lower nSOC contents after percolation with fresh and oxi- dized PyOM, respectively (p < 0.01). The losses of nSOC were signiﬁcantly larger with fresh PyOM than with oxidized PyOM in the sandy topsoil and subsoil (p = 0.01). 4.1 Mobility of PyOM Relative deviations of native soil organic carbon (nSOC) to control of the total soils in soil columns (over all depth) after the percolation for sandy and loamy topsoil and subsoil with addi- tion of fresh and oxidized PyOM (±1 SE). Negative values indicate leaching losses. The signiﬁcance of differences of fresh and oxi- dized PyOM and the signiﬁcance of the deviation from the control are shown with p values. No signiﬁcant differences were found for the loamy soil. Figure 5. Relative deviations of native soil organic carbon (nSOC) Figure 5. Relative deviations of native soil organic carbon (nSOC) to control of the total soils in soil columns (over all depth) after the percolation for sandy and loamy topsoil and subsoil with addi- tion of fresh and oxidized PyOM (±1 SE). Negative values indicate leaching losses. The signiﬁcance of differences of fresh and oxi- dized PyOM and the signiﬁcance of the deviation from the control are shown with p values. No signiﬁcant differences were found for the loamy soil. Figure 5. Relative deviations of native soil organic carbon (nSOC) to control of the total soils in soil columns (over all depth) after the percolation for sandy and loamy topsoil and subsoil with addi- tion of fresh and oxidized PyOM (±1 SE). Negative values indicate leaching losses. The signiﬁcance of differences of fresh and oxi- dized PyOM and the signiﬁcance of the deviation from the control are shown with p values. No signiﬁcant differences were found for the loamy soil. Figure 7. Total mobilized fresh (F) and oxidized (O) PyOM and its relative proportion in the percolates after total percolation and in the soil column in 0–2.3, 2.3–4.6 and 4.6–7.0 cm below the soil– PyOM application layer for the sandy and loamy topsoil and subsoil (±1 SE). The signiﬁcance values are presented in Figs. 2 and 4. Figure 7. Total mobilized fresh (F) and oxidized (O) PyOM and its relative proportion in the percolates after total percolation and in the soil column in 0–2.3, 2.3–4.6 and 4.6–7.0 cm below the soil– PyOM application layer for the sandy and loamy topsoil and subsoil (±1 SE). The signiﬁcance values are presented in Figs. 2 and 4. Figure 6. Relative recovery of fresh (F) and oxidized (O) PyOM-C in fPOM and MAOM fractions in the ﬁrst layer below the soil– PyOM layer (0–2.3 cm) of sandy and loamy topsoil and subsoil (±1 SE). 4.1 Mobility of PyOM The density fractionation of the ﬁrst 0–2.3 cm below the soil– PyOM layer revealed that large proportions of the PyOM- C remained in the light fPOM fraction in the sandy soil (Fig. 6). In the sandy topsoil, 93.3 ± 0.9 % of the PyOM- C was found in the fPOM fraction. The same fraction con- tributed to 86.3±1.7 % of the total PyOM-C in the ﬁrst sandy At the end of the experiment, 3.8 %–10.8 % of the added PyOM moved vertically from its initial location. This in- cludes PyOM recovered in 7 cm of soil below the soil– PyOM application layer and exported PyOM in the per- colates (Fig. 7). Large parts of the mobilized PyOM were https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experimen M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6466 6466 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment Figure 5. Relative deviations of native soil organic carbon (nSOC) to control of the total soils in soil columns (over all depth) after the percolation for sandy and loamy topsoil and subsoil with addi- tion of fresh and oxidized PyOM (±1 SE). Negative values indicate leaching losses. The signiﬁcance of differences of fresh and oxi- dized PyOM and the signiﬁcance of the deviation from the control are shown with p values. No signiﬁcant differences were found for the loamy soil. Figure 7. Total mobilized fresh (F) and oxidized (O) PyOM and its relative proportion in the percolates after total percolation and in the soil column in 0–2.3, 2.3–4.6 and 4.6–7.0 cm below the soil– PyOM application layer for the sandy and loamy topsoil and subsoil (±1 SE). The signiﬁcance values are presented in Figs. 2 and 4. Figure 7. Total mobilized fresh (F) and oxidized (O) PyOM and its relative proportion in the percolates after total percolation and in the soil column in 0–2.3, 2.3–4.6 and 4.6–7.0 cm below the soil– PyOM application layer for the sandy and loamy topsoil and subsoil (±1 SE). The signiﬁcance values are presented in Figs. 2 and 4. Figure 5. 4.2 Dynamic of mobilized PyOM However, mobilized PyOM is most likely less controlled by microbial decomposition due to the high stability of PyOM (Kuzyakov et al., 2014; Singh et al., 2012), allowing a po- tential deeper migration. It needs to be noted that we only included one type of PyOM (ryegrass derived and produced at 450 ◦C), which constrains general conclusions since the chemical and physical properties of PyOM, such as particle size, are highly dependent on the feedstock and production conditions (Lehmann et al., 2015; Saiz et al., 2018). We percolated the soil columns in total with 18 000 Lm−2, which would be equal to a continuous precipitation of 23– 29 years given the mean annual precipitation of the two sites (sandy soil = 620mm and loamy soil = 780mm). Therefore, the percolation applied here over 5 d was conducted at rela- tively high rates. This experimental duration avoided any ad- ditional decomposition within the columns and allowed us to maintain a continuously saturated soil column system. How- ever, given the experimental setup, we were not able to es- timate any PyOM transportation rate under ﬁeld conditions under which the transport is inﬂuenced by seasonal precipi- tation variations and unsaturated conditions. The PyOM-C export was continuous for the sandy top- soil and subsoil, indicating an ongoing mobilization and mi- gration through the coarse soil. Besides this large export of PyOM with the ﬁrst ﬂush, PyOM-C was not detectable in the last percolated fraction of the loamy topsoil. Therefore, large proportions of PyOM were able to migrate through the loamy topsoil with the ﬁrst ﬂush, but with decreasing PyOM-C con- centration, the retention and most likely the sorption to the mineral phase and OM increased. In comparison, the loamy subsoil retained larger proportions of the PyOM mobilized with the ﬁrst ﬂush (ﬂush of 51 %–58 % of the total exported PyOM) but continuously released the retained PyOM into the solution with further percolation. Under ﬁeld conditions, pedoturbation (e.g., due to swelling and shrinking of clay-rich soils) and bioturba- tion would potentially promote the vertical translocation of PyOM (Hobley, 2019; Rumpel et al., 2015). Physical frag- mentation and breakdown of PyOM during environmental aging reduces the particle size, which can increase the ver- tical mobility of PyOM due to decreasing friction of smaller particles during transport and the generation of colloids (Hobley, 2019; Pignatello et al., 2015; Spokas et al., 2014). 4.2 Dynamic of mobilized PyOM relatively limited mobility of PyOM observed under experi- mental and ﬁeld conditions compared to non-pyrolyzed OM. The ﬁrst ﬂush, leached from the columns with 1000 Lm−2, contributed to the highest export of PyOM from the soil columns, and the mobilized amounts decreased with the per- colation for all soils (Fig. 2 and Table S2). We hypothesized a continuous export of PyOM-C and decrease with time, which can be conﬁrmed with our experiment. Our results clearly in- dicate that the ﬁrst ﬂush is a major event of PyOM transport through soil and contributed to 80 %–84 % and 51 %–79 % of total exported PyOM from the sandy and loamy soil, re- spectively. This can be attributed to mobile PyOM fractions which are directly produced during the pyrolysis and easily mobilized with the initial ﬂux (Hilscher et al., 2009). There- fore, the initial ﬂux of PyOM may signiﬁcantly contribute to its total export from soils and its transition to aquatic systems under ﬁeld conditions. Most ﬁeld experiments and observa- tion miss the initial PyOM ﬂux (lateral and vertical) with the ﬁrst rain event after a ﬁre, resulting in a potential underesti- mation of the dissolved transport rates of PyOM (Santos et al., 2017). This may further cause an underestimation of the vertical PyOM transport from depositional landscape posi- tions after a redistribution following an initial lateral mass transport (Rumpel et al., 2015). The export of fresh PyOM with the ﬁrst ﬂush from the subsoils indicates that a large proportion can be mobilized before any aging-associated ox- idation occurs, which can be of signiﬁcance at depositional sites where relatively fresh PyOM can be buried. Don and Kalbitz (2005) reported that fresh and 12-month in situ-incubated litter from a variety of temperate forest trees (e.g., sycamore maple, mountain ash, beech, spruce and pine litter) can release 0.3 %–6.5 % of water-extractable DOC. Liebmann et al. (2020) applied highly 13C labeled beech lit- ter on the surface of a temperate forest ﬂoor and reported that after 22 months 11.2 % of the applied litter migrated within a depth of 0–140 cm, but 87 % of this mobilized lit- ter fraction was found in the upper 5 cm, indicating a minor importance of aboveground litter DOC input in deeper soils. M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6467 4.1 Mobility of PyOM translocated to the ﬁrst 0–2.3 cm below the soil–PyOM layer (3.5 %–10.1 % of added PyOM-C; Fig. 4). The total recovery of PyOM in this layer did not differ signiﬁcantly between the sandy and loamy soil. This indicates a potential accumula- tion close to the transition of the soil–PyOM layer and the upper 0–2.3 cm regardless of the soil texture. The PyOM- C recovered in the deeper soil depth of 2.3–7.0 cm and in the percolates was most likely derived from dissolved and colloidal PyOM. Dissolved and colloidal PyOM is reported to be a major mobile fraction in soils (Wagner et al., 2017, 2018). Abiven et al. (2011) reported that 0.31 wt. %–0.42 wt. % of PyOM was mobilized in a batch experiment without any soil addition as dissolved (< 0.45µm) and colloidal (0.45– 5 µm) forms. In ﬁeld incubation experiments (1 to 2 years), a small proportion of 0.041 %–0.004 % of initially added PyOM was reported to be mobilized vertically to > 15cm depth in dissolved forms (Maestrini et al., 2014; Major et al., 2010). Hilscher and Knicker (2011) reported that 2.3 % of added PyOM migrated to 5 cm depth, and 0.4 % was leached from soil columns (8 cm length) and found in the column outﬂow in a 1-year incubation experiment. We identiﬁed that 0.17 %–0.70 % of the added PyOM-C was exported from the soil columns (> 7cm; Fig. 2). This is in accordance with the Figure 6. Relative recovery of fresh (F) and oxidized (O) PyOM-C in fPOM and MAOM fractions in the ﬁrst layer below the soil– PyOM layer (0–2.3 cm) of sandy and loamy topsoil and subsoil (±1 SE). Biogeosciences, 17, 6457–6474, 2020 https://doi.org/10.5194/bg-17-6457-2020 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 4.4 PyOM retention in soil The majority of the mobilized PyOM was retained in the soil (92.8±1.0%). This indicates the importance of PyOM reten- tion in soils during its migration and thus conﬁrms our third hypothesis. The density fractionation of the ﬁrst soil layer (0– 2.3 cm) below the soil–PyOM layer revealed that most of the translocated PyOM remained as fPOM with 85.6 %–94.5 % and 56.9 %–61.4 % in the sandy and loamy soil, respectively (Fig. 6). More than one-third of the PyOM (40.2 ± 1.8%) in the loamy topsoil and subsoil, 0–2.3 cm below the soil– PyOM layer, however, was found to be association with the mineral phase. This is surprising considering the relatively short interaction time of PyOM and the soil mineral surface and soil aggregation in a saturated column experiment. y In a batch experiment, Abiven et al. (2011) reported an in- crease in water-extractable PyOM fraction by 40–50 times after 10 years of natural aging compared to recent PyOM and a higher aromaticity of the solubilized aged PyOM. This, however, was estimated without any soil interaction of the mobile fraction. Hockaday et al. (2006) provided indirect ev- idence that PyOM in a ﬁre-affected watershed is mainly de- rived from PyOM previously aged in soils and that it is un- dergoing a fractionation during its migration through the soil depending on its initial aromaticity. Velasco-Molina et al. (2013), identiﬁed that PyOM accumulated in tropical deep sombric horizons (> 40cm soil depth) and that this PyOM underwent an oxidation process which increased its content of carboxylic groups. In a recent study, Braun et al. (2020) reported a decreasing aromaticity of water-extractable PyOM extracted from agricultural soils compared to the bulk PyOM and no changes after 3 years of aging under ﬁeld condi- tions. Contrastingly, Wagner et al. (2017) reported an in- creased aromaticity of PyOM exported from soils with aging (> 100years) compared to fresh bulk PyOM. The authors, however, reported that the oxidation alone could not explain the reported higher mobility of PyOM with aging. This high- lights that oxidation is not only increasing its mobility but also its reactivity in soil during aging and its transport. 4.2 Dynamic of mobilized PyOM These colloids tend to become more mobile with aging, de- creasing particle size, and increasing pH, and in the presence of OM, whereas their mobility tends to decrease in the pres- ence of clay minerals, hydrophobic contaminants and high ionic strengths due to aggregation (Castan et al., 2019; Sig- mund et al., 2018). Fragmentation further increases the rel- ative speciﬁc surface area of PyOM and may promote stabi- lization and thus its retention in soils due to increased physic- ochemical interaction with the mineral phase (Singh et al., 2012; Xiao and Pignatello, 2015). The ongoing PyOM export from soils in our experiment could potentially explain the steady, less seasonally affected and from ﬁre history decoupled ﬂux of soil-derived PyOM to rivers as observed in the ﬁeld (Bao et al., 2019; Dittmar et al., 2012a, b; Santos et al., 2017; Wagner et al., 2018). In our experiment, the proportion of percolated PyOM-C of the total percolated C ranged between 2.4 %–17.2 % for the ﬁrst ﬂush and between 0.2 %–2.8 % for the last percolates, as an average of all soils (Table S2). Artiﬁcially produced PyOM is reported to have a higher stability than PyOM natu- rally produced during wildﬁres, which challenges the use of one type as a proxy for the other (Santín et al., 2017). The proportions observed here, however, are in line with globally reported dissolved black carbon proportions on total riverine https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6468 (3 %–15 %) and marine (0.1 %–2.9 %) DOC (Coppola and Druffel, 2016; Jaffé et al., 2013; Jones et al., 2020; Wagner et al., 2018). However, we did not investigate degradation (biotic or abiotic) of mobilized PyOM and nSOC after the export from the soil, which would increase the proportion of PyOM-C on the total DOC. surfaces with the soil mineral phase and the native soil OM. Scanning electron microscopy showed a preferential interac- tion of partially oxidized PyOM and the soil mineral phase (Brodowski et al., 2005). The increased PyOM reactivity and mobility with oxidation can be attributed to the higher abun- dance of functional groups containing hydrogen and oxy- gen (e.g., carboxyl groups, Fig. 1) on the oxidized surface (Knicker, 2011). 4.2 Dynamic of mobilized PyOM This oxidation consequently increased the polarity, which enhances its water solubility and its interac- tion with the mineral phase (Cheng et al., 2008; Fang et al., 2014; Pignatello et al., 2015; Zhao and Zhou, 2019; Zimmer- man, 2010). 4.3 Effect of oxidation on PyOM mobility and reactivity The oxidation signiﬁcantly increased the PyOM mobility and resulted in 2–7 times higher PyOM-C recoveries in the soil in 2.3–7.0 cm (Fig. 4) and 2–3 times higher losses through per- colation (Fig. 2) compared to fresh PyOM (Fig. 7). This con- ﬁrms our second hypothesis that oxidation (aging) is enhanc- ing the mobility of PyOM. The similar quantities of fresh PyOM-C found in the percolates from the sandy and loamy soil indicate that the mobility of fresh PyOM is most likely only partially inﬂuenced by soil texture and rather controlled by the total abundance of mobile compounds originated di- rectly from the pyrolysis. For the oxidized PyOM, however, the exported quantities of PyOM-C revealed a nearly 2-fold higher mobility in the sandy soil than in the loamy soil. This indicates a higher mobility of PyOM with aging in coarse- textured soils and a potentially higher retention of the oxi- dized PyOM in soils with a ﬁner texture. Our results show that the long-term fate of PyOM in soils is highly controlled by its degree of oxidation and thus will change with aging. However, aging will not only increase the PyOM mobility and solubilization but also its reactivity and thus interaction with the soil mineral phase and OM and thus its long-term sequestration. Therefore, future studies should include the effect of aging for more than a few years under ﬁeld conditions. 4.5 Effects of PyOM on nSOC mobility able clay and silt content and may be predominately con- trolled by the physical and chemical interaction rather than biological processes, since these were neglectable in our ex- periment given the short duration of 5 d. This rapid mineral interaction will control the long-term stability of PyOM in soils and requires more research under unsaturated and ﬁeld conditions and also different PyOM feedstocks. The addition of PyOM signiﬁcantly enhanced the total nSOC export in the sandy topsoil and subsoil by 48 %–270 %, which is equivalent to an additional loss of 0.07–0.37 g of nSOC per kilogram of soil (Figs. 3 and 5 and Table S2). The nSOC export from the loamy soil was signiﬁcantly increased compared to the control for the ﬁrst ﬂush (1000 Lm−2) by 56 %–105 % (0.04–0.10 g of carbon per kilogram of soil) but negligible over the whole percolation. Therefore, our fourth hypothesis can be partially accepted, and the effect of PyOM on nSOC mobility was clearly controlled by soil texture and properties of the soil and PyOM. y The higher proportion of PyOM recovered as fPOM in the sandy soil indicates that the PyOM–mineral interac- tion was limited, which is in agreement with the general higher proportion of TOC in fPOM fraction (41 %–73 %) and high sand contents (> 88%; Table 1). In coarse soils, Fe and Al (hydr)oxides are considered to interact and stabilize PyOM and SOC due to its great afﬁnity to OM (Brodowski et al., 2005; Pignatello et al., 2015; Wiesmeier et al., 2019). Slightly more fresh PyOM (p = 0.24) and signiﬁcantly more oxidized PyOM (p = 0.05) was recovered in the MAOM fraction in the sandy subsoil compared to the topsoil. This indicates that the sandy subsoil had a higher potential to re- tain PyOM due to increased mineral interaction compared to the topsoil. p p y With PyOM, the pH of the percolates increased in all soils except of the loamy subsoil, which can further enhance the nSOC mobility (Table S2, Fig. S3). Increases in pH can be associated with a liming effect due to carbonates which are formed during the pyrolysis (Smebye et al., 2016). The pH signiﬁcantly controls the mobility of DOC (Kaiser and Guggenberger, 2000; Kalbitz et al., 2000), and an increase by 0.5 pH units was shown to enhance the DOC export by 50 % (Tipping and Woof, 1990). 4.5 Effects of PyOM on nSOC mobility We found increases in pH by 0.2–0.8 units (Table S2, Fig. S3). The liming effect lasted over the whole percolation for the sandy soil, which also showed a continuously higher export of nSOC with PyOM compared to the control. Therefore, the increasing pH in the sandy soil, with low initial pH values of 3.4–4.1, may have caused a re-mobilization of adsorbed nSOC-derived DOC. The fresh PyOM had a signiﬁcantly higher liming effect as indicated in the ﬁrst ﬂush of the sandy topsoil, as well as the loamy topsoil and subsoil, while the oxidized PyOM caused a higher EC in the ﬁrst ﬂush (Table S2, Fig. S4). This can be attributed to a decarbonatization of PyOM with oxida- tion, which was also observed with the mid-infrared analysis (Fig. 1). Therefore, a liming effect of PyOM on the nSOC mobility will potentially decrease with aging but is substan- tial in coarse soils. Subsoils are unsaturated in OM due to little inputs, such as DOC from upper soil horizons, root-derived and already mi- crobially processed OM, but large mineral surfaces (Kaiser and Kalbitz, 2012; Lützow et al., 2006). Therefore, the prob- ability of mobilized PyOM to interact with the mineral phase is higher in the subsoil, resulting in a higher retention com- pared to topsoils where OM is already occupying sorption sites on the mineral surfaces. The subsoils of the loamy soil contained signiﬁcantly more PyOM at 2.3–7.0 cm depth be- low the soil–PyOM layer compared to the topsoil (Figs. 6 and 7). This was also the case for the last soil layer (4.6–7.0 cm) of the sandy subsoil and topsoil. Therefore, the loamy and sandy subsoils had a higher PyOM retention potential than topsoils, where the nSOC contents were 5–10 times higher. It needs to be noted that the PyOM retained in this depth rep- resented only < 1% of the added PyOM. In addition, we did not include unsaturated ﬂow conditions, which would resem- ble the mobility and retention under ﬁeld conditions. Barnes et al. (2014) reported that wood-derived PyOM (mesquite biochar, 400 ◦C, 4 h) resulted in higher DOC ﬂuxes from organic-poor sandy soil in a soil column experiment (ﬁlled with soil–PyOM mixtures containing 10 wt % PyOM) but attributed this increase to PyOM-derived C and not soil- derived C. . Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experimen 6469 4.4 PyOM retention in soil The soils in our experiment showed large differences in the contents of amorphous Fe(o) and Al(o) and in texture with 13 %–15 % clay and 22 %–23 % silt in the loamy soil com- pared to < 12 % silt and clay combined in the sandy soil (Ta- ble 1). The aromatic compounds of the PyOM can directly interact with the edge functional groups of clay (Joseph et al., 2010; Lehmann et al., 2007; Pignatello et al., 2015). Brodowski et al. (2006) found up to 24 % of PyOM in for- est soils to be occluded in aggregates and concluded that PyOM can act as a binding agent for the aggregate forma- tion. In a ﬁeld incubation experiment of 10 months, Singh et al. (2014) recovered > 25% of the added PyOM in the occluded fraction in temperate forest soils, and Vasilyeva et al. (2011) found up to 70 % of PyOM associated with the mineral fraction after 55 years of bare fallow in Chernozem. Here, the MAOM fractions contain PyOM which is in direct association with the mineral phase and occluded in aggre- gates. Our results show that the PyOM–mineral interaction and occlusions can occur very quickly in soils with reason- The reactivity of PyOM in soils clearly increased with oxi- dation in our experiment and caused a higher retention of ox- idized PyOM in 2.3–7.0 cm soil depth below the soil–PyOM layer in both soils (Fig. 4). This increased retention can be associated with enhanced interaction of the oxidized PyOM https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 4.5 Effects of PyOM on nSOC mobility It is likely that this mobilized nSOC would be adsorbed again during its further transport through the loamy soil (> 7cm). proportions of the mobilized PyOM were retained in the soil and accumulated mainly in particulate form close to the ini- tial layer within the ﬁrst 0–2.3 cm. Less than 1 % of the added PyOM migrated to greater depth and was exported from the soil (> 7cm). Furthermore, the majority of the ex- ported PyOM was mobilized with the ﬁrst ﬂush. Oxidation and thus aging of PyOM signiﬁcantly increased its mobility and also its reactivity, resulting in an overall larger mobiliza- tion but also larger retention in the soil of oxidized PyOM compared to fresh PyOM. Both can be clearly ascribed to the oxidized PyOM surfaces with a higher abundance of oxygen- and hydrogen-containing functional groups. The migration of oxidized PyOM was further largely inﬂuenced by the soil texture, resulting in a higher export from the sandy soil and higher retention in the loamy soil due to an increased associ- ation to the soil mineral phase. proportions of the mobilized PyOM were retained in the soil and accumulated mainly in particulate form close to the ini- tial layer within the ﬁrst 0–2.3 cm. Less than 1 % of the added PyOM migrated to greater depth and was exported from the soil (> 7cm). Furthermore, the majority of the ex- ported PyOM was mobilized with the ﬁrst ﬂush. Oxidation and thus aging of PyOM signiﬁcantly increased its mobility and also its reactivity, resulting in an overall larger mobiliza- tion but also larger retention in the soil of oxidized PyOM compared to fresh PyOM. Both can be clearly ascribed to the oxidized PyOM surfaces with a higher abundance of oxygen- and hydrogen-containing functional groups. The migration of oxidized PyOM was further largely inﬂuenced by the soil texture, resulting in a higher export from the sandy soil and higher retention in the loamy soil due to an increased associ- ation to the soil mineral phase. Dissolved organic matter mobility in sandy soils is mainly controlled by adsorption on Fe and Al (hydr)oxides. High- molecular-weight compounds with aromatic structures are reported to have a higher sorption afﬁnity and can desorb less adsorptive compounds from soil mineral phases (Cow- ard et al., 2019; Eusterhues et al., 2011; Kaiser and Kalbitz, 2012; Kalbitz et al., 2000). 4.5 Effects of PyOM on nSOC mobility This is also shown by a fraction- ation of DOC migration through the soil (Oren and Chefetz, 2012) Recently, Zhang et al. (2020) identiﬁed a higher sorp- tion afﬁnity of dissolved organic matter derived from maize straw PyOM by hydrophobic partition, H bonding and elec- trostatic interactions compared to non-pyrolyzed dissolved organic matter. Therefore, PyOM caused a re-mobilization of nSOC by desorbing it from complexes of the mineral phase and pre-existing OM. Due to a higher abundance of less ox- idized highly aromatic compounds from fresh PyOM com- pared to oxidized PyOM, the desorption of nSOC was more dominant with fresh PyOM, while oxidized PyOM adsorbed mainly on free mineral surfaces due to its higher reactivity. Fresh and oxidized PyOM signiﬁcantly increased the mo- bility of nSOC in the sandy soil and in the ﬁrst ﬂush of the loamy soil but not over the whole experiment. This can be attributed to a higher sorption afﬁnity of high-molecular- weight PyOM compounds to the mineral phase and thus a desorption of already sorbed and mineral-associated nSOC, which will eventually be exposed to microbial degradation. The re-mobilization of nSOC was greater in the sandy sub- soil compared to the topsoil, supporting the concept that sub- soil OM is already microbially processed and its association to the mineral phase is weak (because of low availability of mineral surfaces): hence it can be easily desorbed by younger OM with a higher sorption afﬁnity. Further research is needed to understand the fate of PyOM under unsaturated and ﬁeld conditions as well as larger scales such as pedon and catena, including PyOM from different feedstocks. We identiﬁed that the vertical PyOM mobility is highly dependent on soil properties and the degree of PyOM oxidation (age), which increases not only its mobility, but also reactivity in soils and inﬂuences its effect on nSOC. This will inﬂuence the pyrogenic carbon and nSOC dynamics in the vadose zone and between the terrestrial and aquatic sys- tems. The mobilization effect of PyOM on nSOC was more pro- nounced in the sandy subsoil with 7.8 %–9.9 % of the initial nSOC exported compared to 2.5 %–3.5 % of initial nSOC ex- ported from the topsoil (Fig. 3). 4.5 Effects of PyOM on nSOC mobility The authors found no increase in DOC ﬂux from organic- and clay-rich soil but identiﬁed an increase in the aromaticity of the DOC, and the authors identiﬁed that leach- able PyOM fractions were lost but soil-derived C was re- tained with PyOM. The authors concluded that the net in- crease in DOC export from soils with moderate amounts of clay, silt and OM is limited. Major et al. (2010) reported that wood-derived PyOM amendment (mango wood biochar, 400–600 ◦C, 48 h) in a savanna soil (sandy clay loam) in- creased the ﬂux of non-PyOM-derived particulate organic carbon and DOC by 2.3–4.1 times after 2 years under ﬁeld conditions. However, the authors attributed these increases to a higher belowground net primary production and a cor- The higher retention of PyOM in the subsoils can explain its accumulation in greater soil depth, which is found under ﬁeld conditions (Brodowski et al., 2007; Soucémarianadin et al., 2019). We showed that PyOM can be continuously re- mobilized from subsoils by percolating water and thus can potentially be exported to deeper soil depth or to the ground- water, which would ﬁnally result in the export from terrestrial to aquatic systems such as rivers. Furthermore, it can be con- sidered that large proportions of the mobilized PyOM will not be affected by microbial degradation to the same extent that non-pyrogenic SOC and DOC are during the percolation through the soil (Don and Kalbitz, 2005; Kuzyakov et al., 2014; Tipping et al., 2012). This would result in increasing proportions of PyOM on the total subsoil OM. https://doi.org/10.5194/bg-17-6457-2020 Biogeosciences, 17, 6457–6474, 2020 M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment M. Schiedung et al.: Vertical mobility of pyrogenic organic matter in soils: a column experiment 6470 responding increase in OM input with PyOM. Jones et al. (2012) found no change in the DOC ﬂux after PyOM (wood biochar, 450 ◦C, 48 h) from a sandy clay loam in a 3-year ﬁeld trial. By tracing the highly labeled PyOM-C, our re- sults conﬁrm that PyOM may have a limited effect on the nSOC mobility in ﬁne-textured loamy soils over the long- term since we could not identify signiﬁcant differences in the cumulative nSOC loss from the soil columns with fresh and oxidized PyOM. However, the ﬁrst ﬂush of dissolved PyOM enhanced the loss of nSOC signiﬁcantly in the loamy topsoil and subsoil. 4.5 Effects of PyOM on nSOC mobility This supports the concept of continuous sorption, microbial processing, and desorp- tion during the vertical OM migration and thus an accumu- lation of microbially processed OM in subsoils as described by Kaiser and Kalbitz (2012) as the cascade concept. The binding of microbial processed OM to the mineral surfaces is weaker than for plant-derived compounds (high in aliphatic, aromatic and carboxylic groups), and thus it is more easily mobilized by PyOM with a high sorption afﬁnity towards the mineral surfaces. We found that even a small fraction of mo- bilized PyOM may cause a signiﬁcant mobilization of po- tentially labile nSOC. Even if this effect decreased with oxi- dized PyOM, it indicates a long-term effect on nSOC mobil- ity and inﬂuence of the C cycle of ﬁre-affected soils. Data availability. The data related to this article are available at https://doi.org/10.5281/zenodo.4268490 (Schiedung and Abiven, 2020). Supplement. The supplement related to this article is available on- line at: https://doi.org/10.5194/bg-17-6457-2020-supplement. Author contributions. MS and SA designed the experiment. MS conducted the experiment, analyzed the data and wrote the manuscript. SA, SLB, GS and KK provided input to the data dis- cussion and the manuscript. References Coplen, T. B.: Guidelines and recommended terms for ex- pression of stable-isotope-ratio and gas-ratio measure- ment results, Rapid Commun. Mass Sp., 25, 2538–2560, https://doi.org/10.1002/rcm.5129, 2011. 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https://openalex.org/W3091030324	https://pure.ulster.ac.uk/ws/files/86187893/Main_paper_unblinded_86008527_.pdf	English	null	‘The Lynchpin of the Acute Stroke Service’—An envisioning of the scope and role of the advanced nurse practitioner in stroke care in a qualitative study	Journal of clinical nursing	2,020	cc-by	10,179	General rights Th i ht General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. eneral rights e copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document Unless otherwise stated, users are permitted to download a copy of the output for personal study or non-commercial research and are permitted to freely distribute the URL of the output. They are not permitted to alter, reproduce, distribute or make any commercial use of the output without obtaining the permission of the author(s). If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. If the document is licenced under Creative Commons, the rights of users of the documents can be found at https://creativecommons.org/share-your-work/cclicenses/. Link to publication record in Ulster University Research Portal Publication Status: Published (in print/issue): 01/12/2020 Document Version Author Accepted version Document Licence: CC BY General rights The copyright and moral rights to the output are retained by the output author(s), unless otherwise stated by the document licence. The Lynchpin of the Acute Stroke Service” – An envisioning of the scope and role of the advanced nurse practitioner in stroke care in a qualitative study Laird, LEA., McCauley, C.-O., Ryan, A., & Beattie , A. (2020). The Lynchpin of the Acute Stroke Service” – An envisioning of the scope and role of the advanced nurse practitioner in stroke care in a qualitative study. Journal of Clinical Nursing, 29(23-24), 4795-4805. https://doi.org/10.1111/jocn.15523 Laird, LEA., McCauley, C.-O., Ryan, A., & Beattie , A. (2020). The Lynchpin of the Acute Stroke Service” – An envisioning of the scope and role of the advanced nurse practitioner in stroke care in a qualitative study. Journal of Clinical Nursing, 29(23-24), 4795-4805. https://doi.org/10.1111/jocn.15523 Link to publication record in Ulster University Research Portal Take down policy Take down policy The Research Portal is Ulster University's institutional repository that provides access to Ulster's research outputs. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact pure-support@ulster.ac.uk Download date: 24/10/2024 Abstract Background: Stroke prevalence is rising internationally. Advanced practice nursing is established across many jurisdictions, however its contribution to stroke services is under researched. Aim: To gain insights into the future scope and role of future advanced nurse practitioners in stroke care from the perspectives of key stakeholders. Design: A qualitative descriptive approach Methods: Interviews were conducted in 2019 with a purposive sample of 18 participants, comprising stroke nurses, stroke unit managers, stroke survivors and their family carers, recruited in one UK healthcare trust. The research is reported in line with COREQ. Data were analysed in accordance with an inductive content analysis approach. Results: The abstraction process generated four main themes. These were “The lynchpin of the acute stroke service”, “An expert in stroke care”, “Person and family focussed”, and “Preparation for the role”. Conclusion: These findings offer new perspectives on the potential scope and role of advanced nurse practitioners in stroke service delivery. Further research should focus on how to address the challenges confronted by advanced nurse practitioners when endeavouring to engage in autonomous clinical decision-making. Impact: Study findings may advance post registration education curricula, clinical supervision models and research directions. Relevance to clinical practice: There is support for the implementation of advanced practice nursing in the hyper acute and acute stroke phases of the care pathway. An interprofessional model of clinical supervision has potential to support the developing advanced nurse practitioner in autonomous clinical decision making. Keywords: Advanced Nurse Practitioners, Education, Leadership, Qualitative Approaches, Stroke eywords: Advanced Nurse Practitioners, Education, Leadership, Qualitative Approaches, Stroke Introduction Stroke can affect people of all ages but is more prevalent among adults aged 65 years and older. In tandem with ageing demographic trends and rising prevalence of metabolic syndrome, it is anticipated that the prevalence of stroke will increase globally (GBD 2016 Stroke Collaborators, 2019). In the UK, the expectation is that there will be a 44% rise in the number of people experiencing a new stroke over the next twenty years (Kings College London, 2017). The quality of stroke care varies across the UK (Royal College of Physicians, 2017), and this is despite robust research that demonstrates that access to hyper acute and acute stroke units, and faster intervention of thrombolysis and thrombectomy, generate improved clinical outcomes (Friebel et al., 2018; Hastrup et al., 2018). To date, stroke nurses have been pivotal to the delivery of hyper acute and acute stroke services internationally (Middleton et al., 2015; Kummarg et al., 2018). In addition, research has demonstrated that nurse led protocols for management of pyrexia, hyperglycaemia and swallowing in the acute stroke phase reduce mortality and have potential for long term and sustained benefits for patients (Middleton et al., 2017). This paper presents a qualitative study that addresses the future role and scope of an advanced nurse practitioner in the delivery of stroke services in a region of the UK. Background Advanced practice nursing is firmly established across many jurisdictions, including the US (National Council of State Boards of Nursing APRN Advisory Committee, 2010), Australia (Nursing and Midwifery Board of Australia, 2016) and the UK (Royal College of Nursing [RCN], 2018). Advanced practice nursing defines a higher level of practice demonstrated by advanced clinical skills, experience and knowledge. The advanced nurse practitioner (ANP) is educated to at least Master’s degree (RCN, 2018), and in the US, nurse practitioners must be certified and licensed. In the UK, nurses who meet the required competencies for ANPs will have their advanced status and prescribing authority recorded on the Nursing and Midwifery Council register. There is a paucity of published peer reviewed research nationally and internationally that has addressed advanced practice nursing in the sphere of stroke care. In the grey literature, Minchin and Wensley (2003) reported that the introduction of medical nurse practitioners, defined as experienced registered nurses with further training and competence in assessing, diagnosing and treating complex problems, facilitated timely referral to the specialist stroke team. More recently, Sanders (2019) reported that the implementation of an advanced nurse practitioner programme in one stroke service supported enhanced quality of care. A scoping review undertaken by Wilson & Ashcroft (2018) has described a variety of nurse practitioner roles in stroke care in primary care, acute hospital care and secondary prevention in the community. The four regions of the UK have made a commitment to enhancing and reshaping their stroke services towards centralised models of care (NHS England, 2019; NHS Scotland, 2019; NHS Wales, 2017; Northern Ireland Health and Social Care Board, 2017). There is potential that the implementation of ANPs in stroke care delivery can drive enhancements and efficiencies. ANPs in the UK are supported by standards developed by the Royal College of Nursing (2018), and regional practice frameworks (Northern Ireland Practice Education Council [NIPEC], 2016). Nurse prescribing is a prerequisite skill to advanced nursing practice in the UK, and there is an expectation that ANPs will contribute highly developed assessment, diagnostic, analytical and clinical judgemental skills, and perform many functions previously reserved for doctors (RCN, 2018). This has relevance in stroke care as pharmacological interventions and management are pivotal to the prevention, treatment, and long-term management of stroke. In addition, there is potential for the ANP to expedite the treatment regimens for patients with ischaemic stroke in the hyper acute stroke phase. Methods The study employed a qualitative descriptive approach that utilised semi-structured interviews for the collection of data. The qualitative description is a design that seeks to produce a low inference description of a phenomenon, with the intention to remain close to the original data (Neergaard et al., 2009; Sandelowski, 2000, 2010). With regards to methods, a variation sampling approach is appropriate, in order to gain a broad insight into the phenomenon (Neergaard et al., 2009). In addition, semi-structured interviews are commonly used in data collection, to glean a straightforward description generated from a range of perspectives of individuals and groups (Lambert & Lambert, 2012). The study was conducted in accordance with the consolidated criteria for reporting qualitative research (COREQ supplementary file 1) (Tong et al., 2007). It incorporated a purposive sampling approach to generate perspectives from adults that had experienced a stroke, and their family carers, in addition to senior stroke nurses, specialist stroke nurses, and stroke service managers. Background Whilst the evidence demonstrates that faster time to initiation of lysis treatment, is associated with improved clinical outcomes, it is important to point out that lysis treatment is associated with adverse events for up to 6% of patients (Jahan et al., 2019). To date, there are no ANPs employed in stroke services in Northern Ireland. In a climate of increasing demand and tight fiscal management, nurses with advanced skills have potential to reduce the burden of stroke and make a significant contribution to the delivery of timely, effective and efficient care. There is currently a paucity of published research nationally and internationally that has addressed advanced practice nursing in the sphere of stroke care and there is a lack of clarity around what an ANP role in the delivery of stroke services will encompass. The present study aimed to explore perspectives about the scope and role of future advanced nurse practitioners in the delivery of stroke services from a range of key stakeholders. The purpose of the study was to ascertain the level of support for the role and to develop a substantive description of scope and role to inform curriculum development, service planning and future research. Participants and setting The study was conducted from July 2019 to November 2019 at two hospital sites in one healthcare trust in the UK, and in the homes of stroke survivors and their family carers. The target population for healthcare professionals was senior stroke nurses, specialist stroke nurses, and stroke unit managers employed in the Trust’s stroke service. The inclusion criteria for these healthcare professionals were at least four years’ experience in the delivery of stroke services, and willingness to participate. The target population for service users was adults living at home, that had experienced stroke care in the Trust, as well as their family carers. The inclusion criteria for stroke survivors were age 18 years and older, living at home, experienced a stroke in the previous six months, engagement in community rehabilitation services, and ability and willingness to participate in an interview. The inclusion criteria for family carers were age 18 years and older, with ability and willingness to participate in an interview. The ability to participate in an interview, was construed by ability to engage in the stroke community rehabilitation service after discharge from hospital and confirmed in consultation between the gate keeper appointed to support recruitment and the rehabilitation service managers. The rationale for the mix of participant groups was to gain a breadth of perspectives that could encompass the aspirations for advanced nursing practice in stroke services. It was anticipated this this approach would also identify the challenges and strengths in the existing service, that participants perceived could be addressed and/or built on, in advanced nursing practice. The research invitation and information pack for participants included general information about advanced nursing practice to ensure that all participant groups were sufficiently informed to be able to provide perspectives. The purposive sampling method was used to recruit the participants, with sampling continuing until no new substantive data or new category was acquired. Selection of the participants was based on the variation of samples and the progression of the interviews. Accordingly, a total of 18 individuals provided their written informed consent to participate in the study. These included four adults that had experienced a stroke, and their family carers (n=4), four senior stroke nurses, three specialist stroke nurses, and three stroke unit managers. Participants and setting This study was approved by the Research Ethics Filter Committee of the School of Nursing and School of Health Sciences, University of Ulster, and Northwest Liverpool Central REC 19/NW/0256 in March 2019. The main ethical issues related to voluntariness, consent, confidentiality and protection of data. Prepared letters of invitation and participant information sheets and separate consent forms for stroke service participants, service user participants and carer participants supported voluntariness, informed consent, and right to withdraw from the study. There was full adherence to research governance pertaining to processing, storage and disposal of data. Data collection The data were collected through 18 face to face individual interviews conducted in the time period from 2nd July 2019 to 22nd November 2019, with each lasting between 30 to 55 minutes. The interviews with the healthcare professionals were conducted by the first author in private meeting rooms in Healthcare Trust locations that were convenient to them. The interviews with the adults that had experienced a stroke, and carers were conducted individually in a private room in their own homes. The interviews with the adults that had experienced a stroke were conducted by the first author, and the interviews with the family carers were conducted by the second author. All four authors have expertise in qualitative interviewing and qualitative research. With the permission of the participants, each interview was audio recorded. In addition, field notes during and after each interview were made by the interviewers, to support data analysis. Interview guides were formulated for each participant group and subjected to critical review by two specialist stroke nurses employed in a healthcare trust not involved in this study, academic colleagues in the School of Nursing, and a stroke advocacy group. Constructive feedback informed amendments and the final versions that were used. The interview guides were focussed on perspectives of the participants and their current and past experiences of delivering or receiving stroke care. The series of open general questions were supported by probes designed to clarify responses and to elicit more detailed information and are available in supplementary file 2. Rigour The conduct and presentation of this study was informed by the Consolidated criteria for reporting qualitative research (Tong et al., 2007). Data trustworthiness was assessed by the four criteria recommended by Lincoln and Guba (1988). The credibility and confirmability criteria were achieved through a thorough engagement with the research from conception, through planning, physical engagement in the field for purposive sampling, selecting and recruiting participants, undertaking preliminary data analysis after each interview, ensuring accurate verbatim transcription of the audio files, repeated review of the transcripts and cross checking with field notes, classification of the extracted codes and categories, and maintenance of an audit trail. Furthermore, a summary of the developing analysis and interpretation was shared with two of the senior nurse participants and one service user and carer, and their review confirmed interpretation. Confirmability and dependability of data is supported by the inclusion of raw data (meaning unit quotes), the data analysis and data reduction through presentation of the main themes, sub-themes, and the results. To enhance the level of transferability, the service user participants have been allocated ID codes P1, P2 etc, the carer participants have been allocated ID codes C1, C2 etc, and the healthcare professional participants are coded HP1, HP2 etc. The conduct and presentation of this study was informed by the Consolidated criteria for reporting qualitative research (Tong et al., 2007). Data trustworthiness was assessed by the four criteria recommended by Lincoln and Guba (1988). The credibility and confirmability criteria were achieved through a thorough engagement with the research from conception, through planning, physical engagement in the field for purposive sampling, selecting and recruiting participants, undertaking preliminary data analysis after each interview, ensuring accurate verbatim transcription of the audio files, repeated review of the transcripts and cross checking with field notes, classification of the extracted codes and categories, and maintenance of an audit trail. Furthermore, a summary of the developing analysis and interpretation was shared with two of the senior nurse participants and one service user and carer, and their review confirmed interpretation. Confirmability and dependability of data is supported by the inclusion of raw data (meaning unit quotes), the data analysis and data reduction through presentation of the main themes, sub-themes, and the results. Data Analysis Qualitative content analysis is a systematic means of describing phenomenon (Schreier, 2012). The interview data underwent qualitative content analysis in accordance with the inductive process described by Elo and Kyngas (2008). This method of analysis was chosen, because of the paucity of published research and the lack of clarity pertaining to the role of the ANP role in the delivery of stroke services. The audio interview data were transcribed, and participant names were replaced by pseudonyms. The preparation stage of the analysis involved a review of the transcriptions, followed by repeated reading and study of the text to generate deeper understanding of the link between the data and the scope and role of a future ANP in stroke care. The organisation phase encompassed 1) the selection of meaning units and open codes, 2) the grouping of related codes, 3) categorisation of the groups, and 4) the abstraction process which generated four main themes. This was a progressive process whereby the extracted categories that evolved after successive interview data were analysed until data saturation was reached. The initial data analysis and extraction of meaning units and the grouping of related codes were carried out by the first author. Then the second author reviewed and validated both the transcripts and the codes. A few incompatibilities in coding were identified by the second author, leading to further study of the data on the part of the first author and refinements in the coding and subsequently agreement was reached between the authors. Extracted codes that were identified as similar were classified into categories, and then grouped into sub-themes, and then further grouped into the four main themes. All four members of the research team were involved through discussions and critical appraisal of the analysis, in the formulation of the final list of main themes, and sub-themes. These are presented in table 1. Findings A total of 18 people participated in this study of which four were adults that had experienced a stroke, four were family carers, four were senior staff nurses, three were stroke unit managers, and three were specialist stroke nurses. The participants that had experienced a stroke and their family carers were aged 38-65years and 39-66 years respectively. Three of the four people that had experienced a stroke were women, and two of the four family carers were women. All the stroke nurses and stroke unit managers were women. Analysis of the interview data resulted in four main themes. These were “The Lynchpin of the acute stroke service”, “An expert in stroke care”, “Person and family focussed”, and “Preparation for the role”. Rigour To enhance the level of transferability, the service user participants have been allocated ID codes P1, P2 etc, the carer participants have been allocated ID codes C1, C2 etc, and the healthcare professional participants are coded HP1, HP2 etc. “The lynchpin of the acute stroke service” The nurse being there helped me. I was slow, and a wee bit overwhelmed but it was necessary.” (P1). Nurses appeared to be tuned into the fear experienced by people with a new stroke, and their reflections portrayed a level of confidence that rendered a sense of calm. “You see the fear in their faces. In ED, someone was inserting a cannula, another doing an ECG, another taking bloods, and the consultant was trying to get a cohesive history. I introduced myself and explained that I’ll be with you through this journey. Later I went to see her in the ward, and she recognised me, and when she felt secure enough, she let the tears fall about everything that happened to her in such a short period of time.” (HP4). “You see the fear in their faces. In ED, someone was inserting a cannula, another doing an ECG, another taking bloods, and the consultant was trying to get a cohesive history. I introduced myself and explained that I’ll be with you through this journey. Later I went to see her in the ward, and she recognised me, and when she felt secure enough, she let the tears fall about everything that happened to her in such a short period of time.” (HP4). A senior staff nurse highlighted the negative impact of fear on blood pressure and pulse and provided this as a rationale for the calming intervention of an ANP: “Anyone coming in with a stroke is scared, and fear can exacerbate blood pressure and pulse. If you had the calming influence of one nurse, who says I’ll be there with you.” (HP10). She later conjectured that the ANP role should extend beyond the hyperacute stroke phase and stated: “Ideally you would like the ANP to stay with the patient until they get them settled on the stroke ward.” (HP10). Furthermore, a family carer postulated that one to one support for the patient should be provided for 24 hours “For the first 24 hours, I think that each patient should have a dedicated nurse that is just slowly looking after that one patient and is focused on that one patient.” (C3) The healthcare participants highlighted that expediting the stroke pathway through enhanced coordination would be pivotal to the delivery of evidence-based care. “The lynchpin of the acute stroke service” A specialist stroke nurse explained: “When we get the hyper acute unit, there will be a faster pace in getting the patients in and getting them out…. The ANP will be the one that knows the person with stroke is going to the right place and expedite all their test investigations.” (HP4). It was anticipated that the ANP would generate efficiency: “The ANP will make the journey for the patient as direct as possible, getting the patient in the right place and making their journey as stress-free as possible. Having their scans and doing everything in a timely fashion.” (HP5). “The lynchpin of the acute stroke service” This theme encompasses the scope of the ANP. The healthcare professionals conjectured that the ANP role will predominantly fall within the acute treatment component of the stroke pathway. A specialist stroke nurse stated: “The ANP would be the lynchpin of the acute stroke service.” (HP6). The word lynchpin refers to a vital member of a group or organisation, that holds together the other members or parts for them to function as intended (Merriam-Webster Dictionary, 2020). This definition was reflected in data conveying the importance of the rapid stroke team response, projecting a calming influence in the hyper-acute stroke phase, and enhanced coordination of the stroke pathway. A family carer illuminated the positive impact of early engagement with a senior nurse in the hyperacute stroke phase: “There was a doctor standing behind the nurse in ED, and we told the nurse what had happened. She instantly lifted the phone to the stroke team, and the team came down and my wife was being dealt with in minutes.” (C4). For the stroke team response to be coordinated, timely and effective, the healthcare participants proposed that the ANP should be available as soon as the person with suspected stroke is coming through the hospital door. A senior staff nurse outlined that “the ANP will first see the patients in ED, and she will then follow them in the ward” (HP7). A specialist stroke nurse explained that “They'd be involved primarily with instigating everything that needs to be done for the patient.” (HP1), and another conjectured that placing the ANP at the acute end of the stroke pathway would not only expedite timely initiation of treatment but would lead to better clinical outcomes for patients. “The ANP role should encompass the quick response diagnosis and the initiation of treatment, because with the prescribing and thrombolysis, the scan needs to be read quickly. If we have all that in place, then obviously there will be a better outcome.” (HP1) Stroke is a frightening condition. The data richly illuminated the psychological and emotional context of people and families feeling overwhelmed and fearful in the hyper acute stroke phase. “All the team came in very quickly; everything was done one after the other. I was getting confused at the speed. I was kind of overwhelmed because my brain wasn't taking it all in. The speed was obviously paramount, and it was 100% efficient. “An expert in stroke care” This theme is an acknowledgement that the ANP will be an expert in stroke care. How this expertise will manifest and be experienced by others was captured in data which conveyed bridging the gap between stroke nursing and medicine, clinical competence, sharing knowledge and educating, and the ability to implement change. The healthcare professionals anticipated that the ANP will take responsibility for key aspects of clinical practice that are currently the remit of junior medical staff or physician associates. They were appraising the parameters of the role: “An ANP may be doing more than a junior doctor. I looked at the associate medical role, and the ANP role would not be far off that.” (HP4). Their appraisal supported the understanding that “The ANP will be bridging the gap between stroke nursing and medicine.” (HP8). Whilst they were keen to highlight their accrued competence to undertake full assessments of patients with stroke, it was less clear whether their assessments were supporting autonomy in implementing plans for diagnostics, treatment and care. Interprofessional teamwork supports quality stroke care and underpins clinical confidence. A family carer emphasised that ANPs must be able to convey clinical confidence within the inter professional team, and particularly with medical staff: “The ANP must have a good relationship with the doctor and be more affirmative with doctors if she/he thinks this or that should be done” (C2). The nurses were anticipating the potential for power dynamics to surface and interfere with the ability of ANPs to deliver fully on their role. A senior staff nurse stated that: “The ANP will need to be a confident person to say this is the way we should be doing it, and to stand your ground with medical staff, for yourself and for your nursing staff and for your patients.” (HP10). Stroke care in the region, is supported by an annual professions conference, and close relationships between stroke organisations, universities and healthcare trusts. The healthcare participants shared the expectation that the ANP will keep up to date with emerging research and best practice clinical guidelines in stroke care, and act as an education resource. One nurse stated this as an essential element of the role: “The ANP needs to have knowledge of the best practice and to share it” (HP7). “An expert in stroke care” Another nurse emphasised the ability to educate clinical colleagues: “The ANP needs to be able to pass on that knowledge and expertise to other colleagues.” (HP6). It was further clarified that the ANP’s education role will extend to patients and students. “An ANP is an expert in stroke care and will be the person for communicating up to date methods and treatments and a resource for nurses, patients and students to go to, for information. (HP10). A transformative leadership style was evoked. Perhaps this is not surprising given that the data conveyed a time of flux and impending change: “A big thing is implementing change. There are plans for a hyperactive unit, and my role is going to be changing.” (HP4). The healthcare professionals conveyed a strong desire for the ANP to be the transformational agent, who can engage collaboratively with multi-disciplinary colleagues to overcome challenges: “To be innovative as well, and especially with the medical team, because there can be barriers between the doctors and the nurses.” (HP10). A specialist stroke nurse highlighted an approach that the ANP may use to steer the team towards successful change: “The ANP should be able in conjunction with colleagues, to say this is what we need to do, and this is how we need to do it. Now, how can we achieve it?” (HP6) “The initial days were so important. Your husband's life is in their hands. You look at them to look at you and to find out if there is something that the family can help with.” (C2). “The initial days were so important. Your husband's life is in their hands. You look at them to look at you and to find out if there is something that the family can help with.” (C2). One major benefit of working closely with the person and family is the learning that can be derived about past medical history, co-morbidities and self-management strategies. A significant proportion of people that experience a stroke have comorbidities, such as diabetes, cardiovascular disease, or other long-term conditions. A tendency for healthcare professionals to focus on the stroke diagnosis and give lesser attention to comorbidities was conveyed by the service user and carer participants. The importance of paying attention to comorbidities was stressed. A patient related “I have a chronic back injury. I was telling them, and some of the nurses did not seem to understand. Then there was one nurse, and she pushed to get the medications for me.” (P4). Patients need to feel confident that nurses are not only paying attention to the past medical history, but that they are giving due attention to the treatment of co-existing conditions: “I have diabetes, and the most important thing of concern for me in the early days after my stroke was that the nursing staff didn’t seem to understand my schedules for insulin and eating.” (P2). This statement conveys the understanding that the patient may not be able to start fully engaging with the new stroke diagnosis and care plans until he/she is assured that the treatment regimens for co-existing conditions are put in place to their satisfaction. Stroke affects people in uniquely individual ways. Some stroke symptoms are overt, such as a limb weakness. Other symptoms, such as fatigue, speech and language difficulties, cognitive deficits and difficulties with emotions can present significant challenges to the person but manifest in less apparent ways. The importance of an ANP taking time to listen attentively and to support the formulation of questions and answers in the presence of aphasia, was emphasised by stroke survivors. “The nurse was with me, that first night, and she asked me a question, and then said write down the answer, so I picked up the pen in my right hand, and I couldn’t use my right hand. She then put it in my left and I just scribbled, and she understood what I was asking for. “Person and Family Centred” Stroke is a condition, that affects lives in a most abrupt manner. One third of people with a new stroke will experience depression, and the majority that are discharged from hospital, leave with a disability. This understanding might explain why approaches that serve to actively engage, empower and support the person and family from early in the stroke diagnosis, pervade the data. Nurses conjectured that the values of the ANP will manifest in a desire to take time to share the diagnosis and pertinent information with the patient and family: “I envisage that the ANP will be the type of nurse that wants the patients to get their diagnosis with their families, takes time to sit and explains the details and the impact it may have on them.” (HP10). Furthermore, the service users and carers were anticipating that the ANP will perceive the person living with stroke and the family as integral members of the stroke team and actively encourage their contribution in care planning. A patient explained: “The nurse said to my sister “Is anybody staying tonight?” I think she understood the importance of somebody being there, you know, as my husband had the kids to come home to.” (P3). A carer put forward the view that the establishment of early rapport and trust with close members of the family is not only crucial to wellbeing but is also a way of drawing on their lived experience of caring for their loved one. “Preparation for the role” The fourth theme is an acknowledgement that ANPs will require preparation for the role and encompasses the development of an ANP skills facilitated through clinical education placements, clarification on who should deliver the lysis bolus, and clinical supervision. The healthcare professionals outlined a skill set that comprises history taking, medical assessment and identification of stroke mimics, lysis administration, GTN infusion administration, NIH assessments and interpretation of diagnostics, nurse prescribing, medication reviews, and progress reviews. Nurses were keen to highlight that their skill set was advancing in line with medical advances in stroke treatments. A nurse stated: “Obviously, we do the lysis observations, and we’re getting more GTN infusions. Then you have the fasting blood glucose protocols for people that are nil by mouth. We are getting more advanced and more technical. You need to have your skills set because their condition can change in an instant.” (HP8). When nurses take on more advanced roles, there is a risk that some fundamental elements of care will receive lesser attention. A nurse pointed out that there are consequences if this was to happen: “Don’t forget the fundamental skills too. People do not realise how important the swallow screen is. When we're doing the lysis, we're keeping these patients without swallow screens for longer than necessary.” (HP4). The healthcare professionals identified the emergency department and neurovascular / transient ischaemic attack (TIA) clinics as ideal first placements for ANP trainees to hone their diagnostic skills. It was recommended that trainees should then rotate to placements in hyper-acute and acute stroke unit care, because “it is important to know how hyper acute and acute stroke care is delivered”. (HP9). It was proposed that additional learning could be attained in stroke review clinics, and regional centres of excellence: “Consider placement days in other hospitals as well, to see the excellence that they provide, like the clot retrieval in the regional hospital. Maybe a short placement in the stroke review clinics too.” (HP7). Whilst the healthcare professionals largely conveyed an incremental approach through a series of different clinical learning placements, consideration was also given to a person-centred approach. “The ANP trainee needs to be in the TIA clinics and ED and see the patient from when they come in the door, and then follow them through the whole aspect of lysis treatment. “The initial days were so important. Your husband's life is in their hands. You look at them to look at you and to find out if there is something that the family can help with.” (C2). I felt so much relief that she was able to understand me.” (P3). The healthcare professional, service user and carer participants stressed the importance of and the anticipation that the ANP will take account of the many ways that stroke can affect people, and that this will generate a sensitive communication approach. More specifically, a carer conveyed the concept of ‘tuning in’ to the patient: “They (ANPs) need to understand the way stroke affects people, and the way it can change them. My sister would be much slower in processing conversations and movement. There’s nothing physically showing. The ANP would need to be tuned in. At that time Carol was quite emotional.” (C1). “Preparation for the role” They need to get the whole process of door to needle.” (HP5) An area in need of clarity uncovered in our data, pertained to whether an ANP or a physician will administer the Lysis bolus infusion. Prompt screening for suitability for Lysis treatment, and rapid door to needle times are hallmarks of a modern 24/7 hyper-acute stroke service. A specialist stroke nurse contended: “I can order the diagnostics, the CT scans, and it's a group discussion on the lysis. I'm not saying whether the ANP would want to give the lysis, but you certainly are a big influence on the decision making.” (HP4). The nurses indicated a hesitancy with regards to taking on the lead role for Lysis bolus administration, and that perspective was carried through to ward manager level: “The ANP would have the active part to play with the doctor, who is still administering the bolus. I think that's something that needs to remain with the physician. It’s more the initial assessment that the ANP could have greater involvement, and aid the decision making around that.” (HP2) The healthcare professionals supported the need for a clinical supervision team to aid the development of the ANP. The proposed membership of the team included “an ANP or stroke specialist nurse already in post, and a stroke consultant and someone from diagnostics”. (HP8). A nurse highlighted the contribution that a consultant physician could bring to the team: “A team must include a consultant who sees people with stroke on a regular basis, or a neurologist who would also have the knowledge, experience, and research background.” (HP6). The advantages of widening the team beyond nursing and medical expertise were identified: “We need involvement of radiology because you’ll be examining and interpreting CT scans and MRIs.” (HP4). A ward manager gave consideration for pharmacist involvement: “The medical staff need to be involved, and maybe to an extent input from the pharmacist, because we work closely with the pharmacist on the ward.” (HP3). In addition, nurses pointed out that the supervision team will need support and guidance from the Stroke Lead in the university. This was based on the understanding that “the Stroke Lead will be delivering the education and will have the knowledge to guide the team” (HP4), and that a joint up approach would have most potential for success. Discussion The findings of the present study indicated strong support for advanced nursing practice in the delivery of stroke service. Wilson and Ashcroft’s (2018) scoping review of nurse practitioner roles in stroke care identified a wide variety of different roles undertaken that spanned the entire stroke care pathway, including primary care, pre-hospital care, acute care treatment, and secondary care prevention. This broad scope contrasts with the findings of our study. In our study, the dominant perspective was that the ANP will be the lynchpin of the acute stroke service, with priority roles in instigating the rapid stroke team response. The psychosocial context of people and families feeling overwhelmed and fearful was moderated by nurses introducing themselves and building early rapport. Our findings also differed from Wilson and Ashcroft (2018) with the envisioning that the ANP will expedite timely transit through the stroke pathway. The literature more generally indicates that ANP leadership has a positive impact on service delivery (Vaismoradi et al., 2016; Kraus & DuBois, 2017). However, Kraus and Dubois caution that further research is required to establish the relationship between the leadership practices of APNs and clinical outcomes. The ‘Expert in stroke care’ theme refers to up to date knowledge about stroke treatments, ability to share best practice guidelines, clinical confidence, and transformational leadership. A key challenge is whether the ANP will be able to translate clinical confidence into autonomous clinical decision making. An integrative review of the literature (Heinen et al., 2019) has reported that leadership competencies for advanced nursing practice fall within clinical, professional, health systems. and health policy domains. The potential for power dynamics within the interprofessional team to reduce the scope of ANPs was a key finding in emergency care research undertaken by Boman et al. (2018). The nurse practitioner role was perceived as a threat to colleagues and organizational structures, with the consequence that their autonomous status only pertained to care of individuals with non-urgent clinical needs. Boman et al. concluded that in order to achieve successful implementation of advanced practice nursing, there must be engagement with co-workers as part of the process. Further support for the need to harness interprofessional support and address organisational factors for successful implementation of advanced nursing practice is derived from a qualitative study conducted by Casey et al (2018). The values articulated in our study place the person and family firmly at the centre of stroke care planning. Discussion Patients and family carers understand that the focus of nurses will be on the management of acute stroke, but they are concerned that their co-existing medical conditions, may receive lesser attention. This is an interesting finding, given that research has indicated that diabetes management may be sub-optimal in stroke care (Mitchell et al., 2012). If the ANP utilises approaches that actively involve, support and empower the person and family unit as anticipated, there is potential for agreement about priorities in care and partnership approaches towards improved management of co-morbidities. Healthcare professionals experience challenge in communicating with stroke survivors with speech and cognitive impairments (Clancy et al., 2020), and therefore the training needs of the ANPs in this regard will need to be addressed. More generally, the research that has investigated the nature of the discourse between ANPs and people and their families, demonstrates mixed findings (Jenkins et al., 2018; Siouta et al., 2019). There is evidence that ANPs in primary care are contributing towards quality person-centred care (Jenkins et al., 2018). However, in other spheres of care, a discourse dominated by the biomedical model of care is surfacing. For example, Siouta et al (2019) utilised discourse analysis to investigate the communication style of ANP consultations in nurse-led chemotherapy clinics. What emerged was discourse framed by the biomedical model, with only fragments of the values of person-centredness evident when the ANP was localising the person’s health problems to their sociocultural context. The inter-connectedness of social and personal values and power is manifested in discourse (Foucault, 2002). With the trend towards expanding the scope of advanced nursing practice, the authors of the present study recommend that discourse analysis is afforded greater attention by nurse educators and researchers. Preparation for the ANP role encompasses development of a skill set, clinical education placements, role clarification, and clinical supervision. Our findings indicate that the skill set of stroke nurses in the UK has already evolved, and embraces history taking, medical assessment, National Institute of Health stroke assessment scores, interpretation of diagnostics, treatment and care. In addition, our findings convey that specialist stroke nurses are prescribing, and undertaking medication and progress reviews. What will differentiate the ANP from the beside stroke nurse, will be advanced assessment skills, prescribing and administering the lysis regime, and autonomous clinical decision making, in line with the imperative that ANPs will execute diagnostic, analytical and clinical judgemental skills (NIPEC, 2016). Discussion The literature demonstrates that the implementation of advanced nursing practice in new clinical fields needs careful planning (Boman et al., 2018; Casey et al., 2018). Role clarity is a significant area that must be addressed, before implementation of ANPs (Casey et al., 2018). A finding of the present study was the lack of clarify about whether an ANP or a physician should administer the Lysis bolus infusion in the hyper acute stroke phase. Hesitancy was evident among the nurse participants with regards to taking on this role. It is possible that the ANP prescribing role in the hyper acute stroke phase could expedite the commencement of treatment regimes. There is a narrow time window of opportunity to treat the patient with acute ischemic stroke, for successful outcomes, and this carries risk. Adverse events can occur in up to 7% of patients (Jahan et al., 2019). Even in a tight 30 to 120-minute treatment window, there is pressure to treat early, because faster time from arrival at the hospital door to initiation of lysis treatment, is associated with improved clinical outcomes (Jahan et al., 2019). There are inherent hazards, when making clinical decisions in such a narrow time frame, and this may in part explain the hesitancy among the nurses for this crucial role. Therefore, it is important that the prescribing role of ANPs in the delivery of hyper acute stroke services is developed and supported by Trust protocols, and processes are put in place to ensure that ANP trainees are supported to gain the requisite confidence and competence. A participant in our study cautioned that the ANP should not dismiss fundamental nursing skills in advancement of their role and pointed out that quality stroke care was dependent on fundamental needs of patients being addressed. This is an important consideration given the growing concern that fundamental nursing skills are being de-valued (Feo et al., 2019). A recommendation therefore is that ANPs embrace and advocate for exemplary fundamental skills. The clinical placements proposed for supporting the development of the skill set of the student ANP include the emergency department, TIA clinics, the hyper-acute stroke unit, including access to regional centres of excellence for thrombectomy services, the acute stroke unit, and stroke review clinics. A key finding in the present study was the broad support for an inter professional model of supervision for the developing ANP trainee. Limitations The context of the study was a region in the UK where its healthcare staff and service users are preparing for major reorganisation of stroke services. This was therefore a time of flux. Despite this context, the participants were keen to share their perspectives, and their passion for stroke care was evident in the data. A total of 18 people participated in this study, comprising individuals that had experienced a stroke and their family carers, stroke unit managers, senior staff nurses, and specialist stroke nurses. In total, only three of the participants were male. This gender imbalance was not unanticipated among the stroke service users, given the evidence that women are more likely to experience stroke (Fonseca et al., 2018), nor in a study where most participants are nurses. Despite a slightly smaller sample size than was proposed at the outset of the study, data saturation was achieved, and the analysis was opened to critical dialogue with the team. A summary of the themes generated was presented to a subgroup of four participants and was verified. These measures support the trustworthiness of the results. Discussion A model of interprofessional supervision team has potential to build the quality of collegial cohesion that will be able to address organisational contexts, power dynamics and role clarity (Boman et al., 2018; Casey et al., 2018). Conclusions This qualitative study aimed to address a lack of clarity around the potential scope and role of advanced nurse practitioners in the delivery of stroke services. There was strong support for an advanced nurse practitioner role in the acute stroke phase of the pathway. A challenge that surfaced pertained to how the ANP can be enabled to move from confidence in patient assessment to autonomous clinical decision making, lysis prescribing and administration. Given the hesitancy of nurses to take on a role in administration of the lysis bolus, our study suggests that role clarification will be a key area that needs to be addressed for successful implementation of advanced practice nursing in acute stroke services. Prescribing and administering lysis treatment is not without risk, and ANPs will need clinical supervision and prescribing protocols in place in their Trusts to support them to develop confidence and competence in their role. The fear experienced with a new stroke was strongly conveyed in our study, and in addition, an expectation that advanced nurse practitioners will utilise person centred approaches and demonstrate sensitivity in communicating with the person with stroke and their family carers. The literature indicates a tendency for advanced nurse practitioners to adopt a consultation style that is framed by the biomedical model. We therefore recommend that discourse analysis be incorporated into education programmes for advanced nurse practitioner trainees. Our study supports the premise that advanced nurse practitioners can make a significant contribution to the delivery of timely, effective and efficient stroke care, however further research will be required to establish the relationship between the practices of APNs and clinical outcomes. Relevance to Clinical Practice Nurses are pivotal to the delivery of evidence informed stroke services, and nurse led protocols for the management of people in the acute stroke phase can reduce mortality. There is potential for the implementation of advanced practice nursing in stroke services to drive forward further enhancements in care. This study is relevant to clinical practice because it aimed to clarity the potential scope and role of advanced nurse practitioners in the delivery of stroke services. Our study indicates strong support for the implementation of advanced practice nursing in the hyper-acute and acute stroke phases of the care pathway. Our findings demonstrate that nurses are confident in undertaking a range of assessments of people with acute stroke, but that they will require education and an interprofessional model of clinical supervision in order to develop the confidence and competence for the level of autonomous clinical decision making that is required of an advanced nurse practitioner. The literature indicates that reducing door to needle times is crucial for enhanced clinical outcomes for patients with ischaemic stroke. Our findings indicate that nurses are informing decisions with regards to administration of lysis, but that they are hesitant about taking on a lead role in administering the lysis bolus. It is important that nurses engage with medical staff in open dialogue and discussion and resolve the hesitancy of nurses to undertake this vital role. There is an expectation that advanced nurse practitioners will utilise person centred approaches to actively involve people with stroke and their family carers in their care, however the literature suggests that advanced nurse practitioners have a tendency to adopt a consultation style that is framed by the biomedical model. The consultation style of the developing advanced nurse practitioner is therefore an area that will need to be monitored in education curricula and assessed in clinical practice. Further research will be required to establish the relationship between the practices of APNs and clinical outcomes. What does this paper contribute to the wider global clinical community? • New perspectives on the scope and role of advanced nurse practitioners in stroke service delivery. • There is support for the advanced nurse practitioner role particularly in the acute stroke phase of the pathway. • Our findings indicate a need to clarity the role of advanced nurse practitioners in lysis bolus administration in the hyper acute stroke phase. Relevance to Clinical Practice What does this paper contribute to the wider global clinical community? What does this paper contribute to the wider global clinical community? • New perspectives on the scope and role of advanced nurse practitioners in stroke service delivery. • There is support for the advanced nurse practitioner role particularly in the acute stroke phase of the pathway. • Our findings indicate a need to clarity the role of advanced nurse practitioners in lysis bolus administration in the hyper acute stroke phase. 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Consolidated criteria for reporting qualitative research (COREQ): a 32-item checklist for interviews and focus groups. Int J Qual Health Care. 2007;19(6):349-357. Vaismoradi, M., Griffiths, P., Turunen, H., Jordan, S. 2016. Transformational leadership in nursing and medication safety education: a discussion paper. J Nurs Manag 24 (7); 970-980. doi:10.1111/jonm.12387. Table 1: Main themes, sub-themes and selected meaning units in the envisaging of an advanced nurse practitioner in stroke care. National Health Service Scotland. 2019. Scottish Stroke Improvement Programme 2019 Report. https://www.strokeaudit.scot.nhs.uk/Publications/docs/2019/Scottish-Stroke-Improvement- Programme-2019-National-Report.pdf Main themes Sub-themes Selected meaning unit ‘The lynchpin of the acute stroke service’ The instigator of the rapid stroke team response “The ANP role should encompass the quick response diagnosis and the initiation of treatment” A calming influence in the hyper acute stroke phase “The calming influence of one nurse, who says I’ll be there with you” Expediting the Stroke Pathway “The ANP will make the journey for the patient as direct as possible” ‘An expert in stroke care’ Bridging the gap between stroke nursing and medicine “An ANP will be doing more than a junior doctor” Clinical confidence “The ANPs must be confident in what they are doing” Shares knowledge and educates “The ANP needs to have knowledge of the best practice and to share it” Can implement change “A big thing is implementing change” ‘Person and family focussed’ Actively engages the person and family in care planning “You look at them to look at you and to find out if there is something that the family can help with” Pays attention to the person’s medical history “There was one nurse, and she pushed to get the medications for me” Communicates with sensitivity, anticipating speech, language and cognitive deficits “They need to understand the way stroke affects people, and the way it can change them” ‘Preparation for the role’ The ANP skills set “You need to have your skills set because their condition can change in an instant” Clinical Education Placements “TIA clinics and ED will hone their diagnostic skills” Clarification on who should administer the Lysis bolus “I'm not saying whether the ANP would want to give the lysis” Clinical Supervision “A team must include a consultant who sees people with stroke on a regular basis” e 1: Main themes, sub-themes and selected meaning units in the envisaging of an advanced e practitioner in stroke care.
https://openalex.org/W4254914254	https://europepmc.org/articles/pmc6367807?pdf=render	English	null	Pilot Randomized Controlled Trial Testing the Influence of Front-Of-Pack Sugar Warning Labels on Food Demand	AEA Randomized Controlled Trials	2,019	cc-by	5,984	Ang et al. BMC Public Health (2019) 19:164 https://doi.org/10.1186/s12889-019-6496-8 Ang et al. BMC Public Health (2019) 19:164 https://doi.org/10.1186/s12889-019-6496-8 Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Front-of-pack (FOP) nutrition warning labels to identify potentially harmful foods/beverages have recently been considered in Singapore. The objective of this study was to pilot test two promising FOP warning labels intended to reduce purchases of products high in sugar to determine whether a full scale trial testing one or both these labels using actual purchases is warranted. Methods: Five hundred twelve participants ≥21 years old and residing in Singapore completed all study elements online via the NUSMart Online Grocery Store study website. The study was designed as a Randomized Controlled Trial (RCT) where consumers were randomized and asked to hypothetically shop in one of three versions of an online grocery store; 1) no FOP label (control), 2) a graphical high-in-sugar label shaped like a stop sign, or 3) a text- based warning label. The proportion of labelled products purchased (primary outcome) and all secondary measures of diet quality were calculated using participants’ orders. Ordinary Least Squares (OLS) regression was used to compare purchasing behavior across the three study arms. Results: The proportion of high-in-sugar products selected (i.e., those targeted for labelling) was largest in the no label control arm at 20%. The proportion was a non-statistically significant 2 percentage points lower (P = 0.146) for the high-in-sugar stop-sign label arm and 4 percentage points lower (P < 0.05) in the warning label with deterrent text arm. We could not reject the hypothesis of equal effectiveness of the two warning labels (P = 0.231). Conclusions: Results suggest that the two health warning labels have potential to reduce demand for high-in-sugar products in Singapore. Future studies should test the influence of these labels using actual purchases in efforts to identify whether either labelling strategy should be considered for adoption in the local setting. Trial registration: The American Economic Association’s registry for randomized controlled trials; AEARCTR-0003800. Registered 18 January 2019. Trial registration: The American Economic Association’s registry for randomized controlled trials; AEARCTR-0003800. Registered 18 January 2019. Keywords: Front-of-pack labeling, Nutrition labeling, Warning labels, Sugar, Online grocery store Pilot randomized controlled trial testing the influence of front-of-pack sugar warning labels on food demand Felicia Jia Ler Ang, Sagun Agrawal and Eric A. Finkelstein* Felicia Jia Ler Ang, Sagun Agrawal and Eric A. Finkelstein* Felicia Jia Ler Ang, Sagun Agrawal and Eric A. Finkelstein* Background to decipher for many shoppers and there is scant evi- dence that the panel has helped to curtail rising rates of obesity and NCDs [8–10]. The incidence of obesity, diabetes and other non-com- municable diseases (NCDs) has risen rapidly in recent years. As diet is a key risk factor for the onset of NCDs [1–4], policy-makers have been looking to identify strat- egies aimed at discouraging unhealthy food consumption [5–7]. Most pre-packaged foods and beverages include a nutrition information panel (NIP) to help consumers make healthier purchases. However, the NIP is difficult Singapore, the country of focus for this effort, recog- nized the limitations of the NIP and supplemented it with a simple front-of-pack (FOP) label termed the Health Choice Symbol (HCS). The HCS symbol was first introduced in 2001 and although optional, is strongly encouraged by the Singapore government. It offers the Health Promotion Board’s endorsement for products that are healthier options within a food category, includ- ing options that are Lower in Sugar and Lower in * Correspondence: eric.finkelstein@duke-nus.edu.sg Program in Health Services & Systems Research, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore * Correspondence: eric.finkelstein@duke-nus.edu.sg Program in Health Services & Systems Research, Duke-NUS Medical School, 8 College Road, Singapore 169857, Singapore Ang et al. BMC Public Health (2019) 19:164 Ang et al. BMC Public Health (2019) 19:164 Ang et al. BMC Public Health (2019) 19:164 Page 2 of 8 Page 2 of 8 In this study, we use an experimental web-based grocery store to pilot test two different theory- and evidence-based ‘FOP’ warning labels aimed to reduce purchases of high-in-sugar products, even in the pres- ence of the HCS symbol and NIP. The first label we consider is an English language version similar to the one used in Chile (Fig. 1 Left Panel) that shows a black stop sign (Arm termed SS) with the words high-in-sugar in the center (Fig. 1 Left Panel) [5]. The second label we consider is a text-based health warning label (Arm termed TW), which is similar to labels considered in several municipalities in the US (Fig. 1 Right Panel) and also resembles the warning label on cigarettes, but with- out the accompanying graphics (Fig. 1 Right Panel) [22]. Given the strongly worded language similar to tobacco warning labels that are used in the text-based health warning label, we hypothesized that the proportion of labelled (or targeted for labelling in Control Arm) prod- ucts among respondents would be the largest in Control and smallest in the warning label condition. There are three reasons to assume the text based warning label will show greater effectiveness. First, it is likely to generate greater levels of loss framing through negative termin- ology including ‘warning’ and ‘tooth decay’. Second, because it mirrors tobacco warning labels, the negative association may lead to an implicit bias against purchas- ing. Finally, because of the requirement to include the full message in a readable format, it is larger than the stop sign label and therefore may be more salient to consumers. We hypothesized a similar ordering for secondary outcomes, including total sugar purchased, sugar (g) per dollar spent, and total spending. Saturated Fat. Although this particular label has not been systematically evaluated, FOP nutrition labels appear to outperform other forms of nutritional labels in improving consumers’ ability to find and use nutritional information in purchasing decisions [11–13]. Therefore, the HCS logos may improve a shopper’s ability to identify the healthier products even when the NIP is available on the back of the label [14]. However, these logos appear on only a small percentage of products (roughly 9%) and do not identify the worst offending foods and beverages when it comes to sugar or other harmful ingredients. In this study, we use an experimental web-based grocery store to pilot test two different theory- and evidence-based ‘FOP’ warning labels aimed to reduce purchases of high-in-sugar products, even in the pres- ence of the HCS symbol and NIP. The first label we consider is an English language version similar to the one used in Chile (Fig. 1 Left Panel) that shows a black stop sign (Arm termed SS) with the words high-in-sugar in the center (Fig. 1 Left Panel) [5]. The second label we consider is a text-based health warning label (Arm termed TW), which is similar to labels considered in several municipalities in the US (Fig. 1 Right Panel) and also resembles the warning label on cigarettes, but with- out the accompanying graphics (Fig. 1 Right Panel) [22]. only a small percentage of products (roughly 9 not identify the worst offending foods and bever it comes to sugar or other harmful ingredients. Research has shown that, compared with non labels like the Nutrition Facts Panel, dire semi-directive labels may improve consumers find and understand nutritional information Consumers tend to prefer simple FOP labe appreciate interpretational aids like descriptor codes [11, 18]. However, there is some evid positive FOP labels targeting healthier foods m sufficient to discourage consumption of les alternatives [14, 19]. As a result, health warn have recently been proposed as a complemen native to positive FOP food labels. Studie warnings for tobacco products showed impr sumer education, greater knowledge of health tobacco use, and decreased purchases [20, study showed reduced intention to purchase S presence of SSB warning labels [22]. The suggest that warning labels identifying harmfu ents or adverse effects of certain food product potential to improve diet quality. Therefore, vated by several social psychological theories, loss-framing [23] and theories of risk percep providing a clearly identifiable and salient m consumers on which foods are highest in harm dients could effectively signal which foods to thus further positively alter food purchasing even in the presence of the NIP and HCS l Several empirical studies showing the effect tobacco warning labels and warning labels foods provide additional support for testing s in Singapore [14, 20, 26–28]. Fig. 1 Warning labels Research has shown that, compared with non-directive labels like the Nutrition Facts Panel, directive and semi-directive labels may improve consumers’ ability to find and understand nutritional information [14–17]. Consumers tend to prefer simple FOP labels and to appreciate interpretational aids like descriptors or color codes [11, 18]. However, there is some evidence that positive FOP labels targeting healthier foods may not be sufficient to discourage consumption of less healthy alternatives [14, 19]. As a result, health warning labels have recently been proposed as a complement or alter- native to positive FOP food labels. Studies of text warnings for tobacco products showed improved con- sumer education, greater knowledge of health harms of tobacco use, and decreased purchases [20, 21]. One study showed reduced intention to purchase SSBs in the presence of SSB warning labels [22]. These studies suggest that warning labels identifying harmful ingredi- ents or adverse effects of certain food products have the potential to improve diet quality. Therefore, as moti- vated by several social psychological theories, including loss-framing [23] and theories of risk perception [24], providing a clearly identifiable and salient message to consumers on which foods are highest in harmful ingre- dients could effectively signal which foods to avoid and thus further positively alter food purchasing patterns even in the presence of the NIP and HCS logos [25]. Several empirical studies showing the effectiveness of tobacco warning labels and warning labels on select foods provide additional support for testing such labels in Singapore [14, 20, 26–28]. p y g g p g g Given the strongly worded language similar to tobacco warning labels that are used in the text-based health warning label, we hypothesized that the proportion of labelled (or targeted for labelling in Control Arm) prod- ucts among respondents would be the largest in Control and smallest in the warning label condition. There are three reasons to assume the text based warning label will show greater effectiveness. First, it is likely to generate greater levels of loss framing through negative termin- ology including ‘warning’ and ‘tooth decay’. Second, because it mirrors tobacco warning labels, the negative association may lead to an implicit bias against purchas- ing. Finally, because of the requirement to include the full message in a readable format, it is larger than the stop sign label and therefore may be more salient to consumers. We hypothesized a similar ordering for secondary outcomes, including total sugar purchased, sugar (g) per dollar spent, and total spending. Online grocery store For this pilot and future research studies, an online grocery store (NUSMart Online Grocery Store) was de- veloped. At the time of the pilot, NUSMart contained over 1800 non-fresh food and beverage products com- monly purchased at local markets in Singapore (see Additional file 1). The web store was designed to mirror an actual web-based grocery store in look and feel. It Fig. 1 Warning labels Page 3 of 8 Page 3 of 8 Page 3 of 8 Ang et al. BMC Public Health (2019) 19:164 Ang et al. BMC Public Health contained products across major Food & Beverage categories, including: checkout below or above the minimum or maximum re- spectively. The minimum expenditure was intended to ensure sufficient purchasing data would be collected per sales order and the maximum was intended to ensure that our results were not overly influenced by a few shoppers with very large expenditures. Following the shopping task, participants completed a brief survey and were compensated according to the web panel’s in-house point system. The survey included information on age, gender, height, weight, ethnicity, presence of children in household, and whether the participant is the primary grocery shopper in the household. Beverages Dairy Products Cooking & Baking Frozen & Chilled Products Confectionery Cereals, Bakery & Spreads Beverages Dairy Products Cooking & Baking Frozen & Chilled Products Confectionery Cereals, Bakery & Spreads Beverages Dairy Products Cereals, Bakery & Spreads All products include a picture of the item and current retail price. A subset of qualifying products also have the HCS logo displayed. The store operates similar to other on-line grocery stores, with a cart that fills as consumers shop on the store and the ability to review purchases before checkout. Participants and procedures We powered the study to detect differences of at least 3 percentage points across arms in terms of the percentage of high in sugar products purchased. Assuming a common group standard deviation of 0.83 (based on un- published data from ongoing studies), an alpha of 0.05 and power of 0.8, and adjusted for multiple comparisons, a sample of 170 participants per arm was required to test our hypotheses. Based on this power calculation, 512 participants were recruited anonymously from an on-line panel in August 2017. Potential participants were emailed unique links that directed them to a screener. They were excluded if they were under 21 years of age or a non-resident of Singapore. All others were offered the chance to partici- pate. Those who consented were randomized into 1 of the 3 arms and asked to spend between S$50 (approxi- mately 37 USD) and S$250 (approximately 183 USD) on NUSMart as if it were a real household grocery shopping trip (see Fig. 3 for diagram of study flow). A pop-up message appeared on-screen if they attempted to Data analysis y All analyses on primary and secondary outcomes were conducted using Ordinary Least Squares (OLS) regres- sion. A Generalized Linear Model (GLM) with logit link was also used for analysis of the primary outcome, given it represents a proportion. However, as results were identical to the OLS results, we do not report the GLM results although they are available upon request. Specif- ically, the outcome of interest was regressed on treat- ment indicators for the two labelling conditions, with the Control Arm being the omitted reference group. Results were run on the full shopping basket and separ- ately for beverages given Sugar Sweetened Beverages are a prime target of labelling efforts [29–31]. All regres- sions controlled for age, gender, ethnicity, body mass index (BMI), whether the participant is a primary pur- chaser for the household and has children. All statistical analyses were performed using STATA. Measures The primary outcome of interest is the proportion of high in sugar products purchased, as this is the most direct test of the influence of the labels. However, it is possible that consumers could respond to the labels by purchasing fewer labelled products but not reduce their net sugar intake, partly because a change in the labels could also influence their total spending. Therefore, we also include the following secondary outcomes: We identified products to receive a warning label based on the percentage of sugar as compared to other products within the food category. We chose a within category approach to mirror the approach taken for the HCS program. A food product is classified as high-in- sugar if it is among the top 20% of products with the highest sugar content per 100 g within the food category. A beverage is classified as high-in-sugar if it contains at least 10 g per 100 ml (i.e. 10%) of sugar. Using this ap- proach, 20% of food within each food category and 27% of beverages were defined as high-in-sugar and received the corresponding label for the SS and TW arms. As with the HCS symbol, the labels were displayed at the bottom of the product images (See Fig. 2). Figure 2 shows the storefront and an example of a fictional prod- uct as it appears in each arm of the study. Total sugar purchased (in g) per shopping trip, Sugar purchased per dollar spent (in grams per $), Total spending (in $) given that high-in-sugar prod- ucts tend to be less expensive, and Total expenditure on high-in-sugar products ($). Total expenditure on high-in-sugar products ($). Data screening Table 1 presents the characteristics of the final sample, by allocated Arm. Five hundred twelve participants were part of the final sample for the analysis. The total sample Ang et al. BMC Public Health (2019) 19:164 Page 4 of 8 Ang et al. BMC Public Health Fig. 2 Example of the Pilot-DIET NUSMart storefront with a sample of the warning labels on the same fictional product as it appears in each study arm -DIET NUSMart storefront with a sample of the warning labels on the same fictional product as it appears in each study arm Fig. 2 Example of the Pilot-DIET NUSMart storefront with a sample of the warning labels on the same fictional product as it appears in each study arm Table 3 presents the regression output for each dependent variable when the sample is restricted to beverage purchases made by the 432 participants who purchased beverages. For this restricted sample, the pro- portion of High-in-Sugar products was 33% for the Control arm. The proportion was 6 percentage points lower (P = 0.085) for the SS arm and 11 percentage points lower (P = 0.002) for the TW arm compared to Control. As with the full sample we could not reject the hypothesis of equal effectiveness of the two warning labels (P = 0.172) nor were there any statistically signifi- cant differences across arms in any of the secondary outcomes. was largely Chinese (86.1%) and the mean age was 38.1 years (SD = 11.5). The average BMI was 22.8 (SD = 4.9). The majority (72.9%) reported being the primary grocery shopper for the household, and about half (46.7%) were female. Table 2 presents the regression output for each dependent variable for the full shopping basket. Both SS and TW participants purchased a lower proportion of High-in-Sugar products than Control participants but the difference was only statistically significant for TW (P < 0.01). Differences between SS and TW were not statistically significant (P = 0.231). None of the secondary outcomes (total sugar purchased (g), sugar purchased per dollar spent (g per $), total spending ($) and total expenditure on high-in-sugar products ($)) were statisti- cally different across arms. Higher BMI is associated with a higher proportion of High-in Sugar products purchased (P < 0.01). Older age was associated with higher purchases of sugar (P < 0.01) Ang et al. BMC Public Health (2019) 19:164 Page 5 of 8 Ang et al. BMC Public Health Fig. 3 Participant flow diagram trial in Singapore using actual purchases. Results lend support to proceeding with a full scale trial as one of the two labels generated a statistically significant reduction in labelled products purchased. Our findings are consist- ent with recent studies examining SSB warning labels. A text-only warning label was shown to reduce purchase probabilities of SSBs, reduce perceived product attract- iveness, quality and taste, and reduce perceptions of consumer “coolness” in a hypothetical experiment [32]. Another hypothetical trial focusing on warning labels for SSBs showed that the labels increase parents’ understand- ing of health harms associated with over-consumption of and greater spending (P < 0.01). There were also signifi- cant effects of gender on sugar purchased per dollar spent, with women on average purchasing 1.5 g per dollar less than men (P < 0.01). Chinese respondents also purchased significantly less total sugar (P < 0.05) and sugar per dollar (P < 0.01) than non-Chinese. Discussion For example, the influence of labels may differ when shopping for a few products for immediate consumption, as is likely when shopping in a convenience store, compared to when making larger purchases for foods to last over a longer period of time. Effectiveness may also wane over repeated shops. Hunger and other visceral fac- tors may also mediate the relationship between labels and food purchases regardless of venue and time to consumption. Results could also be influenced by the color, size, placement and implementation strategy (e.g., what percent of products are labelled) of the labels. In this pilot, the two warning labels were of differing sizes because we needed the health warning label to be large enough such that the text could be clearly seen on even small devices. One reason the text based label was more effective may be due to its larger dimensions. Although we could not realistically reduce the size of this label, we could test the effect of a larger stop sign label. Finally, a small sample size could have masked true but relatively small differ- ences in outcomes between arms. Many of these concerns can be addressed by a carefully conducted 3 arm randomized trial using repeated actual, as op- posed to hypothetical, purchases which is now in the planning stages. SSBs and reduce intentions to purchase SSBs for their children [22]. To date, no studies have assessed how the food labelling law in Chile has impacted purchases of sugar or other macronutrients. Although our study only found one of the labels to be effective at influencing purchases of targeted products, a full scale trial including both labels should be pursued given that we could not reject the hypothesis that one label outperformed the other. Moreover, a full scale trial with actual purchases will allow for determining whether label that effectively influences purchasing patterns also leads to improvements in diet quality. This is relevant given that an effective label may not reduce sugar or calories purchased if consumers alter their behavior by purchasing more of unlabeled products. As this is a pilot study, it is subject to several limita- tions. All shopping was hypothetical and may not generalize to actual purchases. Discussion The primary objective of this pilot study was to deter- mine whether one or both of two promising FOP warn- ing labels for high in sugar products would be worth testing further in a large-scale randomized controlled Table 1 Descriptive Statistics of the participants (n = 512) in the Pilot-DIET Study by allocated Arm Variable Mean/Proportion in Sample Arm 1: Control (n = 175) Arm 2: High-in-Sugar Stop Sign Label (n = 167) Arm 3: Warning Label with Deterrent Text (n = 170) Mean (SD) or % Age (SD) 38.1 (11.9) 38.2 (12.2) 37.9 (10.5) BMI (kg/m2) (SD) 22.4 (3.6) 24.0 (6.6) 21.9 (3.9) Female (%) 55.4 38.9 45.3 Ethnicity (% Chinese) 85.1 86.2 87.1 Primary Purchaser (%) 72.6 75.5 70.6 Have Children (%) 42.9 46.7 44.1 Table 1 Descriptive Statistics of the participants (n = 512) in the Pilot-DIET Study by allocated Arm Variable Mean/Proportion in Sample Table 1 Descriptive Statistics of the participants (n = 512) in the Pilot-DIET Study by allocated Arm Page 6 of 8 Ang et al. BMC Public Health (2019) 19:164 Page 6 of 8 Table 2 Estimates of the Impact of Warning Labels on Measures of Diet Quality in the Pilot-DIET Study (N = 512) Dependent Variable Proportion of High-in Sugar Products (%) Total Sugar Purchased (g) Sugar purchased per dollar spent (g / $) Total Dollar Spent ($) Total expenditure on high-in-sugar products ($) Coefficient [Std. Error] Coefficient [Std. Error] Coefficient [Std. Error] Coefficient [Std. Error] Coefficient [Std. Error] Constant 0.08 351.82 7.90 60.76 −4.92 [0.04] [179.93] [1.77] [13.44] [4.82] Stop Sign Label −0.02 48.91 0.51 2.35 0.61 [0.02] [65.44] [0.645] [4.89] [1.75] Warning Label −0.04 * 26.18 0.36 2.01 −1.61 [0.02] [64.55] [0.64] [4.82] [1.73] Age (years) 0.00 10.50 * 0.03 0.79 * 0.27 * [0.00] [2.60] [0.03] [0.19] [0.07] BMI (kg/m2) 0.01 *** 10.20 * 0.10 * −0.15 0.58 * [0.00] [5.82] [0.06] [0.44] [0.16] Female 0.02 −88.05 −1.50 * −2.39 2.22 [0.01] [56.00] [0.55] [4.18] [1.50] Chinese −0.03 * −196.51 −2.45 * −0.60 −3.12 [0.02] [77.35] [0.76] [5.78] [2.07] Primary grocery Purchaser −0.01 −47.61 0.35 −3.24 −0.68 [0.02] [62.21] [0.61] [4.65] [1.67] Has Children 0.01 −7.07 −0.12 1.29 −1.36 [0.02] [60.95] [0.60] [4.55] [1.63] * P < 0.1, P < 0.05, * P < 0.01 effectiveness may be size and venue specific and differ for web versus in store shopping. Authors’ contributions d f h EAF, FJLA conceived of the study. FJLA led data collection and SA data analysis. EAF and FJLA took lead on the writing and all authors did critical review. All authors read and approved the final manuscript. Conclusions Global Asia Institute (grant number AG-2014-001). Neither funder had any role in the study design, data collection, analysis and interpretation of the data; or the writing of the report. The research was conducted independ- ently of the funders. Results from the present pilot suggest that FOP warning labels have the potential to reduce demand for high in sugar products and should be tested via a full scale randomized trial. This test is warranted given that our pilot results suggest that even an effective label that reduces demand for high in sugar products may not generate a reduction in sugar or calories purchased, which is the ultimate goal of the labelling policy. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Additional file Additional file 1: NUSMart product categories in the Pilot-DIET study. NUSMart Category names, number of products per category, and propor- tions of products qualifying for the logos per category. (DOCX 14 kb) Discussion The NUSMart store at the time this study was conducted also had fewer prod- ucts than would appear in a full grocery store but more products than available in most convenience stores and it is likely that the number and type of products will influence the effectiveness of FOP labels. The NUSMart store has been expanded to include over 4000 products, which will allow for a more realistic test of the effect- iveness of these labels. However, it is worth noting that even if shown to be effective (or ineffective), Page 7 of 8 Ang et al. BMC Public Health (2019) 19:164 Page 7 of 8 Table 3 Estimates of the Impact of Warning Labels on Measures of Diet Quality for Beverage Purchases in the Pilot-DIET Study (N = 432) Dependent Variable Proportion of High-in Sugar Products (%) Total Sugar Purchased (g) Sugar purchased per dollar spent (g / $) Total Dollar Spent ($) Total expenditure on high-in- sugar products ($) Coefficient [Std. Error] Coefficient [Std. Error] Coefficient [Std. Error] Coefficient [Std. Error] Coefficient [Std. Error] Constant 0.06 76.47 19.73 12.28 −3.79 [0.10] [132.96] [5.43] [7.10] [3.52] Stop Sign Label −0.06 * 54.97 −0.06 0.57 0.08 [0.03] [44.21] [1.81] [2.36] [1.17] Warning Label −0.11 * −0.48 −2.10 1.52 −0.25 [0.03] [44.22] [1.81] [2.36] [1.17] Age (years) 0.00 * 3.06 * −0.01 0.13 0.20 * [0.00] [1.75] [0.07] [0.09] [0.05] BMI (kg/m2) 0.00 4.62 −0.02 0.00 0.12 [0.00] [4.52] [0.19] [0.24] [0.12] Female 0.08 * −32.72 0.63 −4.16 ** 1.07 [0.03] [38.29] [1.56] [2.05] [1.01] Chinese 0.04 33.14 −2.25 5.12 * 1.38 [0.04] [54.40] [2.22] [2.91] [1.44] Primary grocery Purchaser −0.04 −55.38 −1.09 −0.75 −1.40 [0.03] [41.79] [1.71] [2.23] [1.11] Has Children −0.02 −32.57 −0.29 −1.36 −1.19 [0.03] [41.24] [1.68] [2.20] [1.09] * P < 0.1, P < 0.05, * P < 0.01 Ethics approval and consent to participate This study protocols and procedures are in accordance with the Declaration of Helsinki and were approved by the National University of Singapore Institutional Review Board Reference Code: S-17-172. Written informed con- sent was obtained from all subjects. Abbreviations FOP: Front-of-pack; GLM: Generalized Linear Model; HCS: Healthier choice symbol; NCDs: Non-communicable diseases; NIP: Nutrition information panel; OLS: Ordinary least squares; RCT: Randomized controlled trial; SS: Black stop- sign with the words high-in-sugar; TW: Health warning label with deterring text Consent for publication Not Applicable. Competing interests The authors declare that they have no competing interests. References Global, regional, and National Consumption of sugar-sweetened beverages, fruit juices, and Milk: a systematic assessment of beverage intake in 187 countries. PLoS One. 2015;10(8):e0124845. 5. Corvalan C, Reyes M, Garmendia ML, Uauy R. Structural responses to the obesity and non-communicable diseases epidemic: the Chilean law of food labeling and advertising. Obes Rev. 2013;14(Suppl 2):79–87. 5. Corvalan C, Reyes M, Garmendia ML, Uauy R. 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https://openalex.org/W2884289401	https://repositorium.uni-muenster.de/transfer/miami/0066fee6-8888-45a0-b201-a54584b2cae0	English	null	Visual attention in 5-year-olds from three different cultures	PloS one	2,018	cc-by	12,766	RESEARCH ARTICLE Visual attention in 5-year-olds from three different cultures Moritz Ko¨ster1,2, Shoji Itakura3, Relindis Yovsi4, Joscha Ka¨rtner1 1 Department of Psychology, University of Mu¨nster, Fliednerstraße 21, Mu¨nster, Germany, 2 Institute of Psychology, Free University Berlin, Habelschwerdter Allee 45, Berlin, Germany, 3 Department of Psychology, Graduate School of Letters, Kyoto University, Yoshida-honmachi, Sakyo-ku, Kyoto, Japan, 4 Independent ECD consultant, Brussels, Belgium moritz.koester@fu-berlin.de * moritz.koester@fu-berlin.de a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 Editor: Kim A. Bard, University of Portsmouth, UNITED KINGDOM Received: June 21, 2017 Accepted: June 24, 2018 Published: July 16, 2018 Copyright: © 2018 Ko¨ster et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: The data assessment in Cameroon was partly funded by a DAAD fellowship (German Academic Exchange Service) to MK. The data assessment in Cameroon was partly funded by an Owen Aldis Award (International Society for Human Ethology) to MK. Ruth Baumann received a PROMOS stipend from the University of Mu¨nster. We received financial support by Open Access Publication Fund of University of Mu¨nster. OPEN ACCESS OPEN ACCESS Citation: Ko¨ster M, Itakura S, Yovsi R, Ka¨rtner J (2018) Visual attention in 5-year-olds from three different cultures. PLoS ONE 13(7): e0200239. https://doi.org/10.1371/journal.pone.0200239 Citation: Ko¨ster M, Itakura S, Yovsi R, Ka¨rtner J (2018) Visual attention in 5-year-olds from three different cultures. PLoS ONE 13(7): e0200239. https://doi.org/10.1371/journal.pone.0200239 Citation: Ko¨ster M, Itakura S, Yovsi R, Ka¨rtner J (2018) Visual attention in 5-year-olds from three different cultures. PLoS ONE 13(7): e0200239. https://doi.org/10.1371/journal.pone.0200239 Editor: Kim A. Bard, University of Portsmouth, UNITED KINGDOM Abstract Cognitive processes differ markedly between children from different cultures, with best evi- dence for attention to visual scenes and the activities of others. Children from urban West- ern cultures tend to focus on focal objects, whereas children from urban East-Asian cultures rather attend to contextual elements of a visual scene. Regarding the attention to others’ activities, children from subsistence-based farming communities often observe several activities simultaneously, while children from urban Western contexts focus on activities sequentially. Here we assessed 144 5-year-old children from three prototypical cultural con- texts (urban Germany, rural Cameroon, urban Japan) to investigate variations in attention across a variety of tasks. Attention to the elements of a visual scene was assessed in an optical illusion task, in picture descriptions and an eye-tracking paradigm. Attention to and learning from others’ activities was assessed in a parallel action task and a rule-based game. Some tasks indicated higher context-sensitive attention in urban Japan, while other findings indicated higher context-sensitive attention in urban Germany. Levels of parallel attention and learning from others’ activities were lower in rural Cameroonian children com- pared to the urban samples. Across tasks, the visual attention measures were unrelated. These findings substantiate that culture has a profound influence on early cognitive develop- ment, already in the preschool years. Furthermore, they raise critical questions about the early origins of cultural specificities in attention and the generalizability of attention phenom- ena beyond specific tasks and populations. Visual attention in children from different cultures developed a set of tasks to assess diverse aspects of children’s visual attention and employed these tasks in three prototypical cultural contexts (urban Germany, rural Cameroon, urban Japan). The main objective was to assess multiple indicators of children’s attention to visual scenes and the activities of others in order to shed new light on cultural differences in diverse aspects of visual attention, and how they may be related to each other in the preschool years. Competing interests: The authors have declared that no competing interests exist. Competing interests: The authors have declared that no competing interests exist. Regarding the attention to visual scenes, there is a research tradition that focuses on edu- cated urban middle-class populations in Western and Eastern cultural contexts and describes two prototypical attention styles [2]. An analytic style with a focus on focal objects and their properties (i.e., low context-sensitivity), described for people from Western cultural contexts, and a holistic style with a higher sensitivity for the context and the relations between elements in a scene (i.e., high context-sensitivity), described for East Asian adults. For example, Masuda and Nisbett [5] found that US-Americans tended to report a focal fish swimming in an aquar- ium, while Japanese participants reported more details from the background, including plants and smaller animals, when describing a picture. Similar differences were found in visual atten- tion processes as measured by participants’ gaze behavior [6, 7]. For example, Chua and col- leagues [6] presented pictures with a focal object on a background (e.g. a tiger in the woods) and recorded the gaze behavior of Chinese and US-American university students. They reported that Chinese students spent more time looking on the background than US-Ameri- can students. In the same vain, East Asian participants perceive more relations between objects [8, 9] and are more easily deceived by optical illusions, when adjusting a focal element within a deceptive context [10], compared to Western participants. These attentional phenomena are assumed to index more general attention styles, with higher or lower sensitivity for contextual elements of a visual scene, which explain variation across verbal, non-verbal, and non-seman- tic tasks [1, 2]. Recent studies found a relation between different measures, namely picture descriptions and gaze pattern in adults [11] and a relation between picture descriptions, mne- monic measures and even visual cortical processes in early childhood [12]. Introduction Human basic cognitive processes differ strongly between cultures. A specific interest lies in the developmental origins of cultural differences in visual attention in the childhood years [1–4]. Two major lines of research from the past decades revealed cultural differences in the attention to visual scenes (for reviews, see [1, 2]) and to others’ activities when learning from others (for reviews, see [3, 4]). Thus far, both lines of research were investigated independently and focused on different age groups and different cultural contexts. In the present study, we PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 1 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Visual attention in children from different cultures more time shifting their attention to different simultaneous activities and learning opportuni- ties, compared to children from Euro-American urban middle-class families, who show a more focused, sequential attention pattern when learning from others. For instance, when learning a new activity, triads of 6- to 10-year-olds from Indigenous-heritage regions of Mexico were often keenly observing the model and the other child and distributed their atten- tion between multiple events simultaneously. Euro-American children showed a rather focused attention style, focusing their attention on one event [18–20]. According to Rogoff and colleagues, the keen observation of others’ activities, typical reported for subsistence- based communities, also has implications for observational learning. For example, 5- to 11-year-old Indigenous-heritage children from Guatemala were better at attending to ongoing events, compared to Euro-American children. They were given a distractor toy and were told to wait, while the experimenter demonstrated how to produce a toy animal to another child, Guatemalan children were more often attending to both the distractor toy and the ongoing activity simultaneously and, as a consequence, were better at learning the demonstrated action sequence. Namely, they needed less support when given the chance to reproduce actions on a toy animal one week later [21]. Rogoff also proposes that children in these contexts, keenly attending to the activities of others, are also better at extracting conventional rules and regular- ities by observing others’ activities [3]. Rogoff and colleagues explain these culture-specific attention patterns when observing the activities of others by different types of learning opportunities [22]. Children from Indige- nous-heritage communities typically participate in the life of adults and learn by community participation (keen observation of and pitching into the activities of others). In contrast, learn- ing in the US is often based on direct teaching and segregated from everyday life, which becomes specifically apparent in the context of formal education. Both lines of research describe cultural differences in the way children attend to different aspects of their environment. However, both lines of research are disparate and rely on very different theoretical assumptions and empirical findings from different cultural contexts, com- paring urban Western with urban Eastern cultural contexts or urban Western with rural sub- sistence-based communities. Furthermore, there is evidence that differences in context-sensitive attention are socialized and culturally trans- mitted via verbal learning processes [11, 12]. Cross-cultural differences in holistic and analytic attention emerge roughly between 5 and 7 years of age, with evidence from picture description [13] and optical illusion tasks [11, 14, 15], as well as the Framed Line Test [16, 17]. In a study by Imada and colleagues [13], 6- to 7-year-olds (but not younger children) from Minneapolis, USA, and Kyoto, Japan, showed sig- nificant differences across several indicators from a picture description task and an Ebbin- ghaus illusion. A recent study compared the degree of context-dependent attention in optical illusions in children from more diverse cultural contexts, an urban sample from the United Kingdom, an urban Namibian sample, and a sample of traditional Himba children across a broad age range [14]. The researchers found that participants’ deception to the optical illusions increased in UK children in the early school years, but only between 9 and 10 years in urban Namibian children. Overall, it remained at a very low level in traditional Himba children across the investigated age range, from as early as 3 years of age into adulthood. The authors explained these effects mainly by urban environmental factors as well as different levels of experience with print media. Overall, these findings show that culture-specific attention styles start to emerge in the late preschool years, before the 6th year of age, and further increase in the years thereafter. In another research tradition in developmental psychology, influenced by cultural anthro- pology, Rogoff and colleagues [3, 4] have theorized and documented differences in attention and the consequences for learning by observing the activities of others in urban Western and rural subsistence-based contexts in Central America. The authors describe a distributed atten- tion pattern for children from Indigenous-heritage communities of the Americas, spending PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 2 / 21 The main purpose of the current study is to assess analytic and holistic visual attention [1, 2] together with sequential and distributed attention [3, 4] and potential consequences for learning from others’ activities. Specifically, we were interested in cultural differences in attention to visual scenes and the activities of others which already exist in the preschool years, before children have been influenced by formal schooling. Concerning schooling, it has been argued that it affects visual attention and learning from others in the sense that it leads to more focused and sequential patterns of attention and generally encour- ages direct teaching and learning at the expense of distributed attention to others’ activities [4]. We selected three cultural contexts, each representing an often-studied prototypical cultural contexts. These were urban middle-class samples from Western and East-Asian cultures, a selection informed by the first line of research, and a sample of children living in a subsis- tence-based farming ecology in rural Cameroon, also an often studied and well-described cul- tural context [23]. The characteristics of the latter nicely map onto cultural contexts described by Rogoff, especially in terms of children’s education through participation in communal endeavors and adults’ everyday activities. In developing their argument, Rogoff and colleagues repeatedly draw on findings from Indigenous-heritage, subsistence-based communities out- side the Americas, mainly findings from Sub-Sahara Africa and Asia, with low levels of formal education [3, 4, 23]. These contexts were selected because they capture the essence of those included in previous research (e.g. [1, 3]). The urban German middle-class represents a prototype of an PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 3 / 21 Visual attention in children from different cultures individualistic [24] (or autonomous [25]) cultural context, with small family and household sizes (mainly the nuclear family) and high levels of formal education. Parents are occupied in professional jobs, and their children usually attend the kindergarten. Children furthermore engage in organized hobby groups, and possess a large variety of advanced toys, including computer games. The focus of parental behavior and socialization goals lies on dyadic interac- tions and individual development, such as making choices independently [25, 26]. The life- world of children in Japan looks very similar in terms of the ecology (urbanization, education, wealth, modernity), however, the social structure is often referred to as interdependent. This is, personal relationships and socialization goals are characterized by stable, often life-long social bonds between family members and friends, which come with specific role obligations and entitlements [27], manifesting in an interdependent self-concept [23, 24]. Regarding the environment, Japanese urban contexts are marked by higher complexity [28] (e.g., indicated by pictures of scenes from cities in Japan and the United States). The lifeworld of children from the Nso culture in rural Cameroon differs from those of Western and Eastern urban con- texts in a number of ways. Children grow up in large, extended family settings in subsistence- based villages, dominated by a relational cultural model [23]. Most parents are farmers and engage their children in household tasks and fieldwork from early on [26, 29]. From age 4, most children attend preschool in the mornings, while their parents work on the fields outside the village. Primary socialization goals are obedience and taking over responsibilities associ- ated with social roles in hierarchical social relationships. A large body of research on the cultural differences in human basic cognitive functions focusses on the early school years and the years thereafter. In the present study, we aimed to understand cultural differences in visual attention with no or only minimal schooling experi- ences (i.e., the preschool in Cameroon). Furthermore, we aimed to understand the relation between different tasks on attention to scenes and other’s behavior and the generalization of phenomena beyond previously studied cultural contexts. Towards this end, we adapted and developed a set of tasks to assess attention to visual scenes and others’ behavior in Mu¨nster (urban Germany), Banten (a farming village in rural Cameroon) and Kyoto (urban Japan), at the age of five. To obtain multiple measures for chil- dren’s holistic and analytic attention (i.e., high or low context-sensitivity), we employed classi- cal tasks which assess children’s context-sensitive attention, namely an optical illusion task, an eye-tracking paradigm, and a picture description task. To measure children’s attention to oth- ers’ activities, we measured their learning performance for actions that occur in parallel and by observing the conventions of a rule-based game. First, children saw two action sequences simultaneously (manual production of a toy), their gaze was recorded, and they could repro- duce both action sequences afterwards. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Results We assessed 144 5-year-old children (M = 5;5 years, SD = 0;4 in Germany, M = 5;6 years, SD = 0;4 in Cameroon, and M = 5;8 years, SD = 0;3 in Japan) from Mu¨nster (urban Germany; n = 43), farming villages in Banten near Kumbo (rural Cameroon; n = 52), and Kyoto (urban Japan; n = 49). The age of the children was significantly different between cultures, F(2, 141) = 8.62, p < .001, namely children from Japan were older then children from Germany and Cam- eroon, both \|t\| > 2.53, p < .013. However, overall, age did not correlate with the 14 dependent measures reported throughout the analyses in any of the three cultures (i.e., only 1 out of 42 correlations reached p < .05). Thus, age was not entered as a covariate in the subsequent analy- ses. Proportions of girls and boys (60% girls in Germany, 45% girls in Cameroon, and 52% girls in Japan) did not differ between the three samples, χ2 = 2.22, p >.25. Note that not all children completed the full set of tasks and that, for each task, analyses are based on the complete subsets from each culture (find details about the procedure, analyses, and exclusion criteria in the Materials and Methods section below.) All data reported in the manuscript are available in the Supporting Information (S1 File). Second, children saw two actors playing a rule-based game that included sorting rules (objects to locations) and hand activities, before they played the game with the experimenter to assess their learning performance. Drawing on the two lines of research outlined above, we hypothesized that visual attention measures tend towards the holistic style in Kyoto as compared to Mu¨nster, and, based on the findings from the optical illusion tasks [14, 15], a more object-focused attention style for Ban- ten, compared to children from the urban contexts. Furthermore, we expected higher levels of distributed attention when observing parallel activities in the village of Banten as compared to the Western urban context in Mu¨nster and, possibly, also compared to Japan. Regarding the two different theoretical views on children’s attention across cultures, namely analytic versus holistic and sequential versus distributed attention, we aimed to explore if these measures would be associated with each other across different tasks and domains (i.e., attention to visual scenes and the activities of others), in the preschool years. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 4 / 21 Visual attention in children from different cultures Cultural contexts Parameters of children’s family contexts are summarized in Table 1. There were significant dif- ferences in the number of siblings, with a higher number of siblings in rural Cameroon com- pared to two urban contexts, both \|t\| > 11.62, p < .001, also reflected in the people living in the household, both \|t\| > 13.67, p < .001. In Germany, the majority of mothers and fathers worked in the professional jobs (62.8% and 74.4%, respectively), in Cameroon most parents were farm- ers (mothers: 92.5%, fathers: 89.6%), and in Japan mothers were mostly house wives (41.8%) with husbands being office workers (51.0%). We tested for potential correlations between three socio-demographic variables, which we assessed from all children (number of siblings, people in the household, years of maternal edu- cation, see Table 1). The correlation of these three measures with all 14 dependent measures from the three different tasks (in 3 cultures) revealed that only 8 out of 126 correlations (i.e., 6.35% of all correlations) were significant at the level of p < .05, which corresponds pretty much to the chance level. Thus, these variables were not included as covariates in the subse- quent analyses. This is, since the potential covariates (age of the mother, number of siblings, people in the household, years of maternal education) were not independent of the group vari- able culture (independent variable), and there was no major influence of these potential covar- iates on the dependent variables, the preconditions for an analysis of covariance (ANCOVA) were not met ([30], p. 200). Note. The table presents means with standard deviations in parentheses. Note. The table presents means with standard deviations in parentheses. Optical illusion task We tested children’s deception by context information with an optical illusion task [11–15] for four illusions (two Ebbinghaus illusions, Mu¨ller-Lyer illusion, Sander illusion), see Fig 1A, Table 1. Family contexts. Variables Germany Cameroon Japan F p Age mother (years) 39.9 (5.3) 37.7 (7.7) 40.0 (3.8) 1.11 >.25 Number of siblings 1.1 (0.8) 5.1 (2.1) 1.2 (0.9) 8.62 < .001 People in household 4.0 (1.0) 6.6 (2.0) 4.1 (0.8) 168.32 < .001 Mother, years of school 16.0 (3.1) 7.1 (2.8) 15.5 (1.8) 174.92 < .001 Table 1. Family contexts. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 5 / 21 Visual attention in children from different cultures Fig 1. Optical illusion task. (A) The four optical illusions. The red element had to be adjusted at the side indicated by the black arrow. (B) Group comparisons indicate the results of t-tests, following significant main effects. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi org/10 1371/journal pone 0200239 g001 Fig 1. Optical illusion task. (A) The four optical illusions. The red element had to be adjusted at the side indicated by the black arrow. (B) Group comparisons indicate the results of t-tests, following significant main effects. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g001 each illusion presented twice. Before the illusion task, the same red elements were shown with- out the gray, illusory contextual elements, to obtain a measure of children’s accuracy. In a first step, the illusion scores were entered into a repeated-measures ANOVA with task (4 tasks) as within factor and culture (3 contexts) as between factor. We found a main effect of task, F(3, 414) = 131.59, p < .001, η2 = .49, and culture, F(2, 138) = 11.76, p < .001, η2 = .15, but no culture x task interaction, F(6, 414) = 1.21, p = .301, η2 = .02. Urban German and urban Japanese children were deceived to a higher degree (M = 20.1% and M = 17.7%) than children from rural Cameroon (M = 13.9%), see Fig 1B. Mean deception values, separated by task were: Ebbinghaus, circle: 9.3%; Ebbinghaus, diamond: 8.0%; Mu¨ller-Lyer: 24.1%; Sander: 27.1%. We further assessed the accuracy of the adjustments of the red shapes in control trials, without context. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Optical illusion task Urban German and urban Japanese children were more accurate (M = 5.2% and M = 5.4%) than children from rural Cameroon (M = 9.5%), F(2, 138) = 59.41, p < .001, η2 = .46, see Fig 1B. In a second step, we explored the correlations between the scores of the different illusions, to see if they would assess the same perceptual phenomenon. This revealed that the scores from both versions of the Ebbinghaus illusion were correlated, consistently in all three cultures (urban Germany: r = .44, p = .004; rural Cameroon: r = .23, p = .115; urban Japan: r = .34, p = .015). Likewise, the scores from the Mu¨ller-Lyer and the Sander illusion were correlated (urban Germany: r = .29, p = .063; rural Cameroon: r = .35, p = .014; urban Japan: r = .30, p = .032). All other correlations between the optical illusions were low (urban Germany: both \|r\| < .16, p > .314; rural Cameroon: \|r\| < .11, p > .461; urban Japan: \|r\| < .11, p > .453). Thus, these two pairs of illusions seem to assess independent perceptual processes, which may be described as field dependence (Ebbinghaus illusions) and sensitivity to carpentered corners (Mu¨ller-Lyer and the Sander illusion). 6 / 21 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Visual attention in children from different cultures Fig 2. Picture description task. (A) Children described pictures with a focal object to the experimenter. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g002 Fig 2. Picture description task. (A) Children described pictures with a focal object to the experimenter. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g002 Fig 2. Picture description task. (A) Children described pictures with a focal object to the experimenter. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g002 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Picture description task Another task to assess context-sensitivity in terms of explicit attention to focal and background elements and their features are picture description tasks [11–13]. Here, children described pic- tures with a focal object (animals and means of transport), in front of a simple background (e.g., natural scenes, roads and buildings), see Fig 2A, to the experimenter. The relative number of references to the object, compared to the references to the back- ground (object score) differed between cultures, F(2, 121) = 14.95, p < .001, η2 = .20, and was higher in urban Japanese children, compared to both other samples, see Fig 2B. German chil- dren uttered a higher proportion of relations between the elements of a scene, compared to rural Cameroonian children (M = .057 versus M = .024 relations per picture) and Japanese children, where relations were almost absent (M = .013 relations per picture), F(2, 121) = 7.72, p < .001, η2 = .11. Part of these findings may be explained by differences in how talkative children were, F(2, 121) = 7.63, p < .001, η2 = .11. Picture descriptions were most verbose in urban Germany (M = 3.9 references to object or background and their features), followed by rural Cameroon (3.3 references) and then urban Japan (M = 2.7 references), and differed significantly between Germany and Japan. Correlations between number of references (i.e., volume) and the depen- dent variables (object score and relations) revealed a highly negative correlation between object score and volume across samples, all r < -.75, all p < .001, but no correlation with the number of relations, all r < .15, all p > .353. Therefore, we also looked at the object score for the first reference made by the participants, i.e., the percentage of picture descriptions that started with the object and not the background. Similar to the results of the object score, this analysis revealed a significantly higher proportion of first references to the object in urban PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 7 / 21 Visual attention in children from different cultures Japanese (M = .95) compared to urban German (M = .82) and rural Cameroonian children (M = .85), F(2, 121) = 6.53, p = .002, η2 = .10. Japanese (M = .95) compared to urban German (M = .82) and rural Cameroonian children (M = .85), F(2, 121) = 6.53, p = .002, η2 = .10. Eye-tracking task To assess early visual processing of scenes, similar pictures to those in the picture description task where presented, participants’ gaze behavior was recorded [6, 11]. Children saw real pic- tures which displayed objects in front of a simple background and abstract (non-semantic) pic- tures with artificial objects [31] on abstract backgrounds [32], see Fig 3A. These where included because they were unfamiliar to children from all contexts. In a first step, we entered the proportion of (1) the total looking time spent exploring the object and (2) the latency of the first fixation to the object into two independent ANOVAs with the within factor picture type (natural, abstract) and culture as a between factor. Both ANOVAs revealed a main effect of picture type, latency: F(1, 127) = 207.13, p < .001, η2 = .62, looking time: F(1, 127) = 414.49, p < .001, η2 = .77, and culture, latency: F(2, 127) = 72.60, p < .001, η2 = .53, looking time: F(2, 127) = 46.92, p < .001, η2 = .10, and the looking time on the object (but not the latency score) revealed a culture x picture type interaction, F(2, 127) = 7.70, p < .001, η2 = .11. Given this interaction, we further explored the cultural differences in look- ing time in subsidiary ANOVAs, split for the picture types. For natural pictures, rural Camer- oonian children looked at the objects in each picture longest (object focus: M = 82% of the looking time), compared to urban German children (M = 73%) and urban Japanese children (M = 72%), see Fig 3B, F(2, 127) = 70.17, p < .001, η2 = .53, and, F(2, 127) = 24.46, p < .001, η2 = .28, respectively. For the abstract pictures, there were significant differences in the object focus between cultures, F(2, 127) = 34.85, p < .001, η2 = .35, again, with a higher focus on the object in rural Cameroon, (M = 65%), compared to urban Germany (M = 52%), and urban Fig 3. Visual attention in an eye-tracking task. (A) Children saw natural pictures with a clear object and background, as well as abstract objects background combinations. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g003 Fig 3. Visual attention in an eye-tracking task. Eye-tracking task (A) Children saw natural pictures with a clear object and background, as well as abstract objects background combinations. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g003 Fig 3. Visual attention in an eye-tracking task. (A) Children saw natural pictures with a clear object and background, as well as abstract objects background combinations. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g003 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 8 / 21 Visual attention in children from different cultures Japan (M = 46%). The first fixation onto the object, collapsed over stimulus type, was signifi- cantly later in Japanese (M = 907 ms) compared to German (M = 548 ms) and rural Cameroo- nian children (M = 459 ms), F(2, 127) = 72.60, p < .001. Furthermore, we explored the correlations between the scores of the different measures, to see if they would assess the same perceptual phenomenon. Correlational analyses revealed that all four measures were closely related in urban Germany (all six correlations: \|r\| > .31, p < .059, at least at the level of a trend). However, the correlations were less consistent in rural Cameroon and urban Japan, where correlations were only found between the two measures (Object focus and first fixation) within the two stimulus categories, namely for normal pictures in Japan (r = -.31, p < .042), and for abstract pictures in Cameroon (r = -.51, p < .001) and Japan (r = -.49, p < .001). Parallel actions task We aimed to capture children’s distributed attention, that is, the extent to which children are able to attend to two activities in parallel, more rigorously than previous studies [19–21]. There- fore, we developed a video-based task, with two action sequences shown simultaneously, see Fig 4A and S2 File. As a direct measure of distributed attention, we recorded the gaze behavior of the children and, as an indirect measure of distributed attention, children were given the oppor- tunity to reproduce the two handicrafts afterwards and their performance was coded. The number of attention shifts from one action sequence to the other was higher in urban Japanese, compared to urban German, and compared to rural Cameroonian children, and attention shifts were also significantly more frequent in urban Germany compared to rural Cameroon, see Fig 4B, F(2, 126) = 35.54, p < .001, η2 = .36. Furthermore, rural Cameroonian children focused more on one of the two action sequences (M = 64.9%), compared to the Fig 4. Attention to and reproduction of parallel action sequences. (A) Children saw a video, in which 2 actions sequences were performed in parallel, and their gaze was recorded. Later on the reproduction of actions was analyzed. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. htt //d i /10 1371/j l 0200239 004 Fig 4. Attention to and reproduction of parallel action sequences. (A) Children saw a video, in which 2 actions sequences were performed in parallel, and their gaze was recorded. Later on the reproduction of actions was analyzed. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < 01 p < 001 Bonferroni corrected Fig 4. Attention to and reproduction of parallel action sequences. (A) Children saw a video, in which 2 actions sequences were performed in parallel, and their gaze was recorded. Later on the reproduction of actions was analyzed. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. Fig 4. Attention to and reproduction of parallel action sequences. (A) Children saw a video, in which 2 actions sequences were performed in parallel, and their gaze was recorded. Later on the reproduction of actions was analyzed. Rule-based game The rule-based game was designed to assess the competence to infer conventional rules based on attending others playing a game [1, 2]. Specifically, we wanted to test children’s ability to understand the rules underlying the observed actions of a social game. Children saw a video with two people playing a rule-based game, see Fig 5A, before child and experimenter played the game together. Rural Cameroonian children acquired fewer rules (M = 1.06 remembered rules), compared to German (M = 2.44) and Japanese children (2.48), F(2, 139) = 24.81, p < .001, η2 = .26, see Fig 5B. Action reproduction differed significantly between all three cultural contexts with the highest number of reproduced action by urban Japanese children (M = 2.64), and higher action reproduction skills in urban Germany (M = 2.07), compared to rural Cameroon (1.39), F(2, 139) = 11.74, p < .001, η2 = .15. Both measures were correlated in the rural Cameroonian sample (r = .34, p = .017) but not in the two urban samples (both \|r\| < .03, p > .87). Correlations between the measures between the different tasks Regarding the between-task correlations within each cultural context, there was no consistent correlational pattern. This was indicated by the fact that, first, no single correlation between two measures reached significance across all three contexts and, second, that less than 10% of all correlations (uncorrected) were above \|r\| = .30. Parallel actions task (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g004 PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 9 / 21 Visual attention in children from different cultures urban German (M = 60.0%) and to the Japanese children (M = 59.9%), F(2, 126) = 5.71, p = .004, η2 = .08. With regard to the number of actions reproduced, children from rural Cameroon exhibited fewer of the nine action sequences (M = 1.75) than children from urban Germany (M = 3.65) and Japan (M = 3.44), F(2, 132) = 20.73, p < .001, η2 = .24. However, when looking at the pro- portion of both parallel actions out of all reproduced actions, in the number of all actions, there were no differences between cultural contexts, F(2, 129) = 1.22, p = .300, η2 = .02. Correlational analyses, to see if the different measures would assess similar attention pro- cesses, revealed close associations between the rate of attention shifts and the distribution of attention (Germany: r = -.42, p = .009; Cameroon: r = -.44, p = .002; Japan: r = -.21, p = .164), this is, the more they shifted their gaze between action sequences, the more equal they distrib- uted their gaze time between both actions. Noteworthy, this close association may be due to the close dependency of both measures, with more attention shifts allowing an more equal distribution of attention towards the two action. Furthermore, the number of reproduced actions was closely related to the proportion of parallel actions children reproduced (Germany: r = .74, p < .001; Cameroon: r = .24, p = .108; Japan: r = .62, p < .001). However, there were no correlations between the visual attention measures and the learning measures (all \|r\| > .26, p > .13). Again, this may be because both measures are interdependent, with more actions reproduced leading to higher probability of parallel actions to be reproduced. Discussion p < .05, p < .01, p < .001, Bonferroni corrected. https://doi.org/10.1371/journal.pone.0200239.g005 combined the cultural contrasts of previous studies, namely Western vs. East-Asian urban middle-class and Western urban middle-class vs. Non-Western subsistence-based farming ecologies. This also allowed us to test the consistency of different aspects of children’s attention pattern across tasks and cultures. Taken together, the findings of the present study indicate that basic cognitive functions vary highly between cultures, already in the preschool years. At the same time, as outlined in more detail below, our findings were much more heterogeneous than former literature sug- gested and, despite the significant cross-cultural differences in attention to scenes and others’ activities, the different measures were not related to each other. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Discussion The aim of the present study was to investigate similarities and differences in the visual atten- tion to scenes [1, 2] and to the activities of others [3, 4], in preschool children from different cultural contexts. Towards this end, we investigated these phenomena across several tasks in three prototypical cultures (urban Western, urban Eastern and a rural subsistence-based vil- lage contexts). First, this was to assemble tasks which assess different aspects of children’s visual attention processes, namely analytic and holistic attention [1, 2], as well as sequential and distributed attention [3, 4]. Second, we assessed these tasks in three cultural contexts that PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 10 / 21 Visual attention in children from different cultures Fig 5. Learning conventions in a rule-based game. (A) Children saw a video, in which 2 players played a rule-based game including 4 rules and 4 actions. Later on, their reproduction for the rules and actions was tested, when playing the game with the experimenter. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. https://doi org/10 1371/journal pone 0200239 g005 Fig 5. Learning conventions in a rule-based game. (A) Children saw a video, in which 2 players played a rule-based game including 4 rules and 4 actions. Later on, their reproduction for the rules and actions was tested, when playing the game with the experimenter. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. Fig 5. Learning conventions in a rule-based game. (A) Children saw a video, in which 2 players played a rule-based game including 4 rules and 4 actions. Later on, their reproduction for the rules and actions was tested, when playing the game with the experimenter. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. p < .05, p < .01, p < .001, Bonferroni corrected. Fig 5. Learning conventions in a rule-based game. (A) Children saw a video, in which 2 players played a rule-based game including 4 rules and 4 actions. Later on, their reproduction for the rules and actions was tested, when playing the game with the experimenter. (B) Group comparisons indicate the results of t-tests, following significant ANOVAs. Cultural differences in attention to visual scenes Our main assumption was that Japanese (urban East-Asian) children would show greater lev- els of holistic attention, compared to German (urban Western) children. The results were mixed, some pointing into the expected direction and other pointing into the opposite direc- tion Taken together, like in former studies, the results from the optical illusion task did not dif- fer between cultures and the results from the eye-tracking task show first hints for the classical differences between urban Western and urban Eastern contexts. Furthermore, results were contrary to the classical cultural pattern in the picture description task, pointing towards higher levels of context-sensitive attention in Germany compared to Japan. Furthermore, there were no consistent correlations between tasks in the preschool age, which is also consis- tent with former studies, reporting correlations only for older children and adults [11, 12]. The visual attention of children from rural Cameroon differed from both urban contexts and was characterized by a high object focus across tasks. In this sample, we found the lowest PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 11 / 21 Visual attention in children from different cultures level of deception in the optical illusion task. Furthermore, Cameroonian children made the first saccade on the object most rapidly and spent more time exploring the object than both other samples. In the picture description task, Cameroonian children fell somewhere between the other two samples. The reduced deceivability of rural Cameroonian children is generally in line with ecological accounts of optical illusions, which emphasize the importance of the expe- riences with carpentered corners for the effect of optical illusions like the Mu¨ller-Lyer and the Sander illusion (cf. [33]). Specifically, the present findings complement recent studies con- ducted in another rural village context, namely in traditional Himba people [14], where con- text-sensitivity in the Ebbinghaus task was very low across the whole life span. Thus, cultural differences in context-sensitive attention are present already in the preschool years, when they are not yet found in the classical eastern western comparison (see [13, 16] and above). The higher object focus in the eye-tracking paradigm is consistent with the low level of context sen- sitivity in the illusion tasks. Alternatively, this may also reflect an unfamiliarity-effect, because the objects shown here, both real and abstract, are less common in the Nso children’s lifeworld and may have led to an increased interest. Cultural differences in attention to visual scenes One potential explanation for the low levels of context-sensitivity found in the picture description task would be that this sample of Japanese urban dwelling children were non-tradi- tional or differed in some other way from the typical Japanese samples in other studies [1, 2]. However, the participants came from the same population as in the study by Imada and col- leagues [13], where context-sensitive attention, pooled across several tasks, including picture descriptions, emerged in the school years. Overall, these findings suggest that visual attention towards focal or contextual elements of a scene is influenced by the cultural context. However, in the early childhood years it seems not yet to be a consistent construct that aligns visual attention across different types of tasks (e.g., verbal, non-verbal, semantic, abstract). Potentially, the culture-specific context-sensitivity pattern reported in literature may override the differences identified here later in development [11, 12, 13, 34], possibly via verbally guided learning processes [11, 12, 35], for example, as found in formal schooling. The differences in visual attention identified here, for the preschool years, may be based on different cultural and environmental learning experiences. For exam- ple, they may rather reflect children’s basic familiarity with the stimulus materials and stimuli, rather than culturally transmitted attention styles. In line with this proposal, see recent studies by Ko¨ster and colleagues [11, 12], for similar inconsistencies in context sensitive attention in 5-year-olds, which develop in more coherent pattern from around 7 years of age. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Cultural differences in in attention to others’ activities Regarding children’s attention to others’ activities, we found a different pattern than hypothe- sized. Specifically, in the parallel action task, children from rural Cameroon focused their atten- tion rather on one of two activities and shifted their gaze between scenes at a lower rate than children from both urban contexts. Furthermore, their learning performance was at an overall lower rate. Similarly, in the rule-based game, rural Cameroonian children performed fewer rules and actions compared to urban German and Japanese children. The comparison between urban Germany and urban Japan did not reveal many differences, but there were more frequent shifts of attention in Japan then in Germany, when observing two action sequences in parallel. Interestingly, the attention pattern towards parallel actions, namely a more unilateral atten- tion on one of the two activities in rural Cameroonian children, resembles the more object- focused attention of Cameroonian children found in the visual attention task. Although the attention and learning pattern was different from previous studies [3, 4], it may be speculated that the ability to distribute attention to various aspects of a visual scenes or when observing PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 12 / 21 Visual attention in children from different cultures others’ activities allows children to grasp more details about the activities and conventions of others. Here, this may explain higher scores of distributed attention as well as learning scores in Germany and Japan. However, within cultures, we found no consistent correlations between the tasks assessing the attention to visual scenes and the activities of others, despite moderately large sized samples. Differences in visual attention from a lifeworld perspective Most importantly, the task testing visual attention to scenes employed here were, thus far, mostly applied in Western and Eastern urban contexts, and the materials used to test visual attention to others activities used materials certainly more familiar to German and Japanese children. Furthermore, the form of presentation, namely on a computer screen, was not known to Cameroonian children. Thus the present results, and in particular the learning per- formances for others’ activities have to been interpreted in the light of the very different life- world’s and learning contexts, which children from the different sample reported here grow up in. We outlined the contrast of the lifeworlds of the prototypical contexts chosen here in the introduction. In the tenet outlined above, the manifold variations in children’s visual attention between cultures identified here, are very likely due to the different experiences children make in their environment for their first five years of life. In this sense, we conceptualize early cul- tural differences in visual attention as a results of versatile learning experiences. At least, com- mon dichotomous perspectives, based on one or a few experimental tasks and the comparison of a two cultural contexts, do not account for the variations between and within the cultural contexts identified here. In this sense, the present results also illustrate the great challenges for the development of experimental materials, which may capture different aspects of children’s attention in a cultur- ally fair way. For example, regarding rural Cameroon, we chose very simple materials for the construction of the toys and very basic material and actions in the social game. However, it may still be less common for children from Cameroon to tinker handicrafts or to learn rule- based games or, critically, to acquire novel information from a screen, instead of a live interac- tion. Noteworthy, although the children watched the two tasks simultaneously, they were given the construction tasks sequentially, which may be more typical in the urban contexts. Regarding the pictorial stimuli, Cameroonian children did only recognize and label about half of the objects assessed in the picture description task (and only those were taken into account in the analyses). Similarly, abstract stimuli might be more familiar to children in Mu¨nster and Kyoto. Thus, the present findings on learning from others may demonstrate that cognitive functions are inextricably knit to everyday visual experiences and materials (cf. [36]). Visual attention in children from different cultures culture-specific pattern of attention, as indicated by the correlation of different measures of context-sensitivity found in older children [12] and adults [11]. Specifically, recent findings highlight the role of language in cultural transmission processes [11, 12, 35]. To better under- stand the enculturation process of general attention styles, which may emerge later in life, future studies should be complemented by longitudinal or cross-sectional assessments, across a broader age-range. With regard to the very different lifeworlds of children from the present study, the tasks employed here may rather reflect the activities of urban Western and East-Asian cultures. This had very likely negative consequences for the learning performance of the rural Cameroonian children. Future research may benefit from adapting some of the experimental tasks more closely to the daily activities and the materials in this cultural context. This includes children’s activities and play habits, the materials and pictorial stimuli used, as well as the form of presen- tation. For example, using life interactions to demonstrate activities. It should also be noted that, besides those features of the tasks that assess the attention to the elements of a visual scene and others’ activities, each task requires additional and unique cognitive capacities and skills that may have contributed to cultural differences and compro- mised associations between tasks. For instance, performance in the optical illusion tasks may furthermore depend on inhibitory control, and both the picture description and eye-tracking tasks may depend on the familiarity with the natural, but also the abstract stimuli used. Finally, the reproduction tasks for the observed activates require advanced memory and, in the case of the artcraft, manual skills. Although we tried to keep these requirements as low as possible, we cannot preclude that these have caused some of the inconsistencies found in the present study. Thus, for future research it would be desirable to assess the diverse skills and possibly experi- ences which contribute to the differential response pattern in different tasks in early childhood. Taken together, the present study highlights the general importance (i) to assess common phenomena from cross-cultural Psychology with more diverse populations and tasks, (ii) to analyze changes in a broader age range, in order to understand developmental trajectories, and (iii) to assess more thoroughly and formally specific characteristics of the eco-social envi- ronment and adapt tasks more closely to different lifeworlds. Thus, the present study under- lines the necessity of further research, to elucidate theoretical accounts on ecological and social factors that contribute to inter-individual variations in cognitive development. Conclusion The cultural differences in attention to the elements of a visual scene and the activities of oth- ers in the preschool years, demonstrate early differences in development between cultures, which are more diverse than common theoretical accounts suggest. Human cognitive func- tions and processes align with and are tightly knit to the very specific experiences, such as the learning materials and activities. At least for the phenomenon of context-sensitivity, it may only be later in life that more general attention styles shape visual processes. It is a major chal- lenge for future research to identify the learning mechanisms and socio-ecological factors that give rise to the cultural versatility and culture-specific developmental trajectories of human basic cognitive functions in early childhood and later in life. Future directions By assessing visual attention with multiple tasks and methods and in novel cultures, the pres- ent findings add an important piece of puzzle to the existing literature. Because all measures indicated high cross-cultural variation at an early age already, there does not seem to be much generalizability beyond the specific contexts and tasks. Thus, more generally, the field would benefit from a higher variation of cultural contexts and experimental tasks assessed in these contexts. Beyond the description of variations, it would also be highly valuable to more thor- oughly assess environmental variables and cultural transmission processes (e.g., socialization), which may better elucidate the developmental origins of these variations. We selected the preschool age in the present study to avoid the influence of formal school- ing. This age may yet have been too early for attention processes to develop into a coherent PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 13 / 21 Participants We assessed 144 5-year-old children from three different cultural contexts, selected for their ecosocial profiles. Forty-three children lived in middle-class families from Mu¨nster (urban PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 14 / 21 Visual attention in children from different cultures Germany), 52 children came from small subsistence-based farming villages in Banten near Kumbo (rural Cameroon), and 49 children were from middle-class families in Kyoto (urban Japan). In Cameroonian villages, children were recruited in cooperation with local schools. In Kyoto and Mu¨nster, mothers were contacted via databases from the university. Four additional data assessments from rural Cameroon were not included in the analysis, because children came from a different ethnic group and did not speak Lamnso, the local language. Informed written consent was obtained from each parent, in each context, and children gave informed assent. Furthermore, not all children completed the full set of tasks. The number of missing children is indicated for each task below. Ethics statement This research was conducted in accordance with the Declaration of Helsinki and the Ethical Principles of the German Psychological Society (DGPs), the Association of German Profes- sional Psychologists (BDP), and the American Psychological Association (APA). It involved no invasive or otherwise ethically problematic techniques and no deception (and therefore, according to National jurisdiction, did not require a separate vote by a local Institutional Review Board; see the regulations on freedom of research in the German Constitution (§ 5 (3)), and the German University Law (§ 22)). Stimuli and procedure Children visited the laboratory for one experimental session. In Cameroon the laboratory was set up close to the school, whereas in Japan and Germany children visited the Laboratory of the University with a parent. Because the research focus was also on associations between tasks, but not on mean differences between conditions, assessments follow a fixed order, start- ing a warm-up phase, followed by the optical illusion task, the parallel learning task, the social game task, and finally the eye-tracking and the picture description task. The instructions for each task were read out to the children and children repeated the instructions prior to the start of each task. The stimuli presentation procedures were implemented in psychophysics toolbox (Version 3.0.12, on MATLAB, Version R2013a) and with the respective presentation programs in the eye-tracking tasks (Cameroon and Germany: ExperimentCenter, Version 3.5.169; Japan: Tobii Studio, Version 3.3.2). The presentation was on a notebook display and keyboard in Cameroon and a desktop Monitor and external keyboard in Japan and Germany. However, we used the same size for stimulus across cultural-contexts, 15 inch, at distance of 50–70 cm and we did not note any difficulties in the use of the keyboards in either of the two setups. Optical illusion task. Four different optical illusions (two versions of the Ebbinghaus illu- sion, Mu¨ller-Lyer illusion, Sander illusion; see Fig 1A) were shown twice, resulting in eight tri- als, presented in a randomized order. An adjustable element and a reference element were colored in red and deceiving context elements were colored in gray, see Fig 1C. The red ele- ment that could be adjusted was indicated by a black arrow and could be adjusted between -35 and +35 percent, compared to the size of the reference element via two keys of a keyboard. Children were asked to pay attention to the red elements to the same size as the reference element and to “not pay attention to the context, but only to the red element” (to avoid an rela- tive interpretation of the task instruction). Prior to the actual task, children were instructed carefully and could practice the adjustment of a red element in one training trial. Trials were presented in a randomized order and the side of the context elements was counterbalanced. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Each occurrence of the following categories was coded: (a) References to the focal object and its features (e.g., camel, car is large, looks happy), (b) references to the background (e.g., desert, road) and its features (e.g., is rocky, has green leaves), and (c) any relations that were made between elements within the picture (e.g., is driving on, is looking at). In order to quantify participants’ descriptions of the object compared to the background, we computed an object score: For each trial, all references to the object were divided by the number of all references to the object and the background. Thus, a score of 1 would indicate that a participant only talks about the object, and a score of 0 would indicate that a participant only refers to the background. These scores were averaged over all trials for each participant. Furthermore, we calculated the proportion of relations compared to all (number of relations divided by the sum of all references to the object, the background and all relations) as a second indicator for their context-sensitivity. Finally, we looked at the average volume of references (to object or background) and the proportion of first references, which referred to the object. In Germany and Japan the coding was done by native speakers. In Cameroon the data were translated and transcribed from the local language (Lamnso) to English for later coding. To compute inter-rater reliability in the Japanese sample, 20% of the picture descriptions were translated and transcribed to German by a native Japanese, fluent in German. Inter- rater agreements were assessed for more than 20% of the data (Cohen’s kappas for were: Germany: κ object and features = .82, κ background and features = .79, κ relations = .91; Cameroon: κ object and features = .80, κ background and features = .89, κ relations = .98; Japan: κ object and features = .63, κ background and features = .81, κ relations = .57, the low kappa for relations in the Japanese sam- ple was due to the low number of relations uttered by the children). Visual attention in children from different cultures element. This was to assess participants’ overall accuracy and correct the degree to which chil- dren were deceived by the context [11–13]. Children’s illusion score for each task was computed by subtracting the percent of decep- tion in the optical illusion tasks from the percent of deception in the trials without contextual information (in both conditions, with and without contextual information, we calculated the deception as the difference between the adjusted element and the red reference element). The mean deception over both trials of each illusion in percent (i.e., adjusting the left or the right shape) was used as the context-sensitivity measure for this task. For the overall accuracy, that is the precision of adjustment in trials without context, we averaged the absolute differences between the adjustable and the context element across all trials in the no context condition. Three children were excluded from the analyses of the optical illusion task (Cameroon: n = 2; Japan: n = 1). This was due to very high deviation between the reference and the adjust- able element, indicated by an accuracy value that deviated more than 2 SD from the sample specific mean, indicating difficulties in task comprehension. Picture description task. Twenty real pictures which displayed a focal object (animals and means of transport), in front of a simple background (e.g., natural scenes, roads and build- ings), see Fig 2. Pictures were taken by the first author or downloaded from a public domain database (pixabay.com). Pictures were presented for 15 s each, in a randomized order and sep- arated by a blank screen. Children were instructed to tell the experimenter, what they see on the pictures (exact wording: “. . . tell me everything you see on the pictures”). While children described the pictures, the experimenter listened attentively. The final analyses are based on those 11 out of the initial 20 pictures that the Cameroonian children reliably identify and label. Audio recordings of 15 seconds were taken for each picture and coded in MaxQDA (Ver- sion 12). PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Stimuli and procedure Furthermore, as a control condition, prior to the optical illusion task, children adjusted the size of each pair of red elements for all eight trials without the presence of the gray context PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 15 / 21 Visual attention in children from different cultures Eye-tracking task. Children saw 40 real, semantic pictures which displayed objects in front of a simple background [6, 11], see Fig 3A. These pictures were similar to those of the picture description tasks and taken from the same sources. Furthermore, 40 abstract, non- semantic pictures with abstract objects in front of abstract backgrounds were shown. We used artificial objects from experimental psychology (greebles, fribbles, geons and multipart geons, e.g., [31], taken from an online database: http://wiki.cnbc.cmu.edu/Novel_Objects) and fractal pictures (20 pictures; created with quadrium 2.0, quadrium.en.softonic.com) or details of abstract drawings (20 pictures) as backgrounds, cf. [32]. The pictures of both sets were pre- sented in a randomized order. Trials started with a fixation dot (shown for 1 s), followed by the stimulus (5 s). The instruc- tion for the children was to “. . .look at the pictures attentively. . .”, while the experimenter sat beside them. Participants’ gaze behavior was recorded binocularly, at a sampling rate of 60 Hz or higher. In Cameroon and Germany we used a SMI redm250 (SensoMotoric Instruments GmbH, Tel- tow, Germany), in Japan we used a Tobii X60 (Tobii Technology, Stockholm, Sweden) eye- tracking system. Individual fixations were identified by the respective eye-tracking software (Cameroon and Germany: BeGaze, Version 3.5.101; Japan: Tobii Studio, Version 3.3.2). Fixa- tions were then exported for further analyses in MATLAB (Version 2013a). Areas of interest (AOI) were drawn around the objects of each stimulus using BeGaze (Version 3.5.101) and imported into MATLAB as a template, to assure that AOIs were identical across contexts. To quantify participants’ visual attention to the object, relative to the background, we calcu- lated an object focus score: The duration of all fixations made into the AOI of the object was divided by the duration of all fixations on the picture within the 5 s of stimulus presentation. Thus, a score of 1 would indicate that the participant only looks at the object, whereas a score of 0 would indicate that a participant does only look at the background. As a further measure for participants’ object bias, we looked at the average latency, until the first fixation entered the AOI of the object (cf. [6]). Twenty children were excluded from the analyses of this task, due to technical errors occur- ring during this task (Germany: n = 4; Japan: n = 4), insufficient trials with valid recordings (5 or less recordings; Germany: n = 4; Japan: n = 7), and a child that did not say anything in this task (Cameroon: n = 1). PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 16 / 21 Data from fourteen children were excluded from this task, due to a lack of motivation to participate in both conditions (Germany: n = 1; Japan: n = 4), insufficient trials with valid recordings per condition (i.e., less than 10 trials with more than 1.5 seconds or 5 fixation with recorded gaze behavior, which indicates that children did not attend the stimulus or where staring; Germany: n = 2; Cameroon: n = 1; Japan: n = 3), inconsistent gaze behavior (less than 15% of presentation time used for object exploration in one condition, indicating bad eye- tracking recordings; Germany: n = 2), and technical problems during assessment (Japan: n = 1). Parallel action task. In the parallel action task children saw a video with two action sequences shown in parallel (i.e., the display was split in two halves), see Fig 1D. Specifically, each video displayed a pair of hands producing artificial handicrafts from four different col- ored pellets of modelling clay, two additional materials (e.g., wires or sticks), and two tools (e.g., a pounder or a scarper). In both action sequences, an artificial object was produced in nine action steps. Specifically, each pellet was modified, with a tool or with the hands, before all pellets were assembled, including the two additional materials. The display of the action sequences was precisely timed, such that each action step took 20 seconds, the hands in both sequences started simultaneously and ended each action step simultaneously in their starting position, at the lower edge of the screen. The side of the presentation of the two action sequences was counterbalanced across participants. Children were not told that they would have to reproduce the shown actions, but instructed to watch exactly, what is done in the videos. During the presentation of the video, the gaze behavior was recorded, using the same apparatus as in the eye-tracking task, see above. After PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 17 / 21 Visual attention in children from different cultures the presentation of the video, children were given two trays with the pellets, materials and tools from the presented action sequences, one after another, and were asked to reproduce what they had seen in the video. For each tray children had seven minutes of time, or until the child indicated that they had finished. The experimenter sat beside the child, pretending to read, but gave a series of standardized cues (after one and two minutes: “. . .Try to do exactly what was done in the video.”; after four minutes the experimenter indicated the unused pellets, materials and tools: “Do you remember what was done with [. . .], just do what you think was done”; after 6 minutes: “You have to finish soon. . .”). After seven minutes the tray was taken away and the second tray was handed to the child for another seven minutes. Because pilot data indicated that one of the action sequences was somewhat more difficult to reproduce, all children started with the more difficult object, before they received the tray with the somewhat easier object. This was to avoid stark differences in the number of actions reproduced, and our interest to look at sequences that were displayed in parallel. The analysis focused on, first, children’s gaze behavior when watching the action sequences and, second, their reproduced action steps. After preprocessing the eye-tracking data as in the scene exploration task (see above), we calculated the number of saccades that shifted from one action sequence to the other (e.g., from left to right or vice versa), divided by the overall gaze time in seconds. Furthermore, to estimate the distribution of their attention to both sequences, we computed the proportion of time that children focused on the preferred action sequence (i.e., the action sequence that the child looked at for longer). That is, a score of 1 would indicate that the child only attended to one of the sequences, whereas a score of 0.6 would indicate that the child looked for 60% of the time to the preferred sequence and only 40% of the time to the non-preferred sequence. With regard to children’s ability to reproduce actions of both sequences, we looked at correctly reproduced actions, namely correct tool/material-pellet combinations (e.g., pounder to red pellet), followed by the correct action (e.g., squeezing the red pellet). We looked at the proportion of reproduced actions that were shown in parallel. This is, we divided the number correctly reproduced actions, which were shown in parallel, by the total number of actions that where reproduced. Inter-rater agreements for correctly reproduced actions were assessed for > 20% of the data and Cohen’s kappa for each context was κ > .81. PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 Nine children were excluded from the behavioral analyses of this task, due to a lack of moti- vation to continue with the task or the data assessment (Germany: n = 3; Japan: n = 4), and a technical error occurring during the assessment (Germany: n = 1). Eight additional partici- pants were excluded only from the eye-tracking analyses due to incomplete or insufficient eye- tracking recordings (Germany: n = 4; Japan: n = 4) Rule-based game. In the rule-based game children where shown a video with two people playing a social game (Fig 4A). Each turn of the game comprised taking a card with a certain pattern (e.g., a square), sorting the pattern into an envelope on a cardboard with a certain shape (e.g., into a star shape) and to perform a certain action (e.g., clapping hands). Each of four moves was presented twice in the video. The stimulus videos were videotaped for each context with local research assistants. The timing (20 s per move) and the speech was fully scripted. In particular, the actors labeled what they were doing (“these are stars [. . .], I do clap, clap, clap” ) but did not mention the rules explicitly (e.g., combination of pattern on the card and shape of the envelope). Before the game started, children were instructed to watch carefully and explicitly asked to remember the rules and the actions, because they would play the game with the experimenter later on. After the presentation of the video, the child and the experimenter moved to a table and played the rule-action game. The child started and the cards were sorted in a way that the child drew each pattern first, before the experimenter drew the same pattern. It was then PLOS ONE \| https://doi.org/10.1371/journal.pone.0200239 July 16, 2018 18 / 21 Visual attention in children from different cultures observed, whether the child remembered the rule (pattern on card to shape of envelope alloca- tion) and whether they performed an action that was demonstrated in the video. If the child hesitated and could not remember a rule or an action, the experimenter asked: “Think about it, do you remember where this card was placed/which action was done next. Acknowledgments We would like to thank Chisato Fukuda, Kanako Shirai, Ruth Baumann, Eunis Yovsi, Gaston Funyuy, Marlene Krieger, Marius Gruber, Hanna Sinder, Lena Brentrup, Franca Ohde, Lena Knu¨wer, Sonja Duhe, and Hannah Rohberg for their contributions to task development, data assessment, and data coding. Ruth Baumann received a PROMOS stipend from the University of Mu¨nster. We thank all the children for their participation. Author Contributions Conceptualization: Moritz Ko¨ster, Shoji Itakura, Relindis Yovsi, Joscha Ka¨rtner. Data curation: Moritz Ko¨ster. Formal analysis: Moritz Ko¨ster. Investigation: Moritz Ko¨ster. Methodology: Moritz Ko¨ster, Joscha Ka¨rtner. Project administration: Moritz Ko¨ster, Shoji Itakura, Relindis Yovsi, Joscha Ka¨rtner. Supervision: Shoji Itakura, Joscha Ka¨rtner. Writing – original draft: Moritz Ko¨ster, Joscha Ka¨rtner. Writing – review & editing: Shoji Itakura, Relindis Yovsi. Conceptualization: Moritz Ko¨ster, Shoji Itakura, Relindis Yovsi, Joscha Ka¨rtner. Data curation: Moritz Ko¨ster. Formal analysis: Moritz Ko¨ster. Investigation: Moritz Ko¨ster. Methodology: Moritz Ko¨ster, Joscha Ka¨rtner. Methodology: Moritz Ko¨ster, Joscha Ka¨rtner. Project administration: Moritz Ko¨ster, Shoji Itakura, Relindis Yovsi, Joscha Ka¨rtner. Supervision: Shoji Itakura, Joscha Ka¨rtner. Supervision: Shoji Itakura, Joscha Ka¨rtner. Writing – original draft: Moritz Ko¨ster, Joscha Ka¨rtner. Writing – original draft: Moritz Ko¨ster, Joscha Ka¨rtner. Writing – review & editing: Shoji Itakura, Relindis Yovsi. Writing – review & editing: Shoji Itakura, Relindis Yovsi. Otherwise you may guess and try.” Inter-rater agreements for the comprehension of rules and correctly reproduced actions were assessed for > 20% of the data (Cohen’s kappas for each context was κrule comprehension = 1.0, and κaction reproduction > .91). Two children were excluded from the behavioral analyses of this task, due to a lack of moti- vation to continue the data assessment (Germany: n = 1), and a procedural error by the experi- menter (Germany: n = 1). References Dev Sci. 2013; https://doi.org/10.1111/desc12016 14. Bremner AJ, Doherty MJ, Caparos S, Fockert J, Linnell KJ, Davidoff J. Effects of culture and the urban environment on the development of the Ebbinghaus illusion. 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https://openalex.org/W4361854698	https://hal.science/hal-04059124/document	English	null	Key-Recovery by Side-Channel Information on the Matrix-Vector Product in Code-Based Cryptosystems	Lecture notes in computer science	2,023	cc-by	8,266	To cite this version: Boly Seck, Pierre-Louis Cayrel, Idy Diop, Vlad-Florin Dragoi, Kalen Couzon, et al.. Key-Recovery by Side-Channel Information on the Matrix-Vector Product in Code-Based Cryptosystems. 25th International Conference on Information Security and Cryptology (ICISC 2022), Nov 2022, Séoul, South Korea. pp.219-234, 10.1007/978-3-031-29371-9_11. hal-04059124 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-04059124 https://hal.science/hal-04059124v1 Submitted on 18 Oct 2023 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License Key-Recovery by Side-Channel Information on the Matrix-Vector Product in Code-Based Cryptosystems Boly Seck1,2[0000−0002−2362−601X], Pierre-Louis Cayrel2[0000−0002−6708−868X], Idy Diop1[0000−0002−9143−196X], Vlad-Florin Dragoi4[0000−0002−8673−9097], Kalen Couzon3, Brice Colombier2[0000−0002−6028−3028], and Vincent Grosso2[0000−0002−3874−7527] 1 ESP, Laboratoire d’imagerie médicale et de Bio-informatique, Dakar, Sénégal seck.boly@ugb.edu.sn 2 Univ Lyon, UJM-Saint-Etienne, CNRS, Laboratoire Hubert Curien UMR 5516, F-42023, Saint-Etienne, France pierre.louis.cayrel@univ-st-etienne.fr 3 Univ de Versailles Saint-Quentin, Yvelines, France kalen.couzon@ens.uvsq.fr 4 Faculty of Exact Sciences, Aurel Vlaicu University, Arad, Romania vlad.dragoi@uav.ro pierre.louis.cayrel@univ-st-etienne.fr Univ de Versailles Saint-Quentin, Yvelines, Fran kalen.couzon@ens.uvsq.fr Abstract. The modern security protocols in most of our systems rely primarily on three basic functions of asymmetric cryptography: public key encryption, digital signature, and key exchange. Today, we only do key exchange (TLS 1.3) with the ECDH protocol. The confidentiality is persistent because the session keys are discarded at the end and to certify this key exchange, we sign it with RSA or ECDSA. However, these cryptosystems are at least theoretically attackable in a quantum computer model. Thus the NIST PQC standardization process has given significant momentum to research on code-based public-key cryptosys- tems specifically. Their security is based on the hardness of the syndrome decoding problem. In this article, we first propose a new formalism of the matrix-vector product in based-code cryptography. Second, we present a chosen-ciphertext attack on the first step of Niederreiter decryption by solving the matrix-vector product problem with side-channel infor- mation. Finally, we put this result to recover secret information in code- based cryptosystems including some candidates for the extension of the NIST PQC normalization process. Keywords: · Code-Based Cryptography · Side-Channel Attack · Matrix- Vector Product Problem · NIST PQC Standardization 1 Introduction In recent years, a lot of research has been done on quantum computers [17, 32, 14]. These are computers that exploit the phenomena of quantum mechanics Boly et al. 2 to solve difficult mathematical problems in number theory, such as the Integer Factorization Problem or the Discrete Logarithm Problem. Shor [28] proved that if large-scale quantum computers are built, they will be able to break most of the current asymmetric cryptography like RSA, ECDSA, and ECDH schemes. This would seriously compromise the confidentiality and integrity of all digital communications. Since then, cryptographic community proposed alternative solutions which remain safe in the quantum era. These schemes are called post-quantum resis- tant. In 2016, the National Institute of Standards and Technology (NIST) made a call to the community to propose post-quantum secure solutions for standard- ization. The process consists of several rounds, and only some of the candidates in each round are chosen to enter the next round. The most popular approaches are those based on the search for low-weight words for lattice, the problem of decoding random codes, solving multivariate polynomial systems, isogenies, and hash functions [7, 5]. Lattice-based cryptography has the reputation of being very efficient. Code-based cryptography using some codes is often considered to be already more mature and reliable such as McEliece [23] and Niederreiter [25] cryptosystems. The majority of code-based post-quantum cryptosystems base their security on the classic hard problem in code-based cryptography: the binary Syndrome Decoding Problem (SDP). Informally, for a binary linear code C of length n and dimension k, having a parity-check matrix H, the SDP is defined as follows: given s ∈Fn−k 2 , find a binary vector x having less than t values equal to one, such that Hx = s. The best algorithm to solve this problem in this original version is the Information Set Decoding (ISD) proposed by Prange [27]. The ISD techniques are considered the best strategy for message recovery. It consists, in randomly permuting the matrix H (denote P such a permutation) until the support of the permuted x is included in the set {0, . . . , n −k −1}, i.e., the set where the HP is in upper triangular form. It has been considerably improved since then [3, 18, 19, 21, 22, 29], although the complexity remains exponential in t. 1 Introduction A recent possible solution to solve the syndrome decoding problem is to use Integer Linear Programming (ILP). The idea was first proposed by Feldman [11] and later improved by Feldman et al. [12]. Since the initial problem is nonlinear, some relaxation was proposed in order to decrease the complexity. Most recently, Cayrel et al. [9] showed that the SDP becomes considerably easier to solve if the syndrome is computed in N. They perform a laser fault injection attack on the matrix-vector product when computing the syndrome in the encapsulation of Classic McEliece. This allows them by corrupting some specific instructions during this operation to have a syndrome in N. To solve the syndrome decoding problem in N, they propose to define the SDP as an ILP inspired by the ideas of Tanatmis et al. [33]. The complexity of recovering the secret message from the faulty syndrome is polynomial O(n2) with an optimized version of their algorithm. Afterwards, Colombier et al. [10] proposed to perform a message-recovery attack in Classic McEliece that relies on side-channel information only instead Key-Recovery by Side-Channel Information on CBC 3 of laser fault injection in the previous work[9]. The latter depends on the very strong attacker model and does not apply to optimized implementations of the algorithm that make optimal usage of the machine words capacity. Improvements include the power consumption analysis that is sufficient to obtain an integer syndrome using machine learning techniques. To recover the secret message they use the computationally-efficient score function and known information-set de- coding methods. Contribution: In this work, a key-recovery chosen-ciphertext attack against code- based cryptosystems is performed. We analyze in particular the secret opera- tion of matrix-vector multiplication in Niederreiter decryption using a physical attack. First, we will introduce a new formalism in code-based cryptography. Informally, for z in Fn−k 2 of any weight, the Matrix-Vector Product Problem (MVPP) is defined as follows: given z∗in Nn−k , find S ∈F(n−k)×(n−k) 2 such that SzT = z∗. To get z∗in Nn−k, we will use the same method of the power analysis attack in [10]. This method is based on side-channel analysis using ran- dom forests to recover z∗from the Hamming weight information obtained from the matrix-vector product in the first step of Niederreiter decryption. 1 Introduction Second, we show that if we can construct the matrix Z∗= (z∗ 1, · · · , z∗ n−k) correctly, we can directly find the secret of the cryptosystem. We obtain directly the secret without solving the syndrome decoding problem unlike in [9, 10] and this is applicable for most of the code-based cryptosystems. Organization: The paper is organized as follows. In Section 2, we focus on code- based cryptosystems, and in particular on the results of the NIST PQC com- petition. Section 3 defines the new formalism in code-based cryptography, the Matrix-Vector Product Problem (MVPP). In Section 4, we present our attack on the matrix-vector product in Niederreiter decryption using a side-channel attack. Finally, we conclude this paper in Section 5. 2.1 Encoding Theory The best-known algorithms for solving this problem are all exponential in t. Except if the syndrome is computed in N instead of F2 [9]. Definition 3 (N-SDP). Let H ∈Mn−k,n (N), with hi,j ∈{0, 1} for all i, j, a vector s ∈Nn−k and t ∈N∗. The syndrome decoding problem in N is to find x ∈Nn with xi ∈{0, 1} such that HxT = s and wt(x) ≤t. Definition 3 (N-SDP). Let H ∈Mn−k,n (N), with hi,j ∈{0, 1} for all i, j, a vector s ∈Nn−k and t ∈N∗. The syndrome decoding problem in N is to find x ∈Nn with xi ∈{0, 1} such that HxT = s and wt(x) ≤t. H and x are sampled in the same way as for the binary SDP, only the matrix- vector multiplication operation changes, and thus its result s.i g Thus we define the new problem on the matrix-vector product as follows, Definition 4 (Binay-Matrix-Vector Product Problem (MVPP)). Let z ∈Fn−k 2 of any weight, a vector z∗∈Nn−k. The matrix-vector product problem is to find S ∈F(n−k)×(n−k) such that SzT = z∗. Definition 4 (Binay-Matrix-Vector Product Problem (MVPP)). Let z ∈Fn−k 2 of any weight, a vector z∗∈Nn−k. The matrix-vector product problem is to find S ∈F(n−k)×(n−k) such that SzT = z∗. We can find z∗for side-channel information with power consumption analysis and then with a chosen-ciphertext attack we find S. 2.1 Encoding Theory Notations The following conventions and notations are used. A finite field is denoted by F, and the ring of integers by N. Vectors (column vectors) and matrices are written in bold, e.g., a binary vector of length n is x ∈{0, 1}n, an m × n integer matrix is A = (ai,j)0≤i≤m−1 0≤j≤n−1 ∈Mm,n (N). A[i] denotes the i-th ≤j≤ line of A and a row sub-matrix of A indexed by a set I ⊆{0, . . . , m −1} is denoted by AI, = (ai,j) i∈I 0≤j≤n−1. The same applies to column vectors, i.e., xI is ≤j≤ the sub-vector induced by the set I on x. ≤j≤ the sub-vector induced by the set I on x. Error-Correcting Codes An [n, k] linear code C over Fq is a vector subspace of Fn q , where k, n are positive integers with k < n. The elements of C are called 4 Boly et al. codewords. The support of a codeword Supp(c) is the set of non-zero positions of c. We will represent a code either by its generator matrix G ∈Fk×n q such that its lines form a basis of the vector space C, or by its parity-check matrix, H ∈F(n−k)×n q , where HGT = 0 holds. One of the key elements of decoding is the use of metrics. In the Hamming metric, we consider codes with coefficients in Fq (generally, F2). Definition 1 (Hamming metric). Let x ∈F2, the Hamming weight wt(x) is the number of non null coordinates in x, and the distance between two vectors x and y is the number of non null coordinates in wt(x −y). The hardness of general decoding for a linear code is an NP-complete problem in coding theory [4]. This is the Syndrome Decoding Problem (SDP), which is the hard problem in code-based cryptography. The hardness of general decoding for a linear code is an NP-complete problem in coding theory [4]. This is the Syndrome Decoding Problem (SDP), which is the hard problem in code-based cryptography. Definition 2 (Binary-SDP). Let H ∈F(n−k)×n 2 , a vector s ∈Fn−k 2 and t ∈N. The syndrome decoding problem is to find x ∈Fn 2 such that HxT = s and wt(x) ≤t. The best-known algorithms for solving this problem are all exponential in t. Except if the syndrome is computed in N instead of F2 [9]. 7 https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP. 800-56Br1.pdf 5 https://nvlpubs.nist.gov/nistpubs/FIPS/NIST.FIPS.186-4.pdf 6 2.2 NIST PQC Standardization and Result On July 5, 2022, NIST released the first four winning algorithms from a campaign launched in 2016 to standardize post-quantum cryptographic algorithms. These future standards will be default options for selecting post-quantum algorithms in the majority of security products. Provided that these post-quantum algorithms are also combined with proven classical algorithms through hybrid mechanisms. The main goal of the process started by NIST is to replace three standards that are considered the most vulnerable to quantum attacks, i.e., FIPS 186-45 (for digital signatures), NIST SP 800-56A6, and NIST SP 800-56B7(both for keys 5 https://nvlpubs.nist.gov/nistpubs/FIPS/NIST.FIPS.186-4.pdf 6 6 https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP. 800-56Ar2.pdf 7 https://nvlpubs.nist.gov/nistpubs/SpecialPublications/NIST.SP. 800-56Br1.pdf Key-Recovery by Side-Channel Information on CBC 5 establishment in public-key cryptography). For the first round of this competi- tion, 69 candidates met the minimum criteria and the requirements imposed by NIST. 26 out of 69 were announced on January 30, 2019, for moving to the second round. Of these, 17 are public-key encryption and/or key-establishment schemes and 9 are digital signature schemes. In July 2020, NIST started the third round of this process where only seven finalists were admitted (four PKE/KEM and three signature schemes). In addition to the finalists, eight alternate candidates were selected. The first four algorithms selected are a key establishment algorithm named CRYSTALS-Kyber; and three digital signature algorithms named CRYSTALS- Dilithium, FALCON, and SPHINCS+. The first three of these algorithms are based on structured lattices; the last one, SPHINCS+ is a hash-based signature scheme. These four algorithms will therefore be used as the basis for writing U.S. federal standards. The scope of the NIST announcement is international with strong involvement of the cryptography research community, which will make the future US standards also used as de facto international industry standards. Beside the four winners, an extension of the NIST PQC standardization cam- paign (4th round) is planned for four key establishment algorithms: BIKE [1], HQC [24], Classic McEliece [2] (all three based on error-correcting codes), and SIKE [16] (isogeny graphs-based). Classic McEliece was the first selected final- ist as a key encapsulation mechanism, while BIKE and HQC were alternative candidates. The latter two use special codes to reduce the size of the public key, which is considered the main drawback of code-based cryptosystems. Classic McEliece is a code-based scheme using binary Goppa codes, the same codes that McEliece originally proposed in [23]. 2.2 NIST PQC Standardization and Result During Round 2 the scheme merged with NTS-KEM, which was using the same codes. The Classic McEliece scheme uses the dual of McEliece’s scheme, as proposed by Niederreiter [25], and tightly turns this OW-CPA PKE into an IND-CCA2 KEM. BIKE (Bit Flipping Key Encapsulation) is a key encapsulation mechanism (KEM) based on quasi-cyclic codes with moderate density parity check matrices. The code structure in BIKE is public and allows to reduce the size of the public key. Bit flipping corrects errors by repeatedly flipping the input bits that, given the secret moderate-density parity checks, seem most likely to be errors. HQC (Hamming Quasi-Cyclic) uses error-correcting codes built from Reed- Muller and Reed-Solomon. The public key includes a random h and s = x+h·y, where x, y are secretes and small Hamming weights. The ciphertext includes u = r1 + r2 · h and v = M + s · r2 + e, where r1, r2, e are small Hamming weights and M is a message encoded using an error-correcting code. The receiver computes v−u·y = M+s·r2 +e−u·y, which is close to M since x, y, r1, r2, e are small, and decodes the error-correcting code to recover M. SIKE (Supersingular Isogeny Key Encapsulation) is a key encapsulation mechanism based on the hard problem of pseudo-random walks in supersingular isogeny graphs. SIKE is a relatively new problem in the cryptographic arena and currently undergoing several attacks like its instantiation SIDH (Supersingular Isogeny Diffie–Hellman key exchange protocol) [8, 13, 20, 34, 35]. These are key Boly et al. 6 recovery attacks, reduces of the level security, side-channel attacks, and fault injection. Some of these algorithms could therefore later join the same standardization process as the four algorithms already selected. The final objective of NIST is indeed to be able to standardize a varied range of algorithms to cover a majority of use cases. Most of these constructions based on error-correcting codes use matrix-vector products in the decryption, as in Niederreiter’s scheme (Table 1).ii ( ) The goal of our attack is to find the secrete matrix Q. But first, let’s as- sume that we already have the result of the product Q−1z in N using the same technique as in [10]. 2.2 NIST PQC Standardization and Result Table 1: Niederreiter PKE scheme KeyGen(n, k, t) = (pk, sk) H-parity-check of C that corrects t errors An n × n permutation matrix P An (n −k) × (n −k) invertible matrix Q Compute Hpub = QHP pk = (Hpub, t) sk = (Q, H, P ) Encrypt(m, pk) = z Encode m →e \\ error vector of wt(e) = t z = Hpube Decrypt(z, sk) = m Compute z∗= Q−1z = Sz \\ target of our attack z∗= HP e e∗= Decode(z∗, H) Retrieve m from P −1e∗ Table 1: Niederreiter PKE scheme 3.1 On the Decryption of Niederreiter Our attack on Niederreiter’s decryption is now described. It consists in directly finding the secret matrix Q. In the following, we note S = Q−1 ∈F(n−k)×(n−k) 2 . We assume that, we can recover the result of the matrix-vector product Sz in N (z∗∈Nn−k) with side-channel information as in [10]. Key-Recovery by Side-Channel Information on CBC 7 Profiled side-channel measurements. We performed side-channel measurements during the computation of the product Sz in the Niederreiter decryption imple- mentation. The vector z∗is computed as the matrix-vector product Sz = z∗. We have recorded a single trace that will be sufficient to form the training set for the profiled attack. This trace is composed of n samples and stored as a row vector. We chose the ciphertexts zi in F(n−k) 2 linearly independent as the inputs of the matrix-vector product algorithm. In addition, we stored a second trace, used as a test set when training the classifier. For both traces, we also stored the Hamming weights of the intermediate value resulting from the matrix-vector product Szi. Profiled side-channel measurements. We performed side-channel measurements during the computation of the product Sz in the Niederreiter decryption imple- mentation. The vector z∗is computed as the matrix-vector product Sz = z∗. We have recorded a single trace that will be sufficient to form the training set for the profiled attack. This trace is composed of n samples and stored as a row vector. We chose the ciphertexts zi in F(n−k) 2 linearly independent as the inputs of the matrix-vector product algorithm. In addition, we stored a second trace, used as a test set when training the classifier. For both traces, we also stored the Hamming weights of the intermediate value resulting from the matrix-vector product Szi. After the traces acquisition, we performed an adequate preprocessing for reducing the dimension (eight dimensions since there are nine possible values for the Hamming weight of a byte) of the data by Linear Discriminant Analysis (LDA) to make it easier to handle by the classifier. We chose the random forest algorithm, used previously for side-channel analysis with good results [15], to recover the Hamming weight of z∗. We obtained the Hamming weight of the intermediate value of the prod- uct Szi, we derived the entries of z∗in N with 98.65% accuracy. 1. Can we know if the matrix Z∗is not correct? Boly et al. 8 2. If so, how can we correct the errors and find the secret matrix S? 2. If so, how can we correct the errors and find the secret matrix S? 2. If so, how can we correct the errors and find the secret matrix S? We will discuss question 2 in Section 3.2. For the first point, we assume that ∗∗ ∗ 2. If so, how can we correct the errors and find the secret matrix S? We will discuss question 2 in Section 3.2. For the first point, we assume tha 2. If so, how can we correct the errors and find the secret matrix S? We will discuss question 2 in Section 3.2. For the first point, we assume that in Step 2 we obtain the matrix Z∗∗instead of Z∗. So we have We will discuss question 2 in Section 3.2. For the first point, we assume that in Step 2 we obtain the matrix Z∗∗instead of Z∗. So we have S′ = Z∗∗Z−1 (2) Z∗∗= Z∗+ Er (3) (2) and Z∗∗= Z∗+ Er (3) (3) where Er is an error matrix. How to distinguish S from S′? We know that S is an invertible matrix in F2. Thus it’s enough to look directly at its coefficients and compute its determinant.i We have shown that our attack allows us to directly find the secret matrix S in the case where there is no error in Step 2. Otherwise, we know how to detect it. We have two levels of optimization of this attack either minimize the risk of errors when recovering the matrix Z∗or reduce its coefficients modulo an integer number to speed up the computations. We can choose judiciously the zi at step 1, for example, taking zi of low Hamming weights allows having regular words with a “1” for each zi. This can considerably reduce the risk of errors during the acquisition of the traces. Moreover, in this case, we would have Z = In−k and we obtain the secrete matrix S directly without computing Z−1. We can also suppose that the victim does not accept to decrypt n −k ciphertexts for example, but with the choice of ciphertexts with low regular weights we avoid this problem. We will now see how to correct the errors in the matrix Z∗and find the correct matrix S. correct matrix S. 3.1 On the Decryption of Niederreiter Indeed, the Hamming distance between two consecutive intermediate values is exactly the number of 1’s found in the bitwise AND between the row of the matrix S and the byte of the ciphertext z. Computing the value of the integer z∗entry is equivalent to counting those ones, which in turn is equivalent to summing the Hamming distances between consecutive intermediate values (the absolute value of the difference of their Hamming weights). In our implementation (n = 6, 960, k = 5, 413 and t = 119), the Hamming distance between two consecutive inter- mediate values is small and satisfies the condition in [10, Equation (3)] to recover the entries of z∗in N with good accuracy (82% for Hamming distance). Course of the attack. We propose a chosen-ciphertext attack that essentially consists of 4 steps: ( k) Step 1. We choose the ciphertexts or vectors zi in F(n−k) 2 linearly indepen- dent. We therefore define a matrix Z = (z1, · · · , zn−k) which is invertible. Step 1. We choose the ciphertexts or vectors zi in F(n−k) 2 linearly indepen- dent. We therefore define a matrix Z = (z1, · · · , zn−k) which is invertible. i ( ) Step 2. For each vector zi thanks to the side-channel attack, we have the vector z∗ i = Szi in N (in reality we get the Hamming weight for each component). This gives us a new matrix Z∗= (z∗ 1, · · · , z∗ n−k). ( 1 n k) Step 3. We solve a matrix system SZ = Z∗with S the unknown matrix in F(n−k)×(n−k) 2 . Since Z is invertible, we multiply on the right-hand side by its inverse and we obtain S = Z∗Z−1. (1) (1) Then we just have to read the entries of the right matrix to get the values of S and thus the secret matrix Q. A toy example is discribed in the Appendix A. Then we just have to read the entries of the right matrix to get the values of S and thus the secret matrix Q. A toy example is discribed in the Appendix A. The attack as presented above allows to find the secret matrix directly. How- ever, in Step 2, we can raise two questions: 3.2 Error Correction In this section, we will provide an answer to question 2 and show that we can indeed find the matrix S in some cases. We consider the case where we have Er in the matrix Z∗at Step 2, equations (2) and (3). We consider two assumptions h1 et h2 about Er: 1. The matrix Er has coefficients 0 or 1, (h1). fi 2. The matrix Er has, at most, a 1 on each row, (h2). These two assumptions are not restrictive, we will see that we can deduce the general case and we assume that the error can be controlled to some extent, i.e., Z∗∗does not differ “too much" from Z∗. f According to the above assumptions, there exist two finite sets I and J such that: Er = X (i,j)∈I×J Ei,j (4) (4) with Ei,j the square matrix of order n −k where all coefficients are zero, except the one of row i and column j which is 1. We will need the following lemma: We will need the following lemma: Key-Recovery by Side-Channel Information on CBC 9 Key-Recovery by Side-Channel Information on CBC 9 9 Lemma 1. Let 1 ≤a, b ≤n. Let M = (mi,j) be a square matrix of order n−k. then : Lemma 1. Let 1 ≤a, b ≤n. Let M = (mi,j) be a square matrix of order n−k. then :   Ea,bM =                0 · · · 0 0 · · · 0 ... · · · ... mb,1 · · · mb,n−k ... · · · ... 0 · · · 0 0 · · · 0                ←a-th row Ea,bM =                0 · · · 0 0 · · · 0 ... · · · ... mb,1 · · · mb,n−k ... · · · ... 0 · · · 0 0 · · · 0                ←a-th row       0 · · · 0 0 · · · 0 ... · · · ... 3.2 Error Correction       Ea,bM =           mb,1 · · · mb,n−k ... · · · ... 0 · · · 0 0 · · · 0           ←a-th row In other words: [Ea,bM]i,j = ( 0 if i ̸= a mb,j if i = a To find S despite the error in Z∗. We have To find S despite the error in Z∗. We have SZ = Z∗+ Er = Z∗+ X (i,j)∈I×J Ei,j Hence S = Z∗Z−1 + ErZ−1 = Z∗Z−1 + X (i,j)∈I×J (Ei,jZ−1) From the above we deduce the following theorem: Theorem 1. For any i ∈[1, n −k], there exists j ∈[1, n −k] and ε ∈{0, 1} such that Z∗∗Z−1[i] −εZ−1[j] is binary and Z∗∗Z−1[i] −εZ−1[j] = S[i]. Theorem 1. For any i ∈[1, n −k], there exists j ∈[1, n −k] and ε ∈{0, 1} such that Z∗∗Z−1[i] −εZ−1[j] is binary and Z∗∗Z−1[i] −εZ−1[j] = S[i]. Theorem 1. For any i ∈[1, n −k], there exists j ∈[1, n −k] and ε ∈{0, 1} such that Z∗∗Z−1[i] −εZ−1[j] is binary and Z∗∗Z−1[i] −εZ−1[j] = S[i]. Proof. Let us suppose \|I × J\| = 1, let I × J = (a, b). Then we have S = Z∗Z−1 + Ea,bZ−1. According to Lemma 1, only the line a of Ea,bZ−1 is nonzero. We deduce that for all i ̸= a, According to Lemma 1, only the line a of Ea,bZ−1 is nonzero. We deduce that for all i ̸= a, Z∗∗Z−1[i] = S[i]. It is ,therefore, sufficient to take ε = 0 and any j. According to Lemma 1, It is ,therefore, sufficient to take ε = 0 and any j. According to Lemma 1, Ea,bZ−1[a] = Z−1[b] and so it suffices to take ε = 1 and j = b. and so it suffices to take ε = 1 and j = b. and so it suffices to take ε = 1 and j = b. Let (a, b) ∈I × J, (c, d) ∈(I × J) \ (a, b) and if \|I × J\| ≥2, the hypothesis (h2) implies that c ̸= a. 3.2 Error Correction fi j Let (a, b) ∈I × J, (c, d) ∈(I × J) \ (a, b) and if \|I × J\| ≥2, the hypothesis (h2) implies that c ̸= a. Boly et al. 10 According to Lemma 1, we have According to Lemma 1, we have Ec,dZ−1[a] = 0. Z∗Z−1[a] + X (i,j)∈I×J (Ei,jZ−1[a]) = Z∗Z−1[a] + Ea,bZ−1[a]. Ec,dZ−1[a] = 0. thus Z∗Z−1[a] + X (i,j)∈I×J (Ei,jZ−1[a]) = Z∗Z−1[a] + Ea,bZ−1[a]. Z∗Z−1[a] + X (i,j)∈I×J (Ei,jZ−1[a]) = Z∗Z−1[a] + Ea,bZ−1[a]. We thus have, on each line, at most one contribution. We deduce then 1. if i ̸∈I, then Z∗∗Z−1[i] = S[i], it is enough to take ε = 0 and any j. 2. if i ∈I, then there exists j such that (i, j) ∈I × J, we have Z∗∗Z−1[i] = S[i] + Z−1[j], so it is enough to take the j given previously and ε = 1. We deduce that in some cases, it is possible to find S by the following algorithm 1. Algorithm 1 Finding S with errors in Z∗ Table 2: McEliece PKE scheme KeyGen(n, k, t) = (pk, sk) G-generator matrix of C that corrects t errors An n × n permutation matrix P An k × k invertible matrix S Compute Gpub = SGP pk = (Gpub, t) sk = (S, G, P ) Encrypt(m, pk) = z Encode m →c = mGpub z = c + e \\ e is an error vector of wt(e) = t Decrypt(z, sk) = m Compute z∗= zP −1 z∗= mSG + eP −1 m∗= Decode(z∗, G) Retrieve m from m∗S−1 Table 2: McEliece PKE scheme KeyGen(n, k, t) = (pk, sk) G-generator matrix of C that corrects t errors An n × n permutation matrix P An k × k invertible matrix S Compute Gpub = SGP pk = (Gpub, t) sk = (S, G, P ) Encrypt(m, pk) = z Encode m →c = mGpub z = c + e \\ e is an error vector of wt(e) = t Decrypt(z, sk) = m Compute z∗= zP −1 z∗= mSG + eP −1 m∗= Decode(z∗, G) Retrieve m from m∗S−1 Table 2: McEliece PKE scheme Table 2: McEliece PKE scheme G-generator matrix of C that corrects t errors An n × n permutation matrix P Thus we have the following result : Thus we have the following result : 1. If i ̸∈I, then the sequence (εk) is null and it is enough to take any (jk) and two by two distinct.i 2. If i ∈I, considering the set Gi defined above and according to what precedes, it is enough to take the sequence (εk) constant equal to 1 and the (jk) given by Gi. 2. If i ∈I, considering the set Gi defined above and according to what precedes, it is enough to take the sequence (εk) constant equal to 1 and the (jk) given by Gi. In the case where the matrix Er has negative coefficients or is not binary, we can adapt Theorem 1. It is sufficient to allow the sequence εk to take the value -1 and to be able to subtract (or add) the same row several times. In the case where the matrix Er has negative coefficients or is not binary, we can adapt Theorem 1. It is sufficient to allow the sequence εk to take the value -1 and to be able to subtract (or add) the same row several times. Algorithm 1 Finding S with errors in Z∗ 1: We assume that we can determine the erroneous line(s) of S (for example, S is not binary). 1: We assume that we can determine the erroneous line(s) of S (for example, S is not binary). ) 2: Let Li be an erroneous row of S, we subtract from Li the rows of the matrix Z−1 and keep those that are binary. 2: Let Li be an erroneous row of S, we subtract from Li the rows of the matrix Z−1 and keep those that are binary. 3: We thus obtain a list of possible candidates for S and for each candidate matrix, we compute its determinant to check its invertibility in F2. 4: In particular, if this list is reduced to one element, we obtain S. We will now lighten the assumptions by deleting (h2). According to Theorem 1, we can deduce the general case of our approach. Corollary 1. Let i ∈[1, n−k], let ri be the Hamming weight of the vector Er[i], there exists a sequence (jk)1≤k≤ri of distinct pairwise elements and a sequence (εk)1≤k≤ri such that Z∗∗Z−1[i]− X Z∗∗Z−1[i]− ri X k=1 εkZ−1[jk] is binary and Z∗∗Z−1[i]− ri X k=1 εkZ−1[jk] = S[i]. Proof. We use the same notations as before. We suppose that \|I ×J\| ≥2 (if not, we are in the previous case). Let (a, b) ∈I × J. p ) ( , ) We try to count the couples (a, t) with t in J. There are as many as the amount of “1" on the row Er[a], in other words, there are ra couples (a, t). [ ], , Let Ga = { (a, t) \| t ∈J} = {(a, j1), (a, j2), · · · , (a, jra)}. By Lemma 1 , for all c ̸= a and d ∈J, the a-th row of Ec,dZ−1 is zero. We deduce that S[a] = Z∗∗Z−1[a] = Z∗Z−1[a]+ErZ−1[a] = Z∗Z−1[a]+ X (i,j)∈I×J (Ei,jZ−1)[a] Key-Recovery by Side-Channel Information on CBC 11 = Z∗Z−1[a] + ra X k=1 Z−1[jk]. 3.3 Comparison to other Attacks Recall that the goal of our attack is to find the secret matrix Q in the Niederreiter scheme. It has been shown in various previous works that it is possible to obtain secret information about the decryption in the McEliece scheme (Table 2). Falko Strenzke proposed in [30, 31] two attacks on each of the two calls of the Extended Euclidean Algorithm (EEA) in McEliece decoding with the parameters n = 2, 048 and t = 50. This vulnerability in the Patterson algorithm [26] in the error correction phase allows an attacker to gather information about the secret n×n permutation matrix P through a timing side channel. The first attack [30] targets the second call of the EEA in the Patterson algorithm to determine the polynomials forming the error locator polynomial σ(x). The polynomial of deg(σ(x)) ≤t consists of two polynomials a(x) and b(x) whose degrees have a direct impact on the number of iterations of the EEA. This variation in the number of iterations implies a difference in the execu- tion times and makes possible a timing attack. The attacker creates ciphertext (chosen-ciphertext attack) using random error vectors with Hamming weight wt = 4 and then lets the decryption routine decrypt the ciphertext. It evaluates whether zero or one iteration occurred in the EEA. If an iteration has occurred, deg(b(x)) = 1, nothing is done, and if there are no iterations, deg(b(x)) = 0, the error vector is added as a new row of a matrix over F2 having n columns. Each time a row is added, a Gauss-Jordan elimination is performed and the rank is determined. Once the maximum rank is reached (here 2,036), the attack is com- pleted with 7,848,229 ciphertexts. However, such an approach only recovers the secret permutation matrix P when the Hamming weight of error e is small (2 or 4). The second attack [31] is based on the vulnerabilities that are present in the inversion of the error syndrome through the extended euclidean algorithm (first call in the Patterson algorithm). Strenzke showed the existence of a timing side channel vulnerability in the syndrome inversion that allows the attacker to gain knowledge of the zero-element of the secret support. It is based on the analysis of the key equation to deduce the relations between the degrees of the polynomials involved in it. Algorithm 1 Finding S with errors in Z∗ We notice that, unlike in the previous case, we cannot give an algorithm to determine a list of possible candidates for the matrix S. A similar approach as the one presented above would be too expensive. We do not know, a priori, the number of rows to remove from each erroneous row. Although correcting the error in the matrix Z∗at Step 2 is theoretically possible, it may be difficult in practice. Boly et al. 12 4 Conclusion This article presents a key-recovery attack against the Matrix-Vector Product Problem, which is a new formalism that we introduce in based-code cryptog- raphy. We have also shown that with a side-channel attack on this operation, we can recover secret information on based-code cryptosystems without solv- ing the hardness of the binary syndrome decoding problem. In addition to the side-channel information, we performed a chosen-ciphertext attack, which, with careful choice of the ciphertexts, can find the secret matrix without errors. When noise (errors) is present during the attack, we have shown that in some cases it is possible to find the secret matrix. Our attack can be applied to code-based cryptosystems with matrix-vector product operations. permutation matrix P . The Table 3 give more details on the comparison with Strenzke’s attacks to find the secret matrix P . permutation matrix P . The Table 3 give more details on the comparison with Strenzke’s attacks to find the secret matrix P . permutation matrix P . The Table 3 give more details on the comparison with Strenzke’s attacks to find the secret matrix P . [3] A. Becker, A. Joux, A. May, and A. Meurer. “Decoding Random Binary Linear Codes in 2n/20: How 1 + 1 = 0 Improves Information Set Decod- ing”. In: Annual International Conference on the Theory and Applications of Cryptographic Techniques. Ed. by D. Pointcheval and T. Johansson. Vol. 7237. Lecture Notes in Computer Science. Cambridge, UK: Springer, Apr. 2012, pp. 520–536. 3.3 Comparison to other Attacks As in the first attack, this approach only works for Hamming weights of 2, 4, or 6 of the error vector e to recover the secret permutation matrix P . Despite the improvements in [6], the main problem with these two previous attacks is the number of cases (depending on the Hamming weight of e) that can be exploited to find the secret. In our attack, we have no constraints on the Hamming weights of the ciphertext (or error vector e) to find the secret matrix Q in the case that we correctly construct the matrix Z∗with a random forest. Moreover, we attack the least complex step of Niederreiter decoding (first step). The Niederreiter PKE is slightly different from the McEliece PKE (Table 1 and Table 2). However, here too, an error vector is chosen during the encryption and decryption features, and since these features are the prerequisites of our attack, it is also applicable to McEliece’s PKE. Unlike the attack of Strenzke in [30] we only need 2,048 ciphertexts instead of 7,848,229 to find the secret n × n Key-Recovery by Side-Channel Information on CBC 13 Table 3: Attacks to recover the secret matrix P Attack Hamming weight Number of ciphertexts Target Timing attacks of Strenzke 2, 4 or 6 7,848,229 in [30] EEA in Patterson algorithm Our attack against MVPP no constraints 2,048 First step in McEliece’s decryption Table 3: Attacks to recover the secret matrix P References [1] C Aguilar Melchor, N Aragon, P Barreto, S Bettaieb, L Bidoux, O Blazy, J. 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One chooses here n −k = 3, we have for instance   Z =     1 1 1 1 0 1 1 1 0     its inverse Z−1 =     −1 1 1 1 −1 0 1 0 −1     and Z∗=     2 2 1 1 0 1 2 1 2    . its inverse its inverse and and Z∗=     2 2 1 1 0 1 2 1 2    . From the equation 1, S = Z∗Z−1, we find S =     1 0 1 0 1 0 1 1 0    . From the equation 1, S = Z∗Z−1, we find S =     1 0 1 0 1 0 1 1 0    . Case 2: few errors in Z∗ We keep the same Z, its inverse and Z∗matrices in case 1. According to equation 3, Z∗∗= Z∗+ Er, we have Case 2: few errors in Z∗ We keep the same Z, its inverse and Z∗matrices in case 1. According to equation 3, Z∗∗= Z∗+ Er, we have According to equation 3, Z∗∗= Z∗+ Er, we have Key-Recovery by Side-Channel Information on CBC 17 Er =     1 0 0 0 0 0 0 0 0    . Er =     1 0 0 0 0 0 0 0 0    . By performing the same operation as before, we obtain the following S′ matrix according to equation 2 S′ =     0 1 2 0 1 0 1 1 0    . This matrix S′ is not binary, so we deduce that the first row contains a fault. We then apply the algorithm 1 of Section 3.2 to determine the list of possible candidates for the matrix S S′[1] −Z−1[1] = [1 0 1] S′[1] −Z−1[2] = [−1 2 2] S′[1] −Z−1[3] = [−1 1 3]. Only the first case gives a binary vector, we deduce that S′[1] = S[1] + Z−1[1] and we have directly S. A Simple Version of Our Attack Case 3: errors in Z∗ We consider the error matrix We consider the error matrix Er =     0 0 0 0 1 0 0 0 0    . We then obtain the following matrix S′ =     1 0 1 1 0 0 1 1 0    . Here the matrix is binary, moreover its determinant det(S′) = 1. In this case, we cannot detect that the matrix is not correct.
https://openalex.org/W2894856023	https://europepmc.org/articles/pmc6176114?pdf=render	English	null	A Systematic Review and Meta-Analysis of Alpha Synuclein Auto-Antibodies in Parkinson's Disease	Frontiers in neurology	2,018	cc-by	8,428	SYSTEMATIC REVIEW published: 01 October 2018 doi: 10.3389/fneur.2018.00815 A Systematic Review and Meta-Analysis of Alpha Synuclein Auto-Antibodies in Parkinson’s Disease Kirsten M. Scott 1, Antonina Kouli 1, Su L. Yeoh 2, Menna R. Clatworthy 3† and Caroline H. Williams-Gray 1† 1 Department of Clinical Neurosciences, John van Geest Centre for Brain Repair, University of Cambridge, Cambridge, United Kingdom, 2 University of Cambridge School of Clinical Medicine, Cambridge, United Kingdom, 3 MRC Laboratory of Molecular Biology, Molecular Immunity Unit, Department of Medicine, University of Cambridge, Cambridge, United Kingdom Immune dysfunction has been associated with Parkinson’s disease (PD) and its progression. Antibodies play an important role in both innate and adaptive responses, acting as powerful effector molecules that can propagate inﬂammation by activating innate immune cells. Alpha synuclein binding antibodies have been described in PD patients with conﬂicting associations. In this article, we consider the potential mechanistic basis of alpha synuclein auto-antibody development and function in PD. We present a systematic review and meta-analysis of antibody studies in PD cohorts showing that there is weak evidence for an increase in alpha synuclein auto-antibodies in PD patients particularly in early disease. The conﬁdence with which this conclusion can be drawn is limited by the heterogeneity of the clinical cohorts used, inclusion of unmatched controls, inadequate power and assay related variability. We have therefore made some recommendations for the design of future studies. Edited by: Cristoforo Comi, Università degli Studi del Piemonte Orientale, Italy Reviewed by: Tomasz Brudek, Bispebjerg Hospital, Denmark Natalia P. Rocha, University of Texas Health Science Center at Houston, United States Reviewed by: Tomasz Brudek, Bispebjerg Hospital, Denmark Natalia P. Rocha, University of Texas Health Science Center at Houston, United States Correspondence: Kirsten M. Scott kms61@cam.ac.uk Correspondence: Kirsten M. Scott kms61@cam.ac.uk Correspondence: Kirsten M. Scott kms61@cam.ac.uk Correspondence: Kirsten M. Scott kms61@cam.ac.uk †These authors have contributed equally to this work †These authors have contributed equally to this work Specialty section: This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Neurology Specialty section: This article was submitted to Multiple Sclerosis and Neuroimmunology, a section of the journal Frontiers in Neurology Keywords: antibodies (Abs), alpha synuclein (α syn), auto-antibodies, Parkinson’s disease (PD), peripheral inﬂammation, Fcγ receptor INTRODUCTION Parkinson’s disease (PD) is a neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra resulting in a movement disorder and many non-motor symptoms, including dementia, postural hypotension and gut dysfunction (1). Whilst dopaminergic treatments may alleviate the motor symptoms, there are currently no disease-modifying therapies that slow clinical progression. Received: 10 July 2018 Accepted: 10 September 2018 Published: 01 October 2018 Immune dysfunction has been associated with PD and its progression (2–5) and represents a tractable target for disease modiﬁcation. However, the cellular and molecular mechanisms underpinning this association have yet to be elucidated. Potential pathways include the activation of adaptive immunity via antigen-speciﬁc recognition of alpha-synuclein or non-speciﬁc innate immune activation due to cell damage and death. POTENTIAL MECHANISMS UNDERLYING ANTIBODY GENERATION IN PD of the peripheral immune system to a central nervous system (CNS) antigen. Monomeric alpha synuclein is abundant in the CNS in pre-synaptic terminals of neurons and is also produced by platelets and red blood cells peripherally. Pathological forms of the protein range from soluble oligomers to mature insoluble ﬁbrillar forms (8). Multiple studies have sought to measure alpha synuclein in either the blood or serum [reviewed in (9)] for use as a biomarker. Substantial variation in levels may be a confounding factor in studies measuring alpha synuclein antibodies as these may be undetectable if already bound. B lymphocytes can produce antibodies via T cell-independent (TI) and T cell-dependent pathways (TD) (see Figure 1). TI pathways involve the recognition of multimeric carbohydrate and lipid antigens by the B cell receptor (BCR) or by toll like receptors (TLR) on the cell surface of “B1” cells (or marginal zone B cells in the spleen). This leads to the production of polyreactive IgM that binds with low aﬃnity and can facilitate the removal of blood borne encapsulated organisms (17). Antibodies produced in this context are called “natural antibodies.” Most of the literature on alpha synuclein antibodies suggests that these are natural antibodies (18–21). Natural antibodies are part of innate immune surveillance against pathogens or cell damage and are present from an early point in development (22). They are predominantly IgM but IgG and IgA natural antibodies have also been described (22). Antibodies to alpha synuclein epitopes could be generated via this process. A recent study demonstrated the presence of alpha-synuclein speciﬁc CD4 and CD8 T cells in PD patients, implicating an alpha-synuclein speciﬁc adaptive immune response in disease pathogenesis (10). CD4 T cells orchestrate adaptive immunity, including humoral responses which result in the production of antibodies. Antibodies are powerful immune eﬀector molecules produced by plasma cells, terminally diﬀerentiated B lymphocytes. The most common circulating antibody isotype is IgG, that can readily initiate and propagate inﬂammation by activating complement and engaging cell surface antibody receptors [Fcγ receptors (FcγR)] that are expressed by most innate immune cells. The recent description of alpha synuclein speciﬁc T cells in patients with PD (10) supports the thesis that alpha synuclein antibodies may be generated by a TD response. These antibodies recognize protein antigens and their production requires a cognate interaction between “B2” cells and CD4 T cells. POTENTIAL MECHANISMS UNDERLYING ANTIBODY GENERATION IN PD This facilitates iterative rounds of somatic hypermutation and clonal selection within a germinal center reaction to generate class- switched long lived plasma cells or memory B cells capable of initiating a secondary response upon further encounter of the antigen (22). The plasma cells that arise from this process are able to produce large quantities of speciﬁc, high aﬃnity class-switched antibodies (17). Humoral responses to self-antigen are limited by negative selection of self-reactive clones during B cell development. However, if the self-antigen is modiﬁed suﬃciently, as in the case of alpha synuclein toxic species, and is present in an immunogenic context, such as cell death, some B cell clones may be activated to produce alpha synuclein antibodies. Such an antibody response might change over time; ﬁrstly IgM may dominate, but with progression of the germinal center reaction, there is class switching to IgG or IgE. Secondly, with persistent exposure to neo-antigen, clones with higher levels of somatic hypermutation and higher aﬃnity antibodies would be selected. Thirdly, the overall level of alpha synuclein antibody might change with age, as older age is associated with decreasing antibody response to antigens (e.g., vaccines) and immunosenescence of the B cell compartment (23). Alpha synuclein-speciﬁc IgG antibodies have been described in PD, but their role is unclear with many conﬂicting studies. Publication bias favoring positive ﬁndings in this ﬁeld may also further complicate attempts to unmask a true eﬀect. The presence of alpha synuclein speciﬁc antibodies in early disease could potentially contribute to pathology by exacerbating local inﬂammation in the brain, promoting neuronal damage and causing disease progression. Consistent with this hypothesis, IgG isolated from PD patients and injected into rat substantia nigra causes selective dopaminergic cell death that was absent in animals receiving control IgG (11). There is also attenuation of disease in Fcγ receptor knockout mice receiving PD IgG, conﬁrming that activation of microglia by PD IgG is pathogenic (12). Approximately 30% of dopaminergic cells in the substantia nigra of post-mortem PD brains were bound by IgG highlighting that immunoglobulins do cross the blood brain barrier in PD and may play a role in disease (13). Alternatively, alpha synuclein auto-antibodies may play a protective role, facilitating the clearance of toxic protein species by opsonizing alpha-synuclein for FcγR-mediated uptake by phagocytes. Consistent with this hypothesis, the passive peripheral transfer of alpha-synuclein speciﬁc antibodies in some mouse models of PD improved disease outcomes (14). Citation: Scott KM, Kouli A, Yeoh SL, Clatworthy MR and Williams-Gray CH (2018) A Systematic Review and Meta-Analysis of Alpha Synuclein Auto-Antibodies in Parkinson’s Disease. Front. Neurol. 9:815. doi: 10.3389/fneur.2018.00815 The accumulation of aggregated alpha-synuclein within CNS neurons is the pathological hallmark of PD (6). There is also evidence that misfolded alpha synuclein accumulates in the periphery, for example in the gut, in early stages of disease (7), providing a route for the exposure October 2018 \| Volume 9 \| Article 815 1 Frontiers in Neurology \| www.frontiersin.org Scott et al. Alpha Synuclein Auto-Antibodies in PD Frontiers in Neurology \| www.frontiersin.org POTENTIAL MECHANISMS UNDERLYING ANTIBODY GENERATION IN PD There is evidence that FcγRI and FcγRIIB/C are required for uptake of alpha synuclein by CNS derived cells in culture and that this is mediated by the presence of alpha synuclein speciﬁc antibodies (26). One recent paper suggested that FcγRIIB (a low aﬃnity inhibitory Fc receptor) is not only responsible for the inhibition of phagocytosis of alpha synuclein ﬁbrils (via low aﬃnity binding with the ﬁbrils themselves) but also mediates cell to cell transmission of alpha synuclein (27). In addition, the glycosylation status of immunoglobulin also aﬀects downstream binding and eﬀector function (28). A study investigating the IgG glycome in PD showed signiﬁcant diﬀerences between patients and controls, with the authors concluding that the changes observed in PD may result in enhanced Fcγ RIIIa-mediated antibody-dependent cellular cytotoxity (with the potential to contribute to chronic inﬂammation) (29). SYSTEMATIC REVIEW OF ALPHA SYNUCLEIN ANTIBODY STUDIES IN PD We [AK, SLY and KS] searched the literature for studies published prior to 1st June 2018 using Pubmed, Medline, Frontiers in Neurology \| www.frontiersin.org Cochrane database, Embase, Google scholar and Keele Web of Science. We used the following search terms: “Antibody and Parkinson’s Disease,” “Auto-antibody and Parkinson’s disease,” “Alpha synuclein antibody,” “Alpha synuclein auto-antibody.” To ensure complete study capture we also searched using “Auto- antibody dementia” “Antibody dementia.” Reference lists of the selected papers were also manually searched to identify additional studies. Papers were excluded if they did not involve PD patients, if they did not measure alpha synuclein antibodies and if there was no control group. Otherwise all papers measuring antibodies to alpha synuclein or its epitopes in Parkinson’s disease patients were included in the systematic review. The literature searches were done between 1 May 2018 and 6 June 2018. Summary information from each study was compiled into a table (Table 1). (25). Diﬀerent subclasses of IgG (IgG1, IgG2, IgG3 and IgG4) have diﬀerent aﬃnities for the FcγR on cells which can be either activating or inhibitory [see (25)]. There is evidence that FcγRI and FcγRIIB/C are required for uptake of alpha synuclein by CNS derived cells in culture and that this is mediated by the presence of alpha synuclein speciﬁc antibodies (26). POTENTIAL MECHANISMS UNDERLYING ANTIBODY GENERATION IN PD One recent paper suggested that FcγRIIB (a low aﬃnity inhibitory Fc receptor) is not only responsible for the inhibition of phagocytosis of alpha synuclein ﬁbrils (via low aﬃnity binding with the ﬁbrils themselves) but also mediates cell to cell transmission of alpha synuclein (27). In addition, the glycosylation status of immunoglobulin also aﬀects downstream binding and eﬀector function (28). A study investigating the IgG glycome in PD showed signiﬁcant diﬀerences between patients and controls, with the authors concluding that the changes observed in PD may result in enhanced Fcγ RIIIa-mediated antibody-dependent cellular cytotoxity (with the potential to contribute to chronic inﬂammation) (29). In order to assess whether studies were adequately powered, mean alpha synuclein antibody titres (or optical density) in each group and standard deviations were recorded and used to calculate required sample size to detect a diﬀerence of the magnitude reported. The following formula was used to calculate sample size [modiﬁed from (43)]. October 2018 \| Volume 9 \| Article 815 POTENTIAL MECHANISMS UNDERLYING ANTIBODY GENERATION IN PD Trials of both passive and active immunization therapies targeting alpha synuclein are underway (15, 16). B cell activation to generate plasma cell-producing antibodies generally occurs within secondary lymphoid organs (lymph nodes and spleen) but may also occur in tertiary lymphoid follicles that develop in inﬂamed tissues. The site of B cell activation to generate alpha synuclein-speciﬁc responses is unclear and may be peripheral or within the CNS. Follicles have been described in the meninges of patients with multiple sclerosis (24), with the potential to generate CNS localized antibodies, but whether such structures exist in PD is unknown. Clearly, it is critical to have a better understanding of how alpha synuclein autoantibodies relate to PD and its progression. In particular, there is a need to discern whether they constitute a useful diagnostic or prognostic biomarker or may have potential therapeutic relevance. In this article, we will consider the potential mechanistic basis of their role in PD, present a systemic review of antibody studies in PD cohorts, critically discuss the value and limitations of existing data and make recommendations for future studies. Antibodies bind non-speciﬁcally to Fcγ receptors on other immune cells (e.g., phagocytes, monocytes, dendritic cells) or via engagement of their Fc region with complement components October 2018 \| Volume 9 \| Article 815 2 Scott et al. Alpha Synuclein Auto-Antibodies in PD FIGURE 1 \| Possible T independent and T dependent mechanisms of antibody generation in PD. Microglia and neuron images modiﬁed from templates obtained https://smart.servier.com/smart_image/microglia-3/ under Creative Commons Attribution 3.0 Unported License. TfH, T follicular helper cell; pTfH, peripheral T follicular helper cell; NK, Natural killer; PAMP, pathogen associated molecular motif; DAMP, damage associated molecular motif, TLR, toll like receptor. FIGURE 1 \| Possible T independent and T dependent mechanisms of antibody generation in PD. Microglia and neuron images modiﬁed from templates obtained https://smart.servier.com/smart_image/microglia-3/ under Creative Commons Attribution 3.0 Unported License. TfH, T follicular helper cell; pTfH, peripheral T follicular helper cell; NK, Natural killer; PAMP, pathogen associated molecular motif; DAMP, damage associated molecular motif, TLR, toll like receptor. (25). Diﬀerent subclasses of IgG (IgG1, IgG2, IgG3 and IgG4) have diﬀerent aﬃnities for the FcγR on cells which can be either activating or inhibitory [see (25)]. SYSTEMATIC REVIEW OF ALPHA SYNUCLEIN ANTIBODY STUDIES IN PD nA = κnB and nB = (1 + 1 κ)(Swithinz1 −a 2 + z1 −β µA −µB ) 2 October 2018 \| Volume 9 \| Article 815 nA = κnB and nB = (1 + 1 κ)(Swithinz1 −a 2 + z1 −β µA −µB ) 2 We [AK, SLY and KS] searched the literature for studies published prior to 1st June 2018 using Pubmed, Medline, October 2018 \| Volume 9 \| Article 815 Frontiers in Neurology \| www.frontiersin.org 3 Alpha Synuclein Auto-Antibodies in PD Scott et al. TABLE 1 \| Summary of studies measuring alpha synuclein antibodies in Parkinson’s disease. Paper Method Fluid N (HC = healthy controls) Matched Mean age of PD (SD) Disease duration years (SD) H and Y (SD) Finding (in PD vs. controls) Required N∧∧ <=5 years DD Xu et al. (30) Electrochemical impedance spectroscopy Serum 60 PD, 29 HC Yes 69.4 (SD10.8) 1.4 (1.44) 20 HandY1, 20 HandY2, 20 HandY3 ↑in PD, more in HandY 1 and 2 than controls, no diff betweeen stages 382 Horvath et al. (31)* indirect ELISA Plasma, CSF 20 PD, 20 HC Yes Mild: 65.5 (38–79) Moderate: 67.2 (56–77) 2.8 (1–8) months (<1 year) 1.5 to 2 ↑in PD vs. HC in CSF and plasma Decreased in moderate vs. mild disease N/A Smith et al. (9) ELISA Serum 14 PD, 11 PD syndrome, RBD 10, 9 HC Yes RBD 58 (SD 9), PD 63 (9) Median 3.5 (1–12) 1.3 (range 1–3.5) No difference N/A Gruden et al. (32)* ELISA Serum 32 PD, 26 HC Yes 60.8 (2) 8.6 (3.4)** Subgroup <5 2.1 (0.6) ↑in PD vs. HC, greater difference with monomers than oligomers 23 Shalash et al. (33) ELISA Serum 46 PD, 20 HC Yes 56.26 (SD12.26) 5.2 (3.36) 3 (1.5–3.5 range) ↑in PD vs. HC N/A 7-10 years DD Akhtar et al. (34) ELISA Serum, CSF Serum: 53 PD, 16 HC CSF: 93 PD, 52 HC Both CSF and serum for 24 participants No Serum 70.9 (7) CSF 67.1 (9.4) 7.9 (5) 3 (1–4) (median + range) CSF ↑, serum → 77 Brudek et al. (19) ELISA, MSD Plasma 46 iPD, 46 HC No 62.4 (6.7) 7.9 (5) 2 (median) ↓in PD vs. HC 126 Papachroni et al. (35) Immunoblot Serum 31 iPD, 20 FPD, 26 HC Yes Idiopathic: 65.1 (11.6), Familiial: 66.1 (12.7) Calculated 7.2 iPD, 9.4 FPD 2.4 (FPD), 2.5 iPD ↑in FPD vs. Frontiers in Neurology \| www.frontiersin.org ndard deviation unless otherwise speciﬁed. The table is ordered according to disease duration. Patient and control groups were considered “matched” if there were no signiﬁcant between-group differences . One paper was removed due to cohort overlap (42). N/A was recorded in the sample size column if there was not sufﬁcient data to do the power calculation. Range, SEM, *author overlap, ∧∧in each Swithin = s (n1 −1) S12 + (n2 −1) S22 n1 + n2 −2 n1 = sample size (SS) in patients, n2 = SS in controls S1 = SD in patients, S2 = SD in controls. n1 = sample size (SS) in patients, n2 = SS in controls S1 = SD in patients, S2 = SD in controls. ndard deviation unless otherwise speciﬁed. The table is ordered according to disease duration. Patient and control groups were considered “matched” if there were no signiﬁcant between-group differences . One paper was removed due to cohort overlap (42). N/A was recorded in the sample size column if there was not sufﬁcient data to do the power calculation. Range, SEM, author overlap, ∧∧in each \| Paper Method Fluid N (HC = healthy controls) Matched Mean age of PD (SD) Disease duration years (SD) H and Y (SD) Finding (in PD vs. controls) Required N∧∧ Besong-Agbo et al. (18) ELISA Serum 62 iPD, 46 HC Yes 68.6 (9) 10.2(6) >3 ↓in PD vs. HC 60 Unknown DD Bryan et al. (40) Electrochemical impedance spectroscopy Serum 30 PD, 14 HC No Not reported Not reported 1 to 3 ↑in PD vs. HC, increasing up to HandY 2 then decreasing for HandY 3. N/A Heinzel et al. (20) ELISA Serum, CSF 66 PD, 69 HC (CSF 59 PD and 46 controls) Yes No ages reported Not reported 2 No difference 214 Woulfe et al. (41) ELISA Serum Serum: 28 PD, 19 HC CSF: 4 PD, ﬁve controls Not reported Not reported Not reported Not reported No difference N/A Numbers refer to mean and standard deviation unless otherwise speciﬁed. The table is ordered according to disease duration. Patient and control groups were considered “matched” if there were no signiﬁcant between-group differences in age and gender distributions. One paper was removed due to cohort overlap (42). N/A was recorded in the sample size column if there was not sufﬁcient data to do the power calculation. Range, SEM, author overlap, ∧∧in each group, DD, Disease duration. Numbers refer to mean and standard deviation unless otherwise speciﬁed. The table is ordered according to disease duration. Patient and control groups were considered “matched” if there were no signiﬁcant between-group differences in age and gender distributions. One paper was removed due to cohort overlap (42). N/A was recorded in the sample size column if there was not sufﬁcient data to do the power calculation. Range, SEM, author overlap, ∧∧in each group, DD, Disease duration. Where: K = nA/nB (matching ratio between groups—nA = PD patients, nB = controls) p Swithin = pooled standard deviation across groups α = Type I error (set at 0.05) yp β = Type II error (1-β = power, set at 0.8) β = Type II error (1-β = power, set at 0.8) The pooled within sample standard deviation was calculated to overcome diﬀerences in variation between the groups [from (44)]: Swithin = s (n1 −1) S12 + (n2 −1) S22 n1 + n2 −2 n1 = sample size (SS) in patients, n2 = SS in controls S1 = SD in patients, S2 = SD in controls. SYSTEMATIC REVIEW OF ALPHA SYNUCLEIN ANTIBODY STUDIES IN PD PD or controls N/A Yanamandra et al. (36)** Elisa, western blot, biocore surface plasmon resonance Serum 39 PD, 23 HC Yes 55.7 (10) 7.7 (5.6) HandY1-2 27, HandY 2.5–4 12 ↑PD vs. HC N/A Caggiu et al. (37) ELISA Serum 40 PD, 40 HC Yes 69.8 (7.95) 8.42(4.29) 3.01 (0.88) ↑in PD vs. controls to three peptides (similar to HSV) N/A Maetzler et al., (38) ELISA Serum 93 PD (demented subgroup 31), 194 controls No 68.5(SD9) PDND, 76.7 (SD8) PDD 9.5 (1–26*) 2 (1–4) No difference N/A 10-12 years DD Alvarez-Castelao et al. (39) ELISA immunoblots Plasma 55 iPD, 104 LRRK2 carriers, 85 HC No 67.8 (9.9) iPD 68.37 (10.2) LRRK2 12 (8.7) iPD 13 (11) LRRK2 2.44 (0.8) iPD, 2.55 (0.88) LRRK2 Controls and iPD no difference Using stringent criteria ↑antibodies in LRKK2 pre-manifest N/A ontiers in Neurology \| www.frontiersin.org 4 October 2018 \| Volume 9 October 2018 \| Volume 9 \| Article 815 4 Alpha Synuclein Auto-Antibodies in PD Scott et al. Frontiers in Neurology \| www.frontiersin.org META-ANALYSIS OF ALPHA SYNUCLEIN ANTIBODY STUDIES IN PD We undertook a meta-analysis, stratiﬁed by disease duration given the suggestion in the literature that this is a relevant factor [e.g., (36)]. Studies with mean disease durations of 5.9 years and less were included in an “early disease” meta-analysis and those with disease durations of 7 years or more were included in a “later disease” meta-analysis given the trends noted in the review above and in Table 1. More stringent data quality criteria were adopted for the meta-analysis than for the systematic review described above. Inclusion criteria: i) The study measured antibodies to full length alpha synuclein ii) The antibodies were measured using titres (either relative or absolute) as a continuous measure iii) The study included both idiopathic PD patients and controls iv) The study stipulated a measure of disease duration for the cohort v) The controls were age and gender matched to the patients vi) Antibodies were measured in either serum or plasma If a study had not published appropriate statistical tests to determine whether the controls were matched appropriately to the patients this was performed (independent samples t-test for age; chi-squared test for gender). The study estimates were extracted from the included papers according to the protocol below; Study estimate extraction: Study estimate extraction: i) Means and standard deviations were used as the basis for the study estimates, if reported. ii) If these were not reported, then the median and interquartile ranges were extracted and converted into means and standard deviations using the methodology described in (45) and an online calculator (http://www.comp.hkbu.edu.hk/~ xwan/median2mean.html). iii) If the above estimates were not described in the text then they were estimated from the boxplots or graphs published in the text. October 2018 \| Volume 9 \| Article 815 5 Alpha Synuclein Auto-Antibodies in PD Scott et al. the response to speciﬁc epitopes deemed to be relevant due to their similarity to EBV (37). As all studies used diﬀerent assays and units of measurement, it was not possible to do a direct comparison using the raw unstandardised mean diﬀerence. The study estimates were therefore used to calculate the standardized diﬀerence and the associated variance (yi and vi, respectively) using the metafor package for R in R studio (version 1.0.153), and the following formulas (44): S2 = standard deviation in controls S2 = standard deviation in controls A random eﬀects model was used to assess the overall diﬀerence between patients and controls. Forest plots were generated to show the results graphically. Funnel plots were generated to plot standardized mean diﬀerence (x axis) against standard error (y axis) to assess the impact of publication bias and heterogeneity. The variance of d (referred to as vi) is given by the following formula (see (44) page 27): vi = n1 + n2 n1n2 + d2 2(n1 + n2) Patient age also varies between study cohorts, ranging from a mean of 55.7 (36) to 69.8 [(37); Table 1]. Antibody responses vary with age and gender (47). It is therefore also critical to ensure that patient and control groups are well-matched. Of the 17 studies reviewed, seven either did not report appropriate demographic information or the control group was not matched to the patients. Clinical Heterogeneity There is wide variation in disease stage and duration across studies (see Table 1). Previous studies have noted an increase in early disease e.g., (42). Of the ﬁve papers reporting a mean disease duration of 5 years or less (see Table 1), four report an increase in alpha synuclein antibodies in patients compared to controls (representing a total of 196 patients and 121 controls excluding the ﬁrst Gruden et al paper as described above) (9, 30, 31, 33, 36, 42). Only the smallest of the studies in early PD showed no PD-control diﬀerence (N = 14 PD patients and nine controls) (9). Even taking a conservative interpretation of these results, the larger studies are consistent in reporting an increase in alpha synuclein antibodies in early disease. An additional study for which disease duration was unavailable reported an association with HY disease stage with increasing titres from HY stage 1 to 2, decreasing at stage 3 (40). Alvarez-Castelao et al. found increased alpha synuclein antibodies in LRRK2 carriers vs. controls but not in patients with longer disease durations (>10 years) (39). Other studies have also reported a similar association with HY staging (33, 46). Of six studies with mean disease durations between 7 and 10 years, two studies report a clear increase in patients vs. controls (36, 37). Two further studies show an increase in a subgroup, in familial PD vs. controls (but not idiopathic PD) (35) in one study and in CSF only and not serum in another (34). The two studies that showed either no diﬀerence (38) or a diﬀerence in the opposite direction (19) did not have age and gender matched control groups. In the two studies with disease duration beyond 10 years there was either no diﬀerence (39) or a decrease in patients compared to controls (18). yi = X1 −X2 Swithin Where yi = standardized mean diﬀerence (d) X1 = sample mean in PD patients X1 = sample mean in PD patients X2 = sample mean in controls Swithin = within groups standard deviation, pooled across groups (as used above for the power calculation) Swithin = s (n1 −1) S12 + (n2 −1) S22 n1 + n2 −2 S1 = standard deviation in PD group RESULTS A total of 17 papers met the inclusion and exclusion criteria for the systematic review (Table 1). Eight studies found a statistically signiﬁcant increase in alpha synuclein antibodies in idiopathic PD patients compared to controls (30–33, 36, 37, 40, 42). These studies included a total of 305 patients and 198 controls but two of the papers appear to use overlapping patient samples with identical demographic tables and results ﬁgures and so the second of these was excluded (32, 42). Frontiers in Neurology \| www.frontiersin.org Assay Variability Most studies have made use of custom ELISAs with one study using a commercial ELISA for serum anti-alpha synuclein antibodies (33). Two positive studies by the same group in diﬀerent patient cohorts used electroimpedence spectroscopy (30, 40). Several others used immunoblots or western blots (35, 36, 39). ELISAs are limited by many factors including the requirement for two independent binding events and problems with non-speciﬁc binding (30). There is also variation in conditions between studies, such as buﬀers used, protein coating concentration and temperature of the assay which are particularly relevant for an intrinsically disordered protein, such as alpha- synuclein. Three papers found raised alpha synuclein antibodies in sub- groups of PD patients, either in familial PD (35), pre-manifest LRRK2 carriers (39) or only in CSF and not serum (34). Four studies reported no diﬀerence in peripheral anti-alpha synuclein antibodies (9, 20, 38, 41) and two studies found that alpha synuclein antibodies were decreased in patients vs. controls (18, 19). Importantly the Brudek et al. paper focused on high aﬃnity antibodies only which may underlie the diﬀerence in ﬁndings. Three studies investigated antibodies in CSF as well as in plasma or serum (20, 31, 34) with two of these ﬁnding raised alpha synuclein antibodies in the CSF (31, 34). Most of the alpha synuclein for the use in ELISAs was generated in E. coli in-house, and therefore may not include post-translational modiﬁcations present in mammalian cells (30, 31, 34–36, 38, 40) (with other papers obtaining commercially generated protein). Alvarez-Castelao et al. attempted to replicate All studies investigated the antibody response to full length alpha synuclein apart from the Caggiu et al study that assessed October 2018 \| Volume 9 \| Article 815 Frontiers in Neurology \| www.frontiersin.org 6 Alpha Synuclein Auto-Antibodies in PD Scott et al. their ELISA ﬁndings using immunoblots and identiﬁed that some of the ELISA positive samples were recognizing something other than alpha synuclein (39). This eﬀect disappeared when they introduced an additional puriﬁcation step suggesting the possibility that at least some of the ﬁndings in the literature may be due to interfering antibodies to bacterial toxins rather than to alpha synuclein itself. Antibodies present in serum may also be bound to serum protein (either speciﬁcally or non-speciﬁcally) which may interfere with antibody detection (38). Most of the papers investigated antibody responses to FIGURE 2 \| Flow diagram showing inclusion and exclusion of studies in the meta-analysis. Assay Variability TABLE 2 \| Study estimates, standardized effect sizes (yi) and variance (vi) (“early disease” <5.9 years disease duration). Year Controls PD yi vi Mean SD N Mean SD N Gruden 2011 25.00 50.99 26 310.00 452.55 32 0.83 0.08 Xu 2012 1.24 1.44 29 1.62 2.04 60 0.20 0.05 Smith 2012 0.83 1.13 9 1.06 1.81 14 0.14 0.18 Horvath 2017 5.00 0.67 20 6.50 2.72 20 0.74 0.11 Shalash 2017 0.49 0.69 20 4.39 1.78 46 2.50 0.12 Model results: Estimate = 0.88 (95% CI 0.005–1.17), SE = 0.42, Z = 2.09, p = 0.036. I2 = 89.32%. Q(df = 4) = 33.71, p = 0.0001. TABLE 3 \| Study estimates, standardized effect sizes (yi) and variance (vi) (“later disease,” >7 years disease duration). Study Year Controls PD yi vi Mean SD N Mean SD N Yanamandra 6.7 years 2011 108.67 126.43 23 696.44 821.82 27 0.95 0.09 Yanamandra 9.7 years 2011 108.67 126.43 23 313.11 490.58 12 0.66 0.13 Besong-Agbo 2013 153.5 103.77 46 105.40 85.83 62 −0.51 0.04 Model results: Estimate 0.34 (95% CI −0.57–1.25), SE 0.46, Z = 0.73, p = 0.47. I2 = 87.6%. Q(df = 2) = 19.80, p = 0.0001. FIGURE 2 \| Flow diagram showing inclusion and exclusion of studies in the meta-analysis. FIGURE 2 \| Flow diagram showing inclusion and exclusion of studies in the meta-analysis. TABLE 2 \| Study estimates, standardized effect sizes (yi) and variance (vi) (“early disease” <5.9 years disease duration). TABLE 3 \| Study estimates, standardized effect sizes (yi) and variance (vi) (“later disease,” >7 years disease duration). Study Year Controls PD yi vi Mean SD N Mean SD N Yanamandra 6.7 years 2011 108.67 126.43 23 696.44 821.82 27 0.95 0.09 Yanamandra 9.7 years 2011 108.67 126.43 23 313.11 490.58 12 0.66 0.13 Besong-Agbo 2013 153.5 103.77 46 105.40 85.83 62 −0.51 0.04 Model results: Estimate 0.34 (95% CI −0.57–1.25), SE 0.46, Z = 0.73, p = 0.47. I2 = 87.6%. Q(df = 2) = 19.80, p = 0.0001. TABLE 3 \| Study estimates, standardized effect sizes (yi) and variance (vi) (“later disease,” >7 years disease duration). October 2018 \| Volume 9 \| Article 815 7 Frontiers in Neurology \| www.frontiersin.org Frontiers in Neurology \| www.frontiersin.org Alpha Synuclein Auto-Antibodies in PD Scott et al. Power Lack of adequate power may be an important factor leading to false negative ﬁndings in a number of studies. The largest study included 93 PD patients and 194 controls (38) but unfortunately the controls were not age and gender matched to the patients (see Table 1). Of the 17 studies, seven included appropriate information to calculate power. Of those with incomplete information, this was usually because the data were presented as graphs or as medians and IQ range. The estimated sample sizes required to detect the diﬀerences reported ranged from 23 to 382, with a mean of 147 per group (see Table 1). The only study that was adequately powered was that by Gruden et al. that reported much larger diﬀerence between controls and patients than other studies and is therefore an outlier. Excluding this study, the estimated required sample size per group is between 60 and 382. Three studies were excluded due to a lack of reported disease duration (20, 40, 41); four studies were excluded due to a lack of age and gender matching between patients and controls (19, 34, 38, 39). The Alvarez-Castelao paper did not include published signiﬁcance testing of the age diﬀerence which was therefore done as part of this review. There was FIGURE 4 \| Forest plot showing study effect sizes in later disease (>7 years). FIGURE 5 \| Funnel plot showing standard error vs. standardized mean difference in early disease. FIGURE 4 \| Forest plot showing study effect sizes in later disease (>7 years). Assay Variability monomeric alpha synuclein (which is not necessarily the disease relevant species) with only a minority assessing responses to ﬁbrils, mutated alpha synuclein (36, 39, 42), oligomers or other pathological forms [e.g., phosphorylated alpha synuclein (19) or speciﬁc peptides (37)]. The Brudek et al. paper focused on high aﬃnity antibodies ﬁnding that these were decreased in patients compared to controls which is consistent with them having a role in alpha synuclein clearance. As other studies have investigated the overall antibody response it is not useful to directly compare these. were estimated based on the reported medians and interquartile ranges. The medians and interquartile ranges from the other two papers were estimated from boxplots and subsequently converted to means and standard deviations as described in the methods (9, 33). Study eﬀect size estimates and model results are shown in Table 2. Overall, there is a signiﬁcant increase in antibodies in patients vs. controls across studies (see forest plot in Figure 3) but the eﬀect size is modest (0.88, 95% CI 0.05–1.71, p-value = 0.036). There was signiﬁcant heterogeneity across studies (I2 = 89.32%). Only two of the “later disease” studies (mean disease duration >7 years) met inclusion criteria (18, 36). Means and standard deviations were published in the Besong-Agbo study and therefore used to calculate study estimates. Medians and interquartile ranges were estimated from boxplots in the Yanamandra study which was divided into two subgroups (mean disease duration 6.7 years and mean disease duration 9.7 years as there was no available data for the patient group overall). Means and standard deviations were then derived from this data. Lastly, some of the variation between studies may be due to the use of either serum or plasma (although only two studies used plasma rather than serum, see Table 1). It is possible that factors present in plasma but not in serum (e.g., alpha synuclein produced by platelets) may aﬀect subsequent results and therefore it would be wise to standardize the use of serum across studies. Meta-Analysis There was no overall diﬀerence between groups in this small sample (estimate = 0.34, 95% CI = −0.57–1.24, p = 0.46) and there was also signiﬁcant heterogeneity (I2 = 87.67%). There is a clear need for further studies in this ﬁeld and we recommend that future studies should focus on the following points: Given the signiﬁcant heterogeneity, funnel plots were generated plotting standardized mean diﬀerence on the x axis against standard error on the y axis for studies in the “early disease” group (there were too few in the later disease group to make interpretation of these plots meaningful). The plot is symmetrical around the eﬀect size of 0.88 (z = 0.20, p = 0.84) but shows that two of the studies fall outside of the 95% CI of an assumed true eﬀect (see Figure 5). One of the many explanations for the shape of this plot is the presence of true heterogeneity between studies (both clinical and assay related factors discussed above). If we were able to include more studies in the analysis one would expect, assuming the same true eﬀect, that eﬀect estimates from smaller studies would spread widely along the bottom with those from larger, more powerful studies appearing at the top (see Figure 5). One cannot fully discount the role played by publication bias in this context as positive ﬁndings in this ﬁeld will be more likely to be written up and published than negative results particularly in the context of smaller studies. 1. Appropriate sample size with an absolute minimum of 60 in each group (based on approximate power calculations from existing studies) 2. Well-characterized clinical cohorts with appropriately matched controls using both serum and CSF if possible 3. Longitudinal assessment to measure changes in antibody levels over the course of the disease and relationship with clinical disease progression 4. Study of prodromal PD cohorts to establish whether the antibody response is truly an early feature of the disease 5. Using a robustly validated method (ideally with validation using a second method in the same samples) to measure antibodies including standardization and testing of diﬀerent coating concentrations, buﬀers and assay temperature. 6. Study of epitope-speciﬁc antibodies and Ig subclasses to allow a fuller understanding of the adaptive immune response to PD. Meta-Analysis y All of the “early disease” papers shown in Table 1 met the inclusion criteria (see also ﬂow plot in Figure 2) Means and standard deviations were available from two of the studies (30, 32). The means and standard deviations from Horvath et al. (31) FIGURE 4 \| Forest plot showing study effect sizes in later disease (>7 years). FIGURE 3 \| Forest plot showing study effect sizes in early disease (<5.9 years). FIGURE 3 \| Forest plot showing study effect sizes in early disease (<5.9 years). FIGURE 5 \| Funnel plot showing standard error vs. standardized mean difference in early disease. FIGURE 5 \| Funnel plot showing standard error vs. standardized mean difference in early disease. FIGURE 5 \| Funnel plot showing standard error vs. standardized mean difference in early disease. FIGURE 5 \| Funnel plot showing standard error vs. standardized mean difference in early disease. FIGURE 3 \| Forest plot showing study effect sizes in early disease (<5.9 years). October 2018 \| Volume 9 \| Article 815 Frontiers in Neurology \| www.frontiersin.org 8 Alpha Synuclein Auto-Antibodies in PD Scott et al. a signiﬁcant diﬀerence in age between patients and controls according to a independent samples t-test (idiopathic PD mean 67.81, SD 9.98 and controls mean 61.4, SD 14.7), t[136] = 2.83, p = 0.005). One study was excluded as it only measured antibodies to speciﬁc epitopes of alpha synuclein rather than the entire protein (37) and one other study was excluded because outcomes were recorded as percentage positive on immunoblots (35). diﬃcult to draw any ﬁrm conclusions from this aspect of the study). Hence the value of alpha synuclein auto-antibodies as a diagnostic or prognostic biomarker remains uncertain. Further studies are needed to demonstrate a consistent, reproducible eﬀect in early PD cases vs. controls (or indeed between diﬀerent groups of PD patients), to investigate the speciﬁcity of raised antibody titres in PD vs. other alpha-synucleinopathies, and to track longitudinal changes in antibody titres and their relationship to disease onset and clinical disease progression. The possible utility of using antibody based biomarkers for identifying patients who would potentially beneﬁt from either immune modulating or antibody based therapies is also unknown. The study estimates are shown in Table 3 and the overall random eﬀects model is shown in the forest plot in Figure 4. AUTHOR CONTRIBUTIONS KS designed the study, reviewed the literature, performed the meta-analysis and wrote the ﬁrst draft of the manuscript. SY and AK reviewed the literature, made summary tables and critically reviewed the manuscript. MC and CW-G contributed signiﬁcantly to the design of the study and critically reviewed the ﬁnal manuscript. Whilst the available data does not suggest elevation of alpha synuclein antibodies universally across all stages of PD, it is consistent with the hypothesis that there is an increased antibody response in early disease that wanes during disease progression, which is biologically plausible. According to our meta-analysis the eﬀect size is modest in early disease but the analysis is limited by signiﬁcant study heterogeneity. 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https://openalex.org/W3038086895	https://ijpds.org/article/download/1357/2555	English	null	Why the public need a say in how patient data are Used for Covid-19 Responses	International journal of population data science	2,020	cc-by	1,389	International Journal of Population Data Science (2020) 5:2:01 International Journal of Population Data Science (2020) 5:2:01 Submission History Submitted: 26/05/2019 Accepted: 11/06/2020 Published: 20/06/2020 1Newcastle University, Urban Sciences Building, 1 Science Square, Newcastle upon Tyne, NE4 5TG 2Usher Institute, University of Edinburgh, Old Medical School, Teviot Place, Edinburgh, EH8 9AG 3Imperial College Health Partners, 30 Euston Square, London, NW1 2FB 4Department of Politics, School of Geography, Politics and Sociology, Newcastle University, Newcastle upon Tyne, NE1 7RU 5University of Edinburgh, Chrystal Macmillan Building, Edinburgh, EH8 9LD 6Swansea University Medical School, Singleton Park, Swansea, SA2 8PP 7Centre for Biomedicine, Self and Society, Usher Institute, University of Edinburgh, 23 Buccleuch Place, Edinburgh, EH8 9LN 8Data Science Building, Swansea University Medical School, Singleton Park, Swansea, SA2 8PP 1Newcastle University, Urban Sciences Building, 1 Science Square, Newcastle upon Tyne, NE4 5TG 2Usher Institute, University of Edinburgh, Old Medical School, Teviot Place, Edinburgh, EH8 9AG 3Imperial College Health Partners, 30 Euston Square, London, NW1 2FB 4Department of Politics, School of Geography, Politics and Sociology, Newcastle University, Newcastle upon Tyne, NE1 7RU 5University of Edinburgh, Chrystal Macmillan Building, Edinburgh, EH8 9LD 6Swansea University Medical School, Singleton Park, Swansea, SA2 8PP 7Centre for Biomedicine, Self and Society, Usher Institute, University of Edinburgh, 23 Buccleuch Place, Edinburgh, EH8 9LN 8Data Science Building, Swansea University Medical School, Singleton Park, Swansea, SA2 8PP International Journal of Population Data Science Journal Website: www.ijpds.org Dear Editors, ment and engagement (PI&E) relating to data-intensive health research published in IJPDS last year [2]. This statement was co-authored by 31 international researchers, practitioners and patient representatives from the U.K., Ireland, Australia, Canada, Finland and the Netherlands. It sets out eight prin- ciples to underpin best practice in this ﬁeld and to inform the design, implementation and evaluation of PI&E strategies and activities. The global coronavirus pandemic has clearly demonstrated the great urgency to collect and use patient data eﬀectively to understand, track and manage the spread of Covid-19. The value of patient data in this pandemic is undeniable, however considerations around how – and by whom - such data should be collected, accessed and used, and for what purposes, remain to be fully debated and resolved. Who decides, and how such decisions are made, remain unclear. We argue that, as with all uses of patient data, public engagement and deliberation are essential for good governance and are key to establish and maintain a legitimate social licence for data practices around Covid-19. The principles put forward in the consensus statement are, that public involvement and engagement with data-intensive health research should: 1. Have institutional buy-in; Previous data controversies have clearly demonstrated the importance of establishing a social licence for data practices, and that there can be meaningful diﬀerences between what is legally permissible and what is socially acceptable [1]. The standard response to such controversies has typically been reemphasising commitments to public engagement [2] in order to (re)build or restore public trust [3]. This overlooks the dy- namic nature of public trust, and the importance of ongoing relationships to establish and maintain trust over time. 2. Have clarity of purpose; 3. Be transparent; 4. Involve two-way communication; 5. Be inclusive and accessible to broad publics; 6. Be ongoing; 7. Be designed to produce impact; Commitments to public engagement must go beyond lip service [2] and also need to recognise that not only are there diﬀerent approaches to public engagement, some are more le- gitimate and useful than others. We advocate engaging the public, policy makers and users of patient data in collective de- liberation to enable mutual learning and informed policy mak- ing. This would make the social licence epistemically superior and more legitimate. 8. Be evaluated. https://doi.org/10.23889/ijpds.v5i2.1357 June 2020 © The Authors. Open Access under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/deed.en) ∗Corresponding Author: Email Address: Mhairi.Aitken@newcastle.ac.uk (M Aitken) Why the Public Need a Say in How Patient Data are Used for Covid-19 Responses am-Burley, S2, Darlington, A3, Elstub, S4, Escobar, O5, Jones, KH6, Sethi, N7, and Thompson, R8 Aitken, M1*, Cunningham-Burley, S2, Darlington, A3, Elstub, S4, Escobar, O5, Jones, KH6, Sethi, N Submission History Submitted: 26/05/2019 Accepted: 11/06/2020 Published: 20/06/2020 Yours Sincerely, Dr Mhairi Aitken, Senior Research Associate, Newcastle University Dr Mhairi Aitken, Senior Research Associate, Newcastle University Prof. Sarah Cunningham-Burley, Professor of Medical and Family Sociology / Dean of Molecular, Genetic and Population Health Sciences, Usher Institute, University of Edinburgh Prof. Sarah Cunningham-Burley, Professor of Medical and Family Sociology / Dean of Molec Health Sciences, Usher Institute, University of Edinburgh Prof. Sarah Cunningham-Burley, Professor of Medical and Family Sociology / Dean of Molecular, Genetic and Population Health Sciences, Usher Institute, University of Edinburgh Amy Darlington, Executive Director, Imperial College Health Partners and Engagement Lead for OneLondon Local Health and Care Record Exemplar Dr Stephen Elstub, Senior Lecturer in British Politics, Newcastle University Dr Oliver Escobar, Senior Lecturer in Public Policy, University of Edinburgh Prof. Kerina Jones, Professor of Population Data Science, Swansea University, Dr Nayha Sethi, Chancellor’s Fellow in Data Driven Innovation, Centre for Biomedicine, Self and Society, University of Edinburgh Rachel Thompson, Research Associate, Population Data Science, Swansea University Dr Stephen Elstub, Senior Lecturer in British Politics, Newcastle University Dr Oliver Escobar, Senior Lecturer in Public Policy, University of Edinburgh Prof. Kerina Jones, Professor of Population Data Science, Swansea University, Dr Nayha Sethi, Chancellor’s Fellow in Data Driven Innovation, Centre for Biomedicine, Self and Society, University of Edinburgh Rachel Thompson, Research Associate, Population Data Science, Swansea University Rachel Thompson, Research Associate, Population Data Science, Swansea University Statement of Competing Interests None declared. Aitken, M et al. / International Journal of Population Data Science (2020) 5:2:01 Aitken, M et al. / International Journal of Population Data Science (2020) 5:2:01 already online or have little experience of digital communica- tion. resonates in the current context where it is important to avoid caricaturing “the public” or speculating on how “the public” will respond to particular measures or data practices, but rather to engage diverse publics in consequential deliberation to inform and shape policy responses and data practices. Despite the speed required to deal with the pandemic, it is vital to adhere to these principles not just to do things well in relation to the current crisis, but because current practices, hastily developed, will forge the way for future ways of work- ing. The pandemic brings with it an imperative to realise the value of PI&E in shaping socially acceptable and ethically ro- bust data practices and to raise the proﬁle of PI&E increasing public interest in these activities. This will ensure that the social licence generated will endure because it is based on au- thoritative and authentic deliberation. A number of public engagement initiatives relating to Covid-19 have been announced [4] and innovative approaches are being developed to engage the public in these discussions during lockdown and beyond. Innovation in using digital meth- ods is clearly vital if we are to engage diverse publics at this time in the development and governance of new data initia- tives. We must also strive to be inclusive of those who are not 8. Be evaluated. It is time to reinvigorate these principles so that public en- gagement is not overlooked in the rapid response to COVID- 19. The key premise of the consensus statement is that the public should not be characterised as a problem to be over- come, but a key part of the solution towards establishing so- cially beneﬁcial data-intensive health research for all. This We already have a consensus statement on public involve- https://doi.org/10.23889/ijpds.v5i2.1357 June 2020 © The Authors. Open Access under CC BY 4.0 (https://creativecommons.org/licenses/by/4.0/deed.en) Aitken, M et al. / International Journal of Population Data Science (2020) 5:2:01 4. For examples of public engagement activity in this area see: https://participedia.net/collection/6501 References 1. Carter P, Laurie GT, Dixon-Woods M, ‘The social li- cence for research: Why care.data ran into trouble’ Jour- nal of Medical Ethics 2015; 41: 404-409. doi:https: //doi.org/10.1136/medethics-2014-102374 3. Lawler M, Morris AD, Sullivan R, Birney E, Middleton A, MakaroﬀL, Knoppers BM, Horgan D, Eggermont A. ‘A roadmap for restoring trust in Big Data’. The Lancet: Oncology. 2018 Aug; 19(8): 1014. https: //doi.org/10.1016/S1470-2045(18)30425-X 2. Aitken M, Tully MP, Porteous C, Denegri S, Cunningham-Burley S, et al. ‘Consensus statement on public involvement and engagement with data-intensive health research’. 2019 Feb 12; 4(1). https://doi. org/10.23889/ijpds.v4i1.586 4. For examples of public engagement activity in this area see: https://participedia.net/collection/6501 2
https://openalex.org/W1967060302	https://zenodo.org/records/1925182/files/article.pdf	English	null	A CASE OF FORWARD DISLOCATION OF THE RADIUS AND ULNA WITHOUT FRACTURE OF THE OLECRANON PROCESS OF THE ULNA.	Lancet	1,905	public-domain	1,496	290 Gould and Warren in their " International Text-book of Surgery " refer to this dislocation as " rare and its occurrence without exist- ing fracture has been doubted ; in point of fact the latter complication has been still more rarely recorded." Helferich says : "This is a very rare injury ; it was formerly said it never occurred without simultaneous fracture of olecranon. May be produced by a fall or blow on olecranon while arm is in extreme flexion." Hamilton quotes nine cases and states " that it seems to have been in most cases caused by a fall upon elbow while forearm is forcibly flexed." Cheyne and Burghard state that this dislocation " is sometimes met with ...... as result of a severe blow upon back of flexed elbow." very The patient’s condition having undergone a slight improve- ment 1 left her about 4 A.M., giving instructions that the hypodermic injections should be continued as directed and that I should be sent for immediately if any change for the worse took place. To my surprise I heard no more of the patient until I made my usual visit to the ward on the follow- ing morning at 10 o’clock. She was then resting quietly ; all signs of dyspnoea had disappeared and, except for some facial pallor and slight exhaustion, she seemed to be none the worse for her experience of the early hours. On examina- tion of the chest a few moist râles could still be heard over the affected area while all signs of cardiac dilatation had vanished and at 4 o’clock on the same afternoon the chest, being again examined, this time in company with one of my colleagues, was pronounced by him to be normal. From this time onwards the puerperium was uneventful, save for the development of a small, superficial mammary abscess which rapidly yielded to appropriate treatment, and the patient was allowed up on the twenty-first day and dis- charged herself from the infirmary two days later. upon e bo . Stimson in his book refers to a case which was under his care in 1895. He says : "The elbow is held at a right angle but can be somewhat flexed and extended ; is moveable laterally. The epitrochlea can be plainly felt, also the inner face and edge ot trochlea. 290 290 290 an angle of 1300. It could be slightly flexed and extended and permitted a good deal of lateral movement. The head of the radius and the olecranon process of the ulna could be felt lying on the shaft of the humerus about two and a quarter inches above the condyles. It was possible to feel both condyles of the humerus, the olecranon fossa, the coronoid depression, the trochlea, and the capitellum ; in fact, the lower end of the humerus could be easily grasped in the band. There was distinct lengthening of the forearm. patient complaining of great pain above the right breast and difficulty in breathing) a colourless frothy liquid, at times slightly stained with haemoglobin, had been coughed up in considerable quantities. There was about half a pint in all. Of specific gravity about 1030, it coagulated on boiling, contained no red corpuscles when examined microscopically, but gave the spectroscopic test for oxyhmmoglobin. As the more alarming symptoms seemed to be due to an acute engorgement of the right ventricle a small venesection was performed, about ten ounces of blood being withdrawn, and five minims of solution of strychnine, together with 20 minims of ether purus, were given hypodermically, weak brandy and water being ordered by the mouth, in teaspoon- ful doses, as the respiratory distress rendered swallowing very difficult. t e ba d. e e as d st ct lengthening The points of interest in this case were : (1) its rarity (2) the absence of fracture of the olecranon ; and (3) the maintenance of the relationship between the radius anc ulna. Attention has been called to this in Treves’s " Surgery " by Mr. Marmaduke Sheild who states that " this dislocation u extremely rare, and quotes Canton’s case in which the radius maintained its relationship to the ulna. Looking up the literature on the subject I find that there are about 12 recorded cases. Erichsen states that six cases of this dis. location are on record. Rose and Carless state that fracture of the olecranon always occurs in this dislocation. 290 The overlying flexor muscles of hand having been torn away from humerus, the tip of olecranon is below, and even a little in front of, the trochlea, the inner anterior portion of articular surface of which can also be felt on depressing the skin. The head of radius in front of capitellum ; the forearm is markedly abducted....... The only other reported case is Wright’s. The symptoms were similar." charged infirmary days As regards a diagnosis, dyspnoea coming on suddenly after labour in a healthy woman such as this patient, taken together with the physical signs present during the attack, seemed to me to point to an embolus of uterine origin which had been carried away and become arrested in the pulmonary circulation, while the fact of the signs clearing up so rapidly (all physical signs had disappeared 16 hours after onset) makes it probable that the embolu?, if such it were, must have been composed of air rather than dislodged blood clot. If, however, this diagnosis be admitted as possibly a correct one it seems difficult to imagine how the air was introduced into the uterus, since no intra-uterine manipulations were made nor was a douche given either before or after labour. The point about which I feel most interest is as to whether a hurried expulsion of the afterbirth, such as was effected in this case, could have given rise to the condition described, air presumably being sucked into the vagina as the uterus was released from the somewhat powerful grasp necessary to expel a not sufficiently separated placenta ; and if this be so, why so lengthy an interval as an hour elapsed before the onset of the disquieting symptoms ? Should any of your readers feel sufficiently interested I should be grateful for an expression of opinion upon this, to me, somewhat puzzling experience. Waterford. DISAPPEARANCE OF RODENT ULCER UNDER THE APPLICATION OF THE X RAYS. BY FREDERICK B. JEFFERISS, F.R.C.S. EDIN., M.R.C.S. ENG., L.R.C.P. LOND. BY FREDERICK B. JEFFERISS, F.R.C.S. EDIN., M.R.C.S. ENG., L.R.C.P. LOND. THE successful treatment of rodent ulcer by the applica- tion of the x rays is now well established and many cases are on record where a complete recovery has resulted. The same, however, cannot be said of carcinomata, some cases improv- ing for a time but relapsing again or even getting worse as the treatment is continued. experience. Cambridge Heath, N.E. The patient in the present instance was an elderly woman who suffered from an epithelial growth on the left frontal region. The history was to the effect that about two years before she first came under my notice she observed a small pimple at the site of the growth which she injured by accidently striking it against the mantelpiece causing ulceration. She treated it herself with zinc ointment with- out any benefit and it steadily grew for the two years. When I first saw her the growth was about as big as a five- shilling piece, rounded in shape, and very much raised, the ulcer on the top of the growth being about half an inch above the level of the surrounding skin, so that the whole growth was crateriform in appearance. It was very vascular, several large veins ramifying on the surface, and bleeding was easily induced on manipulation. There was no pain but the growth was very tender and the whole was soft, including the edges of the ulcer, which although heaped up were not indurated ; the mass was freely moveable and none of the neighbouring glands were involved. A diagnosis of rodent ulcer was made but it could not be confirmed as the patient refused to allow a piece to be removed for microscopic examination. A CASE OF FORWARD DISLOCATION OF THE RADIUS AND ULNA WITHOUT FRACTURE OF THE OLECRANON PROCESS OF THE ULNA. BY F. W. STAUNTON, M.B. DUB., HONORARY SURGEON TO WATERFORD COUNTY AND CITY INFIRMARY. THE patient in this case was a man, aged 45 years, whose description of his accident was to the effect that "while walking quickly downstairs he felt as if he had trod on some- thing slippery; his foot went from under him, he twisted round in order to save himself and fell backwards down- stairs, the tip of his left elbow striking the edge of the stair- case first." On examination the elbow was seen to be held at I began the treatment with the x rays on March 5th, 1904, giving two sittings a week of five minutes each. No change
https://openalex.org/W2976397985	https://www.jnwpu.org/articles/jnwpu/pdf/2019/04/jnwpu2019374p846.pdf	Chinese	null	Morphological Control and Simulation of Fractal Growth	Xibei gongye daxue xuebao	2,019	cc-by	483	文章编号：１０００⁃２７５８（２０１９）０４⁃０８４６⁃０５文章编号：１０００⁃２７５８（２０１９）０４⁃０８４６⁃０５分形是现代数学和非线性科学研究领域中一个十分活跃的分支。由于世界的本质是非线性的，因此，分形的应用领域非常广泛，是当前研究的一个热点。而分形生长是分形中重要的研究课题之一，从煤灰微粒的凝聚、闪电的形态、癌细胞的扩散与凝聚，到自然界中植物的生长等复杂而又不规则的实际聚集生长现象，都是分形生长的实际案例。因此，分形生长的理论研究与应用分析具有重要的理论意义和实际应用价值。然而，实际环境中的聚集生长过程总是会受到像磁场、温度等许多复杂因素的影响，从而使得生长粒子的形态变化多端，或者使得生长粒子的生长速度爆发性增长，以至于给自然界和人类社会带来很多负面的影响。因此，实际环境中的聚集生长形态的预测和控制，以及生长速度的控制具有重要的现实意义。态变化进行有效的控制。基于已有ＤＬＡ模型生长速度慢的问题，应用多粒子发射理论，提出一个改进模型———多粒子扩散限制凝聚（ｍｕｌｔｉ⁃ｐａｒｔｉｃｌｅｄｉｆｆｕｓｉｏｎｌｉｍｉｔｅｄａｇｇｒｅｇａ⁃ｔｉｏｎ）模型，简称ＭＤＬＡ模型，探索和研究该模型下分形生长的形态控制和模拟问题，使其产生出实际应用中需要的控制效果。西北工业大学学报西北工业大学学报２０１９年８月第３７卷第４期２０１９年８月第３７卷第４期Ａｕｇ．Ｖｏｌ．３７２０１９Ｎｏ．４Ａｕｇ．Ｖｏｌ．３７２０１９Ｎｏ．４ＪｏｕｒｎａｌｏｆＮｏｒｔｈｗｅｓｔｅｒｎＰｏｌｙｔｅｃｈｎｉｃａｌＵｎｉｖｅｒｓｉｔｙｈｔｔｐｓ：／／ｄｏｉ．ｏｒｇ／１０．１０５１／ｊｎｗｐｕ／２０１９３７４０８４６ｈｔｔｐｓ：／／ｄｏｉ．ｏｒｇ／１０．１０５１／ｊｎｗｐｕ／２０１９３７４０８４６收稿日期：２０１８⁃０７⁃０９　　　　　基金项目：国家自然科学基金（６１０７０２３３）、陕西省自然科学基础研究计划（２０１８ＪＭ６０６６）与国家级大学生创新创业训练计划项目（２０１８１０６９９２９１，２０１９１０６９９１０９１）资助作者简介：周敏（１９６６—），女，西北工业大学副教授、博士，主要从事计算机辅助几何设计与图形图像处理研究。分形生长的形态控制与模拟周敏，张海丽，王奕昊，包小兰（西北工业大学理学院，陕西西安　７１０１２９）分形生长的形态控制与模拟周敏，张海丽，王奕昊，包小兰（西北工业大学理学院，陕西西安　７１０１２９）摘　要：从定量分析的角度将控制的思想和方法引入到分形理论中，基于已有ＤＬＡ生长模型生长速度慢的问题，从改变粒子发射方式和条件出发，应用多粒子发射理论，提出一个改进模型———多粒子扩散限制凝聚模型，简称ＭＤＬＡ模型。讨论了迭代步长和粒子半径对生长形态的影响，并对生长形态进行了定向模拟，实验结果表明了所提出方法的有效性。关　键　词：ＭＤＬＡ模型；源项；分形生长；形态控制关　键　词：ＭＤＬＡ模型；源项；分形生长；形态控制中图分类号：Ｏ２３１．２　　　文献标志码：Ａ　　　文章编关　键　词：ＭＤＬＡ模型；源项；分形生长；形态控制中图分类号：Ｏ２３１．２　　　文献标志码：Ａ　　　文章编１　ＭＤＬＡ生长模型的提出标准ＤＬＡ模型中粒子是从远离中心的某个点逐个发射的，当实际应用中需要生成一个由成千上万个粒子生成的大的团簇时，再用此方法发射粒子，时间效率就很低下。为了加快粒子凝聚速度，本文从改变粒子发射方式和粒子死亡条件的角度出发，提出改进模型⁃ＭＤＬＡ生长模型，该模型与标准ＤＬＡ模型有３个区别：１９８１年美国密捷安大学Ｗｉｔｔｅｎ和Ｓａｎｄｅｒ首次提出了扩散限制凝聚（ｄｉｆｆｕｓｉｏｎｌｉｍｉｔｅｄａｇｇｒｅｇａｔｉｏｎ）的分形生长模型（简称ＤＬＡ模型）［１］，该模型用来描述像悬浮在大气中的煤灰、金属粉末或烟尘的扩散过程。近几年来对分形生长的研究主要体现在２个方面：一是生长模型改进［２⁃５］；二是分形生长的应用［６⁃８］。针对目前国内外分形生长控制研究热点，本文将从定量分析的角度将控制的思想和方法引入到分形理论中，研究对实际环境中的分形生长的形１）粒子不是逐个从矩形边缘发射的，而是被全部初始化；２）粒子运动方向由４个方向变为８个方向；３）粒子消失的条件设定为与其初始位置的距离大于某个常数。ＭＤＬＡ模型的生长规则是由迭代产生的，所以具有分形的自相似性，同时ＭＤＬＡ模型还具有屏蔽收稿日期：２０１８⁃０７⁃０９　　　　　基金项目：国家自然科学基金（６１０７０２３３）、陕西省自然科学基础研究计划（２０１８ＪＭ６０６６）与国家级大学生创新创业训练计划项目（２０１８１０６９９２９１，２０１９１０６９９１０９１）资助作者简介：周敏（１９６６—），女，西北工业大学副教授、博士，主要从事计算机辅助几何设计与图形图像处理研究。收稿日期：２０１８⁃０７⁃０９　　　　　基金项目：国家自然科学基金（６１０７０２３３）、陕西省自然科学基础研究计划（２０１８ＪＭ６０６６）与国家级大学生创新创业训练计划项目（２０１８１０６９９２９１，２０１９１０６９９１０９１）资助基金项目：国家自然科学基金（６１０７０２３３）、陕西省自然科学基础研究计划（２０１８ＪＭ６０６６）与国项目（２０１８１０６９９２９１，２０１９１０６９９１０９１）资助周敏，等：分形生长的形态控制与模拟第４期第４期 · ７４８ · 效应和非整数性。ＭＤＬＡ模型定义了８个方向，即上、下、左、右、左上、左下、右上、右下，粒子向８个方向运动是等可能的，每个方向概率均为１／８。广，粘贴上的概率就大，最终生长的形状应该是接近平行于该方向，表明相同生长条件下，粒子凝聚更快。广，粘贴上的概率就大，最终生长的形状应该是接近平行于该方向，表明相同生长条件下，粒子凝聚更快。ＭＤＬＡ算法是一次性初始化多个粒子，被初始化后的粒子都在做随机布朗运动。只要某个粒子满足与团簇粘贴的条件，该粒子（候选点）就会成为生长簇的一部分，停止运动。因此，候选点的选择不唯一，同时生长点的选择也是随机且不唯一，只要有粒子能够与之粘贴，该粒子就是生长点。因此，在ＭＤＬＡ模型中，同一时刻生长点和候选点的选择都不是唯一的。在ＭＤＬＡ模型中，Ｕ代表粒子的粘贴概率矩阵，当Ｕ值全是１，此时没有任何干扰项，即扩散方程的右端项为０，此时初始化的粒子向各个方向生长的可能性是一样的。这样，粒子在运动过程中，只要满足粘贴条件，该粒子就会成为生长簇的一部分。２　迭代步长对生长形态的影响实验结果表明，ＭＤＬＡ算法要求ｎ个寄存器，记录粒子的当前状态，包括位置、颜色、大小、半径。时间复杂度接近Ｏ（ｎ），以空间换取时间，从而加快粒子的凝聚。当粒子同时满足下面３个条件时，粒子可能会进行更新，继续走下一步：１）粒子没有粘贴在团簇上；２）粒子当前位置与其初始位置的距离小于一个常数；进行更新，继续走下一步：进行更新，继续走下一步：１）粒子没有粘贴在团簇上；粒子当前位置与其初始位１）粒子没有粘贴在团簇上；２）粒子当前位置与其初始位置的距离小于一因ＭＤＬＡ模型只是改变了粒子初始化方式，故也与标准ＤＬＡ模型一样满足Ｌａｐｌａｃｅ方程。求解Ｌａｐｌａｃｅ方程的数值解是相当困难的，尤其当方程的阶数比较大时。因此运用有限差分理论，并结合第一边界条件，把定解问题转化为求未知函数ｕ（ｘ，ｙ）在节点上数值为未知量的线性方程组的解的问题。定义Ａ为系数矩阵，与网格节点的划分和编号方式有关，ｂ为方程右端的常数向量，由边界条件确定，因此系统方程转化为如下线性方程组因ＭＤＬＡ模型只是改变了粒子初始化方式，故也与标准ＤＬＡ模型一样满足Ｌａｐｌａｃｅ方程。求解Ｌａｐｌａｃｅ方程的数值解是相当困难的，尤其当方程的阶数比较大时。因此运用有限差分理论，并结合第一边界条件，把定解问题转化为求未知函数ｕ（ｘ，ｙ）在节点上数值为未知量的线性方程组的解的问题。定义Ａ为系数矩阵，与网格节点的划分和编号方式有关，ｂ为方程右端的常数向量，由边界条件确定，因此系统方程转化为如下线性方程组３）粒子还没有逃出窗口边界。因此，粒子运动步长的改变，使粒子相对容易走３）粒子还没有逃出窗口边界。因此，粒子运动步长的改变，使粒子相对容易走到聚集团旁边或离开凝聚区域，进而决定粒子的运动方向，影响最终生长簇的生长形态。到聚集团旁边或离开凝聚区域，进而决定粒子的运动方向，影响最终生长簇的生长形态。做出以下推测：粒子迭代步长的增大，使粒子更快凝聚或更快停止生长（溢出边界条件或与其最初位置的距离超过一定值）。特别当ｘ，ｙ方向的步长不一致时，分形图形状会发生改变。同时，迭代步长的变化可能会影响分形维数。定义Ａ为系数矩阵，与网格节点的划分和编号方式有关，ｂ为方程右端的常数向量，由边界条件确定，因此系统方程转化为如下线性方程组ＡＵ＝ｂＡＵ＝ｂ式中，Ｕ是扩散方程的解。在平面上，ｕ是ｘ和ｙ的函数，不带源项的扩散系统方程可以写成式中，Ｕ是扩散方程的解。图１表明了相同初始化条件下，粒子在运动的过程中，采用不同迭代步长ｄ得到的单种子生长簇。在平面上，ｕ是ｘ和ｙ的函数，不带源项的扩散系统方程可以写成系统方程可以写成不同迭代步长的生长簇图１　不同迭代步长的生长簇 ∂２ｕ ∂２ｘ＋∂２ｕ ∂２ｙ＝０将平面网格化，令ｘｉ＝ｘ０＋ｉｈｙｊ＝ｙ０＋ｊτ { 　ｉ，ｊ＝０，± １，± ２，… 将平面网格化，令ｘｉ＝ｘ０＋ｉｈｙｊ＝ｙ０＋ｊτ { 　ｉ，ｊ＝０，± １，± ２，… 式中，（ｘ０，ｙ０）是ｘｏｙ平面上的一个随机点，ｈ，τ 分别表示ｘ，ｙ方向步长。由于（ｘ０＋ｉｈ，ｙ０＋ｊτ）＝（ｘｉ，ｙｊ）（ｘｉ，ｙｊ）＝（ｉ，ｊ），（ｘ０，ｙ０）＝（０，０）。（ｘ０＋ｉｈ，ｙ０＋ｊτ）＝（ｘｉ，ｙｊ）表１列出了对应图１ａ）～１ｃ）的生长簇的回旋半径ｒ［９］、粒子数ｎ、分形维数Ｄｆ的变化值。表１说明不同步长生长簇的粒子数和分形维数基本不变，变化的是回旋半径。由此可知，变化的步长在一定程度上会影响生长簇的局部特性，而分形维数并未受到影响。ｊ当ｈ＝τ 时，只要粒子数目确定，粒子随机向周围８个方向运动一步，ＭＤＬＡ生长基本上是一致的。当ｈ＝τ ＝１时，就是各个方向的迭代步长为１的ＭＤＬＡ（云团多粒子凝聚扩散）。４　生长形态的定向模拟，，图２表明了不同坐标方向的不同搜索步长的生长图。图２ａ）表明，当ｘ方向的搜索步长与ｙ方向的步长时相等时，粒子均匀生长。当ｘ方向的搜索步长大于ｙ方向的搜索步长时，粒子有向ｘ方向粘贴的趋势，最终长成侧向“Ｖ”字形（见图２ｂ）），而且某个方向的搜索步长越小，生长簇的分叉越小，方向步长的改变直接影响生长簇的生长形态。下面从改变生长参数入手，如方向概率、粒子初始化位置、粘贴位置、种子数目等，探讨生长簇的形态控制问题。图４模拟的是初始化位置不同，不同形态的生长簇。图中点密集地方代表初始化的粒子，稀疏且出现分叉的点代表生长簇上的点，由图４可知，生长簇的最终形态与初始化状态有关。图２　不同方向不同搜索步长Ｍ的生长图图４　不同初始化方式得到的生长图图２　不同方向不同搜索步长Ｍ的生长图图４　不同初始化方式得到的生长图图３　粒子半径不同的生长簇在ＭＤＬＡ模型中，规定粒子向其周围８个方向在ＭＤＬＡ模型中，规定粒子向其周围８个方向随机运动，假定Ｍ（ｘ），Ｍ（ｙ）分别为ｘ，ｙ坐标方向的搜索步长。搜索步长是粒子在其周围搜索是否存在生长点的一种衡量尺度，步长越大，越有利于生长。图２表明了不同坐标方向的不同搜索步长的生长图。图２ａ）表明，当ｘ方向的搜索步长与ｙ方向的步长时相等时，粒子均匀生长。当ｘ方向的搜索步长大于ｙ方向的搜索步长时，粒子有向ｘ方向粘贴的趋势，最终长成侧向“Ｖ”字形（见图２ｂ）），而且某个方向的搜索步长越小，生长簇的分叉越小，方向步长的改变直接影响生长簇的生长形态。ＭＤＬＡ（云团多粒子凝聚扩散）。当ｈ≠τ 即ｘ，ｙ方向步长不一致时，理论上是哪当ｈ≠τ 即ｘ，ｙ方向步长不一致时，理论上是哪个方向的步长大，粒子在哪个方向上运动的范围就个方向的步长大，粒子在哪个方向上运动的范围就西　北　工　业　大　学　学　报 · ８４８ · 第３７卷表１　图１中生长簇的参数变化值ｒｎＤｆ１６４．７９１４２１２１．６５５１３５．３０１８３０６１．６５７１７２．８８１６３４５１．７２０５　结　论５本文基于已有ＤＬＡ模型，提出了一个改进模型———ＭＤＬＡ模型。从改变模型参数入手，通过分析、观察、对比等手段，研究生长形态的定向控制问题，并用仿真实例表明了控制方法的有效性，为进一步研究分形生长的定向控制奠定了基础。图６　粒子粘贴位置不同的生长簇图６　粒子粘贴位置不同的生长簇图６　粒子粘贴位置不同的生长簇参考文献：３　粒子半径对生长形态的影响下面探究方向概率对生长形态的影响。在ＭＤＬＡ模型中，方向概率是指粒子在任意时刻，任意位置，往某个方向运动的可能性大小。在此模型中方向概率有８个方向，所以粒子朝向某个方向运动的概率是相等的，均为１／８，如果某个方向的方向概率为１说明粒子仅向该方向运动。图５表明了仅方向概率不同时的生长图。图５ａ）中粒子向上的生长概率为１，表示粒子只向上运动，所以粒子的粘贴主要在种子下方，形成一个倒立的“锥形”。而图５ｂ）是各方向的方向概率共同作用的结果，哪个方向的概率大，粒子朝向哪个方向运动的可能性就大，从而定性决定生长簇的大致形态。图５ａ）和图５ｂ）中的生长图方向概率不相同，分形维数Ｄｆ也不相同。３粒子半径代表着粒子大小，粒子在运动过程中可以认为是恒定不变的，默认条件下粒子半径是１，这样当粒子运动步长为１时，相当于走粒子半径大小的距离。那么当粒子运动步长不变时，粒子半径对生长簇的影响是什么呢？下面从实验角度探讨半径Ｒ的作用。当粒子半径Ｒ＝１．０，２．０，３．０，其他条件均相同时生长图如图３所示。实验结果表明，粒子半径越大，生长簇的颜色越深，生长簇看起来越密集，但生长簇上的粒子数并没有变很大，同时发现分形维数基本保持在１．７５左右。由此可以得出，粒子大小影响的是生长簇的局部性质，而对全局性质影响不大，这从分形维数的变化可以看出来。粒子半径代表着粒子大小，粒子在运动过程中可以认为是恒定不变的，默认条件下粒子半径是１，这样当粒子运动步长为１时，相当于走粒子半径大小的距离。那么当粒子运动步长不变时，粒子半径对生长簇的影响是什么呢？下面从实验角度探讨半径Ｒ的作用。当粒子半径Ｒ＝１．０，２．０，３．０，其他条件均相同时生长图如图３所示。实验结果表明，粒子半径越大，生长簇的颜色越深，生长簇看起来越密集，但生长簇上的粒子数并没有变很大，同时发现分形维数基本保持在１．７５左右。由此可以得出，粒子大小影响的是生长簇的局部性质，而对全局性质影响不大，这从分形维数的变化可以看出来。周敏，等：分形生长的形态控制与模拟第４期第４期 · ９４８ · 另外初始化种子数不同，可导致同一时刻可以有多个粒子与团簇发生碰撞，最终表现为生长形态的差异。同时由于分形的非整数性及自相似性，可以推断出其分形维数也不同。图７表明了不同的种子个数的生长簇。图５　方向概率不同得到不同维数Ｄｆ的生长图图７　不同种子数的生长簇图５　方向概率不同得到不同维数Ｄｆ的生长图粒子粘贴条件是粒子能粘贴到生长簇上的条件。首先在整个窗口中初始化所有粒子，所有粒子做随机布朗运动，生长簇上的粒子除了满足基本粘贴条件外，还应满足被预先设定的粘贴位置条件。图６表明了粒子粘贴位置不同而产生的不同的生长簇，生长簇的粒子横坐标ｘ，纵坐标ｙ满足一定的函数关系。图７　不同种子数的生长簇 ©２０１９ＪｏｕｒｎａｌｏｆＮｏｒｔｈｗｅｓｔｅｒｎＰｏｌｙｔｅｃｈｎｉｃａｌＵｎｉｖｅｒｓｉｔｙ．参考文献：［１］　ＳＡＮＤＥＲＬＭ，ＷＩＴＴＥＮＴＡ．Ｄｉｆｕｓｉｏｎ⁃ＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎ：ａＫｉｎｅｍａｔｉｃＣｒｉｔｉｃａｌＰｈｅｎｏｍｅｎｏｎ［Ｊ］．ＰｈｙｓｉｃａｌＲｅｖｉｅｗＬｅｔｔｅｒｓ，１９８１，４７：１４００⁃１４０３［１］　ＳＡＮＤＥＲＬＭ，ＷＩＴＴＥＮＴＡ．Ｄｉｆｕｓｉｏｎ⁃ＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎ：ａＫｉｎｅｍａｔｉｃＣｒｉｔｉｃａｌＰｈｅｎｏｍｅｎｏｎ［Ｊ］．ＰｈｙｓｉｃａｌＲｅｖｉｅｗＬｅｔｔｅｒｓ，１９８１，４７：１４００⁃１４０３［２］　ＢＲＡＧＡＦＬ，ＲＩＢＥＩＲＯＭＳ．ＤｉｆｆｕｓｉｏｎＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎ：ＡｌｇｏｒｉｔｈｍＯｐｔｉｍｉｚａｔｉｏｎＲｅｖｉｓｉｔｅｄ［Ｊ］．ＣｏｍｐｕｔｅｒＰｈｙｓｉｃｓＣｏｍｍｕｎｉ⁃ ｃａｔｉｏｎｓ，２０１１，１８２（８）：１６０２⁃１６０５［２］　ＢＲＡＧＡＦＬ，ＲＩＢＥＩＲＯＭＳ．ＤｉｆｆｕｓｉｏｎＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎ：ＡｌｇｏｒｉｔｈｍＯｐｔｉｍｉｚａｔｉｏｎＲｅｖｉｓｉｔｅｄ［Ｊ］．ＣｏｍｐｕｔｅｒＰｈｙｓｉｃｓＣｏｍｍｕｎｉ⁃ ｃａｔｉｏｎｓ，２０１１，１８２（８）：１６０２⁃１６０５［３］　ＫＡＳＰＥＲＲＫ，ＬＩＬＩＡＮＤＭ，ＪＲＵＵＤＶＡＮＯＭＭＥＮ，ｅｔａｌ．ＯｐｔｉｍｉｚｉｎｇＯｆｆ⁃ＬａｔｔｉｃｅＤｉｆｆｕｓｉｏｎＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎ［Ｊ］．Ｃｏｍｐｕｔ⁃ ｅｒＰｈｙｓｉｃｓＣｏｍｍｕｎｉｃａｔｉｏｎｓ，２０１４，１８５（３）：８４１⁃８４６［３］　ＫＡＳＰＥＲＲＫ，ＬＩＬＩＡＮＤＭ，ＪＲＵＵＤＶＡＮＯＭＭＥＮ，ｅｔａｌ．ＯｐｔｉｍｉｚｉｎｇＯｆｆ⁃ＬａｔｔｉｃｅＤｉｆｆｕｓｉｏｎＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎ［Ｊ］．Ｃｏｍｐｕｔ⁃ ｅｒＰｈｙｓｉｃｓＣｏｍｍｕｎｉｃａｔｉｏｎｓ，２０１４，１８５（３）：８４１⁃８４６［４］　ＢＲＡＧＡＦＬ，ＭＡＴＴＯＳＯＡ，ＡＭＯＲＩＮＶＳ，ｅｔａｌ．ＤｉｆｆｕｓｉｏｎＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎｏｆＰａｒｔｉｃｌｅｓｗｉｔｈＤｉｆｆｅｒｅｎｔＳｉｚｅｓ：ＦｒａｃｔａｌＤｉ⁃ ｍｅｎｓｉｏｎＣｈａｎｇｅｂｙＡｎｉｓｏｔｒｏｐｉｃＧｒｏｗｔｈ［Ｊ］．ＰｈｙｓｉｃａＡ，２０１５，４２９：２８⁃３４［４］　ＢＲＡＧＡＦＬ，ＭＡＴＴＯＳＯＡ，ＡＭＯＲＩＮＶＳ，ｅｔａｌ．ＤｉｆｆｕｓｉｏｎＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎｏｆＰａｒｔｉｃｌｅｓｗｉｔｈＤｉｆｆｅｒｅｎｔＳｉｚｅｓ：ＦｒａｃｔａｌＤｉ⁃ ｍｅｎｓｉｏｎＣｈａｎｇｅｂｙＡｎｉｓｏｔｒｏｐｉｃＧｒｏｗｔｈ［Ｊ］．ＰｈｙｓｉｃａＡ，２０１５，４２９：２８⁃３４［５］　ＧＨＯＳＨＡ，ＢＡＴＡＢＹＡＬＲ，ＤＡＳＧＰ，ｅｔａｌ．ＡｎＥｘｔｅｎｄｅｄＦｒａｃｔａｌＧｒｏｗｔｈＲｅｇｉｍｅｉｎｔｈｅＤｉｆｆｕｓｉｏｎＬｉｍｉｔｅｄＡｇｇｒｅｇａｔｉｏｎＩｎｃｌｕ⁃ ｄｉｎｇＥｄｇｅＤｉｆｆｕｓｉｏｎ［Ｊ］．ＡＩＰＡｄｖａｎｃｅｓ，２０１６，６（１）：１⁃２［６］　ＫＡＴＹＡＬＮ，ＢＡＮＥＲＪＥＥＶ，ＰＵＲＩＳ．ＦｒａｃｔａｌＳｉｇｎａｔｕｒｅｓｉｎＡｎａｌｏｇｓｏｆＩｎｔｅｒｐｌａｎｅｔａｒｙＤｕｓｔＰａｒｔｉｃｌｅｓ［Ｊ］．ＪｏｕｒｎａｌｏｆＱｕａｎｔｉｔａｔｉｖｅＳｐｅｃｔｒｏｓｃｏｐｙａｎｄＲａｄｉａｔｉｖｅＴｒａｎｓｆｅｒ，２０１４，１４６：２９０⁃２９４［７］　ＲＩＳＬＥＲＴ．ＦｏｃｕｓｏｎｔｈｅＰｈｙｓｉｃｓｏｆＣａｎｃｅｒ［Ｊ］．ＮｅｗＪｏｕｒｎａｌｏｆＰｈｙｓｉｃｓ，２０１５，１７（５）：１⁃１３［７］　ＲＩＳＬＥＲＴ．ＦｏｃｕｓｏｎｔｈｅＰｈｙｓｉｃｓｏｆＣａｎｃｅｒ［Ｊ］．ＮｅｗＪｏｕｒｎａｌｏｆＰｈｙｓｉｃｓ，２０１５，１７（５）：１⁃１３西　北　工　业　大　学　学　报第３７卷第３７卷 · ０５８ · ［８］　ＭＡＪＵＭＤＡＲＳ，ＴＲＵＪＩＬＬＯ⁃ＲＥＹＥＳＪ，ＨＥＲＮＡＮＤＥＺ⁃ＶＩＥＺＣＡＳＪＡ，ｅｔａｌ．ＣｅｒｉｕｍＢｉｏｍａｇｎｉｆｉｃａｔｉｏｎｉｎａＴｅｒｒｅｓｔｒｉａｌＦｏｏｄＣｈａｉｎ：ＩｎｆｌｕｅｎｃｅｏｆＰａｒｔｉｃｌｅＳｉｚｅａｎｄＧｒｏｗｔｈＳｔａｇｅ［Ｊ］．ＥｎｖｉｒｏｎｍｅｎｔａｌＳｃｉｅｎｃｅ＆Ｔｅｃｈｎｏｌｏｇｙ，２０１７，５０（１３）：６７８２⁃６７９２［９］　ＭＥＡＫＩＮＰ．Ｄｉｆｆｕｓｉｏｎ⁃ＣｏｎｔｒｏｌｌｅｄＣｌｕｓｔｅｒＦｏｒｍａｔｉｏｎｉｎ２⁃６⁃ＤｉｍｅｎｓｉｏｎａｌＳｐａｃｅ［Ｊ］．ＰｈｙｓｉｃａｌＲｅｖｉｅｗＡ，１９８３，２７（３）：１４９５⁃１５０７［８］　ＭＡＪＵＭＤＡＲＳ，ＴＲＵＪＩＬＬＯ⁃ＲＥＹＥＳＪ，ＨＥＲＮＡＮＤＥＺ⁃ＶＩＥＺＣＡＳＪＡ，ｅｔａｌ．ＣｅｒｉｕｍＢｉｏｍａｇｎｉｆｉｃａｔｉｏｎｉｎａＴｅｒｒｅｓｔｒｉａｌＦｏｏｄＣｈａｉｎ：ＩｎｆｌｕｅｎｃｅｏｆＰａｒｔｉｃｌｅＳｉｚｅａｎｄＧｒｏｗｔｈＳｔａｇｅ［Ｊ］．ＥｎｖｉｒｏｎｍｅｎｔａｌＳｃｉｅｎｃｅ＆Ｔｅｃｈｎｏｌｏｇｙ，２０１７，５０（１３）：６７８２⁃６７９２［８］　ＭＡＪＵＭＤＡＲＳ，ＴＲＵＪＩＬＬＯ⁃ＲＥＹＥＳＪ，ＨＥＲＮＡＮＤＥＺ⁃ＶＩＥＺＣＡＳＪＡ，ｅｔａｌ．ＣｅｒｉｕｍＢｉｏｍａｇｎｉｆｉｃａｔｉｏｎｉｎａＴｅｒｒｅｓｔｒｉａｌＦｏｏｄＣｈａｉｎ：ＩｎｆｌｕｅｎｃｅｏｆＰａｒｔｉｃｌｅＳｉｚｅａｎｄＧｒｏｗｔｈＳｔａｇｅ［Ｊ］．ＥｎｖｉｒｏｎｍｅｎｔａｌＳｃｉｅｎｃｅ＆Ｔｅｃｈｎｏｌｏｇｙ，２０１７，５０（１３）：６７８２⁃６７９２［９］　ＭＥＡＫＩＮＰ．Ｄｉｆｆｕｓｉｏｎ⁃ＣｏｎｔｒｏｌｌｅｄＣｌｕｓｔｅｒＦｏｒｍａｔｉｏｎｉｎ２⁃６⁃ＤｉｍｅｎｓｉｏｎａｌＳｐａｃｅ［Ｊ］．ＰｈｙｓｉｃａｌＲｅｖｉｅｗＡ，１９８３，２７（３）：［９］　ＭＥＡＫＩＮＰ．Ｄｉｆｆｕｓｉｏｎ⁃ＣｏｎｔｒｏｌｌｅｄＣｌｕｓｔｅｒＦｏｒｍａｔｉｏｎｉｎ２⁃６⁃ＤｉｍｅｎｓｉｏｎａｌＳｐａｃｅ［Ｊ］．ＰｈｙｓｉｃａｌＲｅｖｉｅｗＡ，１９８３，２７（３）：１４９５⁃１５０７ＭｏｒｐｈｏｌｏｇｉｃａｌＣｏｎｔｒｏｌａｎｄＳｉｍｕｌａｔｉｏｎｏｆＦｒａｃｔａｌＧｒｏｗｔｈＺＨＯＵＭｉｎ，ＺＨＡＮＧＨａｉｌｉ，ＷＡＮＧＹｉｈａｏ，ＢＡＯＸｉａｏｌａｎ（ＳｃｈｏｏｌｏｆＮａｔｕｒａｌａｎｄＡｐｐｌｉｅｄＳｃｉｅｎｃｅｓ，ＮｏｒｔｈｗｅｓｔｅｒｎＰｏｌｙｔｅｃｈｎｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｘｉ′ａｎ７１０１２９，Ｃｈｉｎａ）ＭｏｒｐｈｏｌｏｇｉｃａｌＣｏｎｔｒｏｌａｎｄＳｉｍｕｌａｔｉｏｎｏｆＦｒａｃｔａｌＧｒｏｗｔｈＺＨＯＵＭｉｎ，ＺＨＡＮＧＨａｉｌｉ，ＷＡＮＧＹｉｈａｏ，ＢＡＯＸｉａｏｌａｎ（ＳｃｈｏｏｌｏｆＮａｔｕｒａｌａｎｄＡｐｐｌｉｅｄＳｃｉｅｎｃｅｓ，ＮｏｒｔｈｗｅｓｔｅｒｎＰｏｌｙｔｅｃｈｎｉｃａｌＵｎｉｖｅｒｓｉｔｙ，Ｘｉ′ａｎ７１０１２９，Ｃｈｉｎａ）ＭｏｒｐｈｏｌｏｇｉｃａｌＣｏｎｔｒｏｌａｎｄＳｉｍｕｌａｔｉｏｎｏｆＦｒａｃｔａｌＧｒｏｗｔＡｂｓｔｒａｃｔ：Ｔｈｅｉｄｅａａｎｄｍｅｔｈｏｄｏｆｃｏｎｔｒｏｌａｒｅｉｎｔｒｏｄｕｃｅｄｉｎｔｏｔｈｅｆｒａｃｔａｌｔｈｅｏｒｙｆｒｏｍｔｈｅｐｅｒｓｐｅｃｔｉｖｅｏｆｑｕａｎｔｉｔａｔｉｖｅａｎａｌｙｓｉｓ．Ｉｎｏｒｄｅｒｔｏｓｐｅｅｄｕｐａｇｇｒｅｇａｔｉｏｎ，ＭＤＬＡｇｒｏｗｔｈｍｏｄｅｌｗｉｔｈｓｏｕｒｃｅｉｔｅｍｓｉｓｐｕｔｆｒｏｗａｒｄｂｙａｌｔｅｒｉｎｇｔｈｅｗａｙｏｆｐａｒｔｉｃｌｅｇｅｎｅｒａｔｉｎｇａｎｄｋｉｌｌｉｎｇｏｆｔｈｅｓｔａｎｄａｒｄＤＬＡｇｒｏｗｔｈｍｏｄｅｌ．Ｔｈｅｅｆｆｅｃｔｏｆｔｈｅｉｔｅｒａｔｉｖｅｓｔｅｐｓｉｚｅａｎｄｐａｒｔｉｃｌｅｒａｄｉｕｓｏｎｔｈｅｇｒｏｗｔｈｍｏｒｐｈｏｌｏｇｙａｒｅｄｉｓｃｕｓｓｅｄ，ａｎｄｔｈｅｇｒｏｗｔｈｍｏｒｐｈｏｌｏｇｙｗａｓｓｉｍｕｌａｔｅｄｄｉｒｅｃｔｉｏｎａｌｌｙ．Ｋｅｙｗｏｒｄｓ：ＭＤＬＡｍｏｄｅｌ；ｓｏｕｒｃｅｉｔｅｍ；ｆｒａｃｔａｌｇｒｏｗｔｈ；ｍｏｒｐｈｏｌｏｇｉｃａｌｃｏｎｔｒｏｌ ©２０１９ＪｏｕｒｎａｌｏｆＮｏｒｔｈｗｅｓｔｅｒｎＰｏｌｙｔｅｃｈｎｉｃａｌＵｎｉｖｅｒｓｉｔｙ．ＴｈｉｓｉｓａｎＯｐｅｎＡｃｃｅｓｓａｒｔｉｃｌｅｄｉｓｔｒｉｂｕｔｅｄｕｎｄｅｒｔｈｅｔｅｒｍｓｏｆｔｈｅＣｒｅａｔｉｖｅＣｏｍｍｏｎｓＡｔｔｒｉｂｕｔｉｏｎＬｉｃｅｎｓｅ（ｈｔｔｐ：／／ｃｒｅａｔｉｖｅｃｏｍｍｏｎｓ．ｏｒｇ／ｌｉｃｅｎｓｅｓ／ｂｙ／４．０），ｗｈｉｃｈｐｅｒｍｉｔｓｕｎｒｅｓｔｒｉｃｔｅｄｕｓｅ，ｄｉｓｔｒｉｂｕｔｉｏｎ，ａｎｄｒｅｐｒｏｄｕｃｔｉｏｎｉｎａｎｙｍｅｄｉｕｍ，ｐｒｏｖｉｄｅｄｔｈｅｏｒｉｇｉｎａｌｗｏｒｋｉｓｐｒｏｐｅｒｌｙｃｉｔｅｄ．
https://openalex.org/W2976125066	http://pdf.blucher.com.br/medicalproceedings/sbr2019/273.pdf	English	null	T-CELL LARGE GRANULAR LYMPHOCYTE LEUKEMIA, A RARE MANIFESTATION OF RHEUMATOID ARTHRITIS: CASE REPORT	Blucher Medical Proceedings	2,019	cc-by	495	T-CELL LARGE GRANULAR LYMPHOCYTE LEUKEMIA, A RARE MANIFESTATION OF RHEUMATOID ARTHRITIS: CASE REPORT T-CELL LARGE GRANULAR LYMPHOCYTE LEUKEMIA, A RARE MANIFESTATION OF RHEUMATOID ARTHRITIS: CASE REPORT Priscila Garcia Câmara Cabral Tavares (Hospital Universitário Walter Cantídio, Universidade Federal do Ceará, Fortaleza, CE, Brasil), Ana Paula de Menezes Cunha (Hospital Universitário Walter Cantídio, Universidade Federal do Ceará, Fortaleza, CE, Brasil), Ruy Sampaio Siqueira Neto (Hospital Universitário Walter Cantídio, Universidade Federal do Ceará, Fortaleza, CE, Brasil), André de Gois Rocha (Hospital Universitário Walter Cantídio, Universidade Federal do Ceará, Fortaleza, CE, Brasil), Leila Patrícia Fonseca Oliveira (Hospital Universitário Walter Cantídio, Universidade Federal do Ceará, Fortaleza, CE, Brasil), Cristiano Nogueira Marques (Hospital Universitário Walter Cantídio, Universidade Federal do Ceará, Fortaleza, CE, Brasil) CASE REPORT Patient, 57 years old, female, from Fortaleza-CE, with rheumatoid arthritis since 1996. Over the years, has evolved with deformities despite optimal use of disease-modifying anti-rheumatic drugs (DMARDs) and corticosteroids. Periodic examinations showed normal levels of blood cells, however, in May, 2016 was detected significant and persistent leucopenia and neutropenia, in addition to recurrent infections. The medications in use were suspended due to the suspicion of myelotoxicity and a hematological investigation was started. On October 31, 2018, after myelogram, bone marrow biopsy, immunohistochemistry and karyotype banding, was diagnosed with large granular T-cell lymphocytic leukemia, CD8 positive, ruling out myelodysplastic syndromes and drug myelotoxicity. Methotrexate (25mg/ day) was started in November 2018 and suspended in early June 2019 due lack of response. It has been decided to treat with rituximab, however is awaiting a urinary infection resolution to begin the use. BACKGROUND Rheumatoid arthritis (RA) is an inflammatory, systemic, autoimmune and chronic disease that affect joints with a destructive potential. It can also affect other organs and systems such as skin, heart, muscles, vessels. A variant of Felty's syndrome has been reported in patients with RA with neutropenia and increased numbers of large granular lymphocytes in blood and bone marrow. T-CELL LARGE GRANULAR LYMPHOCYTE LEUKEMIA, A RARE MANIFESTATION OF RHEUMATOID ARTHRITIS: CASE REPORT T-CELL LARGE GRANULAR LYMPHOCYTE LEUKEMIA, A RARE MANIFESTATION OF RHEUMATOID ARTHRITIS: CASE REPORT CONCLUSION T-Cell Large Granular Lymphocyte (LGL) Leukemia is a rare clonal disorder in which the main clinical manifestations are cytopenias (mainly neutropenia), splenomegaly, and recurrent infections. T cell LGL leukemia can be associated with RA, usually in patients with long-term disease, severe joint damage, and extra-articular manifestations. The diagnosis is based on the discovery of a monoclonal LGG expansion in the peripheral blood and also in the bone marrow with a characteristic immunophenotype (CD3+, CD4-, CD8+, CD16+, CD28- and CD57+). Generally, LGL leukemia has a chronic and indolent course, with an average survival of 10 years. The objectives of LGL leukemia therapy are the normalization of cytopenias and reduction of infectious episodes and also reduce the tumor burden to treat the underlying disease. As in RA, the treatment of choice is with methotrexate at the usual doses, with remission up to 50% of the cases; Alternatives are cyclosporin A, cyclophosphamide and rituximab.
W2337284753.txt	https://europepmc.org/articles/pmc5078010?pdf=render	en		Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides	Oncotarget	2,016	cc-by	7,654	Oncotarget, Vol. 7, No. 22 www.impactjournals.com/oncotarget/ Avoiding drug resistance through extended drug target interfaces: a case for stapled peptides Siau Jia Wei1, Sharon Chee1, Larisa Yurlova3, David Lane1, Chandra Verma2,4,5, Christopher Brown1, Farid Ghadessy1 1 P53 Laboratory (ASTAR), #06-04/05 Neuros, 138648, Singapore 2 Bioinformatics Institute (ASTAR), 07-01 Matrix, 138671, Singapore 3 ChromoTek GmbH, 82152 Planegg-Martinsried, Germany 4 School of Biological Sciences, Nanyang Technological University, 637551, Singapore 5 Department of Biological Sciences, National University of Singapore, 117543, Singapore Correspondence to: Farid Ghadessy, email: fghadessy@p53Lab.a-star.edu.sg Keywords: p53, HDM2, MDM2, stapled peptide, cancer resistance Received: November 11, 2015 Accepted: March 18, 2016 Published: April 4, 2016 ABSTRACT Cancer drugs often fail due to the emergence of clinical resistance. This can manifest through mutations in target proteins that selectively exclude drug binding whilst retaining aberrant function. A priori knowledge of resistance-inducing mutations is therefore important for both drug design and clinical surveillance. Stapled peptides represent a novel class of antagonists capable of inhibiting therapeutically relevant protein-protein interactions. Here, we address the important question of potential resistance to stapled peptide inhibitors. HDM2 is the critical negative regulator of p53, and is often overexpressed in cancers that retain wild-type p53 function. Interrogation of a large collection of randomly mutated HDM2 proteins failed to identify point mutations that could selectively abrogate binding by a stapled peptide inhibitor (PM2). In contrast, the same interrogation methodology has previously uncovered point mutations that selectively inhibit binding by Nutlin, the prototypical small molecule inhibitor of HDM2. Our results demonstrate both the high level of structural p53 mimicry employed by PM2 to engage HDM2, and the potential resilience of stapled peptide antagonists to mutations in target proteins. This inherent feature could reduce clinical resistance should this class of drugs enter the clinic. The stapled peptide PM2/sMTide-02 binds to the highly evolutionary conserved E3 ubiquitin ligase HDM2 [3, 8, 9]. PM2 inhibits HDM2 from targeting p53 for proteosomal degradation via ubiquitination [10–13]. Cell fate in response to a plethora of stress signals is directed by p53 [14, 15]. The pro-apoptotic activity of p53 is compromised in 50% of all cancers via mutation, highlighting its importance [16, 17]. In certain cancers where p53 is not mutated, its function is often mitigated through overexpression of HDM2 [18, 19]. Inhibition of HDM2 by a wide range of antagonists has been shown to lead to increased cellular levels of wild type p53 and cell death [20–23]. In normal cells, HDM2 inhibition can cause reversible arrest, highlighting a possible target for cyclotherapy regimens [24–26]. The prototypical HDM2 antagonist Nutlin and its numerous derivatives comprise a signature chemotope that recapitulates the interaction of 3 INTRODUCTION Stapled peptides are emerging as a robust class of synthetic biologics capable of selectively perturbing protein-protein interactions (PPIs) [1, 2]. The judicious introduction of a chemical tether (the “staple”) linking two amino acid side chains of a peptide can result in prestabilization of the alpha helical conformation favoured in complex formation with a target protein. Importantly, stapling can also impart the desirable drug-like properties of cell penetration, protease resistance and intracellular target engagement on otherwise biologically inert peptides [3, 4]. Stapled peptides targeting numerous intracellular targets have been described, with many employing an all-hydrocarbon olefin stapling moiety [5]. Alternative stapling chemistries have also been reported [6, 7]. www.impactjournals.com/oncotarget 32232 Oncotarget key residues in the N-terminal region of p53 (F19, W23, L26) with a hydrophobic pocket in the N-terminal domain of HDM2 [20, 27–29]. These 3 residues are absolutely required in effective peptidic antagonists including PM2 [30–32]. In vitro selection has identified point mutations in HDM2 that selectively abrogate Nutlin binding, with no loss in interaction with p53 [33]. As small molecule HDM2 inhibitors have only recently entered clinical trials [34–40], it remains to be seen whether this mechanism of drug resistance will be realized in patients with cancers that retain wild-type p53. Ex vivo studies have indicated inactivating p53 mutations and endoreduplication as principal modes of resistance to Nutlin efficacy [38, 41–43]. However, a recent in vivo study using xenograft tumours in mice showed development of resistance to the Nutlin analogue SAR405838 was associated with a point-mutated p53 that still retained activity [23, 44]. Notably, PM2 and several derivatives are able to bind and antagonize Nutlin-resistant HDM2 [45]. This is attributed to the broad, diffuse network of contacts they form with HDM2, which contrasts with the intrinsically limited number of “anchor” points employed by the comparatively small molecule Nutlin [20, 46, 47]. The engagement mode of peptidic antagonists suggests that resistance through point mutation in target proteins is less likely compared to small molecule binders. However, this has yet to be experimentally verified. Here, using the PM2-HDM2 interaction as a model system, we carried out in vitro selections to identify point mutations in the N-terminal domain of HDM2 that would selectively preclude binding of PM2 but not p53. The results show that a significant phenotype is only commensurate with co-loss of p53 binding, and therefore unlikely to occur in cancers that retain p53 function. Peptidic drugs may therefore prove robust antagonists in oncology applications, where clinical resistance is of fundamental importance to the treatment outcome [48, 49]. RESULTS HDM2 variants resistant to PM2 inhibition show reduced p53 binding To evolve PM2-resistant HDM2 we used a previously described method that enabled selection of Nutlin-resistant HDM2 variants (Figure 1) [33, 50, 51]. A library of randomly mutated genes expressing Figure 1: Selection of PM2-resistant HDM2 by in vitro compartmentalisation. 1. HDM2 expression constructs (blue and purple bars) appended with 2CONA p53 response element (“RE”, green) and HA-tag coding sequence (black) and p53 expression construct (red bar) are segregated into aqueous emulsion compartments along with PM2 (cyan helix). Protein expression occurs within compartments. PM2 inhibition of HDM2 results in no HDM2-p53-DNA complex formation (left bubble), whereas resistant HDM2 can form the complex (right bubble). 2–3. The emulsion is broken and complexes captured with anti-HA antibody. DNA encoding resistant HDM2 variants is amplified by PCR. 4. Selectants further evaluated by secondary pull-down assay or subjected to further rounds of selection. www.impactjournals.com/oncotarget 32233 Oncotarget the HDM2 N-terminal domain (with a C-terminal HA tag) and containing a p53 response element (RE) was clonally segregated into the aqueous compartments of a water in oil emulsion along with the p53-expressing gene cassette and PM2. Within each compartment, protein expression occurs, and in the absence of inhibitor, a complex forms between p53, variant HDM2 and the gene encoding the variant HDM2. In the presence of PM2, this complex does not form unless the HDM2 is mutated to exclude PM2, but not p53 binding. Upon disruption of the emulsion, persisting complexes are enriched by immunoprecipitation using magnetic beads coated with anti-HA antibody, and the genes encoding resistant HDM2 variants amplified by PCR for further rounds of selection and/or secondary assays. After 4 rounds of selection, 3 HDM2 variants (C8, C11 and C12) were identified that showed PM2 resistance as judged by pull-down assay using in vitro expressed proteins (Figure 2A). Whilst these appeared significantly resistant to PM2, with little or no reduction in their interaction with p53 in the presence of PM2 (top and second panel), this came at the cost of reduced p53 binding compared to wild type N-terminal domain, particularly for C11 and C12. All selectants showed a high mutational burden, with 9-12 mutations present in each (Figure 3). Six specific mutations were present in more than one selectant (boxed), highly indicative of positive selection. The initial library was made to include the M62A mutation shown to abrogate Nutlin binding. Whilst this mutation in isolation does not affect PM2 binding, it was introduced to bias selections as it removes a sizeable packing interface between PM2 and HDM2 [47] . However, reversion of this mutation in the C8 selectant did not alter the phenotype (Figure 2B), indicating the importance of the other mutations. The C8 selectant showed the strongest resistance phenotype, and therefore all 9 constituent mutations were next analysed as N-terminal single point mutants to assay their relative contributions (Figure 4 and Figure S1A). The mutations generally fell into two groups: a subset that was clearly resistant to PM2 binding albeit at the cost of reduced p53 binding (L34P, Y60C) and a group that retained p53 binding and showed weak resistance to PM2 (F55L, P89S, I99V). Interestingly, with the exception of I99V, all of these mutants displayed Nutlin resistance (Figure 4, panel 2). The Y67H, C77R, and V108A point mutants showed no PM2-resistance phenotype. The reduced binding of the L34P mutant may arise from the significantly reduced expression levels in the assay compared to WT N-terminal domain (Figure 4). However, reduced p53 interaction was also observed in subsequent experiments using the full-length HDM2 point mutant whose expression was comparable to WT, both in vitro and ex vivo (Figure 5, 6B). The mutations displaying the PM2-resistant phenotype behaved similarly when introduced as point mutants into full-length HDM2 and assayed for p53 interaction (Figure 5A, 5B). In the context of full-length protein, only the F55L mutant showed notable Nutlin resistance (Figure 5A). The difference in behavior towards Nutlin binding/inhibition between the N-terminal and full-length proteins possibly results from the secondary p53 interaction site in the acidic domain of HDM2 [52, 53]. We additionally analysed the K36E, Y48C and L54P point mutants derived from the C11 and C12 selectants as these mutations were shared exclusively between them. Only the L54P mutation showed an exceptionally weak resistance phenotype, and this came at the cost of reduced interaction with p53 (Figure S1B). Figure 2: Selected HDM2 variants display in vitro PM2-resistance phenotype. A. In vitro pull-down assay showing reduced inhibition by PM2 (10 µM) to binding of p53 for indicated parental HDM2 variants and WT HDM2 (residues 1-125). Note: exposure time for HDM2 inputs is 8 hours and 1 second for all other panels. B. in vitro pull-down assay showing little impact upon reversion of the M62A mutation to PM2 binding in HDM2-C8 (residues1-125). Blank indicates background p53 binding in absence of HDM2. Note: exposure time for HDM2 inputs (developed using film) is 8 hours and 10 second for all other panels (digitally acquired). www.impactjournals.com/oncotarget 32234 Oncotarget HDM2 variants show weak resistance phenotype in DKO cells activity rising to ~26% for both. In the case of HDM2-C8, initial knockdown of p53 activity was ~ 6-fold reduced, most likely a result of the reduced interaction capability of C8 with p53 (Figure 2). Importantly, addition of PM2 did not result in any significant increase in p53 activity, indicating a resistance phenotype. Similarly, Nutlin had little effect in abrogating the function of C8. Analysis of C8-derived point mutants showed that the L34P and Y60C mutations in isolation could partially recapitulate the parental phenotype (Figure 6B). Both mutants showed We next investigated the behavior of the C8 HDM2 variant ex vivo, using p53/MDM2-null DKO cells. Plasmids encoding p53, HDM2 and a p53-reporter gene were transfected into cells, and transactivation by p53 measured. HDM2 ablated p53 activity to ~ 6% of that observed in the absence of HDM2 co-transfection (Figure 6A). Addition of Nutlin or PM2 inhibited HDM2, with p53 Figure 3: Sequence alignment of selectant HDM2 clones showing PM2 resistance. Mutated residues are highlighted in red, with those present in more than one selectant boxed. The M62A mutation (green) was incorporated into the selection library. Figure 4: PM2 resistance comes at cost of reduced interaction with p53. In vitro pull-down assay showing reduced inhibition by PM2 (10 µM) to indicated point mutants (asterisk) derived from HDM2-C8 (residues 1-125). The point mutants L34P, F55L, Y60C and C77R also show reduced inhibition by Nutlin (10μM). Blank indicates background p53 binding in absence of HDM2. Note: exposure time for HDM2 inputs is 8 hours (developed using film) and 3 minutes for all other panels (digitally acquired). www.impactjournals.com/oncotarget 32235 Oncotarget No significant resistance observed in BHK cells using the fluorescent two-hybrid assay significantly decreased knockdown of p53 activity (~7 fold compared to WT HDM2), again most likely due the reduced interaction observed in vitro (Figures 4, 5), with no rescue observed after addition of either PM2 or Nutlin. The F55L and I99V point mutants displayed a weak resistance phenotype in this assay. Both reduced knockdown of p53 activity (~1.5-fold compared to WT HDM2) and whilst addition of PM2 rescued activity, the magnitude of this was consistently less for both mutants compared to WT HDM2 (~3.2 versus ~ 4-fold)(Figures 6B, 6C). The P89S mutant essentially behaved like wildtype HDM2. Interaction of HDM2-C8 and the L34P, F55L, Y60C point mutants with p53 was further studied using the fluorescent two-hybrid assay (F2H) [54]. This assay facilitates real-time detection of p53-HDM2 interaction in living cells and perturbation by small molecule/peptidic antagonists. It requires expression of the p53 N-terminal domain (residues 1-81) as a fusion with GFP and HDM2 (residues 7-134) as a fusion with RFP in transgenic F2HBHK mammalian cells. As observed for recombinant expression in E.Coli, only the HDM2-F55L mutant could be stably expressed in this system, and the results indicated no significant difference compared to WT for inhibition by both PM2 and Nutlin (Figure S2). The weak difference observed in pull-down, reporter and FP assays is therefore likely to be below the detection threshold of the F2H assay or to be less profound in an intracellular environment. Collectively, these results confirm what is essentially a weak phenotype for the F55L mutation in mammalian cells. The HDM2-F55L variant shows reduced relative binding affinity to PM2 We next used the fluorescence polarisation (FP) assay to measure relative binding affinity of PM2 to recombinantly expressed HDM2 (1-125) and relevant mutants. Only the F55L mutant could be stably expressed in E. coli and purified, and this showed a slightly reduced relative binding affinity to PM2 compared to WT HDM2 (117 ± 30 nM and 53 ± 9 nM respectively), consistent with the in vitro pull-down assays and cell-based reporter assay. Using this assay, binding to the p53 peptide (amino acids 16 to 29 ) was also slightly comprised for F55L compared to WT HDM2 ( 7 ± 1.2 μM and 3 ± 0.7 μM respectively), in this case consistent with the slightly reduced activity of HDM2 F55L on p53 function compared to WT (Figure 6C). DISCUSSION The comparatively larger interaction footprint of a stapled peptide antagonist should impart broad resistance to point mutations. To test this hypothesis, we carried out a high-throughput selection for mutations in the N-terminal domain of HDM2 that inhibit stapled peptide, but not p53 Figure 5: PM2 resistance is also seen when point mutants are introduced into full-length HDM2. A. In vitro pull-down assay showing reduced inhibition by PM2 (10 µM) to indicated C8-derived point mutants (asterisk) present in full-length HDM2 The point mutant F55L also shows reduced inhibition by Nutlin (10μM). Blank indicates background p53 binding in absence of HDM2. B. As in A, additionally showing levels of wild type and indicated HDM2 variants co-eluted off beads after pull-down following mock (panel 3) and PM2 treatment (panel 4). Note exposure time for p53 pull-down in absence of treatment (panel 1) is 5s and 10 minutes for pull-down after PM2 treatment (panel 2, developed using film). Exposure time for HDM2 input and HDM2 (+ indicated variants) eluted off beads after pull-down is 30 seconds (digitally acquired). www.impactjournals.com/oncotarget 32236 Oncotarget binding. The results indicate that whilst PM2-binding can be abrogated by mutation in HDM2, this generally comes at the cost of significantly reduced p53 binding, and hence would be unlikely to occur in cancers where p53 is not frequently inactivated. Note that the residual p53 binding of these mutants is sufficient to withstand the selection conditions employed. This binding may partially result from a secondary interaction site in the C-terminal domain of p53 that interacts with the HMD2 N-terminal domain [55]. The crystal structure of the stapled peptide M06 bound to HDM2-M62A was recently described [47]. As MO6 differs from PM2 by a single amino acid (Figure 7), we have used this structure to further understand the mutations present in HDM2-C8. The mutations L34P and Y60C result in significantly decreased interaction with p53 and resistance to PM2 inhibition (Figures 4, 5, 6B). The side chains of L34 and Y60 pack against numerous hydrophobic residues contributing to the p53-binding hydrophobic cleft (Figure 8). Mutations to less bulky residues (P and C respectively) likely result in altered structural dynamics/conformation of the cleft, causing gross destabilization that inhibits both p53 and PM2 binding. Poor expression yields of proteins containing these mutations (both in vitro and ex vivo) support this notion. A very similar phenotype was observed for the L82P mutation in HDM2 that makes it resistant to Nutlin binding at the cost of reduced p53 binding [33]. The mutations F55L and I99V resulted in a weak PM2 resistance phenotype with p53 binding only slightly impaired (Figures 4-6). F55 resides in the α2 helix (residues 50-65) and its sidechain projects into solution. A. Figure 6: PM2 shows reduced inhibition of selected HDM2 variants in p53/MDM2-null DKO cells. A. Wild-type and HDM2-C8 (full-length) were co-transfected with p53 and p53-reporter gene, and reporter gene activity measured in the presence of PM2 (20 µM) or Nutlin (10 μM). p53 activity is denoted as percentage of that observed when p53-alone co-transfected with reporter gene. Shown below are Western blots indicating expression levels of HDM2 variants and p53 cotransfected into DKO cells. B and C. As in ‘A’, with wild-type HDM2 and indicated HDM2-C8 derived point mutants (full length) co-transfected into DKO cells. p53 activity is denoted as percentage of that observed when p53-alone co-transfected with reporter gene. Shown below are Western blots indicating expression levels of HDM2 variants and p53 cotransfected into DKO cells. (Continued) www.impactjournals.com/oncotarget 32237 Oncotarget B. C. Figure 6 (Continued): B and C. As in ‘A’, with wild-type HDM2 and indicated HDM2-C8 derived point mutants (full length) co-transfected into DKO cells. p53 activity is denoted as percentage of that observed when p53-alone co-transfected with reporter gene. Shown below are Western blots indicating expression levels of HDM2 variants and p53 cotransfected into DKO cells. www.impactjournals.com/oncotarget 32238 Oncotarget When bound to HDM2, the hydrophobic staple moiety of MO6 packs against F55 (Figure 9, left panel). The same interaction is observed in the structure of the SAH-8 stapled peptide bound to HDM2 [56]. As the hydrocarbon staple is the major differentiating factor between p53 and stapled peptide, mutations would be expected to arise that discriminate against it. Mutation to leucine could therefore result in less optimal packing of the staple against the α2 helix. To further test this, we mutated F55 to the less bulky/hydrophobic alanine and carried out in vitro pull down assays. Surprisingly, the results showed this mutation to make little difference towards interaction with p53 or PM2 (Figure S3). In the structure of M06 bound to HDM2-M62A the staple re-orientates itself to overcome loss of a favourable packing interaction with M62. In light of this remarkable plasticity, it is plausible that it can re-orientate to overcome loss of the favourable F55 interaction when mutated to alanine. Mutation to the slightly less bulky, but still hydrophobic leucine may not warrant conformation changes in the staple, instead resulting in slightly reduced packing interactions and the weak resistance phenotype observed. Interaction of p53 with the HDM2 N-terminal domain is mediated by three signature residues in p53 (F19, F23, L26) that interact with discrete pockets in the HDM2 binding cleft [27]. I99 resides in the α2’ helix (residues 95-104) that forms one side of the pocket that accommodates L26 of p53. The side chain of I99 is in close proximity to L26 whose interaction is favoured through hydrophobic interactions. An overlay of the MO6 Figure 7: Stapled peptide recapitulates key p53 signature residues that interact with HDM2 N-terminal domain. Overlay of p53 peptide (green) and MO6 stapled peptide (cyan, with staple moiety in grey) when bound to HDM2 N-terminal domains. The relative configurations of the key F19 and W23 residues are conserved, with some deviation in the orientation of L26. Adapted from 1YCR and 4UMN. Shown below is alignment of p53 peptide, PM2 and MO6, with signature residues shaded and residue differing between PM2 and MO6 highlighted in red. www.impactjournals.com/oncotarget 32239 Oncotarget Figure 8: Projection of HDM2-C8 mutants onto HDM2 N-terminal domain structure. Shown is structure of M06 stapled peptide (cyan, grey) bound to HDM2-M62A N-terminal domain (pink). The residues contributing to the resistance phenotype are coloured yellow, and the rest are purple. Adapted from 4UMN. Figure 9: The staple moiety makes favourable contacts with F55 in the N-terminal domain of HDM2. Left: Overlay of p53 peptide (green) and MO6 stapled peptide (cyan, staple in gray) bound to HDM2 N-terminal domain (magenta, surface representation). The positions of the F55 and I99 residues are indicated in yellow. Right: Same as left, highlighting the relative orientation of the p53 peptide and MO6 stapled peptide L26 side chains in respect to I99 in HDM2. www.impactjournals.com/oncotarget 32240 Oncotarget Primers and the p53 peptides bound to HDM2 shows a relatively extended conformation of the alpha helix in the stapled peptide. This significantly alters the C-alpha position of L26, resulting in an altered side chain projection that is more proximal to I99 (Figure 9; right panel). Upon mutation to valine, the extended strand in p53 containing L26 can more readily adjust its packing arrangements to accommodate this mutation and maintain complementarity with the HDM2 protein surface. The stapled peptide is however more rigid, and has less conformational space open to it. If it were to re-orientate its binding pose to form better contacts with the valine it would likely disrupt favourable contacts elsewhere. Notably, mutation to the smaller alanine residue (I99A) did not lead to any further discrimination, resulting in drastic loss of p53 binding (Figure S4), highlighting the inability of p53 (and most likely PM2) to adjust binding pose to compensate for the loss of a critical hydrophobic interaction. Mutations outside of the N-terminal domain of HDM2 have been shown to allosterically modulate its binding properties [33, 57]. In this study we focused on the N-terminal domain with the aim of exploring as much mutational diversity as possible within the technical confines of the in vitro selection platform employed (~ 1010 variants) [51]. It should be noted that practical limitations do not allow for interrogation of all possible mutational diversity, and that mutations conferring the desired phenotype may have been missed. However, a previous selection using the same methodology readily identified point mutations in the N-terminal domain that selectively abrogated binding by the small molecule Nutlin [33]. In light of both this observation and the exceptional structural mimicry of PM2, it appears highly unlikely that mutation in HDM2 can selectively disrupt PM2 binding. Further studies will confirm whether this observation can be extended to other peptide-protein interactions. Synthetic peptidic ligands are being developed against a range of targets for therapeutic use. The results presented here suggest that this emerging class of drug will enable robust antagonism that is poorly ablated through mutation of the target protein. This is potentially of great relevance to the field of oncology, where clinical resistance poses significant barriers to treatment efficacy. Hdm2-Nter-noHA-R: 5'TACTACCAAGT TCCTGTAGAT -3' HA-F: 5'- TACCCATACGATGTTCCAGATT ACGCTTAA -3' HDMM62A-1: 5’-CTTGGCCAGTATATTGCGAC TAAACGATTATATG-3’ HDMM62A-2: 5’-CATATAATCGTTTAGTCGCAA TATACTGGCCAAG-3’ petF2: 5'- CATCGGTGATGTCGGCGAT -3' petR: 5'- CGGATATAGTTCCTCCTTTCAGCA -3' Hdm2-NdeI: 5’- CACAACATATGTGCAATACCA ACATGTCTGTACC -3’ HA-rev-BamHI: 5'- GCTCTGGATCCTTAAGCGT AATCTGGAACATCGTATGGGTA -3' infus-Mdm2-F: 5'- AAGGAGATATACATATGTGC AATACCAACATG -3' Infus-M62AC8-Nter-R: 5'- TACTGCCAAGTT CCTGTAGATCATGGT -3' M62AC8Hdm2-Nter-F: 5'- ACCATGATCTACAG GAACTTGGCAGTAGTCAATCAGCAGGAATCATC GG -3' petATG-R: 5’-CATATGTATATCTCCTTCTTAAA GTTAAAC-3’ M62AC8-revertQC1: 5'- CTTGGCCAGTGTATTA TGACTAAACGATTACA -3' M62AC8-revertQC2: 5'- TGTAATCGTTTAGTCA TAATACACTGGCCAAG -3' petF3: 5'- ATAGGCGCCAGCAACCGCACCTG -3' mdm2-L34P-QC1: 5'- GGTTAGACCAAAGCCAT TGCCTTTGAAGTTATTAAAGTCTGTTGGTGC -3' mdm2-L34P-QC2: 5'- GCACCAACAGACTTTAAT AACTTCAAAGGCAATGGCTTTGGTCTAACC-3' mdm2-F55L-QC1: 5'- CCTATACTATGAAAGAGG TTCTTCTTTATCTTGGCCAGT -3' mdm2-F55L-QC2: 5'- ACTGGCCAAGATAAAGA AGAACCTCTTTCATAGTATAGG -3' mdm2-Y67H-QC1: 5'- ATGACTAAACGATTACA TGATGAGAAGCAACAACATATTG -3' mdm2-Y67H-QC2: 5'- CAATATGTTGTTGCTTCT CATCATGTAATCGTTTAGTCAT -3' mdm2-C77R-QC1: 5'CAACATATTGTATATcGTTCAAATGATCTTC -3' mdm2-C77R-QC2: 5'- GAAGATCATTTGAACg ATATACAATATGTTG -3' mdm2-L85S-QC1: 5'- GATCTTCTAGGAGATTcG TTTGGCGTGCCAAGC -3' mdm2-L85S-QC2: 5'- GCTTGGCACGCCAAACg AATCTCCTAGAAGATC -3' mdm2-P89S-QC1: 5'- GATTTGTTTGGCGTGtCA AGCTTCTCTGTGAAAGAGC -3' mdm2-P89S-QC2: 5'- GCTCTTTCACAGAGAAG CTTGaCACGCCAAACAAATC -3' mdm2-I99V-QC1: 5'- GCTTCTCTGTGAAAGAGC ACAGGAAAgTATATACCATGATCTACAGG -3' MATERIALS AND METHODS Materials Unless otherwise specified, all oligonucleotides used in this work were from Integrated DNA Technologies, restriction enzymes from NEB and chemical reagents from Sigma. Nutlin was from Calbiochem. Anti-HA and actin antibodies (mouse monoclonal) were purchased from Sigma. Anti-p53 antibody conjugated to horseradish peroxidase (DO1-HRP, mouse monoclonal) was purchased from Santa Cruz. www.impactjournals.com/oncotarget 32241 Oncotarget mdm2-I99V-QC2: 5'- CCTGTAGATCATGGTATAT AcTTTCCTGTGCTCTTTCACAGAGAAGC-3' mdm2-V108A-QC1: 5'- CCATGATCTACAGGAA CTTGGcAGTATACCCATACG -3' mdm2-V108A-QC2: 5'- CGTATGGGTATACTgCC AAGTTCCTGTAGATCATGG -3' mdm2-I99A-QC1: 5'- GAAAGAGCACAGGAAA gcATATACCATGATCTA -3' mdm2-I99A-QC2: 5'- TAGATCATGGTATATgcTT TCCTGTGCTCTTTC-3' mdm2-F55A-QC1: 5'- ATGAAAGAGGTTCTTgcT TATCTTGGCCAGTA -3' mdm2-F55A-QC2: 5'- TACTGGCCAAGATAAgcA AGAACCTCTTTCAT -3' Hdm2-L54P-QC1: 5’-CTATGAAAGAGGTTCcTT TTTATCTTGGCCA-3’ Hdm2-L54P-QC2: 5’-TGGCCAAGATAAAAAgG AACCTCTTTCATAG-3’ Hdm2-Y48C-QC1:5’-CACAAAAAGACACTTgT ACTATGAAAGAGGT-3’ Hdm2-Y48C-QC2: 5’-ACCTCTTTCATAGTAcAA GTGTCTTTTTGTG-3’ Hdm2-K36E-QC1: 5’-AAGCCATTGCTTTTGgAG TTATTAAAGTCTG-3’ Hdm2-K36E-QC2:5’-CAGACTTTAATAACTcCA AAAGCAATGGCTT-3’ NterHDM2 M62AC8 PET22b via mutagenesis with M62AC8-revertQC1 and M62AC8-revertQC2. Single mutant HDM2 clones were also generated by Quikchange mutagenesis of 2ConA HDM2 PET22b or 2ConA NterHDM2 PETb using appropriate primer pairs. The same primers were used to introduce mutations into the parental pCMV-HDM2 mammalian expression construct. In vitro selection of HDM2 variants resistant to PM2 IVT reactions consisting of 0.5μM ZnCl2, 10μM PM2, p53 (10ng in preselection round, 10ng in round 1, 4ng in round 2, 2ng in rounds 3/4), library amplicons (5ng in preselection round, 5ng in round 1, 2ng in round 2, 1ng in round 3/4) in a total volume of 50µL PURExpress® in vitro protein synthesis solution (New England Biolabs) were assembled on ice and emulsified as previously described [50, 51]. After incubation at 37°C, the reactions were centrifuged at 8000rpm for 10mins to separate the aqueous and oil phase. The oil phase was removed and 50uL PBS was added to the pellet of aqueous phase compartments. The compartments were disrupted by four rounds of hexane extraction and the aqueous phase incubated with anti-HA antibody-coated protein G beads (Invitrogen) at 4°C with rotation. During round 4, PM2 (1µM) was added during this step to increase selection stringency. The beads were washed thrice with PBST-0.1%BSA, and thrice with PBS. The beads were resuspended in 20µl water and the protein-protein-DNA complexes eluted by incubation at 95°C for 5mins. The eluates were amplified with petF3 and HArevBamHI during a primary amplification and with Hdm2-NdeI and HArevBamHI during a secondary amplification. The products were cloned back into 2ConA-PET22b via Nde1/ BamHI sites and re-amplified with petF2 and petR for the next round of selection. Vector and HDM2 library construction 2ConA NterHDM2 PET22b was created via inverse PCR with primers Hdm2-Nter-noHA-R and HA-F on 2ConA HDM2 PET22b. Primers HDMM62A-1 and HDMM62A-2 were used in QuikChange mutagenesis on 2ConA NterHDM2 PET22b to create 2ConA NterHdm2M62A PET22b. All of the above mentioned constructs additionally encode a C-terminal HA tag. Error-prone PCR was carried out on both 2ConA NterHDM2 PET22b and 2ConA NterHDM2-M62A PET22b using petF2 and petR and the mutant genes re-amplified with Hdm2-NdeI and HArevBamHI. The libraries were then ligated into 2ConA PET22b via Nde1/ BamHI sites and re-amplified with petF2 and petR to produce library amplicons with T7 promoter and ribosome binding site required for in vitro transcription/translation (IVT), as well as the 2ConA RE site located before the T7 promoter site. p53-PET22b was also amplified with petF2 and petR for IVT of wild-type 53. PM2 resistant parental clones obtained from the selection were amplified with petF2 and petR to create amplicons for secondary assays. M62AC8 parental clone was amplified with infus-Mdm2-F and InfusM62AC8-Nter-R for cloning by infusion into 2ConA HDM2 PET22b that was amplified with M62AC8Hdm2Nter-F and petATG-R to create full length 2ConA HDM2 M62AC8 PET22b. The mutation M62A was also removed from 2ConA HDM2 M62AC8 PET22b or 2ConA www.impactjournals.com/oncotarget Secondary co-immunoprecipitation assay and western blot analysis Protein G beads were incubated with anti-HA (1µg per 10µL beads) for 1 hour in PBST-3%BSA and subsequently washed thrice in PBST-0.1%BSA. IVTexpressed protein was incubated with the beads on a rotator for 30 mins. PM2 was added at 100µM and incubation carried out for 30 mins. IVT-containing secondary protein was added to the mixture and incubation allowed for 1 hour. Beads were finally washed thrice in PBST-0.1%BSA and thrice with PBS, and bound proteins eluted by resuspension in 20µL SDS-PAGE loading buffer and incubation at 95°C for 5 minutes. Where required, blank IVT extract (no template DNA added) was used as control. The eluates were subjected to electrophoresis, transferred to nitrocellulose membranes and probed for p53 with DO1-HRP or for HDM2 with anti-HA antibody 32242 Oncotarget followed by rabbit anti-mouse (Dakocytomation). Image acquisition was carried out using either film or digitally (Odyssey FC, Li-Cor). Un-cropped blot images are shown in Figure S5. cleaved protein eluted off the column with wash buffer. Dialysis into buffer A solution (20 mM Bis-Tris, pH 6.5, 1 mM DTT) using HiPrep 26/10 Desalting column was performed and the protein sample was subsequently loaded onto a cation-exchange Resource S 1 mL column (GE Healthcare) pre-equilibrated in buffer A. Six column volumes of buffer A was used to wash the column and the bound protein was eluted with a linear gradient in buffer comprising 1 M NaCl, 20 mM Bis-Tris pH 6.5, and 1 mM DTT over thirty column volumes. Protein purity as assessed by SDS-PAGE was ~95%, and the proteins were concentrated using Amicon-Ultra (3 kDa MWCO) concentrator (Millipore). Cell culture and reporter assay Mouse embryonic fibroblast p53/Mdm2 doubleknockout (DKO) cells (a kind gift from Guillermina Lozano) [58] were maintained in Dulbecco's modified Eagle's medium (DMEM) with 10% (v/v) foetal calf serum (FCS) and 1% (v/v) penicillin/streptomycin. The cells were seeded at 1.0 × 105 cells/well in 6-well plates, 24 hours prior to transfection. Cells were co-transfected with parental or individual PM2-resistant HDM2 plasmid, p53-pcDNA plasmid, LacZ reporter plasmid and luciferase transfection efficiency plasmid using TurboFect transfection reagent (Thermo Scientific) according to the manufacturer’s instructions. Nutlin and PM2 were added to selected wells at required concentrations 4.5 hours posttransfection. In all cases, the total amount of plasmid DNA transfected per well was equilibrated by addition of the parental vector pcDNA3.1a (+). Fluorescence polarization (FP) assay Fluorescence anisotropy assays were performed as previously described [3]. Titrations of purified wildtype and mutant HDM2 (1–125) were incubated with 50 nM of carboxyfluorescein (FAM) labelled 12-1 peptide (FAM-RFMDYWEGL-NH2) to determine the dissociation constants for the peptide-protein interaction. Apparent Kds of PM2 and human p53 peptide (AcQETFS DLWKLLPEN–NH2) were then determined by competitive fluorescence anisotropy. Titrations of PM2 and human p53 peptide were carried out with a constant concentration of wild-type HDM2 at 150 nM, mutant HDM2-F55L at 200 nM and the labelled peptide at 50 nM. Anisotropy measurements were carried out using the Envision Multilabel Reader (PerkinElmer). All experiments were carried out in PBS (2.7 mM KCl, 137 mM NaCl, 10 mM Na2HPO4 and 2 mM KH2PO4, pH 7.4), 3% DMSO and 0.1% Tween-20 buffer. All titrations were carried out in duplicate (n = 3 to 9 independent titrations). Curve fitting was carried out using Prism 4.0 (GraphPad). β-Galactosidase assay and western blot analysis DKO cells were harvested 24hours post transfection and β-galactosidase activities were assessed using the Dual-light System (Applied Biosystems) according to the manufacturer's protocol. β-galactosidase activity was normalized with luciferase activity for each sample. To check for expression levels of relevant proteins via western blot, 5 µg of the cell lysates were probed for p53 with horseradish peroxidase conjugated DO1 antibody, for HDM2 and actin with anti-HA antibody and AC15 antibody respectively followed by rabbit anti-mouse. F2H co-localization assay Protein expression and purification Plasmids encoding the GFP-tagged bait p53 (amino acids 1-81) fusion protein and different RFP-tagged prey HDM2 (amino acids 7-134) fusion proteins were co-transfected into transgenic F2H-BHK cells (F2HKit Basic, ChromoTek GmbH) [59] in 96 multiwell plates (µClear Greiner Bio-One, Germany) using the Lipofectamine 2000 (Life Technologies) reverse transfection protocol according to manufacturer’s instructions with 0.2 µg DNA and 0.4 µl Lipofectamine 2000 per well. 16 hours after transfection, cells were treated with Nutlin (0-10 µM) or PM2 (0-50 μM) for 6-8 hours in serum-free DMEM at 37°C, 5% CO2. Interaction (%) was determined as the ratio of cells showing colocalization of fluorescent signals at the nuclear spot to the total number of evaluated cells. The INCell Analyzer 1000 with a 20X objective (GE Healthcare) was used for automated image acquisition. Automated image segmentation and analysis was performed with the HDM2 (amino acids 1–125) was cloned as a GSTfusion protein using the pGEX-6P-1 GST expression vector (GE Healthcare). QuikChange site directed mutagenesis (Stratagene) was used to create the mutant HDM2-F55L (amino acids 1–125). The constructs were then transformed into Escherichia coli BL21(DE3) pLysS (Invitrogen) competent cells. Cells were grown in LB medium at 37°C and induced at OD600 nm of 0.6 with 0.5 mM IPTG at 16°C. The cells were harvested by centrifugation after overnight induction, resuspended in binding buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl), and lysed by sonication. The lysate was centrifuged for 60 mins at 19,000xg at 4°C and applied to a 5 mL GSTrap FF column (GE Healthcare) pre-equilibrated in wash buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM DTT). On-column cleavage by PreScission protease (GE Healthcare) was carried out overnight at 4°C and the www.impactjournals.com/oncotarget 32243 Oncotarget corresponding INCell Workstation 3.6 software. At least 100 co-transfected cells were analyzed per well. Titrations were carried out independently (n=2). 5. Kim YW, Grossmann TN and Verdine GL. Synthesis of allhydrocarbon stapled alpha-helical peptides by ring-closing olefin metathesis. Nat Protoc. 2011; 6:761-771. 6. Lau YH, de Andrade P, McKenzie GJ, Venkitaraman AR and Spring DR. Linear aliphatic dialkynes as alternative linkers for double-click stapling of p53-derived peptides. Chembiochem. 2014; 15:2680-2683. Molecular modelling To model the interactions of the N terminal domain of human HDM2 with the peptides (p53/stapled PM2) and Nutlin, the crystal structures of the HDM2-p53 complex [60] (PDB code 1YCR, resolved at 2.6Å), the crystal structure of the stapled peptide M06 bound to HDM2M62A (PDB code 4UMN, resolved at 1.99 Å) [47] and the HDM2-Nutlin complex [61] (PDB code 1RV1, resolved at 2.3Å) were used. 7. Muppidi A, Doi K, Ramil CP, Wang HG and Lin Q. Synthesis of cell-permeable stapled BH3 peptide-based Mcl-1 inhibitors containing simple aryl and vinylaryl crosslinkers. Tetrahedron. 2014; 70:7740-7745. 8. Lane DP, Cheok CF, Brown C, Madhumalar A, Ghadessy FJ and Verma C. Mdm2 and p53 are highly conserved from placozoans to man. Cell Cycle. 2010; 9:540-547. 9. Lane DP, Cheok CF, Brown CJ, Madhumalar A, Ghadessy FJ and Verma C. The Mdm2 and p53 genes are conserved in the Arachnids. Cell Cycle. 2010; 9:748-754. ACKNOWLEDGMENTS We thank Jacqueline Bogner for excellent technical assistance in F2H assay. 10. Honda R, Tanaka H and Yasuda H. Oncoprotein MDM2 is a ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett. 1997; 420:25-27. CONFLICTS OF INTEREST 11. Haupt Y, Maya R, Kazaz A and Oren M. Mdm2 promotes the rapid degradation of p53. Nature. 1997; 387:296-299. 12. Kubbutat MH, Jones SN and Vousden KH. Regulation of p53 stability by Mdm2. Nature. 1997; 387:299-303. The authors declare no conflicts of interest. 13. Momand J, Zambetti GP, Olson DC, George D and Levine AJ. The mdm-2 oncogene product forms a complex with the p53 protein and inhibits p53-mediated transactivation. Cell. 1992; 69:1237-1245. FUNDING This work was funded by the Agency for Science, Technology and Research, Singapore. 14. Kastan MB, Onyekwere O, Sidransky D, Vogelstein B and Craig RW. Participation of p53 protein in the cellular response to DNA damage. Cancer Res. 1991; 51:6304-6311. REFERENCES 15. 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https://openalex.org/W3010812628	https://upcommons.upc.edu/bitstream/2117/337020/1/TPC1de1.pdf	es		Mesógenos alquiloxicianobifenilos : aspectos teóricos y experimentales en las transiciones de fase SmA-N y N-I	null	2,023	cc-by	209	Anexo 1 - J.Phys. Chem. B. l09. 16284-16289 Anexo 1 "Liquid Cristal Binary Mixtures of 8OCB+10OCB. Evidence for a Smectic A-to-Nematic Tricritical Point", J. Phys. Chem. B, 109, 16284-16289 (2005) Atencion¡¡ Las páginas 248 a 254 de la tesis, que contienen el artículo citado deben consultarse en la página web del editor: https://pubs.acs.org/doi/10.1021/jp051957x Anexo 2 - J. Phys. Chem. B. HO. 26194-26203 Anexo 2 "Re-entrant Nematic Behavior in the 7OCB+9OCB Mixtures: Evidence for Multuple Nematic-Smectic Tricritical Points", J. Phys. Chem. B, 110, 26194-26203 (2006) Atencion¡¡ Las páginas 256 a 266 de la tesis, que contienen el artículo citado deben consultarse en la página web del editor: https://pubs.acs.org/doi/10.1021/jp0642286 Anexo 3 - J. Phys. Chem. 8.111. 8974-8984 Anexo 3 "Critical Behavior and Scaling Relationships at the SmAct-N and N-1 Transitions in Nonyloxycyanobiphenyl (9OCB)", J. Phys. Chem. B, 111, 8974-8984 (2007) Atencion¡¡ Las páginas 268 a 280 de la tesis, que contienen el artículo citado deben consultarse en la página web del editor: https://pubs.acs.org/doi/abs/10.1021/jp070689v Anexo 4 - Liquid Crystals.35, 685-703 (2008) Anexo 4 "Overall analysis of first-order phase transitions in some alkyloxycyanobiphenyl liquid crystals", Liquid Crystals, 35, 695-703 (2008) Atencion¡¡ Las páginas 282 a 292 de la tesis, que contienen el artículo citado deben consultarse en la página web del editor: https://www.tandfonline.com/doi/full/10.1080/02678290802120273
https://openalex.org/W4309246739	https://ijgc.bmj.com/content/ijgc/32/12/1568.full.pdf	English	null	Diagnostic accuracy of mutational analysis along the Müllerian tract to detect ovarian cancer	International journal of gynecological cancer	2,022	cc-by	6,044	Original research Original research Original research on October 23, 2024 by http://ijgc.bmj.com/ Int J Gynecol Cancer: first published as 10.1136/ijgc-2022-003911 on 16 November 2022. Downloaded from Diagnostic accuracy of mutational analysis along the Müllerian tract to detect ovarian cancer on October 23, 2024 by guest. Protected by copyright. http://ijgc.bmj.com/ ancer: first published as 10.1136/ijgc-2022-003911 on 16 November 2022. Downloaded from Majke H.D. van Bommel ‍ ‍ ,1 Johanna M.A. Pijnenborg,1 Louis J M van der Putten,1 Johan Bulten,2 Marc P.L.M. Snijders,3 Heidi V.N. Küsters-Vandevelde ‍ ‍ ,4 Sanne Sweegers,2 M. Caroline Vos,5 Marjolein J.L. Ligtenberg,2,6 Astrid Eijkelenboom,2 Joanne A de Hullu,1 Casper Reijnen7 WHAT THIS STUDY ADDS ⇒We found that diagnostic accuracy was low for cer- vicovaginal self-samples, Pap smears, and endo- metrial biopsies. Thus, the samples assessed in this way cannot be used for early detection of ovarian cancer. ⇒We found that diagnostic accuracy was low for cer- vicovaginal self-samples, Pap smears, and endo- metrial biopsies. Thus, the samples assessed in this way cannot be used for early detection of ovarian cancer. WHAT IS ALREADY KNOWN ON THIS TOPIC ►Additional supplemental material is published online only. To view, please visit the journal online (http://dx.doi.org/ 10.1136/ijgc-2022-003911). Objective Ovarian cancer is known for its poor prognosis, which is mainly due to the lack of early symptoms and adequate screening options. In this study we evaluated whether mutational analysis in cervicovaginal and endometrial samples could assist in the detection of ovarian cancer. ⇒DNA variants related to ovarian cancer can be de- tected along the Müllerian tract. In this study we compared DNA pathogenic variants in cervicovag- inal self-samples, Papanicolaou (Pap) smears, and pipelle endometrial biopsies with the pathogenic variants in the surgical specimen in patients with ovarian cancer and control patients. We assessed diagnostic accuracy of detecting ovarian cancer with those samples. ⇒DNA variants related to ovarian cancer can be de- tected along the Müllerian tract. In this study we compared DNA pathogenic variants in cervicovag- inal self-samples, Papanicolaou (Pap) smears, and pipelle endometrial biopsies with the pathogenic variants in the surgical specimen in patients with ovarian cancer and control patients. We assessed diagnostic accuracy of detecting ovarian cancer with those samples. For numbered affiliations see end of article. Methods In this prospective multicenter study, we included patients surgically treated for either (suspicion of) ovarian cancer or for a benign gynecological condition (control group). A cervicovaginal self-sample, a Papanicolaou (Pap) smear, a pipelle endometrial biopsy, and the surgical specimen were analyzed for (potentially) pathogenic variants in eight genes (ARID1A, CTNNB1, KRAS, MTOR, PIK3CA, POLE, PTEN, and TP53) using single- molecule molecular inversion probes. Sensitivity and specificity were calculated to assess diagnostic accuracy. Results Based on surgical histology, our dataset comprised 29 patients with ovarian cancer and 32 controls. In 83% of the patients with ovarian cancer, somatic (potentially) pathogenic variants could be detected in the final surgical specimen, of which 71% included at least a TP53 variant. In 52% of the ovarian cancer patients, such variants could be detected in either the self-sample, Pap smear, or pipelle. The Pap smear yielded the highest diagnostic accuracy with 26% sensitivity (95% CI 10% to 48%). Overall diagnostic accuracy was low and was not improved when including TP53 variants only. Correspondence to Majke H.D. van Bommel, Obstetrics & Gynaecology, Radboudumc, Nijmegen, The Netherlands; majke. vanbommel@radboudumc.nl Correspondence to Majke H.D. van Bommel, Obstetrics & Gynaecology, Radboudumc, Nijmegen, The Netherlands; majke. vanbommel@radboudumc.nl HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE, OR POLICY ⇒This study contributes to further unraveling the on- cogenesis of ovarian carcinoma and assists in re- search regarding the urgently needed detection of ovarian cancer. ⇒This study contributes to further unraveling the on- cogenesis of ovarian carcinoma and assists in re- search regarding the urgently needed detection of ovarian cancer. ducts (fallopian tubes, uterus, upper part of the vagina) with the ovaries secondarily involved. Nowadays, ovarian, fallopian tubal, and/or the peritoneal malig- nancies are considered collectively as ovarian carci- nomas, of which approximately 75% are high-grade serous carcinomas. There is compelling evidence that high-grade serous carcinoma originates in the fallo- pian tubes,4 potentially offering new strategies for ovarian cancer prevention and early detection. Conclusions Mutational analysis in cervicovaginal and endometrial samples has limited accuracy in the detection of ovarian cancer. Future research with cytologic samples analyzed on methylation status or the vaginal microbiome may be relevant. ber 23, 2024 by guest. Protected by copyright. Received 9 August 2022 Accepted 17 October 2022 Published Online First 16 November 2022 Original research smear and found a sensitivity of detecting ovarian cancer of 41%, particularly driven by mutated Tumor Protein 53 (TP53) variants.6 When combining mutational analysis with DNA extracted from a Pap smear with plasma, sensitivity improved to 63%. An intra- uterine brush to sample DNA closer to the primary source increased sensitivity.7 Currently, uterine and tubal lavage to detect early-stage ovarian cancer is being investigated (NCT 02039388). The first results are promising as ovarian cancer cells could be collected in 24 of 30 patients with ovarian cancer, and mainly TP53 mutations could be identified.8i sample (ovarian tissue) (Figure 1). Samples were collected in the aforementioned order by the operating gynecologist on the day of surgery. Demographic information was extracted from medical records. sample (ovarian tissue) (Figure 1). Samples were collected in the aforementioned order by the operating gynecologist on the day of surgery. Demographic information was extracted from medical records. Pathogenic Variant Analysisl The complete workflow is provided in Online supplemental docu- ment 1. Briefly, DNA was extracted from the four specimens and analyzed using single-molecule molecular inversion probes- based sequencing on a NextSeq500 device (Illumina, San Diego, California, USA), as previously described.10 The single-molecule molecular inversion probes were constructed to highlight hotspots in the oncogenes relevant in ovarian and endometrial cancer: Catenin Beta 1 (CTNNB1), Kirsten rat sarcoma virus (KRAS), mammalian target of rapamycin (MTOR), Phosphatidylinositol- 4,5-Bisphosphate 3-Kinase Catalytic Subunit Alpha (PIK3CA), and Polymerase ε (POLE); and all coding and splice site sequences of the tumor suppressor genes: AT-rich interactive domain-containing protein 1A (ARID1A), Phosphatase and tensin homolog (PTEN), and Tumor Protein 53 (TP53). Genes were chosen based on the genetic characteristics of ovarian and endometrial cancer as described in The Cancer Genome Atlas (TCGA).11–13 Variants were categorized according to the following classes: 1, benign; 2, likely benign; 3, variant of unknown significance; 4, likely pathogenic, and 5, path- ogenic. The last three classes were considered (potentially) patho- genic. The above findings support the presence of ovarian cancer cells along the Müllerian tract, which could potentially be detected with minimally invasive sampling methods. Therefore, we investigated the diagnostic accuracy of detecting ovarian cancer by assessing DNA pathogenic variants in cervicovaginal and endometrial samples and comparing them with the pathogenic variants found in the tumor itself. Design and Population This prospective observational multicenter study included consec- utive patients undergoing surgery for high suspicion of ovarian cancer or for a benign gynecological condition (control group) in three Dutch hospitals: Radboud University Medical Center, Nijmegen; Canisius-Wilhelmina Hospital, Nijmegen; and Elisabeth- TweeSteden Hospital, Tilburg. Suspicion was based on a Risk Malignancy Index >200, the presence of ascites, peritoneal depo- sitions, omental cake, or laparoscopic evaluation. Inclusion criteria were adult age and surgery between December 2013 and January 2017 in a participating hospital. Exclusion criteria were a history of pelvic radiotherapy or previous hysterectomy. Ethical approval was obtained in all hospitals (Study Number 2013/451) and each patient signed informed consent. The study was prospectively registered at the Dutch Trial Registry (NTR4299) and performed according to the STARD guidelines for Standards for the Reporting of Diagnostic accuracy studies. Patients with endometrial cancer were included as well. Their results have been published previously.9 on October 23, 2024 by guest. Protec http://ijgc.bmj.com/ November 2022. Downloaded from All surgical ovarian samples and pipelle endometrial biopsies were analyzed for the presence of the (potentially) pathogenic vari- ants with a variant allele frequency of ≥3% and a minimal number of five variant reads (equal to three unique genomic DNA mole- cules). Additionally, the pipelle data were evaluated at the positions with known pathogenic variants in the surgical specimen with a cut-off of five unique variant reads and no minimal variant allele frequency. For evaluation of the Pap smears and self-samples, two independent library preparations were analyzed using a minimal variant allele frequency of 1% as we expected low variant allele frequencies with the samples mainly containing healthy endocer- vical cells and few tumor cells. on October 23, 2024 by guest. Protected by copyright. ttp://ijgc.bmj.com/ Data Collection Baseline data were analyzed descriptively and differences between groups were analyzed using a t-test, Mann–Whitney U test, or χ2 test. To measure diagnostic accuracy, we calculated sensitivity and specificity. First, we calculated the detection rates of (potentially) pathogenic variants per sample among all patients. Second, we measured diagnostic accuracy among all patients using the detec- tion of (potentially) pathogenic variants in their surgical sample as gold standard. Third, we focused on TP53 pathogenic variants as these variants are primarily related to ovarian cancer.14 Four specimens were collected from each patient: cervicovaginal self-sample, Pap smear, pipelle endometrial biopsy, and surgical on October 23, 2024 by guest. Protected by copyright. Figure 1 Study flowchart. est. Protected by copyright. INTRODUCTION Screening for ovarian cancer using transvaginal sonography and cancer antigen 125 (CA125) has been proven ineffective in the general population3 and in women at increased inherited risk.5 Lately, instead of focusing on macroscopic changes, there is increasing interest in detecting microscopic (pre)malignant cells that detach along the Müllerian ducts. Interest in DNA analysis in cytological samples is growing since (cell-free) DNA variants can be detected in cytological samples even without the presence of tumor cells. Kinde et al extracted DNA from a Papanicolaou (Pap) Epithelial ovarian cancer is the most lethal gyne- cological cancer.1 Patients generally present with advanced-stage disease leading to a 5-year survival of approximately 45%,2 mainly due to the absence of early symptoms and reliable screening methods.3 By contrast, survival for the limited number of patients with localized disease is around 92%, suggesting that early detection of epithelial ovarian cancer could substantially improve prognosis.2 uest. Protected by copyright. Epithelial ovarian cancer is thought to develop from tissues embryologically derived from the Müllerian 1568 van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 on October 23, 2024 by guest. Protected by copyright. http://ijgc.bmj.com/ Int J Gynecol Cancer: first published as 10.1136/ijgc-2022-003911 on 16 November 2022. Downloaded from Table 1 Baseline characteristics Ovarian cancer patients (n=29) Controls (n=32) P value Age, years 66 (32–83) 57 (45–82) 0.063 Body mass index, kg/m2 25 (19–32) 23 (20–44) 0.644 CA125, kIU/L 360 (8–2196) 20 (9–142) <0.001 Menopausal status 0.321 Pre-menopausal 4 (14) 8 (25) Post-menopausal 25 (86) 23 (72) Unknown 0 1 (3) Histology High-grade serous 20 (69) Endometrioid 3 (10) Clear cell 1 (3) Malignant mixed Müllerian tumor 1 (3) Mixed* 4 (14) Myoma 9 (28) Cystadenoma ovarii 10 (31) Fibroma/teratoma ovarii 7 (22) Other† 6 (19) FIGO stage NA IA 1 (3) IB 0 IC 2 (7) IIA 2 (7) IIB 2 (7) IIC 0 IIIA 1 (3) IIIB 4 (14) IIIC 14 (48) IV 3 (10) Values presented as median (range) or N (%). Mixed histology included two clear cell/serous, one clear cell/endometrioid, and one serous/endometrioid. †Other histology included one adenomyosis, one inflammation, two prolapse, and two normal. CA125, cancer antigen 125; FIGO, International Federation of Gynecology and Obstetrics. o http://ijgc.bmj.com/ nt J Gynecol Cancer: first published as 10.1136/ijgc-2022-003911 on 16 November 2022. Downloaded from Values presented as median (range) or N (%). Mixed histology included two clear cell/serous, one clear cell/endometrioid, and one serous/endometrioid. †Other histology included one adenomyosis, one inflammation, two prolapse, and two normal. CA125, cancer antigen 125; FIGO, International Federation of Gynecology and Obstetrics. were found to have a KRAS variant in their surgical specimen. Indi- vidual characteristics are shown in Online supplemental table 3. Surgical Specimens In the tumors of 29 patients with ovarian cancers, 79% of the exons had a mean coverage of >250 reads, reflecting a 95% probability of detecting a variant, ‘adequately sequenced’ (see Online supple- mental table 2).10 We detected at least one pathogenic variant in 24 patients (83%). In total, 34 (potentially) pathogenic variants were detected (Figure 2A). Among the 24 patients with ovarian cancer with pathogenic variants, 17 had a TP53 variant (71%). Fifteen of these 17 had high-grade serous carcinoma, one had a clear cell/ endometrioid ovarian cancer, and one a clear cell/serous ovarian cancer. Other detected variants were: PIK3CA (21%), CTNNB1 (14%), PTEN (10%), KRAS (7%), ARID1A (3%), and MTOR (3%) (Table 2). Of the five patients without a detected variant in their surgical sample (17%), three had high-grade serous carcinoma, one had a clear cell carcinoma, and one had clear cell/serous histology. Among the controls, two patients (6%), both with a mucinous cystadenoma, RESULTS Specimens were collected from 37 patients with ovarian cancer and 32 controls. Eight patients with ovarian cancer were excluded because sequencing of the surgical specimen (the gold standard) was unsuccessful: the coverage was too low to detect any potential variant. Thus, 29 patients with ovarian cancer and 32 controls were included. Table 1 shows the characteristics of the patients. Figure 1 Study flowchart. Figure 1 Study flowchart. 1569 van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 Original research Original research Figure 2 Overview of detected (potentially) pathogenic variants in the surgical specimens (A), self-samples (B), Pap smears (C), and pipelle endometrial biopsies (D) and visualization of overlapping and non-overlapping variants between the surgical specimen and self-samples (B), Pap smears (C), and pipelle (D) in patients with ovarian cancer and controls. p jg j jg Figure 2 Overview of detected (potentially) pathogenic variants in the surgical specimens (A), self-samples (B), Pap smears (C), and pipelle endometrial biopsies (D) and visualization of overlapping and non-overlapping variants between the surgical specimen and self-samples (B), Pap smears (C), and pipelle (D) in patients with ovarian cancer and controls. variant)). Two control patients had both a PIK3CA variant solely in their Pap smear. Results of the Pap smears were available for 26 patients with ovarian cancer; exons ‘adequately sequenced’: 98%. Among them, 10 patients (38%) had one or more (potentially) pathogenic variants. In total, we detected 15 variants of which eight were overlapping with the variants in the surgical specimen (53%) (see Figure 2C and Online supplemental table 4). The eight overlapping variants were detected in six patients. Four of these six patients had high- grade serous carcinoma (of which three had a TP53 variant (two stage 3C and one stage 2A) and one had a PIK3CA, KRAS and PTEN variant (high-grade serous carcinoma stage 3C); one had an endometrioid carcinoma stage 2A (and CTNNB1 variant); and one had a clear cell/endometrioid carcinoma stage 1C (and PIK3CA Cervicovaginal Samples (Self-Samples and Pap Smears) Cervicovaginal Samples (Self Samples and Pap Smears) Analysis of the cervicovaginal self-samples of 27 patients with ovarian cancer identified six patients (22%) with a total of seven (potentially) pathogenic variants: PIK3CA (n=3), TP53 (n=2), and ARID1A (n=2); 98% of the exons were ‘adequately sequenced’. When evaluating overlapping variants (variants that were detected in more than one specimen), we found that two variants were found in all samples but the surgical specimen (one TP53 variant, one PIK3CA); and one variant was found in the surgical specimen, self- sample and pipelle but not in the Pap smear (ARID1A). Four vari- ants were solely found in the self-sample (see Figure 2B and Online supplemental table 4). One control had an ARID1A variant in her self-sample that was not detected in her other specimens. uest. Protected by copyright. 1570 van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 on October 23, 2024 by guest. Protected by copyrig http://ijgc.bmj.com/ Int J Gynecol Cancer: first published as 10.1136/ijgc-2022-003911 on 16 November 2022. Downloaded from on October 23, 2024 by guest. Prote http://ijgc.bmj.com/ Int J Gynecol Cancer: first published as 10.1136/ijgc-2022-003911 on 16 November 2022. Downloaded from Table 2 Number of (potentially) pathogenic variants in the various specimens Ovarian cancer patients (n=29) (Potentially) pathogenic variant Self-sample ‍ ‍ (n=27) Pap smear ‍ ‍ (n=26) Pipelle ‍ ‍ (n=23) Surgical sample ‍ ‍ (n=29) TP53 2 (7) 6 (19) 2 (9) 17 (59) PIK3CA 3 (11) 5 (15) 4 (17) 6 (21) CTNNB1 0 1 (4) 0 4 (14) PTEN 0 1 (4) 1 (4) 3 (10) KRAS 0 1 (4) 2 (9) 2 (7) ARID1A 2 (7) 1 (4) 1 (4) 1 (3) MTOR 0 0 0 1 (3) POLE 0 0 0 0 Total number of pathogenic variants 7 15 10 34 Patients without a pathogenic variant 21 (78) 16 (62) 16 (70) 5 (17) Control patients (n=32) (Potentially) pathogenic variant Self-sample ‍ ‍ (n=31) Pap smear ‍ ‍ (n=32) Pipelle ‍ ‍ (n=32) Surgical sample ‍ ‍ (n=31) TP53 0 0 0 0 PIK3CA 0 2 (6) 2 (6) 0 CTNNB1 0 0 0 0 PTEN 0 0 0 0 KRAS 0 0 0 2 (6) ARID1A 1 (3) 0 0 0 MTOR 0 0 0 0 POLE 0 0 0 0 Total number of pathogenic variants Patients without a pathogenic variant 30 (97) 30 (94) 30 (94) 29 (94) Values are presented as number of pathogenic variants (% of patients). Percentages may total >100% as one patient can have multiple variants. Table 2 Number of (potentially) pathogenic variants in the various specimens Ovarian cancer patients (n=29) Table 2 Number of (potentially) pathogenic variants in the various specimens Values are presented as number of pathogenic variants (% of patients). Percentages may total >100% as one patient can have multiple variants. table 4). Two control patients (6%) had a PIK3CA variant which were not found in their other specimens. roughly similar between early and late stage (see Supplementary Document 2). No correlation was found between the variant allele frequency in the ovarian tumor and the likelihood of detecting the variant in any of the sampling methods (data not shown). 2024 by guest. Protected by copyright. Pipelle Endometrial Biopsies A pipelle was available for 23 patients with ovarian cancer; 71% of the exons were ‘adequately sequenced’. Analysis showed seven patients (30%) with a total of 10 pathogenic variants: four PIK3CA, two TP53, two KRAS, one PTEN, and one ARID1A. We found one overlapping variant between the surgical specimens and the pipelles (10%), which was an ARID1A variant in a woman with high- grade serous carcinoma stage 3B who had this variant in all spec- imens but the Pap smear (see Figure 2D and Online supplemental est. Protected by copyright. van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 1571 Original research Results in the Context of Published Literature Results in the Context of Published Literature Table 3 Diagnostic accuracy of various combinations of measurements to detect ovarian cancer Sensitivity Specificity Any (potentially) pathogenic variant (all patients) ‍ ‍ 22 (9 to 43) 97 (83 to 100) ‍ ‍ 38 (20 to 59) 94 (79 to 99) ‍ ‍ 30 (13 to 53) 94 (79 to 99) ‍ ‍ 52 (33 to 71) 88 (71 to 96) (Potentially) pathogenic variants overlapping with the surgical specimen ‍ ‍ 5 (0 to 23) 97 (83 to 100) ‍ ‍ 26 (10 to 48) 93 (80 to 99) ‍ ‍ 6 (0 to 27) 93 (80 to 99) ‍ ‍ 29 (13 to 51) 87 (69 to 96) Diagnostic accuracy includes sensitivity and specificity. Values are presented as percentage (95% CI) Table 3 Diagnostic accuracy of various combinations of measurements to detect ovarian cancer Despite an impressive research effort to improve the therapeutic options for ovarian cancer, the survival rate has barely increased over the past decades.2 Diagnosing epithelial ovarian cancer in an early stage might improve prognosis substantially, emphasizing the need for early detection methods.2 It is notable that early stage detection seemed not to be inferior to late stage detection in our study. Based on anatomical position, one could reason that path- ogenic variants would be more frequently found in endometrial biopsies compared with cervicovaginal samples. Some studies reported on potential precursors of serous epithelial ovarian cancer in the endometrium,15 16 although the fallopian tubes are nowadays considered as the site of origin of mainly serous epithelial ovarian cancer.4 Thus far, no research has been published about sampling the endometrium with a pipelle biopsy to potentially detect ovarian cancer early. However, as we found, mutational analysis of the endometrium obtained via a pipelle seems not to be appropriate for this purpose, although this does not exclude the potential role of the uterus and/or endometrium in early ovarian cancer detec- tion. The lower prevalence of diagnosed pathogenic variants in the pipelle biopsies compared with the cervicovaginal samples could be explained by the fact that endometrial tissue was processed in paraffin, in which DNA preservation is less optimal leading to a lower sequence coverage and thus lower sensitivity. Results in the Context of Published Literature The cervico- vaginal samples were stored in PreservCyt medium, which better maintains DNA stability.17 Future studies could investigate whether the detection rate of pathogenic variants would increase when analyzing DNA from pipelle samples being preserved in PreservCyt medium. Diagnostic accuracy includes sensitivity and specificity. Values are presented as percentage (95% CI) As demonstrated, cytology samples might be more promising in early ovarian cancer detection than endometrial histology samples. This is especially attractive as obtaining cervical cytology samples is highly accepted and less invasive than obtaining endometrial histology. Of the 34 detected variants in the surgical specimens, eight were also found in the Pap smear (24%) whereas we only detected one variant in the cytological cervicovaginal self-sample overlapping with the surgical specimen (3%). Our results are very similar to those of Wang et al,7 who found that 29% of patients with ovarian cancer harbored detectable variants, mostly TP53, in their Pap smears. They also investigated intra-uterine cytology sampling using Tao brushes which could detect a variant in 42% of patients with ovarian cancer. Current research is investigating whether uterine cytology samples, obtained via lavage of the uterine cavity and analyzed with next-generation sequencing, can serve as an early detection method (NCT 02039388). Combining the results of their uterine cytology samples with cytologic assessed pipelle biopsies might show a new insight into the etiology of ovarian cancer. Also, the methylation status of such cytologic samples may contribute/improve ovarian cancer detection. Moreover, Barrett et al recently demonstrated that the DNA methylome in cervical samples can predict the risk of ovarian cancer with about 75% certainty.18 Evaluation of the vaginal microbiome as a possible early detection method could also be promising. The microbiome might impact estrogen metabolism and may influence the risk of ovarian cancer, like exogenous estrogens.19–22 In colorectal oncogenesis the micro- biota seem to play a major role,23 which may also apply to gyneco- logical cancers. methods were 29% (95% CI 13% to 51%) and 87% (95% CI 69% to 96%), respectively. When analyzing TP53 pathogenic variants only, we identified 21 patients with ovarian cancer with 22 TP53 variants, of whom 17 had the TP53 variant in their surgical specimen. Overlapping TP53 variants with the surgical specimen were found in the Pap smear in three patients (sensitivity 18% (95% CI 4% to 43%)). No overlapping TP53 variants were found in the self-samples or the pipelles. Diagnostic Accuracy Among the 29 patients with ovarian cancer, at least one (potentially) pathogenic variant was detected in 83% of the surgical specimens, in 22% of the self-samples, in 38% of the Pap smears, and in 30% of the pipelles. In the patients with ovarian cancer the detection rate for a (potentially) pathogenic variant in any of the sampling methods (the self-sample, Pap smear, or pipelle) was 52%. Among the controls, a false positive variant was detected in 6%, 3%, 6%, and 6%, respectively, of the specimens. Detection rates were uest. Protected by copyright. The diagnostic accuracy of overlapping (potentially) patho- genic variants—for example, a pathogenic variant in minimally one of the sampling methods among patients with a pathogenic variant in their surgical specimen (n=24) and controls without a pathogenic variant in the surgical specimen (n=30)—is shown in Table 3. Sensitivity and specificity for an overlapping patho- genic variant in the surgical specimen and any of the sampling van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 1572 Results in the Context of Published Literature Among controls, no TP53 variants were detected (100% specificity). on October 23, 2024 by guest. Protected by copyright. http://ijgc.bmj.com/ ber 23, 2024 by guest. Protected by copyright. Original research on October 23, 2024 by guest. Protected by copyrig http://ijgc.bmj.com/ Int J Gynecol Cancer: first published as 10.1136/ijgc-2022-003911 on 16 November 2022. Downloaded from Strengths and Weaknesses Our study is the first to sample the endometrium with a pipelle to potentially detect ovarian cancer. We covered most of the Müllerian tract by sampling the endometrium, cervix, and vagina, in addition to the tumor. There are, however, some limitations. A larger sample size would strengthen our results. The low prevalence of (potentially) pathogenic variants overall might be related to insufficiently deep sequencing of some samples, the choice of the library preparation method, and the content of the gene panel, although we expected that the majority would be picked up with this panel. The detection rate may have been higher when the pipelle samples were stored in PreservCyt medium. There might be some false positive vari- ants as healthy persons appear sometimes to have pathogenic variants as well.26 27 Furthermore, the class III pathogenic vari- ants were considered (potentially) pathogenic, although these reflect a minority of all variants. Thus far it is unknown whether or not these variants should be considered pathogenic. Data availability statement Data are available upon reasonable request. All data relevant to the study are included in the article or uploaded as supplementary information. The data supporting the conclusions of this article are available upon reasonable request to the corresponding author. In accordance with the journal’s guidelines, we will provide our data for independent analysis by a selected team by the Editorial Team for the purposes of additional data analysis or for the reproducibility of this study in other centers if such is requested. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. on October 23, 2024 by guest. Protect http://ijgc.bmj.com/ November 2022. Summary of Main Results In this multicenter prospective study we investigated the diag- nostic accuracy of detecting ovarian cancer with mutational anal- ysis in cervicovaginal and endometrial biopsies. We found (poten- tially) pathogenic variants in 83% of the ovarian cancer tumors, of which 71% were TP53 variants. In 52%, a (potentially) pathogenic variant could be detected in either a cervicovaginal self-sample, Pap smear, or pipelle. Sensitivity was low for all sampling methods and remained low when analyzing TP53 variants only. Among the controls, hardly any variants were detected, resulting in very high specificity of all sampling methods. 1573 van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 ORCID iDs Majke H.D. van Bommel http://orcid.org/0000-0002-7562-9861 Heidi V.N. Küsters-Vandevelde http://orcid.org/0000-0002-1012-941X Majke H.D. van Bommel http://orcid.org/0000-0002-7562-9861 Heidi V.N. Küsters-Vandevelde http://orcid.org/0000-0002-1012-941X Strengths and Weaknesses Downloaded from Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. Patient consent for publication Consent obtained directly from patient(s) Patient consent for publication Consent obtained directly from patient(s) Patient consent for publication Consent obtained directly from patient(s) Ethics approval This study involves human participants and was approved by the Medical-Ethical Committee of Arnhem-Nijmegen study number 2013/451. Participants gave informed consent to participate in the study before taking part. Ethics approval This study involves human participants and was approved by the Medical-Ethical Committee of Arnhem-Nijmegen study number 2013/451. Participants gave informed consent to participate in the study before taking part. Provenance and peer review Not commissioned; externally peer reviewed. Provenance and peer review Not commissioned; externally peer reviewed. REFERENCES 1 Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021;71:209–49. 1 Sung H, Ferlay J, Siegel RL, et al. Global cancer statistics 2020: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries. CA Cancer J Clin 2021;71:209–49. 2 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7–34. 2 Siegel RL, Miller KD, Jemal A. Cancer statistics, 2019. CA Cancer J Clin 2019;69:7–34. 3 Menon U, Gentry-Maharaj A, Burnell M, et al. Ovarian cancer population screening and mortality after long-term follow-up in the UK Collaborative Trial of Ovarian Cancer Screening (UKCTOCS): a randomised controlled trial. Lancet 2021;397:2182–93. CONCLUSIONS We investigated whether mutational analysis of samples along the Müllerian tract could be used to detect ovarian cancer. Diag- nostic accuracy with our analysis was low for cervicovaginal self- samples, Pap smears, and endometrial biopsies when comparing the pathogenic variants in the samples to the variants in the tumor itself. Thus, these samples should not be used for (early) ovarian cancer detection. 4 Labidi-Galy SI, Papp E, Hallberg D, et al. High grade serous ovarian carcinomas originate in the fallopian tube. Nat Commun 2017;8:1093.i 5 Hermsen BBJ, Olivier RI, Verheijen RHM, et al. No efficacy of annual gynaecological screening in BRCA1/2 mutation carriers; an observational follow-up study. Br J Cancer 2007;96:1335–42. 6 Kinde I, Bettegowda C, Wang Y, et al. Evaluation of DNA from the Papanicolaou test to detect ovarian and endometrial cancers. Sci Transl Med 2013;5:ra4. 7 Wang Y, Li L, Douville C, et al. Evaluation of liquid from the Papanicolaou test and other liquid biopsies for the detection of endometrial and ovarian cancers. Sci Transl Med 2018;10:eaap8793 doi:10.1126/scitranslmed.aap8793 Implications for Practice and Future Research This study contributes to the unraveling of ovarian cancer etiology and assists in research regarding the urgently needed detection of ovarian cancer. For future research it would be relevant to investi- gate cytology samples acquired along the Müllerian tract, stored in PreservCyt medium, using alternative library preparation methods, expanding the gene panel, and potentially analyzing the methyla- tion status or the vaginal microbiome. van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 Original research 4Pathology, Catharina Hospital, Eindhoven, The Netherlands 5Obstetrics and Gynaecology, Elisabeth-TweeSteden Ziekenhuis, Tilburg, The Netherlands 6Human Genetics, Radboudumc, Nijmegen, The Netherlands 7Radiation Oncology, Radboudumc, Nijmegen, The Netherlands 4Pathology, Catharina Hospital, Eindhoven, The Netherlands 5Obstetrics and Gynaecology, Elisabeth-TweeSteden Ziekenhuis, Tilburg, The Netherlands Considering the oncogenesis of high-grade serous carcinoma, we expected to find more TP53 pathogenic variants. More- over, approximately 90% of all patients with serous epithelial ovarian cancer have a TP53 variant.14 We found a TP53 variant in 59% of patients, which might be explained by tumor hetero- geneity.24 Also, three of our five patients without a pathogenic variant in the surgical specimen had a high-grade serous carci- noma. Further, ARID1A variants were under-represented among patients with clear cell histology based on TCGA, probably because only four patients had (mixed) clear cell histology. Our detection rate may have been higher if we had included genes involved in homologous recombination as these are commonly related with epithelial ovarian cancer. For example, somatic BReast CAncer pathogenic variants can be detected in about 17% of all patients with epithelial ovarian cancer.25 6Human Genetics, Radboudumc, Nijmegen, The Netherlands 7Radiation Oncology, Radboudumc, Nijmegen, The Netherlands Contributors MvB wrote the main manuscript text. MvB, LvdP, MS, HKvdV, ML, AE, JdH, JP, and CR provided the conceptualization and methodology of the study. MvB, LvdP, JB, MS, SS, ML, AE, JdH, JP, and CR were involved in the analysis of the data. CR acted as guarantor. All authors reviewed the manuscript. Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors. Competing interests None declared. i 1Obstetrics & Gynaecology, Radboud Institute for Health Science, Radboudumc, Nijmegen, The Netherlands 2Pathology, Radboudumc, Nijmegen, The Netherlands 3Obstetrics and Gynaecology, Catharina Hospital, Eindhoven, The Netherlands Original research 9 Reijnen C, van der Putten LJM, Bulten J, et al. 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Integrated genomic characterization of endometrial carcinoma. Nature 2013;497:67–73. 22 Nené NR, Reisel D, Leimbach A, et al. Association between the cervicovaginal microbiome, BRCA1 mutation status, and risk of ovarian cancer: a case-control study. Lancet Oncol 2019;20:1171–82. 12 Cancer Genome Atlas Research Network. Integrated genomic analyses of ovarian carcinoma. Nature 2011;474:609–15. 13 cfCG. Ovarian Epithelial Tumor - Serous Ovarian Cancer, 2014. Available: http://www.cbioportal.org/ 23 Abu-Ghazaleh N, Chua WJ, Gopalan V. Intestinal microbiota and its association with colon cancer and red/processed meat consumption. J Gastroenterol Hepatol 2021;36:75–88. 14 Ghezelayagh TS, Pennington KP, Norquist BM, et al. Characterizing TP53 mutations in ovarian carcinomas with and without concurrent BRCA1 or BRCA2 mutations. Gynecol Oncol 2021;160:786–92. 24 Erickson BK, Kinde I, Dobbin ZC, et al. 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Author affiliations i 1Obstetrics & Gynaecology, Radboud Institute for Health Science, Radboudumc, Nijmegen, The Netherlands 2Pathology, Radboudumc, Nijmegen, The Netherlands 3Obstetrics and Gynaecology, Catharina Hospital, Eindhoven, The Netherlands 8 Maritschnegg E, Wang Y, Pecha N, et al. Lavage of the uterine cavity for molecular detection of Müllerian duct carcinomas: a proof-of- concept study. J Clin Oncol 2015;33:4293–300. 1574 van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911 Original research Uterine and tubal lavage for earlier cancer detection using an innovative catheter: a feasibility and safety study. Int J Gynecol Cancer 2018;28:1692–8. 17 Filho AL, Gonçalves AEP, Martinho O, et al. Liquid-based cytology in DNA-based molecular research: viability and potential application. Anal Quant Cytol Histol 2009;31:395–400. 27 Moore L, Leongamornlert D, Coorens THH, et al. The mutational landscape of normal human endometrial epithelium. Nature 2020;580:640–6. on October 23, 2024 by guest. Prot http://ijgc.bmj.com/ n 16 November 2022. Downloaded from 1575 van Bommel MH.D, et al. Int J Gynecol Cancer 2022;32:1568–1575. doi:10.1136/ijgc-2022-003911
https://openalex.org/W4287726205	https://zenodo.org/records/3935541/files/NUTRI-SCORE.pdf	English	null	Report of the Scientific Committee of the Spanish Agency for Food Safety and Nutrition (AESAN) on the implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products	Zenodo (CERN European Organization for Nuclear Research)	2,020	cc-by	9,901	Report of the Scientific Committee of the Spanish Agency for Food Safety and Nutrition (AESAN) on the implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Report of the Scientific Committee of the Spanish Agency for Food Safety and Nutrition (AESAN) on the implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Report of the Scientific Committee of the Spanish Agency for Food Safety and Nutrition (AESAN) on the implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Reference number: AESAN-2020-004 Report approved by the Scientific Committee in its plenary session on 4 March 2020 Working group Montaña Cámara Hurtado (Coordinator), Carlos Alonso Calleja, Rosa María Giner Pons, Elena Gon- zález Fandos, Jordi Mañes Vinuesa, José Alfredo Martínez Hernández, Esther López García, Victoria Moreno-Arribas, María del Puy Portillo Baquedano, David Rodríguez Lázaro, Magdalena Rafecas Martínez, Marta García Solano (AESAN), Enrique Gutiérrez González (AESAN) and Mª José Yusta Boyo (AESAN) Scientific Committee Carlos Alonso Calleja Universidad de León Rosa María Giner Pons Universitat de València Sonia Marín Sillué Universitat de Lleida Magdalena Rafecas Martínez Universitat de Barcelona Montaña Cámara Hurtado Universidad Complutense de Madrid Elena González Fandos Universidad de La Rioja José Alfredo Martínez Hernández Universidad de Navarra David Rodríguez Lázaro Universidad de Burgos Álvaro Daschner Hospital de La Princesa de Madrid María José González Muñoz Universidad de Alcalá de Henares Francisco José Morales Navas Consejo Superior de Investigaciones Científicas Carmen Rubio Armendáriz Universidad de La Laguna Pablo Fernández Escámez Universidad Politécnica de Cartagena Esther López García Universidad Autónoma de Madrid Victoria Moreno Arribas Consejo Superior de Investigaciones Científicas María José Ruiz Leal Universitat de València Carlos Manuel Franco Abuín Universidade de Santiago de Compostela Jordi Mañes Vinuesa Universitat de València María del Puy Portillo Baquedano Universidad del País Vasco Pau Talens Oliag Universitat Politècnica de València Technical Secretary Vicente Calderón Pascual Report of the Scientific Committee of the Spanish Agency for Food Safety and Nutrition (AESAN) on the implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Translated from the original published in the journal: Revista del Comité Científico de la AESAN, 31, pp: 77-97 Reference number: AESAN-2020-004 1 revista del comité científico nº 31 g g p Montaña Cámara Hurtado (Coordinator), Carlos Alonso Calleja, Rosa María Giner Pons, Elena Gon- zález Fandos, Jordi Mañes Vinuesa, José Alfredo Martínez Hernández, Esther López García, Victoria Moreno-Arribas, María del Puy Portillo Baquedano, David Rodríguez Lázaro, Magdalena Rafecas Martínez, Marta García Solano (AESAN), Enrique Gutiérrez González (AESAN) and Mª José Yusta Boyo (AESAN) revista del comité científico nº 31 Scientific Committee Carlos Alonso Calleja Universidad de León Rosa María Giner Pons Universitat de València Sonia Marín Sillué Universitat de Lleida Magdalena Rafecas Martínez Universitat de Barcelona Montaña Cámara Hurtado Universidad Complutense de Madrid Elena González Fandos Universidad de La Rioja José Alfredo Martínez Hernández Universidad de Navarra David Rodríguez Lázaro Universidad de Burgos Álvaro Daschner Hospital de La Princesa de Madrid María José González Muñoz Universidad de Alcalá de Henares Francisco José Morales Navas Consejo Superior de Investigaciones Científicas Carmen Rubio Armendáriz Universidad de La Laguna Pablo Fernández Escámez Universidad Politécnica de Cartagena Esther López García Universidad Autónoma de Madrid Victoria Moreno Arribas Consejo Superior de Investigaciones Científicas María José Ruiz Leal Universitat de València Carlos Manuel Franco Abuín Universidade de Santiago de Compostela Jordi Mañes Vinuesa Universitat de València María del Puy Portillo Baquedano Universidad del País Vasco Pau Talens Oliag Universitat Politècnica de València Technical Secretary Key words Nutri-Score, labelling, FOPL, olive oil. Technical Secretary Key words Nutri-Score, labelling, FOPL, olive oil. Suggested citation AESAN Scientific Committee. (Working group) Cámara, M., Alonso, C., Giner, R.M., González, E., Mañes, J., Martínez, J.A., López, E., Moreno-Arribas, V., Portillo, M.P., Rodríguez, D., Rafecas, M., García, M., Gutiérrez, E. and Yusta, M.J. Informe del Comité Científico de la Agencia Española de Seguridad Alimentaria y Nutrición (AESAN) sobre la aplicación en España del sistema Nutri-Score de información sobre la calidad nutricional de los alimentos. Revista del Comité Científico de la AESAN, 2020, 31, pp: 77-97. Technical Secretary Vicente Calderón Pascual Non-communicable diseases are the main cause of morbidity and mortality in Europe. Imbalances in the intake of salt and some nutrients, particularly fats, sugars or fibre, among others, are the main modifiable risk factors with regard to these diseases. One of the tools of public health policies to promote healthy diets is nutrition labelling, which is regulated in the European Union by Regulation (EU) No. 1169/2011 on the provision of food information to consumers. This regulation enables the possibility of using, in a complementary and voluntary way, a system of front-of-pack nutrition labelling in order to make the use and understanding of the mandatory nutritional information easier for consumers, favouring healthier choices, and encouraging manufacturers to make products with a better nutritional composition. AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Among the front-of-pack nutrition labelling schemes, the Nutri-Score system stands out. It is a graphic system developed in France based on the use of a letters and colours code to inform con- sumers, in a simple way, of the nutritional quality of food and drinks in addition to the mandatory nutritional information set forth by European legislation. The purpose of this report is to respond to the request for the opinion of the Scientific Committee on the suitability of the adaptation of the Nutri-Score system by incorporating the content of olive oil in its algorithm. In answer to the question raised, a review of the front-of-pack nutrition labelling models used in the WHO European Region has been conducted, with a special focus on the model applied in the United Kingdom and Nutri-Score in France. The value scale used is described and the amendments made are explained, including the one corresponding to olive oil. revista del comité científico nº 31 2 revista del comité científico nº 31 The Scientific Committee considers that the Nutri-Score adaptation proposal including olive oil improves the consideration of a product, which presents nutritional benefits based on its oleic acid content, and entails an improvement regarding this front-of-pack nutrition labelling system. It is necessary to keep identifying other aspects that may be incorporated into the Nutri-Score system for the nutritional assessment of foods and drinks usually found in the Spanish diet. Suggested citationi gg AESAN Scientific Committee. (Working group) Cámara, M., Alonso, C., Giner, R.M., González, E., Mañes, J., Martínez, J.A., López, E., Moreno-Arribas, V., Portillo, M.P., Rodríguez, D., Rafecas, M., García, M., Gutiérrez, E. and Yusta, M.J. Informe del Comité Científico de la Agencia Española de Seguridad Alimentaria y Nutrición (AESAN) sobre la aplicación en España del sistema Nutri-Score de información sobre la calidad nutricional de los alimentos. Revista del Comité Científico de la AESAN, 2020, 31, pp: 77-97. AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products 1. Introduction Noncommunicable diseases are the main cause of morbidity and mortality in the World Health Organization (WHO) European Region (WHO, 2018). Diet plays a determining role in the prevention of a number of noncommunicable diseases including cardiovascular diseases, cerebrovascular disease, type 2 diabetes mellitus and different types of cancer. Imbalances in salt intake and some nutrients, in particular fats, sugars and fibre, are the main preventable risk factors for these diseases. In most European Union Member States, governments have approved health policies aimed at promoting healthy diets, tackling growing obesity rates and guaranteeing nutrition and food safety. However, there is a margin for improvement in the exhaustiveness of these policies and their ambi- tion. The European Food and Nutrition Action Plan 2015-2020 (WHO, 2014) and the EU Action Plan on Childhood Obesity 2014-2020 (EU, 2014) promote the development of a series of public health polices through a multidisciplinary focus on all areas of general government, with the involvement of both the private sector and citizens. The objective is to improve the availability, accessibility and appeal of healthy food, with a view towards improving the general quality of the citizens’ diet and, ultimately, the health and well-being of the population. 3 revista del comité científico nº 31 3 revista del comité científico nº 31 To foster healthy eating, promote physical activity and reverse the rising trend in the prevalence of obesity and, with that, substantially reduce the high rates of morbidity and mortality attributable to chronic diseases, Spain has developed the NAOS Strategy for Nutrition, Physical Activity and the Prevention of Obesity. Nutritional labelling is one of the public health policy tools to promote a healthy diet. The European regulation on nutritional information (Regulation (EU) No. 1169/2011 of the European Parliament and of the Council of 25 October 2011 on the provision of food information to consumers) has set, since 2016, the requirements to be met by mandatory nutritional information. It also mentions the possibility of using, on a complementary and voluntary basis, front-of-pack labelling (FOPL) in order to facilitate consumers’ use and understanding of mandatory nutritional information, fostering healthier choices and encouraging manufacturers to produce products with better nutritional composition. Among the different front-of-pack nutrition labelling schemes, the Nutri-Score system stands out. This is a graphic scale developed in France based on the use of a code of letters and colours to inform consumers of the nutritional quality of food and drinks in a simple way in addition to the mandatory nutritional declaration established in the European regulation. The graphic system or Nutri-Score logo consists of five colours (from dark green to dark orange), each associated with a letter (A to E), and each signifying five categories based on the nutritional information (from best to worst, respectively). Classification into one category is the result of the calculation of an algorithm, designed based on public health criteria and scientifically validated. Points are allocated based on the nutritional composition per 100 g of the product. It takes into account nutrients considered “unfavourable” from a nutritional perspective (calories, sugars, satu- rated fatty acids and sodium) and “favourable” (proteins, fibre and percentage of fruits, vegetables, pulses and nuts). A score is assigned depending on the content of each, according to grids included in two different tables. AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products The usage regulation establishes cheeses as a specific case. 3 revista del comité científico nº 31 The limits for specific nutrient con- tent showed on the grids are not modified, but the final score is calculated considering the protein content score in all cases regardless of the sum of the unfavourable points. Other specific cases contained in the regulation are beverages and added fats, for which the content limits for some nutrients showed in the grids, are modified. In Spain, in November 2018, the Minister of Health, Consumer Affairs and Social Welfare published the intention to implement Nutri-Score, it being considered the most suitable front-of-pack labelling system for Spain’s public health objectives. This was a response to the demands of citizens, profes- sionals and the scientific community; the intentions of some Regional Authorities to adopt their own models; and the announcement of the imminent implementation of another model proposed by five food sector multinationals in several countries, including Spain. revista del comité científico nº 31 4 revista del comité científico nº 31 4 Over subsequent months, Santé publique France, the French national public health agency, devel- oped an amendment to allow for the positive consideration of the olive oil content of foods, whose Nutri-Score grading did not differ from other oils with worse nutritional profile. Consequently, in September 2019, an amendment to the decree regulating the use of Nutri-Score in France was published in the Journal Officiel de la Republique Française. This amendment shall apply homogenously across all countries that adopt Nutri-Score. The purpose of this report is to respond to the request for the opinion of the Scientific Committee on the suitability of adapting the Nutri-Score system by incorporating the content of olive oil in its calculation. In response to the question raised, a review of the front-of-pack nutrition labelling models used in the WHO European Region has been conducted, focusing especially on the model applied in the United Kingdom and the Nutri-Score in France. The value scale used is described and the adaptations made are explained, including the one corresponding to olive oil. 2. Font-of-pack labelling models in the WHO European Region Regulation (EU) No. 1169/2011 establishes the possibility of using FOPL, on a complementary and voluntary basis, in addition to the mandatory nutritional information, without replacing it, provided that it complies with the requirements mentioned in said Regulation: it shall not mislead the con- sumer, it shall not be ambiguous or confusing for the consumer and it shall be based on the relevant scientific data (EU, 2011). Therefore, European Union Member States are afforded certain freedom for the development of FOPL, provided it complies with the requirements established in Regulation (EU) No. 1169/2011, which also calls on the European Commission to prepare a report, yet to be published, on the use of additional forms of expression and presentation, with the effect on the internal market and whether further harmonisation is advisable. Awaiting the publication of the European Commission report, various FOPL models have been developed in different European Union countries that can be classified according to different criteria. The FOPL models are primarily split between the non-interpretive models, which provide a summary AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products of the nutritional value of the products to help purchase decisions; and interpretive models, which provide an assessment of the nutritional quality of the product. revista del comité científico nº 31 4 According to data for the WHO European Region, the governments of 15 countries have backed an interpretive FOPL policy, and 13 of them have adopted a logo (Kelly and Jewell, 2018). Most of the logos identify healthy products; however, in three countries these logos also provide information on less healthy components (warning labels in Israel, Nutri-Score in France and the Traffic Light system in the United Kingdom). FOPL that identifies the unfavourable components of food products seems to support consumers better in choosing nutritionally healthy products. The four main interpretive FOPL systems used in the WHO European Region (including European Union Member States for whom compliance with Regulation (EU) No. 1169/2011 is mandatory and countries outside the European Union) are detailed in Table 1 and are the following: 5 revista del comité científico nº 31 5 revista del comité científico nº 31 1. Endorsement logos: based on an assessment of positive products, favourable criteria are taken into account. They will only appear on those products that comply with certain established nutritional standards. Most products, therefore, don’t carry this type of logo. The criteria to define these nutritional standards vary from one logo to another depending on the nutrients and/or ingredients considered, the content limits and measurement units used, the food groups to which they apply and whether the criteria are established for each food group. 1. Endorsement logos: based on an assessment of positive products, favourable criteria are taken into account. They will only appear on those products that comply with certain established nutritional standards. Most products, therefore, don’t carry this type of logo. Th it i t d fi th t iti l t d d f l t th d di nutritional standards. Most products, therefore, don t carry this type of logo. The criteria to define these nutritional standards vary from one logo to another depending on the nutrients and/or ingredients considered, the content limits and measurement units used, the food groups to which they apply and whether the criteria are established for each food group. The inclusion of text to accompany the logo usually improves consumers’ understanding 2. Nutrient-specific warning labels are classified on an unfavourable criteria-based assessment of the product for each nutrient. The result is a negative evaluation. 2. Nutrient-specific warning labels are classified on an unfavourable criteria-based assessment of the product for each nutrient. The result is a negative evaluation. revista del comité científico nº 31 4 This type of logo shows those products that exceed the established limits for certain critical nutrients (salt, saturated fats, sugars, energy, etc.). The criteria may be established at a general level for all products or for specific food groups. The same product may carry more than one warning logo, each relating to a nutrient. This type of logo shows those products that exceed the established limits for certain critical nutrients (salt, saturated fats, sugars, energy, etc.). The criteria may be established at a general level for all products or for specific food groups. The same product may carry more than one warning logo, each relating to a nutrient. 3. Nutrient-specific interpretive system: a positive or negative assessment is obtained for each nutrient. The FOPL in the United Kingdom, provides numerical information on the percentage contribu- tion a food contributes to the recommended daily intake of a nutrient (non-interpretive) and also includes the interpretive information on the content in nutrients using a colour code (positive or negative assessment). 3. Nutrient-specific interpretive system: a positive or negative assessment is obtained for each nutrient. The FOPL in the United Kingdom, provides numerical information on the percentage contribu- 3. Nutrient-specific interpretive system: a positive or negative assessment is obtained for each nutrient. The FOPL in the United Kingdom, provides numerical information on the percentage contribu- tion a food contributes to the recommended daily intake of a nutrient (non-interpretive) and also includes the interpretive information on the content in nutrients using a colour code (positive or negative assessment). 4. Summary indicator system: a general assessment of the product with a positive or negative result, considering favourable and unfavourable criteria. According to some established criteria for content in nutrients and/or ingredients, a score is calculated, considering favourable and unfavourable points. The final score classifies the products across a range of categories associated with a colour, letter etc. from healthiest to least healthy. In the case of Nutri-Score the scoring criteria are the same for all food groups (except for beverages, fats and cheeses). The logo may be displayed on all products. q y p Table 1. revista del comité científico nº 31 4 Summary of the main types of logos present in interpretive FOPL systems used in the WHO European Region Type of logo Name of logo Countries Endorsement logo Choice logo Belgium, Czech Republic and Poland Green endorsement logo Israel Healthy Living Guarantee Mark Croacia Heart Symbol Finland Keyhole logo Denmark, Iceland, Lithuania, Norway and Sweden Protective Food logo (Little Heart logo) Slovenia Nutrient-specifi c warning system Red warning label Israel (mandatory logo) Nutrient-specifi c interpretive system Traffi c Light (colour coding according to % of recommended intake) United Kingdom, Ireland Summary indicator system Nutri-Score France, Belgium Adapted from: Kelly and Jewell (2018). Recently, Italy, in application of Article 35 of Regulation (EC) No. 1169/2011, notifi ed the European Commission (27 January 2020) of the draft ministerial decree which will recommend that food business operators in Italy use an additional form of expressing nutritional information the “NutrInform Battery” Adapted from: Kelly and Jewell (2018). Recently, Italy, in application of Article 35 of Regulation (EC) No. 1169/2011, notifi ed the European Commission (27 January 2020) of the draft ministerial decree which will recommend that food business operators in Italy use an additional form of expressing nutritional information, the “NutrInform Battery”, consisting of the display, within a battery symbol for each nutrient, of the percentage of the recom- AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products mended daily intake for energy value, fats, saturated fats, sugars and salt per portion of the product. To date, there are no studies available on the level of understanding, usefulness for discriminating between foods, the association with health outcomes, consumer preferences or studies on consum- ers of different socioeconomic status in different countries, including Spain, for NutrInform Battery. 3. Nutritional profiles used in the development of FOPL modelsi The different types of FOPL models are developed based on nutritional profiles that allow for the classification of food and drinks according to their nutritional composition using criteria relating to the prevention of illnesses and the promotion of health. 7 revista del comité científico nº 31 For this classification, the nutritional profiles can use specific criteria for each food and drink group or the same criteria that apply to almost all food groups. For this classification, the nutritional profiles can use specific criteria for each food and drink group or the same criteria that apply to almost all food groups. The nutritional profiles used in most FOPL models are differentiated in four key aspects: 1. Nutrients considered: in general, only nutritional information for nutrients considered unfa- vourable are taken into account in terms of their association with noncommunicable diseases, that is, the content in total fats, saturated fats, trans fatty acids, sodium (or salt) and/or added/ free sugars. However, some FOPL models such as Nutri-Score consider favourable nutrients or ingredients in terms of how they relate to health, such as fruit and vegetable content, for the calculation of nutritional quality. 2. Reference units: most FOPL models use units of content in g/100 g or ml/100 ml of the product, although in some specific cases other units are used on a complementary basis. In the United Kingdom, in order to establish the products with high content of a nutrient, if the size of the portion is greater than 100 g, thresholds per portion are applied. In other cases, such as that of the Keyhole logo, nutritional criteria relating to saturated fats apply to the total fat, those relating to total fat to the percentage of total energy and the salt content criteria on per portion basis, for some food groups. In the case of Nutri-Score, the g/100 g reference is generally used, with the exception of the added fats group. In this case, the nutritional criteria relating to saturated fat content are applied to the total fats. 2. Reference units: most FOPL models use units of content in g/100 g or ml/100 ml of the product, although in some specific cases other units are used on a complementary basis. In the United Kingdom, in order to establish the products with high content of a nutrient, if the size of the portion is greater than 100 g, thresholds per portion are applied. 3. Nutritional profiles used in the development of FOPL modelsi portion is greater than 100 g, thresholds per portion are applied. In other cases, such as that of the Keyhole logo, nutritional criteria relating to saturated fats apply to the total fat, those relating to total fat to the percentage of total energy and the salt content criteria on per portion basis, for some food groups. I th f N t i S th /100 f i ll d ith th ti f th In the case of Nutri-Score, the g/100 g reference is generally used, with the exception of the added fats group. In this case, the nutritional criteria relating to saturated fat content are applied to the total fats. 3. Nutritional criteria: the nutritional criteria used to develop a FOPL model generally follow three principal approaches: 3. Nutritional criteria: the nutritional criteria used to develop a FOPL model generally follow three principal approaches: a. Contribution of each nutrient to recommended daily intake. The United Kingdom’s traffic light system shows the contribution of each nutrient to the recommended daily intake, in addition to a colour code for some content limits established for each nutrient. b. Compliance with content limits for each nutrient. The endorsement logos, specific warning labels for each nutrient and the specific logos for each nutrient use content thresholds. The criteria for setting the content limits vary across the different FOPL models. They are generally based on the national or WHO recommended intakes. In the case of the United Kingdom’s traffic light system, limits for green colour are established for nutritional claims in accordance with Regulation (EC) AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products No. 1924/2006 on nutrition and health claims made on foods (EU, 2006); red colour red is applied for all nutrients with content in excess of 25 % of the recommended intakes (30 % if applied per portion), and amber colour amber for intermediate contents. No. 1924/2006 on nutrition and health claims made on foods (EU, 2006); red colour red is applied for all nutrients with content in excess of 25 % of the recommended intakes (30 % if applied per portion), and amber colour amber for intermediate contents. c. Use of an algorithm to obtain a general nutritional assessment. Nutri-Score is a front-of-pack labelling model adopted by France in 2017 (JORF, 2017) and by Belgium from 1 April 2019 (Moniteur Belge, 2019). Nutri-Score is a front-of-pack labelling model adopted by France in 2017 (JORF, 2017) and by Belgium from 1 April 2019 (Moniteur Belge, 2019). It consists of a logo that can be displayed voluntarily on the front of food and drink packaging for which there is an obligation to display the mandatory nutritional information, in accordance with Regulation (EU) No. 1169/2011, without replacing it. Unprocessed products, such as fruit, vegetables or fresh fish are not affected, nor are alcoholic drinks, as they are not obliged to display said labelling. There are five possible variations of the Nutri-Score logo, each consisting of a colour (dark green, light green, yellow, light orange and dark orange) corresponding to a letter (A, B, C, D and E, respec- tively). The products are classified according to their nutritional quality and are assigned one of five variations of the logo: • A (dark green): the most favourable product from a nutritional perspective. • E (dark orange): the least favourable product from a nutritional perspective. The nutritional criteria used in the Nutri-Score model to classify the foods and drinks are based on the algorithm of the British Food Standards Agency’s Nutrient Profile System (FSA-NPS) (FSA, 2011), which was initially developed for the implementation of restriction policies for food and drink television advertising aimed at children, thus producing a dichotomous variable (advertising permitted or not). 3. Nutritional profiles used in the development of FOPL modelsi A single score is obtained using an algorithm with the aim of categorizing foods in accor- dance with their nutritional composition, showing which are the healthiest and least healthy. Nutri-Score uses this focus to categorize foods and drinks into five categories from best to worst nutritional quality. revista del comité científico nº 31 8 revista del comité científico nº 31 8 4. Food groups: in general, the FOPL are applied to packaged products. However, the Keyhole logo used in Nordic countries, the Healthy Living Guarantee Mark in Croatia and Israel’s Green Endorsement are applied to both packaged and unpackaged products. Furthermore, the Keyhole logo and Healthy Living Guarantee Mark exclude products that con- tain artificial sweeteners. 4.1 British Food Standards Agency Nutrient Profile System (FSA-NPS) The nutritional profiles developed by the United Kingdom (FSA-NPS) have also served as the basis of the scoring criteria for the nutritional profiles of Food Standards from Australia and New Zealand, the nutritional profiles model in South Africa and the nutritional profiles model in Ireland. The FSA algorithm consists of a simple scoring system in which points are allocated based on the nutrient content per 100 g of product. The calculation uses tables that connect the nutrient content included in the mandatory nutritional information with a point scale. AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products On the one hand, “A” or “unfavourable” points are obtained based on the content across four components for which excessive consumption is considered unhealthy: energy (kJ/100 g), saturated fat (g/100 g), sugars (g/100 g) and sodium (mg/100 g), among those contained in mandatory nutritional labelling established in Regulation (EU) No. 1169/2011 of the European Union. A point scale from 0 to 10 is applied for each component. Calculation of A points per 100 g of product: Calculation of A points per 100 g of product: A points Calories (kJ) Sugars (g) Saturated fatty acids (g) Sodium (mg)() 0 ≤335 ≤4.5 ≤1 ≤90 1 >335 >4.5 >1 >90 2 >670 >9 >2 >180 3 >1005 >13.5 >3 >270 4 >1340 >18 >4 >360 5 >1675 >22.5 >5 >450 6 >2010 >27 >6 >540 7 >2345 >31 >7 >630 8 >2680 >36 >8 >720 9 >3015 >40 >9 >810 10 >3350 >45 >10 >900 () The sodium content corresponds to the salt content contained in the mandatory declaration divided by 2.5. A points = calorie points [0-10] + sugar points [0-10] + saturated fatty acids points [0-10] + sodium points [0-10] = [0-40] 9 revista del comité científico nº 31 () The sodium content corresponds to the salt content contained in the mandatory declaration divided by 2.5. A points = calorie points [0-10] + sugar points [0-10] + saturated fatty acids points [0-10] + sodium points [0-10] = [0-40] Meanwhile, “C” or “favourable” points are obtained depending on the content in three components whose consumption is recommended for a healthy diet: fruit, vegetables, pulses and nuts (% in weight); fibre (g/100 g) and protein (g/100 g) A points scale from 0 to 5 is applied for each component. 4.1 British Food Standards Agency Nutrient Profile System (FSA-NPS) i Calculation of C points: C points Fruit, vegetables, pulses and nuts (% en peso) Fibre (g) Protein (g) 0 ≤40 ≤0.9 ≤1.6 1 >40 >0.9 >1.6 2 >60 >1.9 >3.2 3 - >2.8 >4.8 4 - >3.7 >6.4 5 >80 >4.7 >8.0 C points = fruit, vegetable, pulses and nut points [0-5] + fibre points [0-5] + protein points [0-5] = [0-15] AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products The final score is obtained by subtracting the C points from the A points, with the exception that if the A score is 11 or higher and the score obtained for the fruit and vegetables content is under 5 then the points for protein content will not be taken into account in the C points calculation. This prevents a high A score resulting from a high content of unfavourable components being compensated by protein content. The final score is obtained by subtracting the C points from the A points, with the exception that if the A score is 11 or higher and the score obtained for the fruit and vegetables content is under 5 then the points for protein content will not be taken into account in the C points calculation. This prevents a high A score resulting from a high content of unfavourable components being compensated by protein content. Calculation of final score: Calculation of final score: • A points ≥ 11 Fruit, vegetable points = 5 → Final score = A points - C points Fruit, vegetable points < 5 → Final score = A points - (fruit and vegetables points [0-5] + fibre points [0-5]) Fruit, vegetable points = 5 → Final score = A points - C points revista del comité científico nº 31 10 revista del comité científico nº 31 • A points < 11 Final score = A points - C points This calculation algorithm (see Annex I) is applied in the same manner for all products, with no spe- cific criteria for food and drink categories. It was initially developed to be applied in the regulation of advertising aimed at children with solid food products with a final score of 4 or more considered less healthy and, therefore, not suitable for use in advertising aimed at children. 4.1 British Food Standards Agency Nutrient Profile System (FSA-NPS) For drinks, products with a score of 1 or higher do not meet the criteria for advertising to children. The FSA algorithm is one of the most studied in the scientific literature and the most frequently validated (Labonté et al., 2018). It has been used extensively internationally for the development of different health policies, such as the regulation of health claims in Australia, New Zealand and South Africa; the implementation of FOPL in Australia, New Zealand and France and the regulation of advertising aimed at children in Ireland. a. FSA-NPS modification for cheese Most cheeses (73.3 %) were allocated the letter E, as in this group the A points are 11 or higher, meaning the protein content was not taken into account in the calculation of the final score. The HCSP took up the opinion of the FSA that proteins are a good indicator of the presence of cal- cium, and, on the other hand, cheeses are an important source of calcium for the French population (Coudray, 2011). Therefore, it was concluded that for the cheese group, the protein content should always be taken into account for the final score, regardless of the value of the A points. • A points ≥ 11 • A points ≥ 11 – Fruit, vegetable points = 5 → Final score = A points - C points – Fruit, vegetable points < 5 → Final score = A points - (fruit, vegetable points [0-5] + fibre points [0-5]) • A points < 11 or for cheeses → Final score = A points - C points development of Nutri-Score Regarding FOPL development, the use of a dichotomous scoring system (with binary scoring implying the notion of good and bad food products) may not be considered appropriate (Julia et al., 2014). Tak- ing this into account, Santé publique France, in collaboration with the University of Paris developed, based on the British FSA-NPS, five categories of nutritional quality for the purposes of ensuring a high discriminating power within each group of foods and drinks, while maintaining a central category to avoid classifying food products as good or bad. To facilitate citizens’ understanding, each category would be represented by a letter and a colour (five variations of the Nutri-Score logo) (Julia et al., 2015a). AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products To validate the FSA algorithm in the French context, the French Higher Council for Public Health (Haut Conseil de la Santé Publique, HCSP) conducted several studies (Julia et al., 2014, 2015b) to assess the capacity to adequately classify food and drinks into five nutritional quality categories in line with France’s food-based dietary guidelines. Using the Open Food Facts database on the composition of foods, which includes food regularly consumed by the French general public, the classification of food and drinks was consistent with nutritional recommendations: 95.4 % of fruit and vegetables were classified into the first quintile of distribution (letter A), while 86 % of “sugary snacks” were classified in the fourth and fifth quintiles (letters D and E). Moreover, variability was observed within each one of the food and drink groups, allowing for the discrimination of nutritional quality between groups of products and within them. However, for three groups of food products: cheeses, drinks and fats, the results of the classification into five categories were not consistent. Therefore, some adaptations to the initial scoring system were applied to ensure greater coherence with France’s dietary guidelines. 11 revista del comité científico nº 31 revista del comité científico nº 31 12 Table 3. Point allocation for fruit and vegetable content for drinks C points Drinks Fruit, vegetables (%) Fibre (g) Protein (g) 0 ≤40 ≤0.9 ≤1.6 1 - >0.9 >1.6 2 >40 >1.9 >3.2 3 - >2.8 >4.8 4 >60 >3.7 >6.4 5 - >4.7 >8.0 10 >80 - - () Sodium content corresponds to the salt content shown in the mandatory declaration divided by 2.5. b. FSA-NPS modification for drinks For drinks, the classification does not reflect French dietary guidelines, given that fruit juices obtained a more favourable score than water despite water being the only recommended drink. Moreover, the variability of the scoring for drinks is very low and it did not allow consistent identification of five categories. The modifications for the drinks group consist of a new table for the allocation of points for energy, sugar and fruit and vegetable content (Tables 2 and 3). AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Table 2. Point allocation for energy and sugar content for drinks per 100 ml A points Drinks Calories (kJ) Drinks Sugars (g) Saturated fatty acids (g) Sodium (mg)() 0 ≤0 ≤0 ≤1 ≤90 1 ≤30 ≤1.5 >1 >90 2 ≤60 ≤3 >2 >180 3 ≤90 ≤4.5 >3 >270 4 ≤120 ≤6 >4 >360 5 ≤150 ≤7.5 >5 >450 6 ≤180 ≤9 >6 >540 7 ≤210 ≤10.5 >7 >630 8 ≤240 ≤12 >8 >720 9 ≤270 ≤13.5 >9 >810 10 >270 >13.5 >10 >900 () S di t t d t th lt t t h i th d t d l ti di id d b 2 5 c. FSA-NPS modifications for fats In terms of fats, score range for saturated fat content did not allow for any discrimination between fats of animal and vegetable origin. 75.1 % of fats were classified with the letter E, regardless of origin. The fact that the maximum score for the content in saturated fat is reached with 10 g saturated fat/100 g of product would explain the lack of a distinction between the different types of fats. The modification applied for the group of fats consists of a new table for allocating points for saturated fats content, which is considered over total fat content (Table 4). The modification applied for the group of fats consists of a new table for allocating points for saturated fats content, which is considered over total fat content (Table 4). Table 4. Point allocation for content in saturated fats A points Calories (kJ) Sugars (g) Specific fat limits SFA/lipids (% in weight) Sodium (mg)() 0 ≤335 ≤4.5 <10 ≤90 1 >335 >4.5 <16 >90 2 >670 >9 <22 >180 3 >1005 >13.5 <28 >270 4 >1340 >18 <34 >360 5 >1675 >22.5 <40 >450 6 >2010 >27 <46 >540 7 >2345 >31 <52 >630 8 >2680 >36 <58 >720 9 >3015 >40 <64 >810 10 >3350 >45 ≥64 >900 () Sodium content corresponds to the salt content shown in the mandatory declaration divided by 2.5. 13 revista del comité científico nº 31 1 This proposal was drafted by the team at the Universidad Rovira i Virgili, Reus, Spain (Dr. Jordi Salas and Dr. Nancy Babio), in collaboration with the INSERM/INRA/University of Paris 13 team, which conducted the scientific validation work on Nutri-Score in relation to health (Dr. Serge Hercberg, Dr. Chantal Julia and Dr. Pilar Galán). d. Classification of food products according to final score obtained Depending on the final score obtained, food products are classified in five categories, each repre- sented by a colour and a letter according to the following table. Table 5. Classification of food products according to Nutri-Score Solid foods (points) Drinks (points) Nutri-Score -15 to 1 Water 0 to 2 ≤1 3 to 10 2 to 5 11 to 18 6 to 9 19 - 40 ≥10 Table 5. Classification of food products according to Nutri-Score Solid foods (points) Drinks (points) Nutri-Score -15 to 1 Water 0 to 2 ≤1 3 to 10 2 to 5 11 to 18 6 to 9 19 - 40 ≥10 AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products With regard to the adoption of Nutri-Score, the development of the five nutritional quality categories and the FSA nutritional profile modifications described above have been evaluated through valida- tion studies of several aspects, providing significant scientific support to this public health initiative adopted at the national level in France (Julia et al., 2014, 2015b) (Szabo de Edelenyi, 2019). Switzerland has already implemented it and Germany has submitted a notification to the European Commission. The Minister of Health, Consumer Affairs and Social Welfare announced in November 2018 Spain’s intention to adopt Nutri-Score. The application of Nutri-Score will be voluntary and its use in Spain shall require the submission of a notification to AESAN. Agri-food companies and distributors will therefore have the option of choosing whether or not to use the logo to inform consumers of the nutritional quality of their products. revista del comité científico nº 31 14 revista del comité científico nº 31 5. Modification of Nutri-Score for the inclusion of olive oil For the successful implementation of Nutri-Score in Spain, AESAN considered necessary to take into account specific aspects of the dietary guidelines given to the Spanish population different from the French guidelines. In this regard, the recommendation to consume olive oil as the main source of monounsaturated fatty acids in the Spanish diet must be noted and a very different nutritional con- sideration must be applied to olive oil as compared to other fats, in light of its status as the principal oil consumed in Spain. 5. Modification of Nutri-Score for the inclusion of olive oil For the successful implementation of Nutri-Score in Spain, AESAN considered necessary to take into account specific aspects of the dietary guidelines given to the Spanish population different from the French guidelines. In this regard, the recommendation to consume olive oil as the main source of monounsaturated fatty acids in the Spanish diet must be noted and a very different nutritional con- sideration must be applied to olive oil as compared to other fats, in light of its status as the principal oil consumed in Spain. The initial proposal integrated the quantity of fruit oil, principally virgin olive oil/extra virgin olive oil (VOO/EVOO) and walnut oil in the component “Fruit, vegetables, pulses and nuts” which is included in the positive points (C points) category for the calculation of the Nutri-Score. Therefore, this com- ponent would be amended to “Fruit, vegetables, pulses, nuts and fruit and nut oils”1. A modification was subsequently proposed which would only apply to olive oil, walnut oil and rapeseed oil, and not all fruit and nut oils. This modification is based on the scientific evidence of the health benefits of consuming olive oil (Reis de Souza, 2013), considering it is the most widely consumed oil in Spain and the fact that rapeseed and walnut oils are also recommended in French dietary guidelines. This modification allows for Nutri-Score to be applied to oil itself in a harmonised and uniform manner across all the countries that adopt the system. Thus, olive oil will obtain the maximum points (content in olive oil of 100 %) and can be distinguished from other oils such as sunflower, palm or coconut oils. This modified proposal includes olive oil and not just VOO/EVOO. This decision is because for the other components in the “Fruit, vegetables, pulse and nuts” group, extraction method is not taken into account in calculating the Nutri-Score. Nevertheless, and even though Nutri-Score does not AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products establish any differences between olive oil on the one hand and virgin and extra virgin olive oil on the other, it must be borne in mind at all times that the latter have a very beneficial impact on cardio- vascular health and risk factors, thanks to their content in components other than oleic acid, such as phenolic compounds. 15 revista del comité científico nº 31 15 revista del comité científico nº 31 Tabla 6. Points table incorporating of olive oil Points Fruit, vegetables, pulses, nuts and olive, walnut and rapeseed oil (% in weight) Fibre (g/100 g) Protein (g/100 g) 0 ≤40 ≤0.9 ≤1.6 1 >40 >0.9 >1.6 2 >60 >1.9 >3.2 3 - >2.8 >4.8 4 - >3.7 >6.4 5 >80 >4.7 >8 Subtotal 0-5 (a) 0-5 (b) 0-5 (c) Total (0-15) P points = (a) + (b) + (c) With this modified Nutri-Score proposal, olive oil, rapeseed oil and walnut oil will benefit from a C Nutri-Score (the most favourable score for an oil). Moreover, products that contain olive oil could also benefit, if the content in fruit, vegetables, pulses and nuts and olive, walnut or rapeseed oil exceeds 40 %. 5. Modification of Nutri-Score for the inclusion of olive oil For the successful implementation of Nutri-Score in Spain, AESAN considered necessary to take into account specific aspects of the dietary guidelines given to the Spanish population different from the French guidelines. In this regard, the recommendation to consume olive oil as the main source of monounsaturated fatty acids in the Spanish diet must be noted and a very different nutritional con- sideration must be applied to olive oil as compared to other fats, in light of its status as the principal oil consumed in Spain. These effects have been described in numerous scientific publications including multi-centre trials, such as the PREDIMED study (Estruch et al., 2018), which associates the Mediterranean diet with a 30 % reduction in major cardiovascular events (heart attack, stroke or death from cardiovascular causes), and meta-analyses than link consumption of extra virgin olive oil with a significant improvement in total cholesterol, HDL cholesterol, inflammatory markers and blood pressure (George et al., 2018). The specific recommendation promoting VOO/EVOO forms part of the communication actions around the dietary guidelines implemented in each country. 5.1 Publication of the rule modifying the calculation of Nutri-Score In accordance with these considerations, on 5 September 2019, the Decree amending the form of complementary display of the nutritional declaration recommended by the State was published in the Journal Officiel de la République Française (JORF, 2019). Along with other modifications, reference to “rapeseed, walnut and olive oils” is included in the “Fruit, vegetables, pulses and nuts” category. Thus, in the calculation of the Nutri-Score, the component considered is “Fruit, vegetables, pulses, nuts and olive, walnut and rapeseed oil”. This involves the updating to the Nutri-Score usage regulation and the frequently asked questions document developed by Santé publique France. AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products nclusions of the Scientific Committee with respect to the suitability of the adaptation of the The conclusions of the Scientific Committee with respect to the suitability of the adaptation of the Nutri-Score system incorporating olive oil content into the calculation are the following: The conclusions of the Scientific Committee with respect to the suitability of the adaptatio Nutri-Score system incorporating olive oil content into the calculation are the following: core system incorporating olive oil content into the calculation are the following: • The European Union has not established a harmonised model for front-of-pack labelling and therefore Member States decide this matter. • Neighbouring countries that currently have some form of front-of-pack labelling are the follow- ing: France, Switzerland and Belgium (Nutri-Score), the United Kingdom (Traffic light), Sweden, Norway, Denmark, Iceland, Lithuania and North Macedonia (Keyhole or Nordic Keyhole) and the Netherlands (FOP Choices). These systems have been the subject of debate within the European Commission over the last 2 years. revista del comité científico nº 31 16 revista del comité científico nº 31 16 • Spain has always expressed a position in favour of harmonisation at EU level for front-of-pack nutritional labelling. • Spain has always expressed a position in favour of harmonisation at EU level for front-of-pack nutritional labelling. • The Scientific Committee considers that AESAN’s Nutri-Score adaptation proposal for the inclu- sion of olive oil in the “Fruit, vegetables, pulses and nuts” group and the resulting positive consideration in the Nutri-Score calculation improves the consideration of a product that has nutritional benefits based on its oleic acid content and constitutes an improvement of said front- of-pack nutrition labelling system. • The Scientific Committee believes that the correct wording to apply to the included oils would be: “olive oil, walnut oil and rapeseed oil” and under no circumstances should reference be made to “olive oils”. • The Scientific Committee suggests that, without prejudice to the numerical calculation, a sepa- rate column be created, not within the current fruit and vegetables column (Table 7). • The Scientific Committee suggests that, without prejudice to the numerical calculation, a sepa- rate column be created, not within the current fruit and vegetables column (Table 7). Table 7. • It is necessary to continue to identify other aspects that may need to be incorporated into the Nutri-Score system for the nutritional assessment of foods and drinks characteristic of the Spanish diet. References Coudray, B. (2011). The Contribution of Dairy Products to Micronutrient Intakes in France. Journal of the Ameri- can College of Nutrition, 30 (5 Suppl 1), pp: 410S-404S. Doi: 10.1080/07315724.2011.10719984. Estruch, R., Ros, E., Salas-Salvadó, J., Covas, M.I., Corella, D., Arós, F., Gómez-Gracia, E., Ruiz-Gutiérrez, V., Fiol, M., Lapetra, J., Lamuela-Raventos, R.M., Serra-Majem, L., Pintó, X., Basora, J., Muñoz, M.A., Sorlí, J.V., Martí- nez, J.A., Fitó, M., Gea, A., Hernán, M.A. and Martínez-González, M.A. PREDIMED Study Investigators. (2018). Primary Prevention of Cardiovascular Disease with a Mediterranean Diet Supplemented with Extra-Virgin Olive Oil or Nuts. The New England Journal of Medicine, 378 (25), e34. https://doi.org/10.1056/NEJMoa1800389. EU (2006). Regulation (EC) No. 1924/2006 of the European Parliament and of the Council of 20 December 2006 on nutrition and health claims made on foods. OJ L 404 of 30 December 2006, pp: 9-25. 17 revista del comité científico nº 31 EU (2011). Regulation (EC) No. 1169/2011 of the European Parliament and of the Council of 25 October 2011on the provision of food information to consumers. OJ L 304 of 22 November 2011, pp: 8-63. EU (2012). Commission Regulation (EU) No. 432/2012 of 16 May 2012 establishing a list of permitted health claims made on foods, other than those referring to the reduction of disease risk and to children’s development and health. OJ L 136 of 25 May 2012, pp: 1-40. EU (2014). EU Action Plan on Childhood Obesity 2014-2020. Available at: https://ec.europa.eu/health/sites/health/ files/nutrition_physical_activity/docs/childhoodobesity_actionplan_2014_2020_en.pdf [accessed: 01-03-20]. EU (2014). EU Action Plan on Childhood Obesity 2014-2020. Available at: https://ec.europa.eu/health/sites/health/ files/nutrition_physical_activity/docs/childhoodobesity_actionplan_2014_2020_en.pdf [accessed: 01-03-20]. FSA (2011). Nutrient profiling Technical Guidance. Available at: https://www.gov.uk/government/publications/ th t i t fili d l [ d 01 03 20] FSA (2011). Nutrient profiling Technical Guidance. Available at: https://www.gov.uk/government/publications/ the-nutrient-profiling-model [accessed: 01-03-20]. George, E.S., Marshall, S., Mayr, H.L., Trakman, G.L., Tatucu-Babet, O.A., Lassemillante, A.C.M., Bramley, A., Reddy, A.J., Forsyth, A., Tierney, A.C., Thomas, C.J., Itsiopoulos, C. and Marx, W. (2018). The effect of high- polyphenol extra virgin olive oil on cardiovascular risk factors: A systematic review and meta-analysis. Cri- tical reviews in food science and nutrition, 59 (17), pp: 2772-2795. https://doi.org/10.1186/1476-511X-13-154. JORF (2017). Journal Officiel de la République Française. Ministère des Solidarités et de la Santé. Arrêté du 31 octobre 2017 fixant la forme de présentation complémentaire à la déclaration nutritionnelle recommandée par l’Etat en application des articles L. 3232-8 et R. 3232-7 du code de la Santé Publique. JORF (2019). nclusions of the Scientific Committee with respect to the suitability of the adaptation of the Suggested scoring table presentation for the inclusion of olive oil in Nutri-Score Points Fruit, vegetables, pulses and nuts (% in weight) Olive oil, walnut oil and rapeseed oil (g/100 g o 100 ml) Fibre (g/100 g) Protein (g/100 g) 0 ≤40 ≤0.9 ≤1.6 1 >40 >0.9 >1.6 2 >60 >1.9 >3.2 3 - >2.8 >4.8 4 - >3.7 >6.4 5 >80 >4.7 >8 Subtotal 0-5 (a) 0-5 (b) 0-5 (c) Total (0-15) P points = (a) + (b) + (c) • It is necessary to continue to identify other aspects that may need to be incorporated into the Nutri-Score system for the nutritional assessment of foods and drinks characteristic of the Spanish diet. • It is necessary to continue to identify other aspects that may need to be incorporated into the Nutri-Score system for the nutritional assessment of foods and drinks characteristic of the Spanish diet. AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products References Journal Officiel de la République Française. Ministère des Solidarités et de la Santé. Arrêté du 30 août 2019 modifiant l’arrêté du 31 octobre 2017 fixant la forme de présentation complémentaire à la décla- ration nutritionnelle recommandée par l’Etat en application des articles L. 3232-8 et R. 3232-7 du code de la Santé Publique. Julia, C., Ducrot, P., Lassale, C., Fézeu, L., Méjean, C., Péneau, S., Touvier, M., Hercberg, S. and Kesse-Guyot, E. (2015a). Prospective associations between a dietary index based on the British Food Standard Agency nu- trient profiling system and 13-year weight gain in the SU.VI.MAX cohort. Preventive Medicine, 81, pp: 189-194. https://doi.org/10.1016/j.ypmed.2015.08.022. Julia, C., Ducrot, P., Péneau, S., Deschamps, V., Méjean, C., Fézeu, L., Touvier, M., Hercberg, S. and Kesse-Guyot, E. (2015b). Discriminating nutritional quality of foods using the 5-Color nutrition label in the French food mar- ket: Consistency with nutritional recommendations. Nutrition Journal, 14, 100. https://doi.org/10.1186/s12937- 015-0090-4. Julia, C., Kesse-Guyot, E., Touvier, M., Méjean, C., Fezeu, L. and Hercberg, S. (2014). Application of the British Food Standards Agency nutrient profiling system in a French food composition database. The British Journal of Nutrition, 112 (10), pp: 1699-1705. https://doi.org/10.1017/S0007114514002761. Kelly, B. and Jewell, J. (2018). What is the evidence on the policy specifications, development processes and effectiveness of existing front-of-pack food labelling policies in the WHO European Region? Copenhagen: WHO Regional Office for Europe; Health Evidence Network (HEN) synthesis report 61. Labonté, M.È., Poon, T., Gladanac, B., Ahmed, M., Franco-Arellano, B., Rayner, M. and L’Abbé, M.R. (2018). Nu- trient Profile Models with Applications in Government-Led Nutrition Policies Aimed at Health Promotion and AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Noncommunicable Disease Prevention: A Systematic Review. Advances in Nutrition, 9 (6), pp: 741-788. https:// doi.org/10.1093/advances/nmy045. Noncommunicable Disease Prevention: A Systematic Review. Advances in Nutrition, 9 (6), pp: 741-788. https:// doi.org/10.1093/advances/nmy045. Moniteur Belge (2019). Service Public Federal Santé Publique, Securite de la Chaine Alimentaire et Environne- ment. 1er Mars 2019. Arrêté royal relatif à l’utilisation du logo «Nutri-Score». Moniteur Belge (2019). Service Public Federal Santé Publique, Securite de la Chaine Alimentaire et Environne- ment. 1er Mars 2019. Arrêté royal relatif à l’utilisation du logo «Nutri-Score». Reis de Souza, P., Crema-Peghini, B., Santana da Silva, J. and Ribeiro Cardoso, C. (2013). An Overview of the Mo- dulatory Effects of Oleic Acid in Health and Disease. References Mini Reviews in Medicinal Chemistry, 13 (2), pp: 201-210. Reis de Souza, P., Crema-Peghini, B., Santana da Silva, J. and Ribeiro Cardoso, C. (2013). An Overview of the Mo- dulatory Effects of Oleic Acid in Health and Disease. Mini Reviews in Medicinal Chemistry, 13 (2), pp: 201-210. Szabo de Edelenyi, F., Egnell, M., Galan, P., Druesne-Pecollo, N., Hercberg, S. and Julia, C. (2019). Ability of the Reis de Souza, P., Crema-Peghini, B., Santana da Silva, J. and Ribeiro Cardoso, C. (2013). An Overview of the Mo- dulatory Effects of Oleic Acid in Health and Disease. Mini Reviews in Medicinal Chemistry, 13 (2), pp: 201-210. Szabo de Edelenyi, F., Egnell, M., Galan, P., Druesne-Pecollo, N., Hercberg, S. and Julia, C. (2019). Ability of the Nutri-Score front-of-pack nutrition label to discriminate the nutritional quality of foods in the German food market and consistency with nutritional recommendations. Archives of Public Health, 77, pp: 28. https://doi. org/10.1186/s13690-019-0357-x. revista del comité científico nº 31 18 WHO (2014). World Health Organization. European Food and Nutrition Action Plan 2015-2020. Available at: http:// www.euro.who.int/en/publications/abstracts/european-food-and-nutrition-action-plan-20152020-2014 [accessed: 01-03-20]. WHO (2014). World Health Organization. European Food and Nutrition Action Plan 2015-2020. Available at: http:// www.euro.who.int/en/publications/abstracts/european-food-and-nutrition-action-plan-20152020-2014 [accessed: 01-03-20]. WHO (2014). World Health Organization. European Food and Nutrition Action Plan 2015-2020. Available at: http:// www.euro.who.int/en/publications/abstracts/european-food-and-nutrition-action-plan-20152020-2014 [accessed: 01-03-20]. WHO (2018). World Health Organization. Non communicable diseases. Available at: https://www.who.int/es/ news-room/fact-sheets/detail/noncommunicable-diseases [accessed: 01-03-20]. WHO (2018). World Health Organization. Non communicable diseases. Available at: https://www.who.int/es/ news-room/fact-sheets/detail/noncommunicable-diseases [accessed: 01-03-20]. AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Annex I. Calculation of Nutri-Score: Flowchart FLOWCHART FOR CALCULATION OF NUTRI-SCORE Food product with mandatory nutritional information and list of ingredients. Calculation of unfavourable points (A POINTS) and favourable points (C POINTS) Unfavourable components (A points): Energy, sugars, saturated fat, salt Favourable components (C points): Fruit, vegetables, pulses and nuts and olive, rapeseed and walnut oil; Proteins; Fibre. Is the food product a drink or added fat? YES NO Final score calculation: ¿A points < 11 or CHEESE product? Points for Fruit, vegetables, nuts, pulses, olive oil, walnut oil, rapeseed oil ≥ 5? References Final score: A points - C points Final score: A i t C i t ( t ti YES NO YES NO GENERAL TABLES for calculation of unfavourable components (A POINTS) and favourable components (C POINTS) TABLES for calculation of unfavourable components (A POINTS) and favourable components (C POINTS C) for DRINKS or ADDED FATS AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products AESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products ESAN Scientific Committee: Implementation in Spain of the Nutri-Score information system regarding the nutritional quality of food products Food product with mandatory nutritional information and list of ingredients. Calculation of unfavourable points (A POINTS) and favourable points (C POINTS) Food product with mandatory nutritional information and list of ingredients. Calculation of unfavourable points (A POINTS) and favourable points (C POINTS) Unfavourable components (A points): Energy, sugars, saturated fat, salt Favourable components (C points): Fruit, vegetables, pulses and nuts and olive, rapeseed and walnut oil; Proteins; Fibre. Is the food product a drink or added fat? YES NO Final score calculation: ¿A points < 11 or CHEESE product? Points for Fruit, vegetables, nuts, pulses, olive oil, walnut oil, rapeseed oil ≥ 5? Final score: A points - C points Final score: A points - C points (not counting points for proteins) Final score: Allocation of a colour and letter YES NO YES NO GENERAL TABLES for calculation of unfavourable components (A POINTS) and favourable components (C POINTS) TABLES for calculation of unfavourable components (A POINTS) and favourable components (C POINTS C) for DRINKS or ADDED FATS 19 revista del comité científico nº 31 p p gy, g , , urable components (C points): Fruit, vegetables, pulses and nuts and olive, rapeseed and walnut oil; eins; Fibre. Is the food product a drink or added fat? NO GENERAL TABLES for calculation of unfavourable components (A POINTS) and favourable components (C POINTS) TABLES for calculation of unfavourable components (A POINTS) and favourable components (C POINTS C) for DRINKS or ADDED FATS Final score calculation: ¿A points < 11 or CHEESE product? Points for Fruit, vegetables, nuts, pulses, olive oil, walnut oil, rapeseed oil ≥ 5? YES Final score: A points - C points YES NO Final score: A points - C points Final score: Allocation of a colour and letter
https://openalex.org/W3089522984	https://ieeexplore.ieee.org/ielx7/6287639/8948470/09200635.pdf	English	null	Robustness of Complex Networks Considering Attack Cost	IEEE access	2,020	cc-by	4,788	I. INTRODUCTION that scale-free networks are robust against random attack but fragile to intentional attack [13]. In such studies, the intentional attack is conducted on hubs, which are supposed to be the most important nodes in the network, such as those with the highest degrees. Obviously, the removal of these nodes makes the network disconnected easily. In comparison, the homogeneous random graph is robust to both random failure and intentional attack. Large-scale infrastructure networks, such as the Internet, transportation networks, power grids play important roles in our society [1]–[3]. In order to improve their performance and avoid unexpected damages, either due to random failures or intentional attacks, it is essential to study the robustness of such infrastructure networks. However, these networks usually have large scales and complex structures, which make the modeling and analysis difﬁcult. In the above studies, it is assumed that the attacker pays the same cost to remove one node, no matter it is the hub or a remote unimportant node. In the real case, however, the removal of different node usually costs differently, and the intentional removal of a hub usually costs much more than the removal of an unimportant node. Based on this fact, some researchers studied different attack strategies with the consideration of attack cost. Zheng et al. [14] found that, when the attack cost is taken into account, the scale-free networks may be robust against intentional attacks. This ﬁnd- ing is quite different from the famous ‘‘robust-yet-fragile’’ property of scale-free networks, as the latter did not con- sider the attack cost. Hong et al. [15] considered different attack strategies including the high-degree removal strategy (HDRS), low-degree removal strategy (LDRS), and the ran- dom removal strategy (RRS), and studied how the total attack cost and network assortativity coefﬁcient affect the performance of different attack strategies. Zhang et al. [16] Recent progress in network science provides an efﬁcient way for the study on the robustness of complex infrastructure networks [4]–[6]. Network science focuses on the structural properties of networks. Although different networks have different properties, they all can be demonstrated by structural elements such as nodes and links. More interestingly, many distinct networks have quite similar structural properties. And it has been shown that these properties are vital to determine the dynamics performed in the network. The study on the robustness of complex networks has attracted much attention in the past two decades [7]–[12]. The associate editor coordinating the review of this manuscript and approving it for publication was Yang Tang . Received August 23, 2020, accepted September 8, 2020, date of publication September 18, 2020, date of current version September 30, 2020. Received August 23, 2020, accepted September 8, 2020, date of publication September 18, 202 date of current version September 30, 2020. Digital Object Identifier 10.1109/ACCESS.2020.3024942 Digital Object Identifier 10.1109/ACCESS.2020.3024942 CHENGWANG WANG AND YONGXIANG XIA , (Senior Member, IEEE) School of Communication Engineering, Hangzhou Dianzi University, Hangzhou 310018, China Corresponding author: Yongxiang Xia (xiayx@hdu.edu.cn) This work was supported in part by the Laboratory of Science and Technology on Integrated Logistics Support, China. CHENGWANG WANG AND YONGXIANG XIA , (Senior Member, IEEE) School of Communication Engineering, Hangzhou Dianzi University, Hangzhou 310018, China Corresponding author: Yongxiang Xia (xiayx@hdu.edu.cn) This work was supported in part by the Laboratory of Science and Technology on Integrated Logistics Support, China. This work was supported in part by the Laboratory of Science and Technology on Integrated Logistics Support, China. ABSTRACT Robustness of complex networks has attracted much attention in diverse disciplines. Most of previous studies did not consider the attack cost. In this paper, the network robustness is studied with the consideration of attack cost, with different complex network models and different robustness indices. It is found that with different cost function and attack budget, the best attack strategy changes. When the budget is low and attacking hubs costs much more than attacking an unimportant node, then the high-degree attack (HDA) strategy performs worse than the low-degree attack (LDA) strategy. On the contrary, HDA is always a better strategy when the budget is high. Therefore, there is an intersection before and after that different attack strategies perform better. The position of this intersection is affected by the network structure, robustness index, cost function and the budget. under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ VO Received August 23, 2020, accepted September 8, 2020, date of publication September 18, 2020, date of current version September 30, 2020. I. INTRODUCTION (b) the budget p at the intersection, as a function of a. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. When considering the attack cost, there are actually two issues. One is the cost to remove an individual node. Obviously, the removal of a hub node should cost more than the removal of an unimportant node. Another issue is the total cost the attacker can pay. In this paper, we will consider both these two issues and study how these two issues affect the robustness of networks. The rest of paper is organized as follows. In section II, the complex network models, the cost formulation and the robustness index are given. In section III, simulated results and discussions are provided. Finally, the work is concluded in section IV. FIGURE 2. The intersection between HDA and LDA curves in figure 1. (a) the relative size of the largest connected component G at the intersection, as a function of a. (b) the budget p at the intersection, as a function of a. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. II. MODEL A. NETWORK MODELS I. INTRODUCTION One common method is that the attacker is supposed to remove a proportion of nodes, and the robustness is measured by the connectivity of the rest network. It has been shown 172398 172398 172398 VOLUME 8, 2020 C. Wang, Y. Xia: Robustness of Complex Networks Considering Attack Cost FIGURE 1. The relative size of the largest connected component G when considering the attack cost. (a) a=0; (b) a=0.1; (c) a=0.2; (d) a=0.3; (e) a=0.4; (f) a=0.5; (g) a=0.6; (h) a=0.7; (i) a=0.8; (j) a=0.9; (k) a=1. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. FIGURE 1. The relative size of the largest connected component G when considering the attack cost. (a) a=0; (b) a=0.1; (c) a=0.2; (d) a=0.3; (e) a=0.4; (f) a=0.5; (g) a=0.6; (h) a=0.7; (i) a=0.8; (j) a=0.9; (k) a=1. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. FIGURE 2. The intersection between HDA and LDA curves in figure 1. (a) the relative size of the largest connected component G at the intersection, as a function of a. (b) the budget p at the intersection, as a function of a. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. designed an optimization algorithm to defend the network from attacks when the cost is considered. When considering the attack cost, there are actually two issues. One is the cost to remove an individual node. Obviously, the removal of a hub node should cost more than the removal of an unimportant node. Another issue is the total cost the attacker can pay. In this paper, we will consider both these two issues and study how these two issues affect the robustness of networks. The rest of paper is organized as follows. In section II, the complex network models, the cost formulation and the robustness index are given. In section III, simulated results and discussions are provided. Finally, the work is concluded in section IV. II. MODEL A. NETWORK MODELS Two network models are considered in this paper. One is the Barabasi-Albert (BA) scale-free network [17]–[19], and the FIGURE 2. The intersection between HDA and LDA curves in figure 1. (a) the relative size of the largest connected component G at the intersection, as a function of a. II. MODEL A. NETWORK MODELS Each point is the average of 10 runs. B. ATTACK COST When the attacker removes a node from the network, a cost has to be paid. Generally speaking, the more important the node is the higher cost the attacker has to pay. In order to investigate the impact of attack cost on the robustness of network, deﬁne the cost to remove node i as Y i = ki P i ki (1) Yi = ka i (2) (2) (1) where a is the cost factor. When a = 0, then the cost to remove different nodes is the same. This is equivalent to the traditional study where no cost is considered. Usually a should be positive, indicating the fact that removing a more important node costs more. where ki is the degree of node i. Repeat this growth process until the number of nodes reaches N. Denote the average degree of node as <k>. 1) BA SCALE-FREE NETWORK Initially there are m0 nodes in the network. They are fully connected from one to another. Then at each time step, one node is added into the network, and this new node is con- nected to m existing nodes. The connection is based on a preferential rule. More speciﬁcally, the probability that a new node is connected to an existing node i is II. MODEL A. NETWORK MODELS designed an optimization algorithm to defend the network from attacks when the cost is considered. designed an optimization algorithm to defend the network from attacks when the cost is considered. Two network models are considered in this paper. One is the Barabasi-Albert (BA) scale-free network [17]–[19], and the Two network models are considered in this paper. One is the Barabasi-Albert (BA) scale-free network [17]–[19], and the 172399 C. Wang, Y. Xia: Robustness of Complex Networks Considering Attack Cost FIGURE 3. The relative size of the largest connected component G when considering the attack cost. (a) a=0; (b) a=0.2; (c) a=0.4; (d) a=0.6; (e) a=0.8; (f) a=1. ER random graph with N=1000 and <k>=4 is considered. Each point is the average of 10 runs. FIGURE 3. The relative size of the largest connected component G when considering the attack cost. (a) a=0; (b) a=0.2; (c) a=0.4; (d) a=0.6; (e) a=0.8; (f) a=1. ER random graph with N=1000 and <k>=4 is considered. Each point is the average of 10 runs. FIGURE 4. The intersection between HDA and LDA curves in figure 3. (a) the relative size of the largest connected component G at the intersection, as a function of a. (b) the budget p at the intersection, as a function of a. ER random graph with N=1000 and <k>=4 is considered. Each point is the average of 10 runs. other is the Erdos-Renyi (ER) random graph [20], [21]. These two network models show quite different characteristics in structure. The BA scale-free network model has the charac- teristics of growth and preferential attachment. As a result, the hub nodes have a large number of connections whereas most of other nodes only have few links. In this way, different nodes take quite different roles in the network. So, the BA scale-free network model generates heterogeneous network. In comparison, the nodes of the ER random graph model are connected with the same probability. Therefore, the ER random graph generates homogeneous network, where all nodes show identical property statistically. These two models are described in detail as follows. FIGURE 4. The intersection between HDA and LDA curves in figure 3. g (a) the relative size of the largest connected component G at the intersection, as a function of a. (b) the budget p at the intersection, as a function of a. ER random graph with N=1000 and <k>=4 is considered. III. SIMULATION RESULTS Figure 1 shows how the relative size of the largest con- nected component G of the BA network changes with the total attack budget p. It shows that, with higher budget, the attacker always can make more serious damage to the network, no matter what kind of attack strategy is applied. On the other hand, different attack strategies do have dif- ference. The best attack strategy is always one of the two intentional attack strategies, not RA. Take subﬁgure (a) as an example. The cost factor a=0 in this subﬁgure, which means that the cost to remove different node is the same. Under this condition, it is obviously better to attack the high-degree nodes, which makes the network break quickly. So HDA performs the best among three strategies. On the contrary, if the removal begins from the low-degree nodes, then most of survival nodes are still connected. So LDA performs badly. The performance of RA is between HDA and LDA. FIGURE 6. The intersection between HDA and LDA curves in figure 5. (a) the network efficiency E at the intersection, as a function of a. (b) the budget p at the intersection, as a function of a. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. 6. The intersection between HDA and LDA curves in figure 5 a higher degree, whereas LDRS deﬁnes a higher probability to remove the node with a lower degree. In comparison, HDA removes nodes strictly in a descending order of degree, and LDA removes nodes strictly in an ascending order of degree. p More interestingly, with the change of cost factor a, RA curve almost unchanged, but the performance of inten- tional attack strategies changes accordingly. Take the last subﬁgure as an example, where a = 1. According to equation (2), the cost to remove a node is proportional to the node’s degree when a = 1. And we know that the degree distribution of BA network is heterogeneous, which means that a few nodes have a large number of links while most of nodes have only few connections. Thus, the cost to remove a hub is many times higher than the cost to remove a normal node. Then, removal of high-degree nodes may be not a good idea especially when the budget is tight. C. ROBUSTNESS INDEX Two indices are considered to show the robustness of net- works. III. SIMULATION RESULTS This is because when the budget is tight, HDA can only remove few hubs, which has litter effect to the whole network. In comparison, LDA can remove a large number of nodes with the same budget. Although each of them is not an important node, the removal of a large number of these unimportant nodes may make the network broken. In the subﬁgure, when the budget p is low, the LDA curve is the lowest among three curves, followed by the RA curve. Of course, if the budget is higher, HDA still has its advantage. Therefore, in the subﬁgure, an intersection can be seen between LDA and HDA curves. This intersection is important, since before and after it different attack strategy takes the lead. y g g In most cases, the attacker has limited budget and can only attack a proportion of nodes, no matter what kind of attack strategy is used. To show the limited budget, we deﬁne p = P i∈Z ka i PN i=1 ka i (3) (3) where Z is the set of removed nodes. p gives the budget the attacker can use. 2) ER RANDOM GRAPH After deﬁning the cost to remove a node, the attacker can conduct the attack and remove nodes. Here we consider three node attack strategies, i.e., HDA, LDA and RA. The detailed attack strategies are described as follows. Initially there are N isolated nodes without any connection between each other. Then the links are randomly added between nodes, until the average degree reaches <k>. 172400 VOLUME 8, 2020 VOLUME 8, 2020 C. Wang, Y. Xia: Robustness of Complex Networks Considering Attack Cost FIGURE 5. The network efficiency E when considering the attack cost. (a) a=0; (b) a=0.1; (c) a=0.2; (d) a=0.3; (e) a=0.4; (f) a=0.5; (g) a=0.6; (h) a=0.7; (i) a=0.8; (j) a=0.9; (k) a=1; (l) a=1.1; (m) a=1.2. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. FIGURE 5. The network efficiency E when considering the attack cost. (a) a=0; (b) a=0.1; (c) a=0.2; (d) a=0.3; (e) a=0.4; (f) a=0.5; (g) a=0.6; (h) a=0.7; (i) a=0.8; (j) a=0.9; (k) a=1; (l) a=1.1; (m) a=1.2. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. 1. High-degree attack (HDA) strategy: Sort the nodes in a descending order of their degrees. Then remove the nodes sequentially. 3. Random attack (RA) strategy: Remove the nodes randomly. 3. Random attack (RA) strategy: Remove the nodes randomly. It should be noted that our deﬁnitions of HDA and LDA are different from those for HDRS and LDRS. In Ref. [15], a node is removed with a probability related to its degree. HDRS deﬁnes a higher probability to remove the node with 2. Low-degree attack (LDA) strategy: Sort the nodes in an ascending order of their degrees. Then remove the nodes sequentially. 172401 VOLUME 8, 2020 C. Wang, Y. Xia: Robustness of Complex Networks Considering Attack Cost FIGURE 6. The intersection between HDA and LDA curves in figure 5. (a) the network efficiency E at the intersection, as a function of a. (b) the budget p at the intersection, as a function of a. BA scale-free network with N=1000 and <k>≈4 is considered. Each point is the average of 10 runs. III. SIMULATION RESULTS 1) THE RELATIVE SIZE OF THE LARGEST CONNECTED COMPONENT After the attack, the network will be disintegrated into pieces. The largest connected component is the connected piece with the largest number of nodes within it. It shows the best part where the revival nodes can still communicate with each other. So its relative size can be used to measure the robust- ness of network [22] G = N ′ N (4) (4) Comparing different subﬁgures in ﬁgure 1, the intersection moves. To show it more clearly, we plot how the values of G and p at the intersection change with a in ﬁgure 2. It can be seen that both of them are monotone. Since the intersection appears when a ≥0.5 in Fig. 1, the curves in Fig. 2 begin at a = 0.5. where N ′ is the number of nodes in the largest connected component after the attack. 2) NETWORK EFFICIENCY The efﬁciency of the network is deﬁned as [23] The efﬁciency of the network is deﬁned as [23] Similar results can be seen in ER random graph, as shown in ﬁgure 3. And since ER random graph is a homogeneous network, the advantage of HDA is taken over more easily as the intersection appears when a is still small. Figure 4 shows how the values of G and p at the intersection change with a. The results in both networks show that LDA leads HDA for a longer time when the cost gap between removal high-degree node and low-degree node is larger (i.e., when a is larger). E = 1 N(N −1) X i̸=j 1 dij (5) (5) where dij is the shortest path length between node i and node j. Compared with the largest connected component, the efﬁ- ciency takes each and every node into account, instead of those in the largest piece. So this index shows the character- istic of the whole survival network after attack. VOLUME 8, 2020 VOLUME 8, 2020 172402 C. Wang, Y. Xia: Robustness of Complex Networks Considering Attack Cost FIGURE 7. The network efficiency E when considering the attack cost. (a) a=0; (b) a=0.2; (c) a=0.4; (d) a=0.6; (e) a=0.8; (f) a=1. ER random graph with N=1000 and <k>=4 is considered. Each point is the average of 10 runs. FIGURE 7. The network efficiency E when considering the attack cost. (a) a=0; (b) a=0.2; (c) a=0.4; (d) a=0.6; (e) a=0.8; (f) a=1. ER random graph with N=1000 and <k>=4 is considered. Each point is the average of 10 runs. IV. CONCLUSION In the study on network robustness, the largest connected component is often used to measure the performance. However, it only shows the largest connected part of the survival network. To show the whole picture, the network efﬁciency is a suitable index, as it takes each and every node into account. Figures 5-8 give the network efﬁciency of BA scale-free network and ER random graph after attack. The intersection between HDA and LDA can be observed in these ﬁgures. Comparing to the intersections in ﬁgures 1-4, the intersections for the network efﬁciency appears late. Take the BA scale-free network as an example. The intersection appears when a is greater than 0.5 for the largest connected component (see ﬁgure 2), whereas it appears only when a is greater than 0.9 for the network efﬁciency (see ﬁgure 6). During 0.5 < a < 0.9, HDA seems to be a better attack strategy if the efﬁciency of the whole network is the key concern, whereas LDA becomes the better strategy if the aim of attack is to reduce the largest connected component. As for RA, the change of a has little impact on the curve. There are different strategies to attack a complex network. Then the key issue is to ﬁnd a better one. From the study in this paper, it is shown that the better strategy may change with conditions such as the cost function for the attack, the budget the attacker has, the network structure the attacker is facing, the performance measure the attacker concerns about, and so on. More speciﬁcally, when the attack cost is taken into account, attacking the network from the hubs may not be always a good strategy, since hubs cost much higher than most of other nodes. 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https://openalex.org/W3035543664	https://pure.eur.nl/ws/files/48449400/Repub_127778_O-A.pdf	English	null	Economic city branding and stakeholder involvement in China: Attempt of a medium-sized city to trigger industrial transformation	Cities	2,020	cc-by	16,908	A R T I C L E I N F O Among known studies of city branding by Chinese megacities to realise urban transformation, there is no ex- plorative study of how smaller Chinese cities engage in city branding and attempt to trigger industrial trans- formation. In response, this article presents an in-depth case study of city branding processes in a medium-sized Chinese city. Roles, resources and interactions among the city's stakeholders are analysed during the brand creation and implementation stages in two different economic city branding projects. The stakeholder in- volvement mechanisms we identify confirm that city branding creation in China primarily follows political rather than business channels. Chinese local public authorities and more specifically key politicians, depart- ments, and public enterprises are core stakeholders in branding creation. However, this leads to challenges in the branding implementation, since key public sector players tend to withdraw themselves when implementation begins, leaving previously uninvolved private (and public) players to implement the brands. The unsuccessful transformation contrasts with those observed in Chinese megacities, where involvement of powerful corpora- tions and support from higher levels of government are both much higher. It appears that the imperative of broad stakeholder involvement to make city branding successful as we know it for Western cities may also apply in medium-sized Chinese cities. Keywords: Economic city branding Industrial transformation Stakeholder involvement Brand creation and implementation China Medium-sized city Wenting Maa,b,⁎, Martin de Jongb,c, Mark de Bruijnea, Daan Schravend a Department of Multi-Actor Systems, Faculty of Technology, Policy & Management, Delft University of Technology, Jaffalaan 5, 2628 BX Delft, the Netherlands b Rotterdam School of Management & Erasmus School of Law, Erasmus University Rotterdam, Burgemeester Oudlaan 50, 3062 PA Rotterdam, the Netherlands c Institute for Global Public Policy, Fudan University, Shanghai 200433, China a Department of Multi-Actor Systems, Faculty of Technology, Policy & Management, Delft University of Technology, Jaffalaan 5, 2628 BX Delft, the Netherlands b Rotterdam School of Management & Erasmus School of Law, Erasmus University Rotterdam, Burgemeester Oudlaan 50, 3062 PA Rotterdam, the Netherlands c Institute for Global Public Policy, Fudan University, Shanghai 200433, China d Section of Infrastructure Design & Management, Faculty of Civil Engineering & Geosciences, Delft University of Technology, Stevinweg 1, 2628 CN Delft, the Netherlands c Institute for Global Public Policy, Fudan University, Shanghai 200433, China d Section of Infrastructure Design & Management, Faculty of Civil Engineering & Geosciences, Delft University of Technology, Stevinweg 1, 2628 CN Delft, the Netherlands d Section of Infrastructure Design & Management, Faculty of Civil Engineering & Geosciences, Delft University of Technology, Stevinweg 1, 2628 CN Delft, the Netherlands p g j Received 24 July 2019; Received in revised form 16 March 2020; Accepted 24 April 2020 0264-2751/ © 2020 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecom m ons.org/licenses/BY/4.0/). ⁎ Corresponding author at: Faculty of Technology, Policy and Management, Delft University of Technology, Delft, the Netherlands. E-mail addresses: w.ma-2@tudelft.nl (W. Ma), w.m.jong@law.eur.nl (M. de Jong), M.L.C.deBruijne@tudelft.nl (M. de Bruijne), D.F.J.Schraven@tudelft.nl (D. Schraven). Cities 105 (2020) 102754 Cities 105 (2020) 102754 1. Introduction 2012; de Jong, 2019; Kavaratzis & Ashworth, 2005; Schuetze & Chelleri, 2016; Zenker & Martin, 2011). identifies the types of stakeholders in Chinese cities, their roles and resources and provides an analytical framework to analyse our em- pirical data. In Section 3, we explain our research process, methodology and the representativeness of Jingmen in the broader population of small and medium-sized cities in China and present detailed informa- tion regarding our fieldwork. Section 4 offers a brief description of Jingmen city, its industrial structure, and a concise history of brand development. In Section 5, a comparative analysis is made of two processes of brand creation and implementation we encountered in Jingmen, which allows us to analyse the roles, resources, and interac- tions between city departments and stakeholders in each project. Fur- thermore, we analyse the barriers encountered during brand im- plementation. We demonstrate that specific stakeholder roles are underrepresented, what resources are missing and subsequently assess the consequences for Jingmen's city branding strategy. Section 6 pro- vides an in-depth discussion about city brand creation and im- plementation. Section 7 concludes by summarizing how mechanisms for stakeholder involvement work in medium-sized Chinese cities, where their weaknesses lie and we offer some suggestions for further study. City branding in the more advanced coastal regions in China have gained some attention in academic studies. For example, Berg and Bjorner (2014) studied city branding in Chinese megacities, including Beijing and Shanghai. City branding in the Pearl River Delta and the Bohai River basin (around Beijing and Tianjin) are similarly discussed (de Jong et al., 2018). Other scholars have studied different aspects of city branding in Chinese settings. For example, Wu (2000) has focused on urban development and place promotion in Shanghai, while Zhu, Qian, and Gao (2011) investigated the globalisation and city image in Guangzhou. Especially, the relationship between large international events and city branding has been extensively studied by hosts of scholars, for example, the Beijing Olympics (Chen, 2015; Zhang & Zhao, 2009), the Shanghai World Expo (Dynon, 2011; Yu, Wang, & Seo, 2012), and the Guangzhou Asian games (Chen, 2012). Obviously, city branding has been widely adopted by Chinese cities as a strategy to achieve a variety of urban development goals. 2. Stakeholder involvement in city branding in Chinese cities 2.1. An economic policy-oriented view of city branding and China's application 2.1. An economic policy-oriented view of city branding and China's application This contribution focuses on city branding activities of a medium- sized Chinese city, Jingmen. Jingmen represents a typical average Chinese city. This type of city faces an increasingly complex mix of economic and environmental problems. On the one hand, governmental officials and departments face a relentless competition for resources, economic activity and residents with other cities (Jessop & Sum, 2000; Xu & Yeh, 2005). On the other hand, policymakers have the unenviable task to ‘reinvent’ and ‘transform’ these cities to become more sustain- able (Joss & Molella, 2013; Ye, 2011; Zhan & de Jong, 2018). Some cities experience this challenge as deeply conflictual, others perceive this as a challenge and consequently embrace city branding as an op- portunity to engage in industrial transformation (Ma, Schraven, Bruijne, Jong, & Lu, 2019). Jingmen belongs to the second category employs city branding strategies to generate industrial transformation (and the promise of economic growth resulting from that). Jingmen thus avoids using the outdated branding label ‘Industrial City’ and ex- plores new brands and underlying economic ‘business models’ in an attempt to generate economic growth. These include brands which focus on general aviation, more specifically navigation equipment (the General Aviation New Town brand) and the local food industry and tourism (small private farmers and local food producers as destinations for food tourism) (China's Agricultural Valley brand). City branding has been intensely studied in both the academic and professional domains in the last ten years, particularly in urban plan- ning and governance (Lucarelli & Berg, 2011; Ma et al., 2019). In this contribution, we consider city branding as “a whole set of actions to build a positive image of the city and communicate it among various target groups via visuals, narratives, and events locally and inter- nationally to gain a competitive advantage over other cities” (Vanolo, 2008, p.371). On the one hand, city branding has a strong connection with local economic development (Anttiroiko, 2014; Blakely & Leigh, 2013; Cleave, Arku, Sadler, & Gilliland, 2016; Rowe, 2009) and re- sponds to global intercity competition (Anttiroiko, 2015). It aims to direct towards business relocation, expansion and investment (Baker, 2012; Harvey, 2013). Many Chinese cities apply city branding to obtain a competitive advantage and improve their economic position (Lu et al., 2017). 1. Introduction However, little is known of what the smaller and lesser known cities located in China's central heartland provinces do in this regard, even though they represent the large majority of Chinese cities and its urban population. This is re- markable since we would argue that these regions and cities are of more importance from a policy perspective than the ‘outliers’ which have been studied so far; megacities in China harbour less than 13% of the overall population in China (NBSPRC, 2017). 2. Stakeholder involvement in city branding in Chinese cities For example, economic city brands are adopted in Greater Pearl River Delta as a policy instrument to trigger economic development (Lu et al., 2017). On the other hand, city branding as a process stimulates and enables cities to experience in-depth urban and industrial trans- formation, economic restructuring and policy change (Boisen, Terlouw, Groote, & Couwenberg, 2018; Joo & Seo, 2018; Ye & Björner, 2018). Thus, in this article, city branding can be understood as an economic policy instrument, covering the industrial dimension of city branding. The design of a city brand and the subsequent implementation of the brand via city branding is influenced by stakeholder involvement (Dinnie, 2010). Effective stakeholder engagement is crucial for the ac- ceptance of city branding and can help fix and convey the image of a place (Kavaratzis, 2012). Stakeholders' attitudes and roles are essential factors in the process of city branding (Stubbs & Warnaby, 2015). However, public sector players are still the most influential stake- holders in China, and other ones tend to be followers rather than par- ticipants (Lu, de Jong, & Chen, 2017). More needs to be known about what roles these stakeholders take on, and to what extent and how they are involved in the city branding process. Ye and Björner (2018) claim that city branding has become a growth-driven urban policy and governance strategy in Chinese mega- cities. In the Chinese context, city brands are widely adopted as brand identities in various plans, such as the Urban Master Plans and Five Year Social-Economic Plans, which provide a local vision on social, economic and industrial development (Lu et al., 2017). National, pro- vincial, and local plans provide the direction for urban development and guide industrial change. Ideally, all the campaigns, promotions and policy actions should be fleshed out in line with these plans.i l Baker (2012) identified the key factors for branding small cities in some countries, such as making a consistent effort to achieve a vision with reality matching the positive expectations. However, research on city branding in Chinese small cities is non-existent. Therefore, this article seeks to explore how a medium-sized Chinese city engages in city branding strategies that help trigger industrial transfor- mation. More specifically, we examine how different stakeholders get in- volved in the city branding creation and implementation phases of developing a city brand. 1. Introduction aforementioned problems are especially poignant in cities in the na- tion's densely populated western and central heartlands, which are less prosperous than those in the coastal provinces. Furthermore, there is intense competition between cities due to their limited access to re- sources (e.g. a highly qualified workforce, financial and cultural facil- ities) and a growing mobility among inhabitants. For these reasons, many local policymakers are exploring new pathways to stimulate economic development and generate a sustainable urban transforma- tion. Many decision-makers have embraced city branding as a key strategy to guide their municipalities' industrial transformation process. City branding is viewed as an essential strategy to remain ‘competitive’ (Kavaratzis, Warnaby, & Ashworth, 2014). Supporters argue that city branding is crucial to realise a metropolitan vision which feeds into sustainable urban transformation (Belloso, 2011; Goess, de Jong, & Meijers, 2016; Rius Ulldemolins, 2014), whereas critics have claimed that city branding is a vague policy term and merely a tool that is employed by local governments to greenwash a city's image (Braun, Chinese cities have experienced unprecedented economic growth rates in the last forty years (Deng, Huang, Rozelle, & Uchida, 2010; Fleisher, Li, & Zhao, 2010; Zhang, Wang, & Wang, 2012). This has brought many positive changes, especially in terms of quality of life and material prosperity for a great many people who live in these cities (de Jong et al., 2018). On the other hand, a variety of social and ecological problems is associated with urbanisation and industrialisation. Ex- amples of social problems include growing (regional) disparity between cities (Fan, Kanbur, & Zhang, 2011; Wang & Fan, 2004) and a growing gap between the wealthiest and the underprivileged living inside these cities (Cai, Wang, & Du, 2002; Fan et al., 2011; Wang & Fan, 2004; Zhang, 2001). Environmental problems which are increasingly plaguing industrialised Chinese cities are traffic congestion, air pollution, and a growing waste-pile (Chien & Wu, 2011; Logan & Molotch, 2007; Shao, Tang, Zhang, & Li, 2006; Yeh, Yang, & Wang, 2015). The ⁎ Corresponding author at: Faculty of Technology, Policy and Management, Delft University of Technology, Delft, the Netherlands. E-mail addresses: w.ma-2@tudelft.nl (W. Ma), w.m.jong@law.eur.nl (M. de Jong), M.L.C.deBruijne@tudelft.nl (M. de Bruijne), D.F.J.Schraven@tudelft.nl (D. Schraven). W. Ma, et al. Cities 105 (2020) 102754 2012; de Jong, 2019; Kavaratzis & Ashworth, 2005; Schuetze & Chelleri, 2016; Zenker & Martin, 2011). 2.2. Stakeholder involvement and Chinese administration characteristics Stakeholder analysis has emerged as an ap- proach to understand policy design and implementation (Bryson, 2004; Freeman, 2010), including in various fields such as place branding (Kavaratzis, 2012; Stubbs & Warnaby, 2015), urban transformation (Höijertz, 2013; Song, Guan, Wang, & Zhao, 2016), urban renewal (Zhuang, Qian, Visscher, Elsinga, & Wu, 2019), sustainable city projects (Zhan, de Jong, & de Bruijn, 2017), environmental marketing (Cronin, Smith, Gleim, Ramirez, & Martinez, 2011; Polonsky, 1995), and public and citizen participation in China (Enserink & Koppenjan, 2007; Li & de Jong, 2017). Hankinson (2004) claims that place branding, in essence, is a set of relationships between stakeholder groups. International scholars claim that stakeholder involvement strengthens the effectiveness of city branding (Klijn, Eshuis, & Braun, 2012). Stakeholder engagement en- riches and deepens the concepts of city branding, introduces new opi- nions, ideas, and perspectives (Dinnie, 2010; Kavaratzis & Kalandides, 2015). Some scholars emphasise the importance of involving profes- sionals in city branding and take a more instrumental approach, while others believe inclusive, citizen-centric and socially-oriented branding to be more suitable for urban governance (Hereźniak, 2017; Joo & Seo, 2018; Paganoni, 2012). Compared with urban development in Western countries, public authorities play a much bigger role in China (Xu & Yeh, 2005). Gov- ernments are the core stakeholders in China as most resources and policy instruments are regulated and controlled by the state (Chen, Sun, Tang, & Wu, 2011). A rich variety of public stakeholders can be iden- tified, ranging from regional and local organisations to sections of state- owned enterprises who operate at the national level. Furthermore, politicians occupy key positions in the aforementioned government organisations, non-profit organisations, and state-owned companies (Li & Zhou, 2005). China's one-party system is organised more or less in parallel to the various government levels, so that party leaders occupy high positions both within the party and in (local) governments and state-owned enterprises. The party is thus involved at multiple-levels and in many aspects of China's societal governance down to cultural and sports organisations (Oi, 1995). However, we have insufficient knowledge of stakeholder involvement in the city branding activities of Chinese medium and small cities. 2.2. Stakeholder involvement and Chinese administration characteristics Section 2 explores the relationship between city branding and local economic development and industrial transformation. The practice of stakeholder involvement in Chinese political systems is explained. It Freeman defined a stakeholder as “any group or individual who can affect or is affected by the achievement of the organisation's objectives” 2 W. Ma, et al. Cities 105 (2020) 102754 No. Stubbs et al.'s structure No. Type of Stakeholders (adjusted) Explanations for the stakeholders Roles Resources 1 Politicians 1 Governmental organisations Politicians Party executives working at different levels of government or core departments, such as party-secretaries and mayors at the provincial and municipal levels • Dominate the branding creation stage • Influence brand creation and implementation, e.g. approve brand creation and push brand implementation • Influence city branding selection and implementation Opinion leaders 2 Governmental organisations Departments and agencies • Different administrative level governments outside the city (top-down hierarchical organisation) • Different governmental departments within a city • Ask approval from higher level governments • Execute, participate, coordinate and monitor the brand implementation process Administrative (hierarchical) power (policy-making); Financial resources; Regulatory power 3 Cultural and sports organisations 4 Promotion agencies 2 Promotion agencies • Governmental media departments • Travel agencies • Publish and deliver information about brands and activities through different channels Media channels and the capability to respond to various audiences 5 Businesses 3 Enterprises • State owned companies at different levels • Local private companies • Invest and operate projects • Provide knowledge, resource, technology, finance support Technology; Finance; Investment opportunities 6 Academic organisations and schools 4 Academic organisations and schools • Local universities, schools, and academic institutions • External experts and academic associations • Provide expert knowledge Knowledge; Policy expertise 7 Infrastructure and transport providers 5 Infrastructure and transport providers Transport Construction & Investment companies • Develop the transport and infrastructure • Participate in the brand implementation Investment & financing 8 Residents 6 Residents Local citizens • Express their opinions and provide feedback via digital media and public media platforms • Receive brand information and participate in branding activities Labour; Opinions 9 Religious organisations (Freeman, 2010, p.46). 3. Research methodology respondents were interviewed more than once (See Appendix A). Most interviewees were employed in local government departments, such as the Jingmen Planning Bureau, Jingmen Planning and Design Institute, Jingmen Daily Media Group and Jingmen Development and Reform Commission (DRC). We also consulted experts at local universities and research institutions; and the leaders of local enterprises about their knowledge about the city branding policies. A case study can be used to investigate a contemporary phenomenon more deeply and to explore the underlying mechanism of this phenomenon (Yin, 2009). It is a deliberate research strategy to describe and explain the creation and implementation processes of city branding in a specific medium-sized city in China. Jingmen is a highly industrialised city and in its planning documents displays a strong willingness to experience economic, industrial and urban transformation. Roughly 600,000 of Jingmen's in- habitants live in the main urban district, which makes Jingmen a typical medium-sized municipality in China in terms of population numbers (SC, 2014). In terms of social and economic development, Jingmen closely re- presents the ‘average’ among Chinese cities. In 2017, its GDP per capita (57,357 RMB) (HBOS, 2018), closely resembles the Chinese national average GDP per capita (59,201 RMB) (NBSPRC, 2018). In 2018, Jingmen's urbanisation rate reached 59.21% (JMBOS, 2019), quite close to the na- tional level (59.58%) (NBSPRC, 2019). Jingmen is one of the 128 “fifth-tier” cities and localities inlands in China, from a grand total of 337 listed cities in 2019, according to a Chinese media investigation (YICAI, 2019). Based on these data, we argue that the city of Jingmen can serve as a representative case to study city branding practices of a large group of cities.i Furthermore, we participated as observers and participants in two meetings of the Jingmen Urban Planning Bureau, and organised a workshop with the local officials and external experts to analyse and discuss Jingmen's approach to city branding projects and industrial transformation (see Appendix A). Five foreign experts from Delft Uni- versity of Technology, four experts from Chinese top universities, and eight PhD students participated in the workshop discussions. In addition, we conducted telephone interviews and email inquiries with officials and experts to obtain updates and additional information. To complement these interviews, we used information from official websites and project documents and publications to understand the urban context (e.g. Jingmen Urban Master Plan). 1 A prefecture-level municipality (Chinese: 地级市) is an administrative division of the People's Republic of China (PRC), ranking below a province and above a county. 2.3. Stakeholder identification via an adopted involvement framework The identification of stakeholders is a crucial step in any stakeholder analysis. A mixture of several techniques will be adopted, including a positional approach to map power or resources of stakeholders (Enserink et al., 2010) and a social participation approach to identify to what extent the stakeholders relate to activities (Bryson, 2004). Table 1 Stakeholder involvement framework in city branding in China. Here, we combine previous studies on stakeholder involvement in place branding (Kavaratzis, 2012; Klijn et al., 2012; Stubbs & Warnaby, 2015), stakeholder involvement in China (Zhan et al., 2017; Zhuang et al., 2019) and Chinese institutional characteristics (Li & Zhou, 2005; Ye, 2013, 2014) to identify the stakeholder structure in city branding processes in Chinese cities. We apply Stubbs & Warnaby's model (2015) to the Chinese context as shown in Table 1 (left side) but make some crucial adjustments to achieve a better fit with Chinese institutional characteristics. Most importantly, the role of politicians should be merged into various governmental organisations and state-owned en- terprises. In China, there are also professional government organisa- tions in charge of organising cultural and sports activities. So we also merge the cultural and sports organisations into government organi- sations. Since religious organisations are rarely involved in the design of city-level policies and city branding, we exclude them from our analysis. We will use our adjusted stakeholder framework to identify and analyse the various (potential) stakeholders in city branding crea- tion and implementation processes in Jingmen in Section 5. 3 W. Ma, et al. Cities 105 (2020) 102754 Goal 1 Identify the various involved stakeholders, their roles and resources in Jingmen’s two industrial brands. Step 1 Stakeholder Identiﬁcation Method 1 Literature review; Interviews; Documents Goal 1 Identify the various involved stakeholders, their roles and resources in Jingmen’s two industrial brands. Step 2 Stakeholder Analysis Goal 2 Analyse and map the interactions in city brand development and implementation among diﬀerent stakeholders; Analyse the barriers and challenges in city brand(ing) implementation. Step 3 Discussion and ﬁndings Goal 3 Compare patterns of stakeholder involvement in city brand creation and implementation across two cases Method 2 Documents; Interviews; Expert panels Method 3 Tabular analysis of roles, resources and barriers across two cases Fig. 1. Research process. 2.3. Stakeholder identification via an adopted involvement framework Method 1 Literature review; Interviews; Documents Step 2 Stakeholder Analysis Goal 3 Compare patterns of stakeholder involvement in city brand creation and implementation across two cases Step 3 Discussion and ﬁndings Fig. 1. Research process. 3. Research methodology Combined with the interview results in Jingmen, official documents, and the suggestions from expert panels, we present our analysis and results in Section 5, especially focusing on the interactions among different stakeholders (see Figs. 5 and 6). Fig. 1 shows the specific research process in this article, which focuses on the involvement of stakeholders in the creation of city brands and their subsequent implementation in city branding activities. In step 1, we identify the various stakeholders that were involved in two recent city branding projects which are indicative of recent attempts at city branding in the city of Jingmen. The stakeholder framework that was developed in Section 2.3 was used to identify relevant stakeholders via interviews and document analysis during our fieldwork in Jingmen. In step 2, we examined additional information about the city branding projects via interviews and official documents to trace brand creation and implementation processes and map the interactions among the different stakeholders. Expert panel discussions were used to complete the in-depth analysis of the city branding projects to subsequently analyse the barriers and challenges experienced in both pro- jects. Finally, in step 3, we summarise the patterns of underlying stake- holder involvement in the city brand creation and implementation phases in a medium-sized Chinese city.i 4.2. Jingmen's city brands: evolution and development shows the distribution of GDP across the three economic sectors in Jingmen from 1949 to 2018 (HBOS, 2018). The relative proportion of the primary sector decreases significantly over time, whereas the con- tribution of the secondary sector increases rapidly from the 1970s and onwards. Since then the secondary industry has maintained its eco- nomic importance in Jingmen's economy until the time of writing (2020). In 2017, the percentages of GDP in Jingmen's three economic sectors were 13.4%, 51.1%, and 35.5% respectively (JMBOS, 2019), which shows that the secondary sector still dominates the city's eco- nomic structure. In contrast, the workforce distribution of the three industrial sectors is 36.7%, 25.1%, and 38.2% respectively (HBOS, 2018). Roughly half of Jingmen's GDP is produced by a quarter of its working population. A substantial proportion of the working population is still employed in the primary sector even though its contribution to the city's GDP is relatively low. Although the tertiary industry experi- enced stable growth over time, its proportional contribution to Jing- men's GDP is still substantially lower than that of the secondary sector. shows the distribution of GDP across the three economic sectors in Jingmen from 1949 to 2018 (HBOS, 2018). The relative proportion of the primary sector decreases significantly over time, whereas the con- tribution of the secondary sector increases rapidly from the 1970s and onwards. Since then the secondary industry has maintained its eco- nomic importance in Jingmen's economy until the time of writing (2020). In 2017, the percentages of GDP in Jingmen's three economic sectors were 13.4%, 51.1%, and 35.5% respectively (JMBOS, 2019), which shows that the secondary sector still dominates the city's eco- nomic structure. In contrast, the workforce distribution of the three industrial sectors is 36.7%, 25.1%, and 38.2% respectively (HBOS, 2018). Roughly half of Jingmen's GDP is produced by a quarter of its working population. A substantial proportion of the working population is still employed in the primary sector even though its contribution to the city's GDP is relatively low. Although the tertiary industry experi- enced stable growth over time, its proportional contribution to Jing- men's GDP is still substantially lower than that of the secondary sector. City brand development in Jingmen has seen some shifts over the previous decades. The evolution of city brand development from the beginning of the 1970s to the current day can be broadly divided into three stages. 4.1. A brief description of Jingmen in China The location of Jingmen in Hubei province is shown in Fig. 2. The land area of Jingmen is 12,404 sq.km. The number of inhabitants in the administrative area is reported as 2,896,500 (JMBOS, 2019). The Gross Domestic Product of Jingmen in 2018 was RMB 184.8 billion. Histori- cally, Jingmen belonged to Jingzhou, another city next to Jingmen (see in Fig. 2). On August 19, 1983, the State Council approved the pro- motion of Jingmen to prefecture-level municipality1 (PGOJM, 2019). Currently, Jingmen promotes the development of equipment manu- facturing, new energy and new materials, renewable resource utilisa- tion and environmental protection, electronic information, health, fine chemicals and processing of agricultural products (PGOJM, 2019). Specifically, we summarise the generic economic and social in- formation from the websites about Jingmen City and the project in- formation of two economic brands: China's Agricultural Valley (CAV), and General Aviation New Town (GANT).i To obtain insight in Jingmen's economic structure, we distinguish the primary, secondary, and tertiary sectors in the economy. Fig. 3 We conducted our fieldwork in Jingmen from November 2018 to January 2019 and conducted semi-structured interviews with re- presentatives of involved stakeholders. In total, 18 respondents were interviewed, among them the CAV and GANT project leaders. Four 4 W. Ma, et al. Fig. 2. Location of Jingmen in Hubei and the PRC. 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 1949 1952 1955 1958 1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003 2006 2009 2012 2015 2018 Primary industry Secondary industry Tertiary industry Fig. 3. Industry's relative contribution to Jingmen's GDP between 1949 and 2018. Cities 105 (2020) 102754 Fig. 2. Location of Jingmen in Hubei and the PRC. Fig. 2. Location of Jingmen in Hubei and the PRC. 0.0 10.0 20.0 30.0 40.0 50.0 60.0 70.0 80.0 90.0 100.0 1949 1952 1955 1958 1961 1964 1967 1970 1973 1976 1979 1982 1985 1988 1991 1994 1997 2000 2003 2006 2009 2012 2015 2018 Primary industry Secondary industry Tertiary industry Fig. 3. Industry's relative contribution to Jingmen's GDP between 1949 and 2018. Primary industry Secondary industry Tertiary industry 4.2. Jingmen's city brands: evolution and development Jingmen's first city brand (Industrial City) was created during the period of the so-called Third Front Movement2 as part of a large-scale national plan to industrialise China's interior. A substantial number of large national companies were (re)located to stimulate economic development (Qian, 2000). As a result of this deliberate 2 The Third Front Movement (Chinese: 三线建设; pinyin: Sānxiàn jiànshè) was a massive industrial development by China in its interior starting in 1964. It involved large-scale investment in national defense, technology, basic in- dustries (including manufacturing, mining, metal, and electricity), transporta- tion and other infrastructures investments. 5 W. Ma, et al. Cities 105 (2020) 102754 stimulated the development of the secondary industry. national industrial policy, Jingmen evolved into an important petro- chemical industrial hub and became well-known as a typical ‘Industrial City’ (interviewee 1). Currently, the Industrial City brand faces serious challenges. Severe environmental pollution presents an increasingly problematic living environment for local residents and challenges Jingmen's attractiveness to prospective city inhabitants (interviewee 1). When it comes to competition with neighbouring cities, the Industrial City brand has become unattractive and lacks power as it represents an image of en- vironmental pollution and bad air quality. However, at the same time, the economic data shows that Jingmen still relies heavily on the sec- ondary industry (interviewee 6). Nonetheless, Jingmen increasingly avoids branding itself as ‘Industrial City’. Jingmen's Industrial City brand was maintained until 2012 when a new city brand - China's Agricultural Valley (CAV), was developed and launched as part of Jingmen's new urban development model. Being mostly located on plain land, Jingmen is an important production base for commodity grain and high-quality rapeseed oil in the country (PGOJM, 2019). The output of cotton, oilseeds, fruits, pigs and aquatic products ranks among the highest in the province (PGOJM, 2019). As part of a deliberate local government policy to stimulate Jingmen's primary sector and economic development in the region of Qujialing3 (see in Fig. 4), China's Agricultural Valley (CAV) brand quickly caught on. The CAV brand was gradually extended from Qu- jialing to encompass Jingmen proper, and was later even used to brand the entire Hubei Province. Spurred on by high levels of attention from leaders of municipal and provincial governments the CAV brand quickly gained popularity as an economic concept, which in turn fur- ther stimulated the developed of CAV into a city brand of Jingmen it- self. 3 Qujialing is the core implementation area of China's Agricultural Valley in Jingmen, as Fig. 4 shown. 5.1.2. Stakeholder identification in CAV i The principal stakeholders, their roles and resources are shown in Table 3. 5.1.3. Stakeholder analysis in CAV brand creation and implementation Fig. 5 (a) and (b) show the interactions between different stake- holders during the creation and implementation phases of CAV city branding. 5.1.3.1. Interactions between stakeholders during the CAV creation phase. The concept of “China's Agricultural Valley (CAV)” originated in a master plan made by two professors at Huazhong University of Science and Technology at the end of 2008 (PGOJM, 2017). The brand was originally designed specifically for Jingmen's Qujialing area, Jingmen (PGOJM, 2017). In 2011, the secretary of the Hubei Provincial Party Committee paid specific attention to the CAV brand after reading the publication ‘Boiled Agricultural Valley’ in the Hebei Daily. The secretary of the Jingmen Municipal Party Committee subsequently conducted research into the Qujialing Management Area in 2011 and gave more detailed instructions which focused on the construction of the CAV. Consequently, Jingmen government agencies such as the Social Science Association, the Jingmen Development and Reform Commission, the Agriculture Bureau, the Tourism Bureau, and the CSBPP in Jingmen conducted a special investigation to study Qujialing and the construction of the CAV brand. Scholars from the Municipal Social Science Association, and experts from local media organisations put forward suggestions on how to turn the CAV concept into a city brand. The Qujialing Management District Government was subsequently selected by the Hubei Provincial Government as a pilot area for the development of an agricultural demonstration zone in 2011. In 2012, the Hubei Provincial Government decided that the ‘China's Agricultural Valley’ brand would be used as a provincial strategy, and would be implemented in Jingmen first. 5.1. Stakeholder involvement in China's Agricultural Valley (CAV) brand 5.1. Stakeholder involvement in China's Agricultural Valley (CAV) brand 5.1.1. General project introduction China's Agricultural Valley (CAV) brand was officially kicked off in 2012 with the Jingmen municipal government as the key driver. It aims to stimulate the development towards a modern agricultural industry in the Jingmen area. China's Agricultural Valley project initially focused on the Quiialing and Pengdun areas (see Fig. 4). The brand concept initially focused on the Qujialing farming culture, the rich and diverse agricultural resources in Jingmen. Table 2 shows more detailed in- formation about project progress. Actually, in addition to the aforementioned brands, the city of Jingmen also boasts being ‘China's Outstanding Tourist City’, ‘Gateway of Hubei’, ‘National Garden City’, ‘National Forest City’, ‘Cultural Jingmen’ and ‘Ecological Jingmen’ as city brands (PGOJM, 2019). However, since this article aims to analyse how Jingmen uses economic brands to achieve industrial transformation. China's Agricultural Valley, Industrial City, and the General Aviation New Town brands represent three typical industrial city brands in Jingmen. Since the building of Industrial City as a brand began in the 1970s further back in time, we were unable to obtain sufficient information about stakeholder engagement during the creation and implementation of this brand. Consequently, this article considers the Industrial City as background for the study of the other two brands in Jingmen. CAV and GANT are thus selected as cases for our analysis of economic city branding stra- tegies in Jingmen which aim to trigger sustainable industrial transfor- mation. 4.2. Jingmen's city brands: evolution and development These reasons push the city of Jingmen to explore new ways of generating deep sustainable industrial transformation. The solution that the Jingmen government has chosen so far is to let the heavy industry survive but downplay the importance of the industrial sector in its ex- ternal communication while promoting new aviation technology and sustainable agriculture as key economic activities. In the next sections, we will analyse the creation and implementation of these two city brands. More recently, in order to realise an industrial upgrade and trans- form the economy, Jingmen has been exploring new options for de- velopment. Jingmen is considered one of five general aviation in- dustrial clusters in China. Being designated as a ‘general aviation industry comprehensive demonstration zone’ enables Jingmen to plan and build a General Aviation New Town (GANT) as part of its ambitious growth plans. The core area in GANT is Zhanghe New District. The core areas of CAV and GANT are shown in Fig. 4. 5. Stakeholder involvement analysis: CAV and GANT In 2014, Jingmen's Development and Reform Commission published the Master Plan of China's Agricultural Valley and in 2017 the Municipal Party Committee Office released the China Agricultural Valley 2025 Construction Plan. 5.1.3.2. Interactions between stakeholders during the CAV implementation phase. The Jingmen Municipal Government further developed the goals of the strategy underlying the CAV brand and promoted its implementation. China Agricultural Valley Construction Leading Group of Jingmen was set up in 2012 as project organisation and charged with the management of the CAV project with Jingmen's mayor in charge. It operated directly under the Jingmen Municipal Government. China Agricultural Valley Office was a coordinating organisation that was set up to help direct the efforts of the Jingmen Municipal Government and its other bureaus. Numerous other governmental organisations execute and participate in projects under the CAV brand. For example, the Jingmen Tourism Bureau publishes ecological tourism information on Jingmen's official website and organises tourism activities, such as the CAV-Shayang Ecological Agriculture Experience. CSBPP organises annual cultural and sports activities, such as “Farmhouse activities” (interviewee 3). In 2014, Jingmen's Development and Reform Commission published the Master Plan of China's Agricultural Valley and in 2017 the Municipal Party Committee Office released the China Agricultural Valley 2025 Construction Plan. was established as a key part of the city's CAV branding strategy. Its equity is entirely held by the Municipal State-owned Assets Supervision and Administration Commission. Its goals are to promote the develop- ment of CAV projects and develop modern agricultural practices (in- terviewee 13). Jingmen Agricultural Valley Investment Co., Ltd. is a wholly-owned subsidiary of Hubei Agricultural Valley Group and spe- cially established to promote the rapid development of the Hubei Agricultural Valley Industrial Group in 2015 (interviewee 14). The two companies are public enterprises that cover financial gaps incurred by the development of CAV through asset management, project investment and other economic activities related to sustainable agriculture. The Pengdun Group is a large private company which participates in the implementation of projects under the CAV brand which have a clear focus on modern agriculture (interviewee 14). The CAV Research Institute of the Hubei Academy of Social Sciences (HAOSS) was formally established by the Jingmen Municipal Government and HAOSS in 2012. It is the first policy research institu- tion to specialise in the development of the CAV. 5. Stakeholder involvement analysis: CAV and GANT From 1964, a large number of military-industrial and national de- fense enterprises were assigned to Jingmen because of the national Third Front Movement policy. These included Sinopec, Hongtu Aircraft Manufacturing Plant, 605 Research Institute, the 330 Cement Plant, various Thermal Power Plants, the Xiangsha Chemical Plant, but also the Dongguang Optoelectronics Factory, and the Jiangbei Foundry. Many petrochemical and phosphate industries companies joined these companies and most are still present in Jingmen today and are part of Jingmen's industrial basis (PGOJM, 2019). However, the governance of these state-owned enterprises is primarily concentrated at the national and provincial levels. From 1986 to 1995, China experienced a major reform of its economic system and a transition from a planned economy to a market-based economy (Qian, 2000). The Industrial City brand was developed in response to the abovementioned urban economic reforms in the national policy environment (interviewee 1; 4). This brand also 6 W. Ma, et al. Cities 105 (2020) 102754 Fig. 4. Core areas of China's Agricultural Valley (CAV) and General Aviation New Town (GANT) projects in Jingmen City: Qujialing and Zhanghe (Source: Jingmen Urban Master Plan). ural Valley (CAV) and General Aviation New Town (GANT) projects in Jingmen City: Qujialing and Zhanghe (Source: Jingmen Fig. 4. Core areas of China's Agricultural Valley (CAV) and General Aviation New Town (GANT) projects in Jingmen City: Qujialing and Zhanghe (Source: Jingmen Urban Master Plan). 5.1.3.2. Interactions between stakeholders during the CAV implementation phase. The Jingmen Municipal Government further developed the goals of the strategy underlying the CAV brand and promoted its implementation. China Agricultural Valley Construction Leading Group of Jingmen was set up in 2012 as project organisation and charged with the management of the CAV project with Jingmen's mayor in charge. It operated directly under the Jingmen Municipal Government. China Agricultural Valley Office was a coordinating organisation that was set up to help direct the efforts of the Jingmen Municipal Government and its other bureaus. Numerous other governmental organisations execute and participate in projects under the CAV brand. For example, the Jingmen Tourism Bureau publishes ecological tourism information on Jingmen's official website and organises tourism activities, such as the CAV-Shayang Ecological Agriculture Experience. CSBPP organises annual cultural and sports activities, such as “Farmhouse activities” (interviewee 3). 5.2.2. Stakeholder identification in General Aviation New Town Ltd. (CIHG), Transport Construction & Investment Co., Ltd. (TCI), and the Transport Bureau (TB2) are the main road infrastructure and transport providers in Jingmen. They are also in charge of the infra- structure and transport construction projects and policies that have been started under the CAV brand, such as rural transportation, and farmland water conservancy projects. i The principal stakeholders, their roles and resources in GANT are shown in Table 5. i The principal stakeholders, their roles and resources in GANT are shown in Table 5. 5.2.3. Stakeholder analysis in GANT brand creation and implementation Fig. 6 (a) and (b) show the interactions between different stake- holders during the creation and implementation phases of GANT city branding. Even though some residents participate in activities under the CAV brand, their participation is limited and communication about CAV is predominantly one-way; the government claims the brand, the re- sidents receive brand information and participate in brand related ac- tivities (interviewee 16). The local farmers in the CAV area are im- portant stakeholders, however, they only indirectly participate in CAV branding related projects. For example, the farmers sell their agri- cultural products to the large trading enterprises. Migrant workers participate in industrial farming work (interviewee 14). However, the farmers' participation has not been particularly massive, nor very pas- sionate (interviewee 6). 5.2.3.1. The interactions between the stakeholders during the GANT creation phase. The origin of the GANT brand can be traced back to Jingmen's industrial development in the period of the Third Front Movement, as some aviation navigation manufacturing and operation facilities were located in the Jingmen area. In 2009, the State Council released a series of low-altitude flight policies to develop the general aviation industry in China. These policies provided the policy environment that enabled provincial and local government officials the opportunity to develop the aviation industry. In 2010, the Development and Reform Commission of the Hubei provincial government approved the “Jingmen Aviation Industrial Park Development Plan”. In doing so, the Hubei Province supported Jingmen in its development of a new city district for the general aviation industry. In August 2014, the Jingmen AVIC Aviation Town Project was signed by Aviation Industry Corporation of China, Ltd. (AVIC) and the Jingmen Municipal Government. The two sides agreed to build the Jingmen General Aviation New Town, a comprehensive demonstration base for general aviation industry development. Municipal government 2015 – Jingmen Agricultural Valley Investment Co., Ltd. was set up in 2015. 5.2.2. Stakeholder identification in General Aviation New Town 5.2.2. Stakeholder identification in General Aviation New Town In 2016, the Office of the State Council published “the introduction of the guidance of on promoting the development of the navigation industry” (SC, 2016), which strengthened the policy context for promoting the national aviation industry via GANT. Above all, GANT as a city brand was built based on the macro policy support from the national, provincial and municipal level governments. 5.2. Stakeholder involvement in the General Aviation New town (GANT) brand 5.2. Stakeholder involvement in the General Aviation New town (GANT) brand Municipal government Municipal government 2008 – The concept of “China's Agricultural Valley (CAV)” originated from a master plan for the Qujialing area, Jingmen. Made by two professors at Huazhong University of Science and Technology at the end of 2008. 2011 – On July 18, 2011, the secretary of the Hubei Provincial Party Committee, gave important instructions on the publication of ‘Boiled Agricultural Valley’ in the Hubei Daily. On July 2011, the secretary and mayor of Jingmen Municipal Government conducted research in the Qujialing Management Area in 2011 and then gave more instructions on CAV. On July 2011, the secretary and mayor of Jingmen Municipal Government conducted research in the Qujialing Management Area in 2011 and then gave more instructions on CAV. In 2011, the Social Science Association, Jingmen's Development and Reform Commission, the Agriculture Bureau, the Tourism Bureau, and the CSBPP conducted a special investigation of Qujialing into the terms of CAV brand construction. The experts and scholars from the Municipal Social Science Association, and local media organisations put forward suggestions on the development of CAV. On September 13, 2011, Jingmen Municipal Party Committee and Jingmen Municipal Government issued a notice on the establishment of the “China's Agricultural Valley” construction work leading group. 2012 – In 2012, the Hubei Provincial Government confirmed the construction of China's Agricultural Valley as a provincial strategy, and it would be implemented in Jingmen. In 2012, the Hubei Agricultural Valley Industrial Group Co., Ltd. was set up to handle CAV strategy development. 2014 – – 2015 – Jingmen Agricultural Valley Investment Co., Ltd. was set up in 2015. 2016 – – 2018 – – In 2014, the Jingmen Development and Reform Commission released the Agricultural Valley Core Plan. In 2014, the Jingmen Development and Reform Commission released the Agricultural Valley Core Plan. In October 2014, the Agricultural Valley Forum was established in the Qujialing Management Area. August 7, Jingmen Municipal Government announces the cooperation with the Chinese Academy of Agricultural Sciences. August 8, Chinese Academy of Agricultural Sciences participates in compiling the China Agricultural Valley 2025 Construction Plan. Jingmen Municipal Party Committee and Municipal Government issue “Opinions on Implementing the Strategy of China's Agricultural Valley”. Jingmen held the 10th China Agriculture Valley·Jingmen Rape Flower Tourism Festival and the 2nd China Agricultural Valley·Jingmen (Zhongxiang) Lagerstroemia Flower Culture Tourism Festival In October 2014, the Agricultural Valley Forum was established in the Qujialing Management Area. 5. Stakeholder involvement analysis: CAV and GANT Jingchu University of Technology applies for research grants and expresses its opinions on the development of the CAV every year. Scholars from the Jingmen Social Science Federation and Jingchu University of Technology advise on branding and promotion to the municipal and provincial governments through expert panels. However, their suggestions are not necessarily adopted (interviewee 15). The Chinese Academy of Agricultural Sciences invited internal and external experts to compile the “China Agricultural Valley 2025 Construction Plan (2016-2025)”. The general media organisation and tourism agencies in Jingmen are the main promotion agencies of the CAV brand. The Daily Media Group publishes the information on CAV via media channels, such as WeChat and newspapers (interviewee 11; 12). Tourism agencies pub- lish farming tourism information to attract outside visitors (interviewee 10). The Jingmen Urban Construction Investment Holding Group Co., In 2012, the Hubei Agricultural Valley Industrial Group Co., Ltd. 7 Table 2 Project progress in China's Agricultural Valley (CAV) 2008–2016. National Provincial government 2008 – The concept of “China's Agricultural Valley (CAV)” originated from a master plan for the Qujialing area, Jingmen. Made by two professors at Huazhong University of Science and Technology at the end of 2008. 2011 – On July 18, 2011, the secretary of the Hubei Provincial Party Committee, gave important instructions on the publication of ‘Boiled Agricultural Valley’ in the Hubei Daily. 2012 – In 2012, the Hubei Provincial Government confirmed the construction of China's Agricultural Valley as a provincial strategy, and it would be implemented in Jingmen. In 2012, the Hubei Agricultural Valley Industrial Group Co., Ltd. was set up to handle CAV strategy development. 2014 – – 2015 – Jingmen Agricultural Valley Investment Co., Ltd. was set up in 2015. 2016 – – 2018 – – W. Ma, et al. Table 2 Project progress in China's Agricultural Valley (CAV) 2008–2016. National Provincial government 2008 – The concept of “China's Agricultural Valley (CAV)” originated from a master plan for the Qujialing area, Jingmen. Made by two professors at Huazhong University of Science and Technology at the end of 2008. 2011 – On July 18, 2011, the secretary of the Hubei Provincial Party Committee, gave important instructions on the publication of ‘Boiled Agricultural Valley’ in the Hubei Daily. 2012 – In 2012, the Hubei Provincial Government confirmed the construction of China's Agricultural Valley as a provincial strategy, and it would be implemented in Jingmen. 5. Stakeholder involvement analysis: CAV and GANT In 2012, the Hubei Agricultural Valley Industrial Group Co., Ltd. was set up to handle CAV strategy development. 2014 – – 2015 – Jingmen Agricultural Valley Investment Co., Ltd. was set up in 2015. 2016 – – 2018 – – W. Ma, et al. Cities 105 (2020) 102754 W. Ma, et al. Municipal government 5.2.1. General project introduction 5.2.1. General project introduction In 2014, the Aviation Industry Corporation of China, Ltd. (AVIC) and the Jingmen Municipal Government reached an agreement on the construction of the Jingmen General Aviation New Town (GANT). The Jingmen Municipal Government initiated this project to stimulate local economic activity and create a new city strategy for urban transfor- mation. General Aviation New Town is aimed at four industries: the development of navigation research and manufacturing, navigation operation and maintenance, emerging navigation services, and navi- gation training. More detailed information about GANT project pro- gress can be found in Table 4. 8 W. Ma, et al. Cities 105 (2020) 102754 Table 3 Stakeholders identification in CAV. No. Type of stakeholders Departments Roles Resources 1 Governmental organisations Politicians The secretary in the Hubei Provincial Government and the mayor in the Jingmen Municipal Government Approve the creation of CAV Push and coordinate CAV projects at the provincial level Opinion leaders Departments and agencies Government at other levels: Hubei Provincial Government (PG) Jingmen Municipal Government (MG) Qujialing Management District Government (QMDG) Implement various brand activities relevant to their tier of government Policy supply and adminis power Departments or agencies inside Jingmen: Jingmen Municipal Party Committee and Propaganda Department (MPCPD) Jingmen Municipal Committee of Rural Office (MCORO) Agricultural Bureau of Jingmen (AB) Tourism Bureau (TB1) Bureau of Culture Sports Broadcasting Press and Publication (CSBPP) Participate in city brand implementation Carry out city branding activities Organise cultural and sports activities within the CAV brand theme 2 Promotion agencies Public Daily Media Group Publish information about CAV projects Media channels Private Tourism agencies Publish tourism information in alignment with the CAV city brand 3 Enterprises Public Agriculture Valley Group Hubei Agriculture Valley Investment Management Corporation Cooperate with investment companies to promote the agriculture industry and to serve project under the CAV city brand; Develop and cultivate modern agricultural practices Financial resources; Project investment Private Pengdun Group Jingmen Baigufeng Technology Co., Ltd. 5.2.1. General project introduction It is the main management department related to the branding of the General Aviation New Town project. The Leading Group of General Aviation Industry was established in 2017 and became the key department for coordinating the GANT related projects with other local organisational departments. Since 2015, the Jingmen Municipal People's Government, the Hubei provincial Tourism Development Committee, the Hubei Provincial Sports Bureau and AVIC General Aircraft Co., Ltd. began to organise annual Jingmen AVIC Flight Conferences (interviewee 9). It is the biggest promotional activity to support the GANT brand. The conference attracts many visitors every year. In 2017, AVIC Town, Zhanghe new district was selected as a pilot 5.2.3.2. The interactions between stakeholders during the GANT implementation phase. In 2013, the Hubei Provincial Government approved the establishment of the Jingmen Zhanghe New District Management Committee (Zhanghe Xinqu Guanweihui). Zhanghe NDMC represents the district in which General Aviation New Town is situated, as shown in Fig. 4. It is the main management department related to the branding of the General Aviation New Town project. The Leading Group of General Aviation Industry was established in 2017 and became the key department for coordinating the GANT related projects with other local organisational departments. Since 2015, the Jingmen Municipal People's Government, the Hubei provincial Tourism Development Committee, the Hubei Provincial Sports Bureau and AVIC General Aircraft Co., Ltd. began to organise annual Jingmen AVIC Flight Conferences (interviewee 9). It is the biggest promotional activity to support the GANT brand. The conference attracts many visitors every year. In 2017, AVIC Town, Zhanghe new district was selected as a pilot project for sports and leisure towns by the State Sports General Administration. That denomination also pushed the development of GANT brand forward. Local travel agencies publish travel information about GANT on their websites to attract outside visitors. The Daily Media Group also publishes news reports to promote GANT. China Aviation Industry General Aircraft Co., Ltd. is a state-owned enterprise with experience in developing the general aviation industry. It was charged with providing technology and expertise to support the GANT projects in Jingmen. In 2017, the Hubei AVIC General Airport Management Company was launched as a flight service operation and management business at Zhanghe Airport. These public enterprises are the key stakeholders in the projects which aim to develop the GANT city brand. 5.2.1. General project introduction Participate and cooperate in the realisation of projects under the CAV city brand 4 Academic organisations and schools Public Jingmen Social Science Federation Jingchu University of Technology Chinese Academy of Agricultural Science Wuhan University of Technology Huazhong Agricultural University Provide suggestions on CAV city brand creation and promotion to the governments; Participate in city branding activities; Participate in the formulation and compilation of the agricultural plans underlying the CAV city brand Knowledge support 5 Infrastructure and transport providers Public Jingmen Urban Construction Investment Holding Group Co., Ltd. (CIHG) Transport Construction & Investment Co., Ltd. (TCI) Jingmen Transport Bureau (TB2) and District Transport Bureau Provide the farming infrastructure and transport in Jingmen and Qujialing Management Area Investment & financing; Construction and operatio 6 Residents Private Farmers in the (CAV) Qujialing Management District Residents in Jingmen City Sell food and agricultural products to enterprises (farmers) Participate in industrial farming (Migrant workers) Participate in CAV city branding activities and provide feedback and opinions about the brand Agricultural product; Labor; Views W. Ma, et al. Cities 105 (2020) 102754 Hubei Provincial Governments; Politicians Jingmen Municipal Governments; Politicians Qujialing Management District Government Diﬀerent departments inside Jingmen (MCORO, AB, TB1, CSBPP ) Jingmen Social Science Federation Local Media Organisation Hubei Provincial Governments; Politicians Jingmen Municipal Governments; Politicians Qujialing Management District Government Diﬀerent departments inside Jingmen (MCORO; AB; TB1; CSBPP ) Jingmen Social Science Federation; Jingchu University of Technology; Chinese Academy of Agricultural Science; Huazhong Agricultural University CAV leader group/CAV oﬃce Hubei Agricultural Valley Investment Management co. LTD; Agricultural Valley Group; Pengdun Group TB2; CIHG; TCI Residents Hierarchical relationships Information deliverer Departmental cooperation Information feedback Legend Governmental organisations Academic institutions Promotion agencies Enterprises Infrastructure and transport providers Residents Colour Public Public+Private Private Local Media Organisation; Tourism Agencies (a) (b) Fig. 5. Interactions between different stakeholders (CAV): (a) brand creation; (b) brand implementation; Jingmen Social Science Federation; Jingchu University of Technology; Chinese Academy of Agricultural Science; Huazhong Agricultural University Hubei Provincial Governments; Politicians Hubei Provincial Governments; Politicians (b) Fig. 5. Interactions between different stakeholders (CAV): (a) brand creation; (b) brand implementation; 5.2.3.2. The interactions between stakeholders during the GANT implementation phase. In 2013, the Hubei Provincial Government approved the establishment of the Jingmen Zhanghe New District Management Committee (Zhanghe Xinqu Guanweihui). Zhanghe NDMC represents the district in which General Aviation New Town is situated, as shown in Fig. 4. 5.2.1. General project introduction Some private tourism companies provide a platform and work with the government to promote the tourism industry.i During the 13th five year plan, Jingchu University of Technology Table 4 j Table 4 Project progress in General Aviation New Town (GANT) 2009–2018. National government Provincial government Municipal government 2009 In 2009, the State Council and the Central Military Commission jointly issued the Opinions on Deepening China's Low-altitude Airspace Management Reform. – – 2012 – The master plan for the construction of Zhanghe New District was approved by the Hubei Provincial Government in August 2012. – 2013 The Civil Aviation Administration of China issued the General Aviation Flight Approval and Management Regulations in November 2013. The Zhanghe New District Management Committee was approved by the Hubei Provincial Government in February 2013. – 2014 – – In August 2014, Jingmen AVIC Aviation Town Project was signed in Beijing. 2015 – – Since 2015, a Jingmen AVIC Flight Conference is held every year. 2016 On May 13, the State Council released the Guidance on Promoting the Development of the General Aviation Industry. – – 2017 – – In 2017, AVIC town, Zhanghe new district was selected as the site for pilot projects for sports and leisure towns by the State Sports General Administration. Although not directly in line with aviation activities, it was seen as strengthening the profile of the area. 2018 – In January 2018, the Hubei Provincial Government Work Report proposed to support development of the general aviation industry. – Municipal government In August 2014, Jingmen AVIC Aviation Town Project was signed in Beijing. Since 2015, a Jingmen AVIC Flight Conference is held every year. In August 2014, Jingmen AVIC Aviation Town Project was signed in Beijing. Since 2015, a Jingmen AVIC Flight Conference is held every year. In 2017, AVIC town, Zhanghe new district was selected as the site for pilot projects for sports and leisure towns by the State Sports General Administration. Although not directly in line with aviation activities, it was seen as strengthening the profile of the area. In January 2018, the Hubei Provincial Government Work Report proposed to support development of the general aviation industry. 10 W. Ma, et al. Cities 105 (2020) 102754 Stakeholder identification in GANT. No. Table 4 j Type of stakeholders Departments Roles Resources 1 Governmental organisations Politicians The main leaders in Jingmen's Municipal Government Approve brand creation and push brand implementation Opinion leaders Departments and agencies Government at other levels: Jingmen Municipal Government (MG); Jingmen Zhanghe District Management Committee (ZDMC); General Aviation Industry Demonstration Zone Office (GAIDZO) Implement the projects and branding activities in different government levels Policy supply and administration power Departments or agencies inside Jingmen: Jingmen Municipal Party Committee and Propaganda Department (MPCPD); Jingmen Development and Reform Commission (DRC); Tourism Bureau (TB1); Planning Bureau (PB); Bureau of Culture Sports Broadcasting Press and Publication (CSBPP) Execute branding activities; Coordinate interests of different departments; Organise activities each year, such as AVIC Flight Conference 2 Promotion agencies Public Daily media Group Publish information about GANT projects Media channels Private Tourism agencies Publish tourism information about GANT and organise the day trip 3 Enterprises Public Aviation Industry Corporation of China, Ltd.; China Aviation Industry General Aircraft Co., Ltd. Provide technical support and relevant expertise for local authorities Financial and technology resources Private Hubei Longhao Flight Training Co., Ltd.; Travel agencies Participate in GANT project 4 Academic organisations and Schools Public Jingmen Social Science Federation; Jingchu University of Technology; Chinese Aviation Society Provide suggestions on city branding to the government; Provide professional talents to the general aviation industry Knowledge support 5 Infrastructure and transport providers Public Jingmen Urban Construction Investment Holding Group Co., Ltd. (CIHG); Zhanghe New district Zhangfu Investment Development Co., Ltd.; Investment Development Co., Ltd.; AVIC Jingmen Development Co., Ltd.; China Special Aircraft Research Institute; Jingmen Traffic Construction Investment Co., Ltd.; Hubei Zhonghang General Airport Management Co., Ltd. Construct Zhanghe New district infrastructure and Zhanghe airport; Invest and finance of infrastructure construction in the Zhanghe New district. Financial and technology resources 6 Residents Private Local residents Participate in GANT city branding activities and provide feedback and opinions about the brand Views 11 W. Ma, et al. Table 4 j Cities 105 (2020) 102754 Hubei Provincial Governments; Politicians Jingmen Municipal Governments; Politicians Jingmen Municipal Governments; Politicians Jingmen Zhanghe New District Management Committee Diﬀerent departments inside Jingmen (DRC; TB1; PB; CSBPP) Jingmen Social Science Federation; Jingchu University of Technology; Chinese Aviation Society Leading Group of General Aviation Industry Integrated Demonstration Zone Aviation Industry Corporation of China, Ltd.; China Aviation Industry General Aircraft Co., Ltd.; Travel agencies Aviation Industry Corporation of China, Ltd.; China Aviation Industry General Aircraft Co., Ltd Diﬀerent Departments in District Level TB2; CIHG; TCI Residents Hierarchical relationships Information deliverer Departmental cooperation Information feedback Legend Governmental organisations Academic institutions Promotion agencies Enterprises Infrastructure and transport providers Residents Colour Public Public+Private Private Local Media Organisations; Tourism Agencies (a) (b) Fig. 6. Interactions between different stakeholders (GANT): (a) brand creation; (b) brand implementation; Jingmen Social Science Federation; Jingchu University of Technology; Chinese Aviation Society Aviation Industry Corporation of China, Ltd China Aviation Industry General Aircraft Co., Ltd.; Travel agencies Jingmen Municipal Governments; Politicians Leading Group of General Aviation Industry Integrated Demonstration Zone Hubei Provincial Governments; Politicians Aviation Industry Corporation of China, Ltd.; China Aviation Industry General Aircraft Co., Ltd (a) (b) Fig. 6. Interactions between different stakeholders (GANT): (a) brand creation; (b) brand implementation; established the School of Aviation and created graduation majors for its students including aircraft manufacturing engineering, avionics, elec- trical technology, and precision molding technology for aviation ma- terials (JUT, 2016). In addition, the Jingmen Municipal Government cooperates with the Chinese Aviation Society in support of talent and technology development in the general aviation industry. However, after some initial success visitor numbers could not be maintained and declined dramatically once the activities were orga- nised (interviewees 2; 3; 18). Obviously, politicians and other depart- ments withdrew their attention and participation during the im- plementation phase. Other types of city branding policy instruments were not developed and implemented. At the same time, based on our observations, we conclude that the level of cooperation and coordina- tion between the various public departments and locally engaged actors seems insufficient. Residential participation and interest in the brands has been low and passive. The participation of private enterprises in the implementation of city branding is weak. Thus, two important stake- holder groups remain uncommitted to the city branding efforts. 5.3. Implementation barriers in Jingmen's city branding We use Table 6 to list the main barriers in Jingmen's city branding implementation process. Weakly represented roles among stakeholders and missing resources in industry, markets, and policy supply are shown which result in a rather unsuccessful implementation of the two brands. Both have so far failed to attract noticeable attention from the targeted groups (i.e. investors, private companies, local farmers, and prospective residents). Table 4 j According to the interviews, many projects were not realised, often- times even failing to kick off for lack of interest among companies and insufficient policy provided by the local government was insufficient (interviewees 8; 7). The two cases show no functioning industrial system or market-oriented operation which would benefit from the brands and so far, none have been established. In terms of infra- structure, there are still many difficulties in rural transportation, farmland water conservancy, and communication. For example, in terms of transportation, Jingmen City is the only prefecture-level city in the Hubei province that does not (yet) have a high-speed railway sta- tion. For these two cases, local government departments still need the technology input and expert advice. However, only a few suggestions from experts were adopted (interviewee 15). Little expertise and tech- nology has been developed which could be translated into brand pro- ducts. Many infrastructure and transport providers participate in the in- frastructure and real estate projects which are all conducted under the GANT brand, especially in key projects, such as the reconstruction and expansion of Zhanghe airport and the roads connecting the new town with the city of Jingmen. The Hubei Jingmen Urban Construction Group is a state-owned company responsible for the construction of infrastructure in the Zhanghe New district. Zhanghe New District Zhangfu Investment Development Co., Ltd., was established in 2012, and is a state-owned enterprise. It is the main financial department for urban infrastructure construction and investment in the Zhanghe New District (ZNDZID, 2019). The China Special Aircraft Research Institute contributes to the Jingmen Zhanghe general airport reconstruction and expansion project. Hubei Zhonghang General Airport Management Co., Ltd. leads the construction of the Jingmen general airport, water terminal, temporary take-off and landing point construction project. f Residents only participate in certain projects and activities, such as AVIC Flight Conference, via their role as visitors. In other words, the role of residents in GANT brand implementation is minimal. 6. Discussion and findings The two economic city brands in Jingmen we investigated represent different choices with regard to the strategy towards industrial trans- formation. The CAV brand, leaning primary on the agricultural sector (potentially) focused on the involvement of a large share of the work- force, but of low added economic value to the Jingmen economy. The other GANT brand aims to trigger the development of an advanced, more sustainable part of the services side of the secondary sector (aviation navigation industry). However in doing so and focusing on aviation technology, the brand focuses on a very tiny percentage of the total Jingmen workforce, however one which could generate a potential high economic value. Still, we found the actual branding creation and implementation processes in both cases to show a very similar pattern. Even though Jingmen still uses CAV as a city brand, political and policy attention for this brand fell dramatically after 2013 because of a change in the secretariat of the Jingmen municipal government (in- terviewees 6; 17). Current politicians and departments are more in- terested in the GANT city brand and its associated projects. A fleeting political attention to branding policies in general and the lack of poli- tical attention to brand implementation can be identified in Jingmen's city branding activities. In these two cases, the responsibility for city branding and promo- tion of the city was placed primarily in the hands of the local media and promotion departments, which dutifully organised some public events. Jingmen's local politicians and senior civil servants seem quite fo- cused and successful in the identification and launching of potential brands which could be used to transform Jingmen. In both cases, they 12 W. Ma, et al. Cities 105 (2020) 102754 The weak roles, missing resources and identified barriers in two branding projects. CAV GANT Barriers Roles Approver or Pusher (governments; politicians) Lack of guidance in attracting investment. Unable to build a vigorous market system for local produce. Limited encouragement of public participation from the government. Insufficient planning and coordination of policy implementation. Focus more on more management than implementation. Lack of political support and interest in implementation Coordinator Dissolution of efforts as CAV office was merged into Jingmen's Agricultural Bureau. No specific departmental responsibility and authority for CAV project implementation. Imperfect communication and coordination. Lack of policy interest; Problematic organisation and coordination Executor Local government departments simply follow higher level government guidelines. 6.2. City branding implementation In Jingmen's cases, only the attention of influential local politicians pushes the implementation of a brand. Effectiveness of a brand ser- iously is tightly related to the span of political attention. According to Kavaratzis' advice (2012), city brands should be recognised and ac- cepted by many different stakeholders and this requires their involve- ment throughout the entire city branding process (Baker, 2012; Kavaratzis, 2012). However, Jingmen's governmental organisations at different levels, state-owned enterprises, public media organisations are the key stakeholders in both instances of city branding that we studied. Small private companies, promotion agencies, academic associations, and residents participate in city branding to a much lesser extent, and predominantly play a role as ‘participant’ or ‘recipient’ in the im- plementation phase. Their absence in the creation stage results in ser- ious consequences during the implementation phase, while the absence of many essential public sector players (especially high level politicians and civil servants) similarly affects implementation negatively. The cases seem to provide evidence that branding processes in Chinese cities lack a coherent and integrated city branding approach. Social media activities (Cleave, Arku, Sadler, & Kyeremeh, 2017; Zhou & Wang, 2014) and mega events (Chen, 2015; Marin-Aguilar & Vila-López, 2014) are broadly adopted by policy makers to brand cities for inside as well as in outside communication. For the implementation of Jingmen's city brands, the local government has placed the respon- sibility primarily in the hands of its media and promotion departments. These agencies utilise general media and the organisation of specific events as key instruments to establish a positive city image. In Jingmen, however, the limited number of activities and their small scale result in limited long-term effects of city promotion activities. In China, only the mega cities are in a position to organise mega events, such as Olympic Games or the G20 summits. And even then, it could be questioned whether these events are followed up by sufficient other activities and projects to really identify long-lasting effects in terms of branding. In contrast, the activities which are organised by small and medium-sized cities tend to be small in scale and weak in effect since they do not have sufficient resources at their disposal to organise mega events. 6. Discussion and findings Local government departments simply follow higher level government guidelines. Passive participation Information deliverers Ineffective brand communication via traditional media channels, like newspapers and TV stations. Only media departments are involved in publication and communication. Limited brand publication and promotion Advisor Policy suggestions are not adopted. Few suggestions for GANT creation and implementation. Weak influence of expert advice Participants Insufficient participation from small private enterprises. Few large and strong enterprises support the brand. Farmers' passion are not mobilized. Farmers' participation is relatively small. Leading (state-owned) companies insufficiently involved. Insufficient participation from private enterprises. Low general public participation. Low private participation Resources Policy Lack of policy support from higher level governments. Insufficient policy supply results in failure during the negotiations. Lack of policy support from national government level. China lacks independent navigation regulations and standards. Insufficient policy supply results in failure during the negotiations. Lack of political interest and support for implementation Market The CAV brand hasn't resulted in a viable economic market in support of providers of local produce. Immature market for general aviation. Overall weak industry. High operation costs of navigation and limited low-altitude airspace inhibit the realisation of a vital market. Lack of viable market (perspective) Media channels Only use of simple and traditional brand promotion channels. Only use of simple and traditional brand promotion channels. Simple and traditional promotion channels Finance Low efficiency of funds Higher operating costs Lack of finance Infrastructure Difficulties in rural transportation, farmland water conservancy, and communications, e.g. Highways need reconstruction to connect Jingmen and the agricultural industrial plants. Insufficient infrastructure; lack of a general airport in the Hubei Province. Lack of involvement in policy implementation Technology, knowledge, and talent Technology development was not transferred into agricultural products. Insufficient technology support for the agricultural sector. Lack of in-depth cooperation with non-public actors. Lack of professional aviation talents Lack of professional input 13 W. Ma, et al. Cities 105 (2020) 102754 Chosen brands are attractive, but have limited impact on adding economic value Extensive political, but limited stakeholder involvement The loss of interest of political elites; private sector stakeholders were not involved in brand selection; and lack of economic clout Lack of professional city branding/ marketing/ promotion departments and experienced staﬀs Brand creation and selection Brand implementation City branding processes Fig. 7. Key findings in Jingmen's city branding processes. Brand mplementation Fig. 7. Key findings in Jingmen's city branding processes. 6.1. City branding selection and creation A successful city brand should be aligned with national and pro- vincial plans and policies, and based on historical and cultural tradi- tions (Han et al., 2018). The brand ideally would take into account the current demographic and economic context, reflect future ambitions of the municipal government, set challenging yet realistic goals, be at- tractive and distinct from its neighbours, adopt and apply the brand for the long term through implementation across the various departments within the municipal government and connect it with other policy measures. If these conditions are fulfilled, a brand is (far more) likely to generate deep sustainable transformation. In Jingmen's cases, local government promotes new aviation technology development and sus- tainable agriculture, which comes across as more attractive and more sustainable. However, the economic city brands that were studied in this research did not add much economic value and employment. Jingmen allows its heavy industry, with all of the environmental pro- blems that come associated with it, to survive and simply downplays the importance of the industrial city in its external communication. Economically speaking the aviation industry and sustainable agri- culture play a far smaller role in Jingmen's local economy than the secondary sector. The manufacturing and petrochemical industries re- main unaffected by the new city branding efforts. Although this is un- derstandable, however, brand selection and creation do not go very far in achieving industrial transformation. We would have to conclude that the current state of affairs in Jingmen is not the most productive one in the long run. Actually, city labels, such as ‘eco city’ and ‘low carbon city’, are widely chosen by China's local governments in their planning contexts since these attractive labels are related to national urban de- velopment programs and influenced by national policies (de Jong et al., 2016; Lu & de Jong, 2019). These reflect cities' development visions and economic restructuring ambitions. However, in many occasions, the selected new economic brands do not meet the actual requirements of urban industrial transformation. Moreover, the new city brands barely distinguish one city from the other in terms of brands. This situation also represents a common phenomenon in many Chinese cities. 6. Discussion and findings show remarkable skill in lining up especially provincial and national governments during the brand construction phase. However, the cases at the same time reveal that after these brands are created, politicians ‘leave’ the implementation to other players such as local government agencies which neither have the powers and authority, nor perhaps the right skills to actually realise the original, ambitious policy goals un- derlying the city brands. Fig. 7 shows the key findings in Jingmen's city branding processes. and city officials. The city branding organisation should integrate the city's stakeholders, such as local government departments, public and private enterprises and residents into participation in city branding management (Zavattaro & Daspit, 2016). However, in contrast to this, we found that the realisation of city branding policies in Jingmen was primarily the result of opaque and deeply political processes in which many, especially private stakeholders are not or only quite passively involved. The cases show how political leaders skillfully identify and select seemingly attractive brands such as ecological agriculture or aviation new town to realise the industrial transformation goals for Jingmen. The branding creation can be successfully built with strong support from political leaders. 6.2. City branding implementation Small cities can only focus on less glamourous activities generating far less According to Baker (2012), a brand that has been developed with broad stakeholder support is more easily accepted and more quickly recognised by those at the receiving end. The brand should be widely embraced by a critical mass of stakeholders (Henninger, Foster, Alevizou, & Frohlich, 2016) within and outside the local government, and enjoy strong support from the political and administrative leaders 14 Cities 105 (2020) 102754 W. Ma, et al. publicity. Furthermore, we find that public information campaigns and advertising are quite passive policy instruments and therefore in themselves insufficiently powerful to singlehandedly implement brands without broader participation. The specific actions which were under- taken to implement the two brands in Jingmen are less impressive than the slogans. Potential branding policy instruments are simply not de- veloped or applied. politicians and public organisations dominate city brand creation phase, but are hardly involved and seem hardly interested in the actual implementation phase. In other words, there remains a wide gulf be- tween ambitious, politically inspired intentions during brand creation and a continuous and viable commitment of the majority of stake- holders during the implementation phase, which is needed to achieve industrial transformation. Long-term political and bureaucratic com- mitment to city branding implementation efforts after the brands are created also seem important for sustained and lasting success in city branding.f The creation and implementation of city branding activities are processes which are meant to unite and align the various city depart- ments and stakeholders in brand management, to have all the noses point in the same direction (Zavattaro & Daspit, 2016). Consistent and clear city brand recognition is the basis for successful city branding (Kavaratzis, 2004), which can only be achieved through the establish- ment of an appropriate city marketing organisational structure. Many international cities set up professional city branding organisations to focus on the creation and implementation of city branding projects; they focus on a specific tourist market and seek to attract growing numbers of visitors (Boisen et al., 2018; Pike, 2012). In Jingmen, the work of city branding is managed by the Municipal Party Committee Propaganda Departments (MPCPD). City branding efforts are primarily implemented by the daily newspaper media and other propaganda departments. Other departments are barely involved in the field of city image enhancement and city brand building. 6.2. City branding implementation There is a lack of pro- fessional staff specialised in city branding and promotion (Fan, 2014). Lack of professional institutions and experienced staff obviously influ- ences the effectiveness of implementation. Chinese city branding practices present a different style than European or American or other Asian ones because of its unique gov- ernance culture, Chinese cities are exceptional in the sense that they do not use the universally acclaimed mechanism of broad stakeholder in- volvement in city branding, which is described in international litera- ture. However, the research findings suggest that private stakeholders such as residents, farmers and private corporations should be en- couraged to participate more fully in city branding creation and im- plementation to convey their opinions and interpretations and turn the city brand into something more than just a craftily politically en- gineered, temporary slogan. The case study findings provide evidence that seems to support the necessity of organising broad stakeholder involvement in place branding process (Baker, 2012; Kavaratzis, 2012). The broad stakeholder involvement also should be applied in China's city branding, which is a finding of great theoretical significance. ii We are well aware that our conclusions are based on the analysis of two city branding policies within one city. However, Jingmen is re- presentative of a large group of Chinese cities. China counts a great many medium-sized Chinese cities like Jingmen which boast high-level ambitions to use economic city brands to transform their urban economies and industrial structure into more sustainable directions. However, as becomes clear from our study, industrial transformation becomes very difficult for these cities. City officials face the daunting task of making this transition happen, with a dominant secondary sector that cannot easily be phased without incurring major economic damage, with major infrastructure issues and a lowly qualified labour force. Current institutional patterns favour politicisation of the branding creation phase, which lacks of engagement from private sector and citizen engagement. The image of city branding in medium-sized cities in China in terms of branding implementation in comparison with more visible and advanced city branding processes of larger megacities on the Eastern shore does not bode well. The outcome of our study is of importance since what actually takes place in Jingmen may reflect the result of industrial transformation patterns in many Chinese medium- sized cities. 6.2. City branding implementation At the same time, we emphasise the need to investigate a greater number of medium-sized cities in future research to verify our findings. City branding is a potentially crucial tool for positive self-re- invention which could be enormously helpful to the majority of Chinese cities. Making this happen requires a major reconsideration of the prevalent institutional and organisational practices through which this is done. 7. Conclusion This article has presented a case study research to analyse economic city brands' creation and implementation processes of a medium-sized city in China. We mapped a stakeholder oriented typology of city branding practices in Chinese medium-sized cities. City branding in China clearly is embedded within a multi-level governance context. Politicians and provincial, municipal and district governments and departments are all involved in branding activities in different stages. The politicians are heavily involved in the brand creation phase and its final approval. They subsequently mobilise public resources and authorities at the local level to push for brand implementation. They engage in specific city branding activities and rely on specific public enterprises to finance and manage projects which are developed as part of a city brand. Different levels of administrative governments and different departments are the implementers of brand projects. They are the specific executive institutions. The special work office or leading group for each brand project coordinates the various interests and ensures cooperation from different departments. The media organisations such as promotion agencies and propaganda or- ganisations are the information deliverers of city branding im- plementation projects. Academic associations and organisations are the advisors for brand selection and implementation. In most occasions, state-owned enterprises, private companies, residents and infra- structure and transport providers are mainly receivers of city branding projects. Author contributions The mechanisms underlying branding creation and implementation in Jingmen that we encountered could serve as a basis to increase our understanding of city branding processes in medium-sized Chinese ci- ties. The creation phase of city branding in medium-sized cities in China is characterised by politically sophisticated processes during which local politicians compete for the acknowledgement and alignment of potential brands for their local city within the various layers of the Chinese government. However, the implementation of the brands is an altogether different story. City brands are more easily created than implemented. Implementation is generally weak because key private sector stakeholders are not involved during the brand creation phase. However, citizens and private sector stakeholders are required for successful city branding implementation. Conversely, powerful Wenting Ma is the main author of this contribution. Martin de Jong is Wenting's promotor and in charge of Jingmen's project; he helped structure the manuscript, edited the texted and strengthened the nar- rative. Mark de Bruijne is Wenting's daily supervisor and in collabora- tion with Daan Schraven, he contributed to the text-editing and added his insights to the article. Martin de Jong holds the academic respon- sibility for the research program in which this paper is embedded. Declaration of competing interest The authors declare no conflict of interest. They conducted an 15 W. Ma, et al. Cities 105 (2020) 102754 Cities 105 (2020) 102754 China Scholarship Council for supporting Wenting Ma's PhD project. They are also indebted to the Erasmus Initiative for the Dynamics of Inclusive Prosperity and Delft University of Technology for the in kind contributions made by the other three authors. The authors express their gratitude to Jingmen's Planning Bureau and Jingmen's Planning and Design Institute for the support and cooperation provided during our fieldwork. The authors thank Yun Song for his help in drawing the figures. China Scholarship Council for supporting Wenting Ma's PhD project. They are also indebted to the Erasmus Initiative for the Dynamics of Inclusive Prosperity and Delft University of Technology for the in kind contributions made by the other three authors. The authors express their gratitude to Jingmen's Planning Bureau and Jingmen's Planning and Design Institute for the support and cooperation provided during our fieldwork. The authors thank Yun Song for his help in drawing the figures. advisory study to Jingmen City about its sustainable urban transition. 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Appendix A Appendix A Table 7 Interviewees in Jingmen in 2018 and 2019. Interviewees in Jingmen in 2018 and 2019. Interviewee no. Departments (the location interview conducted) Position The interview topics 1 Jingmen Planning and Design Institute (PDI) Vice Dean The role of PDI in city branding implementation 2 Jingmen Planning and Design Institute (PDI) Chief Jingmen's city brands and characteristics 3 Jingmen Planning and Design Institute (PDI) Officer The state quo of Jingmen's city branding and promo- tion 4 Jingmen Planning Bureau (JPB) Officer The state quo of Jingmen's city branding and promo- tion 5 Jingmen Development and Reform Commission (JDRC) Chief GANT project implementation and promotion 6 Jingmen Municipal Committee of Rural Office (JMCORO) Chief CAV project implementation and promotion 7 Investment Bureau in AVIC New Town (JIB) Chief The industrial situation in GANT 8 Bureau of Culture Sports Broadcasting Press and Publication (CSBPP) The Chief in Industrial Office The industrial situation in GANT 9 Bureau of Culture Sports Broadcasting Press and Publication (CSBPP) The Chief in Sport Office AVIC conference and GANT construction 10 Jingmen Tourism Bureau (JTB) Chief City branding promotion and tourism information publication 11 The Daily Newspaper Department in Daily Media Group (DMG) Editor A City brand promotion and media channels in Jingmen 12 The Night Newspaper Department in Daily Media Group (DMG) Editor B City brand promotion and media channels in Jingmen 13 Hubei Agriculture Valley Investment Management Corporation (JMCORO) Manager CAV implementation and promotion 14 Hubei Agriculture Valley Investment Management Corporation (JMCORO) Chief CAV implementation and promotion 15 School of Media, Jingchu University of Technology (JUT) Associate Professor City branding communication of Jingmen 16 School of Management, Jingchu University of Technology (JUT) Associate Professor City branding choice and implementation 17 Citizen 1 The characteristics of Jingmen's city branding 18 Citizen 2 The characteristics of Jingmen's city branding Meeting 1 Planning Meeting (JPB) Officers of Jingmen Planning Bureau and Institutes Feedback on the investigation results of Jingmen's city branding Meeting 2 Jingmen Strategy Planning Conference 2049 (PDI) Officers of Jingmen Planning Bureau and Institutes Discussion on Jingmen development strategy in future Workshop Experts Workshop (PDI) Experts and scholars from home and abroad Jingmen's city branding and sustainable urban trans- formation The interview topics efficiency: Evidence from China. 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https://openalex.org/W4312223161	https://link.springer.com/content/pdf/10.1007/s41297-022-00180-w.pdf	English	null	Conceptualisations of curriculum co-creation: ‘it’s not them and us, it’s just us’	Curriculum perspectives	2,022	cc-by	9,274	Curriculum Perspectives (2023) 43:25–37 https://doi.org/10.1007/s41297-022-00180-w Curriculum Perspectives (2023) 43:25–37 https://doi.org/10.1007/s41297-022-00180-w RESEARCH ARTICLE Abstract Building on the growing field of curriculum co-creation in higher education, this research analyses current conceptualisa- tions of this concept based on the perceptions of student and staff co-creation practitioners. It draws on a rigorous review of international curriculum co-creation literature and describes in-depth research on experiences and conceptualisations of fifteen curriculum co-creation initiatives across eight subject areas at five universities in Scotland. Following an inductive analysis, the findings highlight conceptualisations of curriculum co-creation that focus on (A) developing shared values, (B) enhancing creativity through collaboration and (C) negotiating power for mutual benefit. These findings are discussed with respect to how they contribute to a new definition: curriculum co-creation is a relational way of working underpinned by shared responsibility, reciprocity in learning from each other, mutual respect, care, trust and empathy. This values-based, creative process helps staff and students work together to share and negotiate decision-making about aspects of curricula, which often leads to mutual benefits for learners and teachers. This new definition encompasses current conceptualisations of curriculum co-creation in the rapidly-changing higher education sector, with implications for fostering resilient, authentic and meaningful pedagogies that are relevant to today’s challenges in higher education and beyond. Keywords Curriculum co-creation · Curriculum negotiation · Student engagement · Higher education Conceptualisations of curriculum co‑creation: ‘it’s not them and us, it’s just us’ Tanya Lubicz‑Nawrocka1 Received: 29 July 2021 / Revised: 26 October 2022 / Accepted: 2 December 2022 / Published online: 28 December 2022 © The Author(s) 2022 * Tanya Lubicz‑Nawrocka tanya.lubicz-nawrocka@stir.ac.uk 1 The University of Stirling, Stirling, Scotland Introduction the burgeoning literature has been beneficial, there is a lack of clarity about what curriculum co-creation can be, beyond staff and students’ active engagement and collaborations in learning and teaching. Engagement of higher education students and staff is an increasing concern, especially since the COVID-19 pan- demic when many staff and students are now balancing online and in-person learning and teaching with many other responsibilities. One way of addressing this is to find ways to advance engaging, meaningful and authentic forms of learning and teaching such as curriculum co-creation. In the last decade, there has been a dramatic increase in the discourse on curriculum co-creation (Bovill & Woolmer, 2019) and wider students-as-partners initiatives (Healey & Healey, 2018; Mercer-Mapstone et al., 2017). Many terms are often overlapping, including working with students as partners through co-creation/co-design/co-production both of and in the curriculum and/or learning and teaching (Bovill & Woolmer, 2019; Mercer-Mapstone et al., 2017). Although This paper acknowledges the overlaps between curricu- lum co-creation and partnership research, and it focuses on the under-problematised theory and practice of curriculum co-creation. By taking a critical perspective of the litera- ture with respect to a recent study of co-creation examples across the Scottish higher education context, this research contributes to the existing body of knowledge by reconsider- ing definitions of co-creation and adding new perspectives for adopting it in practice. It also explores implications for advancing authentic and meaningful curricula that address the challenges we face in today’s quickly changing world. Whilst there is a wide literature on school curricular development and planning, the term is rarely defined in the higher education context and, even when it is, there are many different interpretations (Fraser & Bosanquet, 2006; Lattuca & Stark, 2009). Drawing on Fraser and Bosanquet’s work (2006), I acknowledge the many conceptualisations of higher education curricula, ranging from product-focused, (0121 3456789) 3 3456789) 3 Curriculum Perspectives (2023) 43:25–37 26 learner empowerment are realised through the use of participatory, transformative and ‘active’ pedagogies... teacher-directed stances to process-focused, student-centred views. How different individuals view the curriculum influ- ences the possibilities that they envision for curriculum co- creation opportunities (Bovill & Woolmer, 2019). Boomer’s work has been particularly generative by suggesting a ‘cur- riculum-as-negotiation’, bringing together teacher-centred and student-centred curriculum design whilst emphasising a process-focused view of the curriculum as a verb, ‘cur- riculuming’ (Boomer, 1992; Green, 2021). Boomer (1992, p. Introduction 14) wrote: Subsequent definitions also emphasise collaboration; for example, a widely cited definition by Bovill et al., (2016, p. 196) states ‘co-creation of learning and teaching occurs when staff and students work collaboratively with one another to create components of curricula and/or pedagogi- cal approaches’. A subsequent definition by Dollinger et al., (2018, p. 210) draws on marketing: ‘value co-creation is the process of students’ feedback, opinions and other resources such as their intellectual capabilities and personalities, inte- grated alongside institutional resources, which can offer mutual value to both students and institutions’.i Negotiating the curriculum means deliberately plan- ning to invite students to contribute to, and to modify, the educational programme, so that they will have a real investment both in the learning journey and in the outcomes. Negotiating also means making explicit, and then confronting, the constraints of the learning context, and the non-negotiable requirements that apply. Definitions of curriculum co-creation often draw on the concept of partnership, with the latter having emerged as the predominant term in the literature encompassing a wide range of curricular and extracurricular initiatives (Cook- Sather et al., 2018b; Mercer-Mapstone et al., 2017). There are many synergies between curriculum co-creation and the concept of pedagogical partnership. This can be seen in how Cook-Sather, Felten, and Bovill—who individu- ally and in collaboration are extremely influential in this field—used the term co-creation when publishing in 2011 and 2016 but referred to partnership in their pivotal 2014 book. Their widely cited definition of student/staff partner- ship is also important and of relevance: ‘a collaborative, reciprocal process through which all participants have the opportunity to contribute equally, although not necessarily in the same ways, to curricular or pedagogical conceptual- ization, decision-making, implementation, investigation or analysis’ (Cook-Sather et al., 2014, pp. 6–7). These authors also describe the important values of respect, reciprocity and shared responsibility. Although both partnership and co-creation initiatives often seek to include diverse student voices whilst enhancing learning and teaching (Cook-Sather et al., 2018a; Lubicz-Nawrocka, 2019a), many partnership examples involve small numbers of already highly engaged students in optional projects in contrast to whole-class co-creation initiatives that are embedded into coursework (Bovill, 2020b). Bovill (2020a) suggested that co-creation and partnership often overlap in their emphasis on demo- cratic values and shared decision-making, although partner- ship can imply a level of equality that some staff find hard to align with the reality of academic structures and constraints. Introduction Boomer’s work has also been influential in acknowledg- ing democratic values and issues of power (Green, 2021) and by setting the scene for more recent literature curriculum studies including those in curriculum co-creation (Breen & Littlejohn, 2000; Bron et al., 2016; Green, 2021). Inspired by the work of Boomer (1992), Fraser and Bosanquet (2006) and Barnett and Coate (2004) in particular, I see the cur- riculum as a creative, student-centred space where staff and students engage in a process of learning and teaching that they continually adapt—and can take up opportunities to co-create within certain constraints—to meet their shared objectives of developing students’ knowledge, skills and wider capabilities. An overview of curriculum co‑creation literature Recent literature was reviewed in a systematic manner to understand the nature of curriculum co-creation princi- ples and practices in higher education, using the Education Resource Information Centre (ERIC) database. The inclu- sion domain included all conceptualisations, practices and outcomes of all forms of co-created academic experiences and only those examples of co-creation that included both students (undergraduate and/or postgraduate) and staff (aca- demic and/or professional services). The search term ‘co- creat*’ yielded 556 publications from 1 January 2014 to 31 December 2019 including keywords such as ‘co-create’, ‘co-creation’ and ‘co-creating’. Of these, 243 sources were excluded when applying the timeframe inclusion criteria, and 111 further sources were excluded when filtering by ‘higher education’. p Curriculum co-creation promotes strong working rela- tionships through collaboration not only between students and staff (Blau & Shamir-Inbal, 2018; Kaur et al., 2019; Lubicz-Nawrocka, 2019b) but also between different stu- dents through peer learning (Bovill, 2020b; Vaughan et al., 2016). This practice can foster staff and students’ senses of belonging (Cook-Sather et al., 2018a; Masika & Jones, 2016) and community (Blau & Shamir-Inbal, 2018; Lubicz- Nawrocka, 2019a). This literature broadly focuses on valuing students’ views and perspectives, but only limited examples explicitly reference democratic values underpinning co-cre- ational relationships (Bergmark & Westman, 2016; Bovill, 2020a; Lubicz-Nawrocka, 2019b). Reviewing the titles and abstracts of the 132 sources that met the inclusion criteria, 61 further sources were excluded due to focusing on non-academic higher education experi- ences, research and/or examples of co-creation between staff in different departments, staff and employers or partners or students with other students. Of the 50 remaining results focusing on co-creation between staff and taught under- graduate or postgraduate students, 34 provided examples of course-level, curricular co-creation and 16 provided theoreti- cal contributions and/or analysed the co-creation of wider learning and teaching initiatives. Additionally, relevant lit- erature found through citations included related topics such as staff-student partnerships and collaborations or participa- tory curriculum development. The literature provides many rich examples of outcomes from curriculum co-creation (including both benefits and challenges), but here I focus on key themes relating to the nature of co-creative practices: the relationships underpinning co-creation and the ways of working during this process. Existing definitions of curriculum co‑creation Existing definitions do not fully capture the intentionality of creating opportunities for cur- riculum co-creation or how these collaborations are enacted when sharing decision-making. Existing definitions of curriculum co‑creation Two of the most widely cited definitions of curriculum co- creation focus on collaboration and partnership between stu- dents and staff in curriculum development activities. Bovill et al., (2011, p. 137) suggest that ‘co-creation of curricula implies students and academic staff working in partnership to create some or all aspects of the planning, implementa- tion and evaluation of the learning experience’. Ryan and Tilbury’s (2013, p. 16) Higher Education Academy report drew on this definition, focusing on knowledge co-creation: The rationale for this study was to explore the extent to which existing definitions reflect recent examples of cur- riculum co-creation in theory and practice, with respect to this burgeoning field of study. The five influential definitions cited above focus on who is involved (students and staff) and what they are doing (collaborating to develop aspects of the curriculum), with only two emphasising why this work is important. The 2011, 2014 and 2016 definitions developed The concept of ‘co-creation’ is used to indicate inter- actions that encourage collaborative and democratic input from students as stakeholders in shaping knowl- edge practices…. The pedagogical ambitions behind 1 3 Curriculum Perspectives (2023) 43:25–37 27 active learning, student-centred learning and inquiry-based learning—because of its collaborative ethos (Bovill, 2020b; Lubicz-Nawrocka, 2019b; Matthews et al., 2018a; Moore- Cherry, 2019). Co-creation critically involves negotiation and collaboration as opposed to full student control over curricula (Cook-Sather et al., 2014), requiring a positive relationship between students and staff (Bovill, 2020a). This aspect of negotiation is important because staff take owner- ship over quality assurance and assessment outcomes, but they can create opportunities for students to share decision- making affecting how or what they learn (Boomer, 1992). Building relationships can promote an open dialogue about meaningful teaching and learning, which helps both students and staff to understand their responsibilities and creates a more equal balance of power between them (Boomer, 1992; Lubicz-Nawrocka, 2019b; Moore-Cherry, 2019). Although previous definitions of curriculum co-creation focus on collaboration between students and staff, they do not high- light negotiation which critically recognises constraints and notions of power (Boomer, 1992). by Bovill, Cook-Sather and Felten (with colleagues Mil- lard and Moore-Cherry in 2016) highlight approaches to developing aspects of the curriculum, and Ryan and Tilbury (2013) and Dollinger et al. (2018) underscore participatory processes of developing knowledge and wider educational experiences, respectively. Ways of working that focus on authentic and enjoyable processes of learning and teaching Curriculum co-creation often presents new ways of work- ing in higher education that can challenge the status quo of academic cultures, hierarchies and norms (Bergmark & Westman, 2016; Bovill et al., 2016). The context-specific, dialogic process underpinning how students and staff share responsibility is now widely recognised (Bovill, 2020b; Dollinger et al., 2018; Healey & Healey, 2018). Co-creation practitioners may engage in creative risk-taking whilst they share power and engage in innovative curriculum design together (Blau & Shamir-Inbal, 2018; Kaur et al., 2019; Lubicz-Nawrocka, 2019a). Curriculum co-creation can promote authenticity by advancing culturally and personally relevant forms of learn- ing and teaching (Backhouse et al., 2019; Temple Clothier & Matheson, 2019), which is especially relevant in the context of large class sizes and digital education. Students’ deep and Sampling of participants During my data collection in 2015–2016, there were few examples in Scotland that staff or students intentionally described with the term ‘curriculum co-creation’. Using criterion sampling focused on cases that fit Bovill et al.’s (2011) necessarily broad definition of curriculum co-crea- tion as collaboration between staff and students on aspects of curricula, I used publications, conference presentations and word of mouth to identify 19 staff co-creators at Scottish universities. Whilst trying to avoid oversampling in terms of their subject area, gender or institution, I invited 15 staff to take part in a semi-structured interview. Thirteen of these staff participated and I used snowball sampling to identify 11 students who had co-created curricula with them. Participants were engaged in 15 curriculum co-creation initiatives across 8 subject areas at the 5 Scottish universi- ties, with several staff involved in more than one form of cur- riculum co-creation. Participants’ subject areas ranged from medicine and veterinary studies to science (geoscience and biology) and social sciences (politics, sociology, social work and education). In alignment with Bovill et al.’s (2011) defi- nition of curriculum co-creation, the 15 initiatives included at least one aspect—and potentially all aspects—of plan- ning (10 instances), implementation (11 instances) and/or evaluation (7 instances) of students’ learning experience (see Table 1). Co-creating curriculum plans can include development of educational resources for future student cohorts or collaboratively planning and often implementing how later parts of course teaching, assessment or marking will take place. Course co-evaluation involves students and staff identifying strengths and areas for course enhancement, often relating to student engagement. Co-creation in plan- ning, implementation and evaluation is often overlapping, as seen in the literature and in my sample. In an example of evaluation and planning (Initiative 2), staff worked with students who had recently completed a course to identify difficult concepts and work together during a summer pro- ject to address them with co-created, multimedia educa- tional resources. Examples of whole-class implementation included co-creating grading criteria (Initiatives 11 and 14), exam questions (Initiative 7) or projects with community partners in service learning and science outreach (Initiatives 4 and 12). Methodological approach My research question lent itself to qualitative methods of investigation and a heuristic inquiry approach to exploring meaning-making in ways that are exploratory, reflexive, relational and participatory (Nevine, 2019; Punch, 2006). This approach was well suited to help me understand the subjective and nuanced nature of student and staff conceptu- alisations of curriculum co-creation. Most previous research either focused on principles of co-creation whilst drawing on a range of international examples or they focused on single case study initiatives that highlighted practices within one course, programme or institution. I felt it would be benefi- cial to identify a diverse variety of curriculum co-creation initiatives within one, discrete higher education context. I selected the Scottish context based on its focus on quality enhancement in learning and teaching. I was not aiming to gather representative data from the sector as a whole but, Research question This research formed part of a larger, doctoral research study to understand the nature of curriculum co-creation principles and practices and how it may advance individu- als’ aims for students in higher education. The aspect of the study explored here focuses on the research question: how do students and staff experience and conceptualise curriculum co-creation? This research was designed to explore whether previous definitions of curriculum co-creation fully captured staff and students’ understandings of what curriculum co- creation looks and feels like, both in examples from recent literature and in practice in one higher education context. Relationships underpinning curriculum co‑creation Curriculum co-creation (like student/staff partnerships) is distinct from other forms of student engagement—including 1 28 Curriculum Perspectives (2023) 43:25–37 active learning is an integral aspect of co-creation of content and pedagogy (Backhouse et al., 2019; Vaughan et al., 2016) as well as assessment (Bovill, 2020b; Doyle et al., 2019). Curriculum co-creation can also help individuals overcome challenges by working collaboratively and resiliently to find authentic solutions that are relevant to their own context (Bergmark & Westman, 2016; Blau & Shamir-Inbal, 2018; Bovill et al., 2016). By fostering more engaging forms of learning and teaching, students and staff tend to find curricu- lum co-creation processes extremely enjoyable, meaningful and rewarding (Dollinger et al., 2018; Kaur et al., 2019; Lubicz-Nawrocka, 2019a; Temple Clothier & Matheson, 2019). The authentic and rewarding process of curriculum co-creation can motivate the deep engagement of both stu- dents and staff to take up these opportunities (Bergmark & Westman, 2016; Lubicz-Nawrocka, 2019b). rather, wanted to focus on in-depth accounts within reason- able resource restrictions. Shared responsibility the voluntary nature of participation were made transparent through using participant information sheets and consent forms. In-depth, semi-structured discussions were held with participants who describing their understandings of the higher education curriculum and of curriculum co-creation, the nature of their curriculum co-creation activities and their experiences including collaborative relationships and ways of working. All data collection was audio-recorded and transcribed. Both student and staff participants described how they cre- ate a new space and ethos for curriculum co-creation to take place where they feel joint ownership over aspects of the curriculum. Staff 1 described the importance of speak- ing openly to develop shared responsibility during curricu- lum co-creation that differs from more traditional teaching: …you have to be very careful not to appear as if you are requiring students to do the work that you should be doing. They have to understand why certain levels of responsibility might be expected of them. Data analysis The extensive qualitative data was analysed whilst draw- ing on a constructivist grounded theory approach, which is an ‘inductive, iterative, interactive and comparative method geared towards theory construction’ (Thornberg & Charmaz, 2012, p. 41). This data analysis method helped me to draw on existing curriculum co-creation literature to identify core themes that were grounded in my data about participants’ conceptualisations of co-creation of the curriculum. I used the NVivo qualitative data analysis software to facilitate sys- tematic coding and analysis of the extensive qualitative data that consisted of 223,000 words of transcriptions. Staff 2 also emphasised that facilitating authentic engagement can motivate students to share responsibil- ity and see it as an opportunity as opposed to a burden, particularly within the context of increasing tuition fees. Others described sharing responsibility in curriculum co-creation as feeling: ‘It’s not them and us, it’s just us’ (Staff 4). Furthermore, Student 11 described the impor- tance of intentionally working towards shared aims: It was about how everybody would come with some skills or some knowledge and it would all go towards one goal. …I think it’s where you know that you can learn from each other and you can move forward in creating something good for both of you, more than just your own individual use. I drew on Pillow’s (2003, p. 181) ‘four reflexive strate- gies—reflexivity as recognition of self; reflexivity as rec- ognition of other; reflexivity as truth; reflexivity as tran- scendence…’. Therefore, I worked to (1) recognise my background and assumptions; (2) acknowledge different forms of power when working with participants—particu- larly students—whilst promoting their agency; (3) strive to achieve a sense of validity for my conclusions through constant comparisons across the body of data from differ- ent participants; and (4) minimise bias by corroborating my findings with wider literature when presenting my findings. The process of developing both individual and col- lective ownership—in not only learning but also in deci- sion-making affecting teaching—is important in sharing responsibility. Whilst staff and student co-creators take responsibility for different aspects of engagement, their joint ownership often increases their intrinsic motivation to work together. Ethics and data collection The research received Level 1 ethical clearance from the Moray House School of Education and Sport (University of Edinburgh) Ethics Committee, and the aims of the study and 3 Curriculum Perspectives (2023) 43:25–37 29 Table 1 Participants’ 15 co-creation projects Initiative number Co-creation project University type Subject type Co-creation types Students participating Staff participating 1 Co-created aspects of a course Research-intensive Arts, humanities and social science Implementation Whole cohort (earning course credit) Academic staff 2 Co-created educational resources Research-intensive Clinical sciences Planning & evaluation Selected student (receiving payment) Academic staff 3 Staff-supported peer teaching across year groups embedded into courses Research-intensive Clinical sciences Planning & implementation Whole cohort (earning course credit) Academic staff 4 Co-created course with com- munity projects Research-intensive Science or engineering Planning & implementation & evaluation Whole cohort (earning course credit) Academic staff 5 Co-created aspects of a course including co-development of resources for a new course Research-intensive Arts, humanities and social science Planning & implementation Whole cohort (earning course credit) Academic staff 6 Co-created educational resources Research-intensive Clinical and veterinary sciences Planning & evaluation Selected student (receiving payment) Academic staff 7 Co-created educational assess- ment Research-intensive Clinical and veterinary sciences Planning & implementation Whole cohort (earning course credit) Academic staff 8 Co-created aspects of a course Teaching-focused Science and engineering Planning & implementation Whole cohort (earning course credit) Academic staff 9 Co-created research Teaching-focused Science and engineering Evaluation Selected students (volunteering and gaining professional development skills) Academic staff 10 Student learning and teaching consultants Teaching-focused Various: arts, humanities and social science/sci- ence and engineering Planning & evaluation Selected students (volunteering and gaining professional development skills) Academic staff and academic developer 11 Co-created aspects of a course including marking criteria Research-intensive Arts, humanities and social science Implementation Whole cohort (earning course credit) Academic staff 12 Co-created community projects with co-assessment by staff and student Research-intensive Arts, humanities and social science Planning & implementation & evaluation Whole cohort (earning course credit) Academic staff 13 Co-created aspects of a course Research-intensive Science and engineering Implementation Whole cohort (earning course credit) Academic staff 14 Co-created aspects of a course including marking criteria Teaching-focused Arts, humanities and social science Implementation Whole cohort (earning course credit) Academic staff 15 Co-created aspects of a course Teaching-focused Arts, humanities and social science Planning & implementation & evaluation Selected students (volunteering and gaining professional development skills) Academic staff and academic developer 1 Curriculum Perspectives (2023) 43:25–37 30 Reciprocity in learning from each other Based on my inductive analysis of the data, the findings highlight conceptualisations of curriculum co-creation that focus on three key aspects of (A) developing shared val- ues, (B) enhancing creativity through collaboration and (C) negotiating power for mutual benefit. Each of these themes is explored below. There are strong overlaps between reciprocity and empa- thy, and respondents spoke of how co-creation helps them to learn from each other to improve curricula. Student 1 described co-creation as: …openness on the part of the teacher, and willingness or being receptive on the students’ front. …Obviously the student has to be conscientious, receptive and will- ing to learn from the expert; but equally the tutor or the professional has to be open to the students’ ideas and willing to have their perceptions changed. Mutual respect and care Many staff and student co-creators emphasised how respect is both a prerequisite for and an outcome of effective cur- riculum co-creation, and care is a related value that goes beyond notions of respectful tolerance. Many participants highlighted the responsibility of staff to respect, support and value students’ contributions throughout curriculum co-creation. This may be because staff often set the tone for the learning environment, and students tend to respect their subject-related and teaching-related expertise. How- ever, participants such as Students 3, 6 and 9 described how co-creating curricula makes them feel respected and valued, in sharp contrast with some of their other academic experiences when they felt staff do not provide adequate support or respect their time or individual learning needs. St ff 3 d ib d t d k l th t Other participants described how curriculum co-creation facilitates empathy not only on the part of students but also for staff. Staff 10 said: I think that the more you engage students in activities like this, the more they empathise with the role that academics play. That comes back to bridging the gap between staff and students, bringing the communities closer together. …It gave them [students] that insight into what it is like from the other side… [For staff] it is learning something that you couldn’t have gained without students’ insight. Learning environments built on trust can support both staff and students to challenge themselves and work collabo- ratively to overcome challenges in curriculum development. For example, Staff 2 reflected: Staff 3 described respect and care as key values that underpin her curriculum co-creation work: It was about showing students that we actually really care about teaching. Our students are often first gen- eration to come to university, and it’s great building their enthusiasm. …They feel their work is valued now. A lot of it is about how we take the constraints that we have to live with and work against them to create those communities of trust and genuine learning. Furthermore, Staff 3 described students’ wider learning: Furthermore, Staff 3 described students’ wider learning: In addition, Student 7 described how her teacher: They gained respect for teaching, and they learnt why decisions are made. They want to know. They gained respect for teaching, and they learnt why decisions are made. They want to know. Developing shared values Respondents outlined five values that they viewed as under- pinning curriculum co-creation: shared responsibility, reci- procity in learning from each other, mutual respect and care and trust and empathy. Each is described briefly below. Similarly, Staff 11 argued that curriculum co-creation can develop reciprocity that can overcome traditional, hierarchi- cal relations: 1 3 Curriculum Perspectives (2023) 43:25–37 31 The lecturers didn’t know the answers. It was the first time a lecturer has ever asked them [students] for their view and basically said ‘well how would you do it? …Your solutions are equally as important’. The themes of respect and care show how staff attitudes can influence students’ intrinsic motivation and success. Trust and empathy It can be challenging for some staff to be open to stu- dents’ ideas about different ways of learning and teaching, and it can be difficult for some students to engage more actively. However, participants described how they worked to solve complex problems together, since curriculum co- creation advances reciprocity between students and staff who bring different knowledge, expertise and skills that are each valued. Many students and staff described developing trust and empathy when curriculum co-creation bridged the aca- demic hierarchies between them. As a foundation, partici- pants highlighted transparency and open communications about expectations, processes and opportunities. Student 3 explained how curriculum co-creation helps students to trust academics and ‘understand the human side of academic staff’, and Student 8 described: I’ve never had to do anything like it before, but I guess that’s a positive thing… It made me appreciate how hard it must be to be a teacher. Mutual respect and care …said in the very beginning when we first all got together, ‘we have students who are studying some- thing. They’re a resource, why doesn’t the community use it?’. I think that’s a great way of looking at it, and it teaches us that we have something to offer. When staff include students in curricular decision- making, trust and empathy can help them see each other as ‘human’ and work together to tackle challenges in cur- riculum co-creation. Staff show students respect by recognising that they can make important contributions to curricular decision-making and enhancement, which can help motivate students’ active engagement. For example, Student 11 said: Negotiating power for mutual benefit It is salient that all participants in this study highlighted that curriculum co-creation was a mutually beneficial experience. In addition to the shared values, creativity and collaboration that are foundational to curriculum co-creation, participants described how they negotiate power in non-traditional stu- dent/teacher relationships. Staff 3 noted that teachers often take the lead initially in curriculum co-creation: Furthermore, Student 8 said: It was, to be honest, the only course where I felt like I was actually making a difference and had the opportu- nity to apply knowledge that I had learned. Enhancing creativity through collaboration It’s a dialogue between the lecturer and the students It’s a dialogue between the lecturer and the students. Student 11 also shared her perspectives on curriculum co-creation: What was interesting was how much the faculty enjoyed it, in terms of really getting engaged and thinking about the issues... It was a great experience. I hadn’t anticipated how much students would have enjoyed it too. I think it took a certain amount of trust on both of our parts… It’s getting into an environment where you are able to really get your creative side out and trying to see what you can do… For this student, engaging in curriculum co-creation means being intrinsically motivated to explore opportuni- ties and push traditional boundaries by creatively developing the curriculum in ways that benefit others. Furthermore, Student 8 said: 1 3 Enhancing creativity through collaboration Several examples of reciprocity and empathy above show how staff and students welcomed different perspectives whilst working collaboratively and creatively. Respond- ents described how co-creation can facilitate creativity and innovation in curriculum design since collaborative I felt valued as a student because I wasn’t [feeling like] just one in thousands. I felt that I could make a differ- ence… 1 3 Curriculum Perspectives (2023) 43:25–37 32 engagement can enhance curiosity, exploration and con- nection-making. For example, Student 5 described ‘bounc- ing ideas off each other’ to advance learning and Staff 2 said: …through regular meetings and communications, it did get to that shared platform of partnership. …I think both partners have to step into something neutral and they’ve got to really think about how they’re doing that. There was… a willingness and positive intent that informed how everyone would do it. In terms of creating learning within a social environ- ment, I think that can be a very constructive, imagina- tive, and creative process and it’s around how we do that collectively… Staff and students’ intentionality in curriculum co-crea- tion sets the tone for adopting new ways of working collabo- ratively. Participants described how students bring different experience and need different levels of support to engage in co-creation, with power dynamics that change based on those engaging and how power ‘oscillates depending on what’s happening… across time and context’ (Staff 1). Many staff spoke about how curriculum co-creation helps them facilitate more vibrant learning environments by draw- ing on the community’s collective intellectual resources and ideas. In addition to the collaborative processes of curriculum co-creation, participants highlighted the development of creative experiences and resources. Staff 7 described: f By negotiating learning and teaching, co-creators can develop a shared sense of purpose and overcome challenges together. Participants acknowledged challenges such as how it can ‘be a bit daunting because you are letting go of some of the power the teacher would have in the classroom’ (Staff 6) and how ‘sometimes we have to work harder as students’ (Student 6). However, participants including Staff 8 high- lighted the enjoyable and rewarding nature of curriculum negotiation: There’s a symbiosis between us and things that are in the ether now that weren’t there before… [it’s] creating learning materials, creating learning experiences: this idea of the whole being more than the sum of its parts. Trust and empathy Shared responsibility, respect, reciprocity and care often support the development of other key values during cur- riculum co-creation including trust and empathy, which can humanise the higher education experience for all involved (Backhouse et al., 2019; Chappell & Craft, 2011; Lubicz- Nawrocka, 2019b). Whilst there is little research on the role of empathy in higher education, Chappell and Craft (2011, p. 365) show how ‘communal creativity is particularly impor- tant to the humanising process and encourages a strong focus on empathy, shared ownership and group identity. … As valuable new ideas emerge from joint embodied thinking and shared struggles, humanising is the process of becoming more humane, an active process of change for the creative group’. Similarly, participants in my study shared how trust and empathy supported co-creators to engage with non-tradi- tional learning and teaching that could be considered a risk for some when sharing power, being open to different ideas, and dealing with potentially unfamiliar experiences. For staff, risks could involve feeling daunted and uncertain about what students’ input will bring in diversifying curricula and ways of working. For students, risks could mean stepping outside of their comfort zone to engage more actively and learn about both the constraints and possibilities associ- ated with curriculum development. Empathy and trust can help staff and students navigate the aforementioned risks and challenges whilst ‘bridging the gap between staff and students, bringing the communities closer together’ (Staff 10) and helping students ‘understand the human side of aca- demic staff’ (Student 3). Building student/staff relationships based on trust is essential when addressing constraints whilst negotiating curriculum development (Boomer, 1992; Green, 2021) and taking on challenges of working in new ways and taking creative, relational approaches to curriculum develop- ment (Bovill, 2020b; Lubicz-Nawrocka, 2019a; Matthews et al., 2018b). However, the importance of trust has not been mentioned in existing definitions of curriculum co-creation. Besides Cook-Sather et al.’s (2014) brief mention of reciprocity in their definition of partnership in learning and teaching, values are not explicitly highlighted in established curriculum co-creation definitions. However, examples above from this study and from the work of Bergmark and Westman (2016) and Chappell and Craft (2011) show how developing shared values reflects the intentionality behind the relationships that are cultivated whilst co-creating cur- ricula, including the foundational mindsets that help par- ticipants feel included and motivated to contribute actively. Discussion I designed this project so I would always have more power in the sense that I was creating its direction and setting it up against expected outcomes… but there is room for all voices in the discussion. The findings show that students and staff engaged in cur- riculum co-creation conceptualise it as a relational pedagogy in which shared values and strong relationships are foun- dational, and it is a way of working to negotiate curricula through a creative, reciprocal process that is likely to benefit students and staff alike. Below, I discuss these key themes and demonstrate analytical generalisation by showing how my findings relate to relevant concepts and theories. I then present a new definition that encompasses current concep- tualisations of curriculum co-creation. Similarly, students described how staff should lead in quality assurance based on their subject and teaching exper- tise, although ‘there is definitely an element for students to come in’ to enhance curriculum development (Student 9). Participants often discussed how power can shift between staff and students throughout their collaboration. Student 3 emphasised that it takes time to build effective relationships: 1 3 1 3 Curriculum Perspectives (2023) 43:25–37 33 resonate with broader collective and community goals of higher education as a social good’ (p. 966). A values‑based, creative process In the findings above, the values of shared responsibility, reciprocity in learning from each other, mutual respect and care and trust and empathy are often central to curriculum co-creation. Although the values underpinning co-creation could vary—based on those contributing, the relationships between them, and the contexts within which they work (Bovill, 2020a; Cook-Sather et al., 2014; Mercer-Mapstone et al., 2017)—what is important is that these values are shared. Cook-Sather et al.’s work (2014, p. 1) was influen- tial in emphasising that ‘partnerships are based on respect, reciprocity and shared responsibility between students and faculty’. They suggested that respect is an attitude of open- ness promoting two-way communication, reciprocity is a way of thinking that advances equity and responsibility is an action of taking ownership (Cook-Sather et al., 2014, pp. 3–5). Participants in my research reinforce the importance of these values and show how they can promote attitudes and actions that demonstrate co-creators’ eagerness to learn from each other’s expertise and diverse perspectives. Care Beyond respect and openness to other views and experi- ences, participants emphasised care and compassion so that student co-creators ‘feel their work is valued now’ (Staff 3), that they ‘have something to offer’ (Student 7) and that they can ‘make a difference’ (Student 11) through their contributions. This theme of care in curriculum co-creation resonates with others’ findings that teachers’ care and com- mitment can influence students’ choices and motivations to engage with learning (Bovill, 2020b; Lubicz-Nawrocka & Bunting, 2019; Noddings, 2005). Furthermore, Noddings (2005) highlighted the importance of teachers developing students’ ‘capacity to care’ (p. 18) for their learning and for others, and Matthews et al. (2018b) showed that care can foster ‘more personally transformative outcomes that Trust and empathy Extending others’ previous work concerning shared respon- sibility, respect and reciprocity I also identified care, trust and empathy as significant shared values in curriculum co- creation. Creativity is another foundational aspect of cur- riculum co-creation processes explored below. Creativity Developing shared values, particularly trust and empathy, is key to advancing non-traditional ways of working dur- ing curriculum co-creation that often fosters creativity and innovation in curriculum development. This is reinforced by the work of Chappell and Craft (2011), referenced above in relation to how empathy and communal creativity can humanise higher education. Furthermore, curriculum co-cre- ation ‘can be a very constructive, imaginative and creative 1 1 3 3 Curriculum Perspectives (2023) 43:25–37 34 process’ (Staff 2) with ‘the whole being more than the sum of its parts’ (Staff 7). Although the words ‘co-creation’ and ‘creativity’ share the same etymology, the importance of creativity in curriculum co-creation has been rarely noted in the literature and is absent from current definitions. I previ- ously demonstrated the importance of recognising creativity as a core aspect of co-creation since it develops adaptability and resilience, and it underpins both the creative process of collaborative learning and also the creative projects and resources that are produced as a result (Lubicz-Nawrocka, 2019a). positive intent’ (Student 3). This finding is congruous with research showing how teachers’ care, commitment to engag- ing and supporting students and open invitations to students to participate have a strong influence on students’ choices and motivations to engage with learning generally (Lubicz- Nawrocka & Bunting, 2019; Noddings, 2005) and with cur- riculum negotiation in particular (Boomer, 1992; Bovill & Woolmer, 2019).i An important finding is that all students participating in a co-created course emphasised that it was the best course across their entire university degree. Although not all indi- viduals who participate in curriculum co-creation may expe- rience benefits and some face challenges, it is important to recognise the potential for mutual benefit. Indeed, both stu- dent and staff participants overwhelmingly described their enjoyment of rewarding experiences that advanced their personal and professional development, which is congruent with the literature (Dickerson et al., 2016; Mercer-Mapstone et al., 2017). For participants, co-creation means learning from each other to ‘move forward in creating something good for both of you’ (Student 11) whilst ‘making a dif- ference’ (Student 8), which is in alignment with literature showing that curriculum co-creation can be transformative (Bergmark & Westman, 2016; Bovill, 2020b; Lubicz-Naw- rocka & Bovill, 2021; Ryan & Tilbury, 2013). Creativity This is espe- cially significant when recognising that the themes under- pinning curriculum co-creation have strong overlaps with notions of excellent teaching and learning (Bovill, 2020b; Dickerson et al., 2016; Lubicz-Nawrocka & Bunting, 2019). It is particularly notable that curriculum co-creation sup- ports students and staff to find authentic solutions that are both relevant to their own context in today’s quickly-chang- ing world (Bergmark & Westman, 2016; Blau & Shamir- Inbal, 2018; Bovill et al., 2016; Lubicz-Nawrocka, 2019a). Wider educational literature describes how negotiated processes of curriculum development can be more inclusive by inviting students to contribute to that creation process (Boomer, 1992; Bron et al., 2016; Green, 2021), and creativ- ity can reframe and enhance current educational practices to engage students who learn in different ways (Gee, 2003). Furthermore, students and staff often take on non-traditional roles and identities whilst co-creating curricula and working in partnership in democratic and egalitarian learning com- munities (Bergmark & Westman, 2016; Lubicz-Nawrocka, 2019b; Matthews et al., 2018a; Mercer-Mapstone et al., 2018). Therefore, it is important to acknowledge the cen- trality of creativity within curriculum co-creation processes. Staff and students sharing and negotiating decision‑making about aspects of curricula, often leading to mutual benefits The process of students and staff developing shared values, such as those detailed above, underpins how they share deci- sion-making and collaboratively negotiate aspects of cur- ricula. For participants, curriculum co-creation means work- ing together towards shared aims, feeling that ‘we are all in this together’ (Staff 8) and that ‘it’s not them and us, it’s just us’ (Staff 4). The process-focused, relational dimension of staff and student collaboration in sharing decision-making is important here, and it is prevalent in the literature that notes the sense of community and belonging that develops during curriculum co-creation (Blau & Shamir-Inbal, 2018; Bovill, 2020b; Cook-Sather et al., 2018a; Kaur et al., 2019; Lubicz-Nawrocka, 2019a; Masika & Jones, 2016).f A new definition of curriculum co‑creation It is important that definitions are broad enough to include a wide range of initiatives across different contexts, yet spe- cific enough to be clear. As described when reviewing exist- ing co-creation definitions, these focus broadly on student/ staff collaborations in developing aspects of the curriculum (Bovill et al., 2011, 2016) or participatory processes that can increase satisfaction (Dollinger et al., 2018; Ryan & Tilbury, 2013). Although Cook-Sather et al. (2014) highlight several important values underpinning student/staff partnerships, the intentionality of developing shared values is lacking from existing definitions of curriculum co-creation since this is critical for strong working relationships.i Co-creators recognised that staff often lead in curricu- lum development through their subject knowledge, teaching expertise and responsibilities for quality assurance; however, staff create opportunities for students to share responsibil- ity over teaching decisions that affect how or what they are learning. It is clear from the results that staff attitudes and behaviours strongly influence students’ engagement with curriculum co-creation by creating ‘room for all voices in the discussion’ (Staff 3) since ‘they’ve got to really think about how they’re doing that… [with] a willingness and Based on key themes that emerged from my findings across the Scottish higher education sector and international 1 3 35 Curriculum Perspectives (2023) 43:25–37 literature, I offer a new definition that extends beyond broad notions of student/staff collaborations in curriculum devel- opment to capture the intentionally fostered values and dynamics as well as the notably positive outcomes: literature, I offer a new definition that extends beyond broad notions of student/staff collaborations in curriculum devel- opment to capture the intentionally fostered values and dynamics as well as the notably positive outcomes: Although I could only include curriculum co-creation initia- tives that were made visible within the sector, there may have been a wider number and variety of co-creation examples of which I was not aware if they were not shared beyond class- room walls. Participants co-creating curricula were not gener- ally representative of the populations of students and staff at Scottish universities since all appeared to be highly engaged and motivated. It is important to note that the vast majority of participants self-selected to engage in curriculum co-creation. The lack of cultural diversity in the selection (or self-selection) of student co-creators has been identified as a challenge across the sector (Mercer-Mapstone et al., 2017), which also appears to be the case in my study. A new definition of curriculum co‑creation Despite these limitations, this study analyses in-depth experiences and conceptualisations of cur- riculum co-creation with the aim of facilitating dialogue about upscaling these initiatives to become more prevalent and inclu- sive. It is important for future research to examine what aspects of support or development are needed to enable both staff and students to engage in curriculum co-creation and to explore further the potential for their mutual benefit.f Curriculum co-creation is a relational way of working underpinned by shared responsibility, reciprocity in learning from each other, mutual respect, care, trust, and empathy. This values-based, creative process helps staff and students work together to share and negotiate decision-making about aspects of curricula, which often leads to mutual benefits for learners and teachers. Whilst each curriculum co-creation initiative is context- dependent and unique based on the individuals involved (Healey & Healey, 2018), the values that underpin co-cre- ators’ relationships may vary and the outcomes may differ. It is also important not to lose sight of how democratically negotiated curricula can challenge the status quo of aca- demic cultures, structures and processes (Bergmark & West- man, 2016; Boomer, 1992; Bovill, 2020b; Lubicz-Nawrocka, 2019b). However, this definition attempts to specify key dimensions of the relational pedagogy that underpins cur- riculum co-creation—resonating with Bovill’s recent work (2020a)—since its affective nature has the transformative capacity to humanise educational experiences when students and staff feel, as one participant said: ‘it’s not them and us, it’s just us’. i It is powerful that students and staff in this study high- lighted the rewarding nature of curriculum co-creation. Although curriculum co-creation is not without its challenges and it may not be appropriate or feasible in every context, it can humanise teaching and learning by empowering both stu- dents and teachers. This is particularly relevant with respect to how the COVID-19 pandemic has provoked new ways of working through increasingly digital and hybrid forms of education. Perhaps we can overcome current challenges by learning from staff and students who embrace creativity and innovation whilst co-creating authentic and meaningful cur- ricula. We have seen how co-creators work towards shared values and aims as they home in on what is important to them in higher education—the mutually beneficial process of staff and students working and learning together. Concluding thoughts It is hoped that a new definition of curriculum co-crea- tion will clarify the concept to support practitioners by extending discourse on this process-based, values-led work that can benefit both staff and students. By analys- ing curriculum co-creation conceptualisations within a single national context with respect to theory and inter- national practice, this study differs from most co-creation research, which focuses either on in-depth analysis at one institution or on selected international case studies. My discussion of each of the themes included in my new defi- nition demonstrates theoretical hybridity by showing how my findings relate to a wide range of established con- cepts. This research demonstrates (A) concept generalisa- tion showing how findings in my study relate to concepts such as care, trust, empathy and creativity that have not been emphasised widely in the co-creation literature and (B) theoretical generalisation showing how results relate to established definitions. This study may also provide opportunities for provocative generalisability if readers are inspired to think about the possibilities for curriculum co-creation in different contexts. 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Open Access This article is licensed under a Creative Commons Attri- bution 4.0 International License, which permits use, sharing, adapta- tion, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. 1 3 Curriculum Perspectives (2023) 43:25–37 36 educational practice in higher education. 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https://openalex.org/W3180303835	https://www.matec-conferences.org/10.1051/matecconf/202134001035/pdf	English	null	Carbon nanomaterials for supercapacitors: two electrode scheme	MATEC web of conferences	2,021	cc-by	2,512	!!! "# !!! "# $% & '$ ()+) $% ,, - % , % ,, # % % -, , $ % $ # ,, & ,, , . , ,$ #/ # ,, ,,$ $ & $ % ,# , % , , 01 % ,, 2 $ , % # $ ,, " ,, $ #/ / & # # % ,, #/ # , % 3 -, , % % $ %4 # 5 $ MATEC Web of Conferences 340, 01035 (2021) MATEC Web of Conferences 340, 01035 (2021) MATEC Web of Conferences 340, 01035 (2021) RKFM 2021 https://doi.org/10.1051/matecconf/202134001035 , 6 ,,$-$7 © The Authors, published by EDP Sciences. This is an open access article distributed under the terms of the Creative Commons Attribution License 4.0 (http://creativecommons.org/licenses/by/4.0/). , 3 8"4 # $ % H '/I* & , # $ , % % 3A8J A8"/: 4 & - % # #/, , ++ K L F * , 34 # @8& & , , , # % 34 # % ,$ / @ J,, , # # @ . & -, # 8 ?/)* " , & , # ,, #6 , % # * B # - # % % 3,,- C , #4 )//, # - # , # $ ,, , 3# 4 & , , # #, * F< ) B " , -, $ 3F4 # & -, # #/ , , , # G G * & # 3) B " H =4 , 6 ,,$-$7 MATEC Web of Conferences 340, 01035 (2021) MATEC Web of Conferences 340, 01035 (2021) RKFM 2021 https://doi.org/10.1051/matecconf/202134001035 8" -, , 8-, , 3894 % 5 % # , / , , 3 : 4 &8" # % % , 3, % ,4 & , # , 3;<4 , =; 01 8" -, , 8-, , 3894 % 5 % # , / , , 3 : 4 &8" # % % , 3, % ,4 & , # , 3;<4 , =; 01 &8" % % % % 3:4 , # 0)1 : # 5 % , >= % ?, # % & ,, , , # @& A #/ 3 $ 8 ' 4 , (; B* # C > =* # C > * # C 9 % % , % % # B>* 3: 4 &8" % % &8" % % % % 3:4 , # 0)1 : # 5 % , >= % ?, # % & ,, , , # @& A #/ 3 $ 8 ' 4 , (*; B # C > =* # C > * # C 9 % % , % % # B>* 3: 4 2 2 MATEC Web of Conferences 340, 01035 (2021) RKFM 2021 https://doi.org/10.1051/matecconf/202134001035 8" $ % & # # $ % 34 % % % % 0=1 & # # # # , % # , % ,, & # $ =B D 3: )4 & # %E $ # . % # ) # C 8" $ % 8" $ % 8" $ % & # # $ % 34 % % % % 0=1 & # # # # , % # , % ,, & # $ =B D 3: )4 & # %E $ # . % # ) # C ! &% # - % 3 3 https://doi.org/10.1051/matecconf/202134001035 MATEC Web of Conferences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eb of Conferences 340, 01035 (2021) RKFM 2021 https://doi.org/10.1051/matecconf/202134001035 B ? " K A F I* 34 ( F $ F $ : : K$ F F9'& 0? F '8&1 34 ! K A & H 9 $ % ,,6 " $ ,, $ " !I( 3*4 9 @$ &$ F F $ K $ F ' 8 "$ O ? " ? I) 3=4 ) " F ?,$ 9 @$ " &/? =I! 34 = ? " O F O$ $ " " P$ F ? ", ", " )= 3!4 4 MATEC Web of Conferences 340, 01035 (2021) MATEC Web of Conferences 340, 01035 (2021) RKFM 2021 https://doi.org/10.1051/matecconf/202134001035 5 5
https://openalex.org/W4210878734	https://www.frontiersin.org/articles/10.3389/fcell.2022.819050/pdf	English	null	Corrigendum: WASp Is Crucial for the Unique Architecture of the Immunological Synapse in Germinal Center B-Cells	Frontiers in cell and developmental biology	2,022	cc-by	689	CORRECTION published: 09 February 2022 doi: 10.3389/fcell.2022.819050 Yanan Li 1,2,3, Anshuman Bhanja 3, Arpita Upadhyaya 4,5, Xiaodong Zhao 1,2* and Wenxia Song 3 1Department of Rheumatology and Immunology, Children’s Hospital of Chongqing Medical University, Chongqing, China, 2Ministry of Education Key Laboratory of Child Development and Disorders, Chongqing Key Laboratory of Child Infection and Immunity, China International Science and Technology Cooperation Base of Child Development and Critical Disorders, National Clinical Research Center for Child Health and Disorders, Chongqing, China, 3Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, College Park, MD, United States, 4Department of Physics, University of Maryland, College Park, College Park, MD, United States, 5Institute for Physical Science and Technology, University of Maryland, College Park, College Park, MD, United States Keywords: B-lymphocytes, germinal center, actin, WASp, signal transduction, immunological synapse Keywords: B-lymphocytes, germinal center, actin, WASp, signal transduction, immunological synapse Correspondence: Xiaodong Zhao zhaoxd530@aliyun.com WASp Is Crucial for the Unique Architecture of the Immunological Synapse in Germinal Center B-Cells by Li, Y., Bhanja, A., Upadhyaya, A., Zhao, X., and Song, W. (2021). Front. Cell Dev. Biol. 9:646077. WASp Is Crucial for the Unique Architecture of the Immunological Synapse in Germinal Center B-Cells by Li, Y., Bhanja, A., Upadhyaya, A., Zhao, X., and Song, W. (2021). Front. Cell Dev. Biol. 9:646077. doi: 10.3389/fcell.2021.646077 Specialty section: This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology Received: 20 November 2021 Accepted: 10 January 2022 Published: 09 February 2022 Specialty section: This article was submitted to Cell Growth and Division, a section of the journal Frontiers in Cell and Developmental Biology by Li, Y., Bhanja, A., Upadhyaya, A., Zhao, X., and Song, W. (2021). Front. Cell Dev. Biol. 9:646077. doi: 10.3389/fcell.2021.646077 There is an error in the Funding statement. The correct funding number for “XZ” is “81620108014.” The authors apologize for this error and state that this does not change the scientiﬁc conclusions of the article in any way. The original article has been updated. Received: 20 November 2021 Accepted: 10 January 2022 Published: 09 February 2022 Received: 20 November 2021 Accepted: 10 January 2022 Published: 09 February 2022 Copyright © 2022 Li, Bhanja, Upadhyaya, Zhao and Song. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Approved by: Frontiers Editorial Ofﬁce, Frontiers Media SA, Switzerland Correspondence: Xiaodong Zhao zhaoxd530@aliyun.com Approved by: Frontiers Editorial Ofﬁce, Frontiers Media SA, Switzerland Li Y, Bhanja A, Upadhyaya A, Zhao X and Song W (2022) Corrigendum: WASp Is Crucial for the Unique Architecture of the Immunological Synapse in Germinal Center B-Cells. Front. Cell Dev. Biol. 10:819050. doi: 10.3389/fcell.2022.819050 Frontiers in Cell and Developmental Biology \| www.frontiersin.org Citation: Publisher’s Note: All claims expressed in this article are solely those of the authors and do not necessarily represent those of their afﬁliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher. Li Y, Bhanja A, Upadhyaya A, Zhao X and Song W (2022) Corrigendum: WASp Is Crucial for the Unique Architecture of the Immunological Synapse in Germinal Center B-Cells. Front. Cell Dev. Biol. 10:819050. doi: 10.3389/fcell.2022.819050 Copyright © 2022 Li, Bhanja, Upadhyaya, Zhao and Song. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. February 2022 \| Volume 10 \| Article 819050 Frontiers in Cell and Developmental Biology \| www.frontiersin.org
https://openalex.org/W3194820553	https://ieeexplore.ieee.org/ielx7/90/9653186/09487018.pdf	English	null	Efficient Link Scheduling Solutions for the Internet of Things Under Rayleigh Fading	IEEE/ACM transactions on networking	2,021	cc-by	14,193	Efﬁcient Link Scheduling Solutions for the Internet of Things Under Rayleigh Fading Kan Yu , Jiguo Yu , Senior Member, IEEE, Member, ACM, Xiuzhen Cheng , Fellow, IEEE, Member, ACM, Dongxiao Yu , Senior Member, IEEE, and Anming Dong , Member, IEEE in wireless networks is one of the effective ways that can help an IoT to realize the connectivity, increase capacity and throughput, and decrease delay. Besides, the interference among signals must be considered when we design link scheduling algorithms. Also, the interference model selection has signiﬁcant effects on the performance and complexity of link scheduling algorithms. Abstract—Link scheduling is an appealing solution for ensur- ing the reliability and latency requirements of Internet of Things (IoT). Most existing results on the link scheduling problem were based on the graph or SINR (Signal-to-Interference-plus- Noise-Ratio) models, which ignored the impact of the random fading gain of the signals strength. In this paper, we address the link scheduling problem under the Rayleigh fading model. Both Shortest Link Scheduling (SLS) and Maximum Link Schedul- ing (MLS) problems are studied. In particular, we show that a set of links can be activated simultaneously under Rayleigh fading model if all link SINR constraints are satisﬁed. Based on the analysis of previous Link Diversity Partition (LDP) algorithm, we propose an Improved LDP (ILDP) algorithm and a centralized algorithm by localizing the global interference (denoted by CLT), building on which we design a distributed CLT algorithm (denoted by RCRDCLT) that converges to a constant approximation factor of the optimum with the time complexity of O(ln n), where n is the number of links. Fur- thermore, executing repeatedly RCRDCLT can solve the SLS with an approximation factor of Θ(ln n). Extensive simulations indicate that CLT is more effective than previous six popular link scheduling algorithms, and RCRDCLT has the lowest time complexity while only losses a constant fraction of the optimum schedule. Generally, the goal of link scheduling problem is two-fold. One is Maximum Link Scheduling (MLS), i.e., maximizing the number of links that can be scheduled concurrently in a single slot. Another is Shortest Link Scheduling (SLS), which aims at scheduling a set of links in the minimum number of slots. Initially, the link scheduling problem is studied from the beginning of the graph model, but the practicability of algorithms designed under this model is limited by its duality. Efﬁcient Link Scheduling Solutions for the Internet of Things Under Rayleigh Fading Subsequently, extensive research on the problem mainly considered the deterministic Signal-to- Interference-plus-Noise-Ratio (SINR) model which deﬁnes the signal strength fades in a ﬁxed speed. In fact, the strength of a signal cannot be deterministic due to the fading characteristic in signal propagation. The Rayleigh fading model is one of more realistic models, and can describe the real-world environ- ments [2]. However, the model leads to the design and analysis of link scheduling algorithms more challenging and difﬁcult in return. Consequently, there are few works focusing on link scheduling subject to Rayleigh fading constraint (e.g., [3]–[6]). Index Terms—Link scheduling, Internet of Things, distributed algorithm, Rayleigh fading, SINR. 2508 2508 IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMBER 2021 I. INTRODUCTION The success probability of a link cannot achieve 1 under the Rayleigh fading model since the channel fading gain makes the strength of received signal uncertain [4]. Nonetheless, it is reasonable to regard a link as being scheduled successfully if its success probability is 1−ϵ at least, where ϵ is an acceptable failure probability. Similar assumption was also applied in the previous works (e.g. [3], [5], [6]). However, their works were unsatisfactory to provide a better scheduling performance which refers to scheduling more concurrent links in a single slot for the MLS, and using less slots to schedule all links for the SLS. F OR an Internet of Things (IoT), interference, connectivity, capacity, throughput, delay, security and so on are most important indices [1]. Wireless networks play a crucial role in IoT and are a fundamental architecture to gather data of IoT devices. Designing the efﬁcient link scheduling algorithms F Manuscript received February 8, 2020; revised April 17, 2021; accepted June 16, 2021; approved by IEEE/ACM TRANSACTIONS ON NETWORK- ING Editor J. Shin. Date of publication July 15, 2021; date of current version December 17, 2021. This work was supported by the National Natural Science Foundation of China under Grant 61771289, Grant 61832012, Grant 61672321, and Grant 61701269. (Corresponding author: Jiguo Yu.) In the current paper, to enhance the scheduling performance further, we present a series of novel and efﬁcient algorithms with lower time complexity. The main contributions of the paper are summarized as follows. Kan Yu is with the School of Computer Science, Qufu Normal University, Rizhao 276826, China (e-mail: kanyu@qfnu.edu.cn). Jiguo Yu is with the School of Computer Science and Technology, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China, and also with the Shandong Laboratory of Computer Networks, Jinan 250014, China (e-mail: jiguoyu@sina.com). Jiguo Yu is with the School of Computer Science and Technology, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China, and also with the Shandong Laboratory of Computer Networks, Jinan 250014, China (e-mail: jiguoyu@sina.com). • Based on the successive interference cancellation (SIC) technique, we improve Link Diversity Partition algo- rithm (ILDP) under Rayleigh fading model, a centralized algorithm for the MLS with low scheduling performance under the deterministic SINR model, which builds the relationship between two models. Speciﬁcally, ILDP gives a method to localize the global interference, which presents a signiﬁcant insight into distributed algorithm design. Manuscript received February 8, 2020; revised April 17, 2021; accepted June 16, 2021; approved by IEEE/ACM TRANSACTIONS ON NETWORK- ING Editor J. Shin. Date of publication July 15, 2021; date of current version December 17, 2021. This work was supported by the National Natural Science Foundation of China under Grant 61771289, Grant 61832012, Grant 61672321, and Grant 61701269. (Corresponding author: Jiguo Yu.) Kan Yu is with the School of Computer Science, Qufu Normal University, Rizhao 276826, China (e-mail: kanyu@qfnu.edu.cn). Jiguo Yu is with the School of Computer Science and Technology, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250353, China, and also with the Shandong Laboratory of Computer Networks, Jinan 250014, China (e-mail: jiguoyu@sina.com). Xiuzhen Cheng and Dongxiao Yu are with the School of Computer Sci- ence and Technology, Shandong University, Qingdao 266510, China (e-mail: xzcheng@sdu.edu.cn; dxyu@sdu.edu.cn). Anming Dong is with School of Computer Science and Technology, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250014, China (e-mail: anmingdong@qlu.edu.cn). This article has supplementary downloadable material available at https://doi.org/10.1109/TNET.2021.3093306, provided by the authors. Digital Object Identiﬁer 10 1109/TNET 2021 3093306 ative Commons Attribution 4.0 License. For more information, see https://creativecommons.org/licenses/by/4.0/ Manuscript received February 8, 2020; revised April 17, 2021; accepted June 16, 2021; approved by IEEE/ACM TRANSACTIONS ON NETWORK- ING Editor J. Shin. Date of publication July 15, 2021; date of current version December 17, 2021. This work was supported by the National Natural Science Foundation of China under Grant 61771289, Grant 61832012, Grant 61672321, and Grant 61701269. (Corresponding author: Jiguo Yu.) This article has supplementary downloadable material available at https://doi.org/10.1109/TNET.2021.3093306, provided by the authors. work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecommons.o Anming Dong is with School of Computer Science and Technology, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250014, China (e-mail: anmingdong@qlu.edu.cn). Xiuzhen Cheng and Dongxiao Yu are with the School of Computer Sci- ence and Technology, Shandong University, Qingdao 266510, China (e-mail: xzcheng@sdu.edu.cn; dxyu@sdu.edu.cn). Kan Yu is with the School of Computer Science, Qufu Normal University, Rizhao 276826, China (e-mail: kanyu@qfnu.edu.cn). II. RELATED WORK The link scheduling problem has been studied extensively in previous literatures. Initially, the problem was consid- ered under the graph model (e.g., [7]–[14]). Afterwards, it was noted that the deterministic SINR model can offer a more realistic representation of interference between signals. Moscibroda and Wattenhofer ﬁrst studied the link scheduling problem under the deterministic SINR model [15]. Then, Goussevskaia et al. gave a NP-hard proof for the link scheduling problem under the SINR model [16]. Subsequently, attentions for designing effective link scheduling algorithms shifted to a more realistic model using SINR, such as centralized (e.g., [15], [17]–[21]) and distributed algorithms (e.g., [22]–[28]). In [4], Dams et al. gave a scheme adapting link scheduling algorithms in the deterministic SINR model to the Rayleigh-fading model, while losing only a factor of O(log∗n)1 in the approximation guarantee, which was reduced to a constant by Halld´orsson and Tonoyan [37]. Signiﬁcantly, Dams et al. showed that success probability of a successful link under the deterministic SINR model is at least 1/e under Rayleigh fading model. Following this work, by utilizing the idea of localizing global interfer- ence under Rayleigh fading model, some centralized and distributed algorithms were proposed for the MLS and SLS (e.g., [3], [5], [6]). In [7], Sharma et al. modeled the interference by k-hop interference models, in which two links within k-hop distance interfere with each other and no two links within k-hops can successfully transmit at the same time. Although these models were too simplistic, they offered some signiﬁcant insights into interference localization and distributed algorithm design under the deterministic SINR model. g In [3], Qiu and Shen proposed centralized and distributed algorithms (LDP and RLE) with O(g(L)) and O(1) perfor- mance guarantees, respectively, where g(L) (called length diversity) is the number of magnitudes of transmission link lengths. In our previous work [5], we solved the MLS by designing a distributed algorithm Syn_DLS to increase relia- bility and latency requirements of IoT. In our another previous work [6], we derived the minimum distance constraint dmin between the interferers and the receiver under the determin- istic SINR model, and designed more effective distributed algorithms (DLS and DDLS) based on random contention resolution, by ignoring interference safely outside dmin as a constant and showing that those interference cannot inﬂuence whether a link is successful or not. YU et al.: EFFICIENT LINK SCHEDULING SOLUTIONS FOR THE INTERNET OF THINGS UNDER RAYLEIGH FADING 2509 • By utilizing interference localization, we then pro- pose a Centralized and Localized Traversal CLT) algo- rithm for the MLS, by proving that the interference beyond a certain range is a constant. Compared with the optimal schedule, CLT gives a constant approxima- tion factor. Combining with random contention resolu- tion, we present a distributed implementation of CLT RCRDCLT) with O(ln n) rounds, where n is the number of links, which improves the performance of our prior works for MLS in [5] and [6]. • By utilizing interference localization, we then pro- pose a Centralized and Localized Traversal CLT) algo- rithm for the MLS, by proving that the interference beyond a certain range is a constant. Compared with the optimal schedule, CLT gives a constant approxima- tion factor. Combining with random contention resolu- tion, we present a distributed implementation of CLT RCRDCLT) with O(ln n) rounds, where n is the number of links, which improves the performance of our prior works for MLS in [5] and [6]. strategy for the MLS [32]. Asgeirsson and Mitra applied the regret-learning to design an O(1)-approximation algo- rithm for uniform power assignment [33]. However, these game-theoretic algorithms take time polynomial of n to converge. In addition, random contention resolution can be implemented to solve the link scheduling problem in a dis- tributed fashion [34]. The idea is that each agent accesses the limited resource in any slot with a certain probability q until its ﬁrst success. In case of a collision, none of the involved agents is successful in this slot. This idea is easily applicable in wireless networks by letting each IoT device transmit its packet in each slot with probability q until the ﬁrst success. For the SLS, Kesselheim and V¨ocking gave a distributed O(log2 n)-approximation algorithm with sublinear power assignment [34], and later resulting in a O(log n)- approximation algorithm by Halld´orsson and Mitra [35]. Similar conclusions were also obtained in works [24] and [36]. In [36], Goussevskaia et al. proposed a centralized algo- rithm (GHW) for the MLS with constant approximation ratio guarantee, and solved the SLS by executing GHW repeatedly with the time complexity of O(n log n). • Executing RCRDCLT repeatedly gives an effective solu- tion for the SLS, and an approximation factor of Θ(ln n) is proposed, which shows that the best performance guarantee for the SLS is logarithmic. 1log∗denotes the iterated-logarithm function, and O(log∗n) is essentially “almost constant.” YU et al.: EFFICIENT LINK SCHEDULING SOLUTIONS FOR THE INTERNET OF THINGS UNDER RAYLEIGH FADING The rest of this paper is organized as follows. Section II provides the related work. In Section III, we present network model and notations. In Section IV, we present our central- ized and distributed link scheduling algorithms with rigorous analyses. Extensive experiments are implemented to evaluate the performances of our designed algorithms in Section V. Finally, we conclude the paper in Section VI. Note that the deterministic SINR model ignores the impact of random fading on the strength of received signal. In realistic environment, a successful link under the deterministic SINR model does not means that the link can be successfully scheduled under the Rayleigh fading model. More important, to enable distributed algorithm designs, predictable interfer- ence control, an open question is whether it is possible to develop a model of measuring interference, which has the locality of the protocol model and high ﬁdelity under the Rayleigh fading. I. INTRODUCTION This article has supplementary downloadable material available at https://doi.org/10.1109/TNET.2021.3093306, provided by the authors. This work is licensed under a Creative Commons Attribution 4.0 License. For more information, see https://creativecom YU et al.: EFFICIENT LINK SCHEDULING SOLUTIONS FOR THE INTERNET OF THINGS UNDER RAYLEIGH FADING III. NETWORK MODEL AND DEFINITION According to the decreasing order of received power, the receiver with SIC technique can decode those stronger sig- nals than its intended signal. If an interferer is stronger enough for the receiver, then it can be decoded ﬁrst and canceled off by this receiver before decoding its intended signal. Finally, the expected signal can be decoded successfully. Given an IoT network consisting of n communication links L = {l1, . . . , ln}, where li represents a transmission from a sender si to a receiver ri. The set of senders is denoted by S = {s1, . . . , sn}. The distance from a sender si to a receiver rj is denoted by dsirj, and dii for short if i = j. j j The wireless propagation is described by attenuation and fading. The Rayleigh fading means that the instantaneous envelope of the received signal follows Rayleigh distribution and its power is an exponentially distributed random vari- able. It is viewed as a reasonable model for tropospheric and ionospheric signal propagation as well as the effect of heavily built-up urban environments on radio signals. Under the above assumptions, if a signal is transmitted by sender si, it will be received by receiver ri with the strength of Pihiid−α ii , where Pi is the transmit power and assumed to be the same for all senders, α is the path-loss exponent, hii is the channel fading gain and follows an exponential distrib- ution with unit mean [3]–[5]. Compared to noise, inter-link interference is the dominating source of limitation on the success probability. Therefore, it is interesting to focus the analysis in the interference-limited regime, which means that the interference dominates the noise since the node density is quite high in large wireless network, and the noise can be neglected [38], [39]. When the received SIR at receiver ri is greater than or equal to γth, ri can correctly decode the signal transmitted by si. Mathematically, In k-SIC, at most k stronger signals can be decoded. The necessary and sufﬁcient conditions for k-SIC at rm is Sim k j=i+1 Sjm + s>k Ssm ≥γth, 1 ≤i ≤k, (3) (3) where Sim is the ith strongest power received at rm [40], [41]. Table I shows some important notations. where Sim is the ith strongest power received at rm [40], [41]. Table I shows some important notations. II. RELATED WORK To localize the global interference under the deterministic SINR model, one of effective methods is partitioning the net- work area into some cells with enough size. By adopting this method, Goussevskaia et al. classiﬁed link classes according to link length, and constructed a feasible schedule for each link class using a greedy strategy [16]. Similar idea was also applied in [18], [22], [29]–[31]. Based on the fact that a short link can tolerate more interference while a long link tolerates less, Huang et al. proposed a novel approximation algorithm for the SLS [30]. Their motivation is that if two links are far away from each other, the interference of one link on the other should be small. By partitioning the links into disjoint local link sets with a certain distance away from each other, such that independent scheduling inside each local link set is possible. Moreover, hexagon partition based link scheduling algorithms were proposed by Yu et al. [18]. Simulations demonstrated that hexagon-partition based link scheduling was more efﬁcient than the square-partition employed by [16] and [31]. In summary, the MLS under the Rayleigh fading model can be simpliﬁed as the following one: given a guarantee requirement of success probability 1 −ϵ, in the determinis- tic SINR model, removing all interfering links within some For distributed algorithm design under the determinis- tic SINR model, Dinitz ﬁrst proposed a regret-learning IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMBER 2021 2510 can be successfully scheduled under Rayleigh fading model if the probability of γi ≥γth is greater than or equal to 1 −ϵ. Similar assumption was also adopted in [3]–[5]. distance (a function of 1 −ϵ) for each selected link, and this link has at least 1 −ϵ success probability under the Rayleigh fading model. The next step that we need to do is to maximize the number of those links. Consequently, it enables us to pay attention to algorithm design and analysis with the consideration of the deterministic SINR model, and apply them to the Rayleigh fading model. To sum up, the difference between [3], [5], [6], [36], and this paper is given as follows. Lemma 1 ([3], [5]): Given a link li and a sender set P ⊂S, the success transmission probability of li is pi = j̸=i,sj∈P 1 1 + dii dsj ri α γth . IV. CENTRALIZED AND DISTRIBUTED ALGORITHMS FOR MLS PROBLEM In this section, we ﬁrst propose an Improved LDP (ILDP) based on 1-SIC by analyzing classic link scheduling algorithm, namely Link Diversity Partition (LDP) [16]. Then utilizing the idea of interference localization, we propose a centralized link scheduling algorithm (denoted by CLT) with a constant approximation factor, and obtain its distributed implementation (denoted by RCRDCLT) with logarithmic rounds. II. RELATED WORK (2) (2) • Compared with [3], [5], and [6], we consider the interference-limited network (i.e., without noise), which simpliﬁes the expression of success probability of a link, and we observe the relationship of distance among links clearly. In addition, SIC is adopted to remove the impact of stronger interfering signal, which can increase success probability of a link. A schedule P is feasible if all senders in P can successfully transmit the message to their intended receivers with proba- bility of at least 1 −ϵ. Deﬁnition 1 (Interference Factor [3]): Deﬁne the interfer- ence factor of si on rj as fsi,rj = ⎧ ⎨ ⎩ ln 1 + djj dsirj α γth if i ̸= j; 0 if i = j. • Compared with [36], Rayleigh fading model is further considered, under which distributed algorithms were pro- posed in [5] and [6] based on idea of interference local- ization. Compared with above scheduling algorithms, by using SIC technique and plane partition, we present a more effective way of interference localization, and the designed distributed algorithm can schedule more successful links in a time slot and use more less number of time slots to ensure that each link has been scheduled at least once. • Compared with [36], Rayleigh fading model is further considered, under which distributed algorithms were pro- posed in [5] and [6] based on idea of interference local- ization. Compared with above scheduling algorithms, by using SIC technique and plane partition, we present a more effective way of interference localization, and the designed distributed algorithm can schedule more successful links in a time slot and use more less number of time slots to ensure that each link has been scheduled at least once. Accordingly, we use fP,rj to denote the interference factor of set P on receiver rj, where fP,rj = si∈P fsi,rj. Accordingly, we use fP,rj to denote the interference factor of set P on receiver rj, where fP,rj = si∈P fsi,rj. Based on the deﬁnition of interference factor, a link li can be regarded as successful under the Rayleigh fading model iff fP,rj ≤γϵ holds, where γϵ = ln 1 1−ϵ . A. An Improved LDP Algorithm LDP is designed for link scheduling under the deterministic SINR [16]. The key idea of LDP is that it ﬁrst classiﬁes the links with similar lengths to one class. For each link class, LDP partitions the whole network area into squares with same size and set neighboring squares to different colors. Such color setting ensures that the links in the same-color squares always have a far enough distance from each other. Then, all the selected links in the same-color squares form a feasible schedule. γi = hii · d−α ii sj∈S,j̸=i hji · d−α sjri ≥γth, (1) γi = hii · d−α ii sj∈S,j̸=i hji · d−α sjri ≥γth, (1) where γth is the decoding threshold [5], [36]. Due to the existence of channel fading gain, a link cannot be successfully scheduled with probability 1 under the Rayleigh fading model [3]–[5]. Therefore, we tolerate an acceptable failure probability ϵ for the transmission. That is, the link li YU et al.: EFFICIENT LINK SCHEDULING SOLUTIONS FOR THE INTERNET OF THINGS UNDER RAYLEIGH FADING 2511 TABLE I NOTATIONS Fig. 1. Partition and two links exist in a same square by using 1-SIC. TABLE I NOTATIONS TABLE I NOTATIONS Fig. 1. Partition and two links exist in a same square by using 1-SIC. and set neighboring squares to different colors. And thus, all the selected links from the same-color squares form a feasible schedule [16], denoted by P. By using 1-SIC, each receiver of a successful link in P can tolerate at most one strongest interferer, when the distance between them satisﬁes the constraint in the following Lemma 2. For instance, assume that lk and li belong to the same link class. As shown in Fig. 1, ri allows to the existence of sk if dskri ≤ρdii. Moreover, rk can also remove the impact of si in the same-color square by 1-SIC and joins into scheduling set, if dsirk ≤ρdkk. In this way, compared with LDP, the size of scheduling set Pildp obtained by ILDP may be doubled in the best case. y y Lemma 2: If two successful links are allowed to exist in the same square, the parameter ρ must satisfy However, it is difﬁcult to apply LDP under the Rayleigh fading model directly. The reason is that, on the one hand, a successful link of deterministic SINR model may fail to be scheduled under the Rayleigh fading model. Algorithm 1 1-SIC Based Improved LDP (ILDP) Algorithm 1 1-SIC Based Improved LDP (ILDP) Algorithm 1 1-SIC Based Improved LDP (ILDP) Algorithm 1 1 SIC Based Improved LDP (ILDP) 1: Initialization: Scheduling set P(k,j) ildp = ∅for link class Lk and color j 2: Input: g(L) link classes {L1, . . . , Lg(L)} 3: for k = 1 to g(L) do 4: Partition the network region into squares of size βk ×βk for link class Lk 5: Color the squares with {1, 2, 3, 4}, s.t. no two adjacent squares have the same-color 6: for j = 1 to 4 do 7: The sender si joins into P(k,j) ildp 8: Find link lk in link class Lk and dkk ≤dii 9: if dskri ≤ρdii and dsirk ≤ρdkk then 10: The sender sk joints into P(k,j) ildp 11: else 12: Remove link lk from Lk 13: end if 14: end for 15: end for 16: return Pildp = arg max P(k,j) ildp , ∀k, j 1: Initialization: Scheduling set P(k,j) ildp = ∅for link class Lk and color j 1: Initialization: Scheduling set P(k,j) ildp = ∅for link class Lk and color j j 2: Input: g(L) link classes {L1, . . . , Lg(L)} 3: for k = 1 to g(L) do j 2: Input: g(L) link classes {L1, . . . , Lg(L)} g Deﬁnition 2: ( [3], [16]) Given a set of links, the link length diversity, denoted by g(L), is deﬁned as 4: Partition the network region into squares of size βk ×βk for link class Lk g(L) = {h\|∃li ∈L : log2(dii/lmin) = h} . (4) (4) 5: Color the squares with {1, 2, 3, 4}, s.t. no two adjacent squares have the same-color 6: for j = 1 to 4 do 7: The sender si joins into P(k,j) ildp 8: Find link lk in link class Lk and dkk ≤dii 9: if dskri ≤ρdii and dsirk ≤ρdkk then 10: The sender sk joints into P(k,j) ildp 11: else 12: Remove link lk from Lk 13: end if 14: end for 15: end for 16: return Pildp = arg max P(k,j) ildp , ∀k, j Namely the diversity g(L) is the number of magnitudes of distances. In real applications, g(L) is usually a small constant [3], [16]. We only consider 1-SIC protocol for analysis simplicity, i.e., at most 1 strong interfering signal can be eliminated. A. An Improved LDP Algorithm On the other hand, for a given link class, the size of all squares is ﬁxed under the deterministic SINR model, while difﬁcult to be estimated once the Rayleigh fading model is considered. In particular, given a successful link in the deterministic SINR model, above failure probability can be upper-bounded. Also, combing with upper bound of interference factor (considering failure probability constraint ϵ), we can calculate a ﬁxed size of each square, which ensures the success probability of each scheduled link in same-color squares is at least 1 −ϵ under the Rayleigh fading model. ρ ≤ γϵ γth · 1 1 + 16 (β−1)α · α−1 α−2 1 α ρ ≤ γϵ γth · 1 1 + 16 (β−1)α · α−1 α−2 1 α ρ ≤ γϵ γth · 1 1 + 16 (β−1)α · α−1 α−2 1 α . The proof of Lemma 2 is shown in Appendix A. The pseudo-code of ILDP is shown in Algorithm 1. he proof of Lemma 2 is shown in Appendix A. Theorem 1: ILDP provides a feasible schedule. The proof of Theorem 1 is shown in Appendix B. The proof of Theorem 1 is shown in Appendix B. Algorithm 2 Centralized and Localized Traversal (CLT) 1: Initialization: Scheduling set Pclt = ∅ Algorithm 2 Centralized and Localized Traversal (CLT) 1: Initialization: Scheduling set Pclt = ∅ 2: Input: Sender set S = {s1, . . . , sn} 3: Output: Pclt 4: while S is not empty do 5: Pick up the sender si in S that has the shortest link length and add it to Pclt 6: Remove each sender sj from S, i.e. dsjri < dmin 7: end while 8: Return Pclt Theorem 2: The approximation factor of ILDP is O(1). Proof: Assume that there are two links in a same square satisfying the distance constraints in lines 8 and 9 of Algorithm 1. Thus, \|Pk ildp\| = 2\|Pk ldp\|. Let Pk opt be the optimal schedule in a link class Lk, Popt = arg max{Pk opt, ∀k}, and Pk ildp = arg max{P(k,j) ildp , ∀j}. According to conclusion in [3], we know \|Pk ldp\| ≥ \|Pk opt\| 4 , then 2: Input: Sender set S = {s1, . . . , sn} 3: Output: Pclt 4: while S is not empty do 4: while S is not empty do 5: Pick up the sender si in S that has the shortest link length and add it to Pclt 6: Remove each sender sj from S, i.e. dsjri < dmin 6: Remove each sender sj from S, i.e. dsjri < dmin 7: end while 4 \|Pildp\| 2 ≥ g(L) k=1 \|Pk ldp\| 4g(L) ≥ g(L) k=1 \|Pk opt\| 8g(L) ≥\|Popt\| 8g(L), 7: end while 8: Return Pclt For link li, we construct the disc centered at receiver ri with the radius of dmin. After executing CLT, all links whose senders, apart from si, are in the disc are removed. This process is repeated until all links have been either scheduled or deleted. The time complexity is O(n log n). (6) where Pildp = arg max{Pk ildp, ∀k}. Therefore, \|Popt\| \|Pildp\| ≤ 8g(L). ( ) In the following, we prove that the schedule Pclt is both feasible (Theorem 4) and efﬁcient, i.e., only a constant factor away from the optimum (Theorem 5). g( ) Theorem 3: For the SLS, executing ILDP repeatedly yields an Θ(ln n) approximation factor in the worst case. y p ( ) Theorem 4: CLT can output a feasible schedule. Proof: Assume that ς is the minimum number of slots in the optimal solution, and then ς = n Popt . Algorithm 2 Centralized and Localized Traversal (CLT) 1: Initialization: Scheduling set Pclt = ∅ Accordingly, dα ii dα sjri ≤ 1 eγϵ −1. Moreover, ln(1 + x) ≥ 1 eγϵ x holds when x ∈[0, eγϵ −1], and dsjri ≤ dsjsi + dii = (1 + k)dii, fsj,ri is lower bounded by Algorithm 2 Centralized and Localized Traversal (CLT) 1: Initialization: Scheduling set Pclt = ∅ Combining with result in Theorem 2, the maximum number of slots needed by ILDP, denoted by ςildp, satisﬁes ςildp ≤8g(L)ς. Proof: The distance between the senders in Pclt \ {si} and ri is at least dmin, the interference factor of Pclt on ri is at most fPclt,ri = sj∈Pclt\{si} ln 1 + dα ii dαsjri γth ≤ +∞ μ=1 1 (μ(β −1))α γth < α(α −2)2 16(α −1)2 γϵ < γϵ, p p ( ) The size of scheduling set obtained by ILDP is at least n ψildp . Then, the number of remaining unscheduled links is at most n 1 − 1 ψildp , and is reduced to n 1 − 1 ψildp s < ne−s/ψildp after s slots. Thus, when s ≥ςildp ln n, all links have been scheduled successfully, and s ψ ≤8g(L) ln n. Moreover, s ψ ≥ ln n since ςildp ≥ς, which gives an Θ(ln n) approximation factor. So far, we have proposed one centralized algorithm for link scheduling problem. Centralized scheduling algorithms require a central coordinator to allocate the link schedules. For a large IoT network, centralized algorithms are vulnerable to single point failure, if the central controller is down, there is no one else to coordinate the resource allocation, and distributed link scheduling algorithms are urgently designed, which only needs the coordination among the nodes in a local neighborhood. Moreover, it is difﬁcult to handle global interference deﬁned by the deterministic SINR model using local distributed algo- rithms, since only considering local interference can lead to incorrect conclusions. From the proof in Theorem 1, we can see that if a link is successfully scheduled, those neighbouring links need to be removed. In other words, whether a link schedule is successful or not only depends on the localization information, which is better for a distributed implementation. based on the following Formula [6], [36] based on the following Formula [6], [36] μ≥dmin 1 μα < α +∞ dmin 1 μα dx, which means that link li can be successfully scheduled. Lemma 3: Let si ∈Pclt. The number of senders in set Pclt\{si} with distance kdii away from si is at most γϵeγϵ γth (1 + k)α. (8) (8) Proof: From deﬁnition 1, we know that interference factor of each sender sj ∈Pclt \ {si} on receiver ri cannot be greater than γϵ. Algorithm 1 1-SIC Based Improved LDP (ILDP) Next, we introduce ILDP based on 1-SIC in detail. This algorithm starts to run by building g(L) disjoint link classes L1, . . . , Lg(L) from L, s.t., if dskri ≤ρdii and dsirk ≤ρdkk then (k j) Remove link lk from Lk Lk = {li ∈L\|2hklmin ≤dii < 2hk+1lmin}. Note that hk is independent of n and can be upper-bounded by a constant if the maximum link length is pre-known. Then, for link class Lk, ILDP partitions the entire network region into squares with size length βk = 2hk+1lminβ, where We show that Pildp is feasible with the consideration of the Rayleigh fading model in the proof of Theorem 1. β = 16 α −2 · γth γε · α −1 α −2 1 α + 1, (5) Theorem 1: ILDP provides a feasible schedule. (5) 2512 IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMBER 2021 Since the interference factor of Pclt \ {si} on ri cannot exceed γϵ, and there are at most γϵeγϵ γth (1 + k)α such senders. Thus, the lemma holds. B. Interference Localization-Based Distributed Algorithm times. On the other hand, denote Nk = 2hk and the senders whose corresponding links are in Lk as the set Sk. The probability that at least two senders in Sk select a same value is at most 1 N 2 k . According to Lemma 4.3 [36], for any red point sj ∈Nr, there exists a subset of blue points G(sj) such that \|G(sj)\| ≥z According to Lemma 4.3 [36], for any red point sj ∈Nr, there exists a subset of blue points G(sj) such that \|G(sj)\| ≥z (z = γϵeγϵ γth (2 + δ)α). Next, we prove that si can be selected by CLT after sj, i.e., dii ≥djj. We can derive that dsisj > (δ + 1) dii. Otherwise the number of senders within distance (δ+1)dii away from si would be greater than γϵeγϵ γth (1+δ+1)α, which contradicts with the conclusion in Lemma 3. Using the triangle inequality, we get Nk Based on the following Lemma, the approximation factor of RCRDCLT will be given in Theorem 6. Lemma 4: [42] Given two disks D1 and D2 of radii R1 and R2, respectively, assume that R1 > R2, we deﬁne χR1,R2 to be the smallest number of disks D2 needed to cover the larger disk D1. It holds that dsjri ≥dsjsi −dii > δdii χR1,R2 ≤2π 3 √ 3 · (R1 + 2R2)2 R2 2 . (12) and and (12) dsirj ≥dsisj −djj > dsisj −dii ≥δdii ≥δdjj, Theorem 6: RCRDCLT only losses a constant fraction of the optimum schedule, i.e., \|Prcrdclt\| ≥ 5 γϵeγϵ γth (2 + δ)α + 1 · 2π 3 √ 3 3 + 1 δ 2 + 1 \|Popt\|. which shows that si should not have been deleted by CLT, resulting in a contradiction. Next, we achieve the distributed implementation of CLT based on the random contention resolution. The main idea is that each sender starts to transmit independently, and it will join into the scheduling set, when there is no interferers within dmin starting to transmit or having been selected into scheduling set. The pseudo-code is given in Algorithm 3. Proof: For any link li, let R1 = dmin+dii and R2 = dmin. From line 6 in Algorithms 2 and 3, there are no interferers within dmin around ri and within dmin + dii around si, as shown in Fig. 2. B. Interference Localization-Based Distributed Algorithm In this subsection, to design distributed algorithm, we ﬁrst propose a centralized and localized link scheduling algorithm for the MLS. In each traversal, the algorithm ﬁrst greedily selects the unselected sender with the shortest link length, since the strength is stronger at close range. Deﬁne dmin as the minimum distance between a non-intended sender and the receiver. And dmin satisﬁes fsj,ri = ln 1 + dα ii dαsjri γth ≥ 1 eγϵ dα ii dαsjri γth ≥ 1 eγϵ 1 (1 + k)α γth. (9) fsj,ri = ln 1 + dα ii dαsjri γth ≥ 1 eγϵ dα ii dαsjri γth ≥ 1 eγϵ 1 (1 + k)α γth. (9) (9) dmin = (β −1)dii (7) (7) Since the interference factor of Pclt \ {si} on ri cannot exceed γϵ, and there are at most γϵeγϵ γth (1 + k)α such senders. Thus, the lemma holds. for link li. That is, the distance between link li and successful links scheduled before and after it is at least dmin. The pseudo-code is given in Algorithm 2. for link li. That is, the distance between link li and successful links scheduled before and after it is at least dmin. The pseudo-code is given in Algorithm 2. YU et al.: EFFICIENT LINK SCHEDULING SOLUTIONS FOR THE INTERNET OF THINGS UNDER RAYLEIGH FADING 2513 Fig. 2. A link li scheduled by RCRDCLT, and those senders which are not successful in RCRDCLT but successful in CLT locate in the gray area. Theorem 5: The cardinality of the difference between Pclt and Popt is bounded by a constant, i.e., \|Popt\| \|Pclt\| ≤5 γϵeγϵ γth (2 + δ)α + 1, where δ = β −1. ) + , β Proof: By contradiction. Suppose that \|Popt \ Pclt\| > 5eγϵ(2 + δ)α γϵ γth \|Pclt\|. (10) (10) We label the set of senders in Popt \ Pclt by blue (i.e., Nb = Popt \ Pclt) and those in Pclt by red (Nr = Pclt). By Lemma 4.3 [36], if \|Nb\| > 5z\|Nr\|, then there is a z-blue-dominant point (sender) si ∈Nb, where z = γϵeγϵ γth (2 + δ)α. (11) (11) Fig. 2. A link li scheduled by RCRDCLT, and those senders which are not successful in RCRDCLT but successful in CLT locate in the gray area. We shall argue that the sender si would have been picked by CLT, leading to a contradiction. B. Interference Localization-Based Distributed Algorithm According to the conclusion in Lemma 4, those links scheduled by CLT cannot be successfully scheduled by RCRDCLT are at most g p g g To model the scenario where the senders with the shorter link length have a priority to transmit, each sender whose link belongs to link class Lk selects randomly a value from range [2hk, 2hk+1] to transmit ﬁrst. In this way, the shorter link is, the faster its sender starts to transmit. On the one hand, the senders in different link classes have different transmission χR1,R2 ≤2π 3 √ 3 3 + 1 δ 2 . That is, \|Pclt\Prcrdclt\| ≤χR1,R2\|Prcrdclt\|. Combining with conclusion in Theorem 5, we get That is, \|Pclt\Prcrdclt\| ≤χR1,R2\|Prcrdclt\|. Combining with conclusion in Theorem 5, we That is, \|Pclt\Prcrdclt\| ≤χR1,R2\|Prcrdclt\|. That is, \|Pclt\Prcrdclt\| ≤χR1,R2\|Prcrdclt\|. Combining with conclusion in Theorem 5, we get , \| clt\ rcrdclt\| ≤χ \| rcrdclt\| Combining with conclusion in Theorem 5, we get \|Popt\| \|Prcrdclt\| ≤ 5γϵeγϵ γth (2 + δ)α + 1 · (χR1,R2 + 1). Algorithm 3 Random Contention Resolution Based Distrib- uted CLT (RCRDCLT) Algorithm 3 Random Contention Resolution Based Distrib- uted CLT (RCRDCLT) Algorithm 3 Random Contention Resolution Based Distrib- uted CLT (RCRDCLT) 1: Initialization: Scheduling set Prcrdclt = ∅ 2: Input: Sender set S = {s1, . . . , sn} 3: Output: Prcrdclt 4: Select ti randomly in range [2hk, 2hk+1] for sender si 5: if Sender si starts to transmit at time ti then 6: if There is no senders starting to transmit or belonging to Prcrdclt within βdii then 7: si joins into Prcrdclt 8: else 9: Quit from current schedule 10: end if 11: end if 12: Output: Prcrdclt Algorithm 3 Random Contention Resolution Based Distrib- uted CLT (RCRDCLT) 1: Initialization: Scheduling set Prcrdclt = ∅ 2: Input: Sender set S = {s1, . . . , sn} 3: Output: Prcrdclt 4: Select ti randomly in range [2hk, 2hk+1] for sender si 5: if Sender si starts to transmit at time ti then 6: if There is no senders starting to transmit or belonging to Prcrdclt within βdii then 7: si joins into Prcrdclt 8: else 9: Quit from current schedule 10: end if 11: end if 12: Output: Prcrdclt 1: Initialization: Scheduling set Prcrdclt = ∅ 2: Input: Sender set S = {s1, . . . B. Interference Localization-Based Distributed Algorithm , sn} 1: Initialization: Scheduling set Prcrdclt = ∅ Theorem 7: Algorithm 3 ends after O(ln n) rounds. heorem 7: Algorithm 3 ends after O(ln n) round Proof: RCRDCLT uses time selector to reduce collisions among different senders and ensure that shorter links have priority to transmit ﬁrst. It can be clearly seen from the above algorithm that the smaller hk is, the sooner senders start to transmit. For sender si in link class Lk, if all senders in Sk \ {si} are away from si at least dmin + dii, it transmits at ti (selecting from [2hk, 2hk+1]) and joins into the schedule P. Otherwise, si should start to transmit ﬁrst, when si selects ti = Nk, the probability that all the senders in Sk start to transmit after or along with si is 1 since they can select any value from [2hk, 2hk+1]. Similarly, when si determines ti = Nk+1, above probability is Nk−1 Nk \|Sk\| . In this way, the probability that the IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMBER 2021 2514 Fig. 3. Random topology. Fig. 4. Cluster topology. sender si starts to transmit before other senders in Sk is sender si starts to transmit before other senders in Sk is sender si starts to transmit before other senders in Sk is sender si starts to transmit before other senders in Sk is p = 1 Nk 1\|Sk\| + · · · + 1 Nk 1 Nk \|Sk\| = 1 Nk ⎛ ⎝ \|Nk\| i=1 i Nk \|Sk\| ⎞ ⎠ ≥ 1 Nk ⎛ ⎝ \|Nk\| j=1 1 j \|Sk\| ⎞ ⎠, j is natural number ≈ζ(\|Sk\|) Nk , where ζ(·) is Riemann zeta function and it is a constant for \|Sk\| > 1 [43], which does not means that si joins into Prcrdclt, once at least one sender in Prcrdclt selects a time as same as ti, they will quit the current schedule. Otherwise, si joints into Prcrdclt. After consuming τ Nk ζ(\|Sk\|) ln n rounds for a constant τ, si either joins into Prcrdclt or quits from current schedule with the high probability, i.e., Fig. 3. Random topology. Fig. 4. Cluster topology. Fig. 4. Cluster topology. 1 − 1 −ζ(\|Sk\|) Nk τ Nk ζ(\|Sk\|) ln n ≥1 −1 nτ . Therefore, the number of rounds for Algorithm 3 is O(ln n). B. Interference Localization-Based Distributed Algorithm ( ) Theorem 8: Executing RCRDCLT repeatedly yields an Θ(ln n) approximation factor for the SLS. ( ) pp Proof: From Theorem 6, we know \|Prcrdclt\| ≥1 ηϵ \|Popt\|, where ηϵ = 2π 3 √ 3 3 + 1 δ 2 + 1 · 5γϵeγϵ γth (2 + δ)α + 1 . where ηϵ = 2π 3 √ 3 3 + 1 δ 2 + 1 · 5γϵeγϵ γth (2 + δ)α + 1 . Fig. 4. Cluster topology. √ Deﬁne ςrcrdclt as the number of rounds to solve the SLS for RCRDCLT. Besides, similar to Theorem 3, ςrcrdclt ≤ηϵς. Any subset of n links contains a scheduling set and its size is at least n ηϵψ. After costing at most ςηϵτ ln n rounds, the number of unsuccessful links is around each of them. The cluster topology aims to simulate a scenario of heterogeneous density distribution. Deﬁne the maximum length of a link as lmax. The simulation was done over 100 different networks. n 1 − 1 ηϵς ψηϵτ ln n < 1, For comparison, we use the centralized one-slot schedul- ing algorithms proposed by Goussevskaia, Halld´orsson and Wattenhofer (GHW) [36], LDP [3], RLE [3], Syn_DLS [5], DLS [6] and DDLS [6] to solve the MLS and SLS. GHW is a simple greedy algorithm, where the links are processed in a non-decreasing order of length, and each link can be scheduled successfully if the affectance of the link, caused by all successful links is less than or equals to the following parameter which shows that all the links are successfully scheduled. which shows that all the links are successfully scheduled. Then approximation factor for the SLS can be bounded by ςrcrdclt ς ≤ςηϵτ ln n ς = ηϵτ ln n. ςrcrdclt ς ≤ςηϵτ ln n ς = ηϵτ ln n. Similar to Theorem 3, ψrcrdclt ψ ≥ln n, resulting in Θ(ln n). c = 1 (2 + max(2, ((23 · 9 + 1)γth α−1 α−2) 1 α )) . V. EVALUATIONS Related parameters in RLE are set to c2 = 0.5 and Related parameters in RLE are set to c2 = 0.5 and Related parameters in RLE are set to c2 = 0.5 and In this section, considering two network topologies with 500 × 500 m2: random and cluster topologies, as shown in Fig. 3 and Fig. 4, the red nodes are senders and the white ones are receivers. We validate the impacts of system parameters on the scheduling performance of designed link scheduling algorithms by MATLAB. In the random topology, the senders and receivers are distributed randomly. In the cluster topology, nc clusters are selected randomly on the plane, and n/nc links are positioned inside disks of radius rc c1 = √ 2 12ζ(α −1)γth γϵ(1 −c2) 1 α + 1, A. MLS Validation in Random Networks Fig. 8. Impact of α with ϵ = 0.1. First, in Fig. 5, we evaluate the inﬂuence of the number of links on performances of CLT, RCRDCLT and centralized and distributed algorithms in [3], [5], [6], [36]. The simulated parameters were set according to Table II. On the one hand, CLT gives a larger enhancement when n increases, which indicates the parameter dmin in Eq. (7) is in effect: compared with LDP, GHW and DLS, the numbers of successful links in a single slot (i.e., single slot scheduling performance) are improved about 174.2%, 90.5%, and 18.4%, respectively. As expected, GHW is not able to schedule more links for a larger n due to the constraint of the parameter c. Even in sparse topology (100 links), CLT and RCRDCLT still compute 1.93 and 1.21 times the number of successful links obtained by GHW. The performance of LDP is poor since the size of each square is relatively larger and more links can located in a same square, i.e., fewer links are scheduled successfully. Setting c2 = 0.5 in RLE, DDLS achieves a better performance than that of RLE due to low efﬁciency of the parameter c2 in RLE. Among all the seven algorithms, CLT schedules the most number of successful links in a single slot for different n in random topology. Fig. 8. Impact of α with ϵ = 0.1. In Fig. 7 and Fig. 8, we analyze the inﬂuence of the path-loss exponent on the schedule performance for above algorithms. The simulation is done with n = 200, other settings keep the same as before. The single slot scheduling performances increase as α increasing. For LDP, the size of each square decreases with α increasing, as shown in Table IV, the probability that only one link exists in a square gets larger, resulting in more successful links. For CLT and RCRDCLT, distance constraint between the interferers and the receiver get smaller over α increasing, more links can join the scheduling set, as shown in Table V. In other words, the impact of α on interfering signal outperforms that of intended signal, a link can be successfully scheduled easily for larger α. Similarly, the performances of RLE and Syn_DLS are also increased with the increment of α, since c1dii used in RLE and dmin used in DLS (or DDLS) decrease as α increases, more links can be scheduled. TABLE II SIMULATED PARAMETERS AND VALUES TABLE II SIMULATED PARAMETERS AND VALUES Fig. 7. Impact of α with ϵ = 0.1. Fig. 7. Impact of α with ϵ = 0.1. respectively. respectively. Moreover, although GHW is an O(1)-approximation algo- rithm and the time complexity is O(n log n), a small schedul- ing set can be obtained due to a lower c. YU et al.: EFFICIENT LINK SCHEDULING SOLUTIONS FOR THE INTERNET OF THINGS UNDER RAYLEIGH FADING 2515 Fig. 5. Impact of n with ϵ = 0.1. Fig. 6. Impact of n with ϵ = 0.1. Fig. 7. Impact of α with ϵ = 0.1. Fig. 8. Impact of α with ϵ = 0.1. Fig. 6. Impact of n with ϵ = 0.1. Fig. 6. Impact of n with ϵ = 0.1. Fig. 7. Impact of α with ϵ = 0.1. TABLE III THE APPROXIMATION FACTOR IN THEOREM 6 MLS Validation in Cluster Networks In this subsection, using the simulated paramet in Table VI, we validate the scheduling performance for abo algorithms in the cluster networks, as shown in Fig. 13-Fig. In Fig. 13 and 14, the scheduling performances of LD GHW, DLS (DDLS), CLT and RCRDCLT increase with increment of Nc. Generally, the more the number of lin in a cluster is, the more interference each link will suff which makes it harder to schedule links in same clust Fig. 9. Impact of lmax with ϵ = 0.1. Fig. 10. Impact of lmax with ϵ = 0.1. with LDP, GHW, and DLS, the single slot scheduling perfor- mances of CLT are improved about 151.6%, 84.2% and 18.8%, respectively, while performance losses of RCRDCLT are sep- arately about 28.8% and 17.4% for α ≥4 when compared with Sys_DLS and DDLS. Moreover, the performance gaps between Syn_DLS and RCRDCLT gets smaller (for instance, RCRDCLT losses 10 links when α = 6 while losses 30 for α = 4), which further validates the effectiveness of dmin in Eq. (7), and RCRDCLT costs more less rounds. The impact of maximum link length on algorithmic per- formance is shown in Fig. 9 and Fig. 10, all algorithm performances decrease when lmax increases. This is because that when a link is selected into scheduling set, it will reject more links since dmin becomes larger over lmax increasing for LDP, RLE, DLS (DDLS), and CLT (RCRDCLT). For GHW and Syn_DLS, the strength of received signal weakens as lmax increasing, resulting in less successful links. From Fig. 9 and Fig. 10, we can see that in all the algorithms, CLT shows the best average scheduling performance, and RCRDCLT cannot deal with the scenarios where communica- tion distance between nodes are larger, it shows relatively good scheduling performance in shorter communication distance (such as 5 and 10). Compared with Syn_DLS and DDLS, performance losses of RCRDCLT are about 26% and 16.2% on the average, but it saves a lot of running time. Fig. 9. Impact of lmax with ϵ = 0.1. Fig. 9. Impact of lmax with ϵ = 0.1. Fig. 10. Impact of lmax with ϵ = 0.1. Fig. 11 and Fig. 12 validate the inﬂuence of ϵ with settings of n = 200. TABLE III THE APPROXIMATION FACTOR IN THEOREM 6 Note that, on the one hand, increasing ϵ improves scheduling performances of LDP, RLE, DLS (or DDLS), CLT and RCRDCLT, the reason is that the size of each square (for LDP), c1dii (for RLE), and dmin (for DLS or DDLS and CLT or RCRDCLT) decrease with ϵ increasing, as shown in Tables IV and V, respectively, resulting in more successful links. On the other hand, scheduling performances of Syn_DLS and GHW are not inﬂuenced by ϵ, the reasons are similar to ones in Fig. 8. Fig. 10. Impact of lmax with ϵ = 0.1. with LDP, GHW, and DLS, the single slot scheduling perfor- mances of CLT are improved about 151.6%, 84.2% and 18.8%, respectively, while performance losses of RCRDCLT are sep- arately about 28.8% and 17.4% for α ≥4 when compared with Sys_DLS and DDLS. Moreover, the performance gaps between Syn_DLS and RCRDCLT gets smaller (for instance, RCRDCLT losses 10 links when α = 6 while losses 30 for α = 4), which further validates the effectiveness of dmin in Eq. (7), and RCRDCLT costs more less rounds. The impact of maximum link length on algorithmic per- formance is shown in Fig. 9 and Fig. 10, all algorithm with LDP, GHW, and DLS, the single slot scheduling perfor- mances of CLT are improved about 151.6%, 84.2% and 18.8%, respectively, while performance losses of RCRDCLT are sep- arately about 28.8% and 17.4% for α ≥4 when compared with Sys_DLS and DDLS. Moreover, the performance gaps between Syn_DLS and RCRDCLT gets smaller (for instance, RCRDCLT losses 10 links when α = 6 while losses 30 for α = 4), which further validates the effectiveness of dmin in Eq. (7), and RCRDCLT costs more less rounds. A. MLS Validation in Random Networks For GHW, the impact of α on interfering signal is larger than that of intended signal. Speciﬁcally, scheduling performance of Syn_DLS is not inﬂuenced by α. This is because that message dissemination among nodes is executed if the distance between nodes is less than 2lmax, changing α cannot change the scheduling performance. A ll h l i h CLT h b i l p gy On the other hand, from Fig. 6 we can see that com- pared with CLT, the single-slot scheduling performance of RCRDCLT decreases about 39.5%, this is because that the distance between the interferer and the receiver in CLT is at least dmin, while RCRDCLT will reject a fraction of successful senders, scheduled by CLT, where the distance between them and the receiver changes from dmin to dmin + 2dii, as shown in Fig. 2. But the performance difference between CLT and RCRDCLT can be upper-bounded, as shown in Theorem 6 and Table III. Although, compared with Syn_DLS and DDLS, the performance decrements of RCRDCLT are about 27.9% and 18.9%, the time complexity of RCRDCLT decreases from O(n ln n) (Syn_DLS and DDLS needed) to O(ln n). Note that if we design distributed CLT based on the idea of DDLS in [6], achieved scheduling performance will be better than those of Syn_DLS and DDLS, resulting in O(n ln n) time complexity. Among all the algorithms, CLT presents the best single slot scheduling performance, and the gap between CLT and RCRDCLT is upper-bounded, as shown in Table III. Compared IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMBER 2021 IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMBER 2021 2516 TABLE III THE APPROXIMATION FACTOR IN THEOREM 6 TABLE IV THE PARAMETER NEEDED β IN EQUATION (5) TABLE V THE MINIMUM DISTANCE dmin WITH lmax = 8 IN EQ. (7) TABLE III THE APPROXIMATION FACTOR IN THEOREM 6 TABLE III THE APPROXIMATION FACTOR IN THEOREM 6 TABLE III THE APPROXIMATION FACTOR IN THEOREM 6 TABLE IV THE PARAMETER NEEDED β IN EQUATION (5) TABLE V THE MINIMUM DISTANCE dmin WITH lmax = 8 IN EQ. (7) Fig. 9. Impact of lmax with ϵ = 0.1. Fig. 10. Impact of lmax with ϵ = 0.1. with LDP, GHW, and DLS, the single slot scheduling perfor- mances of CLT are improved about 151.6%, 84.2% and 18.8%, respectively, while performance losses of RCRDCLT are sep- arately about 28.8% and 17.4% for α ≥4 when compared with Sys_DLS and DDLS. Moreover, the performance gaps between Syn_DLS and RCRDCLT gets smaller (for instance, RCRDCLT losses 10 links when α = 6 while losses 30 for α = 4), which further validates the effectiveness of dmin in Eq. (7), and RCRDCLT costs more less rounds. The impact of maximum link length on algorithmic per- performances decrease when lmax increases. This is becau that when a link is selected into scheduling set, it will rej more links since dmin becomes larger over lmax increas for LDP, RLE, DLS (DDLS), and CLT (RCRDCLT). F GHW and Syn_DLS, the strength of received signal weake as lmax increasing, resulting in less successful links. Fr Fig. 9 and Fig. 10, we can see that in all the algorithm CLT shows the best average scheduling performance, a RCRDCLT cannot deal with the scenarios where communi tion distance between nodes are larger, it shows relatively go scheduling performance in shorter communication distan (such as 5 and 10). Compared with Syn_DLS and DDL performance losses of RCRDCLT are about 26% and 16. on the average, but it saves a lot of running time. Fig. 11 and Fig. 12 validate the inﬂuence of ϵ with settin of n = 200. Note that, on the one hand, increasing ϵ impro scheduling performances of LDP, RLE, DLS (or DDL CLT and RCRDCLT, the reason is that the size of ea square (for LDP), c1dii (for RLE), and dmin (for DLS DDLS and CLT or RCRDCLT) decrease with ϵ increasi as shown in Tables IV and V, respectively, resulting in m successful links. On the other hand, scheduling performan of Syn_DLS and GHW are not inﬂuenced by ϵ, the reaso are similar to ones in Fig. 8. B. B. MLS Validation in Cluster Networks In this subsection, using the simulated parameters in Table VI, we validate the scheduling performance for above algorithms in the cluster networks, as shown in Fig. 13-Fig. 20. In Fig. 13 and 14, the scheduling performances of LDP, GHW, DLS (DDLS), CLT and RCRDCLT increase with the increment of Nc. Generally, the more the number of links in a cluster is, the more interference each link will suffer, which makes it harder to schedule links in same clusters concurrently. Different from all link scheduling algorithms The impact of maximum link length on algorithmic per- formance is shown in Fig. 9 and Fig. 10, all algorithm YU et al.: EFFICIENT LINK SCHEDULING SOLUTIONS FOR THE INTERNET OF THINGS UNDER RAYLEIGH FADING 2517 Fig. 11. Impact of ϵ with n = 200. Fig. 12. Impact of ϵ with n = 200. Fig. 13. Impact of Nc with ϵ = 0.1. Fig. 11. Impact of ϵ with n = 200. Fig. 11. Impact of ϵ with n = 200. Fig. 14. Impact of Nc with ϵ = 0.1. l e y Fig. 14. Impact of Nc with ϵ = 0.1. Fig. 15. Impact of α with ϵ = 0.1. Fig. 16. Impact of α with ϵ = 0.1. Fig. 17. Impact of lmax with ϵ = 0.1. losses about 6 successful links at most in each schedu Syn_DLS cannot present a better scheduling perform cluster networks. In Fig. 21 and Fig. 22, we analyze the inﬂuenc cluster radius with n = 200 and lmax = 10, other pa k h b f I l i i h l Fig. 14. Impact of Nc with ϵ = 0.1. Fig. 11. Impact of ϵ with n = 200. Fig. 12. Impact of ϵ with n = 200. Fig. 15. Impact of α with ϵ = 0.1. Fig. 15. Impact of α with ϵ = 0.1. Fig. 12. Impact of ϵ with n = 200. Fig. 12. Impact of ϵ with n = 200. Fig. 13. Impact of Nc with ϵ = 0.1. Fig. 16. Impact of α with ϵ = 0.1. Fig. 16. Impact of α with ϵ = 0.1. Fig. 17. Impact of lmax with ϵ = 0.1. TABLE VI SIMULATED PARAMETERS AND VALUES Impact of n with ϵ = 0.1. Fig. 25. Impact of α with ϵ = 0.1. expected, LDP, RLE, GHW and DLS cannot compar CLT, while the average performance of RCRDCLT better than that of Syn_DLS, and only increases 7% th of DDLS. On the average, LDP, GHW and DLS compu 2.1 and 1.7 times longer schedule lengths than CLT, s can compute a larger scheduling set, as shown in Fig. Fig. 6. As shown in Fig. 25 and Fig. 26, over α increasi algorithms present more better performance besides Syn The reasons are similar to those in Fig. 7 and Fig. 8, n a larger α results in a smaller distance between the inte and the receiver more links can be scheduled in a tim 2518 Fig. 18. Impact of lmax with ϵ = 0.1. Fig. 19. Impact of ϵ. Fig. 20. Impact of ϵ. Fig. 21. Impact of rc with ϵ = 0.1. example rc = 20), three algorithms in Fig. 22 presen performance, Syn_DLS outperforms than other two o rc increasing. The reason is as the same as described i.e., larger cluster radius makes the distance among the same cluster larger. C. SLS Validation in Random Networks In Fig. 23-Fig. 30, simulated parameters are set a to Table II, we consider the impact of network param the schedule length in random networks IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMBER 2021 2518 Fig. 22. Impact of rc with ϵ = 0.1. Fig. 23. Impact of n with ϵ = 0.1. Fig. 24. Impact of n with ϵ = 0.1. Fig. 25. Impact of α with ϵ = 0.1. expected, LDP, RLE, GHW and DLS cannot comp CLT, while the average performance of RCRDCLT better than that of Syn_DLS, and only increases 7% of DDLS. On the average, LDP, GHW and DLS com 2.1 and 1.7 times longer schedule lengths than CLT Fig. 18. Impact of lmax with ϵ = 0.1. Fig. 22. Impact of rc with ϵ = 0.1. Fig. 18. Impact of lmax with ϵ = 0.1. Fig. 18. Impact of lmax with ϵ = 0.1. Fig. 19. Impact of ϵ. Fig. 20. Impact of ϵ. Fig. 21. Impact of rc with ϵ = 0.1. Fig. 22. Impact of rc with ϵ = 0.1. Fig. 18. Impact of lmax with ϵ = 0.1. Fig. 23. Impact of n with ϵ = 0.1. Fig. 19. TABLE VI SIMULATED PARAMETERS AND VALUES Impact of ϵ. Fig. 23. Impact of n with ϵ = 0.1. Fig. 19. Impact of ϵ. Fig. 24. Impact of n with ϵ = 0.1. Fig. 20. Impact of ϵ. Fig. 24. Impact of n with ϵ = 0.1. Fig. 20. Impact of ϵ. Fig. 25. Impact of α with ϵ = 0.1. Fig. 21. Impact of rc with ϵ = 0.1. Fig. 25. Impact of α with ϵ = 0.1. Fig. 21. Impact of rc with ϵ = 0.1. expected, LDP, RLE, GHW and DLS cannot compare with CLT, while the average performance of RCRDCLT is still better than that of Syn_DLS, and only increases 7% than that of DDLS. On the average, LDP, GHW and DLS compute 2.8, 2.1 and 1.7 times longer schedule lengths than CLT, since it can compute a larger scheduling set, as shown in Fig. 5 and Fig. 6. example rc = 20), three algorithms in Fig. 22 present similar performance, Syn_DLS outperforms than other two ones over rc increasing. The reason is as the same as described as above, i.e., larger cluster radius makes the distance among links in the same cluster larger. TABLE VI SIMULATED PARAMETERS AND VALUES Fig. 17. Impact of lmax with ϵ = 0.1. Fig. 17. Impact of lmax with ϵ = 0.1. losses about 6 successful links at most in each schedule, while Syn_DLS cannot present a better scheduling performance in cluster networks. cited in this paper, CLT can ﬁnd at least two successful links in the same cluster on the average when Nc ≥30, which shows that it can deal with dense cluster networks. Although RCRDCLT presents a relatively poor performance compared with DDLS, it costs more less time complexity and still schedules at least one successful link in each cluster averagely, since cluster radius of rc = 20 separates clusters, RCRDCLT and DDLS can be able to take advantage of this property well, while Sys_DLS cannot, since Syn_DLS will reject those links within 2lmax = 60, the radius of cluster is 20. That is, the more links are in the cluster, the more links are rejected. Moreover, compared with DDLS, RCRDCLT only In Fig. 21 and Fig. 22, we analyze the inﬂuence of the cluster radius with n = 200 and lmax = 10, other parameters keep the same as before. In topologies with larger clusters, scheduling performance of CLT increases signiﬁcantly than other algorithms. Generally, larger cluster radius separates links in the same cluster more easily, which makes it easier to schedule links in the same cluster concurrently. However, LDP, RLE, GHW and DLS cannot be able to take advantage of this property well. Additionally, in the dense networks (for 2518 IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMB Fig. 18. Impact of lmax with ϵ = 0.1. Fig. 19. Impact of ϵ. Fig. 20. Impact of ϵ. Fig. 21. Impact of rc with ϵ = 0.1. example rc = 20), three algorithms in Fig. 22 present similar performance, Syn_DLS outperforms than other two ones over rc increasing. The reason is as the same as described as above, i.e., larger cluster radius makes the distance among links in the same cluster larger. C. SLS Validation in Random Networks In Fig. 23-Fig. 30, simulated parameters are set according to Table II, we consider the impact of network parameters on the schedule length in random networks. In Fig 23 and Fig 24 over n increasing the needed Fig. 22. Impact of rc with ϵ = 0.1. Fig. 23. Impact of n with ϵ = 0.1. Fig. 24. C. SLS Validation in Random Networks As shown in Fig. 25 and Fig. 26, over α increasing, all algorithms present more better performance besides Syn_DLS. The reasons are similar to those in Fig. 7 and Fig. 8, namely a larger α results in a smaller distance between the interferers and the receiver, more links can be scheduled in a time slot and then scheduling all given links needs less slots. When In Fig. 23-Fig. 30, simulated parameters are set according to Table II, we consider the impact of network parameters on the schedule length in random networks. In Fig. 23 and Fig. 24, over n increasing, the needed schedule length increases for all algorithms. As could be YU et al.: EFFICIENT LINK SCHEDULING SOLUTIONS FOR THE INTERNET OF THINGS UNDER RAYLEIGH FADING 2519 F THINGS UNDER RAYLEIGH FADING Fig. 29. Impact of ϵ. Fig. 30. Impact of ϵ. Fig. 31. Impact of rc with ϵ = 0.1. Fig. 32. Impact of rc with ϵ = 0.1. than DDLS and RCRDCLT, but it needs more to schedule all links. Additionally, performance dif schedule length between RCRDCLT and DDLS ge Fig. 26. Impact of α with ϵ = 0.1. Fig. 27. Impact of lmax with ϵ = 0.1. Fig. 28. Impact of lmax with ϵ = 0.1. Fig. 29. Impact of ϵ. Fig. 30. Impact of ϵ. Fig. 31. Impact of rc with ϵ = 0.1. Fig. 26. Impact of α with ϵ = 0.1. Fig. 26. Impact of α with ϵ = 0.1. Fig. 30. Impact of ϵ. Fig. 27. Impact of lmax with ϵ = 0.1. Fig. 30. Impact of ϵ. Fig. 27. Impact of lmax with ϵ = 0.1. Fig. 27. Impact of lmax with ϵ = 0.1. Fig. 31. Impact of rc with ϵ = 0.1. Fig. 28. Impact of lmax with ϵ = 0.1. Fig. 28. Impact of lmax with ϵ = 0.1. Fig. 31. Impact of rc with ϵ = 0.1. Fig. 28. Impact of lmax with ϵ = 0.1. Fig. 32. Impact of rc with ϵ = 0.1. α ≥4, on the average, there’s hardly no difference between RCRDCLT and DDLS (differ by 1%), and they need a shorter schedule length than Syn_DLS. The impacts of the maximum link length on schedule length are given in Fig. 27 and Fig. 28. C. SLS Validation in Random Networks Compared with CLT, averages of 2.5, 2.1 and 1.6 times schedule length are given by LDP, GHW and DLS for different lmax, respectively, since they have lower scheduling performance, as shown in Fig. 9 and Fig. 10. Moreover, Syn_DLS, DDLS and RCRDCLT are particularly affected by lmax compared with other parameters, and RCRDCLT presents the best scheduling performance from the perspective of the average. Fig. 32. Impact of rc with ϵ = 0.1. In Fig. 30, RCRDCLT and Syn_DLS show a similar aver- aged performance in the schedule length (differ by 8%), and obtain a shorter schedule times than DDLS. In addition, CLT is the fastest algorithm to schedule all given links, as shown in Fig. 29. than DDLS and RCRDCLT, but it needs more time slots to schedule all links. Additionally, performance difference in schedule length between RCRDCLT and DDLS gets smaller as rc increases. On the average, RCRDCLT computes only 1.27 times schedule length than DDLS. The impacts of other parameters on the SLS in cluster networks are similar to those in random networks. D. SLS in Cluster Networks In this subsection, simulated parameters are set to those in Table VI, from Fig. 22, Fig. 31 and Fig. 32, we can see that CLT obtains the shortest schedule length among all algorithms, and Syn_DLS cannot deal with cluster networks well, since it schedules more successful links in a time slot REFERENCES [1] Y. Xiao, Y. Jia, C. Liu, X. Cheng, J. Yu, and W. Lv, “Edge computing security: State of the art and challenges,” Proc. IEEE, vol. 107, no. 8, pp. 1608–1631, Aug. 2019. [2] W. Ye and A. M. Haimovich, “Performance of cellular CDMA with cell site antenna arrays, Rayleigh fading, and power control error,” IEEE Trans. Commun., vol. 48, no. 7, pp. 1151–1159, Jul. 2000. Fig. 33. Impact of Nc with ϵ = 0.1. [3] C. Qiu and H. Shen, “Fading-resistant link scheduling in wireless networks,” in Proc. ICPP, 2017, pp. 312–321. Fig. 34. Impact of α with ϵ = 0.1. [4] J. Dams, M. Hoefer, and T. Kesselheim, “Scheduling in wireless net- works with Rayleigh-fading interference,” IEEE Trans. Mobile Comput., vol. 14, no. 7, pp. 1503–1514, Jul. 2015. [5] K. Yu, Y. Wang, J. Yu, D. Yu, X. Cheng, and Z. Shan, “Localized and distributed link scheduling algorithms in IoT under Rayleigh fading,” Comput. Netw., vol. 151, pp. 232–244, Mar. 2019. [6] J. Yu et al., “Efﬁcient link scheduling in wireless networks under Rayleigh-fading and multiuser interference,” IEEE Trans. Wireless Com- mun., vol. 19, no. 8, pp. 5621–5634, Aug. 2020. [7] G. Sharma, N. Shroff, and R. Mazumdar, “On the complexity of schedul- ing in wireless networks,” in Proc. ACM Mobicom, 2006, pp. 227–238. 8 i d Sh ff “ h i f i f h d li [8] X. Lin and N. B. Shroff, “The impact of imperfect scheduling on cross- layer congestion control in wireless networks,” IEEE/ACM Trans. Netw., vol. 14, no. 2, pp. 302–315, Apr. 2006. Fig. 34. Impact of α with ϵ = 0.1. pp p [9] W. Wang, Y. Wang, X. Li, W. Song, and O. Frieder, “Efﬁcient interfer- ence aware tdma link scheduling for static wireless networks,” in Proc. ACM Mobicom, 2006, pp. 262–273. performances of LDP and ILDP increase as Nc (or α) increas- ing, and ILDP can obtain 35.4% and 43.4% enhancements for Nc and α, respectively. The reasons are similar to those in Fig. 13 and Fig. 15. pp [10] A. Krifa, C. Barakat, and T. Spyropoulos, “An optimal joint scheduling and drop policy for delay tolerant networks,” in Proc. IEEE WoWMoM, Jun. 2008, pp. 1–6. [11] C. Joo, X. Lin, and N. B. Shroff, “Understanding the capacity region of the greedy maximal scheduling algorithm in multihop wireless networks,” IEEE/ACM Trans. Netw., vol. 17, no. 4, pp. 1132–1145, Aug. ACKNOWLEDGMENT The authors would like to thank the anonymous reviewers for their extremely useful comments and suggestions. E. Comparison of LDP and ILDP In the end of this section, we compared LDP and ILDP in cluster networks for different Nc and α, respectively, as shown in Fig. 33 and Fig. 34. We can see that scheduling 2520 IEEE/ACM TRANSACTIONS ON NETWORKING, VOL. 29, NO. 6, DECEMBER 2021 Fig. 33. Impact of Nc with ϵ = 0.1. Fig. 34. Impact of α with ϵ = 0.1. Fig. 33. Impact of Nc with ϵ = 0.1. REFERENCES 2009. g g To sum up, the simulations demonstrate that CLT is the best scheduling performance for the MLS and SLS in the random and cluster networks. RCRDCLT, besides having lowest time complexity than Syn_DLS and DDLS, only losses a constant approximation factor compared with the optimal schedule. Moreover, compared with Syn_DLS and DDLS from the perspective of the average, RCRDCLT presents advantages in some speciﬁc scenarios, such as high path-loss and longer communication distance. If we apply the idea of DDLS to design distributed CLT, the scheduling performance obtained will be better than Syn_DLS and DDLS, but the time com- plexity is also increased from O(ln n) to O(n ln n). [12] L. Jiang, D. Shah, J. Shin, and J. Walrand, “Distributed random access algorithm: Scheduling and congestion control,” IEEE Trans. Inf. Theory, vol. 56, no. 12, pp. 6182–6207, Dec. 2010. [13] K. Kar, S. Sarkar, A. Ghavami, and X. Luo, “Delay guarantees for throughput-optimal wireless link scheduling,” IEEE Trans. Autom. Con- trol, vol. 57, no. 11, pp. 2906–2911, Nov. 2012. [14] X. Wang, Z. Li, and J. Wu, “Joint TCP congestion control and CSMA scheduling without message passing,” IEEE Trans. Wireless Commun., vol. 12, no. 12, pp. 6194–6204, Dec. 2013. [15] T. Moscibroda and R. Wattenhofer, “The complexity of connectiv- ity in wireless networks,” in Proc. IEEE INFOCOM, Apr. 2006, pp. 1–13. [16] O. Goussevskaia, Y. Osfwald, and R. Wattenhofer, “Complexity in geometric SINR,” in Proc. ACM Mobihoc, 2007, pp. 1872–1880. VI. CONCLUSION Zheng, J. Zhang, N. Shroff, and C. Joo, “Distributed CSMA algorithms for link scheduling in multihop MIMO networks under SINR model,” IEEE/ACM Transmiss. Netw., vol. 21, no. 3, pp. 1503–1514, Aug. 2013. Jiguo Yu (Senior Member, IEEE) received the Ph.D. degree from the School of Mathematics, Shandong University, Jinan, China, in 2004. He became a Full Professor with the School of Computer Science, Qufu Normal University, Jining, China, in 2007. He is also a Full Professor with the Qilu University of Technology (Shandong Academy of Sciences), Jinan, and the Shandong Computer Science Cen- ter (National Supercomputer Center), Jinan. He is interested in designing and analyzing algorithms for many computationally hard problems in networks. His current research interests include privacy-aware computing, wireless networking, distributed algorithms, peer-to-peer computing, and graph theory. He is a member of the ACM and a Senior Member of the China Computer Federation (CCF). [26] P. Chaporkar, K. Kar, X. Luo, and S. Sarkar, “Throughput and fairness guarantees through maximal scheduling in wireless networks,” IEEE Trans. Inf. Theory, vol. 54, no. 2, pp. 572–594, Feb. 2008. [27] S. P. Weber and J. G. Andrews, “Transmission capacity of wireless networks,” Found. Trends Netw., vol. 5, nos. 2–3, pp. 109–281, 2012. [28] A. Fanghänel, S. Geulen, M. Hoefer, and B. Vöcking, “Online capacity maximization in wireless networks,” J. Scheduling, vol. 16, no. 1, pp. 81–91, Feb. 2013. pp [29] X. Xu, S. Tang, and P. Wan, “Maximum independent set of links under physical interference model,” in Proc. WASA, 2010, pp. 68–74. [30] B. Huang, J. Yu, X. Cheng, H. Chen, and H. Liu, “Scheduling in wireless networks with rayleigh-fading interference,” J. Netw. Comput. Appl., vol. 77, pp. 64–72, Jan. 2017. [31] D. M. Blough, G. Resta, and P. Santi, “Approximation algorithms for wireless link scheduling with SINR-based interference,” IEEE/ACM Trans. Netw., vol. 18, no. 6, pp. 1701–1712, Dec. 2010. Xiuzhen Cheng (Fellow, IEEE) received the M.S. and Ph.D. degrees in computer science from the University of Minnesota–Twin Cities in 2000 and 2002, respectively. She is currently a Professor with the School of Computer Science and Technology, Shandong University. Her current research interests include cyber physical systems, wireless and mobile computing, sensor networking, wireless and mobile security, and algorithm design and analysis. She is a member of the ACM. She received the NSF CAREER Award in 2004. VI. CONCLUSION [17] B. Huang, J. Yu, D. Yu, and C. Ma, “SINR based maximum link scheduling with uniform power in wireless sensor networks,” KSII Trans. Internet Inf. Syst., vol. 8, no. 11, pp. 4050–4067, 2014. In this paper, we proposed two efﬁcient algorithms for the problems of MLS and SLS. To begin with, by constructing the relationships between the acceptable failure probability and the needed SINR constraint in the Rayleigh fading model, we can design link scheduling algorithms under the deterministic SINR model rather than in the Rayleigh fading model directly. Then, we utilized the idea of interference localization to design centralized and distributed algorithms for the MLS, and the proposed algorithms not only localize the global interference, but also costs a logarithmic time complexity. Executing them repeatedly obtained a logarithmic approximation guarantee for the SLS with rigorous analysis. Finally, compared with current popular link scheduling algo- rithms, simulations validated the efﬁciency of our algorithms. Only uniform power assignment was considered, hence one future direction is to consider other methods of power control. Another promising research direction is the dynamic network scenarios where nodes move randomly to broadcast or collect data. [18] J. Yu, B. Huang, X. Cheng, and M. Atiquzzaman, “Shortest link scheduling algorithms in wireless networks under the SINR model,” IEEE Trans. Veh. Technol., vol. 66, no. 3, pp. 2643–2657, Mar. 2017. pp [19] M. Halldörsson, “Wireless scheduling with power control,” in Proc. ESA, 2009, pp. 361–372. [20] A. Fanghänel, T. Kesselheim, H. Räcke, and B. Vöcking, “An improved spine-based infrastructure for routing in ad hoc networks,” in Proc. ACM PODC, 2009, pp. 220–229. [21] A. Fanghänel, T. Kesselheim, and B. Vöcking, “Improved algorithms for latency minimization in wireless networks,” in Proc. ICALP, 2009, pp. 447–458. [22] Y. Zhou, X.-Y. Li, M. Liu, X. Mao, S. Tang, and Z. Li, “Throughput optimizing localized link scheduling for multihop wireless networks under physical interference model,” IEEE Trans. Parallel Distrib. Syst., vol. 25, no. 10, pp. 2708–2720, Oct. 2014. pp [23] L. Le, E. Modiano, C. Joo, and N. 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She is currently a Professor with the School of Computer Science and Technology, Shandong University. Her current research interests include cyber physical systems, wireless and mobile computing, sensor networking, wireless and mobile security, and algorithm design and analysis. She is a member of the ACM. She received the NSF CAREER Award in 2004. She has served on the editorial boards of several technical journals and the technical program com- mittees of various professional conferences/workshops. She has also chaired several international conferences. She worked as the Program Director of the U.S. National Science Foundation (NSF) from April 2006 to October 2006 (full time) and from April 2008 to May 2010 (part time). [32] M. Dinitz, “Distributed algorithms for approximating wireless network capacity,” in Proc. IEEE INFOCOM, Mar. 2010, pp. 1–9. [33] E. I. Asgeirsson and P. Mitra, “On a game theoretic approach to capacity maximization in wireless networks,” in Proc. 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IEEE INFOCOM, Apr. 2009, pp. 1872–1880. Dongxiao Yu (Senior Member, IEEE) received the B.Sc. VI. CONCLUSION degree from the School of Mathematics, Shandong University, Jinan, China, in 2006, and the Ph.D. degree from the Department of Computer Science, The University of Hong Kong, Hong Kong, in 2014. He is currently a Professor with the School of Computer Science and Technology, Shandong University. His research interests include wireless networks and distributed computing. Dongxiao Yu (Senior Member, IEEE) received the B.Sc. degree from the School of Mathematics, Shandong University, Jinan, China, in 2006, and the Ph.D. degree from the Department of Computer Science, The University of Hong Kong, Hong Kong, in 2014. He is currently a Professor with the School of Computer Science and Technology, Shandong University. His research interests include wireless networks and distributed computing. p pp [40] A. Sankararaman and F. Baccelli, “CSMA k-SIC—A class of distributed MAC protocols and their performance evaluation,” in Proc. IEEE INFOCOM, Apr. 2015, pp. 2002–2010. [41] S. Lv, W. Zhuang, X. Wang, and X. Zhou, “Scheduling in wireless ad hoc networks with successive interference cancellation,” in Proc. IEEE INFOCOM, Apr. 2011, pp. 1287–1295. [42] O. Goussevskaia, T. Moscibroda, and R. Wattenhofer, “Local broadcast- ing in the physical interference model,” in Proc. ACM DIALM-POMC, 2008, pp. 35–44. [43] I. Gradshteyn and I. Ryzhik, Table of Integrals, Series, and Products, A. Jeffrey and D. Zwillinger, Eds. Amsterdam, The Netherlands: Else- vier, 2007. Anming Dong (Member, IEEE) received the B.E. degree in electronic information science and technol- ogy from Liaocheng University, Liaocheng, China, in 2004, the M.E. degree in communications and information systems from Lanzhou University, Lanzhou, China, in 2007, and the Ph.D. degree in communications and information systems from Shandong University, Jinan, China, in 2016. He is currently an Associate Professor with the School of Computer Science and Technology, Qilu University of Technology (Shandong Academy of Sciences), Jinan. His research interests include MIMO techniques, wireless communica- tions, wireless networks, and the application of optimization techniques for signal processing and wireless communications. He was a recipient of the Excellent Doctoral Dissertation Awards of Shandong Province, in 2017. Anming Dong (Member, IEEE) received the B.E. degree in electronic information science and technol- ogy from Liaocheng University, Liaocheng, China, in 2004, the M.E. degree in communications and information systems from Lanzhou University, Lanzhou, China, in 2007, and the Ph.D. degree in communications and information systems from Shandong University, Jinan, China, in 2016. VI. CONCLUSION He is currently an Associate Professor with the School of Computer Science and Technology, Qilu University of Technology (Shandong Academy of Sciences), Jinan. His research interests include MIMO techniques, wireless communica- tions, wireless networks, and the application of optimization techniques for signal processing and wireless communications. He was a recipient of the Excellent Doctoral Dissertation Awards of Shandong Province, in 2017. Anming Dong (Member, IEEE) received the B.E. degree in electronic information science and technol- ogy from Liaocheng University, Liaocheng, China, in 2004, the M.E. degree in communications and information systems from Lanzhou University, Lanzhou, China, in 2007, and the Ph.D. degree in communications and information systems from Shandong University, Jinan, China, in 2016. He is currently an Associate Professor with the School of Computer Science and Technology, Qilu University of Technology (Shandong Academy of Sciences), Jinan. His research interests include MIMO techniques, wireless communica- tions, wireless networks, and the application of optimization techniques for signal processing and wireless communications. He was a recipient of the Excellent Doctoral Dissertation Awards of Shandong Province, in 2017. Kan Yu received the M.S. degree from the School of Information Science and Engineering, Qufu Normal University, Rizhao, China, in 2016, and the Ph.D. degree from the College of Computer Science and Engineering, Shandong University of Science and Technology, in 2019. He is currently a Lecturer with the School of Computer Science, Qufu Normal Uni- versity. His main research interests include wireless networks, the IoT security, and distributed algorithm design and analysis. Kan Yu received the M.S. degree from the School of Information Science and Engineering, Qufu Normal University, Rizhao, China, in 2016, and the Ph.D. degree from the College of Computer Science and Engineering, Shandong University of Science and Technology, in 2019. He is currently a Lecturer with the School of Computer Science, Qufu Normal Uni- versity. His main research interests include wireless networks, the IoT security, and distributed algorithm design and analysis.
https://openalex.org/W4365803668	https://journals.openedition.org/rbep/pdf/4033	Portuguese	null	Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença	Revista Brasileira de Estudos da Presença	2,023	cc-by	17,287	Editora Universidade Federal do Rio Grande do Sul (UFRGS) Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Who’s Afraid of Derrida?: Gumbrecht and the metaphysics of presence Qui a Peur de Derrida?: Gumbrecht et la métaphysique de la présence Gustavo Ramos de Souza E-ISSN 2237-2660 OUTROS TEMAS OUTROS TEMAS Refêrencia eletrónica Refêrencia eletrónica Gustavo Ramos de Souza, «Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença», Revista Brasileira de Estudos da Presença [Online], 13 : 2 \| 2023, posto online no dia 20 abril 2023, consultado o 18 agosto 2023. URL: http://journals.openedition.org/rbep/4033 Creative Commons - Atribuição 4.0 Internacional - CC BY 4.0 https://creativecommons.org/licenses/by/4.0/ Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Gustavo Ramos de SouzaI IUniversidade Estadual de Londrina – UEL, Londrina/PR, Brasil RESUMO – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença – O artigo propõe uma reflexão sobre o conceito de presença teorizado por Hans Ulrich Gumbrecht a partir do pensamento de Heidegger, confrontando-o à filosofia de Jacques Derrida, sobretudo em sua desconstrução da metafísica da presença. Assim, não se trata de rastrear a presença heideggeriana ao longo de sua obra nem de defender Derrida das acusações de existencialismo linguístico, mas antes de demonstrar que, ao ter Heidegger como aliado, a cruzada de Gumbrecht contra a metafísica e o campo hermenêutico acaba por restaurá-los mais fortemente, comprometendo-se com aquilo que visa combater. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. ç , g , 3, , , 3 Disponível em: http://dx.doi.org/10.1590/2237-2660126100vs01 Palavras-chave: Hans Ulrich Gumbrecht. Jacques Derrida. Martin Heidegger. Estudos da Pre- sença. Palavras-chave: Hans Ulrich Gumbrecht. Jacques Derrida. Martin Heidegger. Estudos da Pre- sença. ABSTRACT – Who’s Afraid of Derrida?: Gumbrecht and the metaphysics of presence – This paper proposes a reflection on the concept of presence theorized by Hans Ulrich Gumbrecht prem- ised on Heidegger’s thought, contrasting it with the philosophy of Jacques Derrida, especially in his deconstruction of the metaphysics of presence. Accordingly, it is neither a matter of tracing the Heideggerian presence throughout his oeuvre nor of defending Derrida from the accusations of linguistic existentialism, but rather of demonstrating that, by having Heidegger as an ally, Gum- brecht’s crusade against metaphysics and the hermeneutic field ultimately restores them more strongly, thereby he commits to what he aims to oppose. Keywords: Hans Ulrich Gumbrecht. Jacques Derrida. Martin Heidegger. Presence Studies. Introdução Proponho neste artigo um embate em torno do conceito de presença a partir das reflexões de Hans Ulrich Gumbrecht e da desconstrução da meta- física da presença realizada por Jacques Derrida. Assim, não intenciono apresentar o atual estado da arte acerca dos estudos da presença ou as suas contribuições para as ciências humanas e as artes, tampouco de reconhecer a influência da teoria de Gumbrecht, mas sim de problematizar o próprio conceito. Trata-se, nesse sentido, de confrontar os dois autores a partir de suas teorias, de suas obras, buscando aproximações e distanciamentos. Com isso, pretendo demonstrar que, ao se insurgir contra o que chama de “existencialismo linguístico” da desconstrução – a suposta incapacidade da linguagem de se referir ao mundo –, Gumbrecht acaba por recair naquilo que seu conceito de presença visa combater: o “campo hermenêutico”. Isso porque o seu gesto é de mera inversão dos polos, na medida em que sugere uma oposição entre sentido e presença, dando primazia ao segundo e inci- dindo numa espécie de materialismo metafísico, que restaura o “campo her- menêutico” mais fortemente. Uma vez que Gumbrecht apoia-se em Martin Heidegger ao desenvol- ver tal conceito, o objetivo é apresentar, num primeiro momento, a maneira como a filosofia heideggeriana é lida por ele em Production of Presence. Num segundo momento, intenciono mostrar em que medida a desconstrução derridiana já investia contra o pensamento da presença; para Derrida, a obra de Heidegger seria a sua defesa mais profunda. Não se trata, porém, de de- fender Derrida de Gumbrecht, mas sim de colocar os dois autores em diálo- go polêmico, a fim de verificar os limites do conceito de presença. E-ISSN 2237-2660 E-ISSN 2237-2660 Keywords: Hans Ulrich Gumbrecht. Jacques Derrida. Martin Heidegger. Presence Studies. ds: Hans Ulrich Gumbrecht. Jacques Derrida. Martin Heidegger. Presence Studies. RÉSUMÉ – Qui a Peur de Derrida?: Gumbrecht et la métaphysique de la présence – Cet article propose une réflexion sur le concept de présence théorisé par Hans Ulrich Gumbrecht à partir de la pensée de Heidegger, en le confrontant à la philosophie de Jacques Derrida, notamment dans sa déconstruction de la métaphysique de la présence. En ce sens, il ne s'agit pas de suivre les traces de la présence heideggérienne tout au long de son œuvre, ni de défendre Derrida des accusations d' existentialisme linguistique, mais plutôt de démontrer qu'en ayant Heidegger comme allié, la croisade de Gumbrecht contre la métaphysique et le champ de l’herméneutique finit par les restituer plus fortement, se compromettant avec ce qu'il cherche à combattre. Mots-clés: Hans Ulrich Gumbrecht. Jacques Derrida. Martin Heidegger. Études de la Présence. Mots-clés: Hans Ulrich Gumbrecht. Jacques Derrida. Martin Heidegger. Études de la Présence 1 1 ç , g , 3, , , 3 Disponível em: http://dx.doi.org/10.1590/2237-2660126100vs01 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Disponível em: http://seer.ufrgs.br/presenca Ser ou não ser heideggeriano? Em conferência realizada em maio de 1992 na Universidade Estadual do Rio de Janeiro (UERJ), posteriormente publicada no livro Corpo e forma (1998) sob o título O campo não-hermenêutico ou a materialidade da comu- nicação, Gumbrecht traça um breve esquema sobre as características da dita pós-modernidade, a fim de demonstrar a falência do método interpretativo nas humanidades. Após tecer comentários ácidos sobre Wilhelm Dilthey, a quem atribui a “desonra” de ser o fundador da hermenêutica e sistematiza- 2 g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. E ISSN 2237 2660 dor do campo das humanidades, Gumbrecht (1998, p. 141) volta sua aten- ção a Martin Heidegger, que “[...] representa a apoteose do domínio da hermenêutica”. A pretexto de investir contra a hermenêutica, Gumbrecht refere-se di- retamente a Heidegger, tanto que, ao falar sobre a serenidade (Gelassenheit), estado em que os fenômenos revelar-se-iam em sua verdade, ele diz: “[...] condição sem dúvida tributária da perspectiva anti-intelectual do filó- sofo. Afinal, o estado de relaxamento nada tem que ver com o trabalho inte- lectual de buscar a verdade, depurando-a” (Gumbrecht, 1998, p. 142). Evidentemente, ao opor a serenidade a um movimento voluntarista em direção à verdade, Gumbrecht está reafirmando o paradigma sujeito/objeto que fundamenta a hermenêutica, isto é, a concepção de que existe um sujei- to dotado de vontade que desvela uma superfície à procura da verdade. Mas convém assinalar o que Gumbrecht (1998, p. 143) diz em segui- da: “[...] um comentário final sobre o filósofo. Acredito que todo alemão tem a obrigação moral de ser muito crítico com sua obra. Aproveito para es- clarecer o que já se terá entendido: não sou nem um pouco tributário a Heidegger”. Essa obrigação moral diz respeito, é claro, ao envolvimento do filósofo com o regime nazista, devido à sua filiação ao NSDAP, partido de Hitler, dez dias depois de ter tomado posse como reitor na Universidade de Freiburg em 1933. Ou seja, a crítica que faz a Heidegger é tanto de cunho epistemológico, dado o fato de ser um dos principais filósofos da hermenêu- tica, quanto de cunho moral e político em razão dessa nódoa em sua biogra- fia. Embora não seja o meu propósito rastrear uma possível genealogia da presença de Heidegger no pensamento de Gumbrecht, é importante assina- lar que, além de Production of Presence, o filósofo aparece já em textos ante- riores de Gumbrecht: em 1926: Living at the Edge of Time, publicado em 1997, há um capítulo que trata diretamente de Heidegger, Being-in-the- worlds of 1926: Martin Heidegger, Hans Friedrich Blunck, Carl Van Vechten, em que se diz a seu propósito: “[...] talvez toda a contribuição de Heidegger para a filosofia ocidental possa ser lida como originária de uma reação aos ambientes emocional, intelectual e político de 1926”1 (Gumbrecht, 1997, p. 442, tradução nossa). Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. ç g Disponível em: http://seer.ufrgs.br/presenca Nesse mesmo texto, apesar de inserir Heidegger no contexto da “revo- lução conservadora” que desembocou na ideologia nazista, Gumbrecht vai, no entanto, defendê-lo: “[...] essa contaminação não mancha a importância filosófica do pensamento de Heidegger [...]. É claro que Sein und Zeit não pode ser identificado com a ideologia nazista”2 (Gumbrecht, 1997, p. 443, tradução nossa). O filósofo também é assunto de um ensaio publicado em 2000, na re- vista Diacritics: Martin Heidegger and his japanese interlocutors: about a limit of western metaphysics. Ou seja, entre a conferência de 1992 e o livro de 1997, e também em trabalhos subsequentes, a sua posição a respeito de Heidegger sofre uma enorme mudança. Em entrevista concedida à Mariana Lage, publicada na revista Artefilosofia sob o título Da produção de presença ao presente amplo, Gumbrecht (2017, p. 197) afirma que: Até os meus quase quarenta anos (o que significa: quase até o fim do meu período na Alemanha), evitei ler qualquer coisa de Heidegger. [...] As coisas mudaram mais drasticamente depois da minha mudança intelectual e profis- sional para os Estados Unidos, em 1989. Lá, tanto meus alunos quanto meus colegas esperavam, de certa forma ‘natural’, que eu fosse quase um es- pecialista em Heidegger – então prossegui com as minhas leituras. Em al- gum momento (que não associaria a qualquer evento ou livro), os textos Heidegger (especialmente os de depois de 1930) se tornaram mais e mais in- teressantes e desafiadores para mim – até que, em Produção de Presença (pu- blicado em 2004), ele se tornou meu principal autor de referência. Heidegger acaba por se tornar a principal referência de Gumbrecht na sua conceitualização de “presença” em Production of Presence3, publicado 12 anos depois da conferência em que maldizia o filósofo. As fontes potenciais em sua reflexão, a fim de estabelecer uma posição anticartesiana, são encon- tradas na definição aristotélica de signo, na Eucaristia cristã, no teatro me- dieval e na filosofia de Heidegger. Nesse livro, Gumbrecht propõe-se a “sujar as mãos”, a recorrer a con- ceitos obsoletos tais como “substância”, “ser” e “realidade” em sua tentativa de enfrentar o domínio exclusivo da interpretação e da metafísica nas Hu- manidades. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. 3 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Assim, se antes Derrida era tido como aliado devido à sua noção de “exterioridade do significante”5 e Heidegger era encarado com extrema re- serva em razão do seu legado para a hermenêutica, as posições se invertem, ainda que o custo seja recair no “mau gosto intelectual”. A propósito do desconstrucionismo, Gumbrecht (2010, p. 78) diz que o seu sucesso na Academia deve ser atribuído ao fato de que os epígonos de Derrida tacha- vam de “ingênuo” ou “substancialista” quem defendesse a possibilidade de manutenção de um sentido estável, concluindo que “[...] a desconstrução, em grande medida, tem recorrido a um suave terror para consolidar a or- dem vigente nas Humanidades”6. Essa reviravolta epistemológica deve-se, creio, à necessidade de Gumbrecht de defender o conceito de presença, ao passo que os desconstrucionistas colocaram sob suspeita a “metafísica da presença”. Ele diz: “[...] recorrer a esses conceitos é considerado há muito tempo como sintoma de péssimo gosto intelectual nas Humanidades”4 e “[...] acreditar na possibilidade de nos referirmos ao mundo sem ser pelo sentido tornou-se sinônimo do grau mais elevado de ingenuidade filosófica” (Gumbrecht, 2010, p. 77). Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca 4 4 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. via, em vez de Dasein, o conceito que Gumbrecht tenta adequar às suas re- flexões sobre presença é o próprio ser8 e, para tanto, propõe quatro teses so- bre esse conceito em Heidegger. via, em vez de Dasein, o conceito que Gumbrecht tenta adequar às suas re- flexões sobre presença é o próprio ser8 e, para tanto, propõe quatro teses so- bre esse conceito em Heidegger. A primeira tese é a de que o ser toma o lugar da verdade. Mas isso não quer dizer que o ser simplesmente substitui a verdade, porquanto a verdade é algo que acontece – num movimento de revelação e ocultação. Logo, o ser é o que se revela e se oculta ao mesmo tempo no acontecimento da verdade. O ser não é um sentido, mas “pertence à dimensão das coisas”9 (Gum- brecht, 2010, p. 93). Ele prossegue: “[...] se o Ser tem o caráter de coisa, quer dizer que tem substância e, por isso (ao contrário de algo puramente espiritual), ocupa espaço”10 (Gumbrecht, 2010, p. 93). Aqui, é preciso cau- tela, pois, quando Gumbrecht afirma que o ser tem caráter de coisa, possui uma substância e ocupa espaço, podemos ser levados a crer que se trata de algo fixo e simplesmente dado. Contudo, ao retomar o significado originário de ser como ousía, Hei- degger retira o seu aspecto substancial e instala a noção de vigência/presença (anwesen). Esse “ocupar espaço” deve ser entendido, portanto, como existir junto às coisas do mundo, o que caracteriza o próprio modo de ser do Da- sein, ou seja, o ser pertence à dimensão das coisas. Gumbrecht (2010, p. 94), no entanto, se retifica ao dizer em seguida que esse “ocupar espaço im- plica ainda a possibilidade de o Ser revelar um movimento”11. A segunda tese de Gumbrecht diz respeito justamente ao movimento. E o movimento do ser no espaço se revela multidimensional: vertical, hori- zontal e de retirada. A dimensão vertical estaria associada ao movimento do ser em simplesmente estar ali, enquanto a horizontal apontaria para o ser como estando a ser percebido ou oferecendo-se à vista. Por fim, a dimensão de retirada refere-se ao fato de que as coisas que aparecem deixam de ter o aspecto de objeto (Gumbrecht, 2010, p. 94-95). A presença de Heidegger Em O campo não-hermenêutico ou a materialidade da comunicação, ao definir as premissas do campo hermenêutico, Gumbrecht (1998) sintetiza- as em quatro: 1) o sentido tem sua origem no sujeito, e não como qualidade inerente aos objetos; 2) tem-se como parti pris uma distinção radical entre corpo e espírito (tradição cartesiana); 3) o espírito conduz ao sentido; 4) o corpo é um mero instrumento que oculta o sentido. Há, portanto, uma ar- ticulação entre metafísica e hermenêutica, uma vez que, para Gumbrecht, é o cartesianismo que funda a metafísica moderna e serve como base para as ciências do espírito. Assim, para combater a metafísica, recorre a Heidegger, cujo principal alvo é Descartes: “[...] por isso, Ser e tempo apresenta como pecados origi- nais da filosofia moderna o fundamento cartesiano da existência humana no pensamento (e só no pensamento) e as subsequentes dissociações entre a existência humana e o espaço entre a existência humana e a substância”7 (Gumbrecht, 2010, p. 91). O conceito-chave de Heidegger é o Dasein, porque pressupõe, ao con- trário do husserlianismo – que, para superar o cartesianismo, acopla sujeito e objeto, mas suspendendo a existência fática que se realiza no mundo –, que o homem, o ente que nós somos, está desde sempre junto às coisas do mundo: assim, recupera o aspecto de presença em relação às coisas. Toda- 5 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca do dentro do sistema de sentido da cultura, oferecendo-se à percepção e à representação, recaindo na metafísica. Por isso, esse movimento de retirada (desvelamento) revela o ser antes de fazer parte do mundo, ou seja, o ser en- quanto ser: “o Ser só será Ser fora das redes da semântica e de outras distin- ções culturais”13 (Gumbrecht, 2010, p. 96). A terceira tese é a de que, não podendo o Dasein ser definido na cate- goria de sujeito ou subjetividade e sendo o mundo com que está necessari- amente em contato já sempre interpretado, é necessário, para o aconteci- mento da verdade, deixar que as coisas simplesmente aconteçam: para Gumbrecht, essa disposição está associada à serenidade (Gelassenheit). O despertar da serenidade, diz Heidegger (2001, p. 34-35), não parte de nós mesmos, não é provocado, mas sim permitido. Não quer dizer, no entanto, que seja fruto do acaso, uma vez que surge do pensamento ininter- rupto – a despeito de não ter uma finalidade instrumental, como o pensa- mento do cálculo; está, portanto, entre a passividade e a atividade. A sereni- dade para com as coisas (die Gelassenheit zu den dingen) é o “libertar-se do representar transcendental e, assim, um prescindir do querer do horizon- te”14 (Heidegger, 2001, p. 57). Dessa maneira, a serenidade pensa as coisas do mundo em si mesmas antes de qualquer interpretação. A quarta e última tese se refere à obra de arte como lugar privilegiado para o acontecer da verdade. É claro que a obra de arte não é o único espaço em que a revelação do ser acontece, tanto que, em Ser e tempo, Heidegger confere à fala (logos) estatuto especial nesse movimento de encobrir e revelar do ser; porém, a obra de arte seria outra possibilidade. Comentando sobre os conceitos de “mundo” e “terra”, Gumbrecht re- toma a análise de Heidegger sobre um antigo templo grego, que não repre- sentaria nada. Essa retirada constitui o próprio desvelamento da verdade, em que o vertical e o horizontal concor- rem em direções opostas, em movimento centrífugo, a fim de revelar o ser. De acordo com Gumbrecht (2010, p. 95), “[...] o Ser se refere às coi- sas do mundo antes de elas se tornarem parte de uma cultura (ou, para usar a figura retórica do paradoxo, o conceito refere-se às coisas do mundo antes de elas fazerem parte de um mundo)”12. Isso significa que, em suas dimen- sões vertical e horizontal, isto é, estando ali e à vista, o ser pode ser entendi- 6 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Assim, a terra seria a condição de possibilidade para a instalação de um mundo, sendo na interação entre um e outro que pode acontecer a verdade. Quando, no parágrafo 86, Heidegger (2010, p. 117-119) comenta sobre uma pedra, afirma que “ela apenas se mostra quando permanece desvelada e sem esclarecimento”. Esse “mostrar-se”, ainda sem uma explicação criada pela cadeia semântica do mundo, diz respeito à terra, em sua verdade que a revela tal como é. Gumbrecht (2010, p. 100) esclarece: “[...] só a presença de certas coisas [...] abre a possibilidade de aparecerem outras coisas nas suas primitivas qualidades materiais – e esse efeito pode ser visto como uma das maneiras (e como parte) da revelação do seu Ser”15. Enfim, Gumbrecht, a fim de ajustar o conceito heideggeriano de ser à sua reflexão sobre a presença, faz com que as suas quatro teses derivem de uma premissa que se baseia na ideia de que o ser revelado possui o caráter de coisa. Além disso, em sua discussão sobre a obra de arte, o ser guarda se- melhanças com a terra (não entendida como uma coisa estática, mas sim como o surgir e desabrochar). A interpretação de Gumbrecht dá primazia à serenidade como a capacidade de ver as coisas antes de elas integrarem um sistema interpretativo. Serenidade diz respeito, então, à terra, às “coisas en- tendidas independentemente das suas situações culturais específicas”16 (Gumbrecht, 2010, p. 102), em detrimento do mundo, que seria, inversa- mente, as configurações das coisas dentro dessas situações culturais. Em outras palavras, o ser são as coisas do mundo antes de elas se tor- narem mundo, antes de entrarem num sistema de determinações culturais. Mas, por ter caráter de coisa, o ser corresponderia às coisas tangíveis. Julgo que o problema da leitura gumbrechtiana de Heidegger reside em conside- rar: 1) o ser possui caráter de coisa (ding); 2) o ser são as coisas tangíveis; 3) o ser possui substância. Afinal, quando Heidegger empreende a tarefa de chegar ao ser originário da obra de arte a partir da realidade vigente da obra, visando a buscar a coisidade da coisa (Dinghafte des dinges), isto é, a realida- de palpável da obra, ele conclui que a base coisal não pertence ao ser da obra: “[...] erramos o caminho enquanto supusemos que a realidade vigente da obra, em princípio, estaria nessa base coisal” (Heidegger, 2010, p. 93). Vale dizer que, se em Ser e tempo a polarização era feita entre Dasein e mundo, no Heidegger tardio, a partir da virada (die kehre), a dis- puta entre abertura e velamento se estabelece na oposição entre mundo e terra: o mundo seria a atividade humana, os produtos humanos, tendo se estabelecido pelo uso e pela domesticação da terra, enquanto esta seria a ár- vore, a pedra, a grama, a água, os animais se apresentando tais como são: “este surgir e desabrochar em-si e no todo, os gregos denominaram, há mui- to tempo, a physis” (Heidegger, 2010, p. 104). Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca 7 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Em suma, Gumbrecht retoma os conceitos de Heidegger porque supostamente lhe oferecem o arcabouço conceitual contra a visão de mundo metafísica, entendida, grosso modo, como “algo que se encontra além do me- ramente físico”18 (Gumbrecht, 2012, p. 63). O fato é que o ser pertence, sim, à dimensão das coisas, na medida em que as coisas é que permitem o desvelamento da verdade. Porém, isso não significa que possua “caráter de coisa”: isso seria não levar em conta a dife- rença ontológica entre ser e ente. Afinal, o ser só se revela enquanto existên- 8 ç g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Q p Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. E-ISSN 2237-2660 cia, e as coisas não existem, elas simplesmente são, estão dadas – muito em- bora Heidegger assinale que a verdade deva ser sempre arrancada, em pri- meiro lugar, dos entes. E substância implica estase, ao passo que o ser en- quanto ousía, ou anwesen, é presença, vigência, duração. De qualquer forma, a despeito dessas imprecisões, mais adiante, Gum- brecht (2010, p. 104) faz um reparo: apesar do caráter de coisa, o ser tem movimento, tornando “impossível pensar no Ser como algo estável”17. Nes- se sentido, o conceito heideggeriano de ser e o conceito gumbrechtiano de presença guardam afinidades: ambos só são entendidos fora da rede semân- tica do mundo, ambos têm movimento, ambos estão num espaço, ambos são vigência e duração. Ser como presença: anwesen. Assim como Gumbrecht baseia o seu conceito de produção de presen- ça em sua etimologia, ou seja, a tangibilidade do que está à frente em mo- vimento permanente, Heidegger (2010, p. 211) vale-se do sentido grego de thesis como presencializar-se em seu aparecer: “O grego ‘pôr’ se refere a situ- ar como deixar surgir, por exemplo, uma estátua; refere-se a colocar, deposi- tar uma oferenda sagrada. Situar e colocar têm o sentido de trazer para aqui – no desvelado, para diante – na presença, isto quer dizer, deixar-ficar- presente”. Em suma, Gumbrecht retoma os conceitos de Heidegger porque supostamente lhe oferecem o arcabouço conceitual contra a visão de mundo metafísica, entendida, grosso modo, como “algo que se encontra além do me- ramente físico”18 (Gumbrecht, 2012, p. 63). Assim como Gumbrecht baseia o seu conceito de produção de presen- ça em sua etimologia, ou seja, a tangibilidade do que está à frente em mo- vimento permanente, Heidegger (2010, p. 211) vale-se do sentido grego de thesis como presencializar-se em seu aparecer: “O grego ‘pôr’ se refere a situ- ar como deixar surgir, por exemplo, uma estátua; refere-se a colocar, deposi- tar uma oferenda sagrada. Situar e colocar têm o sentido de trazer para aqui – no desvelado, para diante – na presença, isto quer dizer, deixar-ficar- presente”. Em suma, Gumbrecht retoma os conceitos de Heidegger porque supostamente lhe oferecem o arcabouço conceitual contra a visão de mundo metafísica, entendida, grosso modo, como “algo que se encontra além do me- ramente físico”18 (Gumbrecht, 2012, p. 63). – no desvelado, para diante – na presença, isto quer dizer, deixar-ficar- presente”. g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev Bras Estud Presença, Porto Alegre, v 13, n 2, e126100, 2023 Disponível em: http://seer.ufrgs.br/presenca tente’ daquilo que tento colocar em questão sob o título de pensamento da presença”19 (Derrida, 2001a, p. 62). As premissas teóricas de Gumbrecht se baseiam, portanto, na hermenêutica e na metafísica, justamente o que a sua teoria visa combater. No capítulo de Produção de presença em que trata de Heidegger, a fim de relacionar os conceitos de ser e de presença, Gumbrecht (2010, p. 75) faz uma provocação a Derrida ao citar um trecho de Gramato- logia no qual o filósofo escreve que a “era do signo”, tratada como sinônimo de metafísica, talvez nunca venha a ter um fim, embora o seu encerramento histórico esteja traçado. Gumbrecht (2010, p. 77) prossegue: “Derrida nunca se acanhou em inventar novos conceitos, mesmo quando a necessidade de fazê-lo não era muito evidente. Por que estará tão relutante em sugerir um novo conceito que nos permita ‘acabar’ com a era do signo?”20. Ora, a acusação sobre a impertinência da criação de conceitos recai em uma crítica do tipo “Sokal hoax”, que se notabilizou por fazer troça dos jargões pós-estruturalistas. To- davia, não se trata de defender Derrida de Gumbrecht: não é o objetivo des- te artigo. O meu propósito é demonstrar, por meio da filosofia derridiana, em que medida a teoria gumbrechtiana, mediada por Heidegger, reafirma os mesmos pressupostos contra os quais se coloca. De qualquer forma, a despeito da alegação de que Derrida se acanha em acabar com a “era do signo”, para Derrida, só é possível desconstruir a metafísica jogando com seus próprios termos: “é com a ajuda do conceito de signo que se abala a metafísica da presença”21 (Derrida, 1971, p. 233). Nesse sentido, seria não apenas “mau gosto intelectual”, como presume Gumbrecht, mas também quase uma contradição performativa. No ensaio A estrutura, o signo e o jogo no discurso das ciências humanas, Derrida afirma isso com clareza: [...] não tem nenhum sentido abandonar os conceitos da metafísica para aba- lar a metafísica; não dispomos de nenhuma linguagem – de nenhuma sinta- xe e de nenhum léxico – que seja estranho a essa história; não podemos enunciar nenhuma proposição destruidora que não se tenha já visto obriga- da a escorregar para a forma, para a lógica e para as postulações implícitas daquilo mesmo que gostaria de contestar22 (Derrida, 1971, p. 233). Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Derrida e a crítica à metafísica da presença Quando Gumbrecht diz ter mudado de ideia sobre um filósofo cuja obra era tida como moralmente suspeita e a quem dizia não ser nem um pouco tributário, não é, a meu ver, propriamente um problema. O proble- ma reside em retomar um autor cujo método, a hermenêutica (hermenêuti- ca da facticidade, mais precisamente), era antes deplorado, isso porque a sua teoria da produção de presença incorre numa espécie de “realismo ingênuo” e, sobretudo, contribui para a reafirmação do fundamento em que se baseia o próprio campo hermenêutico: a metafísica. Mais do que isso: como bem demonstrou Derrida, a obra de Heide- gger é a apoteose da metafísica ocidental: “[...] tenho por vezes o sentimento de que a problemática heideggeriana é a defesa mais ‘profunda’ e mais ‘po- 9 9 ç g Disponível em: http://seer.ufrgs.br/presenca g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Isso porque pretender abalar a metafísica a partir de um “fora” da me- tafísica significa reivindicar um lugar que a ela seja anterior: um fundamen- to, uma origem fixa, uma arché, um centro que comanda o “dentro”. Os 10 Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Q Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. g Disponível em: http://seer.ufrgs.br/presenca conceitos substancialistas a que Gumbrecht (2010, p. 77) recorre, como “presença”, “realidade” e “ser” – em vez de serem, como ele pretende, possi- bilidades de se referir ao mundo sem ser pelo sentido –, são, na verdade, o que permite falar em sentido (pois é a origem que dá o sentido). Tais con- ceitos caracterizam-se como o centro, porque é o princípio primeiro que rege o jogo de substituição dos conteúdos e dos termos que dele são derivados, visto que, como afirma Derrida (1971, p. 230), “no centro, é proibida a permuta ou a transformação dos elementos”23. Essa imobilidade fundadora que constitui o centro não se encontra, no entanto, no meio do jogo e não é propriamente um centro; antes, está si- multaneamente dentro e fora, é a origem e o fim, ainda que pense a si mes- ma como presença plena e fora do jogo. Assim, quando Gumbrecht assume o conceito de presença ou de realidade anteriores à metafísica, parece esque- cer que esta se caracteriza justamente como aquilo que arroga a posição de anterioridade a todas as coisas, determinando-as. Ou seja, a mera suposição de que há um mundo físico e um mundo não físico e de que aquele precede este é em si metafísica, porquanto está subentendida na própria noção de metafísica a existência de algo físico e de algo não físico, assim como a ideia de presença traz consigo o seu par antitético, a ausência. Só é possível falar em presença porque está previsto tudo aquilo que a presença não é. Explico: a presença só é presença porque não é ausência, mas, para determinar o que é a presença, é necessário estabelecer o que a presença não é. E assim é com todas as oposições binárias: homem e mu- lher, humano e animal, fala e escrita, verdadeiro e falso, dentro e fora, entre outras. A metafísica é o que permite esse jogo de diferenças binárias e, acima de tudo, hierárquicas. Como lembra Derrida (1971, p. 231), “[...] a história da metafísica, como a história do Ocidente, seria a história dessas metáforas e dessas metonímias”24: os sucessivos nomes e formas que o centro – o fora do jogo – recebe no jogo de diferenças. Derrida (1971, p. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev Bras Estud Presença Porto Alegre v 13 n 2 e126100 2023 tos, além do fato de que Derrida lhe dedicou alguns ensaios, bem como o livro Do espírito, Heidegger e a questão, de 1987. Em entrevista concedida a Henri Ronse, que integra o volume de Posições, publicado pela primeira vez em 1972, Derrida assume-se tributário do filósofo alemão, tendo em vista as proximidades entre a destruktion heideggeriana e o seu projeto descons- trucionista: tos, além do fato de que Derrida lhe dedicou alguns ensaios, bem como o livro Do espírito, Heidegger e a questão, de 1987. Em entrevista concedida a Henri Ronse, que integra o volume de Posições, publicado pela primeira vez em 1972, Derrida assume-se tributário do filósofo alemão, tendo em vista as proximidades entre a destruktion heideggeriana e o seu projeto descons- trucionista: Nada do que eu tento fazer seria possível sem a abertura das questões heide- ggerianas. E, antes de tudo, pois aqui nós devemos dizer as coisas muito ra- pidamente, sem atenção àquilo que Heidegger chama de diferença entre o ser e o ente, a diferença ôntico-ontológica tal qual ela permanece, de uma certa maneira, impensada pela filosofia. Mas, apesar dessa dívida para com o pensamento heideggeriano ou, melhor, em razão dessa dívida, tento reco- nhecer no texto heideggeriano – que, como qualquer outro, não é homogê- neo, contínuo, igual, em cada uma de suas partes, à força global e a todas as consequências de suas questões – sinais de pertencimento à metafísica ou àquilo que ele chama de ‘onto-teologia’26 (Derrida, 2001a, p. 16). Nada do que eu tento fazer seria possível sem a abertura das questões heide- ggerianas. E, antes de tudo, pois aqui nós devemos dizer as coisas muito ra- pidamente, sem atenção àquilo que Heidegger chama de diferença entre o ser e o ente, a diferença ôntico-ontológica tal qual ela permanece, de uma certa maneira, impensada pela filosofia. Mas, apesar dessa dívida para com o pensamento heideggeriano ou, melhor, em razão dessa dívida, tento reco- nhecer no texto heideggeriano – que, como qualquer outro, não é homogê- neo, contínuo, igual, em cada uma de suas partes, à força global e a todas as consequências de suas questões – sinais de pertencimento à metafísica ou àquilo que ele chama de ‘onto-teologia’26 (Derrida, 2001a, p. 16). 231) prossegue: Poder-se-ia mostrar que todos os nomes do fundamento, do princípio, ou do centro, sempre designaram a invariante de uma presença (eidos, arquê, te- los, energeia, ousia (essência, existência, substância, sujeito) aletheia, trans- cendentalidade, consciência, Deus, homem etc.)25. Poder-se-ia mostrar que todos os nomes do fundamento, do princípio, ou do centro, sempre designaram a invariante de uma presença (eidos, arquê, te- los, energeia, ousia (essência, existência, substância, sujeito) aletheia, trans- cendentalidade, consciência, Deus, homem etc.)25. Cumpre observar que Derrida se refere a alguns conceitos constantes na filosofia de Heidegger: “ser como presença”, ousía e aletheia. A menção não é gratuita, uma vez que Heidegger é uma figura frequente em seus tex- 11 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. E-ISSN 2237-2660 sua gramatologia. Como assinala Derrida no ensaio A diferença, ao comen- tar o referido trecho de O dito de Anaximandro, para Heidegger, a metafísica esqueceu-se que tinha esquecido da diferença entre ser e ente, apagando o próprio rastro da diferença. Assim, “[...] se admitimos que a diferança [diffé- rance] (é) (ela mesma) diversa da ausência e da presença, se ela é rastro, seria necessário falar aqui, tratando-se do esquecimento da diferença (entre o ser e o ente), de uma desaparição do rastro do rastro”30 (Derrida, 1991, p. 57- 58). Mas o rastro, importa frisar, não possui um próprio, uma propriedade, uma quididade; – do rastro, não se pergunta o que é; – a contrario, o ser, pa- ra Heidegger, por mais que seja uma vigência/presença (anwesenheit), e não uma substância, ainda possuiria algo que permita compreendê-lo enquanto tal, na medida em que seria necessário diferenciá-lo do ente, isto é, o ser não é o ente. Em Gramatologia, Derrida (2013, p. 25) afirma que: A palavra ‘ser’ ou, em todo caso, as palavras que designam nas diferentes lín- guas o sentido do ser, seria com algumas outras, uma ‘palavra originária’ (Urwort), a palavra transcendental que assegura a possibilidade do ser- palavra a todas as outras palavras. Seria pré-compreendida em toda lingua- gem enquanto tal e – esta é a abertura de Sein und Zeit – apenas esta pré- compreensão permitiria abrir a questão do sentido do ser em geral, para além de todas as ontologias regionais e de toda a metafísica: questão que en- ceta a filosofia [...] e se deixa recobrir por ela, questão que Heidegger repete ao lhe submeter a história da metafísica. Não há dúvida de que o sentido do ser não é a palavra ‘ser’ nem o conceito de ser – Heidegger lembra-o sem cessar. Mas, como este sentido não é nada fora da linguagem e da linguagem de palavras, liga-se, senão a tal ou qual palavra, a tal ou qual sistema de lín- guas (concesso non doto), pelo menos à possibilidade da palavra em geral31 (Derrida, 2013, p. 25). Trata-se, portanto, não apenas de uma pré-compreensão ontológica, como o quer Heidegger, mas também de uma compreensão prévia do que seja o ser na linguagem, isto é, no sistema que permite enunciá-lo e formulá- lo enquanto questão: “[...] a questão do ser se une indissoluvelmente, sem lhe reduzir, à pré-compreensão da palavra ser”32 (Derrida, 2013, p. 26). Esses sinais de pertencimento à metafísica podem ser notados tanto nas oposições binárias que operam no texto heideggeriano – ser e ente, au- têntico e inautêntico, verdade como adequação e verdade como desvela- mento, essência e vigência, entre outras – quanto na profunda hierarquiza- ção que subjaz no fato de que o esclarecimento do sentido do ser seria não apenas o propósito mais autêntico da ontologia, mas também a questão que deveria ser primeiramente respondida. Quando Derrida diz que nada do que faz seria possível sem a abertura das questões heideggerianas, sobretudo da diferença ontológica, é importan- te lembrar que, em O dito de Anaximandro, após retomar um dos principais motivos de Ser e tempo, a saber, o de que o esquecimento do ser é o esqueci- mento da diferença entre ser e ente, Heidegger (2002, p. 430) afirma que “[...] a diferença desaparece. Permanece esquecida. Só as coisas que são dife- renciadas, o que está presente e o estar-presente, é que se desvelam – mas não enquanto coisas diferenciadas. Em vez disso, também o vestígio inicial da diferença é apagado”27. Mais adiante: “[...] a clareira da diferença não pode, por isso, significar também que a diferença apareça como diferença”28 (Heidegger, 2002, p. 431). É precisamente ao negar que a diferença apareça enquanto diferença que Heidegger apresenta sinais de pertença à metafísica; e é ao tratar a dife- rença como diferença, ou seja, como différance29, que Derrida desenvolve 12 ç g Disponível em: http://seer.ufrgs.br/presenca Q Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev Bras Estud Presença Porto Alegre v 13 n 2 e126100 2023 E-ISSN 2237-2660 entre a ‘voz do ser’ e a phoné, entre o ‘apelo do ser’ e o som articulado; uma tal ruptura que, ao mesmo tempo confirma uma metáfora fundamental e lança a suspeição sobre ela ao acusar a defasagem metafórica, traduz bem a ambiguidade heideggeriana com respeito à metafísica da presença e ao logo- fonocentrismo. Ela ao mesmo tempo compreendida nestes e os transgride33 (Derrida, 2013, p. 27). Não se trata, no entanto, de dizer que Heidegger clama por um signi- ficado original que se acople ao significante “ser”, quer dizer, um significado puramente imaterial/transcendental. Isso seria de uma incompreensão infi- nita face a uma filosofia extremamente sofisticada, pois o sentido do ser nunca é apenas um significado: “o ser escapa ao movimento do signo”34 (Derrida, 2013, p. 27). O gesto metafísico de Heidegger consiste, mais que na busca pelo no- me único35, na determinação do sentido do ser como presença. O fato é que há algo ainda mais originário que o ser – a despeito de não ser uma origem, um fundamento, e resistir à pergunta “o que é?”: a différance. De acordo com Derrida (1991, p. 56), “uma vez que o ser jamais teve ‘sentido’, jamais foi pensado ou dito senão dissimulando-se no ente, a diferança [différance], de uma certa e muito estranha maneira, (é) mais ‘velha’ do que a diferença ontológica ou que a verdade do ser. É a essa idade que se pode chamar jogo do rastro”36. O fato é que o rastro nunca se apresenta como tal, não possui um “pró- prio”, ele “[...] apaga-se apresentando-se, silencia-se ressoando, como o a es- crevendo-se, inscrevendo a sua pirâmide na diferança [différance]”37 (Derrida, 1991, p. 57); ele “[...] não é mais ideal que real, não mais inteligível que sen- sível, não mais uma significação transparente que uma energia opaca e ne- nhum conceito da metafísica pode descrevê-lo”38 (Derrida, 2013, p. 80). Em suma, “[...] o rastro é verdadeiramente a origem absoluta do sentido em geral. O que vem afirmar mais uma vez que não há origem absoluta do sentido em ge- ral”39 (Derrida, 2013, p. 79-80). Assim, quando Heidegger impõe como tarefa à sua ontologia funda- mental a questão sobre o ser originário, está, na verdade, perseguindo o ras- tro da diferença entre ser e ente, e não o ser. E-ISSN 2237-2660 Com efeito, é o sentido originário da palavra ser que Heidegger intenta res- gatar do esquecimento por meio do desvelamento, a aletheia: É assim que, depois de evocar a ‘voz do ser’, Heidegger lembra que ela é si- lenciosa, muda, insonora, sem palavra, originariamente á-fona (die Gewähr der lautlosen Stimme verboneger Quellen. . .). Não se ouve a voz das fontes. Ruptura entre o sentido originário do ser e a palavra, entre o sentido e a voz, É assim que, depois de evocar a ‘voz do ser’, Heidegger lembra que ela é si- lenciosa, muda, insonora, sem palavra, originariamente á-fona (die Gewähr der lautlosen Stimme verboneger Quellen. . .). Não se ouve a voz das fontes. Ruptura entre o sentido originário do ser e a palavra, entre o sentido e a voz, 13 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Mais ‘velha’ que o próprio ser, uma tal diferança [différance] não tem ne- nhum nome na nossa língua. Mas ‘sabemos já’ que se ela é inominável, não é por provisão, porque a nossa língua não encontrou ainda ou não recebeu este nome, ou porque seria necessário procurá-lo numa outra língua, fora do sistema finito da nossa. É porque não há nome para isso, nem mesmo o de essência ou de ser, nem mesmo o de ‘diferança’ [différance], que não é um nome, que não é uma unidade nominal pura e se desloca sem cessar numa cadeia de substituições diferantes40 (Derrida, 1991, p. 61-62). Se a différance é o próprio jogo que cria as estruturas a que chamamos nomes, sendo o efeito nominal (não se pode falar de nome ou de conceito neste caso) da différance também trazido para dentro do jogo, as palavras “ser”, “essência” e “origem” acabam por se tornar apenas efeitos da différan- ce. Assim, a différance nem mesmo é um nome, “nome único, nem que seja o nome do ser”41 (Derrida, 1991, p. 62). Ou seja, embora Heidegger olhe para o rastro da différance e lhe nomeie ser, o jogo da différance não pode ser nomeado: “[...] o que se escreve diferença [différance] será, portanto, o mo- vimento de jogo que ‘produz’, por meio do que não é simplesmente uma atividade, estas diferenças, estes efeitos de diferença”42 (Derrida, 1991, p. 43). Observa-se que Derrida não fala de efeitos de presença, mas sim de efeitos de diferença; isso porque o verbo diferir tem dois sentidos: de adiar e de discernir. Tendo em vista que o quasi-conceito de différance inscreve-se numa economia da escritura, Derrida utiliza-o na desconstrução do signo e da metafísica da presença. Correntemente, o signo é aquilo que atua vicari- amente, isto é, no lugar da “coisa”, do referente, representando-o em sua ausência: “o signo seria então a presença diferida”43 (Derrida, 1991, p. 40). O signo, portanto, assumiria um caráter provisório e suplementar em rela- ção a essa presença original, estabelecendo uma mediação. E, se o rastro não possui um “próprio”, é o que se apaga ao mostrar-se, não é inteligível nem sensível, is- so significa que não pode ser nomeado: o rastro não tem nome próprio (uma identidade, um próprio). Diz Derrida (1991, p. 61-62): 14 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença différance é o movimento no qual o significado é sempre adiado, nunca pre- sente: A diferança [différance] é o que faz com que o movimento da significação não seja possível a não ser que cada elemento dito ‘presente’, que aparece sobre a cena da presença, se relacione com outra coisa que não ele mesmo, guardando em si a marca do elemento passado e deixando-se já moldar pela marca da sua relação com o elemento futuro, relacionando-se o rastro menos com aquilo a que se chama presente do que àquilo a que se chama passado e constituindo aquilo a que chamamos presente por intermédio dessa relação mesma com o que não é ele próprio: absolutamente não ele próprio, ou seja, nem mesmo um passado ou um futuro como presentes modificados44 (Der- rida, 1991, p. 45). A diferança [différance] é o que faz com que o movimento da significação não seja possível a não ser que cada elemento dito ‘presente’, que aparece sobre a cena da presença, se relacione com outra coisa que não ele mesmo, guardando em si a marca do elemento passado e deixando-se já moldar pela marca da sua relação com o elemento futuro, relacionando-se o rastro menos com aquilo a que se chama presente do que àquilo a que se chama passado e constituindo aquilo a que chamamos presente por intermédio dessa relação mesma com o que não é ele próprio: absolutamente não ele próprio, ou seja, nem mesmo um passado ou um futuro como presentes modificados44 (Der- rida, 1991, p. 45). Isso significa que a différance, ao mesmo tempo que adia o significado, produz diferença entre os outros signos. Não faz mais sentido em falar de signo, já que o signo, na definição agostiniana retomada por Barthes (2007) em Elementos de semiologia, estabelece uma relatio entre dois relata, quais se- jam, o significante e o significado – já que o signo foi sempre compreendido como signo-de: ligando um significante a um significado. Mas, retirado o significado, o campo e o jogo da significação são ampliados indefinidamen- te (Derrida, 1971). Na gramatologia derridiana, “[...] o significante significa de início um significante, e não a coisa mesma nem um significado diretamente apresen- tado”45 (Derrida, 2013, p. 289). Ou melhor, um significante remete sempre a outro significante; o rastro remete a outro rastro. Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença g Disponível em: http://seer.ufrgs.br/presenca Todavia, remetendo a Saussure e às noções de arbitrariedade do signo e de valor diferencial, que são solidárias, Derrida retira as seguintes implica- ções: uma vez que o significado de um signo é sempre arbitrário – ou seja, não há uma relação natural entre significante e significado –, além de nunca presente em si mesmo, dependendo das relações com os outros significados, a materialidade importa menos do que aquilo que está no seu entorno. Se para Saussure é o jogo de diferenças que produz significados, para Derrida a Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. 15 E-ISSN 2237-2660 différance é o movimento no qual o significado é sempre adiado, nunca pre- sente: Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev Bras Estud Presença Porto Alegre v 13 n 2 e126100 2023 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. E-ISSN 2237-2660 de se referir às coisas do mundo, como se o texto estivesse preso numa clau- sura que o isola do mundo exterior, ele parece ignorar a “exterioridade do significante”. A não ser que Gumbrecht entendesse a exterioridade do signi- ficante em oposição a uma suposta internalidade do significado, levando em conta apenas o princípio da arbitrariedade do signo. Decorre daí que, em função de sua crítica à hermenêutica, a qual consistiria em procurar o signi- ficado imaterial em detrimento da superfície material, Gumbrecht promove simplesmente uma inversão, ou seja, dá primazia ao aspecto material; po- rém, a metafísica, segundo Derrida, opera justamente nas oposições biná- rias. Em todo caso, quando Derrida fala sobre a exterioridade do significan- te, está dizendo que a exterioridade é a conditio sine qua non da escritura, pois “[...] não há signo linguístico antes da escritura. Sem esta exterioridade, a própria ideia de signo arruína-se”47 (Derrida, 2013, p. 17). Isso não signi- fica, no entanto, o predomínio do “fora” sobre o “dentro”, ou seja, do su- porte material sobre o sistema interno da língua; na verdade, o movimento de inscrição no suporte traz consigo simultaneamente o jogo da différance. Afinal, se a différance mobiliza no elemento dito presente todas as coisas que não ele mesmo, trazendo o rastro do passado e o do futuro, tal jogo se refere também à imanência da inscrição. Ou melhor: quando escrevo determinado texto, cada traço material (letra, espaçamento, sílabas, palavras etc.) com- porta, em sua “presença”, todos os outros elementos não presentes no mo- mento mesmo da escrita. De acordo com Derrida (2013, p. 56), “[...] antes mesmo de ser ligado à incisão, à gravura, ao desenho ou à letra, a um significante remetendo, em geral, a um significante por ele significado, o conceito de grafia implica, como a possibilidade comum a todos os sistemas de significação, a instância do rastro instituído”48. A instituição do rastro diz respeito à imprensão [em- preinte] na passagem para a forma; por isso, Derrida (2013, p. 77) diz que a différance é a formação da forma: “mas ela é, por outro lado, o ser impresso da imprensão”49. O termo imprensão guarda o sentido de inscrição como corte, traçado, incisão sobre uma superfície. Em outras palavras: ten- do em vista que não há mais um significado estável e fixo – o centro, a pre- sença plena –, o signo que ocuparia o lugar do centro, que supriria a sua au- sência, é substituído por outro signo, outro suplemento. De qualquer for- ma, a presença (o significado, a coisa representada etc.) nunca esteve presen- te no texto: ela é tão somente o rastro de outro rastro. É considerando a noção de rastro que devemos entender a célebre fra- se, tantas vezes incompreendida, de que “[n]ão há fora-de-texto [Il n’y a pas de hors-texte]”46 (Derrida, 2013, p. 194). O texto não se refere a nada que não a ele próprio, ele não avança em direção ao referente ou a um significa- do transcendental. Assim, quando Gumbrecht ironiza o desconstrucionismo, tachando-o de “existencialismo linguístico” que lamenta a incapacidade da linguagem 16 ç g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Q Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. g Disponível em: http://seer.ufrgs.br/presenca Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca ás, os brancos de Mallarmé têm afinidade com a noção de espaçamento de Derrida, pois o espaçamento se refere à pausa, ao branco, à pontuação etc. No domínio do logocentrismo, a omissão da exterioridade do significante foi desde sempre a omissão do espaçamento, da imprensão, do rastro en- quanto rastro. Derrida (2013, p. 79) prossegue: [...] é na zona específica desta imprensão e deste rastro, na temporalidade de um vivido que não é nem no mundo nem num ‘outro mundo’, que não é mais sonoro que luminoso, não mais no tempo que no espaço, que as dife- renças aparecem entre os elementos ou, melhor, produzem-nos, fazem-nos surgir como tais e constituem textos, cadeias e sistemas de rastros. Estas ca- deias e estes sistemas podem-se desenhar somente no tecido deste rastro ou imprensão50. O rastro, portanto, não está no mundo, nem em outro mundo; não es- tá dentro da língua, nem fora dela, não é material, nem imaterial – numa dupla exclusão –; por outro lado, está simultaneamente no mundo e no ou- tro mundo, na língua e fora dela, sendo material e imaterial – numa dupla participação. Depreende-se disso que, assim como não há um fora-de-texto, não há também um dentro-de-texto, pois o movimento da inscrição é o mesmo que produz as diferenças que possibilitam a significação. Isso não significa que haja uma presença irredutível do inscrito, na materialidade do suporte, pois o grafema nunca possui um próprio, uma identidade estável; é a alteridade – aquilo que ele não é – que permite pensá-lo enquanto tal. E essa alteridade exclui a presença plena, chamemo-la de “coisas do mundo” ou de real – até porque o real é apenas um “efeito” da différance, é rastro de outro rastro. Para Derrida (2013, p. 194-195), “[...] nunca houve senão a escritura; nunca houve senão suplementos, significações substitutivas que só puderam surgir numa cadeia de remessas diferenciais, o ‘real’ só sobrevindo, só acrescentando-se ao adquirir sentido a partir de um rastro e de um apelo de suplemento etc.”51 A radicalidade da afirmação de que não há fora-de-texto gera mal- entendidos do tipo: “a dor não é um texto, eu a sinto efetivamente” ou “o real é irredutível ao texto”. A título de ilustração, basta pensarmos no poema Un coup de dés (1897), de Stéphane Mallarmé, porquanto a disposição gráfica das palavras sobre o papel – além da importância dada aos brancos da página – é que di- ta a leitura, ou seja, há uma indiscernibilidade entre o suporte e o texto. Ali- 17 ç g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Gustavo Ramos de Souza Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev Bras Estud Presença, Porto Alegre, v 13, n 2, e126100, 2023 ç g Disponível em: http://seer.ufrgs.br/presenca E-ISSN 2237-2660 E ISSN 2237 2660 (Derrida, 2004, p. 341); 3) a noção de “real” comporta consigo seu par an- titético, quer seja “irreal”, quer seja “ideal”, ou correlatos – ou seja, o real es- taria no domínio da metafísica, pois a estrutura oposicional é, por si só, me- tafísica. Em entrevista a Jean-Louis Houdebine e Guy Scarpetta, de 1971, que integra o volume de Posições, Derrida (2001, p. 72) afirma que “[...] o signi- ficante ‘matéria’ não me parece problemático a não ser no momento em que sua reinscrição não evite que se faça dele um novo princípio fundamental, no momento em que, por uma regressão teórica, ele é reconstituído em ‘significado transcendental’”53. Apesar de ele falar de matéria, poderíamos substitui-la por coisa, realidade, real, presença sensível, referente etc.: o ma- terialismo vulgar promove tão somente uma inversão dos termos metafísicos e os refunda sob nova roupagem. Derrida prossegue: O significado transcendental não é tão-somente o recurso do idealismo no sentido estrito. Ele pode sempre acabar por reafirmar um materialismo me- tafísico. Ele se torna, pois, um referente último, de acordo com a lógica clás- sica implicada por esse valor de referente, ou uma ‘realidade objetiva’ abso- lutamente ‘anterior’ a todo trabalho da marca, um conteúdo semântico ou uma forma de presença que garanta, a partir de seu exterior, o movimento do texto geral54 (Derrida, 2001, p. 72). Ora, o conceito de presença da teoria gumbrechtiana não seria apenas esse momento de inversão da metafísica, isto é, um “materialismo metafísi- co”? Quando Gumbrecht (2010, p. 13-14) fala que a presença não deve ser entendida no sentido temporal, mas sim no espacial, no mundo das coisas e dos objetos, tangível pelas mãos humanas, devendo as “coisas do mundo” ser compreendidas como “uma referência ao desejo dessa imediatez”, não estaria reelaborando a metafísica da presença? A proeminência dada ao su- porte nas “materialidades da comunicação”, em detrimento do sentido (tido como transcendental), não é afim à ideia de que é possível compreen- der/descrever as “coisas mesmas”, a “realidade” etc.? Há, nesse tipo de discussão, ao menos três pro- blemas que não são levados em conta: 1) dor e real são palavras como quaisquer outras: é a linguagem que permite formulá-las; 2) o rastro, como demonstrei longamente, não é pura textualidade; ele só se dá no momento da inscrição: “não há de um lado a linguagem e de outro a realidade”52 18 E-ISSN 2237-2660 (Derrida, 2004, p. 341); 3) a noção de “real” comporta consigo seu par an- titético, quer seja “irreal”, quer seja “ideal”, ou correlatos – ou seja, o real es- taria no domínio da metafísica, pois a estrutura oposicional é, por si só, me- tafísica. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. experiência e permanece na experiência”55 (Husserl, 2006, p. 33). Ou seja, na atitude natural, há a crença sem questionamento de que as coisas e o mundo são tais como se nos apresentam, revelando-se aos nossos cinco sen- tidos. Embora recorra ao ser como presença (anwesenheit) de Heidegger, o conceito de presença de Gumbrecht guarda mais afinidades com a atitude natural do que com a filosofia heideggeriana; afinal, para o filósofo, o ser não é aquilo que está simplesmente dado em sua tangibilidade imediata. Enquanto Gumbrecht (2010, p. 14) destaca o aspecto espacial da presença, afirmando que “[...] uma coisa ‘presente’ deve ser tangível por mãos huma- nas – o que implica, inversamente, que pode ter impacto imediato em cor- pos humanos”56, Heidegger (2012, p. 64) diz: “[...] o ente é entendido em seu ser como ‘vigência’, isto é, a partir de determinado modo do tempo, do atualmente presente”. Assim, em vez da vigência/presença, esse caráter de “tangível por mãos humanas” assemelha-se, senão àquilo que está “diante das mãos” (vorhan- den), ao menos aos instrumentos, à “manualidade” (zuhandenheit), que é quando os entes estão em uso. Como assinala Michael Inwood (2002, p. 113), “[...] vorhandenheit não é o modo primário de ser. O próprio Dasein não é vorhanden, nem o são os artigos de uso ou ‘instrumentos’”57. Contu- do, se admitíssemos com Gumbrecht que o ser como presença pode ser to- mado como a mera tangibilidade do que está à frente, não poderíamos con- tornar o fato de que ainda assim essa presença plena é inacessível. Tudo quanto existe são significantes de significantes, suplementos de suplemen- tos, rastros de rastros, mediação de mediação, véus sob véus, pois a presença é apenas a representação da representação. A despeito da crítica de Derrida à fenomenologia husserliana em A voz e o fenômeno, publicado pela primeira vez em 1967, não se pode negar que a fenomenologia já havia também contestado a suposta objetividade dos mé- todos das ciências empíricas, na medida em que estas assumiriam uma “ati- tude natural”, que se fia na crença de que a experiência é capaz de dar conta da totalidade das coisas do mundo: “o conhecimento natural começa pela 19 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. E-ISSN 2237-2660 vel pela linguagem, ou seja, a única verdade a que temos acesso é a da pró- pria linguagem, sendo injustificada a transposição de uma coisa para esta, como o fazem a lógica e as ciências empíricas. Nietzsche (2007, p. 36) afirma que a verdade nada mais é que “exérci- to de metáforas” e que “[...] a ‘coisa em si’ (ela seria precisamente a pura verdade sem quaisquer consequências) também é, para o criador da lingua- gem, algo totalmente inapreensível e pelo qual nem de longe vale a pena es- forçar-se” (2007, p. 31). Não haveria uma correlação originária entre a me- táfora e coisa representada: mais do que isso, é um erro acreditar que as coi- sas se apresentam a nossa percepção tal como elas são, que temos diante de nós objetos puros, pois as coisas se dissimulam em sua aparência: “[...] vi- vemos somente através de ilusões, sendo que nossa consciência dedilha a su- perfície. Há muita coisa que se esconde diante de nosso olhar” (Nietzsche, 2007, p. 52). O fato é que percebemos somente os efeitos, ainda que não haja uma causa. Não há presença imediata (da coisa, do real etc.), mas apenas relações, mediações: “as propriedades contêm apenas relações” (Nietzsche, 2007, p. 89). Isso significa que os termos que compõem as relações são tão somente relações que se referem a outras relações, não havendo um próprio, um nome único, uma presença. É nesse sentido que devemos compreender o célebre fragmento nietzschiano de A vontade de poder: “[...] contra o positivismo, que fica no fenômeno ‘só há fatos’, eu diria: não, justamente não há fatos, só in- terpretações [Interpretationen]. Não podemos verificar nenhum fato ‘em si’: talvez seja absurdo querer uma tal coisa” (Nietzsche, 2008, p. 260). É a partir dos pressupostos nietzschianos que, no ensaio “Presença na linguagem ou presença contra a linguagem”, Gumbrecht promove o seu “retorno às coisas mesmas”. Ele começa por estabelecer uma oposição entre linguagem e presença, uma vez que a linguagem estaria associada a algo que demanda uma interpretação, a uma atribuição de significados circunscritos às palavras. Ou seja, a linguagem exigiria que se fosse além das palavras ins- critas num suporte material, ultrapassando a superfície física em busca de um significado profundo. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Linguagem e realidade Num texto de 1873 publicado apenas postumamente sob o título So- bre verdade e mentira no sentido extra-moral, Friedrich Nietzsche esboça uma filosofia da linguagem que antecipa muitas das preocupações do chamado pós-estruturalismo (ou filosofia da diferença) e, sobretudo, do desconstruci- onismo derridiano. Contudo, enquanto para Derrida o que chamamos “rea- lidade” seria apenas um jogo de rastros, para Nietzsche as metáforas estão postas de um lado, e a “realidade” de outro. Mas tal realidade é inapreensí- 20 Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. g Disponível em: http://seer.ufrgs.br/presenca Disponível em: http://seer.ufrgs.br/presenca SS 3 660 é identificar de que maneira a presença se insinua no sentido, ou melhor, na linguagem – por isso, a fim de demarcar sua posição teórica, dispara contra o desconstrucionismo derridiano: “[...] encontrei pouco consolo naquilo que gosto de caracterizar como o “existencialismo linguístico” da descons- trução, isto é, os constantes lamento e melancolia (em suas infinitas varia- ções) pela suposta incapacidade da linguagem de se referir aos objetos do mundo”58 (Gumbrecht, 2012, p. 63). Colocando-se contra o paradigma hermenêutico que não cessa de produzir sentido, a despeito da referenciali- dade das coisas do mundo, Gumbrecht parte da premissa de que a presença seria antitética à representação; logo, importa a ele o modo como a presença se insinua na linguagem: isto é, a linguagem fora da representação. Para tan- to, propõe seis tipos de amálgama entre linguagem e presença: 1) a lingua- gem falada é, sobretudo, uma realidade física; 2) a linguagem diz respeito às práticas fundamentais da filologia, sobretudo na função original de curado- ria de textos (desejo de literalmente incorporar textos); 3) qualquer tipo de linguagem é capaz de causar uma experiência estética; 4) a relação entre ex- periência mística e a linguagem do misticismo: “[...] a linguagem mística produz o efeito paradoxal de estimular imaginações que parecem tornar palpável”59 a presença do divino, do sobrenatural (Gumbrecht, 2012, p. 69); 5) a abertura da linguagem ao mundo dos objetos: em vez do paradig- ma da representação, entra em causa a atitude dêitica que usa a linguagem para apontar os objetos, não visando representá-los; 6) a literatura pode ser o lugar da epifania: “[...] na terminologia teológica, o conceito de epifania refere-se à aparição de um objeto, de um objeto que requer espaço, um ob- jeto que está ausente ou presente”60 (Gumbrecht, 2012, p. 70). Em suma, para Gumbrecht, esses tipos de amálgama diferem do paradigma hermenêu- tico da expressão – de um significado espiritual – e instalam a compreensão da linguagem como “morada do ser” ou da presença, porquanto a lingua- gem teria o mesmo status ontológico dos objetos a que remetem. Contrariamente, a hipótese que defendo é que, devido ao fato de partir da noção de que existe uma presença originária, visto que se baseia em Hei- degger, Gumbrecht desconsidera o jogo da différance e reafirma pressupostos metafísicos. Desse modo, linguagem, interpretação e metafísica seriam postas no campo das modernas culturas de sentido, enquanto coisa, ser e matéria in- tegrariam, opostamente, o campo das culturas de presença. A sua pretensão 21 ç g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença E-ISSN 2237-2660 creve e do qual eflui o sentido. Assim é que, dado que a presença surge como antitética à linguagem, os amálgamas que ele sugere entre presença e lingua- gem são de antemão uma impossibilidade, são incomunicáveis. Ora, os três primeiros tipos de amálgama, a despeito de sugerirem uma materialidade pura, uma presença plena aquém do sentido, só podem ser ex- plicados em virtude daquilo que o sentido não é, ou seja, a presença acaba por ser mediada e adiada. Não há realidade física ou experiência estética para além da dobradiça (brisure) entre fora e dentro da linguagem. Mais: ainda que houvesse, seria inapreensível, pois a linguagem é a única realidade que nos é dada a conhecer. Já os outros quatro tipos de amálgama realizam perfei- tamente a metafísica da presença, na medida em que a linguagem seria aquilo que torna presente um referente ausente, ou melhor, o signo substitui vicari- amente a própria coisa – visto que estariam de lados opostos. Contudo, vale recordar, a linguagem refere-se apenas a si mesma, não a algo fora dela. É jus- tamente esse anseio de que o texto transcenda o próprio texto em direção a um significado ou a um referente anteriores a ele que constitui o gesto meta- fisico e, consequentemente, hermenêutico. É por tentar promover uma mera inversão do campo hermenêutico que Gumbrecht o restaura. Não se trata de retratar Derrida perante a acusação de textualismo, mas sim de apontar de que maneira a teoria de Gumbrecht, a despeito de se pretender não- metafísica ou não-hermenêutica, termina por ratificar a metafísica da presen- ça. Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Isso porque já no início afirma: “[...] aquilo que não é linguagem [...] será algo que vim a chamar de ‘presença’”61 (Gumbrecht, 2012, p. 61); com isso, ele corrobora a oposição binária entre algo puramente espiritual de um lado e algo “real” de outro, a saber, o suporte no qual a linguagem se ins- 22 ç g Disponível em: http://seer.ufrgs.br/presenca ç g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. E ISSN 2237 2660 the possibility of referring to the world other than by meaning has become synonymous with the utmost degree of philosophical naïveté” (Gumbrecht, 2004, p. 53). E ISSN 2237 2660 the possibility of referring to the world other than by meaning has become synonymous with the utmost degree of philosophical naïveté” (Gumbrecht, 2004, p. 53). 5 No final da década de 1980, enquanto desenvolvia suas pesquisas sobre as mate- rialidades da comunicação, que culminaram na publicação de Materialities of Communication, Gumbrecht procurava aliados teóricos para defender suas posi- ções: “But we also wanted to see an intellectual ally in Jacques Derrida, who at the beginning of his philosophical trajectory (a good twenty years before our col- loquium) had argued that the systematic bracketing of the ‘exteriority of the sig- nifier’ was one key reason for the devastating dominance, as we were quick to believe, of ‘logo-phonocentrism’ in Western culture” (Gumbrecht, 2004, p. 9). 6 No original em inglês: “[…] deconstruction has thus to a large extent relied on soft terror to shore up the existing order in the humanities” (Gumbrecht, 2004, p. 54). 6 No original em inglês: “[…] deconstruction has thus to a large extent relied on soft terror to shore up the existing order in the humanities” (Gumbrecht, 2004, p. 54). 7 No original em inglês: “[…] this is why Being and Time presents the Cartesian grounding of human existence on thought (and on thought alone) and the subsequent dissociations between human existence and space and between human existence and substance as the original sins of modern philosophy” (Gumbrecht, 2004, p. 66). 7 No original em inglês: “[…] this is why Being and Time presents the Cartesian grounding of human existence on thought (and on thought alone) and the subsequent dissociations between human existence and space and between human existence and substance as the original sins of modern philosophy” (Gumbrecht, 2004, p. 66). 8 No artigo Hans Ulrich Gumbrecht leitor de Martin Heidegger: concepção de pro- dução de presença, Wellington Amâncio da Silva (2017, p. 513) afirma que, em Produção de presença, Gumbrecht faz convergir os conceitos de Dasein e pre- sença. Todavia, na edição norte-americana, Production of presence, de 2004, Gumbrecht (2004, p. 71) opta por manter o conceito heideggeriano no origi- nal em alemão: “[...] the role of Dasein (Heidegger’s word for human existen- ce) in the happening of truth”. Notas 1 No original em inglês: “Perhaps Heidegger’s entire contribution to Western philosophy can be read as originating in a reaction to the emotional, intelectu- al, and political environments of 1926” (Gumbrecht, 1997, p. 442). 2 No original em inglês: “This contamination does not tarnish the philosophical importance of Heidegger's thought […]. Sein und Zeit cannot of course be identified with Nazi ideology” (Gumbrecht, 1997, p. 443). 3 Nota bene: as citações no corpo do texto foram extraídas da edição brasileira de Produção de presença, publicado em 2010 pela editora Contraponto, com tra- dução de Ana Isabel Soares. O original segue em notas de rodapé. 4 No original em inglês: “To use such concepts, however, has long been a symp- tom of despicably bad intellectual taste in the humanities”. […] “to believe in 4 No original em inglês: “To use such concepts, however, has long been a symp- tom of despicably bad intellectual taste in the humanities”. […] “to believe in 23 E-ISSN 2237-2660 the possibility of referring to the world other than by meaning has become synonymous with the utmost degree of philosophical naïveté” (Gumbrecht, 2004, p. 53). Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. ç g Disponível em: http://seer.ufrgs.br/presenca g Disponível em: http://seer.ufrgs.br/presenca g Disponível em: http://seer.ufrgs.br/presenca E, no mesmo parágrafo: “Dasein is being-in- the-world” (2004, p. 71). Na clássica tradução norte-americana de Ser e tempo, Being and Time, de 1962, os tradutores John Mcquarrie e Edward Robinson optam igualmente por não verter para o inglês o conceito de Dasein, preser- vando a palavra alemã. De qualquer forma, ao contrário do que assevera Silva no referido artigo, no trecho em que Gumbrecht estabelece uma aproximação entre ser e presença, fica claro, no original, que ele diz being e não Dasein: “Both concepts, Being and presence, imply substance; both are related to spa- ce; both can be associated with movement” (Gumbrecht, 2004, p. 77). 9 No original em inglês: “[…] belongs to the dimension of things” (Gumbrecht, 2004, p. 68). 24 g Disponível em: http://seer.ufrgs.br/presenca g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. ç g Disponível em: http://seer.ufrgs.br/presenca 21 No original em inglês: “[…] the metaphysics of presence is shaken with the help of the concept of sign” (Derrida, 2001b, p. 354). 21 No original em inglês: “[…] the metaphysics of presence is shaken with the help of the concept of sign” (Derrida, 2001b, p. 354). 22 No original em inglês: “There is no sense in doing without the concepts of me- taphysics in order to shake metaphysics. We have no language – no syntax and no léxicon – which is foreign to this history; we can pronounce not a single destructive proposition which has not already had to slip into the form, the lo- gic, and the implicit postulations of precisely what it seeks to contest” (Derri- da, 2001b, p. 354). 23 No original em inglês: “At the center, the permutation or the transformation of elements (which may of course be structures enclosed within a structure) is forbidden” (Derrida, 2001b, p. 352). 23 No original em inglês: “At the center, the permutation or the transformation of elements (which may of course be structures enclosed within a structure) is forbidden” (Derrida, 2001b, p. 352). 24 No original em inglês: “The history of metaphysics, like the history of the West, is the history of these metaphors and metonymies” (Derrida, 2001b, p. 353). 25 No original em inglês: “It could be shown that all the names related to funda- mentals, to principles, or to the center have always designated an invariable presence – eidos, archè, telos, energeia, ousia (essence, existence, substance, sub- ject) alêtheia, transcendentality, consciousness, God, man, and so forth” (Der- rida, 2001b, p. 353). 26 No original em francês: “Rien de ce que je tente n'aurait été possible sans l'ou- verture des questions heideggeriennes. Et d'abord, puisque nous devons dire ici les choses três vite, sans l'attention à ce que Heidegger appelle la difference en- tre l'être et l'étant, la différence ontico-ontologique telle qu'elle reste d'une cer- taine manière impensée par la philosophie. Mais, malgré cette dette à l'égard de la pensée heideggerienne, ou plutôt en raison de cette dette, je tente de re- connaître, dans le texte heideggerien qui, pas plus qu'un autre, n'est ho- mogène, continu, partout égal à la plus grande force et à toutes les conséquen- ces de ses questions, je tente d'y reconnaître des signes d'appartenance à la mé- taphysique ou à ce qu'il appelle l'onto-théologie” (Derrida, 1972, p. 18-19). Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. g Disponível em: http://seer.ufrgs.br/presenca ç g Disponível em: http://seer.ufrgs.br/presenca 10 No original em inglês: “If Being has the character of a thing, this means that it has substance and that, therefore (and unlike anything purely spiritual), it oc- cupies space” (Gumbrecht, 2004, p. 68). 11 No original em inglês: “[…] occupying space also implies the possibility of Being unfolding a movement” (Gumbrecht, 2004, p. 68). 12 No original em inglês: “Being, I think, refers to the things of the world before they become part of a culture (or, using the rhetorical figure of the paradox, the concept refers to the things of the world before they become part of a world)” (Gumbrecht, 2004, p. 70). 13 No original em inglês: “Being will only be Being outside the networks of se- mantics and other cultural distinctions” (Gumbrecht, 2004, p. 70). 14 No original em inglês: “Releasement is indeed the release of oneself from transcendental re-presentation and so a relinquishing of the willing of a hori- zon” (Heidegger, 1966, p. 79-80). 15 No original em inglês: “Only the presence of certain things […] opens up the possibility of other things appearing in their primordial material qualities–and this effect might be considered as one way (and as a part) of unconcealing their Being” (Gumbrecht, 2004, p. 74). 16 No original em inglês: “[…] seeing things as part of Being, that is, indepen- dently of the shapes imposed upon them by historically specific cultures” (Gumbrecht, 2004, p. 75). 17 No original em inglês: “[…] impossible to think of Being as something stable” (Gumbrecht, 2004, p. 77). 18 No original em inglês: “[…] something beyond the merely physical” (Gum- brecht, 2006, p. 130). 19 No original em francês: “[...] j'ai parfois le sentiment que la problématique heideggerienne est la défense la plus ‘profonde’ et la plus ‘puissante’ de ce que j'essaie de mettre en question, sous le titre de pensée de la présence” (Derrida, 1972, p. 75). 20 No original em inglês: “Derrida has never been shy about inventing new con- cepts, even when the need to do so has not been very obvious. Why is he so hesitant, then, about coming up with some new concept that would allow us to ‘end’ the age of the sign?” (Gumbrecht, 2004, p. 53). 25 27 No original em inglês: “The distinction collapses. It remains forgotten. Al- though the two parties to the distinction, what is present and presencing, re- veal themselves, they do not do so as distinguished. Rather, even the early trace of the distinction is obliterated” (Heidegger, 1984, p. 50-51). 28 No original em inglês: “Illumination of the distinction therefore cannot mean that the distinction appears as a distinction” (Heidegger, 1984, p. 51). 26 But as that sense is nothing outside of language and the language of words, it is tied, if not to a particular word or to a particular system of lan- guage (concesso non dato), at least to the possibility of the word in general” (Derrida, 1997, p. 20-21). 31 No original em inglês: “The word ‘being’, or at any rate the words designating the sense of being in different languages, is, with some others, an ‘originary word’ (‘Urwort’), the transcendental word assuring the possibility of being- word to all other words. As such, it is precomprehended in all language and – this is the opening of Being and Time – only this precomprehension would permit the opening of the question of the sense of being in general, beyond all regional ontologies and all metaphysics: a question that broaches philosophy […] and lets itself be taken over by philosophy, a question that Heidegger re- peats by submitting the history of metaphysics to it. Heidegger reminds us constantly that the sense of being is neither the word ‘being’ nor the concept of being. But as that sense is nothing outside of language and the language of words, it is tied, if not to a particular word or to a particular system of lan- guage (concesso non dato), at least to the possibility of the word in general” (Derrida, 1997, p. 20-21). 32 No original em inglês: “[…] the question of being unites indissolubly with the precomprehension of the word being” (Derrida, 1997, p. 21). 32 No original em inglês: “[…] the question of being unites indissolubly with the precomprehension of the word being” (Derrida, 1997, p. 21). 33 No original em inglês: “It is thus that, after evoking the ‘voice of being’, Hei- degger recalls that it is silent, mute, insonorous, wordless, originarily a-phonic (die Gewähr der lautlosen Stimme verborgener Quellen. . .). The voice of the sources is not heard. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. E-ISSN 2237-2660 29 Conforme Derrida (1991, p. 34-36), “[...] essa diferença gráfica (o a no lugar do e), esta diferença marcada entre duas notações aparentemente vocais, entre duas vogais, permanece puramente gráfica: escreve-se ou lê-se, mas não se ou- ve, não se entende. [...] Sem dúvida este silêncio piramidal da diferença gráfica entre o e e o a só pode funcionar no interior do sistema de escrita fonética e no interior de uma língua ou de uma gramática historialmente associada à escrita fonética bem como a toda a cultura de que ela é inseparável”. Ou seja, a diffé- rance não é uma palavra nem um conceito, mas uma intervenção gráfica, um efeito nominal, que abala o fonologocentrismo. 30 No original em inglês: “If we maintain that differance (is) (itself) other than absence and presence, if it traces, then when it is a matter of the forgetting of the difference (between Being and beings), we would have to speak of a disap- pearance of the trace of the trace” (Derrida, 1982, p. 23-24). 30 No original em inglês: “If we maintain that differance (is) (itself) other than absence and presence, if it traces, then when it is a matter of the forgetting of the difference (between Being and beings), we would have to speak of a disap- pearance of the trace of the trace” (Derrida, 1982, p. 23-24). 31 No original em inglês: “The word ‘being’, or at any rate the words designating the sense of being in different languages, is, with some others, an ‘originary word’ (‘Urwort’), the transcendental word assuring the possibility of being- word to all other words. As such, it is precomprehended in all language and – this is the opening of Being and Time – only this precomprehension would permit the opening of the question of the sense of being in general, beyond all regional ontologies and all metaphysics: a question that broaches philosophy […] and lets itself be taken over by philosophy, a question that Heidegger re- peats by submitting the history of metaphysics to it. Heidegger reminds us constantly that the sense of being is neither the word ‘being’ nor the concept of being. ç g Disponível em: http://seer.ufrgs.br/presenca ggerian situation with respect to the metaphysics of presence and logocentrism. It is at once contained within it and transgresses it” (Derrida, 2001b, p. 22). ggerian situation with respect to the metaphysics of presence and logocentrism. It is at once contained within it and transgresses it” (Derrida, 2001b, p. 22). ggerian situation with respect to the metaphysics of presence and logocentrism. It is at once contained within it and transgresses it” (Derrida, 2001b, p. 22). 34 No original em inglês: “[…] that means that being escapes the movement of the sign” (Derrida, 1997, p. 22). 34 No original em inglês: “[…] that means that being escapes the movement of the sign” (Derrida, 1997, p. 22). 34 No original em inglês: “[…] that means that being escapes the movement of the sign” (Derrida, 1997, p. 22). 35 É importante assinalar que, em um dos textos da chamada viragem [die Kehre], Carta sobre o humanismo, de 1946, o ser guarda familiaridade com a différance derridiana, até mesmo na recusa do nome próprio: “caso o homem encontre, alguma vez, o caminho para a proximidade do ser, então deve antes aprender a existir no inefável [das Namemlose]” (Heidegger, 2005, p. 16). Em tradução di- reta, das Namenlose significa “sem nome”. 36 No original em inglês: “Since Being has never had a ‘meaning’, has never been thought or said as such, except by dissimulating itself in beings, then differen- ce, in a certain and very strange way, (is) ‘older’ than the ontological difference or than the truth of Being. When it has this age it can be called the play of the trace” (Derrida, 1982, p. 22). 37 No original em inglês: “It erases itself in presenting itself, muffles itself in reso- nating, like the a writing itself, inscribing its pyramid in différance” (Derrida, 1982, p. 23). 38 No original em inglês: “the trace is not more ideal than real, not more intelli- gible than sensible, not more a transparent signification than an opaque energy and no concept of metaphysics can describe it” (Derrida, 2001b, p. 65). 39 No original em inglês: “The trace is in fact the absolute origin of sense in gene- ral. Which amounts to saying once again that there is no absolute origin of sense in general” (Derrida, 2001b, p. 65). 40 No original em inglês: “‘Older’ than Being itself, such a différance has no na- me in our language. A rupture between the originary meaning of being and the word, between meaning and the voice, between ‘the voice of being’ and the ‘phonè’, between “the call of being”, and articulated sound; such a rupture, which at once confirms a fundamental metaphor, and renders it suspect by ac- centuating its metaphoric discrepancy, translates the ambiguity of the Heide- 33 No original em inglês: “It is thus that, after evoking the ‘voice of being’, Hei- degger recalls that it is silent, mute, insonorous, wordless, originarily a-phonic (die Gewähr der lautlosen Stimme verborgener Quellen. . .). The voice of the sources is not heard. A rupture between the originary meaning of being and the word, between meaning and the voice, between ‘the voice of being’ and the ‘phonè’, between “the call of being”, and articulated sound; such a rupture, which at once confirms a fundamental metaphor, and renders it suspect by ac- centuating its metaphoric discrepancy, translates the ambiguity of the Heide- 27 g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. ç g Disponível em: http://seer.ufrgs.br/presenca 41 No original em inglês: “There will be no unique name, even if it were the na- me of Being” (Derrida, 1982, p. 27). 42 No original em inglês: “What is written as différance, then, will be the playing movement that ‘produces’ – by means of something that is not simply an acti- vity – these differences, these effects of difference” (Derrida, 1982, p. 11). 43 No original em inglês: “The sign, in this sense, is deferred presence” (Derrida, 1982, p. 9). 44 No original em inglês: “It is because of différance that the movement of signifi- cation possible only if each so-called ‘present’ element, each element appearing on the scene of presence, is related to something other than itself, thereby kee- ping within itself the mark of the past element, and already letting itself be vi- tiated by the mark of its relation to the future element, this trace being related no less to what is called the future than to what is called the past, and constitu- ting what is called the present by means of this very relation to what it is not: what it absolutely is not, not even a past or a future as a modified present” (Derrida, 1982, p. 13). 44 No original em inglês: “It is because of différance that the movement of signifi- cation possible only if each so-called ‘present’ element, each element appearing on the scene of presence, is related to something other than itself, thereby kee- ping within itself the mark of the past element, and already letting itself be vi- tiated by the mark of its relation to the future element, this trace being related no less to what is called the future than to what is called the past, and constitu- ting what is called the present by means of this very relation to what it is not: what it absolutely is not, not even a past or a future as a modified present” (Derrida, 1982, p. 13). 45 No original em inglês: “[…] the signifier first signifies a signifier, and not the thing itself or a directly presented signified” (Derrida, 1997, p. 237). 46 No original em inglês: “[there is no outside-text; il n'y a pas de hors-texte]” (Derrida, 1997, p. 158). 47 No original em inglês: “[…] there is no linguistic sign before writing. Without that exteriority, the very idea of the sign falls into decay” (Derrida, 1997, p. 12). But ‘already know’ that if it is unnameable, it is not provi- sionally so, not because our language has not yet found or received this name, or because we would have to seek it in another language, outside the finite sys- tem of our own. It is rather because there is no name for it at all, not even the name of or of Being, not even that of ‘différance’, which is not a name, which is not a pure nominal unity, and unceasingly dislocates itself in a chain of dif- fering and deferring substitutions” (Derrida, 1982, p. 26). 41 No original em inglês: “There will be no unique name, even if it were the na- me of Being” (Derrida, 1982, p. 27). 41 No original em inglês: “There will be no unique name, even if it were the na- me of Being” (Derrida, 1982, p. 27). 28 Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. ç g Disponível em: http://seer.ufrgs.br/presenca g Disponível em: http://seer.ufrgs.br/presenca 51 No original em inglês: “[…] there have never been anything but supplements, substitutive significations which could only come forth in a chain of differenti- al references, the ‘real’ supervening, and being added only while taking on me- aning from a trace and from an invocation of the supplement, etc.” (Derrida, 1997, p. 159). 51 No original em inglês: “[…] there have never been anything but supplements, substitutive significations which could only come forth in a chain of differenti- al references, the ‘real’ supervening, and being added only while taking on me- aning from a trace and from an invocation of the supplement, etc.” (Derrida, 1997, p. 159). 52 No original em inglês: “No, there isn't language on one side and reality on the other” (Derrida, 2005, p. 146). 52 No original em inglês: “No, there isn't language on one side and reality on the other” (Derrida, 2005, p. 146). 53 No original em francês: “[…] le signifiant ‘matière’ ne me paraît problémati- que qu'au moment où sa réinscription n'éviterait pas d'en faire un nouveau principe fondamental, où, par une régression théorique, on le reconstituerait em ‘signifié transcendantal’” (Derrida, 1972, p. 88). 54 No original em francês: “Le signifié transcendental n'est pas seulement le re- cours de l'idéalisme au sens étroit. Il peut toujours venir rassurer un matéria- lisme métaphysique. Il devient alors un référend ultime, selon la logique classi- que impliquée par cette valeur de référend, ou une ‘réalité objective’ absolu- ment ‘antérieure’ à tout travail de la marque, un contenu sémantique ou une forme de présence garantissant du dehors le mouvement du texte général” (Derrida, 1972, p. 88). 55 No original em inglês: “Natural cognition begins with experience and remains within experience” (Husserl, 1983, p. 5). 56 No original em inglês: “Something that is ‘present’ is supposed to be tangible for human hands, which implies that, conversely, it can have an immediate impact on human bodies” (Gumbrecht, 2004, p. xiii). 57 No original em inglês: “[…] vorhandenheit is not the primary mode of being. Dasein itself is not vorhanden, nor are articles of use or ‘equipment’” (Inwood, 1999, p. 128). 58 No original em inglês: “I have not found much consolation in what I like to cha- racterize as the ‘linguistic existentialism’ of Deconstruction, i.e. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. 48 No original em inglês: “Even before it is linked to incision, engraving, drawing, or the letter, to a signifier referring in general to a signifier signified by it, the concept of the graphie [unit of a possible graphic system] implies the framework of the instituted trace, as the possibility common to all systems of signification” (Derrida, 1997, p. 46). 49 No original em inglês: “But it is on the other hand the being-imprinted of the imprint” (Derrida, 1997, p. 63). 49 No original em inglês: “But it is on the other hand the being-imprinted of the imprint” (Derrida, 1997, p. 63). 50 No original em inglês: “[…] it is in the specific zone of this imprint and this trace, in the temporalization of a lived experience which is neither in the world nor in ‘another world’, which is not more sonorous than luminous, not more in time than in space, that differences appear among the elements or rather produce them, make them emerge as such and constitute the texts, the chains, and the systems of traces. These chains and systems cannot be outlined except in the fabric of this trace or imprint” (Derrida, 1997, p. 65). 50 No original em inglês: “[…] it is in the specific zone of this imprint and this trace, in the temporalization of a lived experience which is neither in the world nor in ‘another world’, which is not more sonorous than luminous, not more in time than in space, that differences appear among the elements or rather produce them, make them emerge as such and constitute the texts, the chains, and the systems of traces. These chains and systems cannot be outlined except in the fabric of this trace or imprint” (Derrida, 1997, p. 65). 29 g Disponível em: http://seer.ufrgs.br/presenca ç g Disponível em: http://seer.ufrgs.br/presenca Referências BARTHES, Roland. Elementos de Semiologia. Tradução: Izidoro Blikstein. São Paulo: Cultrix, 2007. 60 No original em inglês: “In its theological use, the concept of epiphany refers to the appearance of a thing, of a thing that requires space, a thing that is either absent or present” (Gumbrecht, 2006, p. 135). 61 No original em inglês: “[…] which is not language [...] will be what I have come to call ‘presence’” (Gumbrecht, 2006, p. 129). Q p ç Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença the sustained complaint and melancholia (in its endless variations) about the alleged incapacity of language to refer to the things of the world” (Gumbrecht, 2006, p. 130). 59 No original em inglês: “[…] mystical language produces the paradoxal effect of stimulating imaginations that seem to make this very presence palpable” (Gumbrecht, 2006, p. 134). 30 g Disponível em: http://seer.ufrgs.br/presenca ç g Disponível em: http://seer.ufrgs.br/presenca Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. BARTHES, Roland. Elementos de Semiologia. Tradução: Izidoro Blikstein. São Paulo: Cultrix, 2007. DERRIDA, Jacques. A Estrutura, o Signo e o Jogo no Discurso das Ciências Humanas. In: DERRIDA, Jacques. A Escritura e a Diferença. São Paulo: Pers- pectiva, 1971. P. 229-249. DERRIDA, Jacques. Positions. Paris: Les Éditions de Minuit, 1972. DERRIDA, Jacques. Différance. In: DERRIDA, Jacques. Margins of Philoso- phy. Tradução: Alan Bass. Chicago: University of Chicago Press, 1982. P. 1-27. DERRIDA, Jacques. Margens da Filosofia. Tradução: Joaquim Torres Costa e António M. Magalhães. Campinas: Papirus, 1991. DERRIDA, Jacques. Of Grammatology. Tradução: Gayatri Spivak. Baltimore: The Johns Hopkins University Press, 1997. DERRIDA, Jacques. Posições. Tradução: Tomaz Tadeu da Silva. Belo Horizon- te: Autêntica, 2001a. DERRIDA, Jacques. Structure, Sign, and Play in the Discourse of the Human Sciences. In: DERRIDA, Jacques. Writing and Difference. Tradução: Alan Bass. London; New York: Routledge, 2001b. P. 351-370. DERRIDA, Jacques. Outrem É Secreto porque É Outro. In: DERRIDA, Jac- ques. Papel-Máquina. Tradução: Evando Nascimento. São Paulo: Estação Li- berdade, 2004. P. 331-358. DERRIDA, Jacques. Others Are Secret because They Are Other. In: DERRIDA, Jacques. Paper Machine. Tradução: Rachel Bowlby. Stanford: Stanford Univer- sity Press, 2005. P. 136-163. DERRIDA, Jacques. Gramatologia. Tradução: Miriam Chnaiderman e Renato Janine Ribeiro. São Paulo: Perspectiva, 2013. GUMBRECHT, Hans Ulrich. In 1926: living at the edge of time. Cambridge: Harvard University Press, 1997. 31 g Disponível em: http://seer.ufrgs.br/presenca g Disponível em: http://seer.ufrgs.br/presenca g Disponível em: http://seer.ufrgs.br/presenca Q p ç v. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. GUMBRECHT, Hans Ulrich. Corpo e Forma: ensaios para uma crítica não- hermenêutica. Rio de Janeiro: EdUERJ, 1998. GUMBRECHT, Hans Ulrich. Productions of Presence: what meaning cannot convey. Stanford: Stanford University Press, 2004. GUMBRECHT, Hans Ulrich. Presence in Language or Presence Achieved against Language? Revista Brasileira de Literatura Comparada, n. 8, p. 129-137, 2006. GUMBRECHT, Hans Ulrich. Produção de Presença: o que o sentido não con- segue transmitir. Tradução: Ana Isabel Soares. Rio de Janeiro: Contraponto/Ed. PUC-Rio, 2010. GUMBRECHT, Hans Ulrich. Presença na Linguagem ou Presença contra a Lin- guagem. In: GUMBRECHT, Hans Ulrich. Graciosidade e Estagnação: ensaios escolhidos. Tradução: Luciana Villas Boas e Markus Hediger. Rio de Janeiro: Contraponto/Ed. PUC-Rio, 2012. P. 61-74. GUMBRECHT, Hans Ulrich; LAGE, Mariana. Da Produção de Presença ao Presente Amplo: entrevista com Hans Ulrich Gumbrecht. Artefilosofia, n. 22, p. 186-204, 2017. HEIDEGGER, Martin. Discourse on Thinking: a translation of Gelassenheit. Tradu- ção: John M. Anderson and E. Hans Freund. New York: Harper and Row, 1966. HEIDEGGER, Martin. The Anaximander Fragment. In: HEIDEGGER, Mar- tin. Early Greek Thinking: the dawn of Western philosophy. Tradução: David Farrell Krell and Frank A. Capuzzi. New York: HarperCollins Publishers, 1984. P. 13-58. HEIDEGGER, Martin. Serenidade. Tradução: Maria Madalena Andrade e Olga Santos. Lisboa: Instituto Piaget, 2001. HEIDEGGER, Martin. O Dito de Anaximandro. Tradução: João Constâncio. Lisboa: Fundação Calouste Gulbenkian, 2002. HEIDEGGER, Martin. Carta sobre o Humanismo. Tradução: Rubens Eduardo Frias. 2 ed. São Paulo: Centauro, 2005. HEIDEGGER, Martin. A Origem da Obra de Arte. Tradução: Idalina Azevedo e Manuel Antônio de Castro. São Paulo: Edições 70, 2010. HEIDEGGER, Martin. Ser e Tempo. Tradução: Márcia Sá Cavalcante Schuback. 6 ed. Petrópolis: Vozes, 2012. 32 HUSSERL, Edmund. Ideas Pertaining to a Pure Phenomenology and to a Phenomenological Philosophy. Tradução: F. Kersten. The Hague: Martinus Nijhoff Publishers, 1983. HUSSERL, Edmund. Ideias para uma Fenomenologia Pura e para uma Filo- sofia Fenomenológica: introdução geral à fenomenologia. Tradução: Márcio Su- zuki. Aparecida: Ideias e Letras, 2006. INWOOD, Michael. A Heidegger Dictionary. Oxford: Blackwell Publishers, 1999. INWOOD, Michael. Dicionário Heidegger. Tradução: Luísa Buarque de Ho- landa. Rio de Janeiro: Jorge Zahar, 2002. NIETZSCHE, Friedrich. Sobre Verdade e Mentira no Sentido Extra-moral. Tradução: Fernando de Moares Barros. São Paulo: Hedra, 2007. NIETZSCHE, Friedrich. A Vontade de Poder. Tradução: Marcos Sinésio Pereira Fernandes e Francisco José Dias de Moraes. Rio de Janeiro: Contraponto, 2008. SILVA, Wellington Amâncio da. Hans Ulrich Gumbrecht Leitor de Martin Hei- degger: concepção de produção de presença. Revista Brasileira de Estudos da Presença, Porto Alegre, v. 7, n. 3, p. 505-522, set./dez. 2017. Gustavo Ramos de Souza é professor Adjunto A, em regime Doc-CRES, do De- partamento de Letras da Universidade Estadual de Londrina (UEL). Doutorou-se em Letras, pela mesma instituição, em 2019. Desenvolve pesquisas sobre materia- lidades da literatura e intermidialidade. ORCID: https://orcid.org/0000-0001-5164-420X E-mail: avulsoaoavesso@gmail.com Este texto inédito também se encontra publicado em inglês neste número do pe- riódico. Recebido em 25 de julho de 2022 Aceito em 23 de novembro de 2022 Editor responsável: Marcelo de Andrade Pereira Recebido em 25 de julho de 2022 Aceito em 23 de novembro de 2022 Editor responsável: Marcelo de Andrade Pereira Editor responsável: Marcelo de Andrade Pereira Editor responsável: Marcelo de Andrade Pereira Editor responsável: Marcelo de Andrade Pereira Gustavo Ramos de Souza – Quem tem Medo de Derrida?: Gumbrecht e a metafísica da presença Rev. Bras. Estud. Presença, Porto Alegre, v. 13, n. 2, e126100, 2023. Disponível em: http://seer.ufrgs.br/presenca 33
https://openalex.org/W988456295	https://europepmc.org/articles/pmc4452708?pdf=render	English	null	Analysis of [Gossypium capitis-viridis × (G.hirsutum × G.australe)2] Trispecific Hybrid and Selected Characteristics	PloS one	2,015	cc-by	4,611	Analysis of [Gossypium capitis-viridis × (G. hirsutum × G.australe)2] Trispecific Hybrid and Selected Characteristics Di Chen1☯, Yuxiang Wu2☯, Xiling Zhang1, Fuguang Li1 1 Cotton Research Institute, Chinese Academy of Agricultural Science, Anyang, Henan, China, 2 Department of Agriculture, Shanxi Agricultural University, Taigu, Shanxi, China Di Chen1☯, Yuxiang Wu2☯, Xiling Zhang1, Fuguang Li1 1 Cotton Research Institute, Chinese Academy of Agricultural Science, Anyang, Henan, China, 2 Department of Agriculture, Shanxi Agricultural University, Taigu, Shanxi, China ☯These authors contributed equally to this work. * lifuguang2014@hotmail.com (FL); zhangxiling1962@126.com (XZ) ☯These authors contributed equally to this work. * lifuguang2014@hotmail.com (FL); zhangxiling1962@126.com (XZ) Data Availability Statement: All relevant data are within the paper. Data Availability Statement: All relevant data are within the paper. Funding: This research was financially supported by National Natural Science Foundation of China (No. 31301364, No. 31171599), Special fund for basic scientific research business of central public research institutes (1610162014018) and Scientific and technological project in Shanxi province (No. 20130311004-1). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Abstract doi:10.1371/journal.pone.0127023 Academic Editor: Guoping Zhang, Zhejiang University, CHINA Received: September 27, 2014 Accepted: April 11, 2015 Published: June 2, 2015 Copyright: © 2015 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Received: September 27, 2014 Accepted: April 11, 2015 Published: June 2, 2015 Copyright: © 2015 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Copyright: © 2015 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. RESEARCH ARTICLE Abstract Speciation is always a contentious and challenging issue following with the presence of gene flow. In Gossypium, there are many valuable resources and wild diploid cotton espe- cially C and B genome species possess some excellent traits which cultivated cotton al- ways lacks. In order to explore character transferring rule from wild cotton to upland tetraploid cotton, the [G. capitis-viridis × (G. hirsutum × G. australe)2] triple hybrid was syn- thesized by interspecies hybridization and chromosome doubling. Morphology comparisons were measured among this hybrid and its parents. It showed that trispecific hybrid F1 had some intermediate morphological characters like leaf style between its parents and some different characters from its parents, like crawl growth characteristics and two kind flower color. It is highly resistant to insects comparing with other cotton species by four year field investigation. By cytogenetic analysis, triple hybrid was further confirmed by meiosis behav- ior of pollen mother cells. Comparing with regular meiosis of its three parents, it was distin- guished by the occurrence of polyads with various numbers of unbalanced microspores and finally generating various abnormal pollen grains. All this phenomenon results in the sterility of this hybrid. This hybrid was further identified by SSR marker from DNA molecular level. It showed that 98 selected polymorphism primers amplified effective bands in this hy- brids and its parents. The genetic proportion of three parents in this hybrid is 47.8% from G. hirsutum, 14.3% from G. australe, 7.0% from G. capitis-viridis, and 30.9% recombination bands respectively. It was testified that wild genetic material has been transferred into culti- vated cotton and this new germplasm can be incorporated into cotton breeding program. OPEN ACCESS Citation: Chen D, Wu Y, Zhang X, Li F (2015) Analysis of [Gossypium capitis-viridis × (G.hirsutum × G.australe)2] Trispecific Hybrid and Selected Characteristics. PLoS ONE 10(6): e0127023. doi:10.1371/journal.pone.0127023 Academic Editor: Guoping Zhang, Zhejiang University, CHINA Received: September 27, 2014 Accepted: April 11, 2015 Published: June 2, 2015 Copyright: © 2015 Chen et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. OPEN ACCESS Citation: Chen D, Wu Y, Zhang X, Li F (2015) Analysis of [Gossypium capitis-viridis × (G.hirsutum × G.australe)2] Trispecific Hybrid and Selected Characteristics. PLoS ONE 10(6): e0127023. Characterization for Trispecific Hybrid of Gossypium glandless-seed and glanded-plant trait. These Australian cottons of C and B genomes are howev- er phylogenetically remote from upland cotton. The only glanded plants issued from glandless seeds produced this way were hexaploid, pentaploid and multiple addition materials [4] by trans- formation from Australian cottons to upland cotton. A cotton mutant totally devoid of pigment glands discovered by McMichael [5] and subsequently used in several breeding programs to cre- ate cotton commercial varieties with gossypol-free cotton seed. Biotechnological methods such as embryo rescue/ovule culture or interspecific cell fusion are also used to overcome sexual barriers between the cultivated and wild cottons [6]. Yu et al [7] generated fertile somatic hybrids between tetraploid upland cotton and wild cotton G. trilobum by symmetric electrofusion and this hybrid showed strong photosynthesis. The [(G. hirsutum×G. raimondii)2 ×G. sturtianum] triple hybrid was synthesized for selecting genotypes of G. hirsutum genetically balanced and expressing the low gossypol seed and high-gossypol plant trait [4]. Competing Interests: The authors have declared that no competing interests exist. G. hirsutum is the most important cultivated cotton for its fiber in the world. Wang et al [8] recently created a draft sequence of the putative D-genome parent, G. raimondii. It is so benefi- cial for studying cultivated polyploid genomes and to further explore its genome evolution and subgenomes interaction. What is needed is an efficient means of incorporating this diverse and divergent genetic material into upland cotton breeding programs. The present study was initi- ated in order to monitor the introgression of chromosome segments from the wild species G. australe, G. capitis-viridis to cultivated cotton and explore how genomes adjust when they come into contact with each other. Plant materials We chose G. hirsutum TM-1, G. australe, G. capitis-viridis as materials. G. hirsutum is an cultivated tetraploid cotton with genome [AD]1 (2n = 4x = 52), while G. australe and G. capi- tis-viridis are wild diploid cotton species with the genome C and B (2n = 2x = 26). All the wild paternal pollen used for the creation of the trispecific hybrid CHA [G. capitis-viridis ×(G. hirsu- tum ×G. australe)2] in this work, were kindly supplied by the cotton collection of National Wild Cotton Nursery in Sanya of China. Morphology characteristic observation In every growth period of this hybrid, morphology characteristics were observed from its whole plant to leaf style, flower color and performances, pollen grain morphology, its seed set and fertility. Comparisons from different characteristic and performance for this hybrid were also made with its parents. Triple hybrid synthensis Tetraploid G. hirsutum TM-1 was first crossed directly with the diploid parents G. australe, creating a triploid hybrid [ADC]. Allohexaploid was further synthesized by colchicine-dou- bling the sterile intergenomic hybrid G. hirsutum × G. australe. Allopolyploid trispecific CHA hybrid [genome BADC] was finally obtained by allohexaploid crossing with the other wild dip- loid maternal parent G. capitis-viridis according to the pseudophyletic introgression method [9]. Introduction There are abundant germplasms in Gossypium including approximately 46 diploid and 6 poly- ploid species (AADD, 2n = 4x = 52) [1–3]. Diploid wild cotton especially C and B genome species has some excellent traits which are devoid of in cultivated cotton, especially G. capitis-viridis with high quality fiber and a strong resistance to Verticillium and Fusarium wilt, G. australe with the 1 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Meiotic analyses Immature flower buds in different development stages were collected from three parents and this triple hybrid, fixed between 8.00–11.00 am in freshly prepared Carnoy’s solution 2 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Characterization for Trispecific Hybrid of Gossypium (ethanol: acetic acid = 3:1) for 24 h, and then stored at 4°C in 70% EtOH for future studies. Meiotic analysis were carried out on suitable size flower buds. After washing the fixed buds in distilled water, anther squashes were stained on slides in Carbol fuchsin solution and observed under a light microscope. Photographs were taken from freshly prepared slides using an Olym- pus BX60 microscope with automatic camera. Size and sterility of pollen grains from randomly selected anthers were also studied staining with Carbol fuchsin solution. Interpretations were based on analyses of between 15–50 pollen mother cells from the triple hybrid. SSR molecular identification The hybrid identity and its parents was also confirmed by SSR molecular markers. SSR analysis was examined by G. hirsutum, G. australe, allohexaploid, this hybrid and G. capitis-viridis. DNA from each plant was extracted from young leaves using CTAB method and quantified using agarose gel electrophoresis. DNA amplification was carried out in a volume of 10 μL con- taining 1 μL 10×buffer, 1.6 μL Mgcl2 (25 mM), 0.5 μL dNTPs (10mM), 2 μL template DNA (50 ng μL-1), 0.8 μL primers (2.5 μM) (0.4 μL of forward and reverse primer each), 0.1 μL Taq polymerase (5 U/μL, Sangon), 5.6 μL ddH2O. PCR was programmed with an initial denaturing for 3 minutes at 95°C; followed by 30 cycles of pre-denaturation for 3 minute at 94°C; denatur- ation for 40 s at 94°C; annealing at 57°C for 45 s; extension for 1 minutes at 72°C, and a final extension for 5 minutes at 72°C in a DNA Mastercycler, Eppendorf, Germany. PCR product (10 μl) was resolved in PAGE by silver staining for the resolving of the bands. PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Morphology characteristic of this triple hybrid Whole semi-bush plant with crawl growth characteristic and long epidermis hair in the stem, heart and thick leaf style of this triple hybrid. Fig 1. Whole semi-bush plant with crawl growth characteristic and long epidermis hair in the stem, heart and thick leaf style of this triple hybrid. Fig 1. Whole semi-bush plant with crawl growth characteristic and long epidermis hair in the stem, heart and doi:10.1371/journal.pone.0127023.g001 We try to backcross this hybrid with cultivars and hope to recover its fertility incorporating into next cotton breeding program. We will continue to examine the stability of the morphological features and further to check its other excellent agronomic traits of this hybrid in our later study. PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Morphology characteristic of this triple hybrid The hybrid plants were perennial, semi-bush with crawl growth characteristic different from any other parents (Fig 1). It has long epidermis hair in whole plant especially on the stem and two side of leaf following with its wild-type parents G. australe (Fig 1). The shape of leaves is heart style with round edge which is intermediate character between its parents and thicker also different from its three parents (Fig 1). It produced many flower buds, and flowered by pink or white petal with unclear or no speck, sometimes pink and white flower at the same day (Fig 2). It had normal stigma and stamen with yellow pollen grains like its upland parents (Fig 2). But most pollen grains were abnormal resulting in its sterility. For several years this hy- brid had no seed set. Flower performance and leaf characteristic for this hybrid were also evalu- ated and compared with its three parents in Table 1 and Fig 3. The triple hybrid had long epidermis hair on the leaf and stem inheriting from its wild pa- rents. It is highly resistant to insects comparing with other cotton species which always have some damage from cotton worm. In Sanya of China, cotton can grow in whole year because of comfortable hot climate. So cotton is very easy to be harmed by various insects. By four year field investigation from 2010–2014, it was tested that some cultivated cotton species like TM-1, CCRI 12, CCRI 16 were all damaged mostly by various insects like pink worm, cotton boll- worm, aphid, thrips, mite, leaf hopper and stinkbug, whereas this triple hybrid performed high insect resistance especially immune to pink worm, cotton bollworm, aphid and mite under no any protection control. The possible reason is that it inherits the resistant gene from its parents G. capitis-viridis and G. australe. We will further confirm its resistant characteristics and iden- tify this resistant gene in next study. The triple hybrid is also high drought resisting because of its long epidermis hair on the leaf and stem. In addition, the hybrid plants were perennial, semi-bush with crawl growth characteristic different from any other parents. So it can prevent from wind damage suitable for cultivating dwarf and wind resistant species in Hainan of China. 3 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Characterization for Trispecific Hybrid of Gossypium Fig 1. Meiotic Analyses In this study, the meiotic behavior in three parents was quite normal in the first division and completely normal in the second division, generating about 100% of normal tetrads. Observa- tions were also made on chromosome morphology and behavior during meiosis for this triple hybrid. After the occurrence of an irregular first cytokinesis, the meiocytes progressed normally to the second division, generating various numbers of unbalanced microspores mainly because anaphase chromosome movements were blocked. As a consequence of this abnormality in chro- mosome orientation, a few or several micronuclei of various sizes were observed inside the cells in telophase I and II with diads, triads and different polyads (Fig 4). Finally, various abnormal pollen grains were appeared like cracked pollen, big oval-shaped pollen, under-developed pollen PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 4 / 11 Characterization for Trispecific Hybrid of Gossypium Fig 2. Flowers with two kind color of this triple hybrid at the same time (pink and white) and the stigma and stamen with yellow pollen grains of this hybrid. Fig 2. Flowers with two kind color of this triple hybrid at the same time (pink and white) and the stigma and stamen with yellow pollen grains of this h b id Fig 2. Flowers with two kind color of this triple hybrid at the same time (pink and white) and the stigma and stamen with yellow pollen grains of this hybrid. iple hybrid at the same time (pink and white) and the stigma and stamen with yellow pollen grains of this doi:10.1371/journal.pone.0127023.g002 and so on (Fig 5). Only a small portion of pollen grains are normal style. These meiotic observa- tions are similar to those previously reported by Rieseberg [10]. It was further proved that this hybrid is actually wide cross product from different three parents and maybe it has a long way to get into harmonious genome interaction from B, A, D and C. But it gives us suggestive thinking Table 1. Accessions in this study and its characteristics. Accessions Leaf Flower G. hirsutum Big and green Big and yellow G. australe Small and light grey Purple with dark speck Allohexaploid Heart style and grey Big and red BADC hybrid Heart style and thicker White or pink G. capitis-viridis Grey with deep divided Light purple with dark speck doi:10.1371/journal.pone.0127023.t001 Table 1. Accessions in this study and its characteristics. Accessions Leaf Flower G. Meiotic Analyses hirsutum Big and green Big and yellow G. australe Small and light grey Purple with dark speck Allohexaploid Heart style and grey Big and red BADC hybrid Heart style and thicker White or pink G. capitis-viridis Grey with deep divided Light purple with dark speck doi:10.1371/journal.pone.0127023.t001 Table 1. Accessions in this study and its characteristics. Table 1. Accessions in this study and its characteristics. PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 5 / 11 Characterization for Trispecific Hybrid of Gossypium g 3. Flower characteristic and leaf performance comparison for this hybrid with three parents (from up to down: G. hirsutum, G. australe, ohexaploid, G. capitis-viridis and triple hybrid respectively). C a ac e a o o spec c yb d o Gossyp u Fig 3. Flower characteristic and leaf performance comparison for this hybrid with three parents (from up to down: G. hirsutum, G. australe, allohexaploid, G. capitis-viridis and triple hybrid respectively). doi:10.1371/journal.pone.0127023.g003 that it is possible to produce a novel germplasm recombining three parents characters through the production of trispecific hybrid. PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 SSR molecular analysis In this paper, the hybrid and its three parents were analyzed using SSR markers from DNA mo- lecular level. A total of 112 selected polymorphism SSR primers were amplified and 98 pairs were screened effective bands in these four accessions. A total of 230 DNA bands were scored from 98 primers for this hybrid, with loci number ranging from one to six per primer pair. By statistic analysis of amplified bands, the genetic proportion coming from three parents in this hybrid is in Table 2. Among the SSR markers in this trispecific hybrid, 47.8% of them were coming from G. hirsutum, 14.3% from G. australe, 7.0% from G. capitis-viridis. Other 30.9% markers were the new and peculiar ones for this hybrid. Different new polymorphics of molecular markers from its parents resulting from difference of sequences might be due to chromosomal rearrangements in 6 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Characterization for Trispecific Hybrid of Gossypium Fig 4. Abnormal meiosis behavior of the hybrid with several micronuclei of various sizes (different polyads) in telophase II. Fig 4. Abnormal meiosis behavior of the hybrid with several micronuclei of various sizes (different polyads) in telophase II. Fig 4. Abnormal meiosis behavior of the hybrid with several micronuclei of various sizes (different polya doi:10.1371/journal.pone.0127023.g004 this newly formed hybrid [11]. This trispecific hybrid had the characteristic bands of three pa- rents and accordingly had its parent genetic materials from SSR molecule marker analysis. It was also testified that wild genetic material has been transferred into cultivated cotton and this new germplasm can be incorporated into next cotton breeding program. It was also tested that SSR analyses can provide insight into the genome difference in hybrid identification. Our examinations from morphology and cytogenetic analysis in meiosis following with SSR identification showed that this hybrid was actually wide cross product from three parents G. hirsutum, G. australe and G. capitis-viridis. Speciation following with hybridization and Polyploidy Polyploidy is a prominent process in plant evolution. Approximately 70% of angiosperms are thought to have experienced one or more episodes of polyploidy at some point in the past [12]. 7 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Characterization for Trispecific Hybrid of Gossypium Fig 5. Various abnormal pollen grains of this triple hybrid. Fig 5. Various abnormal pollen grains of this triple hybrid. doi:10.1371/journal.pone.0127023.g005 Among the best studied polyploids are many of the world’s leading crops, including cotton, Among the best studied polyploids are many of the world’s leading crops, including cotton, wheat, oat, soybean, peanut, canola, tobacco, coffee, and banana, each of which evolved by the joining of divergent genomes in a common nucleus [13]. The evolution of the genus Gossypium (cotton) has included a very successful experiment in polyploid formation by hybridization be- tween a maternal Old World ‘‘A” genome taxon resembling G. herbaceum (2n = 2x = 26) and paternal New World ‘‘D” genome taxon resembling G. raimondii [14] or G. gossypioides [15] (both 2n = 2x = 26) [16]. Similarly, the reunion of genomes through hybridization and allo- polyploidy is conservatively estimated to account for 2–4% of speciation events in flowering Table 2. The resources of the SSR polymorphism in trispecific hybrid. Materials Triple hybrid G. hirsutum G. australe G. capitis-viridis Recombination SSR bands 230 110 33 16 71 Genetic ratio — 47.8% 14.3% 7.0% 30.9% doi:10.1371/journal.pone.0127023.t002 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 8 / 11 Table 2. The resources of the SSR polymorphism in trispecific hybrid. 8 / 11 Characterization for Trispecific Hybrid of Gossypium plants and 7% in ferns [17], and most plants appear to be paleopolyploids [18]. Hybridization and introgression may play an important role in evolution and often serves as a repair or re- placement strategy, rather than solely as a mechanism for the development and/or acquisition of novel traits [19]. Cells differing only by their ploidy are identical in terms of DNA sequence information and relative gene dosage, and yet are often quite different in terms of physiology, morphology, and behavior [20]. Genetic basis of morphological characteristic The justification for the use of these characters is the assumption that they have a complex ge- netic basis of this triple hybrid. Rieseberg [21] indicates that the genetic basis of morphological differences between wild plant species possibly controlled by a single major locus plus modifi- ers. Vlot et al [22] found a molecular marker locus associated with the major gene controlling pappus part number. Doebley et al [23] also investigated that the genetic control of morpholog- ical traits differentiating maize from its wild ancestor fit the general model of a single major locus with large effects plus modifiers. These contrasts indicate polyploidy speciation in plants is accompanied by a diverse array of molecular evolutionary phenomena, which will vary among both genomic constituents and taxa [24], also between some wide cross hybrids and their parents. A better understanding of the number of loci responsible for species differences would greatly assist in assessing the reliability of particular morphological characters, repro- ductive morphology, frequently employed for phylogenetic and taxonomic inference [21] and hybrids identification. Genome interaction of distant hybrid In most cases, divergent genomes do not coexist peacefully upon first contact and hybrids, if formed at all, are inviable or sterile [25]. Although F1 sterility or inviability is a common feature of wide interspecific crosses, small introgressions often have indiscernible heterozygous effects while being lethal or sterilizing in the homozygous state [26]. Reproductive barrier formation between newly derived hybrid taxa and their parental species represents a major evolutionary hurdle [10]. Rieseberg [27] studies of the genes causing hybrid incompatibilities is that postzy- gotic isolation often as a by-product of adaptation. However, natural selection for viability and fertility provides an effective filter that eliminates gametes and individuals carrying maladap- tive changes and favors those inheriting advantageous changes [18]. The genomic changes ap- peared to be largely complete by the third generation following polyploidy formation, and were accompanied by an increase in seed fertility and a reduction of irregular chromosome pairing in meiosis [28]. In this study, we hope to incorporate G. australe and G. capitis-viridis genetic material into upland cotton with some novel traits from the species, such as insect and disease resistance from G. capitis-viridis and low gossypol seed & high-gossypol plant trait introgressed from G. australe. Its other excellent agronomic traits need our further identification. References 1. Wendel JF, Cronn RC. Polyploidy and the evolutionary history of cotton. Advances in Agronomy. 2003; 78: 139–186. 2. Krapovickas A, Seijo G. Gossypium ekmanianum (Malvaceae), algodon silvestre de la Republica Dominicana. Bonplandia. 2008; 17: 55–63. 3. Rambani A, Page JT, Udall JA. Polyploidy and the petal transcriptome of Gossypium. BMC Plant Biolo- gy. 2014; 14: 3. doi: 10.1186/1471-2229-14-3 PMID: 24393201 4. Benbouza H, Lacape JM, Jacquemin JM, Courtois B, Diouf FBH, Sarr D, et al. Introgression of the low- gossypol seed & high-gossypol plant trait in upland cotton: Analysis of [(Gossypium hirsutum×G. rai- mondii)2 × G. sturtianum] trispecific hybrid and selected derivatives using mapped SSRs. Mol Breed- ing. 2010; 25: 273–286. 5. McMichael SC. Glandless boll in Upland cotton and its use in the study of natural crossing. Agron J. 1954; 46: 527–528. 6. Henn HJ, Wingender R, Schnabl H. Regeneration of fertile interspecific hybrids from cell fusion be- tween Helianthus annuus L. and wild Helianthus species. Plant Cell Rep. 1998; 18: 220–224. 7. Yu XS, Chu BJ, Liu RE, Sun J, Brian JJ, Wang HZ, et al. Characteristics of fertile somatic hybrids of G. hirsutum L. and G. trilobum generated via protoplast fusion. Theor Appl Genet. 2012; 125: 1503–1516. doi: 10.1007/s00122-012-1929-0 PMID: 22777361 8. Wang KB, Wang ZW, Li FG, Ye WW, Wang JY, Song GL, et al. The draft genome of a diploid cotton Gossypium raimondii. Nature genetics. 2012; 1–7. 9. Mergeai G. Introgressions interspe´cifiques chez le cotonnier. Cahiers Agric. 2006; 15:135–143 10. Rieseberg LH. Crossing relationship among ancient and experimental sunflower hybrid linesges. Evo- lution. 2000; 54: 859–865. PMID: 10937259 11. Rieseberg LH, Sinervo B, Linder CR, Ungerer MC, Arias DM. Role of gene interactions in hybrid specia- tion: evidence from ancient and experimental hybrids. Science. 1996; 272: 741–745. PMID: 8662570 12. Masterson J. Stomatal size in fossil plants: evidence for polyploidy in majority of angiosperms. Science. 1994; 264: 421–424. PMID: 17836906 13. Hilu KW. Polyploidy and the evolution of domesticated plants. Am J Bot. 1993; 80: 1494–1499. 14. Wendel JF. New world tetraploid cottons contain old-world cytoplasm. Proc Natl Acad Sci. 1989; 86: 4132–4136. PMID: 16594050 15. Wendel JF, Schnabel A, Seelanan T. An unusual ribosomal DNA sequence from Gossypium gossy- pioides reveals ancient, cryptic, intergenomic introgression. Molecular Phylogenetics and Evolution. 1995; 4: 298–313. PMID: 8845966 16. Jiang CX, Wright RJ, EL-ZIK KM, Paterson AH. Acknowledgments This research was financially supported by National Natural Science Foundation of China (No. 31301364, No. 31171599), Special fund for basic scientific research business of central public research institutes (1610162014018) and Scientific and technological project in Shanxi prov- ince (No. 20130311004–1). 9 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Characterization for Trispecific Hybrid of Gossypium Author Contributions Conceived and designed the experiments: FL. Performed the experiments: DC. Analyzed the data: YW. Contributed reagents/materials/analysis tools: XZ. Wrote the paper: YW. PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 24. Liu B, Brubaker CL, Mergeai G, Cronn RC, Wendel JF. Polyploid formation in cotton is not accompa- nied by rapid genomic changes. Genome. 2001; 44: 321–329. PMID: 11444689 25. Stebbins GL. The inviability, weakness, and sterility of interspecific hybrids. Adv Genet. 1958; 9: 147– 215. PMID: 13520442 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 28. Ozkan H, Levy AA, Feldman M. Allopolyploidy-induced rapid genome evolution in the wheat (Aegilops- Triticum) group. Plant Cell. 2001; 13:1735–1747. PMID: 11487689 References Polyploid formation created unique avenues for re- sponse to selection in Gossypium (cotton). Proc Natl Acad Sci. 1998; 95: 4419–4424. PMID: 9539752 17. Otto SP, Whitton J. Polyploid incidence and evolution. Annu Rev Genet. 2000; 34: 401–437. PMID: 11092833 18. Rieseberg LH. Polyploid evolution: Keeping the peace at genomic reunions. Current Biology. 2001; 11: 925–928. 19. Rieseberg LH Evolution: Replacing genes and traits through hybridization. Current Biology. 2008; 19: 119–122. 20. Hieter P, Griffiths T. Polyploidy-more is more or less. Science. 1999; 285: 210–211. PMID: 10428719 21. Rieseberg LH. The genetic basis of morphological differences between plant species. International Journal of Plant Sciences. 1992; 153: v–vi. 22. Vlot EC, van Houten WHJ, Mauthe S, Bach-mann K. Genetic and nongenetic factors influencing devia- tions from five pappus parts in a hybrid between Mi-croseris douglasii and M. bigelovii (Asteraceae, Lactuceae). In this issue. 1992. 23. Doebley J, Stec A, Wendel J, Edwards M. Genetic and morphological analysis of a maize-teosinte F2 population: implications for the origin of maize. Proc Natl Acad Sci. 1990; 87: 9888–9892. PMID: 11607138 10 / 11 PLOS ONE \| DOI:10.1371/journal.pone.0127023 June 2, 2015 Characterization for Trispecific Hybrid of Gossypium 25. Stebbins GL. The inviability, weakness, and sterility of interspecific hybrids. Adv Genet. 1958; 9: 147– 215. PMID: 13520442 26. Turelli M, Orr HA. Dominance, epistasis and the genetics of postzygotic isolation. Genetics. 2000; 154: 1663–1679. PMID: 10747061 27. Rieseberg LH. Genetics of speciation. Journal of Heredity. 2007; 98: 101–102. 28. Ozkan H, Levy AA, Feldman M. Allopolyploidy-induced rapid genome evolution in the wheat (Aegilops- Triticum) group. Plant Cell. 2001; 13:1735–1747. PMID: 11487689 11 / 11
W4318070780.txt	https://parasitesandvectors.biomedcentral.com/counter/pdf/10.1186/s13071-023-05651-1	en		Leishmania tarentolae: a vaccine platform to target dendritic cells and a surrogate pathogen for next generation vaccine research in leishmaniases and viral infections	Parasites & vectors	2,023	cc-by	9,432	Parasites & Vectors (2023) 16:35 Bandi et al. Parasites & Vectors https://doi.org/10.1186/s13071-023-05651-1 Open Access REVIEW Leishmania tarentolae: a vaccine platform to target dendritic cells and a surrogate pathogen for next generation vaccine research in leishmaniases and viral infections Claudio Bandi1, Jairo Alfonso Mendoza‑Roldan2, Domenico Otranto2, Alessandro Alvaro1, Viviane Noll Louzada‑Flores2, Massimo Pajoro1, Ilaria Varotto‑Boccazzi1, Matteo Brilli1, Alessandro Manenti3, Emanuele Montomoli3,4, Gianvincenzo Zuccotti5,6 and Sara Epis1* Abstract Parasites of the genus Leishmania are unusual unicellular microorganisms in that they are characterized by the capability to subvert in their favor the immune response of mammalian phagocytes, including dendritic cells. Thus, in overt leishmaniasis, dendritic cells and macrophages are converted into a niche for Leishmania spp. in which the parasite, rather than being inactivated and disassembled, survives and replicates. In addition, Leishmania parasites hitchhike onto phagocytic cells, exploiting them as a mode of transport to lymphoid tissues where other phagocytic cells are potentially amenable to parasite colonization. This propensity of Leishmania spp. to target dendritic cells has led some researchers to consider the possibility that the non-pathogenic, reptile-associated Leishmania tarentolae could be exploited as a vaccine platform and vehicle for the production of antigens from different viruses and for the delivery of the antigens to dendritic cells and lymph nodes. In addition, as L. tarentolae can also be regarded as a sur‑ rogate of pathogenic Leishmania parasites, this parasite of reptiles could possibly be developed into a vaccine against human and canine leishmaniases, exploiting its immunological cross-reactivity with other Leishmania species, or, after its engineering, for the expression of antigens from pathogenic species. In this article we review published studies on the use of L. tarentolae as a vaccine platform and vehicle, mainly in the areas of leishmaniases and viral infections. In addition, a short summary of available knowledge on the biology of L. tarentolae is presented, together with informa‑ tion on the use of this microorganism as a micro-factory to produce antigens suitable for the serodiagnosis of viral and parasitic infections. Keywords Protozoa, Leishmaniasis, Antigens, Parasites, Viral vaccines, Immunization *Correspondence: Sara Epis sara.epis@unimi.it 1 Department of Biosciences, Pediatric CRC “Romeo ed Enrica Invernizzi”– University of Milan, Milan, Italy 2 Department of Veterinary Medicine, University of Bari, Valenzano, Italy 3 VisMederi, Siena, Italy 4 Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy 5 Department of Biomedical and Clinical Sciences, Pediatric CRC “Romeo ed Enrica Invernizzi”–University of Milan, Milan, Italy 6 Department of Pediatrics, Ospedale dei Bambini-Buzzi, Milan, Italy © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativeco mmons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Bandi et al. Parasites & Vectors (2023) 16:35 Background Dendritic cells, antigen delivery and Leishmania tarentolae Upon their invasion of vertebrate tissues, infectious microorganisms are typically exposed to the action of phagocytic cells, including dendritic cells (DCs). DCs subsequently transport the engulfed microbes to lymph nodes for processing of the microbial proteins into peptides and for the presentation of peptides to CD4+ T cells. DCs thus act as professional antigen presenting cells (APCs), playing a key role in the initiation of the adaptive immune response [1]. Compared to most pathogens, Leishmania parasites are rather unusual in that they exploit APCs of the myeloid cell line as a favourable habitat for their survival and reproduction [2]. In other words, Leishmania parasites are not only passively Page 2 of 13 exposed to the phagocytic activity of DCs and macrophages but they specifically target phagocytic cells thanks to specific surface receptors. Once engulfed by DCs and macrophages, Leishmania parasites subvert the activation of these cells, inhibiting their oxidative and lytic antimicrobial responses, and use these cells as a mode of transport to the lymph nodes [3]. In summary, Leishmania parasites display one of the desired requirements for a vaccine vehicle: a specific targeting of DCs and lymph nodes (Fig. 1). Despite this feature, however, candidate vaccines against leishmaniases based on the use of whole Leishmania cells, either inactivated or attenuated, have not yet been developed into licensed and available vaccines for human or canine use [4–6]. The major drawbacks Fig. 1 Schematic representation of the cutis of a mammal and a lymph node. A Leishmania-infected Phlebotominae sand fly is also represented (1). A dendritic cell (DC) is represented during the process of phagocytosis of a Leishmania promastigote (2), the transformation of the promastigote into the amastigote stage (3) and then the migration of the infected DC towards the lymph node (4). Inside the lymph node, DCs present the antigens to CD4+ T cells. A neutrophil is also shown as these cells play a role in the first phases of mammalian infection by Leishmania spp (5) Bandi et al. Parasites & Vectors (2023) 16:35 of inactivated anti-Leishmania vaccines can be summarized as follows: (i) the limited immunogenicity of inactivated vaccines, as compared to attenuated ones; (ii) an improper modulation of the immune response, associated with the Th2-biasing properties of pathogenic Leishmania species; and (iii) the limits in the scalability to mass production of inactivated cells from pathogenic Leishmania species, and the logistical difficulties in the wide-scale distribution of the derived vaccine preparations [7]. These drawbacks, all of which are typical of inactivated vaccines—in particular the property of limited immunogenicity—have been overcome in other preparations using attenuated living microbes as vaccines. However, this latter approach entails possible safety issues [8] and even greater logistical difficulties for large-scale vaccine campaigns. In this context, the generation of gene-deleted clones to be used in vaccination programs can minimize the risk of reversal to virulence by attenuated pathogenic Leishmania strains [6]. The limits of inactivated and attenuated vaccines have been circumvented using surrogate pathogens as vaccines. For example, the Bacillus Calmette-Guerin (BCG) vaccine is derived from a cattle strain of the bacterium Mycobacterium bovis and developed as a vaccine against human tuberculosis (caused by Mycobacterium tuberculosis) after serial passages in vitro to achieve its attenuation [9]. An attractive feature of BCG vaccine is that this bacterium is suitable for genetic manipulation and therefore for the production of heterologous proteins. This led to the generation of BCG strains expressing antigens from other microorganisms, that have been assayed as candidate vaccines against a variety of pathogens unrelated with the mycobacteria [10]. Leishmania tarentolae, a parasite infecting reptiles, shares several features with BCG in terms of its potential to be exploited as a surrogate pathogen for use in vaccinations against leishmaniasis [11]. In addition, L. tarentolae has the potential to be developed as a vaccine platform of a wider interest, owing to the suitability of this parasite for genetic manipulation and, therefore, the production of heterologous protein antigens [11, 12]. In this article we review current knowledge on this parasite in relation with its potential use in the development of novel types of vaccines, not only against pathogenic Leishmania species, but also against a variety of viruses. The use of L. tarentolae to produce antigens for serological diagnosis will also be discussed. A schematic representation of the life-cycle of L. tarentolae is presented in Fig. 2. Page 3 of 13 Leishmania tarentolae: natural history and evidence of infectivity in mammalian hosts Although L. tarentolae, the type species of the subgenus Sauroleishmania [13], was described more than one century ago in the gecko Tarentola mauritanica [14], information on its natural history remains limited [11] with respect to other species within the subgenera Leishmania and Viannia. Indeed, the 21 species within the subgenus Sauroleishmania have historically been overlooked in leishmaniasis research, being associated with reptiles (from the families Agamidae, Gekkonidae, Lacertidae, Scincidae and Varanidae) and generally regarded to be non-infectious to mammals. In addition, Sauroleishmania species are transmitted by sand flies of the genus Sergentomyia, which have a primarily herpetophilic feeding behavior [15]. The detection of promastigotes of a protist parasite (Paleoleishmania proterus) mixed with reptilian nucleated blood cells in the midgut lumen of a Palaemyia burmitis female sand fly in mid-Cretaceous amber (approx. 100 MYA) suggested that Sauroleishmania evolved in reptiles [16, 17]. However, phylogenetic analyses placing Sauroleishmania as the sister group of the subgenus Leishmania, after the divergence of Viannia, supported the hypothesis that the ancestor of Sauroleishmania evolved in mammals and then switched to reptiles [12–18]. The distribution of L. tarentolae is limited to the Old World [19] in association with geckoes in Europe, North Africa and the Middle East [20–22]. The tight association between Sauroleishmania spp. and reptilebiting Sergentomyia sand flies [23–26] was challenged by the detection of mammalian blood in these sand fly species [27, 28]. These results also raised questions about the potential role of Sergentomyia sand flies as vectors of Leishmania infantum. Meanwhile, individuals of different species of Phlebotomus (e.g. Phlebotomus perfiliewi, P. perniciosus [29–31]) have been found positive for L. tarentolae, most likely as an effect of their opportunistic feeding behavior, which may be linked with several ecological factors and host availability [32]. These observations are further supported by the experimental infection of Phlebotomus papatasi [33, 34], Ph. perniciosus and Phlebotomus sergenti [34] with Sauroleishmania spp. On these premises, we can conclude that Sergentomyia is not the sole genus of sand flies in which Sauroleishmania can develop and that these Leishmania protozoan parasites could also circulate in mammals. Moreover, laboratory experiments have shown that New World sand flies, such as Lutzomyia longipalpis, are competent as vectors for L. tarentolae [35], thus suggesting the potential circulation of this parasite also in the Americas. In the sand fly vector, Sauroleishmania has traditionally been classified as an hypopylarian microorganism since it develops mainly in the hindgut [36]. However, Bandi et al. Parasites & Vectors (2023) 16:35 Page 4 of 13 Fig. 2 Life-cycle of Leishmania tarentolae in sand flies and vertebrate hosts. In reptiles, amastigote-like forms are known to develop in blood cells, but the details of the life-cycle in reptilian hosts are still unknown. Sand flies ingest infected blood cells, and parasites subsequently differentiate into promastigotes, with a hypopylarian or peripylarian type of development. The transmission to vertebrate hosts is likely to occur, in most cases, through the blood meal or through oral ingestion of the fly. Other modes of transmission have also been suggested, including contact with the mucosae of urine droplets containing the parasites. Urine droplets are indeed produced and released by sand fly females during the blood meal through the prediuresis process. Information on transmission and development in mammals is limited the development of some Sauroleishmania species in the midgut [33, 34, 37] of Ph. papatasi and Ph. perniciosus (i.e. peripylarian or suprapylarian), but not in Ph. sergenti, suggests that Sauroleishmania development is influenced by the insect species [34]. In particular, the presence of L. tarentolae promastigotes in the stomodeal valve of some Phlebotomus species [33, 34] supports the possibility of transmission through the blood meal, whereas the localization in the Malpighian tubules suggests transmission through urine droplets (i.e. the product of prediuresis), bite wounds or contact with the mucosae of the hosts. In addition to the many aspects of the Sauroleishmania—sand fly interactions, the potential infectivity of L. tarentolae for mammals has recently been supported after the molecular detection of DNA from this parasite in a 300-year-old Brazilian mummy [38]. This discovery also raised some questions about the host range and geographical distribution of this species and advocated for specific investigations aimed at defining their ability to infect mammalian hosts. It is interesting to note that another species belonging to the subgenus Sauroleishmania, Leishmania adleri, while primarily associated with ectothermic hosts, may infect humans causing transient skin lesions, similar to those observed in cutaneous leishmaniasis [39], and has also been detected in asymptomatic hamsters and mice [40]. Additionally, under laboratory conditions, L. tarentolae promastigotes differentiate into an amastigote-like form after uptake by mammalian DCs and macrophages [41–44], hinting at the possibility of natural infection in mammals. In Mediterranean countries, human and canine leishmaniases are endemic, and L. tarentolae has been isolated from different species of reptiles [12, 45, 46] and sand flies [28], and its DNA has also been detected by molecular methods in mammals [29, 38, 46, 47]. A recent molecular screening revealed the presence of L. tarentolae DNA in humans and sand flies from central Italy and Linosa island [29, 47]. Importantly, infection by L. tarentolae has also been hypothesized as being associated with a reduction in anti-L. infantum antibody titers in dogs seropositive to Bandi et al. Parasites & Vectors (2023) 16:35 canine leishmaniasis, but clinically healthy [48]. Therefore, in regions where L. tarentolae is highly prevalent in reptile and sand fly populations, the presence of this parasite may represent a factor capable of inhibiting the development of clinical forms of canine leishmaniasis, which is associated with antibody-mediated inflammatory reactions [11]. This possibility is further supported by the finding of Sergentomya minuta as the most abundant sand fly species in canine leishmaniasis endemic areas [31]. In summary, available data confirm the sympatric circulation of L. infantum and L. tarentolae in central and southern Italy, also suggesting that geckoes may be infected with L. infantum. Furthermore, the interactions of these two species of Leishmania in these areas are evidenced in their overlapping circulation in “non-natural” hosts and vectors. Therefore, hosts and vectors that have historically been considered to be non-permissive to the one or the other of the two species could actually play a role in the epidemiological cycle of L. infantum and L. tarentolae. Leishmania tarentolae as a biotech tool: use in the production protein antigens for the serodiagnosis of infectious diseases The classical prokaryotic systems for protein expression and production (e.g. the well-known Escherichia coli system) lack essential components for proper protein folding and eukaryotic-type post-translational modifications. Eukaryotic expression systems have thus been proposed in recent decades, although these are characterized by several limitations, such as slow generation time and high costs of culture maintenance [49]. Leishmania tarentolae has been proposed as an alternative system for protein production that overcomes these limitations since it combines the advantages of both eukaryotic and prokaryotic systems: the maintenance and growth of L. tarentolae is accomplished at a low cost and is easily scalable to industrial production using bioreactors. In addition, L. tarentolae boasts a range of post-translational modifications, including mammalian-type N-glycosylation [50]. A particular focus of research is a strain of L. tarentolae that was originally isolated from lizards and subsequently cultivated in axenic culture for decades. This strain is commercially available as a eukaryotic protein expression platform (In Vivo LEXSY translation system, Jena Bioscience GmbH) and can be used to express both intracellular or secreted proteins under the activity of constitutive or inducible promoters (https://www.jenabiosci ence.com/). This system has successfully been exploited to produce both antigens for sero-diagnostic applications and vaccine development (see following section), as well as a variety of proteins to be used in crystallography. Page 5 of 13 A first obvious application of L. tarentolae is the production of protein antigens from other trypanosomatids that can be used in serological diagnosis. In 2015 Rooney and co-workers published the first study in which L. tarentolae was used to produce antigens from Trypanosoma brucei, with the aim to develop a rapid diagnostic test for African trypanosomiases [51]. Another experimental work exploited L. tarentolae for the expression of the rK39 antigen of L. infantum for serodiagnosis of visceral leishmaniasis (VL); the results showed satisfactory diagnostic accuracy when the antigen was tested on sera from patients with VL [52]. Leishmania tarentolae has also been explored as a means to produce protein antigens for the serodiagnosis of viral infections. The gold standard for serological diagnosis of viral infections implies the use of antigens produced in mammalian cells [53], with all the potential limitations, particularly in terms of fast production and rapid application in the case of emerging epidemics from novel pathogens. Two studies have so far been published on the use of protein antigens produced in L. tarentolae for the diagnosis of viral infections. In the first study, an antigen from the hepatitis E virus capsid protein, produced in L. tarentolae, was successfully used in an enzyme-linked immunosorbent assay (ELISA) for the detection of antiviral antibodies in porcine sera [54]. In a more recent study, antigens from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) were thoroughly tested on sera from humans affected by coronavirus disease 2019 (COVID-19) and found to display diagnostic performances comparable to those achieved by antigens produced in human cells [55]. Recombinant protein yield by L. tarentolae can be improved in bioreactors and altered culture growth conditions. Pion et al. [56], for example, engineered Leishmania for the production of recombinant influenza hemagglutinins in biofermenter culture; changes in the culture volume (2 l) and agitation of the culture led to a tenfold increase in the yield. Taking safety issues into account, the production of recombinant proteins to be used as vaccines or in therapeutic applications should ideally avoid the use of culture media containing components of animal origin [57]. However, promastigotes of L. tarentolae are normally cultivated in liquid media with the addition of blood or animal serum, or other components of animal origin [58]. A commonly used medium is brain heart infusion (BHI) growth medium supplemented with animal serum, but there are alternatives for culturing L. tarentolae. For example, Fritsche et al. [59] tested yeast extract medium, with hemin as the sole component of animal origin. Schneider’s Drosophila medium was also successfully tested for cultivation of Leishmania spp., but always in combination with animal serum. Bandi et al. Parasites & Vectors (2023) 16:35 Finally, a classic study proposed catalase or high concentrations of peroxidase as substitute for hemin in BHI medium [60]. The above efforts to set up optimal media and culture conditions for L. tarentolae cultivation will likely increase its suitability as a tool for protein production at the industrial level, a prerequisite for translational application of this microorganism to produce antigens, vaccines or therapeutics [61]. Leishmania tarentolae as a surrogate of pathogenic leishmaniae in terms of vaccination, and the issue of immune modulation in anti‑Leishmania vaccines The origin of vaccines is traced back to 1796, when Edward Jenner proposed the inoculation of material from cowpox lesions as a treatment to protect humans from smallpox. In modern terminology, the first vaccine comprised a surrogate pathogen (the cowpox virus) that causes only mild infections in humans but nonetheless elicits cross-protective immunity against the human pathogen, namely the smallpox virus [62]. During the first half of the twentieth century, BCG was developed as a surrogate of the human pathogen M. tuberculosis and is still widely used in vaccination campaigns against tuberculosis (see section Background). The use of microorganisms that infect animal species other than humans as surrogates of human pathogens for vaccination targets is thus part of the history of vaccinology and should not be dismissed as an obsolete or useless strategy. Several lines of evidence suggest that L. tarentolae could represent a surrogate pathogen and that it is suitable to be assayed as a vaccine against human and canine leishmaniases. First, cross-protective immunity is well documented in Leishmania infections (for example see [63] and citations therein). The existence of cross-protective immunity in Leishmania species has also been documented at the molecular level [64] or using sub-components of Leishmania cells, as in the LBSap vaccine which, while being composed of antigens from Leishmania braziliensis, is protective against Leishmania infantum [65, 66]. Second, comparative genomics show that L. tarentolae shares approximately 90% of its genes with pathogenic species, including L. infantum and Leishmania major. However, several genes possibly associated with virulence are lacking in L. tarentolae, compared to pathogenic Leishmania species [67], which is reassuring in relation to safety issues. Third, current evidence indicates that L. tarentolae is not pathogenic to mammals, while still being capable of infecting macrophages and DCs, reaching the amastigote state [41, 44, 68]. Finally, the evidence for a circulation of L. tarentolae in dogs, and the absence of any evidence for its association with pathological outcomes, further emphasizes the potential utility of this Page 6 of 13 parasite in anti-Leishmania vaccination (see [11] and section on L. tarentolae natural history and the evidence of infectivity in mammalian hosts). A major issue that should be addressed to determine the potential of L. tarentolae as a vaccine against pathogenic leishmaniae is the type of immune modulation that this reptile parasite determines in mammals. There is a general consensus that hosts displaying classical macrophage activation (M1 activation), with specific polarization of CD4+ T cells towards the Th1 phenotype, are protected against infection and overt disease with most forms of leishmaniases, in both dogs and humans, while hosts displaying alternative macrophage activation (M2 activation) and a Th2-biased response are more exposed to the risk of developing active Leishmania infection and overt disease [69, 70]. For this reason, a central focus in leishmaniasis vaccine research is the identification of adjuvants capable of determining a proper polarization of the immune response towards the M1/Th1 profile [71]. Initial studies, using the murine model, showed that L. tarentolae determines Th1-type cytokine production in vivo [41]. However, most of the subsequent studies suggested that elicitation of a Th1 response requires co-administration of the appropriate adjuvants, or the concomitant expression of immune-modulating molecules [72, 73]. However, the expression of M2 markers in human macrophages, after stimulation with L. tarentolae, was also observed [74]. A recent in vitro study on human DCs reported moderate production of Th1-type cytokines after stimulation by living promastigotes of L. tarentolae and also confirmed the penetration of L. tarentolae into DCs and their maturation, with expression of surface markers of activation, including MHC class II and co-stimulatory molecules [44]. The capacity of DCs to engulf L. tarentolae, the maturation of these cells after exposure to the parasite and evidence for the production, even though moderate, of Th1-associated cytokines are all coherent with the possibility that L. tarentolae possesses some of the characteristics required by an antiLeishmania vaccine. Despite the above theoretical arguments in favor of the potential utility of L. tarentolae as a vaccine against pathogenic Leishmania species, only a few studies have investigated the issue experimentally, always in rodent models. In a first seminal study, intraperitoneal immunization with L. tarentolae was found to achieve a protective immune response against Leishmania donovani in BALB/c mice, associated with a strong Th1 response in the absence of adjuvant [41]; this result was, however, not confirmed by other studies. In that study, the promastigotes of L. tarentolae were not engineered for the expression of any antigen from pathogenic Leishmania Bandi et al. Parasites & Vectors (2023) 16:35 species, nor for any proteins capable of stimulating the immune response, but only for the production of the green fluorescent protein. Live, non-engineered, promastigotes of L. tarentolae have also been shown to provide cross-protective immunity against L. major [75, 76]. The possibility of exploiting wild-type L. tarentolae as a vaccine against dog or human pathogenic Leishmania species is thus worthy of further investigations (see section Concluding remarks). Engineered Sauroleishmania strains as vaccines against pathogenic Leishmania species One possible strategy to increase the immunogenicity of L. tarentolae as a vaccine against human and dog leishmaniases is to engineer this microorganism for the expression of antigens from pathogenic Leishmania species or of immune-modulating proteins associated with M1/Th1 immune activation. Briefly, in anti-leishmania vaccine studies, L. tarentolae has been genetically modified (GM) for the production of the following categories of proteins: (i) antigens from pathogenic Leishmania species; (ii) immune-modulating proteins of human origin; and (iii) antigens or immune-modulating proteins from the sand fly saliva. Strains of L. tarentoale have also been engineered for the production of both immune-modulating proteins and antigens from pathogenic Leishmania species. Studies on GM L. tarentolae in anti-Leishmania vaccination tests are summarized in Table 1. A brief account, focused on protein categories, is also provided in the following paragraphs. Different protein antigens from pathogenic Leishmania species have been investigated in anti-Leishmania vaccine research, with L. tarentolae used as a vehicle for their production and delivery. For example, administration of L. tarentolae engineered to express the A2 antigen from L. donovani provided protection against L. infantum in a murine model of the infection [77]. Other studies using L. tarentolae as a vaccine vehicle exploited the LACK and KMP11 antigens (for the full name of these antigens, see Table 1), that are expressed both in the promastigote and the amastigote stage and are involved in the infection of mammalian hosts [78, 79]. Immunization with L. tarentolae expressing these proteins, co-adjuvated with CpG motifs, achieved a reduction of parasite load by Leishmania major in a murine model [73]. As a third example, live L. tarentolae expressing LPG3 from L. infantum, a protein chaperon involved in the biosynthesis of lipophosphoglycans, induced the expression of high level of interferon gamma (IFN-γ) in the murine model, and partial protection against L. infantum infection, without co-administration of any adjuvant [80]. As already emphasized, L. tarentolae has been exploited not only to express and deliver protein antigens Page 7 of 13 from pathogenic Leishmania parasites, but also for the production and delivery of immune-modulating molecules. Recent findings point to CXCL-10 (C-X-C motif chemokine 10) as a potential tool for immunotherapy of cutaneous leishmaniasis, since this molecule favors a shift towards a Th1 pro-inflammatory response [81]. Indeed, inoculation of L. tarentolae expressing CXCL10 in mice inhibited arginase activity and Th2 cytokine expression towards IFN-γ and nitric oxide production [82]. Another example of an immune-modulating molecule investigated in vaccine research is the human neutrophil peptide (HNP1), which is a member of the large antimicrobial peptides (AMPs) group [83]. In a study published in 2017, a strain of L. tarentolae engineered for the expression of HPN1 from L. major was tested in the murine model; the results showed containment of the parasite load after challenge with L. major itself [84]. The contribution of a vector’s saliva in the immunology of leishmaniases has been extensively been studied in recent decades, with identification of highly immunogenic molecules, such as the PpSP15 protein from Phlebotomus papatasi [85]. Regarding the use of engineered Sauroleishmania in vaccination, the administration of GM L. tarentolae expressing the cysteine proteinases A and B (CPA/CPB) genes, in co-administration with a DNA plasmid coding for the sand fly salivary antigen PpSP15, was shown to be very effective in murine models as a vaccine against L. major [86]. Leishmania tarentolae expressing PpSP15 was also effective in terms of immunization against L. major infection when administered in combination with CpG oligodeoxynucleotides [87]. More recently, the combination of two salivary proteins from the sand fly, PpSP15 and PpSP9, co-expressed and delivered by L. tarentolae, proved to be effective in immunization against two different Leishmania species, L. major and L. tropica [88]. Microbial vaccine vehicles: Leishmania tarentolae as an anti‑viral vaccine At the time of writing this article, four major vaccine technologies or platforms are being used extensively for the production of antiviral vaccines: (i) RNA vaccines delivered through lipid nanoparticles; (ii) adenovirusbased viral vectors; (iii) subunit vaccines, using recombinant protein antigens, produced in eukaryotic expression systems; and (iv) inactivated or attenuated viruses [89]. In addition to these types of platforms, a fifth approach is based on the administration of whole bacterial cells that are engineered for the expression of viral antigens (Fig. 3) [90–92]. The core idea of this strategy is that GM bacteria can be exploited not only as micro-factories to produce the desired antigen, but also as vehicles for the delivery of Bandi et al. Parasites & Vectors (2023) 16:35 Page 8 of 13 Table 1 Use of Leishmania tarentolae as an antigen in vaccination or as a vaccine vehicle, or to produce viral proteins or virus-like particles Type of expressed molecule Whole Lt and/or purified protein Expressed molecule Target infection/disease Animal model Selected references WT Whole Lt // Leishmaniasis Murine [75, 76] GM for: GFP Whole Lt GFP to trace Leishmania tarentolae Leishmaniasis Murine [41] GM for: pLs prot Whole Lt and/or purified protein Leishmaniasis pLs A2 antigen pLs A2 antigen + cysteine proteinases A and B (CPA and CPB without C terminal extension) pLs Leishmania homolog of the receptor for the acti‑ vated C kinase (LACK) + pLs kinetoplastida membrane protein-11 (KMP11) Leishmania infantum lipophosphoglycan 3 (LPG3) pLs cysteine proteinases A and B (CPA, CPB) Murine Hamster [73, 77, 80, 86, 106, 107] GM for: IM prot Whole Lt Interferon-gamma-induced protein 10 (IP-10) or CXCL-10 Human neutrophil pep‑ tide-1 (HNP-1) Immunogenic Phlebotomus papatasi salivary molecule SP15 (PpSP15) Immunogenic Ph. papatasi salivary molecule SP15 (PpSP15) + Phlebotomus sergentii salivary molecule SP9 (PsSP9) Leishmaniasis Murine [82, 84, 87, 88] GM for: IM prot + pLs prot Whole Lt pLs kinetoplastida membrane protein-11 (KMP11) + N-terminal domain of heat shock protein GP96 (NT-GP96) Leishmaniasis Murine [108] GM for: other protozoan protein Whole Lt and/or purified protein Fusion of immunodominant Toxoplasmosis epitopes of 5 Toxoplasma antigens: SAG1-ROP16GRA12-MIC4-M2AP Murine [99] GM for: viral protein Whole Lt and/or purified protein HPV16-L1 SARS-CoV-2 spike protein/ RBD HIV-1 Gag HCV envelope glycopro‑ teins (HCV E1-E2) HPV SARS-CoV-2 HIV HCV Murine Human tissue [42, 98a,100, 109] GM for: IM prot + viral protein Whole Lt and/or purified protein HPV HCV Murine [72, 101] GM for: VLPs VLPs HCV polytope (PT) fused to the N-terminal domain of heat shock protein GP96 (HCV-PT + NT-GP96) (HPVE7 + NT-GP96) + (HPVE7 + CT-GP96) HPV HBV HCV NoV Murine [102–105] HPV late protein 1 (HPV16 L1) HBV small surface antigen (sHBsAg) presenting epitopes derived from the HCV E2 glycoprotein NoV Capsid Prot Bandi et al. Parasites & Vectors (2023) 16:35 Page 9 of 13 Table 1 (continued) GFP Green fluroescent protein, GM genetically modified Leishmania, HBV/HCV hepatitis B/C virus, HIV human immunodeficient virus, HPV human papillomavirus, IM prot immune-modulating protein, Lt Leishmania tarentolae, NoV norovirus, pLs prot pathogenic Leishmania species protein, prot protein, SARS-CoV-2 severe acute respiratory syndrome coronavirus 2, VLPs virus-like particles, WT wild type/non-engineered a A purified recombinant protein and Leishmania cells expressing a recombinant protein were co-administered the antigens themselves, ideally targeting DCs and lymph nodes. These GM bacteria are referred to as bacterial vaccine vehicles, or as living bacterial vehicles, when used without inactivation. In the context of antiviral vaccination, a major limitation with the use of bacteria as antigen vehicles, or even for the simple production of the antigens, is that protein expressed in bacteria are not subjected to post-translational modifications, which are instead characteristic of proteins produced in mammalian cells [93, 94]. Therefore, viral antigens expressed in bacteria are expected to present structural differences in comparison with antigens produced by mammalian cells during viral infections. In addition to bacteria, yeasts have also been proposed as vaccine vehicles for the production and delivery of viral antigens (see, for example, [95]). Indeed, production of protein antigens in yeasts ensures post-translational modification of the antigen, but the glycosylation pattern in yeast-produced proteins is rather different from that of mammalian proteins [96]. Considering the glycosylation issue, L. tarentolae emerges as an alternative solution to produce viral antigens: as outlined above, the glycosylation pattern in this microorganism resembles that of mammals. In addition, both the easy culturing and genetic engineering of L. tarentolae, as well as its propensity to target DCs, make this protozoon an ideal candidate for its use as a microbial antigen vehicle for anti-viral vaccination. We emphasize that the use of GM L. tarentolae as candidate anti-Leishmania vaccines (see above sections) also exploits this microorganism as a vehicle for the antigen. Published studies on the use of L. tarentolae as an antigen vehicle in anti-viral vaccination have so far targeted eight different viruses (Table 1). A first study in this area exploited L. tarentolae for the expression and delivery of the Gag protein from human immunodeficiency virus type 1 (HIV-1). The assays in the murine model led to an effective immune response, with the production of Fig. 3 Use of microbial vectors in vaccination against viruses and other pathogens. A gene coding for an antigen is selected from a pathogen and cloned for its expression into a microbial vehicle (1). Different types of expression could be exploited (2). The selected microbial vehicle is then used for the immunization of the host (3). GM, Genetically modified, WT, wild type Bandi et al. Parasites & Vectors (2023) 16:35 neutralizing antibodies [42]. Another virus that has been targeted in preclinical studies using GM L. tarentolae is human papillomavirus (HPV), in a murine model of the tumor associated with this virus [97]; the results of that study revealed a contained growth of the tumor in vaccinated mice. Finally, GM L. tarentolae expressing the spike protein of SARS-CoV-2 has recently been assayed in mice, and the production of neutralizing antibodies was observed [98]. In the latter study, rectal administration of the engineered strain of L. tarentolae was also shown to be effective in the induction of the neutralizing response, paving the way toward the use of L. tarentolae as an antigen vehicle for mucosal vaccination. In addition to the exploitation of L. tarentolae as an antigen vehicle, this protozoon has also been used to produce viral protein antigens that have been assayed as purified proteins, alone or in combination with GM L. tarentolae strains [e.g. 98], or virus-like particles, that have been assayed in preclinical studies against four different viruses (Table 1). Finally, L. tarentolae has also been investigated for the production of a candidate vaccine against the apicomplexan protozoon Toxoplasma gondii [99]; in that study, a multi-epitope vector-based vaccine, based on immunodominant epitopes of five Toxoplasma antigens, conferred protective immunity against acute toxoplasmosis in BALB/c mice [99]. Concluding remarks Leishmania tarentolae has a great potential as a candidate vaccine, both as non-engineered, WT organism, possibly in combination with adjuvating molecules favoring Th1-biased responses, or after its engineering, for the expression of antigens and/or immune-modulating molecules. However, several research issues still need to be addressed for an effective translation into practice. The use of L. tarentolae as a wild-type whole organism for vaccination of dogs (and possibly humans) will require further studies in rodent models, with also the need to understand whether the parasite can actually replicate within the mammalian host, ensuring adequate stimulation of the immune response, without causing any pathological alteration. In this context, a very important issue is whether wild-type L. tarentolae administered as living cells would benefit from co-administration with an adjuvant. Indeed, co-administration with adjuvants could stimulate an excessive immune response against L. tarentolae, with early inactivation of the parasites, and thus reduced replication. Therefore, investigations on adjuvating strategies capable of favoring the immune response in the Th1 direction while allowing L. tarentolae to undergo a few replication cycles will be of great importance. Regarding the strains of L. tarentolae engineered for vaccination against pathogenic protozoa of Page 10 of 13 the genus Leishmania, viruses or other infectious agents, the effective use of these strains will require addressing the problems related with their status as GM organisms, obtained by incorporating one or more genes for antibiotic resistance. The actual use of these strains could therefore require the removal of resistance genes or the development of metabolic complementation strategies for the selection of transformed strains as an alternative to antibiotic-based selection. More generally, the production of L. tarentolae cells for an effective use in vaccination campaigns will require production to be scaled up to the level of industrial bio-fermenters, with an optimization of the culture media, in the absence of antibiotic pressure (see above). In any case, the increased interest in L. tarentolae, both as a vaccine platform and more generally as a micro-factory to produce recombinant proteins for a variety of applications, will likely lead to an acceleration of research on this organism and to solutions to the above issues. Acknowledgements Figures were created with BioRender.com. CB and SE thank the GSA-IDEA pro‑ ject. CB, SE and DO thank the PNRR project PE-13, INF-ACT One Health Basic and Translational Research Actions addressing Unmet Needs on Emerging Infectious Diseases. Author contributions CB, DO, SE: conceived and designed the structure and overall content of this article. MP, MB, AA, IVB, JAMR, DO, VNLF, AM, GVZ, EM: conducted the literature search, screened articles, extracted data and conceived the figures. CB, SE, JAMR, DO: wrote the manuscript. All authors read and approved the final manuscript. Funding CB and SE received funding from “Fondazione Romeo ed Enrica Invernizzi” (Grant Agreements No. LIB_VT21SEPIS and LIB_VT22CBAND). Availability of data and materials Not applicable. Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Received: 28 November 2022 Accepted: 3 January 2023 References 1. Worbs T, Hammerschmidt SI, Förster R. Dendritic cell migration in health and disease. Nat Rev Immunol. 2017;17:30–48. 2. Martínez-López M, Soto M, Iborra S, Sancho D. Leishmania hijacks myeloid cells for immune escape. Front Microbiol. 2018;9:883. Bandi et al. Parasites & Vectors (2023) 16:35 3. Liu D, Uzonna JE. 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Comparison of protective potency of DNA and live vaccines expressing A2-CPA-CPB-CTE antigens against visceral leishmaniasis in syrian hamster as preliminary study. Iran J Parasitol. 2020;15:383–92. 108. Nasiri V, Dalimi A, Ghaffarifar F, Bolhassani A. Immunogenicity and efficacy of live L. tarentolae expressing KMP11-NTGP96-GFP fusion as a vaccine candidate against experimental visceral Leishmaniasis caused by L. infantum. Iran J Parasitol. 2016;11:144–58. 109. Shirbaghaee Z, Bolhassani A, Mirshafiey A, Motevalli F, Zohrei N. A live vector expressing HPV16 L1 generates an adjuvant-induced antibody response in-vivo. Iran J Cancer Prev. 2015;8:e3991. Page 13 of 13 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub‑ lished maps and institutional affiliations. Ready to submit your research ? 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https://openalex.org/W4232926870	https://rjs.com.ro/articles/2015.4/RJS_2015_4_Art-01.pdf	Romanian, Moldavan	null	null	Romanian Journal of Stomatology	2,015	cc-by	4,762	1 1 PROTETICĂ DENTARĂ REZUMAT Obiective. Scopul acestui studiu a fost verificarea respectării curburii variaţiei cromatice a dinţilor naturali din zona frontală maxilară de către garniturile de dinţi artificiali, precum şi identificarea garniturilor care se apropie cel mai mult de această variaţie. Materiale şi metodă. Un examinator fără deficit de percepţie vizuală şi cu experienţă în utilizarea spectrofotometrelor de uz introral a studiat din punct de vedere cromatic 8 garnituri de dinţi destinate înlocuirii grupului frontal maxilar. (13-23) Patru dintre acestea sunt garnituri de dinţi confecţionaţi din acrilat: Star Lux (Ruthinium Grup, Italia), Vita MTF (Vita, North America), Spofadent Plus (SpofaDent s.a, Cehia) şi Acry Rock (Ruthinium Grup, Italia), 2 garnituri sunt confecţionate din răşină compozită: Ivoclar Ivostar (Ivoclar Vivadent, Lichtenstein) şi Pigeon Summit (Pigeon Dental, China) şi 2 garnituri de dinţi ceramici: Vita Lumin Vacuum (Vita Zahnfabrik,Germania) şi Enta Ceram (Enta B.V., Olanda). ( ) ş ( ) Pentru fiecare dinte din fiecare garnitură s-au efectuat câte 5 măsurători cu aparatul Vita Easy Shade (Vita Zahnfabrik, Germania) şi s-au înregistrat valorile CIE Lab. Rezultatele au fost comparate cu valorile medii obţinute la măsurarea dinţilor naturali. Pentru a calcula variaţia cromatică între diverse elemente de interes în acest studiu, s-a folosit formula consacrată pentru diferenţa relativă de culoare: . (1) ( ) Pentru fiecare garnitură de dinţi s-a observat variaţia culorii între fiecare doi dinţi vecini şi s-a raportat la variaţia culorii dintre perechea echivalentă de dinţi naturali. Diferenţa medie dintre natural şi artificial s-a raportat la valoarea ADA considerată ca limita perceptibilităţii cromatice a ochiului uman (2 unităţi). apo a a a oa ea co s de a ă ca a pe cep b ăţ c o a ce a oc u u u a ( u ăţ ) Rezultate. S-a observat că anumite garnituri de dinţi, pentru anumite grupe de dinţi adiacenţi, au un comportament cromatic comparabil cu cel al dinţilor naturali în limitele perceptibilităţii vizuale, în timp ce altele nu. STUDIU COMPARATIV PRIVIND DISTRIBUŢIA CROMATICĂ LA NIVELUL DINŢILOR FRONTALI MAXILARI DIN GARNITURILE DE DINŢI ARTIFICIALI, RAPORTATĂ LA VARIAŢIA CROMATICĂ A DINŢILOR FRONTALI MAXILARI NATURALI Comparative study regarding the chromatic distribution among anterior maxillary denture teeth in relation to color variation among natural anterior maxillary teeth Asist. Univ. Dr. Andrei Constantinovici1, Prof. Dr. Mihaela Păuna1, Conf. Dr. Oana Cella Andrei1, Şef Lucr. Dr. Titus Farcaşiu1, Asist. Univ. Dr. Adriana Bisoc1, Asist. Univ. Dr. Livia Alice Tănăsescu1, Şef Lucr. Dr. Nicoleta Măru2, Dr. Ionel Iosif3 Dr. Andrei Constantinovici1, Prof. Dr. Mihaela Păuna1, Conf. Dr. Oana Cella Andrei1, Titus Farcaşiu1, Asist. Univ. Dr. Adriana Bisoc1, Asist. Univ. Dr. Livia Alice Tănăsescu1, 1Universitatea de Medicină şi Farmacie „Carol Davila“ Bucureşti, Facultatea de Medicină Dentară, Disciplina Protezare Parţială Mobilizabilă 2Universitatea de Medicină şi Farmacie „Carol Davila“ Bucureşti, Facultatea de Medicină D Disciplina Anatomie 2Universitatea de Medicină şi Farmacie „Carol Davila“ Bucureşti, Facultatea de Medicină Dentară, Disciplina Anatomie 3Centrul Naţional de Supraveghere şi Control al Bolilor Transmisibile, Bucureşti 3Centrul Naţional de Supraveghere şi Control al Bolilor Transmisibile, Bucureşti p Asist. Univ. Dr. Andrei Constantinovici, Bd. Iancu de Hunedoara nr. 35, sc. A, ap. 8, sector 1, Bucureşti, România, cod poştal 011733 E-mail: andrei.constantinovici@gmail.com Autor corespondent: ABSTRACT bjective. The aim of this study was to check whether the ways in which the color of anterior maxillary teeth var- s in artificial dental sets matches the way in which color varies in the anterior maxillary natural dentition. Objective. The aim of this study was to check whether the ways in which the color of anterior maxillary teeth var- ies in artificial dental sets matches the way in which color varies in the anterior maxillary natural dentition. Material and method. One examiner, free of any visual color deficiencies, experienced in the use of intraoral shade matching devices, has studied the chromatic behavior within the maxillary anterior dental group (13-23) for 8 types of artificial denture teeth sets. Four of these sets are made out of acrylic resin: Star Lux (Ruthinium Grup, Italia), Vita MTF (Vita, North America), Spofadent Plus (SpofaDent s.a, Cehia) and Acry Rock (Ruthinium Grup, Italia), 2 sets are made of composite resin: Ivoclar Ivostar (Ivoclar Vivadent, Lichtenstein) and Pigeon Summit (Pigeon Dental, China) and 2 of them are ceramic sets of denture teeth: Vita Lumin Vacuum (Vita Zahnfabrik, Germania) and Enta Ceram (Enta B.V., Olanda). With the help of the Vita Easy Shade (Vita Zahnfabrik – Ger- many), CIELab chromatic parameters have been recorded for each tooth. 5 distinct measurements were car- ried out for every tooth. The results were later compared to the values obtained when measuring color variation in natural anterior maxillary teeth. When calculating the relative color difference between two teeth, the following formula has been used: . (1) For each set of denture teeth, the variation of color between every two adjacent teeth was noted and compared to the color variation between the correspondent natural teeth. The mean difference between these two results was compared to the ADA limit value for human eye perceptibility of color difference (2 units). Results. The following aspects have been noted: some sets of denture teeth, for certain groups of teeth, show a similar chromatic behavior to natural teeth whereas other doesn’t. For the pair of teeth 13/12, only 4 of the studied sets of denture teeth showed an unperceivable difference in color variation when compared to natural dentition (Spofadent Plus, Star Lux, Vita Lumin Vacuum, and Ivoclar Ivostar). Lotul de studiu Au fost studiate 8 garnituri de dinţi artificiali. Patru dintre acestea sunt garnituri de dinţi con- fecţionaţi din acrilat: Star Lux (Ruthinium Grup, Italia), Vita MTF (Vita, North America), Spofadent Plus (SpofaDent s.a, Cehia) şi Acry Rock (Ruthi- nium Grup, Italia), două garnituri sunt confecţionate din răşină compozită: Ivoclar Ivostar (Ivoclar Vivadent Lichtenstein) şi Pigeon Summit (Pigeon Dental, China) şi 2 garnturi de dinţi ceramici: Vita Lumin Vacuum (Vita Zahnfabrik, Germania) şi Enta Ceram (Enta B.V., Olanda). Criteriul de selecţie a garniturilor de dinţi artificiali a fost frecvenţa de utilizare în laboratorul de tehnică dentară al Fa- cultăţii de Medicină Dentară din cadrul UMF „Carol Davila“ Bucureşti în anul 2015, când s-a desfăşurat experimentul ce a stat la baza prezentului studiu. Din cadrul fiecărei garnituri de dinţi arti fi- ciali a prezentat interes strict grupul frontal ma xilar (13-23). Pentru fiecare garnitură de dinţi, fiecare Scopul acestui studiu a fost verificarea respectării curburii variaţiei cromatice a dinţilor naturali din zona frontală maxilară de către garniturile de dinţi artificiali, precum şi identificarea garniturilor care se apropie cel mai mult de această variaţie. INTRODUCERE diului, exminatorul avea o experienţă de 5 ani în utilizarea aparatului Vita Easy Shade (Vita Zahn- fabrik – Germania), precum şi cunoştinţe avansate privind modalităţile de determinare cromatică dentară. Obiectivul oricărei restaurări estetice este re- darea naturalului. (2) În anul 2011, un studiu publi- cat în BMC Oral Health (3) susţine că 89,3% dintre persoanele ches tio nate consideră culoarea drept prin- cipalul element nesatisfăcător al propriilor restaurări dentare, prin urmare aprofundarea studiului culorii în protetica dentară este justificată. De asemenea, căutând în literatura de specialitate nu am găsit alte studii care să compare variaţia cromatică a gar- niturilor de dinţi artificiali cu variaţia cromatică a dinţilor naturali. REZUMAT În cazul perechii 13/12, variaţia este imperceptibilă numai în 4 cazuri (Spofadent Plus, Star Lux, Vita Lumin Vacuum şi Ivoclar Ivostar), în cazul perechii 12/11 acest lucru se întâmplă în 5 cazuri (Star Lux, Spofadent Plus, Vita MTF, Pigeon Summit şi Vita Lumin Vacuum), pentru perechea 11/21 s-au obţinut 5 cazuri (Vita Lumin Vacuum, Acry Rock, Ivoclar Ivostar, Star Lux, Spofadent Plus), în cazul perechii 21/22 am găsit 5 cazuri (Spofadent Plus, Ivoclar Ivostar, Vita MTF, Acry Rock, Star Lux) şi niciun caz pentru perechea 22/23. În limitele acestui studiu am putut afirma că utilizarea garniturii Spofadent Plus duce la obţinerea unor rezultate cromatice cu o variaţie cât mai apropiată de estetica dinţilor naturali. ţ p p ţ Concluzii. Deşi sunt codificate cromatic la fel, garniturile de dinţi artificiali prezintă culori diferite, în funcţie de producător, indiferent de materialul din care sunt confecţionate. Comportamentul cromatic de la un dinte artificial la altul diferă, de asemenea, în funcţie de producător. Domeniul cromaticii dinţilor din garniturile artificiale necesită studiu suplimentar. Concluziile acestui experiment stabilesc o serie de direcţii generale ce pot fi urmate de studii ulterioare, mai aprofundate. Cuvinte cheie: culoare dentară, variaţie cromatică, spectrofotometru, dinţi anteriori, dinţi artificiali REVISTA ROMÂNÅ DE STOMATOLOGIE – VOLUMUL LXI, NR. 4, AN 2015 261 262 REVISTA ROMÂNÅ DE STOMATOLOGIE – VOLUMUL LXI, NR. 4, AN 2015 ABSTRACT 5 is the number of matching sets of denture teeth for the 12/11 pair (Star Lux, Spofadent Plus, Vita MTF, Pigeon Summit, and Vita Lumin Vacuum), 5 sets also matched for the 11/21 pair (Vita Lumin Vacuum, Acry Rock, Ivoclar Ivostar, Star Lux, and Spofadent Plus). For the pair 21/22 5 matching sets were found (Spofadent Plus, Ivoclar Ivostar, Vita MTF, Acry Rock, Star Lux) and no matching sets were found for the 22/23 pair of teeth. Within the limits of this study we can affirm that using the Spofadent Plus set of denture teeth would lead to prosthetic results that show a variation of color within the ante- rior maxillary dental group most similar to natural teeth. Conclusions. Despite the fact that they bare the same name code, different sets of denture teeth produced by different brands have different colors, irrespective of the material they are made of. Chromatic behaviour, from one tooth to the next also differs from one make to another. The field of color in artificial denture teeth requires supple- mentary attention. The conclusions of this study set a general direction that can be followed in future studies. Keywords: dental color, color variation, spectrophotmeter, anterior teeth, artificial teeth Examinatorul Examinatorul a fost un medic dentist, specialist în protetica dentară. Capacitatea de diferenţiere cro matică a examinatorului a fost evaluată în ser- viciul de oftalmologie. În momentul efectuării stu- REVISTA ROMÂNÅ DE STOMATOLOGIE – VOLUMUL LXI, NR. 4, AN 2015 263 din cei 6 dinţi frontali a fost măsurat din punct de ve- dere cromatic cu ajutorul aparatului Vita Easy Shade (Vita Zahnfabrik, Germania). Deşi iniţial instruc- ţiunile de utilizare ale aparatului (4) recomandă utilizarea acestuia numai pentru determinarea cro- matică a dinţilor naturali şi pentru verificarea cro- matică a nuanţei restaurărilor dentare, studii suc- cesive au demonstrat posibilitatea întrebuinţării aparatului şi pentru evaluarea cromatică a dinţilor artificiali din garnituri (5). De asemenea, şi alţi autori susţin posibilitatea utilizării aparatelor de tip spectrofotometru intraoral pentru evluarea atât a dinţilor naturali, cât şi a materialelor de restaurare (6). Pentru fiecare dinte au fost efectuate câte 5 măsurători şi s-au înregistrat variabilele caracteris- tice spaţiului cromatic CIE Lab. Datele au fost sistematizate într-un tabel. Valorile de referinţă, la care s-au raportat aceste măsurători, au fost datele obţinute de autori într-un studiu anterior (7). Pentru a calcula variaţia cromatică între diverse elemente de interes în acest studiu, s-a folosit formula con- sacrată pentru diferenţa relativă de culoare (1). zona frontală maxilară de către garniturile de dinţi artificiali, precum şi identificarea garniturilor care se apropie cel mai mult de această variaţie. Astfel, s-a formulat ipoteza nulă cum că nu există diferenţe între modul în care culoarea variază în grupul frontal maxilar al garniturilor de dinţi studiate şi modul de variaţie a culorii în cadrul dinţilor frontali maxilari ai dentiţiei naturale. Datele au fost anali- zate cu ajutorul software-ului SPSS versiunea 2.2. Distribuţia datelor a fost verificată prin testele Skew ness şi Kurtosis (valoarea lor <2ES) şi prin testul Shapiro-Wilk a cărui valoare p a fost >0,05 (se păs trează ipoteza conform căreia datele sunt uniform distribuite). Totodată, distribuţia datelor poate fi ve rificată şi prin metoda grafică (histograma şi grafice Q-Q Plot). Distribuţia uniformă a datelor permite aplicarea ulterioară a testelor parametrice de semnificaţie statistică. REZULTATE Valorile cromatice Lab* ale perechii de dinţi 13/12 din fiecare garnitură de dinţi artificiali au fost introduse în formula de determinare a diferenţei relative de culoare ∆E. Diferenţele au fost com- parate cu variaţiile culorii între 13 şi 12 în cazul dinţilor naturali. Rezultatele au fost sintetizate într- un tabel (Tabelul 1). Pentru fiecare garnitură de dinţi s-a observat variaţia culorii între fiecare doi dinţi vecini şi s-a raportat la variaţia culorii dintre perechea echi- valentă de dinţi naturali: de exemplu, variaţia dintre 13 şi 12 din garnitura Acry Rock a fost comparată cu variaţia cromatică dintre 13 şi 12 în cazul dinţilor naturali ş.a.m.d. Aceleaşi calcule au fost efectuate pentru fiecare pereche de dinţi adiacenţi: 12/11, 11/21, 21/22, 22/23. Rezultatele obţinute au fost următoarele (Tabelele 2-5): Metode statistice Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 11-21 VITA LUMIN VACUUM 5 1.99 -0.020 0.586 0.973 -1.169 1.129 2 11-21 Acry Rock 5 2.23 -0.260 .629 0.682 -1.492 0.972 3 11-21 IVOCLAR IVOSTAR 5 5 -0.450 0.589 0.45 -1.604 0.704 4 11-21 Star Lux 5 2.62 -0.65 0.601 0.288 -1.829 0.529 5 11-21 ENTA CERAM 5 1.2 0.950 0.586 0.115 -0.199 2.099 6 11-21 Spofadent Plus 5 3.83 -1860 0.867 0.039 -3.559 -0.161 7 11-21 Vita MFT 5 3.72 -3750 0.717 0.001 -5.155 -2.345 TABELUL 4. Perechea 21/22 Nr. Culoare naturală nr=30/4.56 Nr. Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 21-22 Spofadent Plus 5 4.46 0.100 1.467 0.946 -2.775 2.975 2 21-22 IVOCLAR IVOSTAR 5 3.47 1.090 1.414 0.446 -1.681 3.861 3 21-22 Vita MFT 5 3.22 1.340 1.408 0.348 -1.420 4.100 4 21-22 Acry Rock 5 2.86 1.700 1.483 0.260 -1.206 4.606 5 21-22 Star Lux 5 2.67 1.890 1.404 0.187 -0.862 4.642 6 21-22 VITA LUMIN VACUUM 5 2.38 2.180 1.405 0.130 -0.575 4.935 7 21-22 PIGEON SUMMIT 5 2.06 2.500 1.404 0.084 -0.252 5.252 8 21-22 ENTA CERAM 5 1.25 3.310 1.405 0.025 0.557 6.063 TABELUL 5. Perechea 22/23 Nr. Culoare naturală nr=30/6.8 Nr. Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 22-23 Star Lux 5 4.23 2.570 1.213 0.042 0.193 4.947 2 22-23 Spofadent Plus 5 3.71 3.090 1.226 0.017 0.688 5.492 3 22-23 Vita MFT 5 3.28 3.520 1.221 0.007 1.127 5.913 4 22-23 VITA LUMIN VACUUM 5 3.08 3.720 1.210 0.004 1.349 6.091 5 22-23 PIGEON SUMMIT 5 2.91 3.890 1.234 0.003 1.471 6.309 6 22-23 Acry Rock 5 2.5 4.300 1.228 0.001 1.894 6.706 7 22-23 ENTA CERAM 5 1.39 5.410 1.211 0.005 3.037 7.783 8 22-23 IVOCLAR IVOSTAR 5 1 5.800 1.213 0.005 3.422 8.178 TABELUL 3. Perechea 11/21 Nr. Culoare naturală nr=30/1.97 Nr. Metode statistice Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 11-21 VITA LUMIN VACUUM 5 1.99 -0.020 0.586 0.973 -1.169 1.129 2 11-21 Acry Rock 5 2.23 -0.260 .629 0.682 -1.492 0.972 3 11-21 IVOCLAR IVOSTAR 5 5 -0.450 0.589 0.45 -1.604 0.704 4 11-21 Star Lux 5 2.62 -0.65 0.601 0.288 -1.829 0.529 5 11-21 ENTA CERAM 5 1.2 0.950 0.586 0.115 -0.199 2.099 6 11-21 Spofadent Plus 5 3.83 -1860 0.867 0.039 -3.559 -0.161 7 11-21 Vita MFT 5 3.72 -3750 0.717 0.001 -5.155 -2.345 TABELUL 3. Perechea 11/21 TABELUL 4. Perechea 21/22 Nr. Culoare naturală nr=30/4.56 Nr. Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 21-22 Spofadent Plus 5 4.46 0.100 1.467 0.946 -2.775 2.975 2 21-22 IVOCLAR IVOSTAR 5 3.47 1.090 1.414 0.446 -1.681 3.861 3 21-22 Vita MFT 5 3.22 1.340 1.408 0.348 -1.420 4.100 4 21-22 Acry Rock 5 2.86 1.700 1.483 0.260 -1.206 4.606 5 21-22 Star Lux 5 2.67 1.890 1.404 0.187 -0.862 4.642 6 21-22 VITA LUMIN VACUUM 5 2.38 2.180 1.405 0.130 -0.575 4.935 7 21-22 PIGEON SUMMIT 5 2.06 2.500 1.404 0.084 -0.252 5.252 8 21-22 ENTA CERAM 5 1.25 3.310 1.405 0.025 0.557 6.063 TABELUL 4. Perechea 21/22 TABELUL 5. Perechea 22/23 Nr. Culoare naturală nr=30/6.8 Nr. Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 22-23 Star Lux 5 4.23 2.570 1.213 0.042 0.193 4.947 2 22-23 Spofadent Plus 5 3.71 3.090 1.226 0.017 0.688 5.492 3 22-23 Vita MFT 5 3.28 3.520 1.221 0.007 1.127 5.913 4 22-23 VITA LUMIN VACUUM 5 3.08 3.720 1.210 0.004 1.349 6.091 5 22-23 PIGEON SUMMIT 5 2.91 3.890 1.234 0.003 1.471 6.309 6 22-23 Acry Rock 5 2.5 4.300 1.228 0.001 1.894 6.706 7 22-23 ENTA CERAM 5 1.39 5.410 1.211 0.005 3.037 7.783 8 22-23 IVOCLAR IVOSTAR 5 1 5.800 1.213 0.005 3.422 8.178 TABELUL 5. Perechea 22/23 Metode statistice Scopul acestui studiu a fost verificarea respectării curburii variaţiei cromatice a dinţilor naturali din TABELUL 1. Perechea 13-12 Nr. Culoare naturală nr=30/6,33 Nr. Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 13-12 SPOFADENT PLUS 5 6.630 -0.300 1.501 0.843 -3.241 2.641 2 13-12 STAR LUX 5 7.810 -1.480 1.320 0.27 -4.067 1.107 3 13-12 VITA LUMIN VACUUM 5 4.63 1.7 1.289 0.196 -0.827 4.227 4 13-12 IVOCLAR IVOSTAR 5 4.37 1.960 1.371 0.162 -0.728 4.648 5 13-12 PIGEON SUMMIT 5 3.3 3.030 1.393 0.037 0.3 5.76 6 13-12 ACRY ROCK 5 3.1 3.230 1.287 0.017 0.707 5.753 7 13-12 VITA MFT 5 1.980 4.350 1.294 0.002 1.814 6.886 8 13-12 ENTA CERAM 5 1 5.330 1.286 0.001 2.809 7.851 TABELUL 2. Perechea 12/11 Nr. Culoare naturală nr=30/4,16 Nr. Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 12-11 Vita MFT 5 3.71 0.450 1.187 0.707 -1.877 2.777 2 12-11 Star Lux 5 4.66 -0.500 1.119 0.658 -2.693 1.693 3 12-11 PIGEON SUMMIT 5 3.11 1.050 1.221 0.396 -1.343 3.443 4 12-11 VITA LUMIN VACUUM 5 2.57 1.590 1.111 0.162 -0.587 3.767 5 12-11 Spofadent Plus 5 5.84 -1.680 1.307 0.208 -4.242 0.882 6 12-11 IVOCLAR IVOSTAR 5 1.73 2.430 1.114 0.036 0.246 4.614 7 12-11 ENTA CERAM 5 1.58 2.580 1.117 0.027 0.390 4.770 8 12-11 Acry Rock 5 0.91 3.250 1.112 0.006 1.070 5.430 TABELUL 2. Perechea 12/11 Nr. Culoare naturală nr=30/4,16 Nr. Media Diferenţa medie Deviaţia standard p Intervale de încredere 95% pentru diferenţă 1 12-11 Vita MFT 5 3.71 0.450 1.187 0.707 -1.877 2.777 2 12-11 Star Lux 5 4.66 -0.500 1.119 0.658 -2.693 1.693 3 12-11 PIGEON SUMMIT 5 3.11 1.050 1.221 0.396 -1.343 3.443 4 12-11 VITA LUMIN VACUUM 5 2.57 1.590 1.111 0.162 -0.587 3.767 5 12-11 Spofadent Plus 5 5.84 -1.680 1.307 0.208 -4.242 0.882 6 12-11 IVOCLAR IVOSTAR 5 1.73 2.430 1.114 0.036 0.246 4.614 7 12-11 ENTA CERAM 5 1.58 2.580 1.117 0.027 0.390 4.770 8 12-11 Acry Rock 5 0.91 3.250 1.112 0.006 1.070 5.430 REVISTA ROMÂNÅ DE STOMATOLOGIE – VOLUMUL LXI, NR. 4, AN 2015 264 TABELUL 3. Perechea 11/21 Nr. Culoare naturală nr=30/1.97 Nr. DISCUŢII Pentru perechea de dinţi 12-11, variaţia cro- matică diferă imperceptibil de modelul dinţilor na- turali în 5 cazuri (Star Lux, Spofadent Plus, Vita MTF, Pigeon Summit şi Vita Lumin Vacuum). În limitele acestui studiu, în cazul perechii 12-11, apropierea maximă de comportamentul cromatic al dinţilor naturali o are garnitură Vita MTF, iar cel mai puţin apropiat se comportă garnitura Acry Rock. Relevanţă statistică s-a obţinut numai în cazul garniturilor Ivoclar Ivostar, Enta Ceram şi Acr Rock (p<0,05). Ţinându-se cont şi de relevanţa statistică, se poate afirma clar că garniturile Ivoclar Ivostar, Enta Ceram şi Acry Rock nu vor genera rezultate restauratorii cromatice conform curbei naturale de variaţie a culorii, întrucât duc la obţinerea unui ∆E mai mare decât valoarea perceptibilă, iar rezultatele sunt relevante statistic. De asemenea, în limitele acestui studiu, judecând conform principiului că o garnitură de dinţi are o variaţie cromatică cât mai apropiată de cea a dinţilor naturali, cu cât se obţine un ∆E sub 2 pentru cât mai multe perechi de dinţi din cadrul aceleiaşi garnituri, putem afirma că ordinea descrescătoare a garnitu- rilor este: Spofadent Plus (4 rezultate sub limita perceptibilităţii, din care 1 relevant statistic), Star Lux (4 rezultate sub limita perceptibilităţii, niciunul relevant statistic), Vita Lumin Vacuum şi Ivoclar Ivostar (fiecare cu câte 3 rezultate sub limita per- ceptibilităţii, niciunul relevant statistic), Pigeon Summit, Acry Rock şi Vita MTF (fiecare cu câte 2 rezultate sub limita perceptibilităţii, toate fără re- levanţă statistică) şi Enta Ceram, cu un singur re- zultat sub limita perceptibilităţii, fără relevanţă statistică. În cazul perechii 11-21, variaţia cromatică diferă imperceptibil de modelul dinţilor naturali în 5 cazuri (Vita Lumin Vacuum, Acry Rock, Ivoclar Ivostar, Star Lux, Spofadent Plus). În limitele aces- tui studiu, în cazul perechii 11-21, apropierea ma- ximă de comportamentul cromatic al dinţilor natu- rali o are garnitura Vita Lumin Vacuum, iar cel mai atipic se comportă garnitura Pigeon Summit. Rele- vanţa statistică s-a obţinut numai în cazul garni- turilor Vita MTF şi Spofadent Plus; ţinându-se cont şi de relevanţa statistică, se poate afirma clar că Vita MTF nu va genera rezultate cromatice cu o variaţie apropiată de cea a dinţilor naturali, întrucât duce la obţinerea unui ∆E mai mare de 2, iar re- zultatele sunt relevante statistic, în timp ce garnitura Spofadent Plus va reuşi să reproducă variaţia cro- matică a dinţilor naturali, întrucât valorile finale ∆E sunt mici, relevanţa statistică fiind, de asemenea, obţinută (p<0,05). DISCUŢII Prin urmare, se observă că, dintre garniturile stu- diate, variaţia cromatică dintre 13-12 diferă imper- ceptibil de modul în care variază culoarea dintre 13 şi 12 la dinţii naturali în numai 4 cazuri (Spofadent Plus, Star Lux, Vita Lumin Vacuum şi Ivoclar Ivo- star). Un lucru demn de subliniat este relevanţa statistică a rezultatelor. Se observă o relevanţă sta- tistică clar subliniată numai în cazul garniturilor de pe poziţiile 5, 6, 7, 8 (p<0,05). În limitele acestui stu diu, în cazul perechii 13-12, apropierea maximă de modul de variaţie cromatică al dinţilor naturali o are garnitura Spofadent Plus, iar cel mai îndepărtat comportament cromatic îl prezintă garnitura Enta Ceram. Ţinându-se cont şi de relevanţa statistică, se poate afirma clar că garniturile Pigeon Summit, Acry Rock, Vita MFT şi Enta Ceram nu generează În tabele, câmpul „Diferenţa medie“ reprezintă diferenţa medie între valoarea ∆E a fiecărei perechi de dinţi adiacenţi din fiecare garnitură de dinţi şi valoarea în cazul dinţilor naturali echivalenţi. Tabelul este ordonat crescător în funcţie de valoarea absolută a acestui câmp. Cu cât „mean difference“ este mai aproape de 0, cu atât variaţia cromatică a elementelor respective este mai apropiată de curbu- ra de variaţie în cazul dinţilor naturali. S-a raportat, de asemenea, această valoare la valoarea 2, care este limita acceptabilităţii vizuale a diferenţei cro- matice pentru doi dinţi naturali susţinută de ADA (8), deşi există şi studii care susţin că 1,6 este va- loarea diferenţei de culoare perceptibilă de ochiul uman. (9) REVISTA ROMÂNÅ DE STOMATOLOGIE – VOLUMUL LXI, NR. 4, AN 2015 265 În cazul ultimei perechi de dinţi, 22-23, variaţia cromatică în cazul tuturor garniturilor depăşeşte valoarea perceptibilităţii umane susţinută de ADA (2 unităţi). În limitele acestui studiu, în cazul pe- rechii 11-21, apropierea maximă o are cheia Star Lux, în timp ce garnitura Ivoclar Ivostar se comportă cel mai atipic. Relevanţa statistică nu s-a obţinut pentru nicio garnitură, ceea ce ar însemna că în cazul acestei perechi de dinţi, nicio garnitură nu ar duce la obţinerea unor rezultate conform cromaticii dinţilor naturali, întrucât duc la obţinerea unui ∆E mai mare decât valoarea perceptibilă, iar rezultatele sunt relevante statistic. rezultate cromatice asemănătoare cu aspectul den- tar natural, întrucât duc la obţinerea unui ∆E mai mare de valoarea perceptibilă, iar rezultatele sunt relevante statistic. DISCUŢII Neobţinerea unui rezultat semnificativ statistic pentru toate garniturile de dinţi se poate pune pe seama unui număr prea mic de măsurători, ceea ce motivează continuarea studiului pe un lot mai mare şi cu un număr mai mare de măsurători pentru fie- care tip de garnitură în parte. De asemenea, atrage atenţia rezultatul obţinut pentru perechea 22-23, care se diferenţiază de celelalte perechi de dinţi, întrucât niciuna din garnituri nu duce la obţinerea unui rezultat conform cu variaţia naturală. De ase- menea, surprinzătoare este şi lipsa de simetrie în rezultate între perechea 13-12 şi 22-23 care, repre- zentând practic dinţii omologi, ar fi trebuit să dea rezultate asemănătoare. ţ La perechea 21-22, s-au obţinut valori impercep- tibile de variaţie cromatică în 5 cazuri (Spofadent Plus, Ivoclar Ivostar, Vita MTF, Acry Rock, Star Lux). În limitele acestui studiu, în cazul perechii 21-22, apropierea maximă de variaţia cromatică a dinţilor naturali o are garnitura Spofadent Plus, în timp ce garnitura Enta Ceram se comportă cel mai diferit. Relevanţa statistică s-a obţinut numai în ca- zul garniturilor Pigeon Summit şi Enta Ceram; ţinându-se cont şi de relevanţa statistică, se poate afirma clar Pigeon Summit şi Enta Ceram nu vor genera rezultate restauratorii cromatice conform curbei naturale de variaţie a culorii, întrucât duc la obţinerea unui ∆E mai mare decât valoarea percep- tibilă, iar rezultatele sunt relevante statistic. CONCLUZII Deşi sunt codificate cromatic la fel, garniturile de dinţi artificiali prezintă culori diferite, în funcţie de producător. Acest lucru se întâmplă indiferent de materialul din care este confecţionată garnitura de dinţi (răşină acrilică, răşină compozită sau cera- mică). Prin urmare, pentru rezultate estetice optime trebuie folosit acelaşi sistem de la faza de determinare REVISTA ROMÂNÅ DE STOMATOLOGIE – VOLUMUL LXI, NR. 4, AN 2015 266 cromatică până în faza de livrare a protezei finale (ex.: cheie de culori/sistem de comunicare cu tehni- cianul/garnituri de dinţi unitare, de la acelaşi produ- cător). Domeniul cromaticii dinţilor din garniturile arti- ficiale necesită studiu suplimentar. Neobţinerea unor rezultate uniform semnificative statistic deno- tă necesitatea unei analize mai profunde a acestui subiect, cu realizarea unui număr şi mai mare de măsurători. Concluziile acestui experiment stabi- lesc o serie de direcţii generale ce pot fi urmate de studii ulterioare, mai aprofundate. Comportamentul cromatic de la un dinte arti- ficial la altul diferă, de asemenea, în funcţie de producător. În limitele acestui studiu, garnitura cu o variaţie cromatică ce se apropie cel mai mult de cea a dinţilor naturali este Spofadent Plus; prin urmare, utilizarea acestei garnituri ar duce la obţinerea unor rezultate cât mai apropiate de estetica naturală. 7. Constantinovici A., Păuna M., Andrei O.C., Farcasiu T., Iosif I. – Studiu privind variaţia cromatică a grupului frontal superior în funcţie de vârsta pacienţilor, Ro J Stomatol 2015; 61:126-130 9. Nagai I., Yoshida A., Sakai M., Kristiansen J., Da Silva J.D. – Clinical evaluation of perceptibility of color differences between natural teeth and all-ceramic crowns. J Dent. 2009; 37(1):57-63 1. Seghi R.R., Johnston W.M., O’Brien W.J. – Spectrophotometric analysis of color differences between porcelain system, J Prosthet Dent 2011; 56:35-40 6. Johnston W.M. – Color Measurement în Dentistry, J. Pros Dent 2009; 37:2-26 2. Ancowitz S. – Esthetic removable partial dentures. Gen Dent 2004; 52(5):452-9 1. 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https://openalex.org/W2526857517	https://lirias.kuleuven.be/bitstream/123456789/569320/1/Poire%20J%20Hematol%20Oncol%202017.pdf	English	null	Allogeneic Stem Cell Transplantation in Adult Patients with Acute Myeloid Leukemia and 17p Abnormalities in First Complete Remission: A Study from the Acute Leukemia Working Party (ALWP) of the European Society of Blood and Marrow Transplantation (EBMT)	Blood	2,015	cc-by	10,275	Allogeneic stem cell transplantation in adult patients with acute myeloid leukaemia and 17p abnormalities in first complete remission: a study from the Acute Leukemia Working Party (ALWP) of the European Society for Blood and Marrow Transplantation (EBMT) Xavier Poiré1, Myriam Labopin2,15, Johan Maertens3, Ibrahim Yakoub-Agha4, Didier Blaise5, Norbert Ifrah6, Gérard Socié7, Tobias Gedde-Dhal8, Nicolaas Schaap9, Jan J. Cornelissen10, Stéphane Vigouroux11, Jaime Sanz12, Lucienne Michaux13, Jordi Esteve14†, Mohamad Mohty2,15† and Arnon Nagler2,16† Poiré et al. Journal of Hematology & Oncology (2017) 10:20 DOI 10.1186/s13045-017-0393-3 RESEARCH Open Access Allogeneic stem cell transplantation in adult patients with acute myeloid leukaemia and 17p abnormalities in first complete remission: a study from the Acute Leukemia Working Party (ALWP) of the European Society for Blood and Marrow Transplantation (EBMT) Xavier Poiré1, Myriam Labopin2,15, Johan Maertens3, Ibrahim Yakoub-Agha4, Didier Blaise5, Norbert Ifrah6, Gérard Socié7, Tobias Gedde-Dhal8, Nicolaas Schaap9, Jan J. Cornelissen10, Stéphane Vigouroux11, Jaime Sanz12, Lucienne Michaux13, Jordi Esteve14†, Mohamad Mohty2,15† and Arnon Nagler2,16† © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Data collection and patient selection The data on patients over 18 years of age with a diagno- sis of de novo or secondary AML transplanted with a re- lated or unrelated donor were available from the EBMT registry. The latter is a voluntary working group of more than 450 transplant centres reporting regularly on their transplant activity. Only patients having available cyto- genetics and transplanted between 1 January 2000 and 31 December 2013 have been selected. Patients with sec- ond allo-SCT have been excluded as well as those re- ceiving a haplo-identical transplantation. Audits are routinely performed to insure the quality of the reported data. All patients provided informed consent on the use of their data in retrospective studies. The Review Board of EBMT approved this study. We identified a dataset of 10,799 patients with 5495 patients displaying an abnor- mal karyotype. All cytogenetic abnormalities have been carefully reviewed by two physicians (Xavier Poiré and Lucienne Michaux). Most centres report conventional karyotype and a few report also FISH results. Cytogen- etic results found in the registry are complete or often partial depending on the reporting center. Based on available data, we kept for further analysis only patients for whom data were sufficient to confirm the presence of abn(17p). Abn(17p) were defined as loss of 17p13 (TP53 locus) such as monosomy 17, deletion (17p), iso- chromosome 17q (i(17q)), addition (17p) or other abnor- malities that disrupt the 17p13 locus. Only one center reported a patient with TP53 mutation. Those selected patients have been further categorised as CK, MK, pres- ence of monosomy 7, presence of loss of 5q and/or pres- ence of a inversion of chromosome 3 (inv(3)). CK has been defined as the presence of 3 or more cytogenetic abnormalities. MK has been defined as two or more autosomal monosomies or one autosomal monosomy in combination with at least one structural chromosomal abnormality. A total of 139 patients from 78 centres met the criteria and have been selected for further analysis. M l bl i di i i (MAC) h b d fi d TP53 is located in 17p13 chromosomal region and is one of the major tumour suppressor genes, often inacti- vated by deletion and/or mutation in many tumours [18]. It has been described in 10 to 15% of AML pa- tients, with an increased frequency in elderly patients and secondary AML [19]. Abstract Journal of Hematology & Oncology (2017) 10:20 Page 2 of 10 Page 2 of 10 Background Thus, long-term disease control is observed in less than 5% of the patients harbouring the TP53 mutations with conventional chemotherapy [25, 26]. Molecular screen- ing for TP53 mutations is not routinely performed, and loss or disruption of 17p13 (17p abnormalities, abn(17p)) is usually identified by FISH analysis [27]. In this context, the potential capability of allo-SCT to over- come the dismal prognosis of abn(17p) AML is of great interest, scarcely explored until now. A first report from Mohr et al. described the outcome of 47 allografted pa- tients and did not show a different outcome compared to non-transplanted patients, raising the hypothesis of a lack of sensitivity of this entity to the potential benefit of graft-versus-leukaemia effect [28]. This detrimental ef- fect of abn(17p) on allo-SCT outcomes has been con- firmed in another report with an event-free survival (EFS) of only 11% due to a very high incidence of relapse g Allogeneic stem cell transplantation (allo-SCT) is now a standard approach recommended for patients with high- risk acute myeloid leukaemia (AML) in remission [1, 2]. High-risk AML is mainly defined by the presence of de- termined poor-risk cytogenetic abnormalities at diagno- sis together with specific mutational events [3–6]. In general, conventional post-remission high-dose chemo- therapy is not capable to eradicate the initiating stem cell leukemic clone of high-risk AML, harbouring strong chemoresistance mechanisms [7], and only the potent graft-versus-leukaemia arising after allo-SCT may over- come the poor prognosis of these high-risk AML sub- types [8]. Indeed, several reports have confirmed the significant advantage of allo-SCT in high-risk AML, especially when performed early in the course of the disease [9–11]. Among the heterogeneous group of high-risk AML, prognosis can be further stratified based on specific genetic abnormalities, and the potential benefit of allo-SCT differs between these diverse AML subtypes [12–16]. While, it is still questionable if distinct genetic abnormalities with a known worse outcome like complex karyotype (CK) and monosomal karyotype (MK) AML will get the same benefit from allo-SCT [17]. Background [17]. A recent report from Middeke et al. described 201 patients with abn(17p) AML transplanted during the past decade, showing an overall EFS of only 12%, with a slight better outcome among the 84 patients allografted in first complete remission (3-year EFS 18 vs 7%) p < 0.001) [29]. The purpose of the current study was to ex- plore the potential role of early-phase allo-SCT in abn(17p) AML in the multicenter, registry context of EBMT, with the aim to identify specific subsets of pa- tients who could benefit from the procedure. g Allogeneic stem cell transplantation (allo-SCT) is now a standard approach recommended for patients with high- risk acute myeloid leukaemia (AML) in remission [1, 2]. High-risk AML is mainly defined by the presence of de- termined poor-risk cytogenetic abnormalities at diagno- sis together with specific mutational events [3–6]. In general, conventional post-remission high-dose chemo- therapy is not capable to eradicate the initiating stem cell leukemic clone of high-risk AML, harbouring strong chemoresistance mechanisms [7], and only the potent graft-versus-leukaemia arising after allo-SCT may over- come the poor prognosis of these high-risk AML sub- types [8]. Indeed, several reports have confirmed the significant advantage of allo-SCT in high-risk AML, especially when performed early in the course of the disease [9–11]. Among the heterogeneous group of high-risk AML, prognosis can be further stratified based on specific genetic abnormalities, and the potential benefit of allo-SCT differs between these diverse AML subtypes [12–16]. While, it is still questionable if distinct genetic abnormalities with a known worse outcome like complex karyotype (CK) and monosomal karyotype (MK) AML will get the same benefit from allo-SCT [17]. TP53 is located in 17p13 chromosomal region and is one of the major tumour suppressor genes, often inacti- vated by deletion and/or mutation in many tumours [18]. It has been described in 10 to 15% of AML pa- tients, with an increased frequency in elderly patients and secondary AML [19]. TP53 inactivation is associated in AML with a significantly lower response to intensive chemotherapy, translating into a poor outcome [20]. Al- though TP53 mutations/deletions show a high correl- ation with complex karyotype in AML [21–23], TP53 mutations and/or loss have emerged as a strong and in- dependent prognostic marker of very poor outcomes re- gardless of associated cytogenetic abnormalities [24, 25]. Abstract Background: Acute myeloid leukaemia (AML) with 17p abnormalities (abn(17p)) carries a very poor prognosis due to high refractoriness to conventional chemotherapy, and allogeneic stem cell transplantation (allo-SCT) appears as the only potential curative option. Methods: To address outcomes after allo-SCT in patients with abn(17p), we retrospectively analysed de novo or secondary AML undergoing SCT between 2000 and 2013 from the EBMT registry. Results: One hundred thirty-nine patients with confirmed abn(17p) have been selected. At the time of transplant, one hundred twenty-five were in first remission (CR1). Median age was 54 years old. Abn(17p) was associated with a monosomal karyotype in 83% of patients, complex karyotype in 91%, monosomy 5 or 5q deletion (-5/5q-) in 55%, monosomy 7 (-7) in 39% and both -5/5q and -7 in 27%. Seventy-three patients (59%) had a reduced-intensity conditioning regimen. The 2-year overall survival (OS) and leukaemia-free survival (LFS) were 28 and 24%, respectively. The 2-year non-relapse mortality (NRM) was 15%, and 2-year relapse incidence (RI) was 61%. The cumulative incidence of grade II to IV acute graft-versus-host disease (GvHD) was 24% and that of chronic GvHD was 21%. In multivariate analysis, the presence of a -5/5q- in addition to abn(17p) was significantly and independently associated with worse OS, LFS and higher RI. Age and donor types did not correlate with outcome. Conditioning intensity was not statistically associated with OS, LFS and NRM when adjusted for patients’ age. Conclusions: In contrast to the dismal prognosis reported for AML patients harbouring abn(17p) undergoing conventional chemotherapy, allogeneic SCT provides responses in about 25% of those patients transplanted in CR1. Keywords: Acute myeloid leukaemia, 17p abnormalities, Stem cell transplantation, Survival, First remission * Correspondence: Xavier.Poire@uclouvain.be †Equal contributors 1Section of Hematology, Department of Medicine, Cliniques Universitaires Saint-Luc, 10, avenue Hippocrate, 1200 Brussels, Belgium Full list of author information is available at the end of the article © The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 10 Poiré et al. Patients’ characteristics A total of 139 patients with abn(17p) have been iden- tified. There were 125 patients transplanted in first complete remission (CR1), while only 14 patients were transplanted in second remission (CR2). Because of the small number of patients transplanted in CR2, further analysis has been focused on CR1 patients. A detailed table of the different abn(17p) is available as a Additional file 1: Table S1. A total of 125 patients with abn(17p) transplanted in CR1 have been analysed in November 2015. The median follow-up of the cohort was 21 months (ranging 3– 146 months). The median age at transplantation was 54 years old (ranging 18–69 years old). The median year of transplantation was 2009 (ranging 2000–2013). The median time from diagnosis to CR1 was 57 days (ran- ging 18–170 days), and median time from CR1 to trans- plantation was 82 days (ranging 11–286 days). For 81 patients, we had information about the number of in- duction courses to reach CR1. Fifty-two had just one course, 27 had 2 and 3 needed 3 rounds of chemother- apy. Most patients were de novo AML (85%) and only 19 patients corresponded to secondary AML, seven of them arising from an antecedent myelodysplastic syn- drome. The majority of patients were male (57%) and were transplanted with a Karnofsky performance status of more than 90% (70%). A sibling donor was used in 48% and an unrelated donor in 43% (10/10: N = 23 (64%); 9/10: N = 13 (33%), missing: N = 36) whereas a cord blood was used in 10 patients. Source of stem cell was mostly peripheral blood (76%). CMV status was positive in 68% of the patients and also 68% of the do- nors. Fifty-one patients received a MAC and 74 patients a RIC. In patients less than 50 years old, only 14 patients (26.4%) received a RIC but this number increased up to 60 (83.3%) in patients over 50 years old (p < 10−5). Most frequent MAC were the combination of cyclophospha- mide and busulfan (N = 19) followed by the association of total body irradiation (TBI) with cyclophosphamide (N = 14). Most RIC were fludarabine and TBI (N = 25) closely followed by fludarabine and busulfan (N = 24). Seven patients received the sequential FLAMSA-RIC ap- proach [33]. In vivo T cell depletion has been used in 64 patients. Data collection and patient selection TP53 inactivation is associated in AML with a significantly lower response to intensive chemotherapy, translating into a poor outcome [20]. Al- though TP53 mutations/deletions show a high correl- ation with complex karyotype in AML [21–23], TP53 mutations and/or loss have emerged as a strong and in- dependent prognostic marker of very poor outcomes re- gardless of associated cytogenetic abnormalities [24, 25]. Thus, long-term disease control is observed in less than 5% of the patients harbouring the TP53 mutations with conventional chemotherapy [25, 26]. Molecular screen- ing for TP53 mutations is not routinely performed, and loss or disruption of 17p13 (17p abnormalities, abn(17p)) is usually identified by FISH analysis [27]. In this context, the potential capability of allo-SCT to over- come the dismal prognosis of abn(17p) AML is of great interest, scarcely explored until now. A first report from Mohr et al. described the outcome of 47 allografted pa- tients and did not show a different outcome compared to non-transplanted patients, raising the hypothesis of a lack of sensitivity of this entity to the potential benefit of graft-versus-leukaemia effect [28]. This detrimental ef- fect of abn(17p) on allo-SCT outcomes has been con- firmed in another report with an event-free survival (EFS) of only 11% due to a very high incidence of relapse Myeloablative conditioning (MAC) has been defined as a regimen including total body irradiation (TBI) of more than 8 Gy or a busulfan dose of more than 10 mg/kg. Reduced- intensity conditioning (RIC) includes intermediate doses of Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Page 3 of 10 Page 3 of 10 Page 3 of 10 alkylating agents such as 8–10 mg/kg busulfan, 80– 140 mg/m2 melphalan, 600–1200 mg/m2 cyclophospha- mide or 5–10 mg/kg thiotepa, and/or low-dose TBI (<3 Gy). Patients’ characteristics Among those, 47 patients received anti- thymocyte globulin (ATG) and 17 patients alemtuzu- mab. Regarding associated cytogenetic categories, most Data collection and patient selection The following variables have been selected and in- cluded in the analysis: year of transplantation, age, gender, status at transplantation, time to diagnosis to complete re- mission, time to complete remission to allo-SCT, number of induction courses, type of conditioning regimen, in vivo T cell depletion, type of T cell depletion, cytomegalovirus (CMV) status of donor and recipient, donor type, source of stem cells, Karnofsky performance status at transplantation, engraftment, presence of acute and chronic graft-versus- host disease (GvHD), grade of acute GvHD, presence of CK, MK, monosomy 7, loss of 5q and/or inv(3), cause of death. HLA typing was determined at 10 loci (A, B, C, DRB1, DQB1) by high-resolution techniques, although not all the centres report complete data on HLA. All unrelated donors were defined as HLA matched (10/10) or mis- matched at 1 locus (9/10). Additional data have been col- lected on the therapy of relapsing patients when available. HLA data on cord blood (CB) were not captured in this study. Methods and definitions were similar to other stud- ies performed by the Acute Leukemia Working Party of the EBMT [30–32]. All tests were two-sided. The type 1 error rate was fixed at 0.05 for determination of factors associated with time to event outcomes. Statistical analyses were per- formed with SPSS 19 (SPSS Inc./IBM, Armonk, NY) and R 3.0.1 (R Development Core Team, Vienna, Austria) software packages. Statistical analysis and endpoint definitions Endpoints included leukaemia-free survival (LFS), re- lapse incidence (RI), non-relapse mortality (NRM), overall survival (OS), acute and chronic GVHD and GVHD-free/relapse-free survival (GRFS). All outcomes were measured from the time of stem cell infusion. LFS was defined as survival without relapse; patients alive without relapse were censored at the time of last con- tact. OS was based on death from any cause. NRM was defined as death without previous relapse. GRFS was defined as survival without grade 3–4 acute GVHD, ex- tensive chronic GVHD, relapse or death. Surviving pa- tients were censored at the time of last contact. The probabilities of OS and LFS were calculated by the Kaplan-Meier test, and those of acute and chronic GVHD, NRM, and relapse by the cumulative incidence estimator to accommodate competing risks. Results are expressed with a 95% confidence interval (CI). For NRM, relapse was the competing risk, and for relapse, the competing risk was NRM. For acute and chronic GVHD, death without the event and relapse were the competing risks. For all prognostic analyses, continuous variables were categorised and the median was used as a cut-off point. A Cox proportional hazards model was used for multi- variate regression. Factors associated with a p value less than 0.05 by univariate analysis were included in the model. Results were expressed as hazard ratio (HR) with 95% confidence interval Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Page 4 of 10 Page 4 of 10 patients carried also a CK (N = 98) or a MK (N = 86). An inv(3) was present in only 3 patients. Monosomy 7 (-7) was seen in 41 patients and a monosomy 5 or a loss of 5q (-5/5q-) in 58 patients. Both -7 and -5/5q- were present together in 28 patients. Table 1 summarises the patients’ characteristics. Engraftment and graft-versus-host disease Engraftment and graft-versus-host disease Engraftment and graft-versus-host disease Engraftment was successful in 117 patients (94%). Six patients showed a graft failure and 1 patient lost his graft. The cumulative incidence of grade II to IV acute GvHD was 24%. The 2-year cumulative incidence of chronic GvHD was only 21% [95% CI 14.2–29.5] (Fig. 1). This low incidence is explained because many patients relapsed before developing chronic GvHD. Statistical analysis and endpoint definitions A Cox proportional hazards model including age, donor type, use of ATG during the conditioning regimen, source of stem cells and conditioning intensity has been performed for cGvHD (Table 2). Only the use of ATG during the conditioning regimen was signifi- cantly associated with less chronic GvHD (HR 0.33, 95% CI 0.11–0.97; p = 0.04), whereas donor other than an HLA-matched sibling showed just a trend towards more chronic GvHD (p = 0.07). Table 1 Patients’ characteristics (N = 125) Median age at SCT (range) 53.6 years old (18–69) Median follow-up (range) 21 months (3.3–146) Interval between diagnosis and CR1 (range) 56.5 days (18–170) Intervals from CR1 to SCT (range) 81.5 days (11–286) Median year of SCT 2009 (2000–2013) Secondary AML, N (%) 19 (15.2%) CMV+ patient, N (%) 84 (68.3%) CMV+ donor, N (%) 65 (68.3%) Karnofsky >90% at SCT, N (%) 83 (70.3%) Gender, N (%) Male 71 (57%) Female 54 (43%) Donor type, N (%) Sibling 60 (48%) Unrelated 54 (43.2%) Cord blood 10 (8%) Source of SC, N (%) BM 19 (15.2%) PB 95 (76%) CB 10 (8%) Conditioning regimen, N (%) MAC 51 (41%) RIC 73 (59%) In vivo T cell depletion, N (%) 64 (51%) ATG 47 (38%) Alemtuzumab 17 (14%) Monosomal karyotype, N (%) 86 (82.7%) Missing, N 21 Complex karyotype, N (%) 98 (90.7%) Missing, N 17 Inv(3), N (%) 3 (2.9%) Missing, N 20 -7, N (%) 41 (39%) Missing, N 20 -5/5q-, N (%) 58 (55.2%) Missing, N 20 Both -7 and -5/5q-, N (%) 28 (26.9%) Abbreviations; N number, CR1 first complete remission, SCT stem cell transplantation, AML acute myeloid leukaemia, CMV cytomegalovirus, BM bone PB i h l bl d CB d bl d MAC l bl i di i i Table 1 Patients’ characteristics (N = 125) Table 1 Patients’ characteristics (N = 125) Non-relapse mortality and relapse incidence The 2-year cumulative incidence of NRM was 15% [95% CI 8.9–21.8] as illustrated in Fig. 2a. None of the ana- lysed variables (i.e. conditioning intensity, in vivo T cell depletion, use of ATG, age, donor or patient gender, fe- male donor to male recipient, Karnofsky performance status, donor type, number of induction course, CK, MK, -7, -5/5q- or both -7 and -5/5q-) were significantly associated with NRM neither in univariate analysis nor in multivariate analysis. Seventy-six patients relapsed at a median interval of 4 months from allo-SCT (range 0.2–92.8 months) trans- lating into a 2-year cumulative incidence of relapse of 61.3% [95% CI 51.5–69.7] as illustrated in Fig. 2b. The 2 factors significantly associated with a higher RI were conditioning intensity and presence of -5/5q-. On the Fig. 1 Cumulative incidence of chronic GvHD. The 2-year cumulative incidence of chronic GvHD was 21% [95% CI 14.2–29.5] Fig. 1 Cumulative incidence of chronic GvHD. The 2-year cumulative incidence of chronic GvHD was 21% [95% CI 14.2–29.5] Abbreviations; N number, CR1 first complete remission, SCT stem cell transplantation, AML acute myeloid leukaemia, CMV cytomegalovirus, BM bone marrow, PB peripheral blood, CB cord blood, MAC myeloablative conditioning, RIC reduced-intensity conditioning, ATG anti-thymocyte globulin Abbreviations; N number, CR1 first complete remission, SCT stem cell transplantation, AML acute myeloid leukaemia, CMV cytomegalovirus, BM bone marrow, PB peripheral blood, CB cord blood, MAC myeloablative conditioning, RIC reduced-intensity conditioning, ATG anti-thymocyte globulin Fig. 1 Cumulative incidence of chronic GvHD. The 2-year cumulative incidence of chronic GvHD was 21% [95% CI 14.2–29.5] Fig. 1 Cumulative incidence of chronic GvHD. The 2-year cumulative incidence of chronic GvHD was 21% [95% CI 14.2–29.5] Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Page 5 of 10 Table 2 Multivariate analysis using a Cox proportional hazards model, N = 96. Chronic GvHD p HR 95% CI Age ≥50 years old 0.41 1.51 0.56 4.06 Donor other than MSD 0.07 2.39 0.92 6.2 ATG vs No 0.04 0.33 0.11 0.97 PB vs BM 0.89 0.92 0.31 2.71 RIC vs MAC 0.12 2.34 0.8 6.86 Abbreviations: MSD matched sibling donor, ATG anti-thymocyte globulins, PB peripheral blood, BM bone marrow, RIC reduced-intensity conditioning, MAC myeloablative conditioning, HR hazard ratio, CI confidence interval Table 2 Multivariate analysis using a Cox proportional hazards model, N = 96. Chronic GvHD 55.4–80.9]) compared to patients without -5/5q- (51% [95% CI 35.4–65.4], p = 0.03). Non-relapse mortality and relapse incidence Patients’ age above 50 years old and MK showed only a trend towards a higher RI (p = 0.06 and p = 0.05, respectively). The number of induc- tion courses to reach CR1 did not impact on relapse rate. In multivariate analysis, only the presence of -5/5q- kept its significant impact on RI (p = 0.03) while condi- tioning intensity, age and MK did not show a significant impact on relapse risk (Table 3). Among the 56 relapsed patients with available information, 16 patients received donor leukocyte infusion (DLI). Thirteen of them re- ceived a second allo-SCT thereafter and 2 additional pa- tients received a second allo-SCT as the only cell-based therapy for relapse. The 2-year probabilities of OS were 8.4% after DLI [95% CI 0–24.1] and 20% after second allo-SCT [95% CI 0–55.1]. Abbreviations: MSD matched sibling donor, ATG anti-thymocyte globulins, PB peripheral blood, BM bone marrow, RIC reduced-intensity conditioning, MAC myeloablative conditioning, HR hazard ratio, CI confidence interval contrary, in vivo T cell depletion was not associated with higher relapse rate. The relapse incidence was 53% [95% CI 37–66.2] after a MAC and 68% [95% CI 55.2–78.2] when a RIC was used (p = 0.01). The presence of -5/5q- was associated with a significant higher RI (70% [95% CI Survival The 2-year probability of OS was 28% [95% CI 19.7– 37.1] (Fig. 3a). In univariate analysis, factors significantly associated with a worse OS were RIC, older age, MK and presence of -5/5q-. Monosomy 7 showed only a trend towards a decreased OS (p = 0.06 and p = 0.08, re- spectively). Thus, the 2-year probability of OS was 40% [95% CI 25–55] after a MAC and 21% after a RIC [95% CI 10–31] (p < 0.005). Patients above 50 years old had a worse OS (22%, [95% CI 12–36]) than younger patients (39%, [95% CI 23–54], p < 0.005). Given the strong inter- action between use of RIC and older age, as previously described, conditioning intensity did not show any im- pact on OS when adjusted for age. Concerning associ- ated cytogenetic categories, patients harbouring a MK had a decreased 2-year OS (19%, [95% CI 17–36]) com- pared to patients without this cytogenetic abnormality Fig. 2 Non-relapse mortality (NRM) (a) and relapse incidence (RI) (b). The 2-year cumulative incidence of NRM was 15% [95% CI 8.9–21.8] (a) and the 2-year cumulative incidence of relapse was 61.3% [95% CI 51.5–69.7] (b) Fig. 2 Non-relapse mortality (NRM) (a) and relapse incidence (RI) (b). The 2-year cumulative incidence of NRM was 15% [95% CI 8.9–21.8] (a) and the 2-year cumulative incidence of relapse was 61.3% [95% CI 51.5–69.7] (b) Table 3 Multivariate analysis using a Cox proportional hazards model, N = 90. Only variables with p < 0.05 in univariate analysis. LFS, OS and RI LFS, OS and RI p HR 95% CI LFS Age ≥50 years old 0.48 1.23 0.69 2.20 RIC vs MAC 0.13 1.54 0.89 2.68 MK 0.21 1.57 0.77 3.19 Monosomy 5q 0.02 1.83 1.09 3.07 OS Age ≥50 years old 0.35 1.37 0.71 2.64 RIC vs MAC 0.07 1.75 0.95 3.24 MK 0.14 1.79 0.82 3.91 Monosomy 5q 0.01 2.02 1.18 3.47 RI Age ≥50 years old 0.54 1.21 0.65 2.25 RIC vs MAC 0.13 1.58 0.87 2.88 Monosomy 5q 0.03 1.84 1.06 3.19 Abbreviations: N number, LFS leukaemia-free survival, OS overall survival, RI re- lapse incidence, HR hazard ratio, CI confidence interval, MAC myeloablative conditioning, RIC reduced-intensity conditioning, MK monosomal karyotype Fig. 2 Non-relapse mortality (NRM) (a) and relapse incidence (RI) (b). Survival The deleterious impact of mono5 on LFS was independent of presence of additional mono7, with a 2-year LFS of 11% [95% CI 0–23] (mono5 without mono7) and 13% [95% CI 0–27] (mono5 with mono7) vs 38% [95% CI 9–67] (absence of mono5 with mono7) and 37% [95% CI 20–53] (absence of both abnormalities, p = 0.007) (b) (58%, [95% CI 34–82], p = 0.005) as well as patient with concomitant -5/5q- (12%, [95% CI 2–22] vs 44%, [95% CI 28–59], p < 0.005). Taking together -7 and -5/5q- sta- tus, absence of -5/5q- was associated to a better out- come (2-year OS 47% [95% CI 30–65] and 31% [95% CI 0–63] in patients without and with concomitant -7, re- spectively) compared to the subset of patients harbour- ing -5/5q- (2-year OS 16% [95% CI 1–31] and 10% [95% CI 0–22], according to simultaneous -7 or not, respect- ively) (Fig. 4a). Thus, in multivariate analysis, the pres- ence of -5/5q- was significantly associated with a decreased OS (HR 2.02; 95% CI 1.2–3.5, p = 0.01), whereas RIC was associated with a trend towards a worse OS (p = 0.07) (Table 3). Causes of death were dis- ease related in 63 patients, infections in 13, GvHD in 11, haemorrhage in 1 and others in 2 patients. (58%, [95% CI 34–82], p = 0.005) as well as patient with concomitant -5/5q- (12%, [95% CI 2–22] vs 44%, [95% CI 28–59], p < 0.005). Taking together -7 and -5/5q- sta- tus, absence of -5/5q- was associated to a better out- come (2-year OS 47% [95% CI 30–65] and 31% [95% CI 0–63] in patients without and with concomitant -7, re- spectively) compared to the subset of patients harbour- ing -5/5q- (2-year OS 16% [95% CI 1–31] and 10% [95% CI 0–22], according to simultaneous -7 or not, respect- ively) (Fig. 4a). Thus, in multivariate analysis, the pres- ence of -5/5q- was significantly associated with a decreased OS (HR 2.02; 95% CI 1.2–3.5, p = 0.01), whereas RIC was associated with a trend towards a worse OS (p = 0.07) (Table 3). Causes of death were dis- ease related in 63 patients, infections in 13, GvHD in 11, haemorrhage in 1 and others in 2 patients. Survival Taking together -7 and -5/5q- sta- tus, absence of -5/5q- was associated to a better out- come (2-year OS 47% [95% CI 30–65] and 31% [95% CI 0–63] in patients without and with concomitant -7, re- spectively) compared to the subset of patients harbour- ing -5/5q- (2-year OS 16% [95% CI 1–31] and 10% [95% CI 0–22], according to simultaneous -7 or not, respect- ively) (Fig. 4a). Thus, in multivariate analysis, the pres- ence of -5/5q- was significantly associated with a decreased OS (HR 2.02; 95% CI 1.2–3.5, p = 0.01), whereas RIC was associated with a trend towards a worse OS (p = 0.07) (Table 3). Causes of death were dis- ease related in 63 patients, infections in 13, GvHD in 11, haemorrhage in 1 and others in 2 patients. The 2-year probability of LFS was 24% [95% CI 15 7 31 9] as illustrated in Fig 3b In univariate Fig. 3 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b). In the whole cohort, the 2-year probability of OS was 28% [95% CI 19.7–37.1] (a) and the 2-year probability of LFS was 24% [95% CI 15.7–31.9] (b) Fig. 3 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b). In the whole cohort, the 2-year probability of OS was 28% [95% CI 19.7–37.1] (a) and the 2-year probability of LFS was 24% [95% CI 15.7–31.9] (b) Fig. 3 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b). In the whole cohort, the 2-year probability of OS was 28% [95% CI 19.7–37.1] (a) and the 2-year probability of LFS was 24% [95% CI 15.7–31.9] (b) Fig. 4 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b) by cytogenetics subgroup. Mono5 refers as the presence of mono- somy 5 or loss of 5q and mono7 refers as the presence of mono- somy 7. Absence of Mono5 was associated to a better OS (2-year OS 47% [95% CI 30–65] and 31% [95% CI 0–63] in patients without and with mono7, respectively) compared to the subset of patients har- bouring mono5 (2-year OS: 16% [95% CI 1–31] and 10% [95% CI 0– 22], according to simultaneous mono7 or not, respectively) (a). Survival In univariate analysis, decreased LFS was significantly associated with RIC (2-year LFS of 20% [95% CI 8–29] and 30% [95% CI 16–44] after RIC and MAC allo-SCT, respect- ively, p = 0.01), age above 50 years old (2-year LFS: 20% [95% CI 10–30] vs 29% [95% CI 15–43], p < 0.005), MK (2-year LFS 17% [95% CI 8–25] vs 49% [95% CI 25–72], p = 0.02), and presence of -5/5q-). The deleterious impact of 5q loss was independent of presence of additional -7, with a 2-year LFS of 11% [95% CI 0–23] (-5/5q- without -7) and 13% [95% CI Fig. 3 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b). In the whole cohort, the 2-year probability of OS was 28% [95% CI 19.7–37.1] (a) and the 2-year probability of LFS was 24% [95% CI 15.7–31.9] (b) Fig. 4 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b) by cytogenetics subgroup. Mono5 refers as the presence of mono- somy 5 or loss of 5q and mono7 refers as the presence of mono- somy 7. Absence of Mono5 was associated to a better OS (2-year OS 47% [95% CI 30–65] and 31% [95% CI 0–63] in patients without and with mono7, respectively) compared to the subset of patients har- bouring mono5 (2-year OS: 16% [95% CI 1–31] and 10% [95% CI 0– 22], according to simultaneous mono7 or not, respectively) (a). The deleterious impact of mono5 on LFS was independent of presence of additional mono7, with a 2-year LFS of 11% [95% CI 0–23] (mono5 without mono7) and 13% [95% CI 0–27] (mono5 with mono7) vs 38% [95% CI 9–67] (absence of mono5 with mono7) and 37% [95% CI 20–53] (absence of both abnormalities, p = 0.007) (b) Fig. 3 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b). In the whole cohort, the 2-year probability of OS was 28% [95% CI 19.7–37.1] (a) and the 2-year probability of LFS was 24% [95% CI 15.7–31.9] (b) Fig. 3 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b). In the whole cohort, the 2-year probability of OS was 28% [95% CI 19.7–37.1] (a) and the 2-year probability of LFS was 24% [95% CI 15.7–31.9] (b) (58%, [95% CI 34–82], p = 0.005) as well as patient with concomitant -5/5q- (12%, [95% CI 2–22] vs 44%, [95% CI 28–59], p < 0.005). Survival The 2-year cumulative incidence of NRM was 15% [95% CI 8.9–21.8] (a) and the 2-year cumulative incidence of relapse was 61.3% [95% CI 51.5–69.7] (b) Abbreviations: N number, LFS leukaemia-free survival, OS overall survival, RI re- lapse incidence, HR hazard ratio, CI confidence interval, MAC myeloablative conditioning, RIC reduced-intensity conditioning, MK monosomal karyotype Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Page 6 of 10 Fig. 4 Overall survival (OS) (a) and leukaemia-free survival (LFS) (b) by cytogenetics subgroup. Mono5 refers as the presence of mono- somy 5 or loss of 5q and mono7 refers as the presence of mono- somy 7. Absence of Mono5 was associated to a better OS (2-year OS 47% [95% CI 30–65] and 31% [95% CI 0–63] in patients without and with mono7, respectively) compared to the subset of patients har- bouring mono5 (2-year OS: 16% [95% CI 1–31] and 10% [95% CI 0– 22], according to simultaneous mono7 or not, respectively) (a). The deleterious impact of mono5 on LFS was independent of presence of additional mono7, with a 2-year LFS of 11% [95% CI 0–23] (mono5 without mono7) and 13% [95% CI 0–27] (mono5 with mono7) vs 38% [95% CI 9–67] (absence of mono5 with mono7) and 37% [95% CI 20–53] (absence of both abnormalities, p = 0.007) (b) (58%, [95% CI 34–82], p = 0.005) as well as patient with concomitant -5/5q- (12%, [95% CI 2–22] vs 44%, [95% CI 28–59], p < 0.005). Taking together -7 and -5/5q- sta- tus, absence of -5/5q- was associated to a better out- come (2-year OS 47% [95% CI 30–65] and 31% [95% CI 0–63] in patients without and with concomitant -7, re- spectively) compared to the subset of patients harbour- ing -5/5q- (2-year OS 16% [95% CI 1–31] and 10% [95% CI 0–22], according to simultaneous -7 or not, respect- ively) (Fig. 4a). Thus, in multivariate analysis, the pres- ence of -5/5q- was significantly associated with a decreased OS (HR 2.02; 95% CI 1.2–3.5, p = 0.01), whereas RIC was associated with a trend towards a worse OS (p = 0.07) (Table 3). Causes of death were dis- ease related in 63 patients, infections in 13, GvHD in 11, haemorrhage in 1 and others in 2 patients. The 2-year probability of LFS was 24% [95% CI 15.7–31.9] as illustrated in Fig. 3b. Survival analysis, decreased LFS was significantly associated with RIC (2-year LFS of 20% [95% CI 8–29] and 30% [95% CI 16–44] after RIC and MAC allo-SCT, respect- ively, p = 0.01), age above 50 years old (2-year LFS: 20% [95% CI 10–30] vs 29% [95% CI 15–43], p < 0.005), MK (2-year LFS 17% [95% CI 8–25] vs 49% [95% CI 25–72], p = 0.02), and presence of -5/5q-). The deleterious impact of 5q loss was independent of presence of additional -7, with a 2-year LFS of 11% [95% CI 0–23] (-5/5q- without -7) and 13% [95% CI The 2-year probability of LFS was 24% [95% CI 15.7–31.9] as illustrated in Fig. 3b. In univariate Page 7 of 10 Page 7 of 10 Poiré et al. Journal of Hematology & Oncology (2017) 10:20 0–27] (-5/5q- with -7) vs 38% [95% CI 9–67] (absence of -5/5q- with -7) and 37% [95% CI 20–53] (absence of both abnormalities, p = 0.007; Fig. 4b). In vivo T cell depletion and the use of ATG were not significantly associated with worse LFS. In multivariate analysis, only the presence of -5/5q- remained significantly as- sociated with a decreased LFS (p = 0.02) (Table 3). 0–27] (-5/5q- with -7) vs 38% [95% CI 9–67] (absence of -5/5q- with -7) and 37% [95% CI 20–53] (absence of both abnormalities, p = 0.007; Fig. 4b). In vivo T cell depletion and the use of ATG were not significantly associated with worse LFS. In multivariate analysis, only the presence of -5/5q- remained significantly as- sociated with a decreased LFS (p = 0.02) (Table 3). Nonetheless, the study provides evidences of the urgent unmet need to develop novel strategies for these pa- tients. Different transplant modalities, concerning donor source or conditioning regimen, did not have a major impact on transplant outcome in our study, and future improvement attempts must explore pre- and post- transplant interventions, together with innovative modi- fications of allo-SCT conditioning regimen. The presence of chronic GvHD was associated with significantly decreased risk of RI, but resulted in higher NRM, and worse OS and LFS (p < 0.005) (Table 4). The 2-year probability of GvHD and relapse-free survival (GRFS) was 16% [95% CI 9–23]. Among the 10 patients transplanted with CB, we found similar 2-year OS and LFS of 27% [95% CI 0–56] and 27% [95% CI 2–58], re- spectively. Survival However, these results confirm the role of allo-SCT as a reasonable op- tion for the subset of patients achieving sufficient cytore- duction at the time of transplantation. Abn(17p) are highly represented in overlapping cytogenetically very high-risk AML, such as MK and CK, and might partici- pate in the underlying mechanisms responsible of the their refractoriness to standard intensive AML therapy. Table 4 Multivariate analysis using a Cox proportional hazards model. Impact of cGvHD on outcomes (time-dependant variable) p HR 95% CI RI <10−4 0.76 0.69 0.85 NRM <10−4 2.92 2.62 3.25 LFS <10−4 1.45 1.35 1.56 OS <10−4 1.25 1.17 1.35 Abbreviations: N number, LFS leukaemia-free survival, OS overall survival, RI relapse incidence, NRM non-relapse mortality, HR hazard ratio, CI confidence interval Survival No significant differences were found with the other patients (p = 0.95 and p = 0.81, respectively). In this small cohort, four out of them showed the combin- ation of abn(17p) and -5/5q-. Our results are quite comparable to those reported by Middeke et al. [29]. The 2-year LFS and OS of 24 and 28%, respectively, in our cohort are more favourable compared to the previous retrospective study from Mohr et al., based on 47 transplanted patients, which did not show any advantage compared to conventional therapy, with a 4-year probability of survival for the entire cohort of only 4% [28]. Relapse was the main cause of treatment failure, achieving 70% at 2 years after RIC conditioning, and these relapses occurred at a median interval from transplant of 4 months, indicating the need of imple- menting early interventions in the post-transplant period to prevent relapse. Notably, the current results in pa- tients with AML harbouring abn(17p) are similar to those observed with MK and CK AML. In those studies, an independent effect of abn(17p) has not been found [13–15]. In fact, genomic losses of 17p, together with losses of 5q and 7q, and gains 11q and 8q, are the most frequent cytogenetic abnormalities described in CK [34]. Nevertheless, our study cohort represents a more homo- geneous population than the one addressed in the stud- ies evaluating MK and CK AML. On the other hand, we were not able to find a significant effect of the presence of MK, probably because 83% of patients displayed MK at diagnosis. p q Discussion P53 loss of function resulting from chromosomal losses of 17p region and TP53 gene mutations result in marked chemorefractoriness and very poor prognosis, with virtu- ally incurability for most patients treated with conven- tional AML chemotherapy [24, 25, 28, 29]. The present study focused on the capability of allo-SCT to circum- vent this dismal prognosis. In patients allografted in CR1, LFS at 2 years was 24%, suggesting the potential curability of a proportion of these patients with this ap- proach, and the existence of a potent graft-versus- tumour effect capable to sustain response. Our cohort might correspond to a highly selected patient popula- tion, with some degree of chemosensitivity sufficient to achieve an initial response, and is therefore not repre- sentative of the whole abn(17p) AML. Discussion P53 loss of function resulting from chromosomal losses of 17p region and TP53 gene mutations result in marked chemorefractoriness and very poor prognosis, with virtu- ally incurability for most patients treated with conven- tional AML chemotherapy [24, 25, 28, 29]. The present study focused on the capability of allo-SCT to circum- vent this dismal prognosis. In patients allografted in CR1, LFS at 2 years was 24%, suggesting the potential curability of a proportion of these patients with this ap- proach, and the existence of a potent graft-versus- tumour effect capable to sustain response. Our cohort might correspond to a highly selected patient popula- tion, with some degree of chemosensitivity sufficient to achieve an initial response, and is therefore not repre- sentative of the whole abn(17p) AML. However, these results confirm the role of allo-SCT as a reasonable op- tion for the subset of patients achieving sufficient cytore- duction at the time of transplantation. Abn(17p) are highly represented in overlapping cytogenetically very high-risk AML, such as MK and CK, and might partici- pate in the underlying mechanisms responsible of the their refractoriness to standard intensive AML therapy. NRM was only 17%, probably reflecting the positive selection effect in this population, enriched with respon- sive patients to previous chemotherapy. In fact, these 139 patients represent only 1.3% of 10,799 patients with an available karyotype in the EBMT database, a lower proportion than the expected rate of 5–10% in general AML population [19]. These 1.3% of patients refer only to the proportion of abn(17p) AML patients who were in remission and fit enough to survive until the trans- plantation procedure. Lower intensity conditioning regi- mens were associated to a higher relapse risk, up to 70%, in the univariate analysis, an association not con- firmed in the multivariate analysis adjusted for other variable such as concomitant presence of 5q loss and age. Nonetheless, the effect of different regimens aimed to enhance antitumour effect without increasing toxicity must also be explored in the next future. Table 4 Multivariate analysis using a Cox proportional hazards model. Discussion Impact of cGvHD on outcomes (time-dependant variable) p HR 95% CI RI <10−4 0.76 0.69 0.85 NRM <10−4 2.92 2.62 3.25 LFS <10−4 1.45 1.35 1.56 OS <10−4 1.25 1.17 1.35 Abbreviations: N number, LFS leukaemia-free survival, OS overall survival, RI relapse incidence, NRM non-relapse mortality, HR hazard ratio, CI confidence interval Table 4 Multivariate analysis using a Cox proportional hazards model. Impact of cGvHD on outcomes (time-dependant variable) Table 4 Multivariate analysis using a Cox proportional hazards model. Impact of cGvHD on outcomes (time-dependant variable) variable) p HR 95% CI RI <10−4 0.76 0.69 0.85 NRM <10−4 2.92 2.62 3.25 LFS <10−4 1.45 1.35 1.56 OS <10−4 1.25 1.17 1.35 Presence of chronic GvHD, analysed as time- dependant variable, was independently associated with a lower relapse risk (HR 0.76), supporting the existence of a genuine and potent graft-versus-leukaemia. This Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Page 8 of 10 Page 8 of 10 antitumour effect of chronic GvHD, nonetheless, did not result in a neat benefit due to its association to a higher NRM translating into worse OS and LFS. Recognition of a potential graft-versus-leukaemia effect in abn(17p) AML would give the basis to develop strategies aimed to harness this alloimmune effect in the early post- transplant period, such as early withdrawal of immuno- suppression or administration of prophylactic donor leukocyte infusion [35, 36]. Post-transplant administra- tion azacytidine might contribute to stimulate the anti- tumour donor graft effect by enhancing the expression of tumour and minor histocompatibility antigens, with the theoretical added advantage of avoiding an increased GvHD rate by expansion of T regulatory cell population. Several studies have demonstrated feasibility of azacyti- dine during the post-transplant period, and the correl- ation of the expansion determined cytotoxic T cell subsets against tumour antigens with a lower relapse in- cidence, but the clinical benefit of such strategy should be further proven [37–40]. Other innovative donor cell strategies such as NK cell infusion or Cytokine-induced killer population, with a theoretical lower potential of GvHD induction, must be of high interest in this setting [41–43]. Anyhow, based on the very short median time to relapse, post-transplantation interventions should be given early as a prophylactic or maintenance strategy. Vosaroxin [44, 45] is a quinolone derivative reported to be TP53 independent and shows some clinical benefit in combination with high-dose cytarabine in relapsed pa- tients. Availability of data and materials The dataset analysed in the present study is avalaible in the EBMT registry. The dataset analysed in the present study is avalaible in the EBMT registry. Funding Funding This retrospective study is supported by the EBMT. g This retrospective study is supported by the EBMT. Authors’ contributions XP designed the study. XP and LM reviewed the cytogenetic data. XP, ML, JE and AN analysed the data. XP wrote the paper. JE, MM and AN reviewed the paper. JM, IYA, DB, NI, GS, TGD, NS, JC, SV and JS provide the data. JM, IYA, DB, NI, GS, TGD, NS, JC, SV and JS approved the manuscript. All authors read and approved the final manuscript. Additional file Additional file 1: Table S1. (DOCX 13 kb) Additional file 1: Table S1. (DOCX 13 kb) Conclusions All Allo-SCT arises as the best therapeutic option to im- prove survival in selected patients harbouring abn(17p) and achieving CR after frontline chemotherapy, espe- cially in the absence of -5/5q-. Nonetheless, clinical benefit of allo-HCT remains very limited, followed by a high relapse incidence. Recognition of a potential graft- versus-leukaemia effect preventing relapse in some pa- tients gives the rationale basis for the development of early chemotherapy-based or cell-based strategies to pre- vent relapse and therefore to increase the potential benefit of allo-SCT in these patients. Discussion Currently, it is completely unknown if this agent, administered prior to allo-SCT, might result into im- proved outcome after allo-SCT, but it might constitute a model to bring abn(17p) AML with better response to allo-SCT. apoptosis and with increased genomic instability [48]. This translates into chemoresistance and worse out- comes confirmed in our study with patients harbouring both abn(17p) and -5/5q-. In this subgroup of patients, the benefit of allo-SCT appears very limited and new therapeutic strategies are strongly warranted. On the contrary, patients with abn(17p) without -5/5q- showed a relative good outcome after allo-SCT with a 2-year probability of LFS of 37–38%. Abbreviations -5/5q-: Monosomy 5 or loss of 5q-; -7: Monosomy 7; Abn(17p): 17p abnormalities; Allo-SCT: Allogeneic stem cell transplantation; AML: Acute myeloid leukaemia; ATG: Anti-thymocyte globulin; CB: Cord blood; CI: Confidence interval; CK: Complex caryotype; CMV: Cytomegalovirus; CR1: First complete remission; CR2: Second complete remission; DLI: Donor leukocyte infusion; EBMT: European Society of Blood and Marrow Transplantation; EFS: Event-free survival; GRFS: GvHD-free/relapse-free survival; GvHD: Graft-versus-host disease; HLA: Human leukocyte antigen; HR: Hazard ratio; Inv(3): Inversion of chromosome 3; LFS: Leukaemia-free survival; MAC: Myeloablative conditioning; MK: Monosomal karyotype; NRM: Non-relapse mortality; OS: Overall survival; RI: Relapse incidence; RIC: Reduced-intensity conditioning; TBI: Total body irradiation Additional chromosomal 5q loss conferred an even worse outcome in this cohort of patients, with an in- creased relapse risk and 2-year OS and LFS of only 10 and 11%, respectively, regardless the presence of con- comitant monosomy 7. It was the only independent prognostic factor in this patient population. While the biological basis accounting for this combined deleterious effect is mostly unknown, TP53 mutations have been fre- quently observed in association with loss of 5q, up to 80% of cases in some series, suggesting cooperation be- tween TP53 mutations and loss of putative tumour sup- pressor genes localised in 5q region [46–48]. Previous reports supported this hypothesis that multiple candi- date genes localised on 5q cooperate with TP53 muta- tions in the pathogenesis of myelodysplastic syndrome or AML [49–51]. Many genes on 5q have been pro- posed, but recently, haploinsufficiency of ERG1 and APC in combination with the early acquisition of TP53 muta- tions have emerged as a potential mechanism leading to the development of a leukemic clone resistant to References 1. Koreth J, Schlenk R, Kopecky KJ, et al. 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Fenaux P, Preudhomme C, Lai JL, et al. Cytogenetics and their prognostic value in de novo acute myeloid leukaemia: a report on 283 cases. Br J Haematol. 1989; 73:61–7. Ethics approval and consent to participate All retrospective studies performed by the EBMT are centrally approved by the EBMT board. 13. Cornelissen JJ, Breems D, van Putten WL, et al. Comparative analysis of the value of allogeneic hematopoietic stem-cell transplantation in acute myeloid leukemia with monosomal karyotype versus other cytogenetic risk categories. J Clin Oncol. 2012;30:2140–6. All retrospective studies performed by the EBMT are centrally approved by the EBMT board. Competing interests Th h d l h The authors declare that they have no competing interests. The authors declare that they have no competing interests. Page 9 of 10 Page 9 of 10 Page 9 of 10 Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Consent for publication 12. Ferrant A, Labopin M, Frassoni F, et al. Karyotype in acute myeloblastic leukemia: prognostic significance for bone marrow transplantation in first remission: a European Group for Blood and Marrow Transplantation study. Acute Leukemia Working Party of the European Group for Blood and Marrow Transplantation (EBMT). Blood. 1997;90:2931–8. All patients included in the EBMT registry database have previously consented to be part anonymously to the registry. References Br J Haematol. 2016;172:914–22. 5. Dohner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373:1136–52. 6. Grimwade D, Ivey A, Huntly BJ. Molecular landscape of acute myeloid leukemia in younger adults and its clinical relevance. Blood. 2016;127:29–41. 28. Mohr B, Schetelig J, Schafer-Eckart K, et al. Impact of allogeneic haematopoietic stem cell transplantation in patients with abnl(17p) acute myeloid leukaemia. Br J Haematol. 2013;161:237–44. 7. Schoch C, Kern W, Kohlmann A, et al. Acute myeloid leukemia with a complex aberrant karyotype is a distinct biological entity characterized by genomic imbalances and a specific gene expression profile. Genes Chromosomes Cancer. 2005;43:227–38. 29. Middeke JM, Fang M, Cornelissen JJ, et al. Outcome of patients with abnl(17p) acute myeloid leukemia after allogeneic hematopoietic stem cell transplantation. Blood. 2014;123:2960–7. 8. Baron F, Labopin M, Niederwieser D, et al. Impact of graft-versus-host disease after reduced-intensity conditioning allogeneic stem cell transplantation for acute myeloid leukemia: a report from the Acute Leukemia Working Party of the European group for blood and marrow transplantation. Leukemia. 2012;26:2462–8. 30. Rubio MT, Savani BN, Labopin M, et al. The impact of HLA-matching on reduced intensity conditioning regimen unrelated donor allogeneic stem cell transplantation for acute myeloid leukemia in patients above 50 years—a report from the EBMT acute leukemia working party. J Hematol Oncol. 2016;9:65. 31. Ruggeri A, Battipaglia G, Labopin M, et al. Unrelated donor versus matched sibling donor in adults with acute myeloid leukemia in first relapse: an ALWP-EBMT study. J Hematol Oncol. 2016;9:89. 9. Schlenk RF, Dohner K, Mack S, et al. Prospective evaluation of allogeneic hematopoietic stem-cell transplantation from matched related and matched unrelated donors in younger adults with high-risk acute myeloid leukemia: German-Austrian trial AMLHD98A. J Clin Oncol. 2010;28:4642–8. 32. Saraceni F, Labopin M, Gorin NC, et al. Matched and mismatched unrelated donor compared to autologous stem cell transplantation for acute myeloid leukemia in first complete remission: a retrospective, propensity score- weighted analysis from the ALWP of the EBMT. J Hematol Oncol. 2016;9:79. 10. Stelljes M, Beelen DW, Braess J, et al. Allogeneic transplantation as post-remission therapy for cytogenetically high-risk acute myeloid leukemia: landmark analysis from a single prospective multicenter trial. Haematologica. 2011;96:972–9. 33. Schmid C, Schleuning M, Hentrich M, et al. High antileukemic efficacy of an intermediate intensity conditioning regimen for allogeneic stem cell transplantation in patients with high-risk acute myeloid leukemia in first complete remission. Bone Marrow Transplant. 2008;41:721–7. 11. References Versluis J, Hazenberg CL, Passweg JR, et al. Post-remission treatment with allogeneic stem cell transplantation in patients aged 60 years and older with acute myeloid leukaemia: a time-dependent analysis. Lancet Haematol. 2015;2:e427–36. Page 10 of 10 Poiré et al. Journal of Hematology & Oncology (2017) 10:20 Poiré et al. Journal of Hematology & Oncology (2017) 10:20 34. Rucker FG, Bullinger L, Schwaenen C, et al. Disclosure of candidate genes in acute myeloid leukemia with complex karyotypes using microarray-based molecular characterization. J Clin Oncol. 2006;24:3887–94. 35. Schmid C, Schleuning M, Ledderose G, et al. Sequential regimen of chemotherapy, reduced-intensity conditioning for allogeneic stem-cell transplantation, and prophylactic donor lymphocyte transfusion in high-risk acute myeloid leukemia and myelodysplastic syndrome. J Clin Oncol. 2005;23:5675–87. 36. Yan CH, Liu DH, Liu KY, et al. Risk stratification-directed donor lymphocyte infusion could reduce relapse of standard-risk acute leukemia patients after allogeneic hematopoietic stem cell transplantation. Blood. 2012;119:3256–62. 37. Bally C, Ades L, Renneville A, et al. Prognostic value of TP53 gene mutations in myelodysplastic syndromes and acute myeloid leukemia treated with azacitidine. Leuk Res. 2014;38:751–5. 38. Goodyear OC, Dennis M, Jilani NY, et al. Azacitidine augments expansion of regulatory T cells after allogeneic stem cell transplantation in patients with acute myeloid leukemia (AML). Blood. 2012;119:3361–9. 39. Platzbecker U, Wermke M, Radke J, et al. Azacitidine for treatment of imminent relapse in MDS or AML patients after allogeneic HSCT: results of the RELAZA trial. Leukemia. 2012;26:381–9. 40. Craddock C, Labopin M, Robin M, et al. Clinical activity of azacitidine in patients who relapse after allogeneic stem cell transplantation for acute myeloid leukemia. Haematologica. 2016;101(7):879–83. 41. Lim O, Jung MY, Hwang YK, et al. Present and future of allogeneic natural killer cell therapy. Front Immunol. 2015;6:286. 41. Lim O, Jung MY, Hwang YK, et al. Present and future of allogeneic natural killer cell therapy. Front Immunol. 2015;6:286. 42. Pittari G, Filippini P, Gentilcore G, et al. Revving up natural killer cells and cytokine-induced killer cells against hematological malignancies. Front Immunol. 2015;6:230. 43. Stern M, Passweg JR, Meyer-Monard S, et al. Pre-emptive immunotherapy with purified natural killer cells after haploidentical SCT: a prospective phase II study in two centers. Bone Marrow Transplant. 2013;48:433–8. 44. Hotinski AK, Lewis ID, Ross DM. Vosaroxin is a novel topoisomerase-II inhibitor with efficacy in relapsed and refractory acute myeloid leukaemia. Expert Opin Pharmacother. 2015;16:1395–402. 45. Walsby EJ, Coles SJ, Knapper S, et al. References The topoisomerase II inhibitor voreloxin causes cell cycle arrest and apoptosis in myeloid leukemia cells and acts in synergy with cytarabine. Haematologica. 2011;96:393–9. 46. Castro PD, Liang JC, Nagarajan L. Deletions of chromosome 5q13.3 and 17p loci cooperate in myeloid neoplasms. Blood. 2000;95:2138–43. 47. Kulasekararaj AG, Smith AE, Mian SA, et al. TP53 mutations in myelodysplastic syndrome are strongly correlated with aberrations of chromosome 5, and correlate with adverse prognosis. Br J Haematol. 2013;160:660–72. 48. Stoddart A, Fernald AA, Wang J, et al. Haploinsufficiency of del(5q) genes, Egr1 and Apc, cooperate with Tp53 loss to induce acute myeloid leukemia in mice. Blood. 2014;123:1069–78. 49. Bejar R, Stevenson K, Abdel-Wahab O, et al. Clinical effect of point mutations in myelodysplastic syndromes. N Engl J Med. 2011;364:2496–506. 50. Shih AH, Chung SS, Dolezal EK, et al. Mutational analysis of therapy-related myelodysplastic syndromes and acute myelogenous leukemia. Haematologica. 2013;98:908–12. 50. Shih AH, Chung SS, Dolezal EK, et al. Mutational analysis of therapy-related myelodysplastic syndromes and acute myelogenous leukemia. Haematologica. 2013;98:908–12. 51. Walter MJ, Shen D, Shao J, et al. Clonal diversity of recurrently mutated genes in myelodysplastic syndromes. Leukemia. 2013;27:1275–82. 51. Walter MJ, Shen D, Shao J, et al. Clonal diversity of recurrently mutated genes in myelodysplastic syndromes. Leukemia. 2013;27:1275–82. Poiré et al. 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https://openalex.org/W4244443666	https://www.researchsquare.com/article/rs-46879/latest.pdf	English	null	Effectiveness of An Integrated Multidisciplinary Geriatric Rehabilitation Programme for Older Persons with Stroke: A Multicentre Randomised Controlled Trial	Research Square (Research Square)	2,020	cc-by	8,252	Effectiveness of an integrated multidisciplinary geriatric rehabilitation programme for older persons with stroke: a multicentre randomised controlled trial Tom Vluggen (  t.vluggen@maast Universiteit Maastricht Jolanda van Haastregt Universiteit Maastricht Frans Tan Universiteit Maastricht Jeanine Verbunt Universiteit Maastricht Caroline van Heugten Universiteit Maastricht Jos Schols Universiteit Maastricht Tom Vluggen (  t.vluggen@maastrichtuniversity.nl ) Universiteit Maastricht Jolanda van Haastregt Universiteit Maastricht Frans Tan Universiteit Maastricht Jeanine Verbunt Universiteit Maastricht Caroline van Heugten Universiteit Maastricht Jos Schols Universiteit Maastricht Tom Vluggen (  t.vluggen@maastrichtuniversity.nl ) Universiteit Maastricht Research article License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published on February 23rd, 2021. See the published version at https://doi.org/10.1186/s12877-021-02082-4. Page 1/23 Abstract Background: Almost half of the stroke patients admitted to geriatric rehabilitation has persisting problems after discharge. Currently, there is no evidence based geriatric rehabilitation programme available for older stroke patients, combining inpatient rehabilitation with adequate ambulatory aftercare in the community. Therefore, we developed an integrated multidisciplinary rehabilitation programme that includes aftercare for older persons with stroke. We evaluated the effectiveness of this newly developed rehabilitation programme in comparison to usual care. Methods: A multicentre randomised controlled trial was conducted in eight geriatric rehabilitation stroke units and their collaborating partners in primary care. The study population involved stroke patients and their informal caregivers who were aged 65 or over, living in the community before admission to geriatric rehabilitation, and expected to be able to return home after discharge. The programme consisted of three modules: inpatient neurorehabilitation, home-based self-management training, and stroke education. For patients, daily activity (FAI) was assessed as primary outcome and functional dependence (Katz-15), perceived quality of life (SSQoL) and social participation (IPA) as secondary outcomes. Additionally, among informal caregivers perceived care burden (self-rated burden VAS), objective care burden (Erasmus iBMG), and quality of life (CarerQol), were assessed as secondary outcomes. Results: In total 190 patients and 172 informal caregivers were included. Mean age of the patients in the intervention group was 78.9 years (SD= 7.0) and in the usual care group 79.0 years (SD=6.5). Significant favourable effects for the programme were observed for the subscale autonomy outdoors of the IPA (-2.15, P=.047, and for the informal caregivers perceived care burden (1.23, P=.048. For the primary outcome daily activity and the other secondary outcomes, no significant effects were observed. Conclusion: The integrated multidisciplinary programme had no effect on daily activity of older stroke patients. However, patients participating in the programme had a higher level of perceived autonomy of outdoor activities and their informal caregivers perceived a lower care burden. The programme might be promising in providing adequate (after)care, although adaptation of the programme is recommended to increase its feasibility and improve its effects. Background Stroke is one of the leading causes of death and a major cause of disability worldwide. Because of the aging population stroke is highly prevalent and can have a major impact on daily functioning and quality of life1,2. Page 2/23 In the Netherlands, each year about 40% of the older persons who suffer from acute stroke are admitted to an intermediate care facility for geriatric rehabilitation after a period of hospitalisation3-5. About half of the older stroke patients who are discharged home after geriatric rehabilitation still experience serious impairments in daily functioning and social participation, caused by severe cognitive and functional incapacities6 . In patients who are socially inactive and are lacking appropriate coping skills, these impairments can lead to a substantial decrease in quality of life and depression7. Most older persons who are admitted to geriatric rehabilitation have multimorbidity that can interfere with rehabilitation and therefore may influence outcomes negatively. Besides a negative impact on patients, stroke and multimorbidity may also increase the burden of care perceived by informal caregivers which may also result in a decrease in their quality of life7,8. Eventually, when the burden for the informal caregiver becomes too high, this may result in permanent admission of the patient to a long-term care facility. In the Netherlands, stroke care for older patients is organised in stroke services aiming to realise more integrated care. This trend has led to a reduction in mortality, a decrease in admissions to long-term institutional care, more satisfaction among patients and caregivers, and more cost-effectiveness9. Although stroke care has achieved these quality improvements, sufficient aftercare after inpatient geriatric rehabilitation is often lacking in usual care, or when available, is too fragmented which makes it difficult to support patients and their informal caregivers in dealing with stroke related problems at home after discharge from rehabilitation. In the Netherlands, stroke care for older patients is organised in stroke services aiming to realise more integrated care. This trend has led to a reduction in mortality, a decrease in admissions to long-term institutional care, more satisfaction among patients and caregivers, and more cost-effectiveness9. Although stroke care has achieved these quality improvements, sufficient aftercare after inpatient geriatric rehabilitation is often lacking in usual care, or when available, is too fragmented which makes it difficult to support patients and their informal caregivers in dealing with stroke related problems at home after discharge from rehabilitation. Background Therefore, it seems important that older stroke patients and their caregivers, receive a rehabilitation treatment that includes tailor-made aftercare after discharge from geriatric rehabilitation to facilitate the transition to the home situation and to support patients and their caregivers in coping with the patients’ residual impairments in daily life. Training older patients and their caregivers in effective coping skills to manage their impairments might contribute to living independently in the community and staying socially active as long as possible. In addition, adequate aftercare may prevent negative long-term consequences such as decrease in daily activity level, depression and postpone admission to a long-term care facility10,11. Therefore, stroke rehabilitation should include structural follow-up treatment in the patients’ home environment to improve functional independence of patients, to train patients in coping strategies to increase the adaptation skills to manage the remaining physical, cognitive and/or psychosocial impairments and improve quality of life, and to provide support for the informal caregiver to decrease the burden of care12,13. Currently, there is no effective and well-organised aftercare programme available for older stroke patients admitted to geriatric rehabilitation14. Therefore, we developed an integrated multidisciplinary geriatric rehabilitation programme that includes aftercare for older persons with stroke. It aims to facilitate early discharge if possible, to train patients and informal caregivers to cope with the residual impairments by enhancing self-management, to optimise the level of participation after rehabilitation, and to provide support at home after discharge from rehabilitation. The aim of this study was to evaluate the effects of this integrated programme as compared with usual care on the primary outcome daily activity level, and on the secondary outcomes functional independence, perceived quality of life and social participation of patients, and perceived care burden, objective care burden, and quality of life of their informal caregivers. Methods Page 3/23 Page 3/23 Study design The design of this study was a two-arm multicenter randomised controlled trial with patients allocated to either the integrated programme or usual care. The study was conducted in eight geriatric rehabilitation units for patients with stroke. More specific information about the methodology of the study can be obtained from protocol article published earlier15. This study adheres to CONSORT guidelines for Randomized Controlled Trials. The protocol of this study was registered with the International Standard Randomised Controlled Trial Register Number (ISRCTN62286281), and The Dutch Trial Register (NTR2412). Study population The study population involved stroke patients and their informal caregivers who were admitted to one of the eight participating geriatric rehabilitation stroke units after hospital discharge. The study population was restricted to patients aged 65 or over, living in the community before admission to geriatric rehabilitation, and expected to be able to return home after discharge. Inclusion started directly after admission to the geriatric rehabilitation unit. At admission the rehabilitation team under the responsibility of an elderly care physician, conducted a comprehensive geriatric assessment to determine if the patient was expected to return home after discharge. Based on this assessment (in combination with the other eligibility criteria), patients were included in the study. Patients who did not give informed consent for participation, or were medically unstable and thereby not able to start rehabilitation, were excluded. In addition, the primary informal caregiver of each participating patient was invited to participate in the study. A person is considered to be the primary informal caregiver in case the patient indicates him/her as the person mostly involved in informal care activities for this patient. The multidisciplinary teams of the participating geriatric rehabilitation units received a three hour training which included the important key elements of the intervention protocol. During the study, the participating multidisciplinary teams were responsible for checking which admitted patients fulfilled the inclusion criteria of the study. To calculate the sample size, data from earlier research was used. Based on the Frenchay Activity Index score as primary outcome variable16, the assumed clinically relevant difference in activity level of two stroke populations had to be at least 3.5. Based on a power of 0.8 and an alpha of 0.05, the study would need a sample size of 102 patients in each group. With an expected drop-out during follow-up estimated at approximately 25%, each group should include 128 participants. In total 256 participants were needed for the study. Organisation of the integrated programme The integrated programme consisted of three care modules; 1) inpatient neurorehabilitation treatment; 2) home-based self-management training for patient and informal caregiver; and 3) stroke education for patient and informal caregiver. Table 1 presents both the integrated multidisciplinary geriatric programme and usual care. The treatment progress was evaluated in monthly multidisciplinary team meetings for every individual patient. All communication and information by the care professionals about the patient and informal caregiver was conducted by using a shared electronic patient record, which was specifically developed for this study. To optimise care by facilitating faster discharge and to give support after discharge a stroke care coordinator was introduced in all participating rehabilitation teams. The total programme duration, including all three modules, varied between 2 to 6 months, depending on the care needs of the patient. All care professionals of the participating stroke teams were trained in conducting the programme according to protocol15. Randomisation After inclusion, all patients and their informal caregivers of each participating nursing stroke unit were randomised on patient level by an independent research assistant. The randomisation procedure was conducted by a computerised block randomization schedule using IBM SPSS software version 19.0 (10 patients per block) to allocate the included patients to the intervention or usual care group. Patients allocated to the intervention group received the integrated programme and patients allocated to the usual Page 4/23 Page 4/23 care group received care as usual. Data were collected by research assistants who were blinded for treatment allocation. Because of study characteristics, blinding of patients, informal caregivers and care professionals involved was not possible. care group received care as usual. Data were collected by research assistants who were blinded for treatment allocation. Because of study characteristics, blinding of patients, informal caregivers and care professionals involved was not possible. The stroke care coordinator When the patient was admitted to the geriatric rehabilitation unit, the stroke care coordinator was introduced. The stroke care coordinator facilitated the transition of nursing home rehabilitation care services to community care by supporting the collaboration between the multidisciplinary stroke team of the nursing home and the community health services, namely community nurses, paramedical professionals and the general practitioner. After discharge, the coordinator conducted home visits, supports the general practitioner by organising multidisciplinary stroke team meetings and guided the patient and informal care giver in learning to apply self-management principles. At the start of geriatric rehabilitation, the coordinator had an introduction meeting with both the patient and informal caregiver. In this meeting, the coordinator provided general information about the rehabilitation programme. Furthermore, during the rehabilitation process the coordinator facilitated the transition of the patient from inpatient geriatric rehabilitation care to home-based care by supporting the collaboration between the multidisciplinary stroke team of the geriatric rehabilitation unit, community health services and general practitioner. After discharge, the coordinator conducted at least two home visits, organised multidisciplinary stroke team meetings in the community and supported the patient and informal caregiver in practicing self-management skills at home. Module 1: inpatient neurorehabilitation treatment for patients Module 1: inpatient neurorehabilitation treatment for patients Page 5/23 The first module focused on (re)learning the abilities needed for individual patients to function as independently as possible in their own home environment. At the start of this module, an individual treatment plan was made together with the patient including the development of rehabilitation goals facilitating the transition from in-patient to home-based rehabilitation care and to guide further rehabilitation at the patient’s home. To make rehabilitation goals more measurable during inpatient rehabilitation, the principles of the Goal Attainment Scaling (GAS) method were used. GAS is a methodology, which is shown to be appropriate for developing rehabilitation goals among older persons16. To facilitate transition to the home environment, during the stay at the geriatric rehabilitation unit, an occupational or physical therapist, depending on the rehabilitation goals, trained with the patients at least twice in their own home environment. These training sessions were done to optimise recovery, to train specific functional skills at home to increase independence, and to check if any home adjustments were needed before discharge. Module 2: home-based self-management training for patient and informal caregiver The second module started directly after discharge to the home environme learning to cope with residual cognitive and functional impairments as a r coordinator trained patients and caregivers to improve their coping strateg techniques. This training which included formulating rehabilitation goals f action plans, were based on the basic principles of self-management and solving skills and participation13,17. If necessary, patients could still receiv rehabilitation treatment by a physical or occupational therapist, with the in treatment sessions should take place in the patient’s home. If home treatm patient could receive this treatment in a day care facility or private therapy module was also provided by the professionals of the regional multidiscip professionals of the geriatric rehabilitation unit and community health car The second module started directly after discharge to the home environment. Treatment focused on learning to cope with residual cognitive and functional impairments as a result of stroke. The stroke care coordinator trained patients and caregivers to improve their coping strategies and empowerment techniques. This training which included formulating rehabilitation goals for the patients and making action plans, were based on the basic principles of self-management and aimed to increase problem- solving skills and participation13,17. If necessary, patients could still receive ambulatory follow-up rehabilitation treatment by a physical or occupational therapist, with the intention that at least half of the treatment sessions should take place in the patient’s home. If home treatment was not possible, the patient could receive this treatment in a day care facility or private therapy practice. The training in this module was also provided by the professionals of the regional multidisciplinary team consisting of professionals of the geriatric rehabilitation unit and community health care. The stroke care coordinator The training programme within this module was conducted by a multidisciplinary stroke team consisting of professionals working at the geriatric rehabilitation unit of the nursing home. The stroke rehabilitation team included an elderly care physician, a physical therapist, an occupational therapist, a speech therapist, a (neuro)psychologist and a stroke coordinator. Module 3: stroke education for patient and informal caregiver The third module was a short stroke education course for patients and their involved informal caregivers. The course consisted of four education sessions of two hours each with the focus on respectively the psychological and emotional consequences of stroke, perceived problems during independent living and participation in societal activities, and on the role of the informal caregiver. The course was provided by a (neuro)psychologist, two volunteers of the Dutch Stroke Patient Association and Informal Caregivers Association, and a social worker. The stroke coordinator invited the patients and informal caregivers to participate in the course. In two of the four meetings patients and informal caregivers were divided in two separate groups, to provide them the opportunity to express their problems and concerns more freely and share experiences with other patients/caregivers. separate groups, to provide them the opportunity to express their problems and concerns more freely and share experiences with other patients/caregivers. Primary outcome measure An overview of the primary and secondary outcome measurements per time point is presented in table 2. Primary outcome measure was daily activity of patients measured by means of the Frenchay Activity Index (FAI) a 15-items activity scale (range 15-60 with higher scores indicating better functioning)21. The outcome of the FAI at baseline (i.e. at admission to the geriatric rehabilitation unit) was based on the activity level of patients 3 months before stroke occurred, as estimated by the patient. Follow-up measurements of the FAI were conducted after 6 and 12 months of follow-up. Usual care In the Netherlands, usual care for older people with a stroke that need inpatient-rehabilitation consists of multidisciplinary neurorehabilitation on a geriatric rehabilitation unit. After discharge, there is in general no coordinated multidisciplinary aftercare for patient and informal caregiver. Most care programs vary in content and are in general more focused on the recovery of the patient and limited on the needs of the informal caregiver. After discharge, the follow-up care is usually provided by monodisciplinary community services, with no multidisciplinary approach. In general, there is no additional involvement anymore of the stroke rehabilitation team of the Table 1 Integrated multidisciplinary geriatric rehabilitation programme and usual care Integrated programme Usual care Care content Multidisciplinary stroke team + + Care based on Dutch stroke guidelines + + Tailored approach with Goal Attainment Scaling + - Self-management + - Stroke education + - Home therapy during nursing home admission + - Multidisciplinary outpatient rehabilitation + - Home visits of stroke care coordinator + - Care organisation Stroke care coordinator + - Multidisciplinary team meetings in nursing home + + Multidisciplinary team meetings after discharge + - Electronic patient record + - Page 7/23 The following background characteristics were measured in both patients and informal caregivers: age, gender, level of education, marital status, living situation, and relationship between patient and informal caregiver. In addition, cognitive functioning of patients was measured at baseline by means of the Mini Mental State Examination (consisting of 11-items, range 0-30 with higher scores indicating better functioning)18-20. Secondary outcome measures Secondary outcome measures for patients were functional dependence measured by the Katz-15 (consisting of 15 items (range 0-15 with lower scores indicating a higher level of independence)22, perceived quality of life measured by the Stroke Specific Quality of Life scale (SSQoL) a 49-items quality of life scale which contains two subscales “Physical functioning” consisting of 27 items (range 27-135) and “Psychosocial functioning” consisting of 22 items (range 22-110). For both subscales of the SS-QOL, a lower score indicates a lower perceived quality of life23. The outcome measure social participation was measured by two subscales “Autonomy outdoors” consisting of 5 items (range 0-20) and “Social relations” consisting of 7 items (range 0-28)) of the Impact on Participation and Autonomy (IPA)24. For both IPA subscales a lower score indicates a better participation level. The other subscales of the IPA were excluded because of the overlap with items of the FAI, Katz-15, and SSQoL. The Katz-15, IPA, and SSQol were measured at admission to the geriatric rehabilitation unit, and after 6 and 12 months of follow-up. Secondary outcome measures in informal caregivers involved the perceived care burden measured by means of the Self-Rated Burden VAS scale (10 points likert scale with lower scores indicating less care burden) and the Carer Quality of Life scale (7 items with lower scores indicating less care burden)25, objective care burden measured by means of the Erasmus iBMG (4-items care burden scale; item 1 “time spent on helping patient with ADL-activities”, item 2 “time spent on helping patient with personal care”, item 3 “time spent on helping patient with moving outside”, item 4 “time spent by other informal caregivers or volunteers on helping patient”)26. The total amount of time spent on the four items indicates the dependence of help by the informal caregiver. All secondary outcome measures were measured at admission to the geriatric rehabilitation unit, and after 6 and 12 months of follow-up. Data collection Data for the effect evaluation was collected by face-to-face interviews among patients and self-reported questionnaires among informal caregivers (see table 2). Research assistants conducted the interviews in the geriatric rehabilitation unit and at the patient’s home and provided the self-administered questionnaires to caregivers at baseline, after 6 months and after 12 months. All data was gathered between 2010 and 2015. Ethics The medical ethics committee of Maastricht University Medical Centre (MUMC+), the Netherlands, approved this study. The medical ethics committee of Maastricht University Medical Centre (MUMC+), the Netherlands, approved this study. Secondary outcome measures Page 8/23 Table 2 Overview of all outcome measures per time point Subject Outcome measures Measurement scale Number of items Time point T0 T1 T2 Patient Primary outcome measure Activity level after stroke Frenchay Activity Index 15 FI FI FI Secondary outcome measures Level of functioning Katz-15 15 FI FI FI Stroke specific quality of life Stroke Specific Quality of Life Questionnaire 49 FI FI FI Social participation Impact on Participation and Autonomy (subscales autonomy outdoors and social life and relationships) 12 FI FI FI Informal caregiver Secondary outcome measures Perceived care burden Self-Rated Burden VAS 10 SQ SQ SQ Carer Quality of life Carer Qol 7 SQ SQ SQ Objective care load Erasmus iBMG 4 SQ SQ SQ Background characteristics Background characteristics patient - 10/5/5 FI FI FI Background characteristics informal Caregiver - 8/7/7 SQ SQ SQ Cognitive functioning patient Mini Mental State Examination 11 FI - - T0 = at baseline, T1 = after 6 months, T2 = after 12 months, FI = face-to-face interview, SQ = self-report questionnaire. Data collection Statistical Analyses Background characteristics of the patients and informal caregivers were checked for meaningful imbalance, analysed and described by using descriptive statistics.Analyses of the difference between primary and secondary outcomes for intervention group and usual care group were performed according to the intention-to-treat principle (with possible covariates taken into account in case of observed imbalance in baseline characteristics),including all valid data of all available participants, regardless of whether they received the (complete) programme. A two-level linear regression analysis was performed to calculate differences between the intervention and usual care group with regard to primary and secondary outcome measures. In the analyses level one was the repeated measures and level two was the patients. In all analyses, P=< .05 was considered statistically significant. All statistical analyses were conducted using IBM SPSS software version 25 for Windows by a researcher who was blinded for treatment allocation. Background characteristics Background characteristics Table 3 shows the background characteristics of patients and informal caregivers. In total 190 patients (mean age: 78.9 years) and 172 informal caregivers (mean age: 60.8 years) were included. Of these 190 patients 99 patients were randomised to the intervention group and 91 patients to the usual care group. Figure 1 shows the flowchart of the patient sample. Of the 172 informal caregivers 90 were randomised to the intervention group and 82 to the usual care. In both groups most informal caregivers were female. More than half of the patients lived alone, and the most common relationship between informal caregiver and the patient was a parent-child (in law) Page 10/23 relationship. The analyses showed only an imbalance in gender (this outcome measure was included as a covariate), but further no meaningful imbalance between all other outcome measures of the intervention and usual care group at baseline. Background characteristics Page 11/23 Table 3 Table 3 Baseline characteristics of patients and informal caregivers Baseline characteristics Scores (N) Patients (N = 189) Intervention group (N = 97) Usual care group (N = 92) Background characteristics Mean age (SD) 78.8 (6.3) 78.8 (7.0) Female gender N (%) 68 (69.4) 46 (51.1) Mean cognitive status (MMSE) (SD) 21.9 (5.2) 22.0 (4.1) Maried with a partner N (%) 39 (40) 43 (48) Living situation • Independent alone N (%) 53 (54.1) 43 (47.8) • Independent with others N (%) 45 (45.9) 47 (52.2) Outcome measurement at baseline Observed mean (SD) Primary outcome Frenchay Activity Index (FAI) 40.2 (8.8) 38.8 (7.3) Secondary outcome Impact on Participation and Activity (IPA) • Autonomy outdoors 15.4 (4.4) 14.8 (4.3) • Relationship 15.3 (3.9) 15.8 (3.1) Katz-15 6.0 (4.0) 6.5 (3.3) Stroke Specific Quality of Life (SSQoL) • Subscale physical functioning 99.6 (20.8) 97.1 (21.3) • Subscale psychosocial functioning 77.2 (16.8) 76.2 (16.9) Informal caregivers (N = 170) Intervention group (N = 89) Usual care group (N = 81) Background characteristics Mean age (SD) 61.0 (13.5) 60.5 (13.5) Female gender N (%) 53 (59.6) 57 (70.4) Relationship with patient Husband wife life partner N (%) 28 (31 5) 32 (39 5) Baseline characteristics of patients and informal caregivers stics Scores (N) Page 12/23 Baseline characteristics Scores (N) Sister, brother, brother in law, sister in law N (%) 3 (3.4) 5 (6.2) Daughter (in law), son (in law) N (%) 55 (61.8) 39 (48.1) Other N (%) 3 (3.4) 5 (6.2) Outcome measurement at baseline Observed mean (SD) Carer Quality of Life 85.9 (12.7) 82.5 (14.7) Erasmus iBMG (time spended on helping patient) Item 1: helping patient with ADL-activities 0.5 (0.4) 0.5 (0.4) Item 2: helping patient with personal care 0.8 (0.3) 0.8 (0.4) Item 3: helping patient with moving outside 0.5 (0.4) 0.5 (0.4) Item 4: help by other informal caregivers or volunteers 0.7 (0.4) 0.7 (0.4) Self-Rated Burden VAS 4.0 (2.4) 4.3 (2.4) Effects of the integrated programme Patients Patients Results of the two-level multilevel analysis on patient level are presented in table 4. The results show that the intervention had no effect on the primary outcome daily activity as measured with the FAI (-1.69, p=.368). Results of the two-level multilevel analysis on patient level are presented in table 4. The results show that the intervention had no effect on the primary outcome daily activity as measured with the FAI (-1.69, p=.368). The analyses did show a significant favourable effect for the intervention on the subscale “Autonomy outdoors” of the IPA scale (-2.15, p= .047). All other secondary outcome measures i.e. subscale “Social relations” of the IPA scale (.60, p=.560), Katz-15 questionnaire (-.69, p=.372), subscale “Physical functioning” of the SSQoL scale (3.08, p=.476), and the subscale “Psychosocial functioning” of the SSQoL scale (8.45, p= .054) showed no significant effects.. Page 13/23 Page 13/23 Table 4 Effects on primary and secondary outcomes in patients Variable 6 months follow-up observed mean (SD) Group effect P- value 95% CI ICC Primary outcome Intervention group Usual care group Frenchay Activity Index (FAI) 29.7 (8.9) 29.1 (9.1) -1.69 .368 -5.39– 2.00 .74 Secondary outcomes Impact on Participation and Activity (IPA) • Autonomy outdoors 14.2 (4.1) 14.6 (4.2) -2.15 .047 -4.27 – − .03 .55 • Social relations 15.2 (3.4) 16.0 (4.1) .60 .560 -1.43– 2.63 .41 Katz-15 5.9 (3.5) 6.0 (4.0) − .69 .372 -2.22 − .83 .69 Stroke Specific Quality of Life (SSQoL) • Subscale physical functioning 102.2 (19.9) 99.2 (22.5) 3.08 .476 -5.41– 11.56 .71 • Subscale psychosocial functioning 81.3 (17.1) 78.7 (18.0) 8.45 .054 − .14– 17.03 .64 Informal caregivers Table 5 presents the results of the effect of the intervention on the informal caregiver. The results show that the intervention had a significant favourable effect on the Self-Rated Burden vas scale ( 1.23, p=.048), but no effects on the other outcome measures Carer Quality of Life questionnaire (3.54, p=.323), and Erasmus iBMG; item 1) helping patient with ADL-activities (.09, p=.447), item 2) helping patient with personal care (.09, p=.380), item 3) helping patient with moving outside (-.03, p=.784), item 4) help by other informal caregivers of volunteers (.03, p=.767). Table 5 presents the results of the effect of the intervention on the informal caregiver. The results show that the intervention had a significant favourable effect on the Self-Rated Burden vas scale ( 1.23, p=.048), but no effects on the other outcome measures Carer Quality of Life questionnaire (3.54, p=.323), and Erasmus iBMG; item 1) helping patient with ADL-activities (.09, p=.447), item 2) helping patient with personal care (.09, p=.380), item 3) helping patient with moving outside (-.03, p=.784), item 4) help by other informal caregivers of volunteers (.03, p=.767). Page 14/23 Table 5 Effects on the outcomes in informal caregivers Variable 6 months follow-up observed mean (SD) Group effect P- value 95% CI ICC Intervention group Usual care group Carer Quality of Life 85.3 (11.6) 82.9 (13.9) 3.54 .323 -3.50– 10.58 .41 Erasmus iBMG (time spended on helping patient) Item 1: helping patient with ADL- activities 0.4 (0.4) 0.4 (0.4) .09 .447 − .15 – .34 .49 Item 2: helping patient with personal care 0.8 (0.4) 0.8 (0.4) .09 .380 − .12 – .30 .44 Item 3: helping patient with moving outside 0.4 (0.3) 0.5 (0.4) − .03 .784 − .25 – .19 .38 Item 4: help by other informal caregivers or volunteers 0.6 (0.4) 0.6 (0.4) .03 .767 − .18 – .24 .55 Self-Rated Burden VAS 4.3 (2.3) 4.0 (2.0) 1.23 .048 − .02 – 2.48 .43 Table 5 Table 5 Effects on the outcomes in informal caregivers Discussion Based on our results, the increased level of autonomy outdoo seems to indicate that despite the lack of increase in the actu by the FAI, the level of (outdoor) activities is more in accordan patients. An explanation for this finding could be that the self programme may have improved the coping skills of patients a them to have more realistic expectations about the patients’ o related to outdoor activities, is an important finding, as De Gra pay more attention to the social participation of stroke survivo restrictions in participation were perceived in comparison to y stroke30. Furthermore, increased attention for participation ma symptoms after stroke31. Based on our results, the increased level of autonomy outdoors of the patients receiving the programme, seems to indicate that despite the lack of increase in the actual frequency of daily activity as measured by the FAI, the level of (outdoor) activities is more in accordance with the needs and wishes of the patients. An explanation for this finding could be that the self-management component of the programme may have improved the coping skills of patients and their informal caregivers, and helped them to have more realistic expectations about the patients’ outdoor activities. The increase in autonomy related to outdoor activities, is an important finding, as De Graaf and colleagues emphasised the need to pay more attention to the social participation of stroke survivors aged over 70 years, since more restrictions in participation were perceived in comparison to younger stroke survivors one year after stroke30. Furthermore, increased attention for participation may also contribute to preventing depressive symptoms after stroke31. With regard to the informal caregivers, the integrated programme resulted in a significant reduction in the perceived care burden of the informal caregiver. This may indicate that elements of the integrated programme, such as consultation with the stroke coordinator and stroke education, may support informal caregivers in accomplishing their supporting role. This is in accordance with the results of a review of Visser-Meily and colleagues32 who concluded that counselling programs which focus on the problems of the informal caregiver, instead of (only) on the problems of the patients, appear to have the most favourable outcomes. In our programme, the problems and experiences of the informal caregiver were explicitly addressed in different modules. This study is one of few studies that focusses on improving stroke rehabilitation and aftercare for frail older stroke patients and their informal caregivers. Discussion The results of this study show that the integrated multidisciplinary geriatric rehabilitation programme for older patients with stroke had no significant effect on the primary outcome daily activity as compared to usual care. With regard to the secondary outcomes, the programme showed favourable effects on the patients’ outdoor autonomy and the perceived care burden of their informal caregivers. For the other secondary outcomes, no significant intervention effects were observed. The lack of effect of the programme on daily activity and most secondary outcome measures might be explained by several reasons. First, the process evaluation which was performed alongside the trial, revealed that part of patients and informal caregivers did not receive all key elements of the programme27. Although almost all patients formulated rehabilitation goals, the GAS method was only used among two thirds of the patients. In addition, the percentage of therapy sessions performed in the patients’ home environment was lower than planned, and only about a quarter of the patients and informal caregivers attended the education sessions. Furthermore, the self-management training was considered by the care professionals as rather complex and difficult to apply for frail older persons, because it was complicated for the patients to develop and carry out action plans by themselves27. As it is widely recognised that in complex interventions often not all aspects of the intervention are completely Page 15/23 Page 15/23 performed according to protocol and that adaptation to local circumstances may be necessary28, it is important to improve the feasibility of the integrated programme by tailoring the goal attainment scaling, self-management training and education sessions more optimally to the population of frail older stroke patients27. In addition the training of care professionals in conducting the programme could be improved. However, despite this, the majority of patients, informal caregivers and care professionals indicated the beneficial aspects of the programme 27. Second, a review of Fens and colleagues29 performed in 2013 evaluating the effectiveness of multidisciplinary interventions for stroke patients living in the community after being discharged home after hospitalization or inpatient rehabilitation, showed that none of the 11 studies that assessed daily activities reported a favourable effect of the intervention on this outcome. Although these multidisciplinary interventions included different combinations of elements, it clearly shows that improving daily activity among community living stroke patients is very complex, which is also confirmed by the results of our trial. Discussion However, this study has several limitations. First, we did not reach our inclusion goal of 256 patients, although we took all possible and necessary actions (i.e. extending inclusion period, extending the number of nursing homes) to increase the number of patients. This may have underpowered our multilevel analyses. However, the estimated difference between Page 16/23 Page 16/23 intervention and usual care group on our primary outcome daily activity (i.e. 1.69) is below the minimal effect that is still considered clinically relevant (i.e.3.5). Therefore, it is unlikely that including the intended number of patients would have resulted in a statistically significant effect on our primary outcome. However, for the psychosocial functioning subscale of the Stroke Specific Quality of life scale (p=.054) accounts that a higher power may have resulted in a statistical significant favourable effect for patients in the intervention group on this subscale. Second, because we randomised on patient level and not on nursing home level, care professionals treated both people in the intervention group and usual care group. Therefore, it is possible that treatment for persons in the usual care group was contaminated with elements of the programme which may have led to an underestimation of the effects of the programme. Although a number of elements of the programme were exclusively available for persons in the intervention group (such as the meetings with the stroke care coordinator, the multidisciplinary outpatient rehabilitation, and the stroke education course), it is still possible that other elements of the intervention were also applied among persons in the usual care group. However, we have tried to reduce this risk of contamination by emphasizing during the training of the care professionals that the programme elements should exclusively be applied in the intervention group. In addition we repeatedly checked whether contamination has occurred during regular visits of the research team to the participating organisations. During these visits care professionals confirmed that the intervention was only applied to persons in the intervention group. Furthermore, after the intervention period, we checked during a group interview with a sample of the participating care professionals whether contamination had occurred, which was not the case according to the care professionals. Third, patients, caregivers and care professionals could not be blinded for treatment allocation, which might have created some bias. Discussion However, in order to reduce the risk of any additional bias, the outcome measurements were performed by research assistants who were blinded for treatment allocation, and the same accounts for the statistical analyses. Fourth, the baseline measurement of the FAI was based on the activity level of patients three months before stroke occurred, as estimated by the patient. It is possible that this resulted in recall bias. However, it is likely that this accounts for patients in both the usual care and intervention group, which makes it unlikely that is has influenced our results. Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Conclusion This study shows that an integrated multidisciplinary rehabilitation programme for frail older patients with stroke and their caregivers had no effect on the activity level of these patients. However, the intervention did show a significant favourable effect on autonomy regarding outdoor activities as perceived by the patients. Furthermore, we found also a significant favourable effect on the perceived care burden of informal caregivers. Based on these results, the programme might be considered promising in providing adequate aftercare. However, adaptation of the programme is recommended to Page 17/23 increase its feasibility and to improve its favourable effects for patients and informal caregivers. More research is needed to increase knowledge and evidence of effective methods to increase daily activity level in (older) patients with stroke. increase its feasibility and to improve its favourable effects for patients and informal caregivers. More research is needed to increase knowledge and evidence of effective methods to increase daily activity level in (older) patients with stroke. Consent for publication Not applicable. Competing interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Funding This study is funded with a grant (grant number:313070301) from the Netherlands Organisation for Health Research and Development (ZonMw) as part of the National Care for the Elderly Programme, which aims to improve the quality of care for elderly persons by means of developing integrated health care that is adjusted to the individual needs of the elderly persons. The funding organizations had no role in the design, methods, analysis, preparation, or approval of the paper. The views expressed in this publication are those of the authors and not necessarily those of the Netherlands Organization for Health Research and Development (ZonMw). Ethics approval and consent to participate Ethical approval was provided by the medical ethics committee of Maastricht University Medical Centre (MUMC+), the Netherlands (ISRCTN62286281, NTR2412). All participants including patients and informal caregivers gave all written informed consent to take part in the study. An overseeing elderly care physician has ruled out that all adult patients and informal caregivers have been deemed capable of ethically and medically consenting for their participation in the research presented in his manuscript. Authors’ contributions TV, JvH, JV and JS were involved in obtaining research funding, the design of the intervention, the protocol training, acquisitions of subjects, data collection, and data analysis. TV wrote drafts of the manuscript. JvH and FT supervised data analysis. TV, JvH, JV, FT, CvH, and JS were involved in the Page 18/23 Page 18/23 Page 18/23 interpretation of the results. JvH, JV, and JS supervised the project. All authors read, critically reviewed and approved the final manuscript. interpretation of the results. JvH, JV, and JS supervised the project. All authors read, critically reviewed and approved the final manuscript. Authors’ information 1Department of Health Services Research, Maastricht University, Maastricht, the Netherlands; 2Care and Public Health Research Institute, Maastricht University, Maastricht, The Netherlands; 3Department of methodology and statistics, Maastricht University, Maastricht, The Netherlands;4Adelante, Centre of Expertise in Rehabilitation and Audiology, Hoensbroek, The Netherlands;5Department of Rehabilitation Medicine, Maastricht University, Maastricht, The Netherlands; 6Department of Neuropsychology and Psychopharmacology, Maastricht University, Maastricht, The Netherlands;7School for Mental Health and Neuroscience, Maastricht University, Maastricht, The Netherlands . Acknowledgements Firstly, we would like to thank all patients and informal caregivers for participating in this study. Furthermore, we are grateful to all care professionals who were involved in developing and delivering the intervention, and who participated in the study. 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Visser-Meily A, van Heugten C, Post M, Schepers V, Lindeman E. Intervention studies for caregivers of stroke survivors: a critical review. Patient Educ Couns 2005; 56: 257–67. Figures Page 21/23 Page 21/23 Figure 1 Figure 1 flowchart of patients through the study Figure 1 flowchart of patients through the study Figure 1 flowchart of patients through the study Page 22/23 Page 22/23 Supplementary Files This is a list of supplementary files associated with this preprint. Click to download. CONSORT2010Checklist.doc CONSORT2010Checklist.doc Page 23/23
https://openalex.org/W2799449856	https://dash.harvard.edu/bitstream/1/37068179/1/5924739.pdf	English	null	Microfluidic Organ-on-a-Chip Models of Human Intestine	CMGH	2,018	cc-by	8,763	Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of-use#LAA Permanent link http://nrs.harvard.edu/urn-3:HUL.InstRepos:37068179 Published Version doi:10.1016/j.jcmgh.2017.12.010 Published Version doi:10.1016/j.jcmgh.2017.12.010 Citation Bein, Amir, Woojung Shin, Sasan Jalili-Firoozinezhad, Min Hee Park, Alexandra Sontheimer- Phelps, Alessio Tovaglieri, Angeliki Chalkiadaki, Hyun Jung Kim, and Donald E. Ingber. 2018. “Microfluidic Organ-on-a-Chip Models of Human Intestine.” Cellular and Molecular Gastroenterology and Hepatology 5 (4): 659-668. doi:10.1016/j.jcmgh.2017.12.010. http:// dx.doi.org/10.1016/j.jcmgh.2017.12.010. Share Your Story The Harvard community has made this article openly available. Please share how this access benefits you. Submit a story . REVIEW Microﬂuidic Organ-on-a-Chip Models of Human Intestine Amir Bein,1,a Woojung Shin,2,a Sasan Jalili-Firoozinezhad,1,3 Min Hee Park,2 Alexandra Sontheimer-Phelps,1,4 Alessio Tovaglieri,1,5 Angeliki Chalkiadaki,1 Hyun Jung Kim,2 and Donald E. Ingber1,6,7 Microﬂuidic Organ-on-a-Chip Models of Human Intestine Amir Bein,1,a Woojung Shin,2,a Sasan Jalili-Firoozinezhad,1,3 Min Hee Park,2 Alexandra Sontheimer-Phelps,1,4 Alessio Tovaglieri,1,5 Angeliki Chalkiadaki,1 Hyun Jung Kim,2 and Donald E. Ingber1,6,7 1Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, Massachusetts; 2Department of Biomedical Engineering, The University of Texas at Austin, Austin, Texas; 3Department of Bioengineering and iBB - Institute for Bioengineering and Biosciences, Instituto Superior Técnico, University of Lisbon, Lisbon, Portugal; 4Graduate Program, Faculty of Biology, University of Freiburg, Freiburg, Germany; 5Graduate Program, Department of Health Sciences and Technology, ETH Zurich, Zurich, Switzerland; 6Vascular Biology Program and Department of Surgery, Boston Children’s Hospital and Harvard Medical School, Boston, Massachusetts; and 7Harvard John A. Paulson School of Engineering and Applied Sciences, Cambridge, Massachusetts metabolites (eg, short-chain fatty acids) have been recently shown to play a central role in the maintenance of intestinal health, immune modulation, and the develop- ment of both enteral and nonenteral diseases.10,11 How- ever, analysis of gut microbiome interactions with human intestinal cells has been limited to genetic or meta- genomics analysis because it has not been possible to coculture these microbes with living epithelium for more than about 1 day using conventional culture models or even more sophisticated intestinal organoid cultures. Thus, there have been great efforts to develop experi- mental in vitro or ex vivo models of human intestine that permit analysis of intestinal pathophysiology both in the presence and absence of living microbiome. SUMMARY Organs-on-chips are microﬂuidic cell culture systems that recapitulate the structure, function, physiology, and pathol- ogy of living human organs in vitro. In this article, we review recent development of various human intestine-on-a-chip models and their potential value for disease modeling, drug discovery, and personalized medicine. Microﬂuidic organ-on-a-chip models of human intestine have been developed and used to study intestinal physi- ology and pathophysiology. In this article, we review this ﬁeld and describe how microﬂuidic Intestine Chips offer new capabilities not possible with conventional culture systems or organoid cultures, including the ability to analyze contributions of individual cellular, chemical, and physical control parameters one-at-a-time; to coculture human intestinal cells with commensal microbiome for extended times; and to create human-relevant disease models. We also discuss potential future applications of human Intestine Chips, including how they might be used for drug development and personalized medicine. (Cell Mol Gastroenterol Hepatol 2018;5:659–668; https://doi.org/ 10.1016/j.jcmgh.2017.12.010) The most common in vitro intestine models used to study barrier function or model drug absorption involve culturing an established human intestinal epithelial cell line (eg, Caco-212,13 or HT-2914,15 cells) on extracellular matrix (ECM)-coated, porous membranes within Transwell insert culture devices. Although these models are most commonly used by the pharmaceutical industry, this 2-dimensional (2D) culture format fails to recapitulate physiological 3- dimensional (3D) intestinal cell and tissue morphology or re-establish other key intestinal differentiated functions (eg, mucus production, villi formation, cytochrome P-450-based drug metabolism).16,17 These conventional static models also cannot support the coculture of commensal micro- biome with human intestinal cells, which are critical for gut physiology,16 because the bacteria rapidly overgrow and contaminate the human cell cultures within a day. Several ex vivo models, such as the everted sac18 or the Ussing Keywords: Organs-on-Chips; Gut-on-a-Chip; Intestine-on-a-Chip; Microﬂuidic. T T he major organ function of the human intestine is to carry out digestion, absorption, secretion, and motility,1 in addition to establishing a protective epithelial barrier between this digestive environment and the body. Accessibility Accessibility aAuthors contributed equally. Abbreviations used in this paper: ECM, extracellular matrix; IBD, inﬂammatory bowel disease; PD, pharmacodynamics; PDMS, polydimethylsiloxane; PK, pharmacokinetics; 3D, 3-dimensional. Most current article © 2018 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X https://doi.org/10.1016/j.jcmgh.2017.12.010 Bein et al 660 Cellular and Molecular Gastroenterology and Hepatology Vol. 5, No. 4 chamber,19,20 have been developed for drug transport as- says; however, their expected lifespan (<8 hours) is not sufﬁcient to enable many studies on normal intestinal physiology, develop intestinal disease models, or study clinically relevant host-microbiome crosstalk. also do not experience ﬂuid ﬂows and cyclic mechanical deformations similar to those experienced in a peristalsing intestine that contribute signiﬁcantly to intestinal health and function. Furthermore, because each enteroid forms a closed lumen when cultured within surrounding ECM gel, it is experimentally difﬁcult to sample or manipulate luminal components (eg, microbial cells, nutrients, drugs, or toxins). This structure also signiﬁcantly limits the ability of re- searchers to study many critical intestinal functions (eg, absorption, drug PK, or drug metabolism), in addition to critical host-microbiome interactions.26 y Although it had been technically challenging to culture primary human intestinal epithelial cells, intestinal 3D organoid cultures derived from either intestinal crypts containing endogenous intestine cells or from induced pluripotent stem cells have revolutionized the ﬁeld by maintaining stem cell niches and supporting differentiation of various differentiated intestinal epithelial cell subtypes in vitro.21,22 When cultured within a 3D ECM gel in medium containing Wnt, R-spondin, noggin, and other growth fac- tors, small intestinal organoids (enteroids) also spontane- ously undergo villus-crypt morphologic organization and intestinal histogenesis.22 Each organoid line derived from an intestinal tissue biopsy of an individual patient can be grown, frozen, and revived for multiple reuses, which can potentially be used to establish biobanks23,24 and develop multiplexed screening platforms for validating new drug candidates and to advance personalized medicine.25 How- ever, organoids are also limited in that they lack other supporting cell and tissue types found within the living in- testine, such as endothelium-lined blood vessels and im- mune cells, which are important for drug transport, pharmacokinetic (PK) analysis, and disease modeling. They These challenges have recently been overcome by the development of microﬂuidic Organ Chip models of human intestine. SUMMARY In addition, intestines regulate systemic physiology by metabolizing drugs2; communicate with other organs, such as the liver3,4 and pancreas,5 via portal ﬂow; and they contain an enteric nervous system that forms a part of the gut-brain axis.6,7 The intestine is also the major site at which commensal microbes of the gut microbiome live and interact with gut lymphoid tissues and the host immune system, which contributes signiﬁcantly to intestinal homeostasis.8,9 For example, the gut microbiome and its aAuthors contributed equally. Abbreviations used in this paper: ECM, extracellular matrix; IBD, inﬂammatory bowel disease; PD, pharmacodynamics; PDMS, polydimethylsiloxane; PK, pharmacokinetics; 3D, 3-dimensional. Most current article © 2018 The Authors. Published by Elsevier Inc. on behalf of the AGA Institute. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 2352-345X https://doi.org/10.1016/j.jcmgh.2017.12.010 Bein et al Organ Chips are microﬂuidic cell culture devices, originally fabricated using methods adapted from computer microchip manufacturing (eg, soft lithography), which contain continuously perfused chambers inhabited by living cells arranged to simulate tissue- and organ-level physi- ology.27 Over the past 5 years, Organ Chip models of in- testine have been engineered with increasing complexity that also include neighboring channels lined by human microvascular endothelium, and commensal microbes, im- mune cells, and pathogenic bacteria, and some permit application of cyclic mechanical forces that mimic peristalsis-like deformations experienced by living intestine in vivo (Figure 1). Next are review various types of engi- neered in vitro models that emulate the structure, function, Figure 1. The mechanically active human Gut Chip. (A) Human villus intestinal epithelium and vascular endothelium are lined on opposite sides of a ﬂexible porous membrane under ﬂuid ﬂows and peristalsis-like strains. A zoom-in schematic shows the intestinal microenvironment undergoing complex crosstalk between commensal gut microbiome, bacterial pathogens, and immune cells in parenchymal and vascular channels, respectively. Figure modiﬁed with permission from Reference 75. (B) Villus morphogenesis of human Caco-2 intestinal epithelium in the Gut Chip under physiologically controlled motions and ﬂow. Figure modiﬁed with permission from References 17 and 42. (C) An overlaid image of the coculture of green ﬂuorescent protein–labeled Escherichia coli and microengineered villi in the Gut Chip. Bars ¼ 50 mm. Figure 1. The mechanically active human Gut Chip. (A) Human villus intestinal epithelium and vascular endothelium are lined on opposite sides of a ﬂexible porous membrane under ﬂuid ﬂows and peristalsis-like strains. A zoom-in schematic shows the intestinal microenvironment undergoing complex crosstalk between commensal gut microbiome, bacterial pathogens, and immune cells in parenchymal and vascular channels, respectively. Figure modiﬁed with permission from Reference 75. (B) Villus morphogenesis of human Caco-2 intestinal epithelium in the Gut Chip under physiologically controlled motions and ﬂow. Figure modiﬁed with permission from References 17 and 42. (C) An overlaid image of the coculture of green ﬂuorescent protein–labeled Escherichia coli and microengineered villi in the Gut Chip. Bars ¼ 50 mm. Microﬂuidic Human Intestine Models 2018 661 physiology, and pathology of the living human intestine (Table 1). Also considered are the implications of this work for development of more complex disease models, drug development, and personalized medicine in the future. Mechanically Active Gut Chip Model Most of the Intestine Chips contain 2 hollow channels separated by a common porous, ECM-coated polyester or polycarbonate membrane, which had immortalized human intestinal epithelial cells cultured on 1 of its surfaces.30 The epithelial monolayer formed in this device could be probed from both the apical and basal sides of the epithelium, and this enabled quantiﬁcation of tight junction barrier func- tion31,32 and absorption of nutrients33 and drugs.30,34 Some devices integrated multiple chambers with different types of cells to measure PK and pharmacodynamics (PD) properties of drug compounds in vitro.35–37 However, the cells were seeded at a low density in these studies and they were perfused only through the apical channel; as a result, the monolayer of the immortalized intestinal cells could only be cultured for a limited time (<5 days). A more recent study demonstrated real-time barrier integrity assessment capa- bilities in a membrane-free culture of perfused intestinal epithelium tubes.38 To better mimic the 3D form of human intestine using cell monolayers cultured under microﬂuidic ﬂow, micro- molding methods have been used to form polymeric scaf- folds (eg, Ca-alginate or collagen gel) into villus-shaped structures.39,40 Culturing human Caco-2 intestinal epithe- lial cells on these crenulated surfaces was shown to pro- mote formation of a similarly crenulated epithelium, and this was accompanied by increased absorption of drug compounds and improved cellular metabolic enzyme cyto- chrome P-450 activity in response to apical ﬂuid shear stress.39 However, this design did not enable analysis of intestinal barrier function because the solid polymer mate- rial blocked the abluminal surface of the epithelium. Bein et al mechanical deformations similar to those associated with peristalsis that can critically inﬂuence the microbial growth in the intestine.42 This experimental model is also limited by the physical separation of microbial and host epithelial cells by a nanoporous membrane, and the fact that the cocultures can only survive for short times (<24 hours), which may restrict its use for long-term proﬁling of host-microbial in- teractions and intestinal pathophysiology. Microﬂuidic Intestine Chip Models Microﬂuidic devices containing hollow microchannels less than 1 mm in width support laminar ﬂuid ﬂow and control of nanoliter to microliter scale ﬂuid volumes, and thus, they are amenable to use for culture of living cells. By using a syringe or a peristaltic pump, culture medium may be perfused at desired ﬂow rates through each micro- channel, which can mimic the dynamic ranges of ﬂuid ﬂows and associated shear stresses on the cell surface that are observed in the human intestinal lumen,28,29 and in the blood capillaries. This ﬂuidic control also enables delivery of nutrients, growth factors, drug compounds, or even toxins to the intestinal epithelium grown on the microﬂuidic channels in a highly regulated spatiotemporal manner. Mechanically Active Gut Chip Model A more sophisticated, microﬂuidic 2-channel Gut Chip model has been developed that enables human intestinal epithelium, capillary endothelium, immune cells, and even commensal microbial cells to grow, coexist, and interact while experiencing physiologically relevant ﬂuid ﬂow and peristalsis-like mechanical deformations in vitro.42 We use herein “Gut Chip” to refer to previously published human intestine model using Caco-2 cells that used this term.16,17,42 A more sophisticated, microﬂuidic 2-channel Gut Chip model has been developed that enables human intestinal epithelium, capillary endothelium, immune cells, and even commensal microbial cells to grow, coexist, and interact while experiencing physiologically relevant ﬂuid ﬂow and peristalsis-like mechanical deformations in vitro.42 We use herein “Gut Chip” to refer to previously published human intestine model using Caco-2 cells that used this term.16,17,42 The Gut Chip is made of a ﬂexible, gas-permeable, silicone polymer (polydimethylsiloxane [PDMS]) that is crystal clear so that it allows high-resolution imaging by phase contrast, differential interference contrast, or immunoﬂuorescence confocal microscopy. It contains 2 parallel microchannels (<1 mm wide) separated by a thin (w20 mm), ECM-coated, ﬂexible, porous PDMS membrane, and it is surrounded on both sides by hollow, full height, side chambers (Figure 1A).16,17,42,43 Intestinal epithelial cells are cultured on the top surface of the membrane within the upper parenchymal channel, and microvascular endothelial cells are grown on the lower surface of the same membrane within the lower vascular channel to recreate the intestinal tissue-tissue interface. Importantly, pneumatic application of cyclic suction to the hollow side chambers results in outward deformation of the vertical side walls and attached horizontal ECM-coated membrane with the adherent cell layers, thereby mimicking cyclic mechanical deformations similar to those experienced by the intestinal tissues during peristalsis (Figure 1A). Mechanically Active Gut Chip Model 4 Bein et al 662 Model TEER Absorption Coculture Microbiome Differentiation Peristalsis Drug metabolism Crypt-villus axis Oxygen modulation Disease modeling Static Transwell Yes12 Yes12,13 No Yes14,15 (<24 h) No No No No No No Organoid No Yes95 No Yes96 (<1 h) Yes21 No Yes97 Yes21 No Yes22 Ex vivo Yes19,98 Yes19,20 No Yes97 (<3 h) Yes19 No Yes100 Yes19 Yes99 Yes98 Scaffold No No No No Yes40 No No Yes40 No No Microﬂuidic 2-channel Yes32 Yes30 Yes37 No No No Yes39 Yes39 No No Ex vivo No Yes99 No No Yes101 No No Yes101 No Yes101 Multichannel (HuMiX) Yes41 No No Yes41 (<24 h) No No No No Yes41 No Gut Chip Yes16,42 Yes16 No Yes16,42 (>7 d) Yes17,42 Yes16,17,42 Yes16,17 Yes16,17,42 No Yes42 Model TEER Absorption Coculture Microbiome Differentiation Peristalsis Drug metabolism Crypt-villus axis Oxygen modulation Disease modeling Static Transwell Yes12 Yes12,13 No Yes14,15 (<24 h) No No No No No No Organoid No Yes95 No Yes96 (<1 h) Yes21 No Yes97 Yes21 No Yes22 Ex vivo Yes19,98 Yes19,20 No Yes97 (<3 h) Yes19 No Yes100 Yes19 Yes99 Yes98 Scaffold No No No No Yes40 No No Yes40 No No Microﬂuidic 2-channel Yes32 Yes30 Yes37 No No No Yes39 Yes39 No No Ex vivo No Yes99 No No Yes101 No No Yes101 No Yes101 Multichannel (HuMiX) Yes41 No No Yes41 (<24 h) No No No No Yes41 No Gut Chip Yes16,42 Yes16 No Yes16,42 (>7 d) Yes17,42 Yes16,17,42 Yes16,17 Yes16,17,42 No Yes42 TEER, transepithelial electrical resistance. within the lumen of the parenchymal channel for weeks in vitro without compromising barrier integrity or intestinal cell function (Figure 1C).16,42 In fact, barrier function was shown to increase when the intestinal epithelium was cocultured with L rhamnosus GG.16 In addition, tran- scriptomic analyses targeting approximately 23,000 human genes revealed that the in vivo relevant ﬂuid ﬂow and physical deformations dramatically changed the gene expression proﬁles compared with the static Transwell cultures, and that mechanically active Gut Chips that also contained a mixture of commensal microbes (VSL#3 pro- biotic formulation) showed the highest level of genetic similarity to normal human ileum. Taken together, these data suggest that the Gut Chip that emulates the dynamic human intestinal microenvironment more faithfully mimics intestinal function than previously described in vitro intestine models. Mechanically Active Gut Chip Model Pathogenic bacteria, such as enteroinvasive E coli, also can be integrated into this model system, with or without commensal microbes of the normal microbiome, to study how interplay between these microbes and human intestinal epithelial cells contributes to intestinal homeostasis and intestinal disease development.42 Indeed, the Gut Chip was used to demonstrate complex immune-microbiome inﬂammatory interactions germane to chronic inﬂammatory diseases, such as inﬂammatory bowel disease (IBD).42 Addition of lipopolysaccharide endotoxin to the luminal microchannel also was shown to induce secre- tion of the proinﬂammatory cytokines, interleukin-1b, -6, -8, and tumor necrosis factor-a into the lower vascular channel. Moreover, this was accompanied by increased expression of intercellular adhesion molecule-1 on the endothelium, villus blunting, and compromise of intestinal barrier function, but only in the presence of white blood cells (eg, peripheral blood mononuclear cells) that were infused through the capillary microchannel. Moreover, additional experiments conﬁrmed that all 4 cytokines were required to be present at the concentrations measured within the capillary channel to induce this intestinal injury response. Interestingly, addi- tional studies revealed that cessation of cyclic peristalsis- like mechanical motions caused an increase in bacterial overgrowth in the Gut Chip, which closely resembled the small intestinal bacterial overgrowth that can result from cessation of peristalsis in patients with ileus. Thus, by being able to control ﬂow independently of mechanical de- formations, this study revealed that the past clinical assumption that small intestinal bacterial overgrowth re- sults from cessation of ﬂuid ﬂow when peristalsis is inhibited44 is incorrect; instead, it is the lack of mechanical deformations that drives this bacterial overgrowth response. Thus, the innovative modularity of the microﬂuidic Gut Chip (Figure 1A) can be leveraged to gain new insights into in- testinal pathophysiology, and to understand disease mech- anisms in ways that are not possible using conventional in vitro gut models or simpler microﬂuidic Intestine Chips. Mechanically Active Gut Chip Model Under these physiological conditions, human Caco-2 in- testinal epithelial cells, which characteristically grow as ﬂattened cells within a planar monolayer in conventional 2-dimensional cultures, spontaneously undergo villus morphogenesis inside this mechanically dynamic Gut Chip (Figure 1B).17 The microengineered villi are lined by all 4 lineages of differentiated small intestinal cells (absorptive, goblet, enteroendocrine, and Paneth), which exhibit columnar cell morphology similar to that observed in the living intestine.17 Intestinal villus morphogenesis on-chip is also accompanied by establishment of a crypt-villus axis, including restriction of proliferative cells to the basal crypts and their upward migration, drug metabolizing activity, mucus production, and glucose reuptake.17 A multichannel Intestine Chip called HuMiX also has been described that separates a luminal microbial compartment from layers of Caco-2 intestinal epithelium by nanoporous membranes.41 This model was shown to facil- itate survival of a host-microbiome ecosystem containing Caco-2 cells and anaerobic human gut bacteria (eg, Bacter- oides caccae) with constant perfusion of culture medium.41 However, these studies were carried out in the absence of Importantly, because the Gut Chip has continuous ﬂuid ﬂow, villi formation, and mucus production, it is also possible to coculture living commensal microbes (eg, Lactobacillus rhamnosus GG)16 or the VSL#3 clinical probiotic formulation containing 8 different microbial strains42) in direct contact with the intestinal epithelial cells Cellular and Molecular Gastroenterology and Hepatology Vol. 5, No. 4 Cellular and Molecular Gastroenterology and Hepatology Vol. 5, No. Limitations of Human Intestine Chip Models To overcome these challenges, human enteroids47,48 or colonoids49 created from patient biopsies have been enzy- matically fragmented and the released intestinal stem cells plated on Transwell inserts to create primary human in- testinal monolayers, which permit sampling from both the apical and basolateral sides in vitro. These models showed establishment of intact polarized monolayers with apical villin and basolateral Naþ/Kþ-ATPase expression47 and successful cultivation of multiple human norovirus strains in the presence of bile.48 Although microﬂuidic Intestine Chip models faithfully mimic many different phenotypes and responses of human intestine, they only incorporate limited components of the 4-layered intestinal wall, and these missing features might play a signiﬁcant role in some disorders. For example, smooth muscle cells express Toll-like receptors 1–9 and can regulate neuronal integrity by modulating glial-derived neurotrophic factor production.56 This is important because the enteric nervous system regulates intestinal secretion, blood ﬂow, and gut motility.57 Subtle alterations in transmitter production and release have been linked to functional and inﬂammatory diseases of the gastrointestinal tract.58–60 Thus, adding more components, such as muscle and nervous system cells, should be considered in the future for relevant applications. Although static Transwell cultures offer better access to theapicallumenof theseorganoids, they still lack other organ- level features of the intestine, including an endothelium-lined vascular compartment, ﬂow, cyclic mechanical deformation, or immune cells, which have been shown to be important for mimicking intestinal physiology and pathophysiology in the human Gut Chip.42 Organoid cultures also have been modiﬁed to address some of these challenges. For example, application of mechanical forces to ECMs containing organoids has shown to modulate their phenotype and inﬂuence primary intestine cell differentiation.50 In addition, miniaturized bioreactors have been developed to generate ﬂuid ﬂows that facilitate nutrient and oxygen uptake, and ﬂuid shear stresses that convey physiologically relevant mechanical signals to cells in organoid cultures.51 Although PDMS holds many favorable properties for manufacturing microﬂuidic organ devices, it also has the po- tential drawback of adsorbing small and hydrophobic mole- cules.61,62 To overcome this, surface modiﬁcation of PDMS or alternative polymeric (eg, polyurethane, styrenic block co- polymers)63 or ECM-based materials64 could potentially be used in studies designed to predict drug bioavailability or absorption. Limitations of Human Intestine Chip Models Alternatively, when using Organ Chips to model drug PK and PD, PDMS adsorption can be taken into account using computational modeling for most compounds.65 Most recently, organoid and organ chip approaches have been combined to develop a microﬂuidic primary human Intestine Chip model. Human enteroids cultured from pa- tient duodenal biopsies were enzymatically fragmented and the released intestinal stem cells were plated on the ECM- coated porous membrane of a 2-channel PDMS micro- ﬂuidic device with primary human intestinal microvascular endothelial cells on the opposite side of the same membrane within a parallel channel.52 Much like the Caco-2 cell-lined Gut Chip, with application of ﬂuid ﬂow and peristalsis-like deformations, the intestinal epithelial cells undergo villus histogenesis with multilineage differentiation. Interestingly, although this differentiation process is similar to that observed within the intestinal organoids, transcriptomic analysis revealed that the mechanically active Intestine Chip more closely mimics the proliferation and host defense response to infection functions of the human duodenum from which the organoids were originally isolated. In addition, most of the past work on Intestine Chips has beencarriedout inacademiclaboratories,andthus,theresults and robustness of the models can vary from laboratory to laboratory. However, the recent emergence of multiple com- panies that are beginning to commercialize Organ Chip tech- nology offers great hope that issues relating to scale up of manufacturing, robustness, cost, and ease of use will be resolvedovertime.Thesechallengesmustbeovercomebefore human Intestine Chips become a common tool used in aca- demic and industrial research laboratories around the world. Microﬂuidic Human Intestine Models Microﬂuidic Human Intestine Models 663 shown to mimic many physiological and pathophysiological functions of the human intestine, they are still limited by the fact that they use immortalized cells that were originally isolated from a human tumor. For example, these cultures might be particularly difﬁcult to use in studies where genome ﬁdelity is important (eg, analysis of intestinal can- cer formation). In vitro intestine models have been created using human fetal intestinal tissue explants, but they dete- riorate within 24 hours of culture.45,46 Human intestinal organoids offer another way to study normal human intes- tinal stem cell differentiation and villus morphogenesis in vitro; however, they do not recreate a physiologically relevant physical microenvironment (ﬂuid ﬂow, peristalsis- like deformations) and their enclosed lumens limit their value for transport studies and coculture with pathogens. shown to mimic many physiological and pathophysiological functions of the human intestine, they are still limited by the fact that they use immortalized cells that were originally isolated from a human tumor. For example, these cultures might be particularly difﬁcult to use in studies where genome ﬁdelity is important (eg, analysis of intestinal can- cer formation). In vitro intestine models have been created using human fetal intestinal tissue explants, but they dete- riorate within 24 hours of culture.45,46 Human intestinal organoids offer another way to study normal human intes- tinal stem cell differentiation and villus morphogenesis in vitro; however, they do not recreate a physiologically relevant physical microenvironment (ﬂuid ﬂow, peristalsis- like deformations) and their enclosed lumens limit their value for transport studies and coculture with pathogens. Thus, this primary Intestine Chip may be useful as a research tool for many applications where normal intestinal function is crucial, including studies of metabolism, nutrition, and cancer progression, as well as drug absorption and PK/ PD. It also may offer a novel method for emerging disciplines, such as personalized precision medicine,53,54 which are in constant search for faithful and robust tools for disease modeling and drug discovery. Genetic polymorphisms in drug transporters, drug-metabolizing enzymes, and drug targets have been linked to the efﬁcacy, dosage, and toxicity proﬁle in humans.55 Hence, possible applications for this model range from studying speciﬁc pathologic mechanisms (eg, inﬂam- mation, infection, and nutrient malabsorption) to drug dis- covery in disease-speciﬁc and patient-speciﬁc contexts. Human Primary Intestine Models Although human Intestine Chips lined by established intestinal epithelial cell lines (eg, Caco-2 cells) have been 2018 Intestine Chip Models for Developmental Therapeutics human Gut Chip was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells and to analyze how gut microbiome, inﬂammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inﬂammation, which are associated with IBD.42,66 Recent studies suggest that the same Gut Chip model can be used to model the radiation injury response of human intestine (eg, cell death, villus blunting, compromised bar- rier function) when exposed to g-radiation, and prevention of these responses using a potential prophylactic radiation countermeasure drug.67 In addition, the recently developed primary human Intestine Chip also has been used to model differential sensitivity of intestinal responses to bacteria with different levels of virulence (Kasendra et al, unpub- lished data). human Gut Chip was used to coculture multiple commensal microbes in contact with living human intestinal epithelial cells and to analyze how gut microbiome, inﬂammatory cells, and peristalsis-associated mechanical deformations independently contribute to intestinal bacterial overgrowth and inﬂammation, which are associated with IBD.42,66 Recent studies suggest that the same Gut Chip model can be used to model the radiation injury response of human intestine (eg, cell death, villus blunting, compromised bar- rier function) when exposed to g-radiation, and prevention of these responses using a potential prophylactic radiation countermeasure drug.67 In addition, the recently developed primary human Intestine Chip also has been used to model differential sensitivity of intestinal responses to bacteria with different levels of virulence (Kasendra et al, unpub- lished data). Intestine Chip–based disease models can be exploited to identify new therapeutic targets, repurpose existing drugs, test new therapies, and carry out PK/PD testing in vitro. Although extensive testing with conventional culture models and animal experiments are commonly carried out before performing clinical trials, these models often fail to predict efﬁcacy and safety of novel drugs, which results in loss of enormous amounts of money, time, and effort.80 In addition, drug toxicities and efﬁcacies greatly vary depending on the individual patient, given both genetic81 and environmental inﬂuences (eg, nutrients,82,83 drug intake,83,84 gut microbiome85,86). To meet this challenge, there has been increased interest in pursuing a personalized medicine approach to develop therapeutics that are tailored for an individual patient’s genetic background. Primary in- testine models can facilitate this approach by engineering artiﬁcial intestine microenvironments using the patient’s own cells (epithelial, endothelial, immune, connective tissue, or neural) and gut microbiome. Intestine Chip Models for Developmental Therapeutics Because the intestine is a major site for drug metabolism (both by cytochrome P-450 enzymes in intestinal epithelial cells and by the gut micro- biome87), creation of Intestine Chips can facilitate study of drug breakdown both alone and when coupled to other Organ Chips that also contribute to drug metabolism (eg, Liver Chip) and clearance (eg, Kidney Chip) in a human Body-on-a-Chip conﬁguration.36,37 A potentially important application of the microﬂuidic Intestine Chip models is to study various types of enterop- athies for which current in vitro models have only a limited ability to recapitulate, such as those associated with envi- ronmental enteric dysfunction,68 colorectal cancer,69 cystic ﬁbrosis,70 bacterial infectious diseases,71 and celiac dis- ease.72 We believe the likelihood that Intestine Chips will be able to meet this challenge is high given the successful modeling of other complex diseases (eg, pulmonary edema,73 asthma,74 chronic obstructive pulmonary dis- ease,74,75 or Barth syndrome76) with other microﬂuidic Organ Chips. Moreover, we have previously demonstrated that the Gut Chip can be used to model key features of human intestinal diseases, including IBD and small intesti- nal bacterial overgrowth caused by ileus.42 Importantly, the gut microbiome in a mouse or other laboratory animal is remarkably different from that in a human (eg, w85% of gut microbiome that colonizes in human does not exist in mice88), and thus, creation of hu- man Intestine Chips with human microbiome could have a major positive impact on this ﬁeld. Intestine Chips that contain human intestinal microbiome should better reca- pitulate metabolism of oral drugs, and hence, testing of novel drug compounds on these platforms could lead to results that inform clinical trials design in a helpful way. We envision that integrating patient-speciﬁc intestinal organo- ids, local immune cells, and the commensal microbiome into Intestine Chips will create a highly deﬁned and controllable translation platform that should accelerate discovery of new drug and personalized precision therapeutics. Bein et al Intestine Chip Models for Developmental Therapeutics Disease Models Using Human Intestine Chips Only a small body of work has been published on disease models using microﬂuidic intestine devices. The 2-channel Cellular and Molecular Gastroenterology and Hepatology Vol. 5, No. 4 664 Microﬂuidic Human Intestine Models Microﬂuidic Human Intestine Models 2018 665 6.Cryan JF, Dinan TG. Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour. Nat Rev Neurosci 2012;13:701–712. with pathogenic E. coli in this model.42 Thus, the Intestine Chip approach also may be useful for discovery of new microbiome-based therapeutics, such as genetically engi- neered commensal bacteria.92 By integrating circulating and organ-speciﬁc immune cells into Intestine Chips, they might be useful for in vitro development of new mucosal vaccines.93,94 7.Mayer EA. Gut feelings: the emerging biology of gut–- brain communication. Nat Rev Neurosci 2011; 12:453–466. 8.Garrett WS, Gordon JI, Glimcher LH. Homeostasis and inﬂammation in the intestine. Cell 2010;140:859–870. 9.Round JL, Mazmanian SK. The gut microbiome shapes intestinal immune responses during health and disease. Nat Rev Immunol 2009;9:313–323. Intestine Chip Models for Developmental Therapeutics In addition to recapitulating disease phenotypes, human Organ Chips offer a unique way to get insight into the mo- lecular, genetic, and biophysical basis of disease based on their ability to incorporate different levels of cellular complexity, and to independently control various critical parameters (eg, chemical gradients, mechanical forces, cell types, and ECM).42,77 In past Gut Chip disease modeling studies, induction of the pathologic phenotype was accomplished by exposure to speciﬁc pathogenic organisms, toxins, or inﬂammatory me- diators (eg, cytokines), with combination of 2 or more of these factors being required to produce disease responses.42 For example, both lipopolysaccharide endotoxin and im- mune cells had to be added simultaneously in the Gut Chip to produce villus blunting and compromise intestinal bar- rier function, and neither could induce this response on their own.42 Inclusion of additional components, such as microbiome,16,42 pathogenic bacteria,42 mechanical cues,17,42 ECM, connective tissue cells, neuronal cells, organ- speciﬁc immune cells, and/or hormonal signals might be needed to model other speciﬁc phenotypes that result from complex interplay among several signaling pathways. This goal also might be achieved by creating Intestine Chips with cells isolated from intestinal biopsies taken from patients with speciﬁc diseases. This approach also could facilitate development of robust personalized disease models for improved drug screening and matching.78,79 It is important to note that commensal microbes also can be developed as probiotic therapeutics for treatment of human intestinal diseases. For example, fecal microbiota transplantation containing commensal microbes has already been approved by the US Food and Drug Administration for treatment of Clostridium difﬁcile infections by restoring the normal gut microbiome in these patients.89,90 Probiotic formulations also have been used to treat inﬂammation associated with IBD, although the effectiveness and mech- anism of action have not been fully validated.91 Importantly, the probiotic L rhamnosus GG was shown to increase in- testinal barrier function when cocultured in the lumen of the intestinal epithelial channel of the human Gut Chip,16 and the VSL#3 probiotic formulation suppressed villus blunting and loss of barrier function induced by infection Conclusions In this article, we reviewed recently developed micro- ﬂuidic human Intestine Chip models and described the advantages they offer relative to commonly used 2D and 3D in vitro culture systems, including their ability to more closely emulate the structure, function, physiology, and pa- thology of the living human intestine. Microﬂuidic Organ Chip systems provide unparalleled independent control over multiple key biologic, chemical, molecular, cellular and mechanical parameters within the intestinal microenviron- ment, thereby enabling researchers to apply a synthetic biology approach at the cell, tissue, and organ levels that can lead to new insights into intestinal physiology and disease mechanisms. By harnessing these unique capabilities, In- testine Chips can be applied to analyze the molecular pro- cesses underlying various enteropathies, and to advance development of new therapies. Creation of personalized Intestine Chips containing stem cells, microbiome, and im- mune cells from the same patient will offer a powerful new approach to deﬁne patient-speciﬁc drug responses and toxicities, and hence to advance precision medicine. 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https://openalex.org/W2808375196	http://mural.maynoothuniversity.ie/13224/1/MLV_biology_RecBCD.pdf	English	null	RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome amplification	Nucleic acids research	2,018	cc-by	12,562	ABSTRACT mologous recombination (e.g. (3)) and chromosome segre- gation (e.g. (4)). Importantly, genome instability per se need not be detrimental as aneuploidy is found in human hep- atocytes (5), aneuploidy and copy number variations are found in neurons (6), and mosaic aneuploidy contributes to adaptability in organisms such as Leishmania (7). Since re- combination involves DNA synthesis and can lead to chro- mosomal amplification and duplication via recombination- dependent replication in prokaryotes and break-induced replication in eukaryotes (8–11), it is interesting to consider how segregation errors resulting in aneuploidy and copy number variation may arise from improper interactions be- tween recombination and DNA replication. DNA double-strand break (DSB) repair is critical for cell survival. A diverse range of organisms from bac- teria to humans rely on homologous recombination for accurate DSB repair. This requires both coordi- nate action of the two ends of a DSB and stringent control of the resultant DNA replication to prevent unwarranted DNA ampliﬁcation and aneuploidy. In Escherichia coli, RecBCD enzyme is responsible for the initial steps of homologous recombination. Pre- vious work has revealed recD mutants to be nuclease defective but recombination proﬁcient. Despite this proﬁciency, we show here that a recD null mutant is defective for the repair of a two-ended DSB and that this defect is associated with unregulated chromo- some ampliﬁcation and defective chromosome seg- regation. Our results demonstrate that RecBCD plays an important role in avoiding this ampliﬁcation by coordinating the two recombining ends in a manner that prevents divergent replication forks progressing away from the DSB site. p The recombination and DNA replication systems of Es- cherichia coli can provide insight into how chromosomes are handled correctly to avoid segregation errors and how such errors can arise when chromosomes are handled incorrectly. Escherichia coli DNA replication is initiated at a unique origin (oriC) and proceeds bi-directionally around the cir- cular chromosome until the two replication forks meet in the terminus region (12). The two replicated copies of the chromosome are then segregated accurately to the daugh- ter cells. We have developed a system where we can induce a replication-dependent DNA double-strand break (DSB) in the lacZ gene approximately half way around the chro- mosome on the right replichore (13). ABSTRACT The DSB is generated by SbcCD-mediated cleavage of a DNA hairpin formed by a long palindrome on one of the sister chromosomes dur- ing replication and is accurately repaired by homologous recombination using the un-cleaved sister chromosome as a template. The palindrome-induced DSB repair (DSBR) re- action is remarkably efficient, causing a fitness loss of only 0.6% per generation in fast-growing cells (14). DNA re- pair is absolutely dependent on the action of RecBCD and RecA mediated homologous recombination causing recB C⃝The Author(s) 2018. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Published online 13 June 2018 Published online 13 June 2018 6670–6682 Nucleic Acids Research, 2018, Vol. 46, No. 13 doi: 10.1093/nar/gky463 Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, King’s Buildings, Mayﬁeld Road, Edinburgh EH9 3FF UK Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, King’s Bu Edinburgh EH9 3FF, UK ogy, School of Biological Sciences, University of Edinburgh, King’s Buildings, Mayﬁeld Roa F UK Downloaded from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on 11 August 2020 eceived December 18, 2017; Revised May 01, 2018; Editorial Decision May 07, 2018; Accepted June 08, 2018 Martin A. White, Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, MA 02138, USA. Manuel A. Lopez-Vernaza, Department of Biology, Maynooth University, Ireland. To whom correspondence should be addressed. Tel: +44 131 650 5373; Fax: +44 131 650 6556; Email: d.leach@ed.ac.uk Present addresses: To whom correspondence should be addressed. Tel: +44 131 650 5373; Fax: +44 131 650 6556; Email: d.leach@ed.ac.uk Present addresses: Martin A. White, Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, MA 02138, USA Manuel A. Lopez-Vernaza, Department of Biology, Maynooth University, Ireland. To whom correspondence should be addressed. Tel: +44 131 650 5373; Fax: +44 131 650 6556; Email: d.leach@ed.ac.uk To whom correspondence should be addressed. Tel: +44 131 650 5373; Fax: +44 131 650 6556; Email: d.leach@ed.ac.uk Present addresses: Martin A. White, Department of Molecular and Cellular Biology, Harvard University, 52 Oxford Street, Cambridge, MA 02138, USA RecBCD coordinates repair of two ends at a DNA double-strand break, preventing aberrant chromosome ampliﬁcation Martin A. White, Benura Azeroglu, Manuel A. Lopez-Vernaza, A. M. Mahedi Hasan and David R. F. Leach* Institute of Cell Biology School of Biological Sciences University of Edinburgh King’s Buildings Mayﬁeld Road Martin A. White, Benura Azeroglu, Manuel A. Lopez-Vernaza, A. M. Mahedi Hasan and David R. F. Leach* David R. F. Leach* Institute of Cell Biology, School of Biological Sciences, University of Edinburgh, King’s Buildings, Mayﬁeld Road, Edinburgh EH9 3FF, UK RecA ChIP-seq Cells were fixed by the addition of formaldehyde (final con- centration 1%) for 10 min at 22.5◦C to crosslink proteins to DNA. Crosslinking was quenched by the addition of 0.5 M glycine. Cells were then collected by centrifugation at 1500 × g for 7 min before washing three times in ice- cold 1× PBS and re-suspending in 250 l of ChIP buffer (10 ml ChIP buffer consists of 200 mM Tris–HCl (pH 8.0), 600 mM NaCl, 4% Triton X and 1 cOmplete™protease inhibitor cocktail EDTA-free tablet). Samples were then sonicated using a Diagenode Bioruptor® at 30 s intervals for 10 min at high amplitude. After sonication, 350 l of ChIP buffer was added to each sample and the samples gently mixed by pipetting. Immunoprecipitation was per- formed overnight at 4◦C using 1/100 anti-RecA antibody Chromosome marker frequency analysis A detailed description of materials and experimental methodology can be found in supplementary file 1. A brief summary is provided below. gDNA was isolated from 20 ml cultures using a Wiz- ard Genomic DNA Purification Kit following manufac- turer’s guidelines. Purified gDNA was treated with the supplied RNase for 50 min and rehydrated overnight in the supplied TE buffer at 4◦C. Three units of the RNase blend Riboshredder was then added to further destroy con- taminating RNA. Samples were then purified again by phenol/chloroform extraction and ethanol precipitation. Libraries were prepared from the gDNA by Edinburgh Genomics using an Illumina TruSeq DNA Sample Prep kit. Edinburgh Genomics subsequently obtained paired- end reads of the samples using an Illumina HiSeq 2000 plat- form. Two biological repeats of each strain were acquired. Data were analysed as described in supplementary file 1. 2D gel electrophoresis and Southern blotting gDNA was isolated as described above for PFGE (‘plug’ method), except cells were concentrated to an OD600 of 100 in TEN buffer prior to mixing with a 0.8% low melting point agarose in TEN buffer solution. Prior to restriction digest, gDNA (in plugs) was washed six times in appropriate re- striction buffer for 1 h at room temperature with agitation, prior to overnight digestion at 37◦C in restriction buffer with 600 units of restriction enzyme. Digested DNA was loaded onto a 0.4% agarose gel in 1× TBE (89 mM Tris- borate, 2 mM EDTA) at 1 V/cm for 24 h at 4◦C. Lanes con- taining the separated DNA were cut out, rotated 90◦and cast in a new gel composed of 1% agarose in 1× TBE sup- plemented with 0.3 g ml−1 ethidium bromide. This gel (the second dimension) was ran at 6 V/cm for 15 h in circulat- ing 1x TBE buffer supplemented with 0.3 g ml−1 ethid- ium bromide at 4◦C. DNA was then transferred to a pos- itively charged nylon membrane by Southern blotting and cross-linked to the membrane using UV-light. Chromosome fragments of interest were detected using 32P -dATP incor- porated radiolabeled DNA probes as described for PFGE. Data were analysed as described in supplementary file 1. In this work, we have analysed the behaviour of a recD null mutant that is expected to repair DSBs using RecBC enzyme. Previous studies have revealed that recD mu- tants are recombination proficient and even have a hyper- recombination (hyper-rec) phenotype in some assays (21) despite being nuclease defective (22). However, here we show that recD mutants are repair deficient when subjected to two-ended palindrome induced DSBs. Furthermore, we show that cells lacking RecD are unable to coordinate the two ends of the DSB and this results in aberrant DNA repli- cation and defective chromosome segregation. Bacterial strains and growth conditions All bacteria used in this study were derived from the non- pathogenic E. coli K12 strain BW27784. Expression of SbcCD and I-SceI was repressed or induced by the addi- tion of glucose or arabinose at the indicated concentration. PsfiA-gfp expression and total cellular DNA content were as- sayed by flow cytometry following 2 h of growth in the pres- ence of arabinose/glucose. For all other experiments (except growth curve, Supplementary Figure S1 and cellular viabil- ity Figure 1A), cells were sampled following 1 h of growth in the presence of arabinose/glucose. All materials used in this study are listed in Supplementary Table S1. INTRODUCTION Organisms as distantly related as humans and bacteria have evolved sophisticated mechanisms to ensure the stable in- heritance of their genomes through successive generations. However, genome instability does occur and takes the form of mutations, chromosome rearrangements, and both gene and whole chromosome number variations (aneuploidy). Genome instability is an enabling characteristic of cancer (1) and a direct cause of several human syndromes (e.g. Down syndrome and Klinefelter syndrome). These have been attributed to aberrant DNA replication (e.g. (2)), ho- Nucleic Acids Research, 2018, Vol. 46, No. 13 6671 0.3 M NaCl, 0.03 M Na citrate and 0.1% SDS, and then for 30 min in a solution of 0.075 M NaCl, 0.0075 M sodium citrate and 0.1% SDS. 0.3 M NaCl, 0.03 M Na citrate and 0.1% SDS, and then for 30 min in a solution of 0.075 M NaCl, 0.0075 M sodium citrate and 0.1% SDS. and recA mutants to die when subjected to palindrome- induced DSBs (13). The heterotrimeric RecBCD complex is a highly rapid and processive DNA helicase-nuclease that generates ss- DNA at broken DNA ends upon which it actively loads RecA (15), the enzyme that catalyses DNA sequence homology-dependent strand exchange. RecB and RecD are helicases with opposite polarity (RecB, 3′ to 5′; RecD, 5′ to 3′). In addition to its helicase activity, RecB also possesses nuclease activity. This activity requires an interaction with RecD and is modulated upon binding of RecC to a polar oc- tomeric sequence (5′-GCTGGTGG) known as Chi (16,17). RecBC can form a complex in the absence of RecD in vitro. In comparison to RecBCD, RecBC has reduced processivity (18), lacks exonuclease activity (19) and constitutively loads RecA independently of Chi (20). PFGE and Southern blotting (B, D) Quantitative assessment of the number of viable cells (the number of colony forming units per 1 ml of culture normalised to an optical density at 600 nm of 1) following 60 min of exponential growth with (B) SbcCD either expressed or not; (D) I-SceI either expressed or not. (C) Cultures of strains containing the DNA palindrome or not were serially diluted and grown overnight under either conditions where SbcCD was repressed following, or not, exposure to ultraviolet (UV)-light. Data for A) and C) are representative experiments; data for B) and D) are represented as mean ± S.E.M., n = 3. No difference in viability was detected between the recD+ I-SceI+ I-SceIcs+ and recD−I-SceI+ I-SceIcs+ samples (two-sample t test, P = 0.68). See also Supplementary Figure S1. Figure 1. RecD is required for cells to survive certain forms of DNA damage. (A) Cultures of strains containing the DNA palindrome or not were serially diluted and grown overnight under either conditions where SbcCD was induced (SbcCD+), or repressed (SbcCD−). Colony formation at higher dilution (lower OD600) indicates increased cellular viability. (B, D) Quantitative assessment of the number of viable cells (the number of colony forming units per 1 ml of culture normalised to an optical density at 600 nm of 1) following 60 min of exponential growth with (B) SbcCD either expressed or not; (D) I-SceI either expressed or not. (C) Cultures of strains containing the DNA palindrome or not were serially diluted and grown overnight under either conditions where SbcCD was repressed following, or not, exposure to ultraviolet (UV)-light. Data for A) and C) are representative experiments; data for B) and D) are represented as mean ± S.E.M., n = 3. No difference in viability was detected between the recD+ I-SceI+ I-SceIcs+ and recD−I-SceI+ I-SceIcs+ samples (two-sample t test, P = 0.68). See also Supplementary Figure S1. (Abcam, ab63797). Immunoprecipitated samples were then incubated with Protein G Dynabeads® for 2 h with rota- tion at room temperature. All samples were washed three times with 1× PBS + 0.02% Tween-20 before re- suspend- ing the Protein G Dynabeads® in 200 l of TE buffer + 1% SDS. 100 l of TE buffer + 1% SDS were added to the input samples and all samples were then incubated at 65◦C for 10 h to reverse the formaldehyde cross-links. DNA was isolated using the MinElute PCR purification kit according to manufacturer’s instructions. PFGE and Southern blotting DNA was eluted in 100 l of TE buffer using a two-step elution. Samples were stored at –20◦C. Two biological repeats of each strain were acquired. Libraries of the immunoprecipitated DNA were made using NEBNext® ChIP-Seq library preparation kit. Briefly, the samples were first subjected to end repair to fill in ssDNA overhangs, remove 3′ phosphates and phosphorylate the 5′ ends of DNA. Klenow exo- was used to adenylate the 3′ ends of the DNA and NEBNext DNA adaptors (provided in the NEBNext Multiplex Oligos for Illumina kit) were lig- ated using T4 DNA ligase. After each step, the DNA was purified using the Qiagen MinElute PCR purification kit according to the manufacturer’s instructions. After adap- tor ligation, the adaptor-modified DNA fragments were en- riched by PCR using primers (provided in the NEBNext Multiplex Oligos for Illumina kit) corresponding to the be- ginning of each adaptor. Finally, agarose gel electrophoresis was used to size select adaptor-ligated DNA with an aver- age size of approximately 300 bp. All samples were quanti- fied on a Bioanalyzer (Agilent) before being sequenced on either an Illumina® HiSeq 2500 (for DL4184, DL4201 and DL5699) or HiSeq 4000 (for DL6204) by Edinburgh Ge- nomics. Data were analysed as described in supplementary file 1. PFGE and Southern blotting Genomic DNA (gDNA) in agarose plugs was digested with I-SceI and fragments separated using a CHEF-DR II PFGE (Biorad) machine at 6 V/cm for 10 h in 0.5× TBE buffer at 4◦C. DNA was then transferred to a positively charged nylon membrane by Southern blotting and cross- linked to the membrane using UV-light. Chromosome frag- ments of interest were detected using 32P -dATP incorpo- rated radiolabeled DNA probes. Probes were hybridized to membranes overnight at 65◦C in 10 ml of Church-Gilbert buffer (7% SDS, 0.5 M NaH2PO4, 1 mM EDTA, 1% BSA), then washed twice at 60◦C, first for 15 min in a solution of 6672 Nucleic Acids Research, 2018, Vol. 46, No. 13 1. RecD is required for cells to survive certain forms of DNA damage. (A) Cultures of strains containing the DNA palindrome or not were serially d and grown overnight under either conditions where SbcCD was induced (SbcCD+), or repressed (SbcCD−). Colony formation at higher dilution OD600) indicates increased cellular viability. (B, D) Quantitative assessment of the number of viable cells (the number of colony forming units per 1 ulture normalised to an optical density at 600 nm of 1) following 60 min of exponential growth with (B) SbcCD either expressed or not; (D) I-SceI expressed or not. (C) Cultures of strains containing the DNA palindrome or not were serially diluted and grown overnight under either conditions SbcCD was repressed following, or not, exposure to ultraviolet (UV)-light. Data for A) and C) are representative experiments; data for B) and D) resented as mean ± S.E.M., n = 3. No difference in viability was detected between the recD+ I-SceI+ I-SceIcs+ and recD−I-SceI+ I-SceIcs+ samples ample t test, P = 0.68). See also Supplementary Figure S1. Downloaded from https://academic.oup.com/nar/article/46/13/6670/5036844 by gue rom https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on 11 August 2020 Figure 1. RecD is required for cells to survive certain forms of DNA damage. (A) Cultures of strains containing the DNA palindrome or not were serially diluted and grown overnight under either conditions where SbcCD was induced (SbcCD+), or repressed (SbcCD−). Colony formation at higher dilution (lower OD600) indicates increased cellular viability. All three subunits of the RecBCD complex are required to survive cleavage of a DNA hairpin by SbcCD nuclease All three subunits of the RecBCD complex are required to survive cleavage of a DNA hairpin by SbcCD nuclease ( g ) A key intermediate in the DSB repair reaction, RecA pro- tein bound to ssDNA, catalyses auto-cleavage of the tran- scriptional repressor LexA (24), resulting in the DNA dam- age response (known as the SOS response in E. coli). Induc- tion of the SOS response can be detected using a reporter consisting of the green fluorescence protein gene placed un- der the SOS-inducible promoter PsfiA (25). To test if induc- tion of a DSB induced the SOS response in the absence of RecD, we integrated a copy of this reporter into the E. coli chromosome and quantified cellular GFP fluorescence by flow cytometry. Consistent with a similar assay using a PsfiA-gfp reporter on a plasmid (14), cleavage of the hair- pin by SbcCD in RecBCD+ cells resulted in a highly repro- ducible increase in GFP expression (Figure 2E). This DSB- dependent increase in cellular GFP accumulation was more dramatic, and possibly multi-modal, in the recD back- ground (Figure 2E). In contrast to recB and recC mutants that are known to be defective in homologous recombination (e.g. (13)), recD mutants have been characterised as recombination profi- cient (or even hyper-rec) in P1 transduction, Hfr mating and red gam bacteriophage lambda crosses (21). It was there- fore surprising that all three subunits of the RecBCD com- plex were required for cells to survive in the presence of an SbcCD-induced DSB (Figure 1A). In contrast to cul- tures of RecBCD+ cells, 1 h of SbcCD expression in cul- tures of recD mutants harbouring a 246 bp interrupted DNA palindrome in the lacZ gene of the E. coli chromo- some resulted in a 96% decrease in the number of viable cells (Figure 1B); after 2 h of SbcCD expression cell mass ceased to increase (Supplementary Figure S1). Consistent with published data on recD mutants, these recD cells (un- like the recB and recC cells) were able to survive DNA damage induced by ultra-violet light (Figure 1C), indicat- ing that the observed DNA repair defect is not a result of the recD allele used in this study. Site-specific DSBs in- duced at the same lacZ locus by the homing endonuclease I-SceI resulted in an approximate 10-fold decrease in via- bility of both RecBCD+ and recD cells (Figure 1D). All three subunits of the RecBCD complex are required to survive cleavage of a DNA hairpin by SbcCD nuclease The recD cells were not found to be more sensitive to an I-SceI induced DSB than RecBCD+ cells (P = 0.68, two-sample t- test). Flow cytometry To determine SOS induction from PsfiA-gfp, GFP fluores- cence was measured using an Apogee A50 flow cytometer. Either two or three biological repeats of each strain and condition were acquired. Relative cellular DNA contents were measured in fixed cells by adding 1 ml of culture to 8 ml of 100% ethanol. Fixed cells were collected by centrifu- Nucleic Acids Research, 2018, Vol. 46, No. 13 6673 Nucleic Acids Research, 2018, Vol. 46, No. 13 6673 gation, washed twice in 1× PBS and re-suspended in 400 l of 1× PBS. RNA was then degraded and DNA stained by the addition of 100 l of a 1× PBS, 50 g ml−1 propidium iodide, 500 l ml−1 RNaseA solution. Propidium iodide flu- orescence (a measure of DNA content) was measured using an Apogee A50 flow cytometer. Three biological repeats of each strain and condition were acquired. and chromosome organization by wide-field fluorescence imaging of strains harbouring an array of tetO and an ar- ray of lacO sequences on either side of the palindrome inte- gration site lacZ (23) and expressing TetR-YFP and LacI- CFP from a constitutive promoter integrated in the chro- mosome as well as HupA-mCherry (a non-sequence spe- cific DNA binding protein) expressed from the native hupA locus (Figure 2A). In contrast to RecBCD+ cells, the DSB resulted in a large increase in the variability of cell size (Fig- ure 2B), the number of visibly segregated lacZ loci (Figure 2C) and altered nucleoid morphology (Supplementary Fig- ure S2). Cells also lacked the regular repeating pattern of twin YFP/CFP foci observed in controls (Figure 2A and Supplementary Figure S2) and often contained invagina- tions (division septa) that were clearly displaced from mid- cell (e.g. Figure 2A). Although complex and variable in na- ture, these cellular phenotypes are all consistent with defec- tive chromosome segregation. Microscopy Cells were grown in M9 minimal growth media supple- mented with 0.2% glycerol and 1 ng ml−1 anhydrotetracy- cline at 37◦C. 10 l of cell culture was mounted on a pad of 1% agarose.H2O, covered with #1.5 coverslip and im- aged by widefield fluorescence microscopy at a resolution of 100 nm X, 100 nm Y, 200 nm Z using a Zeiss Axiovert 200 fluorescence microscope equipped with a 100× Objec- tive NA1.4 phase objective with a 1.6× Optivar, Photomet- rics Evolve 512 EMCCD camera, Xenon light source and piezo stage. The microscope was controlled using Meta- morph software. Acquired images were deconvolved using Autoquant X2 and visualized and processed using FIJI. Three biological repeats were acquired for each strain. DNA synthesis is an essential intermediate stage of DSBR. Total cellular DNA content was assayed by flow cytometry following fixation, RNase treatment and propid- ium iodide staining. Hairpin cleavage by SbcCD had no no- table effect on cellular DNA content in RecBCD+ samples but resulted in a very broad distribution of DNA content in recD cells, most of which possessed significantly more DNA than recD cells that did not experience an induced DSB (Figure 2D). Broken DNA ends are recombinogenic in the absence of RecD To test if a specific recombination intermediate accumu- lated in recD cells subject to the DSB, we examined a 174 kb region flanking the DSB locus (lacZ) by pulsed-field gel electrophoresis (PFGE) and Southern blotting (Figure 3A). This large region of chromosomal DNA was selected due to the high processivity of RecBCD. In the presence of RecBCD, the DSB is repaired quickly and efficiently with no detectable alteration in DNA state at this level of reso- lution (Figure 3B, (13)). In contrast, in the absence of ei- ther RecB or RecC proteins, linear intact DNA was lost in response to the DSB and broken DNA accumulated as a range of sizes (Figure 3B, (13)). In the absence of RecD protein, cells subject to the DSB also accumulated broken DNA, but in a more discreet range of sizes, particularly with DSB repair in the absence of RecD results in asymmetric cell divisions, defective chromosome segregation, increased cellu- lar DNA content and strong induction of the DNA damage response To test the physiological consequence of the RecBC- dependent DSB repair, we assessed cellular morphology 6674 Nucleic Acids Research, 2018, Vol. 46, No. 13 2. DSB repair in the absence of RecD results in altered cellular physiology. (A) Micrographs of RecBCD+ and recD cells containing or not the palindrome (Pal) following 60 min of SbcCD expression. Cells harboured an array of tetO and lacO sequences on either side of the palindrome tion site (lacZ) and expressed TetR-YFP (green) and LacI-CFP (magenta) from a constitutive promoter. Scale bar shows 5 m. (B) Distribution engths and the coefficient of variation (CV). Data are for 3 biological repeats combined. (C) Histogram showing the distribution of number of ble LacI-CFP and TetR-YFP foci per cell and the coefficient of variation (CV). Data are the cumulative data from 3 biological repeats. (D) Cellular ontent of >100,000 cells was measured by flow cytometry following 2 hours of exponential growth with SbcCD expression either induced or ed in cells containing the palindrome or not. Data is from a single representative experiment. (E) Expression of green fluorescence protein (GFP) e DNA-damage inducible promoter PsfiA as measured by flow cytometry following two hours of exponential growth with SbcCD either expressed At least 100 000 cells were measured for each sample. Data is from a single representative experiment. Broken DNA ends are recombinogenic in the absence of RecD For (B–E), data are displayed as a probability ution function (PDF) to normalize for differences in total number of analysed cells. See also Supplementary Figure S2. Downloaded from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on 11 August 2020 Downloaded from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on rom https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on 11 August 2020 Figure 2. DSB repair in the absence of RecD results in altered cellular physiology. (A) Micrographs of RecBCD+ and recD cells containing or not the DNA palindrome (Pal) following 60 min of SbcCD expression. Cells harboured an array of tetO and lacO sequences on either side of the palindrome integration site (lacZ) and expressed TetR-YFP (green) and LacI-CFP (magenta) from a constitutive promoter. Scale bar shows 5 m. (B) Distribution of cell lengths and the coefficient of variation (CV). Data are for 3 biological repeats combined. (C) Histogram showing the distribution of number of detectable LacI-CFP and TetR-YFP foci per cell and the coefficient of variation (CV). Data are the cumulative data from 3 biological repeats. (D) Cellular DNA content of >100,000 cells was measured by flow cytometry following 2 hours of exponential growth with SbcCD expression either induced or repressed in cells containing the palindrome or not. Data is from a single representative experiment. (E) Expression of green fluorescence protein (GFP) from the DNA-damage inducible promoter PsfiA as measured by flow cytometry following two hours of exponential growth with SbcCD either expressed or not. At least 100 000 cells were measured for each sample. Data is from a single representative experiment. For (B–E), data are displayed as a probability distribution function (PDF) to normalize for differences in total number of analysed cells. See also Supplementary Figure S2. Nucleic Acids Research, 2018, Vol. 46, No. 13 6675 Figure 3. RecD is not required for recombination intermediate formation. (A, B and D) Analysis of the state of a 174 Kb DNA fragment DSB locus (intact, broken or branched) by pulsed-field gel electrophoresis and Southern blot, probed for both sides of the DSB locus (prox to the origin of replication oriC) following induction of SbcCD expression for 60 min. DNA from a strain harbouring an I-SceI cleavage sit which the palindrome is integrated was digested with I-SceI in vitro to act as marker for broken DNA (‘in vitro digest’). In vitro digestion wi in low frequency cleavage at an endogenous chromosomal site (I-SceIcs). Broken DNA ends are recombinogenic in the absence of RecD (C) Normalized enrichment of DNA in an 80 kb region cont locus following RecA chromatin immunoprecipitation (ChIP) in RecBCD+ and recD mutants with and without a palindrome at lacZ, follo of SbcCD expression for 60 min. To account for variability in sequencing depth, data were normalized using a median of ratios scaling f was smoothed using a loess filter with a 2 kb window. All panels show results from single, representative experiments in which DNA repli from left to right. See also Supplementary Figure S3. Downloaded from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on 11 August 2020 Figure 3. RecD is not required for recombination intermediate formation. (A, B and D) Analysis of the state of a 174 Kb DNA fragment containing the DSB locus (intact, broken or branched) by pulsed-field gel electrophoresis and Southern blot, probed for both sides of the DSB locus (proximal and distal to the origin of replication oriC) following induction of SbcCD expression for 60 min. DNA from a strain harbouring an I-SceI cleavage site in the locus in which the palindrome is integrated was digested with I-SceI in vitro to act as marker for broken DNA (‘in vitro digest’). In vitro digestion with I-SceI results in low frequency cleavage at an endogenous chromosomal site (I-SceIcs). (C) Normalized enrichment of DNA in an 80 kb region containing the DSB locus following RecA chromatin immunoprecipitation (ChIP) in RecBCD+ and recD mutants with and without a palindrome at lacZ, following induction of SbcCD expression for 60 min. To account for variability in sequencing depth, data were normalized using a median of ratios scaling factor. The data was smoothed using a loess filter with a 2 kb window. All panels show results from single, representative experiments in which DNA replication proceeds from left to right. See also Supplementary Figure S3. Figure 3. RecD is not required for recombination intermediate formation. (A, B and D) Analysis of the state of a 174 Kb DNA fragment containing the DSB locus (intact, broken or branched) by pulsed-field gel electrophoresis and Southern blot, probed for both sides of the DSB locus (proximal and distal to the origin of replication oriC) following induction of SbcCD expression for 60 min. Broken DNA ends are recombinogenic in the absence of RecD DNA from a strain harbouring an I-SceI cleavage site in the locus in which the palindrome is integrated was digested with I-SceI in vitro to act as marker for broken DNA (‘in vitro digest’). In vitro digestion with I-SceI results in low frequency cleavage at an endogenous chromosomal site (I-SceIcs*). (C) Normalized enrichment of DNA in an 80 kb region containing the DSB locus following RecA chromatin immunoprecipitation (ChIP) in RecBCD+ and recD mutants with and without a palindrome at lacZ, following induction of SbcCD expression for 60 min. To account for variability in sequencing depth, data were normalized using a median of ratios scaling factor. The data was smoothed using a loess filter with a 2 kb window. All panels show results from single, representative experiments in which DNA replication proceeds from left to right. See also Supplementary Figure S3. 6676 Nucleic Acids Research, 2018, Vol. 46, No. 13 regards to the DSB end corresponding the oriC proximal side of the DSB locus (Figure 3B). In contrast to recB and recC mutants, a significant proportion of DNA isolated from recD cells failed to exit the wells of the pulsed-field gels (Figure 3B), indicating the accumulation of branched DNA molecules. (Figure 3D). Compared to DNA isolated form recD sin- gle mutant cells, there was a notable decrease in the rela- tive amount of both linear intact DNA and broken DNA in DNA isolated form recD ruvAB cells, with a concomi- tant relative increase in the amount of branched DNA (Fig- ure 3D). This indicates that in the absence of RecD, DSB ends are processed by homologous recombination and pro- ceed at least to the stage of Holliday junction formation. In order to recombine, RecA must be loaded onto the broken DNA ends. Chromosomal binding of RecA was as- sayed by RecA chromatin immunoprecipitation (ChIP) and whole genome sequencing. In the absence of hairpin cleav- age by SbcCD, and irrespective of the presence or absence of RecD, DNA corresponding to a highly expressed gen- tamycin resistance cassette that was inserted into the cynX gene of the strains used in this study (13), was enriched following RecA ChIP (Figure 3C). RecA ChIP was previ- ously shown to result in a general enrichment of highly ex- pressed genes (26) and may be caused by true RecA bind- ing, a bias in crosslinking efficiency (27), or some other un- known reason. DSB repair in the absence of RecD results in aberrant chro- mosome amplification The recD specific increase in total cellular DNA content following induction of the DSB (Figure 2D) could be caused by ongoing timely DNA replication initiation at oriC in the absence of cellular division and/or aberrant DNA repli- cation initiation events. The DNA replication profile was assayed by marker frequency analysis (MFA) of DNA ex- tracted from asynchronous, exponentially growing cultures of cells. In the absence of hairpin cleavage by SbcCD, and irrespective of the presence or absence of RecD, relative copy number peaked close to the single origin of replication oriC and decayed on either side before reaching a minimum close to a sequence known as dif that sits approximately half way along the circular chromosome from oriC (Figure 4A, B). This observation is consistent with bi-directional replication originating at oriC and holds true for DNA ex- tracted from RecBCD+ cells undergoing hairpin cleavage by SbcCD (28), Figure 4B. In contrast, DNA extracted from recD cells undergoing hairpin cleavage by SbcCD showed a maximal peak that maps close to the DSB locus (Figure 4B). This DSB-dependent alteration in relative DNA abun- dance is most clearly visualised by normalising the results obtained for cultures subject to the DSB by the results ob- tained for an isogenic strain not subject to the DSB (Fig- ure 4C). This analysis showed a DSB-dependent increase in DNA that extends on one side from the palindrome to a se- quence known as terB in the chromosome terminus and on the other side from the palindrome to the rrnE operon, with a dip observed at the rrnH operon (Figure 4D). terB belongs to family of DNA sequences in the E. coli genome that bind Tus protein and blocks DNA replication in a polar manner (12). rrnH and rrnE are two of the seven heavily transcribed E. coli ribosomal RNA operons that are all transcribed in the same direction as DNA replication originating from oriC. These results are consistent with a DSB-dependent initiation of replication in recD cells that can extend from one side of the break before being blocked by terB and on the other side of the break before being blocked first by rrnH and subsequently by rrnE. This DSB-dependent amplifica- tion was not observed in cultures of RecBCD+ cells (Figure 4B, Supplementary Figure S4, (28)). Following RecA-catalysed strand exchange, homolo- gous recombination in E. Broken DNA ends are recombinogenic in the absence of RecD In contrast, in response to hairpin cleavage by SbcCD, and again irrespective of the presence (28) or absence of RecD, RecA ChIP-seq revealed RecA binding to the DNA flanking both sides of the DSB locus (Figure 3C and Supplementary Figure S3) indicating that recD mutants are capable of loading RecA onto both ends of the DSB at the population level. Given that RecBC and the ATP bind- ing mutant RecBCDK177Q are approximately 10 times less processive than RecBCD in vitro (18,29,30), we were sur- prised that approximately ±20 kb of DNA from the palin- drome was enriched for RecA binding in both RecBCD+ and recD strains (Figure 3C). Unlike RecBCD+ cultures, RecA binding was detected between the palindrome and the nearest active Chi sites when RecA ChIP was performed on recD cultures (Figure 3C), consistent with the ability of RecBC to load RecA in a Chi-independent manner. There was a close correlation between the peak of binding and the location of the nearest active Chi sequences for both the RecBCD+ and recD samples (Figure 3C). However, in the case of the recD mutant this close correlation was coinci- dental as the peak of RecA enrichment was not influenced by the position of Chi. It is also noticeable that the amount of RecA bound to the two sides of the DSB is more equal in the recD mutant than in the RecBCD+ cells. This may reflect a higher probability, in the presence of fully active RecBCD enzyme, of degrading the origin distal arm of the break all the way to the replication fork converting a two- ended break to a one-ended break. This binding of RecA to the chromosome in response to the induced DSB is suf- ficient to induce the DNA damage response (Figure 2E). DSB repair in the absence of RecD results in aberrant chro- mosome amplification coli chromosome with relative positions of loci of interest (oriC, rrnE, lacZ, dif, terB and the classically defined genomic position 0). (B) Marker Frequency Analysis of DNA isolated from recD+ and recD cells containing or not the palindrome (Pal) following 1 h of exponential growth with induced SbcCD expression. Data are the mean of two biological repeats, error bars show the range. Data was averaged using a 10 kb window. (C and D) DSB-dependent change in relative DNA abundance isolated from recD cells. Data values for recD SbcCD+ Pal−and recD SbcCD+ Pal+ are the average of two biological repeats normalized by the total number of mapped reads. Data was averaged using either a 10 kb window (C), or a 1 kb window (D). Figure 4. In the absence of RecD, cleavage of the palindrome by SbcCD results in an ectopic origin of chromosomal DNA replication. (A) Diagram of the single, circular 4.6 Mb E. coli chromosome with relative positions of loci of interest (oriC, rrnE, lacZ, dif, terB and the classically defined genomic position 0). (B) Marker Frequency Analysis of DNA isolated from recD+ and recD cells containing or not the palindrome (Pal) following 1 h of exponential growth with induced SbcCD expression. Data are the mean of two biological repeats, error bars show the range. Data was averaged using a 10 kb window. (C and D) DSB-dependent change in relative DNA abundance isolated from recD cells. Data values for recD SbcCD+ Pal−and recD SbcCD+ Pal+ are the average of two biological repeats normalized by the total number of mapped reads. Data was averaged using either a 10 kb window (C), or a 1 kb window (D). with MFA analysis (Figure 4C), this blocked DNA repli- cation species was substantially more abundant in DNA isolated from recD cells subject to the DSB than DNA isolated from RecBCD+ cells subject to the DSB (Figure 5E). RecA ChIP of DNA isolated from recD cells sub- ject to hairpin cleavage by SbcCD showed a peak of enrich- ment, oriC-distal to the rrnH locus that was not observed in RecBCD+ cells or recD cells not subject to hairpin cleav- age by SbcCD (Figure 5F), suggesting that DNA replica- tion forks moving from the palindrome towards oriC are blocked at rrnH and processed by homologous recombina- tion. DSB repair in the absence of RecD results in aberrant chro- mosome amplification coli results in the formation of branched 4-strand DNA structures known as Holliday junctions that are resolved by the Holliday junction re- solvase complex RuvABC (31). If the cleaved chromosomes of recD cells were proceeding to Holliday junction forma- tion, then inactivation of RuvABC (e.g. by deletion of the ruvAB operon) would result in branched DNA that would fail to exit the wells of gels subject to PFGE (31). Consis- tent with previous reports and the accumulation of Holliday junctions, DNA isolated from ruvAB cells subject to hair- pin cleavage by SbcCD showed a reduction in intact, linear DNA and a concomitant increase in branched DNA species DNA replication intermediates can be isolated by 2D gel electrophoresis and the direction of replication deter- mined by integration of an appropriately oriented ectopic terB sequence (Figure 5A) (28). 2D gel analysis of a 7.7 kb DNA fragment containing the DSB locus showed a DSB- dependent increase in DNA replication intermediates in RecBCD+ cells (Figure 5B). Integration of an ectopic terB sequence (oriented to block DNA replication proceeding Nucleic Acids Research, 2018, Vol. 46, No. 13 6677 Figure 4. In the absence of RecD, cleavage of the palindrome by SbcCD results in an ectopic origin of chromosomal DNA replication. (A) Diagram of the single, circular 4.6 Mb E. coli chromosome with relative positions of loci of interest (oriC, rrnE, lacZ, dif, terB and the classically defined genomic position 0). (B) Marker Frequency Analysis of DNA isolated from recD+ and recD cells containing or not the palindrome (Pal) following 1 h of exponential growth with induced SbcCD expression. Data are the mean of two biological repeats, error bars show the range. Data was averaged using a 10 kb window. (C and D) DSB-dependent change in relative DNA abundance isolated from recD cells. Data values for recD SbcCD+ Pal−and recD SbcCD+ Pal+ are the average of two biological repeats normalized by the total number of mapped reads. Data was averaged using either a 10 kb window (C), or a 1 kb window (D). Downloaded from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on 11 August 2020 Downloaded from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on 11 August 2020 Figure 4. In the absence of RecD, cleavage of the palindrome by SbcCD results in an ectopic origin of chromosomal DNA replication. (A) Diagram of the single, circular 4.6 Mb E. DSB repair in the absence of RecD results in aberrant chro- mosome amplification Finally, replication intermediates were also detected at ykgP::terB::eaeH progressing towards oriC in response towards the origin of replication, oriC) into this fragment (lacI::terB, Figure 5A) resulted in accumulation of a specific DNA replication intermediate that was dependent on the presence of a palindrome at lacZ (Figure 5C). This indicates that in the presence of RecBCD, cells that have been sub- ject to hairpin cleavage by SbcCD can initiate DNA repli- cation with the origin distal end of the DSB and that this replication can elongate towards oriC. 2D gel electrophore- sis of DNA isolated from strains with an ectopic terB se- quence integrated 55 KB upstream of the DSB locus (to- wards oriC, ykgP::terB::eaeH, Figure 5D) indicated that in a fraction of RecBCD+ cells, this replication can elongate at least 55 KB from the DSB site (Figure 5E). Consistent 6678 Nucleic Acids Research, 2018, Vol. 46, No. 13 6678 Nucleic Acids Research, 2018, Vol. 46, No. 13 to I-SceI induced DSBs (Figure 5G). In contrast to SbcCD- mediated DSBs, this replication was easily detected in DNA isolated from RecBCD+ cells and only mildly increased by the absence of RecD. The percentage of radiolabelled probe detected at stalled fork was 2.9 ± 0.1% in DNA extracted from RecBCD+ cells compared to 4.3 ± 1.1% in DNA ex- tracted from recD cells (n = 3, ± standard error of the mean). DISCUSSION Coordination between the two ends of DNA double-strand breaks (DSBs) is critical for their accurate repair. The two ends must invade an appropriate single region of DNA ho- mology. Furthermore, a processive DNA replication fork must not be set up at one end that moves past the location of the other end before it has also been synapsed. Invasion of the second end after the passage of the first replication fork can result in two outwardly facing, divergent forks with the potential to over-replicate part, or all, of the chromo- some. In eukaryotic cells, resection of the DNA at the site of a DSB occurs mainly on the 5′ DNA strand leaving the 3′ end close to the site of the break. It seems that coordi- nation of the two ends may involve limited DNA synthesis from the first invading end to extend the D-loop, followed by second-end capture. Second-end capture prevents break- induced replication and promotes gene conversion through the operation of a recombination execution checkpoint that senses the proximity and orientation of the two recombin- ing ends (32). The situation in E. coli is different as both the 3′ and 5′ ended DNA strands at the DSB are unwound (and under some conditions degraded) until a Chi site is recog- nised whereupon a cleaved 3′ ended strand binds RecA and initiates strand invasion and synapsis with the homologous template (16). Given that appropriately oriented Chi sites are likely to be separated from each other by many thou- sands of base pairs and each recombining end is understood to set up a processive replication fork to restore any missing DNA, it is remarkable that the two ends are well coordi- nated. How this coordination is achieved is unknown, but Figure 5. DNA synthesis progressing towards the origin of replication is detected following induction of a DNA double-strand break. (A) Diagram showing the location and orientation of lacI::terB. (B) and (C) analysis of DNA structure within a 7 Kb fragment containing the palindrome integra- tion site and either an ectopic terB sequence (lacI::terB) oriented to block DNA replication proceeding towards oriC (C) or not (B) by 2D gel elec- trophoresis in RecBCD+ cells containing or not the palindrome in lacZ fol- lowing 1h of growth with induced SbcCD expression. (D) Diagram show- ing the location and orientation of ykgP::terB::eaeH. showing the location and orientation of lacI::terB. (B) and (C) analysis of DNA structure within a 7 Kb fragment containing the palindrome integra- tion site and either an ectopic terB sequence (lacI::terB) oriented to block DNA replication proceeding towards oriC (C) or not (B) by 2D gel elec- trophoresis in RecBCD+ cells containing or not the palindrome in lacZ fol- lowing 1h of growth with induced SbcCD expression. (D) Diagram show- ing the location and orientation of ykgP::terB::eaeH. (E and G) Analy- sis of DNA structure by 2D gel electrophoresis of a chromosome frag- ment located 55 Kb oriC proximal to lacZ and containing an ectopic terB site (ykgP::terB::eaeH) that blocks DNA replication proceeding towards oriC. (E) DNA isolated from RecBCD+ and recD cells containing the palindrome in lacZ following 1h of growth with SbcCD either expressed (SbcCD+) or repressed (SbcCD−). (G) DNA isolated from RecBCD+ and recD cells containing an I-SceI cleavage site (I-SceIcs+) in lacZ follow- ing 1h of growth with I-SceI expression either induced (I-SceI+) or not (I- SceI−). Green arrows indicate the location were linear DNA fragments mi- grate. Purple arrows indicate the location were branched DNA molecules blocked at terB migrate. (F) Normalised enrichment of DNA in a 50 kb region containing the rrnH locus following RecA chromatin immunopre- cipitation (ChIP). Data were both normalized using a median of ratios scaling factor and smoothed using a 1 kb loess filter. Data for all panels is representative from one biological repeat. DISCUSSION (E and G) Analy- sis of DNA structure by 2D gel electrophoresis of a chromosome frag- ment located 55 Kb oriC proximal to lacZ and containing an ectopic terB site (ykgP::terB::eaeH) that blocks DNA replication proceeding towards oriC. (E) DNA isolated from RecBCD+ and recD cells containing the palindrome in lacZ following 1h of growth with SbcCD either expressed (SbcCD+) or repressed (SbcCD−). (G) DNA isolated from RecBCD+ and recD cells containing an I-SceI cleavage site (I-SceIcs+) in lacZ follow- ing 1h of growth with I-SceI expression either induced (I-SceI+) or not (I- SceI−). Green arrows indicate the location were linear DNA fragments mi- grate. Purple arrows indicate the location were branched DNA molecules blocked at terB migrate. (F) Normalised enrichment of DNA in a 50 kb region containing the rrnH locus following RecA chromatin immunopre- cipitation (ChIP). Data were both normalized using a median of ratios scaling factor and smoothed using a 1 kb loess filter. Data for all panels is representative from one biological repeat. Figure 5. DNA synthesis progressing towards the origin of replication is detected following induction of a DNA double-strand break. (A) Diagram Figure 5. DNA synthesis progressing towards the origin of replication is detected following induction of a DNA double-strand break. (A) Diagram Nucleic Acids Research, 2018, Vol. 46, No. 13 6679 we show here that it is deregulated in a recD mutant im- plying that fully functional RecBCD enzyme is required for good coordination of ends. It is interesting to note, given the appearance that prokaryotic and eukaryotic systems might operate differently, that mec1 (and mec1 tel1) mutants of Saccharomyces cerevisiae, that are defective for the DNA damage checkpoint, frequently become disomic for chro- mosome VIII in a reaction suppressed by overexpression of the DNA2 gene encoding an exonuclease/helicase impli- cated in end resection during recombination (33). The simi- larities of the roles of RecBCD and DNA2 in end resection and their proposed roles in limiting over-replication leading to aneuploidy are intriguing. of the DSB in a recD mutant strain consistent with aberrant replication (Figure 4C). Aberrant replication is confirmed by 2-D gel analysis of chromosomes containing an ectopic terB site, providing evidence of DNA replication forks pro- gressing towards the origin of replication at a site 55 Kb origin-proximal of the DSBR event (Figure 5C) that lead to DNA double-strand end formation and RecA binding adjacent to the rrnH operon (Figure 5D). recD mutants repair and re-cleave DNA at the site of a DNA palindrome From a genetic perspective a recD null mutant is defec- tive for the repair of palindrome-induced DSBs (Figure 1). This is surprising given the observation that in all other as- says performed to date recD mutants have been found to be recombination proficient (21). However, when we anal- ysed the behaviour of this mutant on pulsed field gels, we observed that the situation was more complicated. DNA fragments, un-cleaved DNA and branched molecules all accumulated in a recD mutant, following formation of a palindrome-induced break (Figure 3A). Fragments corre- sponding to palindrome cleavage could readily be detected as sharp bands by Southern blotting and these accumulated at 60 minutes of SbcCD induction. This was different from the situation observed for a recB mutant, where the cleaved DNA fragments were further degraded (presumably by one or more of the numerous nucleases in E. coli cells) between 30 and 60 min of induction creating a notably smeared band (13). This, coupled with the observation of little or no loss of the intact un-cleaved fragment and an accumulation of hybridising material in the wells of the gel suggested that repair and re-cleavage were ongoing in the recD mutant (Figure 6). That repair is indeed ongoing in the absence of RecD was confirmed by observing a loss of cleaved prod- ucts and un-cleaved DNA, accompanied by an accumula- tion of branched molecules in a recD ruvAB mutant under- going DSBR (Figure 3D). This repair and re-cleavage leads to RecA binding to DNA ends (Figure 3C), a stronger SOS response (Figure 2E) and an accumulation of extra DNA per cell that is readily detected by flow cytometry (Figure 2D). RecA binding reveals extensive unwinding by RecBC enzyme and DSBR at the rrnH locus consistent with replication fork breakage RecA ChIP-seq has revealed the pattern of RecA dis- tribution at a DSB catalysed by RecBC enzyme (Fig- ure 3C). RecA binding rises directly from the site of the double-strand break as expected for an enzyme lacking Chi-regulated exonuclease activity (16). The distribution of RecA binding at the lacZ locus where the palindrome- induced DSB is located is surprisingly similar in overall aspect to that observed in a RecBCD+ strain, including a correlation of peak DNA enrichment with the nearest active Chi sequences (in this case, an artificial array of three closely spaced Chi sequences (23) integrated 1.5 Kb either side of the DSB locus). This suggests that the un- winding catalysed by RecBC enzyme covers approximately the same genomic region as unwinding by RecBCD enzyme. The recD DSB-specific enrichment at the rrnH locus (Fig- ure 5D) and corresponding loss of DNA sequence by MFA analysis (Figure 4D) is consistent with recombination fol- lowing replication fork reversal as was previously shown to occur at inverted rrn operons (34). RecBCD enzyme co-ordinates the two ends at the site of DSBR The reason that recD mutants re-cleave their chromo- somes at the site of a palindrome, whereas recBCD+ cells do not, is unknown. However, it should be noted that un- coordinated repair is predicted to allow repair forks to pass though the palindrome in one (or other) direction as is the case for normal replication forks that lead to hairpin cleav- age by SbcCD. This raises the hypothesis that the lack of palindrome re-cleavage in recBCD+ cells reflects some as- pect of the coordination of DNA ends. The lack of co-ordination between the two ends of the DSB that we propose to occur at a higher frequency in recD mutants (Figure 6), leads to aberrant amplification of the chromosome in a reaction that would lead to three progeny chromosomes from two parental chromosomes if replica- tion were allowed to progress around the genome. However, over-replication extends only as far as the rrnE operon on the origin-proximal side of the break and to terB on the origin-distal side of the break. This means that the two fully replicated chromosomes are connected via a bridge of DNA extending for up to one half of the length of the genome. This bridge would become extended to twice the length of the entire chromosome by the next rounds of replication DISCUSSION It is interesting to note that the accumulation of DNA in a recD mutant following attempted DSBR is higher on the origin-distal side of the break than on the origin-proximal side of the break. This suggests a higher frequency of invasion of the origin proximal end than the origin-distal end followed by re-cleavage and re-invasion setting up multiple forks follow- ing each other towards the terminus. The reason for this bias towards recombination of the origin-proximal end is cur- rently unknown. However, this is the DNA end that needs to recombine in order to restore a replication fork progress- ing in the correct direction towards the terminus of the chro- mosome and the cell may favour this reaction (Figure 6). recD mutants repair and re-cleave DNA at the site of a DNA palindrome Aberrant DNA replication is detected in a recD mutant un- dergoing DSBR MFA of DNA undergoing palindrome-induced DSBR has revealed an accumulation of DNA sequence on both sides 6680 Nucleic Acids Research, 2018, Vol. 46, No. 13 Figure 6. Model for RecBCD-mediated control of DSB-induced DNA synthesis. In the presence of RecBCD, the two ends at the site of a DSB undergo coordinated invasion of the sister chromosome to generate two convergent replication forks that are responsible for the replacement of genetic information lost during end processing. If one of the convergent forks is established before the other and causes the second fork to be reversed, the extruded double- strand end is readily degraded by RecBCD to restore a replication fork. By contrast in the absence of RecD, RecBC either catalyses uncoordinated end invasion or, if end invasion is coordinated, cannot deal with the subsequent fork reversal of one of the convergent forks. Cleavage by RuvABC of the Holliday junction caused by fork reversal that restores one of the ends converting coordinated end invasion to uncoordinated end invasion. In either scenario of uncoordinated end invasion in the absence of RecD, the palindrome is re-cleaved, the chromosome is amplified and viability is lost. Downloaded from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on from https://academic.oup.com/nar/article/46/13/6670/5036844 by guest on 11 August 2020 Figure 6. Model for RecBCD-mediated control of DSB-induced DNA synthesis. In the presence of RecBCD, the two ends at the site of a DSB undergo coordinated invasion of the sister chromosome to generate two convergent replication forks that are responsible for the replacement of genetic information lost during end processing. If one of the convergent forks is established before the other and causes the second fork to be reversed, the extruded double- strand end is readily degraded by RecBCD to restore a replication fork. By contrast in the absence of RecD, RecBC either catalyses uncoordinated end invasion or, if end invasion is coordinated, cannot deal with the subsequent fork reversal of one of the convergent forks. Cleavage by RuvABC of the Holliday junction caused by fork reversal that restores one of the ends converting coordinated end invasion to uncoordinated end invasion. In either scenario of uncoordinated end invasion in the absence of RecD, the palindrome is re-cleaved, the chromosome is amplified and viability is lost. from oriC if the chromosome did not suffer further cleav- age events. Aberrant DNA replication is detected in a recD mutant un- dergoing DSBR This is ef- fectively uncoordinated recombination of the two ends al- beit mediated via the regeneration of one end following an initially coordinated reaction. In a wild type cell, RecBCD enzyme would rapidly digest the double-strand end gen- erated by RFR, regenerating a replication fork structure and thereby prevent uncoordinated recombination. Further work will be required to distinguish between these possibil- ities and to understand how RecBCD enzyme ensures the proper coordination of ends. from oriC if the chromosome did not suffer further cleav- age events. However, re-cleavage of the palindrome has been demonstrated during repair (see above) and new rounds of cleavage are expected to be associated with new rounds of replication. These further cleavage events and associated uncoordinated repair events will further interlink the chro- mosomes in complex ways that will impede accurate seg- regation. If the chromosomes are to segregate, any bridge DNA will need to be broken at some point along its length leading to chromosomes with partially replicated tails. Al- ternatively, segregation is blocked and this may be the major contributor to the cell death phenotype observed in the recD mutant. Indeed, recD cells show clear indication of defec- tive chromosome segregation (Figure 2A and Supplemen- tary Figure S2). Over twenty years ago Tokyo Kogoma and colleagues observed that recD mutants exhibited an elevated frequency of induced stable DNA replication (35). Ko- goma hypothesised that this was because of un-coordinated DSBR leading to chromosome over-replication (36). This is precisely what we have seen here, except that the initial replication associated with the uncoordinated repair does not extend all the way around the chromosome but is ef- fectively blocked by the rrnH operon and by terB and that further cleavage and uncoordinated repair events increase the complexity of the chromosomal products. first scenario, one end invades before the other because of a missing property of the RecBC enzyme. What this might be is unknown but it is intriguing to note that both monomeric and dimeric forms of RecBCD are evident following pu- rification of the protein (37). Furthermore the RecD2 he- licase of Deinococcus radiodurans dimerises (38) suggesting that the dimeric form of RecBCD might be mediated via the RecD subunit. Could the dimerization of two RecBCD monomers each engaged with one end mediate their coordi- nation? Aberrant DNA replication is detected in a recD mutant un- dergoing DSBR However, re-cleavage of the palindrome has been demonstrated during repair (see above) and new rounds of cleavage are expected to be associated with new rounds of replication. These further cleavage events and associated uncoordinated repair events will further interlink the chro- mosomes in complex ways that will impede accurate seg- regation. If the chromosomes are to segregate, any bridge DNA will need to be broken at some point along its length leading to chromosomes with partially replicated tails. Al- ternatively, segregation is blocked and this may be the major contributor to the cell death phenotype observed in the recD mutant. Indeed, recD cells show clear indication of defec- tive chromosome segregation (Figure 2A and Supplemen- tary Figure S2). Over twenty years ago Tokyo Kogoma and colleagues observed that recD mutants exhibited an elevated frequency of induced stable DNA replication (35). Ko- goma hypothesised that this was because of un-coordinated DSBR leading to chromosome over-replication (36). This is precisely what we have seen here, except that the initial replication associated with the uncoordinated repair does not extend all the way around the chromosome but is ef- fectively blocked by the rrnH operon and by terB and that further cleavage and uncoordinated repair events increase the complexity of the chromosomal products. first scenario, one end invades before the other because of a missing property of the RecBC enzyme. What this might be is unknown but it is intriguing to note that both monomeric and dimeric forms of RecBCD are evident following pu- rification of the protein (37). Furthermore the RecD2 he- licase of Deinococcus radiodurans dimerises (38) suggesting that the dimeric form of RecBCD might be mediated via the RecD subunit. Could the dimerization of two RecBCD monomers each engaged with one end mediate their coordi- nation? In the second scenario, the two ends invade in a co- ordinated manner but one assembles a replisome before the other. As the active replication fork approaches the inactive one, the latter undergoes a replication fork reversal (RFR) reaction. This might be catalysed by positive supercoiling ahead of the active fork (39) or by RuvAB action (40). Fol- lowing RFR, RuvC cleaves the Holliday junction that has formed and the second end is thus regenerated. This end is now free to recombine with the intact replicated DNA to generate the outward directed replication forks. Aberrant DNA replication is detected in a recD mutant un- dergoing DSBR It is therefore possible that we detected RecBC-mediated reactions in recD+ cells due to the multitude of DSBs created by I-SceI cleavage. An additional reason for the loss of viability of the recD mu- tant cells undergoing DSBR following palindrome cleavage is likely to be palindrome re-cleavage during repair. This will compound the problems associated with the chromosomal consequences of uncoordinated DSBR by creating further rounds of uncoordinated DSBR. be possible is attested to by the copying of entire yeast chromosome arms by break-induced replication (10,43). Alternatively, if a partially over-replicated chromosome is inherited this could lead to copy number variation by further processing to incorporate the over-replicated section of DNA into the genome. ACKNOWLEDGEMENTS We thank John Eykelenboom and Cheryl Lowans for pre- liminary data, Julia Mawer for construction of strains DL4193 and DL4196 and help with Southern blots, Rachael Stobbs for help with viability tests and PFGE experiments, and Edinburgh Genomics for the whole genome sequencing. Edinburgh Genomics is partly sup- ported through core grants from Natural Environment Re- search Council [R8/H10/56]; Medical Research Council [MR/K001744/1]; and Biotechnology and Biological Sci- ences Research Council [BB/J004243/1]. [ / / ] Author contributions: Conceptualization, M.A.W. and D.R.F.L.; Methodology, M.A.W. and D.R.F.L.; Investiga- tion, M.A.W., B.A., M.A.L. and A.M.M.H; Resources, D.R.F.L.; Writing – Original Draft, M.A.W., B.A. and D.R.F.L.; Writing – Review & Editing, M.A.W., B.A. and D.R.F.L.; Visualization, M.A.W. and D.R.F.L.; Supervi- sion, D.R.F.L.; Funding Acquisition, D.R.F.L. 3/6670/5036844 by guest on 11 August 2020 DATA AVAILABILITY The data files used for chromosome marker frequency anal- ysis have been deposited in the NCBI GEO database un- der accession number is GSE107973. The data files used for RecA ChIP-seq analysis have been deposited in the NCBI GEO database under accession number GSE107972. Supplementary Data are available at NAR Online. Supplementary Data are available at NAR Online. Supplementary Data are available at NAR Online. General conclusions and perspectives The coordination of the two DNA ends during DSBR is clearly de-regulated in a recD mutant implying that RecBCD enzyme plays an important role in the coordina- tion of ends during recombination in E. coli. The conse- quence of this de-regulation is the amplification of ∼30% of the E. coli chromosome. Were the replication forks caus- ing this over-replication not blocked by ter sites on one side and oppositely oriented rrn operons on the other, replica- tion would run around the chromosome generating three chromosomes from two parents. As it is, the two result- ing chromosomes are initially linked by a partially repli- cated chromosomal section. Further replication forks orig- inating from oriC will convert this structure to one where two circular chromosomes are joined by a double chromo- some length linker. Re-cleavage of the palindrome and fur- ther rounds of uncoordinated repair have the potential to increase the complexity of these chromosomal interconnec- tions. This situation will lead to problems for segregation that are likely to cause chromosome breakage and repair in the next cell cycle or simply to cell death. FUNDING Medical Research Council [G0901622 and MR/M019 160/1 to D.R.F.L.]; Human Frontiers Science Program [LT000927/2013 to M.A.W.]. Funding for open access charge: RC UK funding to University.l Medical Research Council [G0901622 and MR/M019 160/1 to D.R.F.L.]; Human Frontiers Science Program [LT000927/2013 to M.A.W.]. Funding for open access charge: RC UK funding to University.l Conflict of interest statement. None declared. Aberrant DNA replication is detected in a recD mutant un- dergoing DSBR In the second scenario, the two ends invade in a co- ordinated manner but one assembles a replisome before the other. As the active replication fork approaches the inactive one, the latter undergoes a replication fork reversal (RFR) reaction. This might be catalysed by positive supercoiling ahead of the active fork (39) or by RuvAB action (40). Fol- lowing RFR, RuvC cleaves the Holliday junction that has formed and the second end is thus regenerated. This end is now free to recombine with the intact replicated DNA to generate the outward directed replication forks. This is ef- fectively uncoordinated recombination of the two ends al- beit mediated via the regeneration of one end following an initially coordinated reaction. In a wild type cell, RecBCD enzyme would rapidly digest the double-strand end gen- erated by RFR, regenerating a replication fork structure and thereby prevent uncoordinated recombination. Further work will be required to distinguish between these possibil- ities and to understand how RecBCD enzyme ensures the proper coordination of ends. In Figure 6, we have suggested two stages at which the dis- coordination of DNA ends may occur. Either the two DNA ends in the recD mutant lack some property essential for coordination or one of the ends is regenerated in the recD mutant following an initially coordinated reaction. In the It surprised us that a recD null mutant was defective for DSBR at the site of a chromosomal palindrome when previ- Nucleic Acids Research, 2018, Vol. 46, No. 13 6681 Nucleic Acids Research, 2018, Vol. 46, No. 13 6681 ous assays for recombination by P1 transduction, Hfr mat- ing, bacteriophage lambda red gam crosses and sensitiv- ity to UV irradiation indicated no defect or even a hyper- rec phenotype (21). However, it is interesting to note that all of these reactions are effectively one-ended recombina- tion events where the coordination of two ends is clearly not required. By contrast, log-phase cells of a recD mu- tant have been shown to be sensitive to gamma irradia- tion, which is known to generate two-ended DNA breaks (41). These observations are fully consistent with an im- portant role for RecBCD enzyme in the coordination of two recombining ends. I-SceI cleavage of chromosomes also leads to two-ended DSBs. Aberrant DNA replication is detected in a recD mutant un- dergoing DSBR However, since I-SceI cleaves the DNA in a sequence dependent manner, it can poten- tially cleave all the sister and cousin chromosomes in a cell as well actively cleave the products of any repair re- action. This complicates the interpretation of any DSBR events observed. In contrast to DNA hairpin cleavage by SbcCD, we detected a loss in viability and a high rate of aberrant DSB-induced chromosomal replication in both RecBCD+ and recD mutant cells (Figures 1D and 5E). Ad- ditionally, RecBCD is maintained at a low copy number per cell (16), and RecD molecules are inactivated follow- ing interaction with Chi (42). It is therefore possible that we detected RecBC-mediated reactions in recD+ cells due to the multitude of DSBs created by I-SceI cleavage. An additional reason for the loss of viability of the recD mu- tant cells undergoing DSBR following palindrome cleavage is likely to be palindrome re-cleavage during repair. This will compound the problems associated with the chromosomal consequences of uncoordinated DSBR by creating further rounds of uncoordinated DSBR. ous assays for recombination by P1 transduction, Hfr mat- ing, bacteriophage lambda red gam crosses and sensitiv- ity to UV irradiation indicated no defect or even a hyper- rec phenotype (21). However, it is interesting to note that all of these reactions are effectively one-ended recombina- tion events where the coordination of two ends is clearly not required. 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https://openalex.org/W2888880859	https://hal.science/hal-02191838/file/diagne_22513.pdf	English	null	Comparative Analysis of Ranging Protocols for Localization by UWB in Outdoor	Wireless sensor network	2,018	cc-by	6,011	To cite this version: Salick Diagne, Thierry Val, Abdou Karim Farota, Bouya Diop. Comparative Analysis of Rang- ing Protocols for Localization by UWB in Outdoor. Wireless Sensor Network, 2018, 10 (5), pp.0. 10.4236/wsn.2018.105006. hal-02191838 Comparative Analysis of Ranging Protocols for Localization by UWB in Outdoor To cite this version: Salick Diagne, Thierry Val, Abdou Karim Farota, Bouya Diop. Comparative Analysis of Rang- ing Protocols for Localization by UWB in Outdoor. Wireless Sensor Network, 2018, 10 (5), pp.0. 10.4236/wsn.2018.105006. hal-02191838 HAL Id: hal-02191838 https://hal.science/hal-02191838v1 Submitted on 23 Jul 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Keywords Outdoor Location, UWB, Ranging, ToF, TWR, DecaWiNo This is an author’s version published in: http://oatao.univ-toulouse.fr/22513 To cite this version: Diagne, Salick and Val, Thierry and Farota, Abdou Karim and Diop, Bouya Comparative Analysis of Ranging Protocols for Localization by UWB in Outdoor. (2018) Wireless Sensor Network, 10 (5). ISSN 1945-3078 Official URL DOI : https://doi.org/10.4236/wsn.2018.105006 Open Archive Toulouse Archive Ouverte OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible OATAO is an open access repository that collects the work of Toulouse researchers and makes it freely available over the web where possible Any correspondence concerning this service should be sent to the repository administrator: tech-oatao@listes-diff.inp-toulouse.fr Abstract The rapid evolution of technology in the field of wireless telecommunications and micro components using MEMS technologies (Micro-electromechanical systems) has contributed to the expansion and rapid development of wireless sensor networks (WSN). This rapid development has contributed to the ap- pearance of sensor and actuator networks (WSAN) or even to the Internet of Things with DL-IoT (Device Layer-Internet of Things). This rapid evolution of WSN is due to the enthusiasm generated by this last in industry and re- search. This new technology is used in several applications, particularly in the outdoor location of communicating nodes. The process of distance calcula- tion between nodes (ranging) is a primordial phase for a precise location of these nodes. This paper presents the result of measurements does with three ranging protocols (TWR, TWR_Skew and SDS-TWR) implemented on De- caWiNo nodes. DecaWiNo nodes use the Ultra-Wide Band (UWB) radio links, proposed by the IEEE 802.15.4 standard amendment of the year 2007, which provides a high-performance ranging by ToF (Time of Flight) [1] [2]. The results are very promising with precision errors of the order of 50 cm over 20 meters. Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). http://creativecommons.org/licenses/by/4.0/ Open Access Open Access Comparative Analysis of Ranging Protocols for Localization by UWB in Outdoor Salick Diagne1, Thierry Val2, Abdou Karim Farota1, Bouya Diop1 1Laboratoire des Sciences de l’Atmosphère et des Océans, UFR SAT/UGB, Saint-Louis, Sénégal 2Institut de Recherche en Informatique de Toulouse, Université de Toulouse, Blagnac, France Salick Diagne1, Thierry Val2, Abdou Karim Farota1, Bouya Diop1 1Laboratoire des Sciences de l’Atmosphère et des Océans, UFR SAT/UGB, Saint-Louis, Sénégal 2Institut de Recherche en Informatique de Toulouse, Université de Toulouse, Blagnac, France Copyright © 2018 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY 4.0). h // i /li /b /4 0 1. Introduction We notice in recent years, agriculture increasingly oriented towards a wide- spread use of new technologies. We no longer talk about the mechanization of agriculture, but rather a smart agriculture. This type of agriculture uses WSAN technology to improve crop yields, in particular by controlling the humidity DOI: 10.4236/wsn.2018.105006 May 30, 2018 S. Diagne et al. level of the plants [3] or for a much more precise irrigation [4]. Its field of appli- cation also covers intrusion detection, or more recently the mobile target track- ing, especially with military security. Our goal is to use this technology in mobile target tracking but applied to ag- riculture for crop protection. This idea was born from an observation made from the destruction of crops in the paddy fields of West Africa, particularly those of Senegal. Indeed, the destruction of agricultural crops by grain-eating birds has always been a fundamental problem, especially in West Africa. According to [5], these birds, such as quelea, the golden sparrows, village weaver with a strong beak, cause very serious damage that can be estimated each year to several hundred thousand tons. They attack rice, maize, sorghum and wheat by eating the mature and milky grains. This is how we asked ourselves this fundamental question that guided our ap- proach: Why not use the advantages offered by this new technology (DL-IoT) to help these farmers to protect their crops without harming the birds? However, to achieve this, one of the first functions that this network must ac- complish is to be able to efficiently locate its own nodes. This first function of our network is also the subject of this document in which we make the compara- tive study of some protocols of distance calculation between nodes (ranging) by ToF based on the UWB, in order to find the best protocol suited for our applica- tion type. This work emphasizes the metric performances of DecaWiNo nodes in outdoor. In fact, these performances were until then studied in indoor [6], thus this result obtained will complete the DecaWiNos metric study with the main protocols using the ToF for a more general use of these nodes (Indoor and Outdoor). So, after this introduction, we will first present the most traditional ranging protocols namely TWR, TWR_Skew and SDS-TWR. 1. Introduction In the following we will present in Section 3 titled Outdoor measurements, the material used for meas- urements in particular the DecaWiNos nodes, the procedures and measurement results of the various protocols as well as their interpretation. Then in Section 4, we will discuss the results obtained. And finally, the conclusion will allow us to emphasize the possibilities of using our results as well as the research perspec- tives arising from this work. 2. The Ranging Protocols For most sensor and actuator network applications, the event detected by a node is useful only if the information about its geographical location is provided. For this, the network needs to locate its nodes. So many algorithms and localization techniques in outdoor have been developed. These algorithms and localization techniques use a variety of communication technologies, offering to users adapted location systems according to the criteria required by the different S. Diagne et al. DL-IoT type applications. These criteria are often the range of communication, the transmission rate or the energy consumption. Technologies such as infrared, Bluetooth, Zigbee or UWB are used in this sense by localization systems. The most frequent localization techniques in literature can be classified into two groups: Range-Free localization and Range-Based localization. The Range-Free technique, like those of the DV-Hop family, is based on to- pology and connectivity information, assuming an isotropic network where the number of jumps between the nodes is proportional to their distance. But this solution is not very accurate (because of the malfunctions and the not guaran- teed network connectivity). The Range-Based localization, uses the distance (or angle) measured between two nodes (Ranging) to determine the position of a node. There are several techniques to measure the distance (or angle) between two nodes: The Time of Flight (ToF), the Time Difference of Arrival (TDoA), the Angle of Arrival (AoA), and the Received Signal Strength Indicator (RSSI). In ToF or ToA (Time of Arrival), we exploit the relationship between distance travelled by the signal and the time taken. For this technique, synchronization between transmitter and receiver is often required. The TDoA uses the same principle as ToA with some differences to measure the distance between two close nodes. It uses the time difference taken by two signals sent by the same node and whose propagation speed is different. In gen- eral, we use ultrasound (V 340 m/s in air at 15˚C) or sound waves (C 3.108 m/s) to have this difference. This technique has drawbacks related to the energy consumption (because of the two signals emitted), the short range and its limited use in outdoor due to ultrasound for example. The technique based on the AoA allows the location of a node by measuring the arrival angle of a signal. In this technique, the nodes measuring the angle are equipped with small antennas or ultrasonic receiver. 2. The Ranging Protocols The receiving node uses the arrival time or signal phase to calculate the arrival angle. These ultrasonic antennas and receivers (imposing small distances between receiving nodes) constitute the major disadvantage in terms of cost and size for this method. Finally, for the RSSI technique that uses the physical characteristic of the radio channel (the RSSI signal strength). In theory more the distance between two nodes increases more the signal is attenuated (the RSSI power indicator de- creases). Thus, it is possible for a receiving node to calculate the distance that separates it from a transmitting node based on the power of the received signal. This method has an advantage over the cost since the nodes are already equipped with RF devices. But it is not very precise compared to other methods. Because of its sensitivity to noise, interference, asymmetry of the communica- tion link (no-conformity of the RSSI), obstacles and types of antennas capable caused errors exceeding 50% of the range of the communication device [7]. This method has an advantage over the cost since the nodes are already equipped with RF devices. But it is not very precise compared to other methods. Because of its sensitivity to noise, interference, asymmetry of the communica- tion link (no-conformity of the RSSI), obstacles and types of antennas capable caused errors exceeding 50% of the range of the communication device [7]. Although requiring a perfect synchronism of the transmitting and receiving S. Diagne et al. nodes, the technique based on the ToF is much more precise than the other techniques especially with the use of the UWB technology (ensuring an accept- able synchronization). Thus, several works propose methods of distance calculation between nodes (Ranging) based on the ToF. These methods of calculation often called Ranging protocols are presented in the following sections. 2.1. TWR (To Way Ranging) This is the simplest protocol using two-node messages and taking the clock from one of the nodes as a reference. Because most of the time, nodes in communica- tion are often asynchronous. The TWR protocol is based on a set of three mes- sages, two of which, (START and ACK) necessary for stamping and obtaining time-of-flight measurement information (Figure 1). In this protocol, the node A, initiator of the exchange sends its first frame (START) and marks its time of emission t1. On receipt of this frame, the node B in turn marks the reception time t2 of this frame and after a certain time sends an acknowledgment frame (ACK) while marking its sending time t3. The node A, on receipt of the ACK frame, marks the reception time t4. The four time stamps are collected on the node A side after receiving the DATA_REPLY frame containing the tags t2 and t3 of the node B. This last frame will allow node A to calculate the flight time noted in Equation (1) and distance in Equation (2). 4 1 3 2 TWR ToF 2 t t t t (1) 8 TWR Distance ToF , with 3.10 C C \| (2) 4 1 3 2 TWR ToF 2 t t t t (1) (1) (2) (1) 8 TWR Distance ToF , with 3.10 C C \| (2) Figure 1. TWR protocol performing ToF measurement between two “asynchronous” nodes. Figure 1. TWR protocol performing ToF measurement between two “asynchronous” nodes. S. Diagne et al. The principal disadvantage of this protocol is the drift of the clocks which causes temporal imprecision. Indeed, the two clocks of the nodes A and B are not based on the same quartz, they cannot be perfectly synchronous. Thus, an overestimation or underestimation of the measurement of the flight time could lead to an absurd location of the nodes. 2.2. TWR_Skew The TWR_Skew is a protocol based on the simple TWR, it is presented in [8]. Its objective was to evaluate the impact of TWR ranging accuracy in normal traffic by introducing a variable artificial delay in order to compensate for imprecision due to delay. This delay introduced between the messages START and ACK ac- tually represents the delay introduced by the usual traffic especially the method of access to the medium. The authors of [8] propose a correction of the introduced error, based on the parameter k which represents the ratio of the frequency fB of the clock of the node B and the frequency fA of the clock of the node A. By introducing this pa- rameter k into Equation (1), this results in a new general ToF equation: 4 1 3 2 TWR_Skew ToF , with 1 2 t t k t t k \| (3) (3) The communication modules that we use with DecaWiNo and also used in [8] offer functionality, the Clock Offset evaluating the difference in frequency be- tween the two clocks. This difference called Skew is the origin of the name of this protocol. It is affirmed in [8] that this correction makes it possible to correct both the drift errors introduced by the temperature variations and those due to delays between messages (Figure 2). 2.3. The SDS-TWR Protocol (Symmetrical Double-Side Two-Way Ranging) In order to reduce or eliminate the error introduced by the clock drift, another protocol symmetries the ranging session by adding an additional message to the classical session of the TWR protocol: this is the SDS-TWR protocol. 1) The Double-sided component: The protocol is based on two symmetrical sessions of the TWR protocol (TWRA−B and TWRB−A). 4 1 3 2 A B A B : TWR ToF 2 t t t t (4) 6 3 5 4 B A B A : TWR ToF 2 t t t t (5) (4) (5) 2) The Symmetrical component: the response time between the two nodes is assumed to be identical (TR−A = TR−B, with TR = Response Time) (Figure 3). Each node marks its own transmit and receive stamps. All the timestamps will be collected on the side of the node A after the reception of the DATA_REPLY S. Diagne et al. Figure 2. Ranging protocol session TWR_Skew. Figure 3. SDS-TWR protocol performing ToF measurement between two “asynchro nous” nodes. Figure 2. Ranging protocol session TWR_Skew. Figure 2. Ranging protocol session TWR_Skew. Figure 2. Ranging protocol session TWR_Skew. Figure 3. SDS-TWR protocol performing ToF measurement between two “asynchro- nous” nodes. Figure 3. SDS-TWR protocol performing ToF measurement between two “asynchro- nous” nodes. message containing the instants t2, t3 and t6 of the node B. Calculations of flight time and distance are obtained by using Equation (6) and Equation (7). 4 1 3 2 6 3 5 4 SDS TWR ToF 4 t t t t t t t t (6) SDS TWR Distance ToF C (7) (6) (7) Because of the frequency shift at the nodes, the return times for the response of A and B are evaluated differently. An average of two “round trip” times of A S. Diagne et al. and B will allow reducing the error induced ranging. This specificity of the pro- tocol will therefore reduce the difference between the actual distance and that which will be measured by the SDSTWR protocol without completely eliminat- ing it [8]. 3. Outdoor Measurements Often, simple analyses based on simulation software are enough to provide a re- sult on the performance of a given system. However, these programs are based on too idealistic models that do not often consider the imperfections and physi- cal realities of the material and the measurement environment. When the system to be studied is a WSAN, the fact that these parameters are not considered may lead to a different operation from the deployment than that provided by the simulation software. Thus, certainly, the best approach would be to perform analyses on real nodes (sensors and actuators) and in a real physical environment. This explains why our analysis approach is based on measurements with real nodes. 3.1.1. DecaWiNo Node DecaWiNo is part of the family of WiNo nodes (Wireless Node) [9] developed at IRIT by the RMESS team. The WiNo family is based on the Arduino environ- ment and its components are developed in Open Hardware, allowing to setting up a wide variety of WiNos (see Figure 5). Table 1 summarizes the principal features of the developed WiNo nodes. As we said earlier in this study, we will use DecaWiNo. This type of WiNo is composed of an Arduino board (Teensy 3.2) and the DecaWave DWM 1000 module with a UWB transceiver and an antenna. For node management, the DecaDuino library was developed by the RMESS team in the Arduino environ- ment (Figure 6). 3.3. Measurement Results We present in the following sections the results obtained with the various pro- tocols mentioned as well as their graphical representation. 3.2. Measurement Procedures For each of the three protocols, we carried out measurements on 19 points, and on each point 100 measurements were collected which makes a total of 5700 measurements. Measurements are made in external environment, considering the two nodes in line of sight (LOS) and always starting for each protocol with a taking at 0.5 m. After this, we start from 1 m to 5 m in steps of 1 m. Then 5 m to 30 m in steps of 2 m and always pass by the point 20 m. 3.1. Description of Measurement Equipment For the measurements, we use two DecaWiNo nodes: the first fixed and located at a height of 1.5 m from the ground, the second always at the same height is linked to a mobile pole. The pole here serves only to vary the distance between our two nodes. See Figure 4. Figure 4. Principle of taking measurements. (a) Fixed DecaWiNo; (b) DecaWiNo carried by the mobile pol. Figure 4. Principle of taking measurements. (a) Fixed DecaWiNo; (b) DecaWiNo carried by the mobile pol. S. Diagne et al. 3.3.1. Ranging by TWR Simple We summarize in Table 2 the measurements made with the TWR protocol. However, with the number of takes made: 100 for each point, we will only give Figure 5. The WiNos nodes. (a) WiNoRF22; (b) TeensyWiNo; (c) DecaWiNo and (d) Wi- NoIR. Figure 5. The WiNos nodes. (a) WiNoRF22; (b) TeensyWiNo; (c) DecaWiNo and (d) Wi- NoIR. S. Diagne et al. g Table 1. Characteristics of WiNos developed [8]. WiNoRF22 TeensyWiNo DecaWino WiNoVW WiNoLoRa WiNoIR CPU/RAM/Flash ARM Cortex M4 (32 bits) 72 MHz, 64 kB RAM, 256 Kb Flash (PJRC Teensy 3.1) Transceiver HopeRF RFM22b: 200 - 900 MHz, 1 - 125 kbps, GFSK/FSK/OOK, +20 dBm DWM 1000: UWB IEEE 802.15.4 Variés RFM95 RF22 + Diodes IR Librairie RadioHead DecaDuino VirtualWire RadioHead IrRemote Capteurs Température, Luminosité Température, Luminosité, Baromètre, Magnétomètre, Accéléromètre, Gyromètre Température, Luminosité Usage WSN, IoT IoT avec Ranging, localisation en intérieur, courte portée Communication très bas débit sur médiums non conventionnels Longue portée, très bas débit Disponibilité DIY snootlab.com DIY (Do It Yourself, à assembler soi-même) Table 2. Summary of TWR measures (m). Real Distance Average distance TWR Average error Minimum Error Maximum error 0.5 0.65 0.15 0.07 0.19 1 1.19 0.19 0.15 0.23 2 2.1591 0.1591 0.12 0.19 3 3.14 0.14 0.09 0.2 4 4.12 0.12 0.06 0.16 5 5.20 0.20 0.16 0.25 7 7.04 0.04 −0.01 0.07 9 9.00 −0.00 −0.05 0.03 11 10.9481 −0.05 −0.1 0.03 13 12.87 −0.13 −0.19 −0.08 15 14.72 −0.28 −0.33 −0.23 17 16.62 −0.38 −0.43 −0.32 19 18.47 −0.53 −0.57 −0.48 20 19.49 −0.51 −0.56 −0.46 22 21.48 − 0.52 −0.56 −0.48 24 23.33 −0.67 − 0.74 −0.58 26 25.21 −0.79 −0.84 −0.71 28 27.08 −0.92 −0.98 −0.86 30 28.89 −1.11 −1.18 −1.05 Table 1. Characteristics of WiNos developed [8]. Table 1. Characteristics of WiNos developed [8]. WiNoRF22 TeensyWiNo DecaWino WiNoVW WiNoLoRa WiNoIR CPU/RAM/Flash ARM Cortex M4 (32 bits) 72 MHz, 64 kB RAM, 256 Kb Flash (PJRC Teensy 3.1) Transceiver HopeRF RFM22b: 200 - 900 MHz, 1 - 125 kbps, GFSK/FSK/OOK, +20 dBm DWM 1000: UWB IEEE 802.15.4 Variés RFM95 RF22 + Diodes IR Librairie RadioHead DecaDuino VirtualWire RadioHead IrRemote Capteurs Température, Luminosité Température, Luminosité, Baromètre, Magnétomètre, Accéléromètre, Gyromètre Température, Luminosité Usage WSN, IoT IoT avec Ranging, localisation en intérieur, courte portée Communication très bas débit sur médiums non conventionnels Longue portée, très bas débit Disponibilité DIY snootlab.com DIY (Do It Yourself, à assembler soi-même) Table 2. 3.3.1. Ranging by TWR Simple Summary of TWR measures (m). Real Distance Average distance TWR Average error Minimum Error Maximum error 0.5 0.65 0.15 0.07 0.19 1 1.19 0.19 0.15 0.23 2 2.1591 0.1591 0.12 0.19 3 3.14 0.14 0.09 0.2 4 4.12 0.12 0.06 0.16 5 5.20 0.20 0.16 0.25 7 7.04 0.04 −0.01 0.07 9 9.00 −0.00 −0.05 0.03 11 10.9481 −0.05 −0.1 0.03 13 12.87 −0.13 −0.19 −0.08 15 14.72 −0.28 −0.33 −0.23 17 16.62 −0.38 −0.43 −0.32 19 18.47 −0.53 −0.57 −0.48 20 19.49 −0.51 −0.56 −0.46 22 21.48 − 0.52 −0.56 −0.48 24 23.33 −0.67 − 0.74 −0.58 26 25.21 −0.79 −0.84 −0.71 28 27.08 −0.92 −0.98 −0.86 30 28.89 −1.11 −1.18 −1.05 Table 2. Summary of TWR measures (m). Real Distance Average distance TWR Average error Minimum Error Maximum error 0.5 0.65 0.15 0.07 0.19 1 1.19 0.19 0.15 0.23 2 2.1591 0.1591 0.12 0.19 3 3.14 0.14 0.09 0.2 4 4.12 0.12 0.06 0.16 5 5.20 0.20 0.16 0.25 7 7.04 0.04 −0.01 0.07 9 9.00 −0.00 −0.05 0.03 11 10.9481 −0.05 −0.1 0.03 13 12.87 −0.13 −0.19 −0.08 15 14.72 −0.28 −0.33 −0.23 17 16.62 −0.38 −0.43 −0.32 19 18.47 −0.53 −0.57 −0.48 20 19.49 −0.51 −0.56 −0.46 22 21.48 − 0.52 −0.56 −0.48 24 23.33 −0.67 − 0.74 −0.58 26 25.21 −0.79 −0.84 −0.71 28 27.08 −0.92 −0.98 −0.86 30 28.89 −1.11 −1.18 −1.05 Table 2. Summary of TWR measures (m). Real Distance Average distance TWR Average error Minimum Error Maximum error S. Diagne et al. Figure 6. Components of DecaWiNo. Figure 6. Components of DecaWiNo. Figure 6. Components of DecaWiNo. the average value measured by the TWR protocol for each point as well as the average, minimum and maximum errors. The representation of the error according to the real distance (Figure 7) shows in a general way that the absolute average error varies from 0 to 1.10 m with extremums ranging from 1 cm to 1.18 m over a real distance of 0.5 to 30 m. In particular, for real distances with less than 14 m, the average error does not exceed 20 cm and the maximum error is 25 cm. These average and maximum absolute errors are around 50 cm over distances between 14 and 22 meters. And for distances between 22 and 30 meters, these values are around 60 centimetres for the average value and 58 to 98 centimetres for the extremums. 3.3.1. Ranging by TWR Simple And finally, we find that the error committed by the TWR is greater than 1 meter for the real distances greater than or equal to 30 meters. We also notice that for distances of less than 9 meters, the TWR overestimates the actual distance. 3.3.2. Ranging by TWR_Skew Under the same conditions and measurement parameters as those of the TWR, we performed measurements with the TWR_Skew protocol. The results are summarized in Table 3 and graphically represented in Figure 8. We note here that the average error varies generally from 20 cm to 1.5 m with absolute extreme values ranging from 37 cm to 1.63 m over real distances from 0.5 to 30 meters. In particular, it can be seen on the one hand that for real distances of less than 13 meters the absolute errors (means and extremums) do not exceed 50 cm. And on the other hand, that the absolute average error evolves rapidly from 50 cm to 1.50 m with absolute extremums of 60 cm to 1.63 m over distances between 13 and 30 meters. We can also notice that with the TWR_Skew protocol, the actual distances are in general underestimates. 3.3.3. Ranging by SDS_TWR Real Distance Average distance TWR Average error Minimum error Maximum error 0.5 0.29 −0.21 −0.25 −0.17 1 0.76 −0.24 −0.27 −0.21 2 1.7743 −0.2257 −0.26 −0.19 3 2.76 −0.24 −0.28 −0.19 4 3.74 −0.26 −0.3 −0.21 5 4.67 −0.33 −0.36 −0.3 7 6.64 −0.36 −0.39 −0.33 9 8.57 −0.43 −0.47 −0.39 11 10.5078 −0.49 −0.56 −0.38 13 12.43 −0.57 −0.62 −0.53 15 14.32 −0.68 −0.71 −0.64 17 16.22 −0.78 −0.82 −0.73 19 18.11 −0.89 −0.92 −0.86 20 19.09 −0.91 −0.94 −0.85 22 20.94 −1.06 −1.10 −1.03 24 −22.97 −1.04 −1.15 −0.87 26 24.68 −1.32 −1.43 −1.21 28 28.63 −1.25 −1.30 −1.19 30 28.63 −1.37 −1.42 −1.31 these absolute average errors evolve rapidly from 55 cm for a real distance of 13 meters to 1.35 m for a real distance of 30 meters, with extremums ranging from 53 cm to 1.43 m respectively. The SDS-TWR protocol always gives an underes- Figure 8. Representation of TWR Skew error depending on the distance. Table 4. Summary of SDS-TWR measurements (m). Real Distance Average distance TWR Average error Minimum error Maximum error 0.5 0.29 −0.21 −0.25 −0.17 1 0.76 −0.24 −0.27 −0.21 2 1.7743 −0.2257 −0.26 −0.19 3 2.76 −0.24 −0.28 −0.19 4 3.74 −0.26 −0.3 −0.21 5 4.67 −0.33 −0.36 −0.3 7 6.64 −0.36 −0.39 −0.33 9 8.57 −0.43 −0.47 −0.39 11 10.5078 −0.49 −0.56 −0.38 13 12.43 −0.57 −0.62 −0.53 15 14.32 −0.68 −0.71 −0.64 17 16.22 −0.78 −0.82 −0.73 19 18.11 −0.89 −0.92 −0.86 20 19.09 −0.91 −0.94 −0.85 22 20.94 −1.06 −1.10 −1.03 24 −22.97 −1.04 −1.15 −0.87 26 24.68 −1.32 −1.43 −1.21 28 28.63 −1.25 −1.30 −1.19 30 28.63 −1.37 −1.42 −1.31 these absolute average errors evolve rapidly from 55 cm for a real distance of 13 meters to 1.35 m for a real distance of 30 meters, with extremums ranging from 53 cm to 1.43 m respectively. The SDS-TWR protocol always gives an underes- i d l f h l di Figure 8. Representation of TWR Skew error depending on the distance. Figure 8. Representation of TWR Skew error depending on the distance. p g Table 4. Summary of SDS-TWR measurements (m). 3.3.3. Ranging by SDS_TWR Still in the same conditions as previously experiments with TWR and TWR_Skew protocols, we performed measurements with the SDS_TWR protocol. The re- sults obtained are recorded in Table 4 and represented in Figure 9. The average absolute error varies here from 20 cm to 1.30 m with extremums of 17 cm to 1.43 m over distances of 0.5 to 30 meters. S. Diagne et al. Figure 7. Representation of the TWR error depending the distance. Figure 7. Representation of the TWR error depending the distance. Figure 7. Representation of the TWR error depending the distance. Figure 7. Representation of the TWR error depending the distance. Table 3. Summary of TWR_Skew Measures (m). Table 3. Summary of TWR_Skew Measures (m). Real Distance Average distance TWR Average error Minimum Error Maximum error 0.5 0.26 −0.24 −0.31 −0.19 1 0.80 −0.20 −0.28 −0.13 2 1.7757 −0.2243 −0.34 −0.15 3 2.77 −0.23 −0.37 −0.12 4 3.76 −0.24 −0.33 −0.13 5 4.83 −0.17 −0.26 −0.1 7 6.66 −0.34 −0.43 −0.26 9 8.61 −0.39 −0.50 −0.30 11 10.6028 −0.40 −0.48 −0.31 13 12.48 −0.52 −0.60 −0.44 15 14.32 −0.68 −0.80 −0.59 17 16.26 −0.74 −0.86 −0.63 19 18.09 −0.91 −0.99 −0.78 20 19.11 −0.89 −1.00 −0.81 22 21.09 −0.91 −1.03 −0.83 24 22.94 −1.06 −1.23 −0.92 26 24.83 −1.17 −1.26 −1.07 28 28.50 −1.32 −1.46 −1.18 30 28.50 −1.50 −1.63 −1.36 Table 3. Summary of TWR_Skew Measures (m). Real Distance Average distance TWR Average error Minimum Error Maximum error 0.5 0.26 −0.24 −0.31 −0.19 1 0.80 −0.20 −0.28 −0.13 2 1.7757 −0.2243 −0.34 −0.15 3 2.77 −0.23 −0.37 −0.12 4 3.76 −0.24 −0.33 −0.13 5 4.83 −0.17 −0.26 −0.1 7 6.66 −0.34 −0.43 −0.26 9 8.61 −0.39 −0.50 −0.30 11 10.6028 −0.40 −0.48 −0.31 13 12.48 −0.52 −0.60 −0.44 15 14.32 −0.68 −0.80 −0.59 17 16.26 −0.74 −0.86 −0.63 19 18.09 −0.91 −0.99 −0.78 20 19.11 −0.89 −1.00 −0.81 22 21.09 −0.91 −1.03 −0.83 24 22.94 −1.06 −1.23 −0.92 26 24.83 −1.17 −1.26 −1.07 28 28.50 −1.32 −1.46 −1.18 30 28.50 −1.50 −1.63 −1.36 As for the TWR_Skew, we also note that for distances less than 13 meters the absolute errors (means and extremums) do not exceed 50 cm. Beyond 13 meters, S. Diagne et al. g Figure 8. Representation of TWR Skew error depending on the distance. Table 4. Summary of SDS-TWR measurements (m). 3.4. Discussion The observation of the average errors shows that the errors made by the TWR_Skew and SDS_TWR protocols are quite close. Even though the TWR_Skew error is slightly smaller for distances less than 28 meters, the error committed by these two protocols exceeds 50 cm for the real distances greater than 13 meters. On the other hand, for the TWR protocol, the error made here is much smaller, going up to an absolute distance of 50 cm from that made by the TWR_Skew protocol. In addition, for this protocol, the error does not exceed 50 cm for distances less than or equal to 22 meters (see Figure 10). This result ob- tained with the TWR protocol seems very interesting especially for applications where the distance between the nodes does not exceed 22 meters and a precision of the order of 20 cm (for distances less than 14 meters) or 50 cm maximum (for distances between 14 and 22 meters). Thus, given the number of measurements taken per point, we can say that comparatively the TWR ranging protocol is better than the TWR_Skew and SDS_TWR ranging protocols in outdoor; even though these last two protocols have been established to correct the mistakes of TWR. But the validation tests of the correction of TWR errors by the TWR_Skew and SDS_TWR have been done until then in indoor environment with a lot of reflections because of the walls and ceiling, and low ranges not exceeding 10 meters. 3.3.3. Ranging by SDS_TWR Real Distance Average distance TWR Average error Minimum error Maximum error 0.5 0.29 −0.21 −0.25 −0.17 1 0.76 −0.24 −0.27 −0.21 2 1.7743 −0.2257 −0.26 −0.19 3 2.76 −0.24 −0.28 −0.19 4 3.74 −0.26 −0.3 −0.21 5 4.67 −0.33 −0.36 −0.3 7 6.64 −0.36 −0.39 −0.33 9 8.57 −0.43 −0.47 −0.39 11 10.5078 −0.49 −0.56 −0.38 13 12.43 −0.57 −0.62 −0.53 15 14.32 −0.68 −0.71 −0.64 17 16.22 −0.78 −0.82 −0.73 19 18.11 −0.89 −0.92 −0.86 20 19.09 −0.91 −0.94 −0.85 22 20.94 −1.06 −1.10 −1.03 24 −22.97 −1.04 −1.15 −0.87 26 24.68 −1.32 −1.43 −1.21 28 28.63 −1.25 −1.30 −1.19 30 28.63 −1.37 −1.42 −1.31 these absolute average errors evolve rapidly from 55 cm for a real distance of 13 meters to 1.35 m for a real distance of 30 meters, with extremums ranging from 53 cm to 1.43 m respectively. The SDS-TWR protocol always gives an underes- timated value of the real distance. S. Diagne et al. Figure 9. Representation of SDS_TWR error depending on the distance. Figure 9. Representation of SDS_TWR error depending on the distance. 4. Conclusion In this paper, we have implemented three ToF ranging protocols (TWR, TWR_Skew and SDS-TWR) on DecaWiNo nodes incorporating UWB technol- ogy. These nodes allowed us to make ranging measurements whose graphical S. Diagne et al. Figure 10. Error representation of different protocols depending on the real distance. -1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0 0.2 0.4 0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 Error (m) Real distance (m) TWR TWR_Skew SDS_TWR Figure 10. Error representation of different protocols depending on the real distance. interpretation allowed to showing the individual performances of the various protocols. These performances offer us several perspectives, first of all on the integration of the TWR protocol on our application whose maximum distance between nodes is 20 meters; since this protocol is the one that better estimates the dis- tance between nodes among the three studied in this document. But also, per- spectives on the search for mechanisms that can help reduce the error commit- ted by the TWR protocol, as well as the study of the impact of the error made by this protocol on the accuracy of mobile target location algorithms. Acknowledgements The authors would like to thank CEA-MITIC of the UFR SAT at the University Gaston Berger of Saint-Louis/Senegal for his financial support for the travel to Toulouse (in France) and the publication of this work. Conflicts of Interest The authors declare no conflicts of interest regarding the publication of this pa- per. References [1] Kobenan IgnaceKossonou (2014) Étude d’un système de localisation 3-D haute précision basé sur les techniques de transmission Ultra Large Bande à basse consommation d’énergie pour les objets mobiles communicants. Electronique Un- iversité de Valenciennes et du Hainaut-Cambrésis, Valenciennes. [2] Fofana, N.I., Van Bossche, A. and Val, T. (2014) Etude des couches MAC dédiées à l’UWB. 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https://openalex.org/W4200256472	https://jurnal.staithawalib.ac.id/index.php/syiar/article/download/37/48	Indonesian	null	Peran Dan Fungsi Public Relation Dalam Mempertahankan Citra Positif Portal Berita Online Antaranews.Com	Syiar	2,021	cc-by	3,874	PERAN DAN FUNGSI PUBLIC RELATION DALAM MEMPERTAHANKAN CITRA POSITIF PORTAL BERITA ONLINE ANTARANEWS.COM (Studi Kasus LKBN ANTARA Biro Lampung) Dede Mercy Rolando1, Tri Adellia2, Nuril Maulana Alifia Aziz3, Galuh Dwi Kartika Wicaksono4 Universitas Islam Negeri Raden Intan Lampung 1dedemercyrolando@gmail.com, 2triadellia10@gmail.com, 3nurilmaulana67@gmail.com, 4galuhdwi1305@gmail.com Universitas Islam Negeri Raden Intan Lampung 1dedemercyrolando@gmail.com, 2triadellia10@gmail.com, 3nurilmaulana67@gmail.com, 4galuhdwi1305@gmail.com SYIAR \| Jurnal Komunikasi dan Penyiaran Islam Volume 1 (2) (2021) 77-86 e-ISSN 2808-7941 https://jurnal.staithawalib.ac.id/index.php/syiar/article/view/37 DOI : https://doi.org/10.54150/syiar.v1i2.37 Kata Kunci: Public Relation, Antaranews.com, Citra Positif, Peran dan Kedudukan ABSTRAK Penelitian ini bertujuan untuk mengetahui bagaimana peran dan fungsi Publik Relation di LKBN ANTARA Biro Lampung dalam mempertahan citra positif dimata publik. Guna mengkaji penelitian ini, penulis menggunakan metode penelitian kualitatif deskriftif dengan pengumpulan data berupa wawancara, dokumentasi dan kepustakaan. Adapun subjek penelitian ini ialah praktisi Public Relation di dalam LKBN Anatara biro Lampung. Penelitian ini melihat dampak kemajuan teknologi telah mendisrupsi media massa konvensional, sehingga saluran berita kini terkonvergensi dalam kanal-kanal online. Namun karena kemudahan yang ditawarkan oleh media online, membuat informasi yang disampaikan tidak dapat difilter dengan baik. Banyaknya informasi yang bersifat anonymous, mengakibatkan banyak terbesarnya berita hoax di media online. Hal ini membuat menurunnya kredibilitas sebuah portal berita online dimata publik. Bahkan sebagian masyarakat meragukan keakuratan berita yang tersedia di kanal-kanal online. Public Relation di LKBN ANTARA Biro Lampung berperan sebagai Communication Technician, Communication Failitator dan Problem Solving Facilitator. Dan Public Relation disini juga bertindak sebagai fungsi manajemen dan fungsi komunikasi. 77 Syiar\| Jurnal Komunikasi dan Penyiaran Islam 77 Syiar\| Jurnal Komunikasi dan Penyiaran Islam 77 Syiar\| Jurnal Komunikasi dan Penyiaran Islam Peran dan Fungsi Public Relation….. 78 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Keywords: Public Relation, Anataranews.com, Positive Image, Role and Fungtion A. PENDAHULUAN Kehadiran teknologi informasi dan komunikasi dalam perusahaan pers telah mendisrupsi media massa konvensional. Dimana seluruh saluran-saluran berita, kini terkonvergensi dalam kanal-kanal online. Sebagai konsekuensi, ketika ingin mempertahankan eksistensinya sebagai pelayan informasi publik, media massa harus mengubahnya ke dalam format digital (AR, 2018). Selain interaktivitas yang ditawarkan oleh media pemberitaan baru, karakteristik yang tidak kalah penting yaitu digitalisasi (Nasrullah, 2013). Tidak dapat dipungkiri, bahwa media penyiaran dengan platform digital memiliki banyak kelebihan dibandingkan media analog atau konvensional. Selain membuka jalur komunikasi dua arah, media baru era informasi juga memiliki performa kualitas tayangan serta sebaran yang lebih luas. Karakter terakhir yang diusung adalah Audience Generated, bahwa media baru memungkinkan khalayak mendistribusikan konten yang mereka himpun sendiri (Bagdikian, 2004; Straubhaar & LaRose, 2006). Namun karena kemudahan yang ditawarkan oleh media online dalam penyampaian informasi kepada publik, membuat informasi yang disampaikan tidak dapat difilter dengan baik(Fakhruroji, 2017; Nasrullah, 2013). Banyaknya informasi yang bersifat anonymous, mengakibatkan banyak terbesarnya berita hoax di media online. Hoax dapat diartikan sebagai informasi atau berita yang berisi hal-hal yang belum pasti atau benar-benar bukan merupakan fakta yang terjadi. Menjamurnya berita hoax yang beredar, membuat menurunnya kredibilitas sebuah portal berita online dimata publik. Bahkan sebagian masyarakat meragukan keakuratan berita yang tersedia di kanal-kanal online. Oleh karena itu, tentu ini menjadi tantangan bagi seorang Public Relation dalam sebuah portal berita online, untuk meyakinkan pembaca tentang keakuratan berita-berita yang disampaikan melalui situs online miliknya. Berangkat dari permasalahan tersebut, sehingga membuat penulis tertarik untuk mengkaji lebih dalam tentang bagaimana peran dan fungsi Public Relation di LKBN ANTARA Biro Lampung dalam mempertahankan citra positifnya dikalangan para pembaca. Penelitian ini bertujuan untuk melihat bagaimana praktisi public relation dalam mempertahankan citra positif pada portal berita online antaranews.com. ABSTRACT This study aims to determine how the role and function of Public Relation in LKBN ANTARA Bureau Lampung in maintaining a positive image in the eyes of the public. In order to examine this research, the author uses descriptive qualitative research methods with data collection in the form of interviews, documentation and literature. The subject of this research is Public Relation practitioners in LKBN Anatara Lampung bureau. This study looks at the impact of technological advances that have disrupted conventional mass media, so that news channels are now converged into online channels. However, due to the convenience offered by online media, the information submitted cannot be filtered properly. The amount of information that is anonymous has resulted in the largest number of hoax news in online media. This reduces the credibility of an online news portal in the eyes of the public. Even some people doubt the accuracy of the news available on online channels. Public Relation at LKBN ANTARA Bureau Lampung acts as a Communication Technician, Communication Facilitator and ProblemSolving Facilitator. And Public Relation here also acts as a management function and communication function. 78 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Dede Mercy Rolando1, Tri Adellia2, Nuril Maulana Alifia Aziz3, Galuh Dwi Kartika Wicaksono4 B. METODE PENELITIAN Jenis penilitian ini adalah kualitatif. Penelitian kualitatif adalah suatu proses penelitian untuk memahami masalah-masalah manusia atau sosial dengan menciptakan gambaran menyeluruh dan kompleks yang disajikan dengan kata-kata, melaporkan pandangan terinci yang diperoleh dari sumber informasi, serta dilakukan dalam latar belakang atau setting yang alamiah (Bungin, 2007; Creswell, 2010; J. Moleong, 1989). Penelitian kualitatif merupakan penelitian yang bersifat deskriptif dan cenderung menggunakan analisis. Sedangkan teknik pengumpulan data yang penulis gunakan dalam penelitian ini adalah wawancara, dokumentasi dan kepustakaan. Wawancara adalah proses komunikasi yang dilakukan dengan memberikan 79 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Peran dan Fungsi Public Relation….. pertanyaan yang dilakukan secara verbal kepada orang-orang yang dianggap dapat memberikan informasi atau penjelasan yang diperlukan (N. K. & Y. S. L. Denzin, 2009). Penulis menggunakan teknik wawancara baku terbuka, wawancara ini dilaksanakan dengan menggunakan seperangkat pertanyaan baku. Karena pertimbangan situasi dan waktu, pelaksanaan wawancara dilakukan melalui media komunikasi whatsapp. Penulis mengirimkan daftar pertanyaan kepada LKBN Biro Lampung, melalui perwakilan Public Relation yang menjabat sebagai divisi Iklan/Pemasaran portal sekaligus reporter. Dokumentasi adalah salah satu metode pengumupul data yang digunakan dalam metodologi penelitian social (Bungin, 2013). Jenis dokumen yang peneliti gunakan ialah dokumentasi sumber eksteren yang masuk kedalam dokumen resmi sebagai bahan untuk menelaah objek penelitian (Bungin, 2007; N. K. Denzin & Yvonna S, 2009). Data dokumentasi yang penulis gunakan dalam hal ini ialah berupa data struktur organisasi miliknya LKBN ANTARA dan data kepustakaan berupa jurnal- jurnal ilmiah pendukung penelitian ini. Analisis data yang penulis gunakan ialah model interaktif dari Miles & Huberman dimana terdapat tiga sub proses yang saling berkaitan yaitu tahap pengumpulan data, persisnya pada saat menentukan rancangan dan perencanaan penelitian, sewaktu proses pengumpulan data dan analisis awal, serta setelah tahap pengumpulan data akhir (N. K. Denzin & Yvonna S, 2009). Guna memperkuat data yang telah penulis dapatkan, penulis melakukan pemeriksaan terhadap keabsahan data yang juga merupakan dasar penyanggah pada peneltian kualitatif (J. Moleong, 1989). Keabsahan data ini dilakukan untuk membuktikan penelitian yang dilakukan benar- benar merupakan penelitian ilmiah. Uji keabsahan data dalam penelitian ini meliputi uji credibility, transferability, dependability dan confirmability (Sugiyono, 2017). C. HASIL DAN PEMBAHASAN 1. Ruang Lingkup Kedudukan Antaranews.com Sebagai Portal Resmi Berita a. Sejarah Terbentuknya antaranews.com Seiring berjalannya waktu, Antara menerbitkan www.antaranews.com sebagai portal resmi yang juga bergerak untuk menyajikan berita. Antaranews.com merupakan perubahan dari antaranews.co.id sejak bulan Juni 2009. Dari segi isi, situsnya lebih serius dibandingkan antara.co.id, namun ada beberapa isi yang belum ada di antaranews.com. Ketika itu beritanya lebih pendek, tidak semua berita masuk, dan jumlahnya sedikit. Berita hanya bisa di akses sampai di lead saja, karena berikut pengakses harus membayar. Kemudian pada tahun 2005, mulai ada sedikit perubahan dipemberitaan, artinya berita semakin banyak dikeluarkan. Kemudian pada tahun 2008 mulai dipermanis dengan keberadaan foto-foto diberita hingga sekarang. 1. Ruang Lingkup Kedudukan Antaranews.com Sebagai Portal Resmi Berita 80 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam 1. Ruang Lingkup Kedudukan Antaranews.com Sebagai Portal Resmi Berita a. Sejarah Terbentuknya antaranews.com j y Seiring berjalannya waktu, Antara menerbitkan www.antaranews.com sebagai portal resmi yang juga bergerak untuk menyajikan berita. Antaranews.com merupakan perubahan dari antaranews.co.id sejak bulan Juni 2009. Dari segi isi, situsnya lebih serius dibandingkan antara.co.id, namun ada beberapa isi yang belum ada di antaranews.com. Ketika itu beritanya lebih pendek, tidak semua berita masuk, dan jumlahnya sedikit. Berita hanya bisa di akses sampai di lead saja, karena berikut pengakses harus membayar. Kemudian pada tahun 2005, mulai ada sedikit perubahan dipemberitaan, artinya berita semakin banyak dikeluarkan. Kemudian pada tahun 2008 mulai dipermanis dengan keberadaan foto-foto diberita hingga sekarang. Diterbitkannya antaranews.com tidak terlepas dari keberadaan Antara sebagai kantor berita. Awalnya, berita yang disajikan memang berasal dari cetak, namun seiring berjalannya waktu terjadi perubahan, berita tidak semua berasal dari cetak. Hal tersebut karena adanya kebijakan yang berbeda diantara 80 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Dede Mercy Rolando1, Tri Adellia2, Nuril Maulana Alifia Aziz3, Galuh Dwi Kartika Wicaksono4 keduanya. Antara redaksi cetak dan redaksi online (web), berbeda manajemen, dengan sistem pemberitaan yang tentu saja terdapat perbedaan didalmnya. Bisa dikatakan bahwa berita yang ada dicetak tidak semuanya sama atau dimasukkan ke dalam portal www.antaranews.com. Karena setiap hari, redaksi cetak dan online memiliki tim lapangan masing-masing untuk mencari berita. b. Visi Misi LKBN ANTARA Visi dan misi bagi sebuah oraganisasi merupakan arah dan tujuan yang hendak dicapai oleh organisasi tersebut. Visi dan misi merupakan gambaran tentang program yang akan dilaksanakan oleh suatu organisasi sehingga suatu organisasi akan lebih mudah dalam menentukan program kerja sesuai dengan tujuan organisasi tersebut.Visi Kantor Berita Antara adalah, menjadi kantor berita berkelas dunia melalui penyediaan jasa berbagai produk mutimedia, untuk mewujudkanmasyarakat berbasis pengetahun, yang didukung oleh tata usaha perusahaan yang baik dan berstandar internasional. Sedangkan Misi perusahaan, merupakan sesuatu yang harus sejalan dengan Visi yang ada, Visi LKBN ANTARA yaitu: 1) Memperkuat marwah LKBN ANTARA sebagai sebuah kantor berita serta perusahaan multimedia yang modern. 2) Mengembangkan jurnalisme Indonesia yang mendidik, mencerahkan, dan memberdayakan dalam bingkai Negara Kesatuan Republik Indonesia. 3) Menyediakan produk serta jasa informasi dan komunikasi yang akurat, terpercaya serta menguntungkan di bidang multimedia. 4) Mengembangkan perusahaan yang modern dan berkesinambungan sehingga dapat memberikan kesejahteraan kepada para stakeholder-nya. 4) Mengembangkan perusahaan yang modern dan berkesinambungan sehingga dapat memberikan kesejahteraan kepada para stakeholder-nya. 81 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam c. 82 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Jenis Aktivitas 1. Menerima informasi dari kantor pusat ANTARANEWS Jakarta untuk diinformasikan kepada publik internal ANTARANEWS Biro Lampung 2. Mengelola berbagai teknologi komunikasi terkini, seperti VSAT dan DVB, serta berbagai teknologi berbasis Internet, seperti situs web, email, dan ftp (file transfer protocol), yang digunakan untuk mempublikasikan konten multimedia. 3. Membuat inovasi dan kreasi dalam bidang layanan web dan produk berbasis internet 4. Memproduksi konten multimedia sehingga bisa didistribusikan kepada publik secara luas dengan media internet, baik itu artikel, news realese, maupun dokumentasi hasil kegiatan. 5. Melakukan koordinasi dengan divisi dan departemen di Direktorat Pemberitaan, khususnya untuk merencanakan, membuat dan menyiarkan berita dan informasi, sesuai dengan kebijakan yang ditetapkan Direktur Pemberitaanya. 6. Menjamin kuantitas dan kualitas layanan berita yang diproduksi, agar sesuai dengan standar pembuatan dan pelayanan berita yang telah ditetapkan. 6. Menjamin kuantitas dan kualitas layanan berita yang diproduksi, agar sesuai dengan standar pembuatan dan pelayanan berita yang telah ditetapkan. 7. Mewakili LKBN ANTARA dalam setiap pertemuan dengan pihak internal 7. Mewakili LKBN ANTARA dalam setiap pertemuan dengan pihak internal 8. Mengadakan rapat redaksi rutin untuk membahas rencana berita yang akan diliput. 8. Mengadakan rapat redaksi rutin untuk membahas rencana berita yang akan diliput. 9. Menyediakan layanan ralat, koreksi, dan hak jawab untuk pembaca. Berdasarkan tabel 1 di atas dari gambaran aktivitas Public Relation yang ada di LKBN ANTARA biro Lampung, dapat disimpulkan bahwa Public Relation disini memiliki dua peran utama. Public Relation di LKBN ANTARA Biro Lampung mempunyai peran manajerial sebagai praktisi yang berfungsi melaksanakan perencanaan terkait penentuan konten media yang akan dipublikasi secara luas kepada publik melalui media internet. Sedangkan secara teknis Public Relation memiliki peran untuk memproduksi konten media yang akan disitribusikan kepada publik, yaitu seperti menulis artikel, news realese, maupun 9. Menyediakan layanan ralat, koreksi, dan hak jawab untuk pembaca. Berdasarkan tabel 1 di atas dari gambaran aktivitas Public Relation yang ada di LKBN ANTARA biro Lampung, dapat disimpulkan bahwa Public Relation disini memiliki dua peran utama. Public Relation di LKBN ANTARA Biro Lampung mempunyai peran manajerial sebagai praktisi yang berfungsi melaksanakan perencanaan terkait penentuan konten media yang akan dipublikasi secara luas kepada publik melalui media internet. Sedangkan secara teknis Public Relation memiliki peran untuk memproduksi konten media yang akan disitribusikan kepada publik, yaitu seperti menulis artikel, news realese, maupun 9. Menyediakan layanan ralat, koreksi, dan hak jawab untuk pembaca. 1. Ruang Lingkup Kedudukan Antaranews.com Sebagai Portal Resmi Berita Tim Redaksi LKBN ANTARA Biro Lampung EDITOR Budi Santoso Budiman Edy Supriatna Triono Subagyo Agus Wira Sukarta REPORTER Dian Hadiatna Ruth Intan Sozometa Kanafi Damiri Hendra Muklasin Emir Fajar Saputra Ardiansyah PORTAL MARKETING ADS Emir Fajar Saputra Samino Angga Permana ADMINISTRATION AND FINANCE Emir Fajar Saputra Samino Angga Permana TECHNICAL SERVICE IT ASMEN: Catur Ujianto WEBMASTERS: Erwin Catur Kurniawan SuhermanSutikno SEO & ADS: Arina Suwanto p g EDITOR Budi Santoso Budiman Edy Supriatna Triono Subagyo Agus Wira Sukarta TECHNICAL SERVICE ADMINISTRATION AND FINANCE Emir Fajar Saputra Samino Angga Permana REPORTER Dian Hadiatna Ruth Intan Sozometa Kanafi Damiri Hendra Muklasin Emir Fajar Saputra Ardiansyah IT ASMEN: Catur Ujianto PORTAL MARKETING ADS Emir Fajar Saputra Samino Angga Permana SEO & ADS: Arina Suwanto 81 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Peran dan Fungsi Public Relation….. Gambar 1. Struktur Organisasi LKBN ANTARA biro Lampung 2. Kedudukan Aktivitas Praktisi Public Relation LKBN ANTARA Biro Lampung Berikut ini adalah beberapa aktivitas Public Relation yang ada di LKBN ANTARANEWS Biro Lampung, diantaranya: 2. Kedudukan Aktivitas Praktisi Public Relation LKBN ANTARA Biro Lampung Berikut ini adalah beberapa aktivitas Public Relation yang ada di LKBN ANTARANEWS Biro Lampung, diantaranya: Jenis Aktivitas Berdasarkan tabel 1 di atas dari gambaran aktivitas Public Relation yang ada di LKBN ANTARA biro Lampung, dapat disimpulkan bahwa Public Relation disini memiliki dua peran utama. Public Relation di LKBN ANTARA Biro Lampung mempunyai peran manajerial sebagai praktisi yang berfungsi melaksanakan perencanaan terkait penentuan konten media yang akan dipublikasi secara luas kepada publik melalui media internet. Sedangkan secara teknis Public Relation memiliki peran untuk memproduksi konten media yang akan disitribusikan kepada publik, yaitu seperti menulis artikel, news realese, maupun 82 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Dede Mercy Rolando1, Tri Adellia2, Nuril Maulana Alifia Aziz3, Galuh Dwi Kartika Wicaksono4 dokumentasi hasil kegiatan dalam bentuk video atau foto (Primarni, 2015). Secara struktural, LKBN ANTARA Biro Lampung memang tidak menempatkan Public Relation dalam divisi khusus Humas/PR. Melainkan dalam pelaksanaannya, Public Relation berada dibagian umum yang sekaligus dijabat oleh kepala biro LKBN ANTARA Lampung. Hal ini dikarenakan selaku pimpinan yang membawahi beberapa tim redaksi memiliki peran sebagai Public Relation itu sendiri. Disamping menjalankan tugas pokok sesuai jobdesknya, pimpinan dan seluruh tim redaksi juga mempunyai tugas utama yaitu mempertahankan citra positif LKBN ANTARANEWS Biro Lampung dimata publik. dokumentasi hasil kegiatan dalam bentuk video atau foto (Primarni, 2015). dokumentasi hasil kegiatan dalam bentuk video atau foto (Primarni, 2015). Secara struktural, LKBN ANTARA Biro Lampung memang tidak menempatkan Public Relation dalam divisi khusus Humas/PR. Melainkan dalam pelaksanaannya, Public Relation berada dibagian umum yang sekaligus dijabat oleh kepala biro LKBN ANTARA Lampung. Hal ini dikarenakan selaku pimpinan yang membawahi beberapa tim redaksi memiliki peran sebagai Public Relation itu sendiri. Disamping menjalankan tugas pokok sesuai jobdesknya, pimpinan dan seluruh tim redaksi juga mempunyai tugas utama yaitu mempertahankan citra positif LKBN ANTARANEWS Biro Lampung dimata publik. . Analisis Peran dan Fungsi dari Public Relation Dalam Mempertahankan Citra Positif Portal Berita Antaranews.com Biro Lampung Positif Portal Berita Antaranews.com Biro Lampung Public relation merupakan perantara antara pimpinan organisasi dengan publik-publiknya. Baik dalam upaya membina hubungan baik atau menumbuhkan citra positif dengan masyarakat internal maupun external (F. Rachmad, 2000; Nova, 2009; Patricia J, 2006; Rosady Ruslan, 2003). Peran public relation pada umumnya diyakini sebagai fasilitator, teknisi komunikasi, problem solving hingga sebagai menejemen organisasi. Penelitian lain (Rahmawati, 2014) menyebutkan bahwa sejatinya peran serta kedudukan public relation ialah kegiatan komunikasi guna menciptakan citra positif dari mitra organisasi atas dasar menghormati kepentingan bersama. Peran public relation dalam portal Antaranew.com sejatinya selaras dengan peran public relation yang diusung oleh Dozier & Broom dalam bukunya (Rosady Ruslan, 2003) diantaranya sebagai communications fasilitator, problem solving process fasilitator, communication technician dan expert preciber. Praktisi Public Relation memiliki peran yang sangat vital di portal ANTARA, diantaranya: Peran utama Public Relation disini adalah sebagai Communication Techinician (Teknisi Komunikasi). Sebagaimana hal ini sejalan dengan kegiatan utama perusahaan pers tersebut, yaitu melakukan segala bentuk kegiatan jurnalistik. Dalam hal ini, Praktisi Public Relation juga bertindak sebagai Journalist in resident yang menyediakan layanan teknis komunikasi atau dikenal dengan method of communication. Peran Public Relation LKBN ANTARA sebagai teknisi komunikasi diantaranya yaitu mengelola berbagai teknologi komunikasi terkini berbasis internet, seperti pengembangkan situs web yang digunakan untuk mempublikasikan setiap konten multimedia. Kemudian juga memanfaatkan seluruh teknologi tersebut untuk memberikan informasi tentang segala bentuk kegiatan LKBN ANTARA kepada masyarakat, hingga menyebarluaskan informasi tersebut ke seluruh penjuru Indonesia dan belahan dunia. Kegiatan lainnya seperti menulis artikel, news realese, maupun dokumentasi hasil kegiatan dalam bentuk video atau foto. Hal ini dilakukan sebagai strategi Public Relation dalam meningkatkan kredibilitas LKBN ANTARANEWS. Public Relation di LKBN ANTARA juga memiliki peran sebagai Communication Facilitator (Fasilitator Komunikasi), praktisi Public Relation disini 83 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam 83 Peran dan Fungsi Public Relation….. 84 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam dokumentasi hasil kegiatan dalam bentuk video atau foto (Primarni, 2015). 84 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Dede Mercy Rolando1, Tri Adellia2, Nuril Maulana Alifia Aziz3, Galuh Dwi Kartika Wicaksono4 Penelitian ini didukung oleh penelitian (Ishak, 2012) yang mengatakan bahwa kegiatan komunikasi yang bersifat strategis tentu nya perlu dijalankan oleh praktisi public relation guna organisasi dapat berjalan dengan baik. Dalam praktiknya Public Relation disini mengelola dan menyediakan berbagai layanan komunikasi berbasis internet seperti web, email, dan media lain. Sehingga, sangatlah jelas bahwa Public Relation bukan sekedar fungsi teknis tetapi merupakan fungsi manajerial yang bertanggungjawab atas terselenggaranya hubungan yang signifikan antara organisasi dengan publik (stakeholder). Public Relation adalah sebuah fungsi strategi di tingkat korporasi. Public Relation adalah jembatan, pembangun dan pemelihara hubungan yang baik antara organisasi dengan publiknya, sehingga diharapkan dapat menguatkan dan terbentuk citra positif dari organisasi tersebut. D. SIMPULAN Dampak kemajuan teknologi telah mendisrupsi media massa konvensional, sehingga saluran berita kini terkonvergensi dalam kanal-kanal online. Namun karena kemudahan yang ditawarkan oleh media online, membuat informasi yang disampaikan tidak dapat difilter dengan baik. Banyaknya informasi yang bersifat anonymous, mengakibatkan banyak terbesarnya berita hoax di media online. Hal ini membuat menurunnya kredibilitas sebuah portal berita online dimata publik. Bahkan sebagian masyarakat meragukan keakuratan berita yang tersedia di kanal-kanal online. Penulis melakukan analisa kualitatif dengan mengumpulkan data sekunder melalui teknik wawancara baku terbuka. Untuk mengetahui bagaimana peran dan fungsi Publik Relations di LKBN ANTARA Biro Lampung dalam mempertahan citra positif dimata publik. Hasilnya, Public Relation di LKBN ANTARA Biro Lampung berperan sebagai Communication Technician, Communication Failitator dan Problem Solving Facilitator. Dan Public Relation disini juga bertindak sebagai fungsi manajemen dan fungsi komunikasi. dokumentasi hasil kegiatan dalam bentuk video atau foto (Primarni, 2015). bertindak sebagai mediator atau komunikator yang membuat adanya komunikasi timbal balik antara Media ANTARA dengan publiknya. Diantara bentuk kegiatan Public Relation yang berkaitan dengan peran ini yaitu Praktisi Public Relation menjadi pusat informasi bagi pihak internal. Public Reations selalu berusaha agar setiap tim redaksi media ANTARA selalu menerima informasi terbaru, baik informasi yang berasal dari Kantor Pusat maupun informasi yang berasal dari publik eksternal. Public Relation juga menjadi fasilitator komunikasi antara perusahaan dengan masyarakat. Dalam pelaksanaannya, peran Public Relation disini yaitu dengan menyediakan layanan ralat, koreksi, dan hak jawab. Layanan ini disediakan untuk mengetahui dan membantu pembaca apabila memiliki tanggapan, koreksi terhadap konten media yang disediakan oleh LKBN ANTARANEWS. Peran ini sangat penting karena sejatinya para praktisi public relation dituntut untuk menjadi mediator untuk membantu pihak menejemen dalam hal endengar aoa yang diinginkan ataupun diharapkan oleh publiknya (Anggraini & Setyanto, 2019). Selain kedua peran diatas Public Relation di LKBN ANTARA juga berperan sebagai Fasilitator Pemecah Masalah (Problem Solving Process Facilitator) sekaligus Penasehat ahli (Expert Presciber) (Sarinah, 2017). Apabila terdapat permasalahan di LKBN ANTARANEWS, Public Relation bertindak sebagai penegah. Misalnya terjadi permasalahan tentang adanya konten pemberitaan, terdapat informasi yang kurang tepat sehingga pihak yang bersangkutan merasa keberatan dengan hal tersebut(Nova, 2009). Disinilah peran Public Relation sebagai penengah antara pihak LKBN ANTARA dengan pembaca. Public Relation bertindak mencarikan solusi awal dengan memberikan layanan ralat, koreksi, dan hak jawab untuk mengetahui apa tanggapan dan koreksi dari pihak yang bersangkutan(Respati, 2014). Sehingga nanti informasi tersebut disampaikan kepada pihak LKBN ANTARA, agar mengkaji kembali konten pemberitaan yang dimaksud. Jika memang terdapat kesalahan pihak LKBN ANTARA akan kembali meralat dan mengoreksi konten pemberitaan yang sudah dipublikasikan. Sehingga dalam hal ini sejatinya Public Relation merangkap sebagai fungsi manajemen berkaitan dengan bagaimana sebuah organisasi menyusun kebijakan sehingga memperlihatkan sebuah kinerja yang bertanggungjawab. Ini berkaitan dengan kenyataan bahwa penampilan yang bertanggungjawab merupakan dasar penerimaan publik terhadap sebuah organisasi. Praktisi Public Relationdi LKBN ANTARA Biro Lampung dalam konteks Public Relation sebagai fungsi manajemen,dalam praktiknya yaitu membantu pihak organisasi ketika menghadapi permasalahan dengan menjadi penengah antara organisasi dan publiknya, untuk kemudian mencarikan solusi dari permasalahan tersebut sebagai bentuk tanggungjawab. Perlu dipahami bahwa kegiatan utama Public Relation adalah melakukan komunikasi. Public Relation sebagai fungsi komunikasi di LKBN ANTARANEWS Lampung menjadi staff khusus yang melayani para pemimpin organisasi, khususnya dalam membantu organisasi berkomunikasi dengan publik-publiknya. 85 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam 86 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam DAFTAR PUSTAKA Anggraini, C., & Setyanto, Y. (2019). Peranan Public Relations dlam Memperkuat Eksistensi Ramayana. Jurnal Prologia, 3(2). AR, M. F. (2018). Sejarah Media: Transformasi, Pemanfaatan, Dan Tantangan. UB Press. Bagdikian, B. H. (2004). The New Media Monopoly: A Completely Revised and Updated Edition With Seven New Chapters (20th ed). Beacon Press. Bungin, B. (2007). Penelitian Kualitatif : Komunikasi, Ekonomi, Kebijakan Publik, dan Ilmu Sosial Lainnya (Edisi Ke-2). Kencana Prenada Media Group. Bungin, B. (2013). Sosiologi Komunikasi: Teori, Paradigma, dan DiskursusTeknologi Komunikasi di Masyarakat (Cet-6). Kencana Prenada Media Group. 85 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam Peran dan Fungsi Public Relation….. Creswell, J. W. (2010). Research Design: Pendekatan Kualitatif, Kuantitatif dan Mixed. Pustaka Pelajar. Denzin, N. K. & Y. S. L. (2009). Handbook of Qualitative Research (Terj. Dariyatno (ed.)). Pustaka Pelajar. Denzin, N. K., & Yvonna S, L. (2009). Handbook Of Qualitative Research (Terjemahan Dariyatno). Pustaka Pelajar. F. Rachmad. (2000). Public Relations dalam Teori dan Praktek. Gramedia Pustaka Utama. Fakhruroji, M. (2017). Dakwah di Era Media Baru: Teori dan Aktivisme Dakwah di Internet. Simbiosa Rekatama Media. Ishak, A. (2012). Peran Public Relations dalam Komunikasi Organisasi. Jurnal Komunikasi, 1(4), 373–380. J. Moleong, L. (1989). Metode Penelitian Kualitatif. Remaja Rosdakarya. Nasrullah, R. (2013). Cybermedia. IDEA Press. J. Moleong, L. (1989). Metode Penelitian Kualitatif. Remaja Rosdakarya. Nasrullah, R. (2013). Cybermedia. IDEA Press. Nova, F. (2009). CRISIS PUBLIC RELATIONS: Bagaimana Public Relations Menangani Krisis Perusahaan. Grasindo. Patricia J. (2006). Etika Public Relations: Panduan Praktik Terbaik (Terj). Esensi Erlangga Group. Primarni, A. (2015). Reposisi Peran dan Fungsi Strategis Public Relations Dalam Organisasi Pendidikan. Jurnal Lentera Komunikasi, 1(1). Rahmawati, Y. (2014). MENEJEMEN PUBLIC RELATIONS SEBAGAI ALAT ETIKA KOMUNIKASI DALAM BISNIS ISLAM. Salam: Jurnal Filsafat Dan Budaya Hukum, 2(1). Respati, W. (2014). Transformasi Media Massa Menuju Era Masyarakat Informasi di Indonesia. Jurnal Humaniora, 5(1). Rosady Ruslan. (2003). Metode Penelitian Public Relations dan Komunikasi. Gramedia Pustaka Utama. arinah, M. (2017). Pengantar Manajemen. Deepublish. Straubhaar, J., & LaRose, R. (2006). Media Now: Understanding Media, Culture and Technology. Thomson Wadsworth. Sugiyono. (2017). Metode Penelitian Kuantitatif, Kualitatif dan R&D (Cet ke-26). Alfabeta. 86 \| Syiar \| Jurnal Komunikasi dan Penyiaran Islam
https://openalex.org/W2890466532	https://europepmc.org/articles/pmc6164946?pdf=render	English	null	Damage Identification for Underground Structure Based on Frequency Response Function	Sensors	2,018	cc-by	12,430	Received: 19 August 2018; Accepted: 7 September 2018; Published: 10 September 2018 Abstract: Damage identiﬁcation that is based on modal analysis is widely used in traditional structural damage identiﬁcation. However, modal analysis is difﬁcult in high damping structures and modal concentrated structures. Unlike approaches based on modal analysis, damage identiﬁcation based on the frequency response function allows for the avoidance of error and easy veriﬁcation through other test points. An updating algorithm is devised is this study by utilizing the frequency response function together with the dynamic reduction with respect to the selected design parameters. Numerical results indicate that the method can be used to deﬁne multiple parameters with large variation and incomplete measurement data and is robust against measurement noise. With the purpose of avoiding the occurrence of resonance and gaining additional information, the trial and error method has been used to choose a proper frequency. Furthermore, an experimental scale model in a soil box is subjected to the excitation of moving load to validate the effectiveness of the damage identiﬁcation approach. The improved damage identiﬁcation method for underground structures, which is based on the analysis of the frequency response function, can be adopted as an efﬁcient and functional damage identiﬁcation tool. Keywords: damage identiﬁcation; frequency response function; model updating; scale model experiment; soil box sensors sensors sensors 1. Introduction Structural damage is caused by changes in the material or geometric properties of systems and it is a general problem in the structural design phase and during the operation of structures, especially large and complex civil urban underground infrastructures. Traditionally, the complexity of the underground environment and the degradation failure of materials are the main causes of damage, which affects the service performance of underground structures [1–5]. In previous research, numerous approaches have been developed for identifying damages in structures [6–8]. These approaches can be broadly classiﬁed into global and local techniques [9–12]. Dynamic and static measures can be used for damage identiﬁcation [13–15], and damage identiﬁcation based on modal analysis is the most widely adopted method [16–18]. Palaez and Krawczuk [19] proposed a promising damage identiﬁcation approach that is based on structural vibration. Juan and Hojjat [20] proposed an approach that is based on vibration and enables real-time monitoring. Qu and Chen [21] derived a seismic damage diagnosis method for frame structures while using an artiﬁcial neural network (ANN). Yang and Liu [22] proposed a structural damage identiﬁcation approach with ﬂexibility. Several methods have been proposed due to differences in parameters. The equilateral triangle resonator (ETR) index, which was proposed by Zong and Huang [23], is the most commonly adopted method. Meanwhile, Cha [24] and Moradipour et al. [25] utilized modal strain Sensors 2018, 18, 3033; doi:10.3390/s18093033 www.mdpi.com/journal/sensors 2 of 20 2 of 20 Sensors 2018, 18, 3033 energy sensitivity for damage identiﬁcation in civil structures. TaeKim et al. [26] and Feng et al. [27, 28] proposed damage detection applications utilizing mode shapes, and some progress has been made. Sensitivity approaches generate the objective function for the ﬁrst-order Taylor expansion. This approach was used by Yi [29] to analyze the relationship between vibration characteristics and stiffness reduction. Damage identiﬁcation that is based on statistical information has been proposed [30–33]. Compared with modal parameters based method, the FRF (Frequency Response Function) based method [34] proposed by some researchers [35,36] has received more attention and applications [37–40] in recent years due to its advantage, as follows: At ﬁrst, the measured FRF data can be utilized directly without transformation. In some software, the calculation of modal parameters is based on the measured FRF data. What is more, the modal analysis is more complex, and the error might arise from the modal identiﬁcation. The identiﬁcation error might greater than the modelling error. 1. Introduction Secondly, the FRF can be measured in more locations of the structure and taken as an objective so it can provide more data. Tranxuan [41] detected the structural damage using measured frequency response function data. Crema and Mastroddi [42] proposed a general procedure based directly on the measured frequency response function data, which can update numerical spatial model or directly to identify and quantify possible damage and failure of the structure. Zou [43] presented an approach for structural damage identiﬁcation that is based on frequency response function and genetic algorithm (GA). Khoshnoudian and Esfandiari [44] developed a global algorithm for damage assessment of structures based on a parameter estimation method whole using the ﬁnite element and measured the modal response of the structure. Yang and Song [45] veriﬁed a method of truss damage identiﬁcation is proposed based on the principal component analysis and frequency response functions. What is more, damage identiﬁcation based on the frequency response function allows the avoidance of error and easy veriﬁcation through other test points [46,47]. This technique is commonly used in light structures, mini-type structures, and bridges and is rarely adopted for long-lining underground structures. This study devised an algorithm that utilizes the frequency response function together with the dynamic reduction with respect to the selected design parameters (Section 2). Section 3 presents an experiment on a scaled aluminum tube model that was conducted to validate the effectiveness of the damage identiﬁcation approach; the model was used to simulate a long-lining underground structure subjected to the excitation of moving load. Meanwhile, the results of the experiment and simulation were compared theoretically and experimentally. The comparison indicated that the improved damage identiﬁcation, which was based on the analysis of the frequency response function, for long-lining underground structures could be applied as an efﬁcient and functional damage identiﬁcation tool. 2. Damage Identiﬁcation Theory based on Acceleration Frequency Response Function First, parameter estimation expressions are established by the acceleration frequency response ction, which is calculated by experimental measurement and model calculation. First, parameter estimation expressions are established by the acceleration frequency response function, which is calculated by experimental measurement and model calculation. The dynamic behavior of a multi-degree-of-freedom structure that is excited by a set of multiforces is described by the following matrix equation: y p The dynamic behavior of a multi-degree-of-freedom structure that is excited by a set of multiforces escribed by the following matrix equation: Mx′′ + Cx′ + Kx = F, (1) (1) where M, C, and K are the mass, damping, and stiffness matrices of the structure, respectively; and x, x′, and x′′ are the displacement, velocity, and acceleration of the structure, respectively. where M, C, and K are the mass, damping, and stiffness matrices of the structure, respectively; and x, x′, and x′′ are the displacement, velocity, and acceleration of the structure, respectively. By substituting x = xeiωt into Equation (1), the function in the frequency domain can be obtained, as follows: −Mω2xeiωt + Cωixeiωt + Kxeiωt = F0 sin ωt. (2) (2) Frequency response function is the ratio of the input and output of the viscous damping system, which has 3n degrees-of-freedom, with harmonic excitation. The input is the harmonic excitation, Frequency response function is the ratio of the input and output of the viscous damping system, which has 3n degrees-of-freedom, with harmonic excitation. The input is the harmonic excitation, 3 of 20 Sensors 2018, 18, 3033 and the output is the acceleration response. The frequency response function of structure can be expressed in Equation (2). ( ) H(ω) = F(ω) X(ω), (3) (3) where X(ω) is the steady acceleration response, H(ω) is the acceleration frequency response function, and F(ω) is the harmonic excitation. With substitution of x = xeiωt and Equations (1) into (3), the ﬁnite element analysis (FEA) model and the acceleration frequency response function matrix are expressed as Equations (4) and (5), respectively. 1 Hs(ω) = [Ms −iCs ω −Ks ω2 ] −1 , (4) He(ω) = [Me −iCe ω −Ke ω2 ] −1 , (5) (4) (5) where Ms, Cs, and Ks are the mass, damping, and stiffness matrices of the structure by simulation, respectively; Me, Ce, and Ke are the mass, damping, and stiffness matrices of the structure by experiment, respectively; and, ω is the excitation frequency. 2. Damage Identiﬁcation Theory based on Acceleration Frequency Response Function where Ms, Cs, and Ks are the mass, damping, and stiffness matrices of the structure by simulation, respectively; Me, Ce, and Ke are the mass, damping, and stiffness matrices of the structure by experiment, respectively; and, ω is the excitation frequency. The damping matrix has only a small effect on the damage and this part can be thus commonly ignored. Equations (4) and (5) can be simpliﬁed as Equations (6) and (7), respectively. Hs(ω) = [Ms −Ks ω2 ] −1 , (6) Hs(ω) = [Ms −Ks ω2 ] −1 , (6) He(ω) = [Me −Ke ω2 ] −1 . (7) (6) He(ω) = [Me −Ke ω2 ] −1 . (7) (7) In the case of a damaged structure, the stiffness matrices after damage according to Equations (6) and (7), is commonly expressed, as follows: In the case of a damaged structure, the stiffness matrices after damage according to Equations (6) and (7), is commonly expressed, as follows: ∆K = Ks −Ke = (ω2M −ω2M + Ks −Ke) = (−ω2M + Ks) −(−ω2M + Ke) = ω2(−Hs(ω)−1 + He(ω)−1) , (8) (8) where Ks is the stiffness matrices before damage, Ke is the stiffness matrices after damage, and ∆K is the variation in the stiffness matrices after damage. Furthermore, the damping matrices are commonly considered constant with the damage that occurs, and the degradation of mass (∆M) is regarded as zero [48–50]. where Ks is the stiffness matrices before damage, Ke is the stiffness matrices after damage, and ∆K is the variation in the stiffness matrices after damage. Furthermore, the damping matrices are commonly considered constant with the damage that occurs, and the degradation of mass (∆M) is regarded as zero [48–50]. However, in this calculation of H−1, a difﬁcult problem is that all of the frequency response function values cannot be measured. Therefore, Equations (6) and (7) can be transformed, as follows: Hs(ω)Ks = ω2Hs(ω)Ms −ω2I, (9) He(ω)Ke = ω2He(ω)Me −ω2I, (10) (9) (10) where I is identity matrix with 1’s on diagonal line and zeros elsewhere. Take row x for example, the equation above can be considered, as foll where I is identity matrix with 1’s on diagonal line and zeros elsewhere. T k f l th ti b b id d f ll where I is identity matrix with 1’s on diagonal line and zeros elsewhere. 2. Damage Identiﬁcation Theory based on Acceleration Frequency Response Function . . . . . H1n(ωn)   e   M11 M12 M21 M22 . . . M1n . . . M2n . . . . . . Mn1 Mn2 . . . . . . Mnn  −   1 0 1 0 . . . 0 . . . 0 . . . . . . 1 0 . . . . . . 0            . (12) (12) Due to the requirement of calculation in this paper, the ﬁrst two rows and two columns Due to the requirement of calculation in this paper, the ﬁrst two rows and two columns " K11 K12 K21 K22 # of stiffness matrix   K11 K12 K21 K22 . . . K1n . . . K2n . . . . . . Kn1 Kn2 . . . . . . Knn  are extracted in Equation (12).  n1 n2 nn  Moreover, because the symmetry of the simpliﬁed stiffness matrix, the number of unknown parameters can be reduced, " K11 K21 # was adopted. Therefore, Equation (12) can be simpliﬁed into Equation (13).   H12(ω1) H12(ω1) H11(ω2) H12(ω2) H11(ω3) H12(ω3) . . . . . . H11(ωn) H12(ωn)   " K11 K21 # =   ω2 1H11(ω1)M11 −ω2 1 ω2 2H11(ω2)M21 −ω2 2 ω2 3H11(ω3)M31 −ω2 3 . . . ω2 nH11(ωn)Mn1 −ω2 n   . (13) Moreover, because the symmetry of the simpliﬁed stiffness matrix, the number of unknown parameters can be reduced, " K11 K21 # was adopted. Therefore, Equation (12) can be simpliﬁed into Equation (13).   H12(ω1) H12(ω1) H11(ω2) H12(ω2) H11(ω3) H12(ω3) . . . . . . H11(ωn) H12(ωn)   " K11 K21 # =   ω2 1H11(ω1)M11 −ω2 1 ω2 2H11(ω2)M21 −ω2 2 ω2 3H11(ω3)M31 −ω2 3 . . . ω2 nH11(ωn)Mn1 −ω2 n   . (13)   H12(ω1) H12(ω1) H11(ω2) H12(ω2) H11(ω3) H12(ω3) . . . . . . H11(ωn) H12(ωn)   " K11 K21 # =   ω2 1H11(ω1)M11 −ω2 1 ω2 2H11(ω2)M21 −ω2 2 ω2 3H11(ω3)M31 −ω2 3 . . . ω2 nH11(ωn)Mn1 −ω2 n   . 2. Damage Identiﬁcation Theory based on Acceleration Frequency Response Function where I is identity matrix with 1’s on diagonal line and zeros elsewhere. Take row x for example, the equation above can be considered, as follows: Hx1, Hx2, Hx3 . . . . . . Hxn]e(Ke)n×n = ω2[Hx1, Hx2, Hx3 . . . . . . Hxn]e(M)n×n −ω2[0, 0, . . . , 0, 1, 0, . . . , 0, 0], (11 [Hx1, Hx2, Hx3 . . . . . . Hxn]e(Ke)n×n = ω2[Hx1, Hx2, Hx3 . . . . . . Hxn]e(M)n×n −ω2[0, 0, . . . , 0, 1, 0, . . . , 0, 0], (11) where [0, 0, . . . , 0, 1, 0, . . . , 0, 0] in Equation (11) is a row matrix with 1’s on the x-th element and zeros elsewhere. (11) [Hx1, Hx2, Hx3 . . . . . . Hxn]e(Ke)n×n = ω [Hx1, Hx2, Hx3 . . . . . . Hxn]e(M)n×n −ω [0, 0, . . . , 0, 1, 0, . . . , 0, 0], (11) where [0, 0, . . . , 0, 1, 0, . . . , 0, 0] in Equation (11) is a row matrix with 1’s on the x-th element and zeros elsewhere. where [0, 0, . . . , 0, 1, 0, . . . , 0, 0] in Equation (11) is a row matrix with 1’s on the x-th element and zeros elsewhere. Sensors 2018, 18, 3033 4 of 20 Hi(ω) changes with parameter ω. Therefore, a new matrix can be created only by the ﬁrst line of every He(ω). Equation (11) can be written, as follows:   H11(ω1) H12(ω1) H11(ω2) H12(ω2) . . . H1n(ω1) . . . H1n(ω2) . . . . . . H11(ωn) H12(ωn) . . . . . . H1n(ωn)   e   K11 K12 K21 K22 . . . K1n . . . K2n . . . . . . Kn1 Kn2 . . . . . . Knn   =   ω2 1 0 0 ω2 2 . . . 0 . . . 0 . . . . . . 0 0 . . . . . . ω2 n              H11(ω1) H12(ω1) H11(ω2) H12(ω2) . . . H1n(ω1) . . . H1n(ω2) . . . . . . H11(ωn) H12(ωn) . 2. Damage Identiﬁcation Theory based on Acceleration Frequency Response Function (13) (13) However, several of the values of Hi(ω) are nearly the same due to the same size and material of the element in practice, thereby possibly affecting damage identiﬁcation. To avoid this phenomenon, Equation (8) can be expressed, as follows: He(ω)∆K = ω2(−He(ω)Hs(ω)−1 + I). (14) (14) Hs(ω)−1 can be calculated using Equation (6). Similarly, a new matrix can be created from the ﬁrst row of every He(ω). Equation (14) can be written, as follows: Hs(ω)−1 can be calculated using Equation (6). Similarly, a new matrix can be created from the ﬁrst row of every He(ω). Equation (14) can be written, as follows: [H11(ω1) H12(ω1) . . . H1n(ω1)]e   ∆K11 ∆K12 ∆K21 ∆K22 . . . ∆K1n . . . ∆K2n . . . . . . ∆Kn1 ∆Kn2 . . . . . . ∆Knn   = ω2          [1 0 . . . 0] −[H11(ω1) H12(ω1) . . . H1n(ω1)]e   H11 H12 H21 H22 . . . H1n . . . H2n . . . . . . Hn1 Hn2 . . . . . . Hnn   s          . (15) (15) The degradation of stiffness (∆K) that is caused by damage is commonly simulated by ∆EI in actual projects [51]. The degradation of stiffness (∆K) that is caused by damage is commonly simulated by ∆EI in actual projects [51]. The degradation of stiffness (∆K) that is caused by damage is commonly simulated by ∆EI in actual projects [51]. p j Therefore, Equation (11) can be written, as follows: Therefore, Equation (11) can be written, as follows: [Hx1, Hx2, Hx3 . . . . . . Hxn]e(Ks) × ∆(EI) = ω2[Hx1, Hx2, Hx3 . . . . . . Hxn]e([H]s)−1 −ω2[0, 0 . . . 1, 0 . . . 0, 0], (16) 1 K Hx1, Hx2, Hx3 . . . . . . Hxn]e(Ks) × ∆(EI) = ω2[Hx1, Hx2, Hx3 . . . . . . Hxn]e([H]s)−1 −ω2[0, 0 . . . 1, 0 . . . 0, 0], (16 (16) where ([H]s)−1 = Ms −Ks ω2 . where [Ks] is the stiffness matrix calculated by the ﬁnite element model and is a known quantity. where ([H]s)−1 = Ms −Ks ω2 . 2. Damage Identiﬁcation Theory based on Acceleration Frequency Response Function where [Ks] is the stiffness matrix calculated by the ﬁnite element model and is a known quantity. where ([H]s)−1 = Ms −Ks ω2 . where [Ks] is the stiffness matrix calculated by the ﬁnite element model and is a known quantity. 5 of 20 model Sensors 2018, 18, 3033 where [ ] ( ) s and is a known qua ∆K can be calculated after the appearance of ∆(EI). Therefore, the location of the damage can be easily evaluated. In this paper, the Monkey algorithm was utilized as a optimization algorithm to ﬁt He(ω) and Ht(ω). Monkey Algorithm (MA) is an intelligent optimization algorithm, which is put forward by Zhao and Tang [52], is used to solve multivariable optimization and multimodal function optimization problem. In recent years, many scientists put into the related study of Monkey Algorithm. The establishment of a gas ﬁlling station model is shown in the work by Zhao [53], and an intrusion detection technology is worked by Zhang and Sun [54]. Yi has studied on sensor placement on Canton Tower for health monitoring using asynchronous-climb monkey algorithm [55]. What is more, to overcome the limitations overcome the limitations of the monkey algorithm, a reprogram monkey algorithm program is improved by Wang [56]. The algorithm is been inspired by the monkey climbing process in a given ﬁeld that has many mountains, which simulated by the climbing, watching, and jumping of the monkey in order to ﬁnd the highest mountaintop. The corresponding search process is as followed: climbing is used to search the local optimal solution of the current location; watching is used to search a better solution of the adjacent of the current location; and, jumping is used to reach a new location which can avoid the local optimum. This algorithm is a process of constantly looking for the optimal solution. Monkey Algorithm was used to identify the change in stiffness, which the error requirements is 0.1% in this paper. Meanwhile, assume that the group number of monkey is 5, and the number of each group is 4. Analysis at the situation when the step size defaults to 1. The process of damage identiﬁcation that is based on the acceleration frequency response function is shown in Figure 1. K Δ can be calculated after the appearance of ( ) EI Δ . Therefore, the location of the damage can be easily evaluated. 2. Damage Identiﬁcation Theory based on Acceleration Frequency Response Function In this paper, the Monkey algorithm was utilized as a optimization algorithm to fit ( ) e H ω and ( ) t H ω . Monkey Algorithm (MA) is an intelligent optimization algorithm, which is put forward by Zhao and Tang [52], is used to solve multivariable optimization and multimodal function optimization problem. In recent years, many scientists put into the related study of Monkey Algorithm. The establishment of a gas filling station model is shown in the work by Zhao [53], and an intrusion detection technology is worked by Zhang and Sun [54]. Yi has studied on sensor placement on Canton Tower for health monitoring using asynchronous-climb monkey algorithm [55]. What is more, to overcome the limitations overcome the limitations of the monkey algorithm, a reprogram monkey algorithm program is improved by Wang [56]. The algorithm is been inspired by the monkey climbing process in a given field that has many mountains, which simulated by the climbing, watching, and jumping of the monkey in order to find the highest mountaintop. The corresponding search process is as followed: climbing is used to search the local optimal solution of the current location; watching is used to search a better solution of the adjacent of the current location; and, jumping is used to reach a new location which can avoid the local optimum. This algorithm is a process of constantly looking for the optimal solution. Monkey Algorithm was used to identify the change in stiffness, which the error requirements is 0.1% in this paper. Meanwhile, assume that the group number of monkey is 5, and the number of each group is 4. Analysis at the situation when the step size defaults to 1. The process of damage identification that is based on the acceleration frequency response function is shown in Figure 1. Figure 1. Process of damage identification based on frequency response function. Figure 1. Process of damage identiﬁcation based on frequency response function. Figure 1. Process of damage identification based on frequency response function. Figure 1. Process of damage identiﬁcation based on frequency response function. 3 1 Similarity Analysis 3.1. Similarity Analysis 3.1. Similarity Analysis Knowledge of similitude rules is essential for satisfying the fundamental conditions in planning the scaled shield tunnel model testing, because the scaled model must behave in a manner similar to th t t Knowledge of similitude rules is essential for satisfying the fundamental conditions in planning the scaled shield tunnel model testing, because the scaled model must behave in a manner similar to the prototype. the prototype. Models in previous studies fall into two categories of vibration research. The first category considers the entire vibrating structure a rigid body. In the second group, the model in which Models in previous studies fall into two categories of vibration research. The ﬁrst category considers the entire vibrating structure a rigid body. In the second group, the model in which vibrations occur in the structure is with respect to its stiffness. The vibration of a scaled shield tunnel model in a soil box with damage belongs to the second research category. Sensors 2018, 18, 3033 6 of 20 The scaled shield tunnel model in this study is a nonlinear elastic model because it involves the shield tunnel and soil structures. Three requirements must be satisﬁed simultaneously in designing the scaled shield tunnel model. (1) Geometry (2) Mass and mass distribution (3) Gravity and inertial forces With the scaled shield tunnel model structure considered to be an inﬁnite Timoshenko beam, the deformation equation is expressed, as follows: 1 r = M EI , (17) (17) where r is the radius of curvature, E is Young’s modulus, and I is the moment of inertia. rp rm = Mm Mp · Ep Em · Ip Im , (18) (18) where subscripts m and p indicate the model and the prototype, respectively. According to Froude’s scaling law [57], Equation (18) can be simpliﬁed to the following: where subscripts m and p indicate the model and the prototype, respectively. According to Froude’s scaling law [57], Equation (18) can be simpliﬁed to the following: Im Ip = Lm Lp 5 · Ep Em , (19) (19) with I ∝L4, where Lm and Lp are the characteristic length of the model and the prototype, respectively. Equation (19) can be written, as follows: Lp Lm = Ep Em . 3 1 Similarity Analysis 3.1. Similarity Analysis (20) (20) The ratio of the similarity of the shield tunnel structural and soil inertia forces, which are indicated by Fss and Fs, must be the same in the model and the prototype. The ratio of the structural inertia force Fss can be expressed, as follows: Fss = ma, (21) (21) Fss = ma, (21 where m is the mass and a is the acceleration. Thus, re m is the mass and a is the acceleration. Thus, where m is the mass and a is the acceleration. Thus, where m is the mass and a is the acceleration. Thus, Fss ∝ρssL4 t2 , (22) (22) where ρss is the shield tunnel structure density and t is the characteristic time. t is directly proportional to L t and thus can be written as follows: where ρss is the shield tunnel structure density and t is the characteristic time. t is directly proportional to L t and thus can be written as follows: Fss ∝ρssL2v2, (23) (23) where v is the velocity of moving load. where v is the velocity of moving load. where v is the velocity of moving load. where v is the velocity of moving load. The requirement for similarity is thus as follows: The requirement for similarity is thus as follows: Fss Fs ∝ρssL2v2 ρsL2v2 = constant, (24) (24) where ρs is the soil density. Meanwhile, moving load scaling should also be considered in this study because it is the source of the main excitation that acts on the shield tunnel model. The scaling law for the situation in this work is also considered the Froude scaling law, which dictates that the scales for the time and velocity were equal to the square root of the length scale. All of the parameters for the shield tunnel model 7 of 20 7 of 20 Sensors 2018, 18, 3033 are shown in Table 1. However, using the same scale for the entire model will result in an excessively small model and hence a conﬂict with the scale of the density. Similarly, the scale of density may be remarkably small, thereby potentially causing the instability between the shield model and the soil. To avoid these problems, aluminum model pipes are used in this experiment, and their properties are listed in Table 1. The stiffness of the shield tunnel model and the soil box parameters are also shown in Table 1. 3 1 Similarity Analysis 3.1. Similarity Analysis The sand was considered a part of the shield tunnel system in the soil box. Fine yellow sand is used in the tests for this study. Table 1. The properties of aluminum model pipes considered in experimental veriﬁcation. Item Scale Prototype Desired Model Adopted Model Soil model Length 1 30 135 m 4500 mm 4300 mm Height 1 30 30 m 1000 mm 800 mm Depth 1 30 30 m 1000 mm 800 mm Bulk density 1 1 1800 kg/m3 1800 kg/m3 1800 kg/m3 Shield tunnel model Inner diameter 1 30 5.4 m 180 mm 160 mm Outer diameter 1 30 6 m 200 mm 180 mm Length 1 30 135 m 4500 mm 4300 mm 3.2. Description of Tested Structures Table 1. The properties of aluminum model pipes considered in experimental veriﬁcation. 3.2. Description of Tested Structures 3.2. Description of Tested Structures (b) (b) segments are tighten by the bolts against the flange; (a) (a) Figure 3. Sketch of aluminum pipe model: (a) (b) sensor (b) y the b (a) of alu Figure 3. Sketch of aluminum pipe model: (a) segments are tighten by the bolts against the flange; (b) sensor. Figure 3. Sketch of aluminum pipe model: (a) segments are tighten by the bolts against the ﬂange; (b) sensor. ( ) 200 10 10 160 10 120 10 280 ( ) 200 10 10 160 10 120 10 280 (a) 200 10 10 160 10 120 10 280 R80 R90 unit:mm (a) (b) Fi 4 Sh d i i f ti f l i t d l ( it ) ( ) id i (b) 200 10 10 160 10 120 10 280 R80 R90 unit:mm (a) (b) Figure 4. Shape and size information of aluminum segment model (unit: mm): (a) side view; (b) front view Figure 4. Shape and size information of aluminum segment model (unit: mm): (a) side view; (b) front view. R80 R90 unit:mm (b) R80 R90 unit:mm (b) (b) (a) (a) (b) Figure 4. Shape and size information of aluminum segment model (unit: mm): (a) side view; (b) f i Figure 4. Shape and size information of aluminum segment model (unit: mm): (a) side view; (b) front view. Figure 4. Shape and size information of aluminum segment model (unit: mm): (a) side view; (b) front view. Two damage types were involved in the study, namely, bar and block damages. Varying damage severities were applied to Groups A and B, which were used to simulate crack and peeling-off damages, respectively. Each damage was located at the top of the aluminum pipe. In Group A, the bending rigidity of the bar damage components was reduced by 5% and 10% (The size of the four damages are 120 mm 20 mm × , 240 mm 20 mm × ), as shown in Figure 5. The same was performed on Group B (The size of the four damages are 50 mm 50 mm × , 70 mm 70 mm × ), as shown in Figure front view. Two damage types were involved in the study, namely, bar and block damages. Varying damage severities were applied to Groups A and B, which were used to simulate crack and peeling-off damages, respectively. Each damage was located at the top of the aluminum pipe. 3.2. Description of Tested Structures The proposed damage identiﬁcation procedure was veriﬁed with real measurement data from moving load vibration tests. Shield tunnel structures are affected by components in the complex environment, such as the surrounding soil and groundwater. Thus, the experimental tests were conducted with a simply scaled aluminum pipe model. The model, which was subjected to the excitation of moving load with all of the designed sensor arrangement scenarios, was placed in the soil box for the segment of shield tunnel for structural damage identiﬁcation analysis. Although the tests were not performed in a soil environment, the testing data provided information that demonstrated the reliability of the proposed damage index in detecting damage. In this experiment, aluminum and yellow sand were selected to represent concrete and soil, respectively. The experimental setup for the dynamic tests of the aluminum pipe model is shown in Figure 2. Eighteen cases for the scaled shield tunnel model with different damage types were tested and monitored by using acceleration sensors, as shown in Figure 3. During testing, the entire structure in the model vibrated as a rigid body. Each segment with a ﬂange structure that formed the entire tunnel structure measured approximately 140 mm in length, 180 mm in outer diameter, and 160 mm in inner diameter. The segments installed were tightened by bolts to seal the gasket and washer against the ﬂange, as depicted in Figure 3. Detailed size information about the experimental model is shown in Figure 4. In the model design, each segment is equivalent to a circle, which has a stiffness reduction according to the area and the moment of inertia. Sensors 2018, 18, x 8 of 21 Figure 2. Experimental setup for dynamic tests of aluminum pipe model. Figure 2. Experimental setup for dynamic tests of aluminum pipe model. Figure 2. Experimental setup for dynamic tests of aluminum pipe model. Figure 2. Experimental setup for dynamic tests of aluminum pipe model. 8 of 20 8 of 20 Sensors 2018, 18, 3033 (a) (b) Figure 3. Sketch of aluminum pipe model: (a) segments are tighten by the bolts against the flange; (b) sensor. Figure 3. Sketch of aluminum pipe model: (a) segments are tighten by the bolts against the ﬂange; (b) sensor. (a) (b) Figure 3. Sketch of aluminum pipe model: (a) segments are tighten by the bolts against the flange; (b) sensor. 3.2. Description of Tested Structures In Group A, the bending rigidity of the bar damage components was reduced by 5% and 10% (The size of the four damages are 120 mm 20 mm × , 240 mm 20 mm × ), as shown in Figure 5. The same was performed on Group B (The size of the four damages are 50 mm 50 mm × , 70 mm 70 mm × ), as shown in Figure Two damage types were involved in the study, namely, bar and block damages. Varying damage severities were applied to Groups A and B, which were used to simulate crack and peeling-off damages, respectively. Each damage was located at the top of the aluminum pipe. In Group A, the bending rigidity of the bar damage components was reduced by 5% and 10% (The size of the four damages are 120 mm × 20 mm, 240 mm × 20 mm), as shown in Figure 5. The same was performed on Group B (The size of the four damages are 50 mm × 50 mm, 70 mm × 70 mm), as shown in Figure 6. Nine cases were considered for each group. An undamaged case, four single-damage cases, three double-damage cases, and one triple-damage case were set. The details of the two groups are tabulated in Tables 2 and 3. Sensors 2018, 18, x 9 of 21 6. Nine cases were considered for each group. An undamaged case, four single-damage cases, three double-damage cases, and one triple-damage case were set. The details of the two groups are tabulated in Tables 2 and 3. (a) (b) Figure 5. Bar damage scenarios: (a) 5% damage; (b) 10% damage. Figure 5. Bar damage scenarios: (a) 5% damage; (b) 10% damage. (b) (a) (b) (a) Figure 5. Bar damage scenarios: (a) 5% damage; (b) 10% damage. Figure 5. Bar damage scenarios: (a) 5% damage; (b) 10% damage. 9 of 20 Sensors 2018, 18, 3033 (a) (b) Figure 6. Block damage scenarios: (a) 5% damage; and, (b) 10% damage. Figure 6. Block damage scenarios: (a) 5% damage; and, (b) 10% damage. (b) (a) (b) (a) Figure 6. Block damage scenarios: (a) 5% damage; and, (b) 10% damage. Figure 6. Block damage scenarios: (a) 5% damage; and, (b) 10% damage. Table 2. Damage cases of Groups A and B considered in experimental verification. Table 2. Damage cases of Groups A and B considered in experimental veriﬁcation. 3.2. Description of Tested Structures Label Segment No. Damage Location Group A Group B Damage Severity Damage Scenario Damage Type Damage Scenario Damage Type 0 Null Null UDMa Bar UDMb Block Null 1 S10 x = 9 m~10 m DMG1a bar DMG1b block 5% 2 S10 x = 9 m~10 m DMG2a bar DMG2b block 10% 3 S15 x = 14 m~15 m DMG3a bar DMG3b block 5% 4 S20 x = 19 m~20 m DMG4a bar DMG4b block 5% 5 S10 x = 9 m~10 m DMG5a bar DMG5b block 5% S20 x = 19 m~20 m bar block 5% 6 S10 x = 9 m~10 m DMG6a bar DMG6b block 5% S20 x = 19 m~20 m bar block 10% 7 S10 x = 9 m~10 m DMG7a bar DMG7b block 5% S15 x = 14 m~15 m bar block 5% 8 S10 x = 9 m~10 m DMG8a bar DMG8b block 5% S15 x = 14 m~15 m bar block 5% S20 x = 19 m~20 m bar block 5% Label Segment No. Damage Location Group A Group B Damage Severity Damage Scenario Damage Type Damage Scenario Damage Type 0 Null Null UDMa Bar UDMb Block Null 1 S10 x = 9 m~10 m DMG1a bar DMG1b block 5% 2 S10 x = 9 m~10 m DMG2a bar DMG2b block 10% 3 S15 x = 14 m~15 m DMG3a bar DMG3b block 5% 4 S20 x = 19 m~20 m DMG4a bar DMG4b block 5% 5 S10 x = 9 m~10 m DMG5a bar DMG5b block 5% S20 x = 19 m~20 m bar block 5% 6 S10 x = 9 m~10 m DMG6a bar DMG6b block 5% S20 x = 19 m~20 m bar block 10% 7 S10 x = 9 m~10 m DMG7a bar DMG7b block 5% S15 x = 14 m~15 m bar block 5% 8 S10 x = 9 m~10 m DMG8a bar DMG8b block 5% S15 x = 14 m~15 m bar block 5% S20 x = 19 m~20 m bar block 5% Table 3. Speciﬁcations of wireless accelerometer. Range (g) ±5 Sensitivity (mV/g) 400 Frequency Response (Hz) 0–600 Output Noise, Differential [Root Mean Square, typical] (µg/ √ Hz) 7 Max Shock [(0.1 ms)] (g) 2000 Non-Linearity (±90%FSO) 0.5% Bias Tem. 3.2. Description of Tested Structures Shift (T = −55 ◦C to + 125 ◦C) 0.0001 g/◦C Type Micro-machined capacitive sense element Excitation Voltage (VDC) +5.0 VDC (±0.025 VDC) Output Impedance 50 Ω Operating Current 15 mA @ 5.0 VDC Differential Output ±2.0VDC FSO Cross Axis Sensitivity <= 3% Operating Temperature −55 ◦C to + 125 ◦C Damping Nitrogen Gas Damped Overal Size (A × B × C) 49.5 mm × 12.4 mm × 20.3 mm Weight (g) 54 Housing 6061 Aluminunum, IP67 rated Cable BDI-RC-187 (specify length) Table 3. Speciﬁcations of wireless accelerometer. Range (g) ±5 Sensitivity (mV/g) 400 Frequency Response (Hz) 0–600 Output Noise, Differential [Root Mean Square, typical] (µg/ √ Hz) 7 Max Shock [(0.1 ms)] (g) 2000 Non-Linearity (±90%FSO) 0.5% Bias Tem. Shift (T = −55 ◦C to + 125 ◦C) 0.0001 g/◦C Type Micro-machined capacitive sense element Excitation Voltage (VDC) +5.0 VDC (±0.025 VDC) Output Impedance 50 Ω Operating Current 15 mA @ 5.0 VDC Differential Output ±2.0VDC FSO Cross Axis Sensitivity <= 3% Operating Temperature −55 ◦C to + 125 ◦C Damping Nitrogen Gas Damped Overal Size (A × B × C) 49.5 mm × 12.4 mm × 20.3 mm Weight (g) 54 Housing 6061 Aluminunum, IP67 rated Cable BDI-RC-187 (specify length) Table 3. Speciﬁcations of wireless accelerometer. Sensors 2018, 18, 3033 10 of 20 d In the experiment, 30 aluminum segments were used to assemble the long-lining shield tunnel structure whose total length was 4200 mm, as shown in Figure 7. Meanwhile, the wireless test system STS LIVE (Bridge Diagnostics, Inc, Louisville, CO, USA) was used to measure and collect the real-time accelerator signals of each sensor. The speciﬁcations of the wireless accelerometer are shown in Table 3. Fifteen wireless sensors were evenly distributed on the pipe model. In the experiment, 30 aluminum segments were used to assemble the long-lining shield tunnel structure whose total length was 4200 mm, as shown in Figure 7. Meanwhile, the wireless test system STS LIVE (Bridge Diagnostics, Inc, Louisville, CO, USA) was used to measure and collect the real- time accelerator signals of each sensor. The specifications of the wireless accelerometer are shown in Table 3. Fifteen wireless sensors were evenly distributed on the pipe model. A moving load was used to simulate the train load in the tunnel. The simulated moving load consisted of six carriages, and each carriage had two axes with a spacing of 100 mm. 3.2. Description of Tested Structures The traction device, which could provide stable power, was used to pull the train load travelling in the tunnel. The power of the traction device was 25 watts, which was rotated at 125 revolutions per minute. The moving load and traction device are shown in Figure 8. The sampling frequency was 1000 Hz. “Same measurements” were taken 10 times in order to avoid mistakes and accidental errors. The number of aluminum pipes is shown in Figure 7. A moving load was used to simulate the train load in the tunnel. The simulated moving load consisted of six carriages, and each carriage had two axes with a spacing of 100 mm. The traction device, which could provide stable power, was used to pull the train load travelling in the tunnel. The power of the traction device was 25 watts, which was rotated at 125 revolutions per minute. The moving load and traction device are shown in Figure 8. The sampling frequency was 1000 Hz. “Same measurements” were taken 10 times in order to avoid mistakes and accidental errors. The number of aluminum pipes is shown in Figure 7. According to the testing condition, the structure was simpliﬁed to a beam, which meant that the response of all the nodes at the same cross section were the same. The ﬁnite element model of the test was simpliﬁed as a beam structure with 30 elements, as shown in Figure 9. In addition, the experiment was conducted within a short time to ensure accuracy. Temperature and humidity were relatively stable, and no interference was caused by external excitation sources. According to the testing condition, the structure was simplified to a beam, which meant that the response of all the nodes at the same cross section were the same. The finite element model of the test was simplified as a beam structure with 30 elements, as shown in Figure 9. In addition, the experiment was conducted within a short time to ensure accuracy. Temperature and humidity were relatively stable, and no interference was caused by external excitation sources. 1 2 3 4 5 6 7 8 9 10 24 ...... 25 26 27 28 29 30 Figure 7. Number of aluminum pipes. Figure 7. Number of aluminum pipes. 18, 18, x 11 (a) (b) Figure 8. Simulated train load: (a) moving train; (b) traction device. Figure 8. 3.2. Description of Tested Structures Simulated train load: (a) moving train; (b) traction device. 8, 18, x 11 (a) (b) Figure 8. Simulated train load: (a) moving train; (b) traction device. 1 2 3 4 5 6 7 8 9 10 24 ...... 25 26 27 28 29 30 Figure 7. Number of aluminum pipes. 8, x 8, x Figure 7. Number of aluminum pipes. Number of aluminum pipes. (b) (b) Figure 7. N (a) (a) (b) (b) (a) (a) Figure 8. Simulated train load: (a) moving train; (b) traction device. Figure 8. Simulated train load: (a) moving train; (b) traction device. Figure 8. Simulated train load: (a) moving train; (b) traction device. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Fi 9 Si lifi d fi it l t d l 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Fi Si lifi d fi i l d l Figure 9. Simpliﬁed ﬁnite element model. Figure Figure 3.3. Experimental Results and Analysis Figure Figure 3.3. Experimental Results and Analysis 3.3. Experimental Results and Analysis Real-time acceleration data were obtained by the wireless node and a wireless test system. To limit the length of this paper, only parts of the results collected by the typical acceleration sensor are 3.3. Experimental Results and Analysis Real-time acceleration data were obtained by the wireless node and a wireless test system. To limit the length of this paper only parts of the results collected by the typical acceleration sensor are Real-time acceleration data were obtained by the wireless node and a wireless test system. To limit the length of this paper, only parts of the results collected by the typical acceleration sensor are shown here. limit the length of this paper, only parts of the results collected by the typical acceleration sensor are shown here. First, the frequency response function of the undamaged structure was tested. In the experimental tests, the speed of the moving load is 2 m/s . The scaling law for the situation in this limit the length of this paper, only parts of the results collected by the typical acceleration sensor are shown here. First, the frequency response function of the undamaged structure was tested. In the experimental tests, the speed of the moving load is 2 m/s . The scaling law for the situation in this First, the frequency response function of the undamaged structure was tested. In the experimental tests, the speed of the moving load is 2 m/s. The scaling law for the situation in this work is also considered the Froude scaling law, which dictates that the scales for the time and velocity should be 11 of 20 (25) Sensors 2018, 18, 3033 equal to the square root of the length scale. According to the reference [39], equivalent node load can be expressed, as follows: √ where ( ) , P x ω is the moving load in frequency domain; s is the Laplace parameter; F is the moving load The frequency response can be obtained with ( ) P x ω The frequency response of the P(x, ω) = F ∆T e−s −v+ √ v2+2ax a , (25) ponse can be obtained with ( ) , P x ω . The frequency response of the onsisted of six carriages, is shown in Figure 10. Figure Figure 3.3. Experimental Results and Analysis (25) of the where P(x, ω) is the moving load in frequency domain; s is the Laplace parameter; F is the moving load. The frequency response can be obtained with P(x, ω). The frequency response of the simulated moving load, which consisted of six carriages, is shown in Figure 10. The acceleration frequency responses of the two damages are shown in the Figures 11 and 12, and the frequency response function of the two groups are shown in Figures 13 and 14. Due to limitation of pages, only some representative cases are presented in Figures 11–14. where P(x, ω) is the moving load in frequency domain; s is the Laplace parameter; F is the moving load. The frequency response can be obtained with P(x, ω). The frequency response of the simulated moving load, which consisted of six carriages, is shown in Figure 10. The acceleration frequency responses of the two damages are shown in the Figures 11 and 12, and the frequency response function of the two groups are shown in Figures 13 and 14. Due to limitation of pages, only some representative cases are presented in Figures 11–14. Figure 10. Moving load frequency response. Moving load frequency response(N) Figure 10. Moving load frequency response. Figure 10. Moving load frequency response Figure 10. Moving load frequency response. The acceleration frequency responses of the two damages are shown in the Figures 11 and 12, and the frequency response function of the two groups are shown in Figures 13 and 14. Due to limitation of pages, only some representative cases are presented in Figures 11–14. Sensors 2018, 18, x 12 of 21 (a) (b) (c) (d) Figure 11. Acceleration frequency response of Group A: (a) UDMa; (b) DMG1a; (c) DMG5a; and, (d) DMG8a. Acceleration frequency response Acceleration frequency response Acceleration frequency response Acceleration frequency response Figure 11. Acceleration frequency response of Group A: (a) UDMa; (b) DMG1a; (c) DMG5a; and, (d) DMG8a. (b) Acceleration frequency response (a) Acceleration frequency response (b) (a) (d) Acceleration frequency response (c) Acceleration frequency response (d) (c) Figure 11. Acceleration frequency response of Group A: (a) UDMa; (b) DMG1a; (c) DMG5a; and, (d) DMG8a. Figure 11. Acceleration frequency response of Group A: (a) UDMa; (b) DMG1a; (c) DMG5a; and, (d) DMG8a. 12 of 20 Sensors 2018, 18, 3033 (a) (b) (c) (d) Figure 12. Acceleration frequency response of Group B. (a) UDMb; (c) DMG5b; and, (d) DMG8b. Figure Figure 3.3. Experimental Results and Analysis Acceleration frequency response Acceleration frequency response Acceleration frequency response Acceleration frequency response Figure 12. Acceleration frequency response of Group B. (a) UDMb; (c) DMG5b; and, (d) DMG8b. Sensors 2018, 18, x 13 of 21 (a) Acceleration frequency response (b) Acceleration frequency response (a) (b) (d) Acceleration frequency response (c) Acceleration frequency response (c) (d) Figure 12. Acceleration frequency response of Group B. (a) UDMb; (c) DMG5b; and, (d) DMG8b. Figure 12. Acceleration frequency response of Group B. (a) UDMb; (c) DMG5b; and, (d) DMG8b. Sensors 2018, 18, x 13 of 21 (a) (b) (c) (d) (e) Figure 13. Frequency response function of Group A: (a) UDMa; (b) DMG1a; (c) DMG5a; (d) DMG8a; and, (e) all cases. Frequency response function Frequency response function Frequency response function Frequency response function Frequency response function Figure 13. Frequency response function of Group A: (a) UDMa; (b) DMG1a; (c) DMG5a; (d) DMG8a; and, (e) all cases. (b) Frequency response function (a) Frequency response function (b) (a) (c) Frequency response function (d) Frequency response function (d) (e) Frequency response function (e) Figure 13. Frequency response function of Group A: (a) UDMa; (b) DMG1a; (c) DMG5a; (d) DMG8a; and, (e) all cases. Figure 13. Frequency response function of Group A: (a) UDMa; (b) DMG1a; (c) DMG5a; (d) DMG8a; and, (e) all cases. 13 of 20 G8a; Sensors 2018, 18, 3033 Figure 13. Fre a d (e) all a ensors 2018, 18, 3033 13 of and, (e) all cases. (a) (b) Frequency response function Frequency response function ensors 2018, 18, x 14 o (c) (d) (e) Figure 14. Frequency response function of Group B: (a) UDMb; (b) DMG1b; (c) DMG5b; (d) DMG8b; and, (e) all cases. Frequency response function Frequency response function Frequency response function Figure 14. Frequency response function of Group B: (a) UDMb; (b) DMG1b; (c) DMG5b; (d) DMG8b; and, (e) all cases. (b) Frequency response function 1 (a) Frequency response function rs 2018, 18, x (b) (a) (c) Frequency response function (d) Frequency response function (d) (c) (e) Frequency response function (e) Figure 14. Frequency response function of Group B: (a) UDMb; (b) DMG1b; (c) DMG5b; (d) DMG8b; and, (e) all cases. Figure 14. Frequency response function of Group B: (a) UDMb; (b) DMG1b; (c) DMG5b; (d) DMG8b; and, (e) all cases. The results of the frequency response function are shown in Figures 13 and 14. Undamaged cases a and b had the same situation. Figure Figure 3.3. Experimental Results and Analysis Stiffness change of partial unit of case 1a. Element Number Undamaged EIu Damaged EId ∆EI ∆EI EIu (%) 5 2.09 × 109 2.03 × 109 6.48 × 107 0.031 7 2.38 × 109 2.35 × 109 3.33 × 107 0.014 10 2.21 × 109 1.73 × 109 4.81 × 108 0.218 13 2.53 × 109 2.43 × 109 9.87 × 107 0.039 14 2.49 × 109 2.44 × 109 4.73 × 107 0.019 17 1.89 × 109 1.68 × 109 2.08 × 107 0.011 20 2.07 × 109 1.98 × 109 8.90 × 107 0.043 The variation ratios of EI obtained for each damage case of Groups A and B are shown in Figures 15 and 16, respectively. Application of the proposed method revealed that the variation ratio of EI (∆EI) of the damaged element showed a noticeably increasing trend. The degradation of stiffness that is caused by damage is commonly simulated by ∆EI in actual projects. The testing result indicated that the numerical model was correct. The variation ratios of EI were observed exactly at the damage element locations in single- and multi-damage situations. Furthermore, the bending rigidity of element 10 in case 1a was reduced by 5%. For example, in Group A, the bending rigidity of element 10 was reduced by 10% in case 2a. Meanwhile, the bending rigidity values of elements 15 and 20 were reduced by 5% in cases 3a and 4a, respectively, and a 5% measurement noise was considered in all of the cases. Figures 15 and 16 indicate that the variation ratios of EI at the damaged element were ampliﬁed obviously. For example, in case 1a, the variation ratio of EI of the damaged element was 0.21, which largely outweighed those of the other elements. This ﬁnding demonstrated the feasibility and accuracy of using the proposed approach in identifying the damage locations in a single-damage situation. Moreover, the variation ratio may also be used for quantifying the damage. In multi-damage situations, the variation ratio tended to increase in comparison with that in the single-damage case. This result may be attributed to the interaction between damages. A comparison of cases 5a and 7a showed that the shorter the distance between the two damage locations, the greater the inﬂuence. The damage detection results for elements 10, 15, and 20 under different damage cases are shown in Figure 17. Figure Figure 3.3. Experimental Results and Analysis Model updating was used as described in Section 2. To limit the length of the paper, only parts of the results are shown here. The data of the undamaged case (which can be considered the simulation model) and damage case 1a are depicted here to illustrate the effect of damage identification. The modified EI values of the undamaged case and damaged degree of case 1a are presented in Tables 4 and 5, respectively. Under the undamaged circumstances, the model had been modified, which is shown as Table 4. By using the modified EI as a benchmark of undamaged structure, the experimental data which can obtain damaged d EI was adopted to identify damage which shown as Table 5. In particular, EI EI Δ is the percentage of damage degree. The results of the frequency response function are shown in Figures 13 and 14. Undamaged cases a and b had the same situation. Model updating was used as described in Section 2. To limit the length of the paper, only parts of the results are shown here. The data of the undamaged case (which can be considered the simulation model) and damage case 1a are depicted here to illustrate the effect of damage identiﬁcation. The modiﬁed EI values of the undamaged case and damaged degree of case 1a are presented in Tables 4 and 5, respectively. Under the undamaged circumstances, the model had been modiﬁed, which is shown as Table 4. By using the modiﬁed EI as a benchmark of undamaged structure, the experimental data which can obtain damaged EId was adopted to identify damage which shown as Table 5. In particular, ∆EI EIu is the percentage of damage degree. 14 of 20 Sensors 2018, 18, 3033 Table 4. Stiffness change of partial unit of undamaged case. Table 4. Stiffness change of partial unit of undamaged case. Element Number Unmodiﬁed EI Modiﬁed EI ∆EI 5 2.71 × 109 2.09 × 109 6.20 × 108 7 2.71 × 109 2.38 × 109 3.33 × 108 10 2.71 × 109 2.21 × 109 5.00 × 108 13 2.71 × 109 2.53 × 109 1.80 × 108 14 2.71 × 109 2.49 × 109 2.11 × 108 17 2.71 × 109 1.89 × 109 8.25 × 108 20 2.71 × 109 2.07 × 109 6.40 × 108 Table 5. Stiffness change of partial unit of case 1a. Table 5. Figure Figure 3.3. Experimental Results and Analysis As Figure 17a shows, the variation ratios of EI of the damaged elements were signiﬁcantly larger than those in the undamaged case. This ﬁnding indicated the effective detection of the introduced damage. In case 6a, elements 10 and 20 had damage severities of 5% and 10%, respectively, and element 15 had no damage, as shown in Figure 17a. The damage index of element 15 showed no noticeable change in comparison with that in the undamaged case. However, the damage index values of damaged elements 10 and 20 showed signiﬁcant increases when compared with those in the undamaged state. A comparison of cases 5a and 6a showed that the damage magnitude has only a small inﬂuence on the effect of damage identiﬁcation. Meanwhile, a comparison of cases 5a and 7a indicated that the shorter the distance between two damage locations, the greater the inﬂuence. Figure 17b shows the situation of Group B. The proposed approach was appropriate for detecting the damage severity in the single- and multi-damage situations. This method is thus attractive for detecting scouring damage in shield tunnels because various uncertainties and noises 15 of 20 Sensors 2018, 18, 3033 are involved in the modeling of shield tunnel structures and measuring their dynamic responses. However, the quantitative relation between the simulated damage and the actual damage is unclear and it should be discussed further. Sensors 2018, 18, x 16 of 21 , q g g it should be discussed further. Sensors 2018, 18, x 16 o (a) (b) (c) (d) (e) (f) (g) (h) Figure 15. Damage location results under moving load of Group A: (a) DMG1a; (b) DMG2a; (c) DMG3a; (d) DMG4a; (e) DMG5a; (f) DMG6a; (g) DMG7a; and, (h) DMG8a. Figure 15. Damage location results under moving load of Group A: (a) DMG1a; (b) DMG2a; (c) DMG3 (d) DMG4a; (e) DMG5a; (f) DMG6a; (g) DMG7a; and, (h) DMG8a. (b) (a) (b) (a) (c) (d) (d) (c) (f) (e) (f) (e) (g) (h) (h) (g) Figure 15. Damage location results under moving load of Group A: (a) DMG1a; (b) DMG2a; (c) DMG3a; (d) DMG4a; (e) DMG5a; (f) DMG6a; (g) DMG7a; and, (h) DMG8a. Figure 15. Damage location results under moving load of Group A: (a) DMG1a; (b) DMG2a; (c) DMG3a; (d) DMG4a; (e) DMG5a; (f) DMG6a; (g) DMG7a; and, (h) DMG8a. 4. Discussion and Conclusions 4. Discussion and Conclusions This work investigated the feasibility and effectiveness of using the acceleration frequency function to identify the structural damage in underground tunnel structures. Numerical and experimental studies were conducted. An aluminum pipe model with a simulated underground boundary condition was designed and tested in a large soil box. Different damage scenarios were simulated by cutting the pipe models into various damage sizes. The experimental studies performed covered the accelerated frequency response function used for damage identification in underground structures. The stiffness loss K Δ could be calculated after the appearance of ( ) EI Δ . Therefore, the location of the damage could be easily judged. Meanwhile, the damage magnitude had less influence on the effect of damage identification. Moreover, the shorter the distance between two damage locations, the greater the influence. Damage identification for underground structures based on the analysis of the frequency response function can be adopted as an efficient and functional damage identification tool in practical applications. However, the quantitative relation between the simulated da a e a d the a tual da a e hould be di u ed fu the This work investigated the feasibility and effectiveness of using the acceleration frequency function to identify the structural damage in underground tunnel structures. Numerical and experimental studies were conducted. An aluminum pipe model with a simulated underground boundary condition was designed and tested in a large soil box. Different damage scenarios were simulated by cutting the pipe models into various damage sizes. The experimental studies performed covered the accelerated frequency response function used for damage identiﬁcation in underground structures. The stiffness loss ∆K could be calculated after the appearance of ∆(EI). Therefore, the location of the damage could be easily judged. Meanwhile, the damage magnitude had less inﬂuence on the effect of damage identiﬁcation. Moreover, the shorter the distance between two damage locations, the greater the inﬂuence. Damage identiﬁcation for underground structures based on the analysis of the frequency response function can be adopted as an efﬁcient and functional damage identiﬁcation tool in practical applications. However, the quantitative relation between the simulated damage and the actual damage should be discussed further. g g Author Contributions: Conceptualization, H.L. Methodology, H.L. and S.W.; Software, S.W.; Validation, S.W.; Formal Analysis, S.W.; Investigation, S.W.; Resources, H.L. 4. Discussion and Conclusions 4. Discussion and Conclusions and X.L.; Data Curation, X.L.; Writing-Original Draft Preparation S W ; Writing-Review & Editing H L and X L ; Visualization H Z ; Supervision H Z ; Project Author Contributions: Conceptualization, H.L. Methodology, H.L. and S.W.; Software, S.W.; Validation, S.W.; Formal Analysis, S.W.; Investigation, S.W.; Resources, H.L. and X.L.; Data Curation, X.L.; Writing-Original Draft Preparation, S.W.; Writing-Review & Editing, H.L. and X.L.; Visualization, H.Z.; Supervision, H.Z.; Project Administration, H.L. and H.Z.; Funding Acquisition, H.L. and H.Z. g g Author Contributions: Conceptualization, H.L. Methodology, H.L. and S.W.; Software, S.W.; Validation, S.W.; Formal Analysis, S.W.; Investigation, S.W.; Resources, H.L. and X.L.; Data Curation, X.L.; Writing-Original Draft Preparation S W ; Writing-Review & Editing H L and X L ; Visualization H Z ; Supervision H Z ; Project Author Contributions: Conceptualization, H.L. Methodology, H.L. and S.W.; Software, S.W.; Validation, S.W.; Formal Analysis, S.W.; Investigation, S.W.; Resources, H.L. and X.L.; Data Curation, X.L.; Writing-Original Draft Preparation, S.W.; Writing-Review & Editing, H.L. and X.L.; Visualization, H.Z.; Supervision, H.Z.; Project Administration, H.L. and H.Z.; Funding Acquisition, H.L. and H.Z. epa atio , S.W.; W iti g eview & Editi g, . . a d . .; Visua i atio , . .; Supe visio , . .; oject Administration, H.L. and H.Z.; Funding Acquisition, H.L. and H.Z. Funding: This research was funded by [the Natural Science Fund of China] grant number [51578260]; [the Funding: This research was funded by [the Natural Science Fund of China] grant number [51578260]; [the Natural Science Fund of China] grant number [51578261]; [Major technical innovation projects in Hubei] grant number [2017ACA183]; [Outstanding youth project of Hubei Natural Science Foundation] grant number [2017CFA074]. Natural Science Fund of China] grant number [51578261]; [Maj number [2017ACA183] [Outstanding youth project of Hube Conﬂicts of Interest: The authors declare no conﬂict of interest. Natural Science Fund of China] grant number [51578261]; [Major technical innovation projects in Hubei] number [2017ACA183]; [Outstanding youth project of Hubei Natural Science Foundation] grant nu Conﬂicts of Interest: The authors declare no conﬂict of interest. Figure Figure 3.3. Experimental Results and Analysis 16 of 20 17 of 21 Sensors 2018, 18, 3033 Sensors 2018 18 x nsors 2018, 18, 3033 16 o ensors 2018, 18, x 17 (a) (b) (c) (d) (e) (f) (g) (h) Figure 16. Damage location results under moving load of Group B: (a) DMG1b; (b) DMG2b; (c) DMG3b; (d) DMG4b; (e) DMG5b; (f) DMG6b; (g) DMG7b; and, (h) DMG8b. Figure 16. Damage location results under moving load of Group B: (a) DMG1b; (b) DMG2b; (c) DMG3b; (d) DMG4b; (e) DMG5b; (f) DMG6b; (g) DMG7b; and, (h) DMG8b. (b) (a) (a) (b) (c) (d) (d) (c) (f) (e) (f) (e) (g) (h) (h) (g) Figure 16. Damage location results under moving load of Group B: (a) DMG1b; (b) DMG2b; (c) DMG3b; (d) DMG4b; (e) DMG5b; (f) DMG6b; (g) DMG7b; and, (h) DMG8b. Figure 16. Damage location results under moving load of Group B: (a) DMG1b; (b) DMG2b; (c) DMG3b; (d) DMG4b; (e) DMG5b; (f) DMG6b; (g) DMG7b; and, (h) DMG8b. 17 of 20 18 of 21 Sensors 2018, 18, 3033 Sensors 2018 18 x (b) (a) (a) (b) Figure 17. Damage location results of all cases: (a) Group A; and, (b) Group B. Figure 17. Damage location results of all cases: (a) Group A; and, (b) Group B. (a) (a) Figure 17. Damage location results of all cases: (a) Group A; and, (b) Group B Figure 17. Damage location results of all cases: (a) Group A; and, (b) Group B. [2017CFA07 C fli f References Conflicts of Interest: The authors declare no conflict of interest. References 1. Feng, L.; Yi, X.H.; Zhu, D.P.; Xie, X.Y.; Wang, Y. 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https://openalex.org/W2147132534	https://link.springer.com/content/pdf/10.1007/s11673-014-9564-x.pdf	English	null	East–West Differences in Perception of Brain Death	Journal of bioethical inquiry	2,014	public-domain	11,515	East–West Differences in Perception of Brain Death Review of History, Current Understandings, and Directions for Future Research Qing Yang & Geoffrey Miller Received: 6 July 2013 /Accepted: 5 December 2013 /Published online: 24 July 2014 # Journal of Bioethical Inquiry Pty Ltd 2014 Abstract The concept of brain death as equivalent to cardiopulmonary death was initially conceived following developments in neuroscience, critical care, and trans- plant technology. It is now a routine part of medicine in Western countries, including the United States. In con- trast, Eastern countries have been reluctant to incorporate brain death into legislation and medical practice. Several countries, most notably China, still lack laws recognizing brain death and national medical standards for making the diagnosis. The perception is that Asians are less likely to approve of brain death or organ transplant from brain dead donors. Cultural and religious traditions have been referenced to explain this apparent difference. In the West, the status of the brain as home to the soul in Enlightenment philosophy, combined with pragmatism and utilitarianism, supports the concept of brain death. In the East, the integration of body with spirit and nature in Buddhist and folk beliefs, along with the Confucian social structure that builds upon interpersonal relation- ships, argues against brain death. However, it is unclear whether these reasoning strategies are explicitly used when families and medical providers are faced with acknowledging brain death. Their decisions are more likely to involve a prioritization of values and a rationalization of intuitive responses. Why and whether there might be differences between East and West in the acceptance of the brain death concept requires further empirical testing, which would help inform policy- making and facilitate communication between providers and patients from different cultural and ethnic backgrounds. Keywords Brain death . Culturaldifferences . Religion . Philosophy. Asia Keywords Brain death . Culturaldifferences . Religion . Philosophy. Asia Bioethical Inquiry (2015) 12:211–225 DOI 10.1007/s11673-014-9564-x Bioethical Inquiry (2015) 12:211–225 DOI 10.1007/s11673-014-9564-x ORIGINAL RESEARCH Introduction Human death triggers a series of emotional, social, and legal ramifications such as grieving, funeral arrange- ments, division of estate, succession of public office, discontinuation of resuscitative efforts, and removal of organs. It is thus a required social necessity for there to be agreement on when death has occurred and when we can declare that death has occurred (Miller 2011). The determination of death and its declaration relies as much on biological knowledge as on social consensus and communal acceptance. For the majority of history, death has been identified by the irreversible absence of a heartbeat and respiration. Brain death, defined as the permanent and irreversible loss of function in the brain as a whole or in the brain stem in some jurisdictions (such as the United Kingdom), emerged as an alternative criterion in the mid-20th century. The brain death doc- trine serves two purposes: (1) the disposition of venti- lated patients with the diagnosis and (2) the potential for Q. Yang Department of Pediatrics, Yale University School of Medicine, New Haven, CT, USA G. Miller (*) Yale University and Yale Bioethics Center, New Haven, CT, USA e-mail: geoffrey.miller@yale.edu Bioethical Inquiry (2015) 12:211–225 212 then to allow a declaration of death when irreversible total brain failure is reliably determined might be viewed as a value judgment. But it is not for the physi- cian to enter into a philosophical discussion on the meaning of death with the patient’s relatives. We recog- nize that the physician’s skillful communication often follows the logic of: (1) There is no evidence of func- tional brain activity; (2) this condition is permanent; and (3) this allows a declaration of death. To some this supports a fiction. It also leaves the physician open to accusations of paternalism and being disingenuous. Lederer writes: “[P]hysicians would benefit from a more coherent and defensible ethical account of vital organ donation” (Lederer 2008, 3). organ donation from an individual with a beating heart (Truog 1997). It was not introduced as an attempt to redefine death, but as a way of determining death that is acceptable as equivalent to the cardiopulmonary criteri- on, especially when the use of ventilators obscures accurate evaluation of cardiopulmonary function (Bee- cher 1968; President’s Commission 1981; Miller and Ashwal 2010). Introduction Since the middle of the last century, it has been widely validated and become a routine part of medical practice in the United States as well as other Western countries (Gardiner et al. 2012; Wijdicks 2002). Ethical issues associated with the concept of brain death have been extensively reviewed elsewhere (for example, President’s Council 2011). These include the role of the brain in defining the biological and philo- sophical meanings of life, the value of brain death as either a real physiological state or an artificial construct, the truthfulness of the belief that equates brain death with the end of life, the justice of minimizing futile medical care, and the social utility of providing organs for transplant. Although it is not the aim of this article to justify the use of the brain death concept, we will briefly review, from the start, some of the ethical arguments concerning its application. We suggest that the acceptance of the notion of brain death may rest on, among other factors, the fiduciary relationship between physicians, patients, and their sur- rogates, as well as the fiduciary relationship between physicians and the state. If the surrogates trust the physi- cians and their methods and trust that death is inevitable, they are more likely to accept a declaration of death when informed that total brain failure or brain death has oc- curred. The arrangement for organ donation, although an important consideration, is not the only good that can occur with the acceptance of the brain death doctrine. It allows the removal of artificial ventilation and the ar- rangements for the procedures that take place following death. This practice of the brain death doctrine also depends on the fiduciary relationship between the state and the medical profession. For example, in the United States, the state, through the legislature, acknowledges death when there is irreversible cessation of either car- diopulmonary function or whole brain function, includ- ing the brain stem. The determination of these conditions is the prerogative of the medical profession. So it can be claimed that the acceptance of a declaration of death when irreversible whole brain failure occurs is a matter of public policy and a socio-medico-legal contrivance (Nair-Collins 2010). Introduction In other cases—such as brain tumors, skull fractures, or intracranial hemor- rhage—the patient ultimately succumbed to circulatory arrest hours following respiratory arrest (Horsley 1894). The first electroencephalography (EEG) recordings in humans coincided with the discovery that death on the cellular level was attributed to the loss of the cell mem- brane electrical potential (Crile, Telkes, and Rowland 1930). Radiographic cerebral angiograms further corre- lated the fall in EEG activity to lack of blood flow to the brain (Crafoord 1939). These technological advances established ways in which death of the nervous system could be detected and characterized. Wertheimer icono- clastically proposed to equate this form of death to the conventional cardiopulmonary death (Wertheimer, Jouvet, and Descotes 1959). about how cultural contexts influence the perception and application of brain death, because unlike the uni- versally upheld cardiopulmonary standard, practice of the neurological standard of death lacks a global con- sensus (Wijdicks 2002). We aim to provide an exposi- tory comparison of the reception and adaptation of brain death in two culturally distinct societies: East and West. y For the purpose of this study, we define the two cultural camps with somewhat arbitrarily demarcated but readily recognizable geographical areas. We think of the West as North America, Western and Northern Europe (original members of the European Union), plus Australia and New Zealand. These 22 countries are largely populated by Caucasian diasporas and share a strong philosophical and political platform stemming from Greek, Roman, and Enlightenment traditions. On the other hand, by East we mean East and Southeast Asia, including China, Japan, South Korea, Taiwan, and the constituents of Association of Southeast Asian Na- tions (ASEAN). These 14 countries contain Chinese diasporas and have been for the majority of their history under the influence of a cultural heritage that has devel- oped independent of the West. Larger cohorts of putative brain dead patients were not reported until intensive care units (ICU) sprang up in hospitals across Europe and North America (Lofstedt and von Reis 1956). Initially developed during the polio epidemics, artificial ventilation—negative pressure chambers, positive pressure bag-valve systems, and fi- nally mechanical ventilators—was used to sustain pa- tients with respiratory failure (Puri, Puri, and Dellinger 2009; Settergren 2003). It was noted that a particular group of patients, usually with primary neurological pathology, never regained spontaneous respiration (Lofstedt and von Reis 1956). Introduction But there are strong arguments to support the forgoing of life-sustaining treatments from individuals with permanent whole brain failure, and it is neither disrespectful to the individual nor a great moral harm to declare death when this irreversible state is recognized. Overall, mainstream Western society ac- knowledges that the concept of brain death is morally reasonable, operationally useful, and not intuitively of- fensive (Miller 2011; Gardiner et al. 2012). H f i f f th di i th thi l When we consider the concept of brain death as equivalent to cardiopulmonary death, there are valid criticisms that highlight the flaws in the brain death doctrine (Shewmon 2001; Nair-Collins 2010). Most people do not have difficulty in appreciating that death exists. The debate concerns when it occurs, how it is recognized, and when and how it may be declared. In the biological and medical realm we debate the nature and recognition of death, and in the sociocultural, polit- ical, and legal realms it is the acceptability of the decla- ration of death that comes into play. In the case of brain death, those involved are asked to accept that an indi- vidual with an irreversible loss of functional whole brain activity would be dead without artificial ventilation, and spontaneous ventilation will inevitably fail. The skepti- cal may say that the individual is not yet dead, but will be once the artificial ventilation is removed. Or they may not believe that the individual is dead by what they can see and feel, which is not a corpse, and they would be correct. The features that determine when brain death may be declared are not identical to how a typical individual views a biological death. It is for this reason that, in the United States, the President’s Council on Bioethics recommended the use of the term “total brain failure” rather than brain death (2011). The judgment Here we refrain from further discussion on the ethical righteousness of the concept. Instead we are curious Bioethical Inquiry (2015) 12:211–225 213 care, and organ transplant (Table 4). As early as the late 19th century, several neurosurgeons observed a cessa- tion of respiration with continued heartbeat in patients with increased intracranial pressure (ICP) (Settergren 2003). In some cases—such as brain abscesses—the respiratory arrest was temporary. Introduction They lacked cranial nerve reflexes, required norepinephrine infusion to maintain blood pressure, failed to show intracranial filling on carotid angiography, and did not exhibit any EEG ac- tivity in the cortex or diencephalon (Lofstedt and von Reis 1956; Mollaret and Goulon 1959; Wertheimer, Jouvet, and Descotes 1959). If ventilation was stopped, they rapidly developed cardiac arrest. Even with contin- ued artificial ventilation, the majority of these patients had cardiac arrest within days (Jennett, Gleave, and Wilson 1981). In a 1959 landmark paper, Mollaret and Goulon termed this state of irreversible coma and apnea “coma dépassé” (Mollaret and Goulon 1959). Following a description of the medical and historical backgrounds, we embark on an inquiry into whether and why there is a difference between the acceptance of brain death in the East and the West. We examine the legal status of brain death, the existence and stringency of medical guidelines for declaring brain death, and the actual incidence of confirmed brain death diagnoses and brain dead organ donors. We also summarize previous studies surveying public opinions about brain death in Eastern and Western countries. Next we review current understandings regarding the source of such behavioral differences, which have focused on the philosophical and religious arguments deemed characteristic of each culture, in support for or against brain death. Lastly, we speculate additional explanations for the observed dif- ferences beyond the perspicuous cultural traditions. We offer suggestions for ways to investigate these new hypotheses and briefly address how improved knowl- edge in this topic can benefit policy-making and pro- mote culturally sensible medical practice. Historical Perspective Advances in organ transplant called upon the appli- cability of the brain death concept to medical practice. Following Carrel’s improved vascular anastomosis, or- gan transplant stumbled through decades of Evolution of the brain death concept is fueled by prog- ress in three areas of medicine: neuroscience, critical Bioethical Inquiry (2015) 12:211–225 214 unsuccessful attempts until 1954, when the kidney transplant between a pair of identical twins unveiled the crucial role of immunity and tissue typing (Starzl 1964). Development of immunosuppressive therapy, including calcineurin inhibitors, enabled solid organ transplant to take off. Successful homologous transplan- tations of liver (Starzl et al. 1963), lung (Hardy and Webb 1963), and pancreas (Kelly et al. 1967) occurred within a few years of each other. Enthusiasm and antic- ipation culminated in the climactic first successful heart transplant (Barnard 1967), a procedure soon replicated throughout the developed world. Even though Barnard’s donor, who was presumed brain dead, was disconnected from the ventilator and sustained several minutes of cardiac arrest before organ retrieval, discussions for using brain dead patients as organ sources was inevita- ble. Indeed, a few years earlier, Alexandre had per- formed the first kidney transplant from a “heart-beating” donor determined to be brain dead based on Mollaret and Goulon’s criteria (Machado 2005). Graft ischemia and time to graft function proved superior to cardiac death donors. A sense of urgency surged regarding the status of the brain dead and a consensus was reached quickly in European and North American medical com- munities. Brain death was officially recognized as hu- man death by the World Medial Association which convened in Sydney in 1968 (Korein 1978; Machado et al. 2007b). This recognition justified the withdrawal of mechanical ventilation. The continuation of respira- tory support was recommended only for perfusing or- gans awaiting procurement (Settergren 2003). In the same year, an Ad Hoc Committee at Harvard published the medical criteria that have guided brain death diag- nosis ever since (Beecher 1968). took the first step to legally permit brain death in the United States. The law was introduced by a physician- legislator and approved without substantial debate (Foley 2011). Finland and Portugal pioneered the Euro- pean trend in 1971 (Haupt and Rudolf 1999; Wijdicks 2002). Following a report written by President Regan’s Bioethics Commission in 1981, the Uniform Determi- nation of Death Act (UDDA) was drafted and approved by all U.S. states. Historical Perspective In the same year, an Ad Hoc Committee at Harvard published the medical criteria that have guided brain death diag- nosis ever since (Beecher 1968). In recent years, debates about brain death in the West have re-emerged and involve the question of whether it is ever sufficient to declare someone dead based on a Fig. 1 East lags behind the West in terms of the timing of brain death legislation Although organ transplant gave impetus to the estab- lishment of the brain death standard, some argue that the intersection between the two was by and large coinci- dental (Machado et al. 2007a; Settergren 2003). Others perceive brain death as an artificial entity solely con- structed for the sake of organ procurement (Sharp 2006; Greenberg 2001). Historical Perspective Authors of the report metaphorically described the brain-oriented criteria as a new window to access the “deeper and more complex reality” of death when technology undermines the validity of traditional vital signs (President’s Commission 1981, 33). The United Kingdom focused on the role of the brain stem in maintaining life-sustaining functions and adopted a standard where death could be declared with “perma- nent functional death of the brain stem” regardless of cortical activities (Anonymous 1976, 1187; Pallis 1982). By the early 1990s, all 22 Western countries had enacted brain death laws and issued medical guide- lines on brain death determination (Haupt and Rudolf 1999) (Fig. 1). Other world regions with a strong West- ern legacy, such as Latin America, also have legislated in favor of brain death (Escudero et al. 2009). unsuccessful attempts until 1954, when the kidney transplant between a pair of identical twins unveiled the crucial role of immunity and tissue typing (Starzl 1964). Development of immunosuppressive therapy, including calcineurin inhibitors, enabled solid organ transplant to take off. Successful homologous transplan- tations of liver (Starzl et al. 1963), lung (Hardy and Webb 1963), and pancreas (Kelly et al. 1967) occurred within a few years of each other. Enthusiasm and antic- ipation culminated in the climactic first successful heart transplant (Barnard 1967), a procedure soon replicated throughout the developed world. Even though Barnard’s donor, who was presumed brain dead, was disconnected from the ventilator and sustained several minutes of cardiac arrest before organ retrieval, discussions for using brain dead patients as organ sources was inevita- ble. Indeed, a few years earlier, Alexandre had per- formed the first kidney transplant from a “heart-beating” donor determined to be brain dead based on Mollaret and Goulon’s criteria (Machado 2005). Graft ischemia and time to graft function proved superior to cardiac death donors. A sense of urgency surged regarding the status of the brain dead and a consensus was reached quickly in European and North American medical com- munities. Brain death was officially recognized as hu- man death by the World Medial Association which convened in Sydney in 1968 (Korein 1978; Machado et al. 2007b). This recognition justified the withdrawal of mechanical ventilation. The continuation of respira- tory support was recommended only for perfusing or- gans awaiting procurement (Settergren 2003). Reception of Brain Death in East and West Another stream of criticism con- cerns the rare but well-documented cases of “chronic brain death” (Shewmon 1998b). Patients, who were often children or infants when declared brain dead, have continued to subsist on ventilatory and nutritional sup- port for weeks, even years, suggesting preservation of coordinated functioning in multiple organ systems. President Obama’s Council on Bioethics revisited brain death in 2008. The Council refuted cortical death on the grounds that such a proposal would generate two deaths—death of the personhood and death of the hu- man organism (President’s Council 2011). The Council upheld the present brain death concept, preferring to use the term “total brain failure” and affirming that loss of both consciousness and spontaneous breathing consti- tutes death. The brain dead state represents the lack of the need, ability, and drive to be receptive to the world and perform self-preserving measures as a whole organ- ism. The Council did not consider patients in “chronic brain death” alive and argued for the withdrawal of interventions. Thus opinions challenging the brain death criteria remain but are in the minority in the West. A closer look at the guidelines of individual countries suggests that Asians have stricter criteria for brain death determination (Haupt and Rudolf 1999; Wijdicks 2002; Devathasan 1985; Kadir 2006; Chen 2000). Seventy- five percent of Eastern countries require two or more physicians to conduct the brain death examination, com- pared to 45 percent of Western countries. Guidelines from the East also tend to require physicians trained in neurological or intensive care specialties and confirma- tion by physicians not directly involved in the care of the particular patient. All of the eight Eastern countries with guidelines have specified a mandatory observation time between the first and second brain death examinations. The average length is 13.5±3.15 hours (mean±standard error of measurement), the shortest being six hours. In the West, 14 countries have mandated observation time, averaging 7.0±1.56 hours, the shortest being two hours. If the countries that do not mandate observation time were included in the calculation, the average would be 4.45±1.29 hours, roughly one-third of what is required in the East. Interestingly, the portion of countries man- dating confirmatory tests for brain death diagnosis was the same in East and West. This may stem from limita- tions in the availability of technology and trained per- sonnel to carry out these tests. Reception of Brain Death in East and West After publication of the Harvard Report, brain death was accepted into Western medicine with relatively little controversy (Wikler 1993; Bowman and Richard 2003; Ohnuki-Tierney et al. 1994). In 1970, Kansas Fig. 1 East lags behind the West in terms of the timing of brain death legislation Bioethical Inquiry (2015) 12:211–225 215 defunct cerebral cortex leading to a social or philosoph- ical death. Proponents argue that permanent loss of higher brain functions, particularly consciousness and mental capacities, should constitute human death (Devettere 1990; Ray 1991). However, it seems clear that the burial or cremation of a body with spontaneous respiration and a heartbeat is not acceptable, neither is abolishing the dead donor rule, an ethical code requiring donors to be declared dead before organ harvest (Truog and Robinson 2003). Another stream of criticism con- cerns the rare but well-documented cases of “chronic brain death” (Shewmon 1998b). Patients, who were often children or infants when declared brain dead, have continued to subsist on ventilatory and nutritional sup- port for weeks, even years, suggesting preservation of coordinated functioning in multiple organ systems. President Obama’s Council on Bioethics revisited brain death in 2008. The Council refuted cortical death on the grounds that such a proposal would generate two deaths—death of the personhood and death of the hu- man organism (President’s Council 2011). The Council upheld the present brain death concept, preferring to use the term “total brain failure” and affirming that loss of both consciousness and spontaneous breathing consti- tutes death. The brain dead state represents the lack of the need, ability, and drive to be receptive to the world and perform self-preserving measures as a whole organ- ism. The Council did not consider patients in “chronic brain death” alive and argued for the withdrawal of interventions. Thus opinions challenging the brain death criteria remain but are in the minority in the West. defunct cerebral cortex leading to a social or philosoph- ical death. Proponents argue that permanent loss of higher brain functions, particularly consciousness and mental capacities, should constitute human death (Devettere 1990; Ray 1991). However, it seems clear that the burial or cremation of a body with spontaneous respiration and a heartbeat is not acceptable, neither is abolishing the dead donor rule, an ethical code requiring donors to be declared dead before organ harvest (Truog and Robinson 2003). Reception of Brain Death in East and West Overall, this apparent tighter stringency in the East may reflect a more cautious stance by the medical community toward brain death. Fewer brain deaths have been confirmed in the East than the West. In the United States, it is estimated that 15,000–20,000 people are declared dead by the brain death criteria each year, equivalent to an incidence rate of 48–64 per million population and accounting for about 1 percent of all death (Swerdlow 2004). In the United Kingdom, the most recent available data showed 992 confirmed brain deaths in 2010, corresponding to 16 per million (Murphy and Smith 2012). In the same year, 32 people were declared brain dead in Japan, a mere 0.25 per million (Committee on Organ Transplant 2012). Of note, this was already a significant increase compared to seven brain deaths in the previous year, due to a legislative revision in early 2010, which expanded the brain death criteria to children and relaxed the re- quirement for familial consent (Ikka and Ikegaya 2010). Even though China does not yet legally recognize brain death, a televised brain death exam on a 61-year-old Wuhan man with brain stem hemorrhage in 2003 was reported as the first officially diagnosed case (Song 2003; Niu 2011). Between 2003 and 2009, approxi- mately 200 brain death diagnoses based on individual In contrast, there has been a marked delay in legally recognizing brain death as human death in the East. Malaysia was the first to pass a brain death legislation in 1993, 25 years after publication of the Harvard Report (Wijdicks 2002). Japan, while as technologically ad- vanced as its Western trade partners, did not have a brain death law until 1997. During Japan’s three decades of debate, several medical teams were accused of murder for performing transplants using brain dead donors (Feldman 1988; Ohnuki-Tierney et al. 1994; Watanabe 2000). The law was a compromise acknowledging brain death only as a premise for organ donation. As of 2013, only six of the 14 Eastern countries legally recognize brain death. Eight have diagnostic guidelines issued by national medical authorities. Six countries, including China, the most populous country and most powerful economic player in Asia, have neither a law nor a guideline. Bioethical Inquiry (2015) 12:211–225 216 countries (43 percent vs. 60–71 percent) (Minemura, Yamaoka, and Yoshino 2010). Reception of Brain Death in East and West A similar study in Ning- bo, China, showed a 33 percent approval rate for brain death (Yu and Xing 2009). A significant fraction of the Asian respondents—29 percent in Japan and 57 percent in China, compared to 6–19 percent of Westerners sur- veyed concurrently—either had not heard of brain death or were not sure what the term meant. Consistent with the above trend, 98 percent of 1,351 randomly selected Ohio residents in the United States have heard of brain death (Siminoff, Burant, and Youngner 2004). Only 16 percent of this study population thought someone de- clared brain dead was alive. Furthermore, this study included three clinical scenarios representing brain death, coma, and persistent vegetative state (PVS) and asked the respondents to make decisions about declara- tion of death, withdrawal of life support, and organ donation; 86 percent correctly identified a patient meet- ing the brain death criteria as dead. hospital protocols were reported, averaging 30 annually, or 0.02 per million (Zhang et al. 2011). hospital protocols were reported, averaging 30 annually, or 0.02 per million (Zhang et al. 2011). In the United States, among those declared brain dead each year 10,500–13,800 are estimated to have no contraindication to organ donation, and more than half become donors (Sheehy et al. 2003). The number of deceased donors in 2011 was 8,126, with 7,701, or 87 percent, being donations after brain death (UNOS 2013; European Committee on Organ Transplantation 2012). In the United Kingdom, 652 brain dead donors were documented in fiscal year 2011–2012, accounting for 60 percent of all deceased donors (NHS Blood and Transplant 2012). Japan’s first official brain dead patient was a 40-year-old Kochi Prefecture woman with a rup- tured brain aneurysm in 1999, two years after the pass- ing of the brain death law (Kochi Shinbunsha 2000). In the 13 years that followed, Japan has had a total of 198 brain death diagnoses, 197 of which led to organ pro- curement (Committee on Organ Transplant 2012). The peculiar legal standard that necessitates brain death ex- amination solely as a premise to organ donation explains the exceptionally high conversion rate (Bagheri 2003). Over this time period, brain dead donors initially accounted for less than 5 percent of the entire deceased donor pool. The proportion dramatically increased to 40 percent after the 2010 revision (Committee on Organ Transplant 2012). Reception of Brain Death in East and West In China, 61 brain dead donors were reported between 2003 and 2009 (Zhang et al. 2011). Donations after brain death (DBD) account for a negli- gible portion of all deceased donors for organ transplant in China, where death-sentenced prisoners serve as the main organ source (Chen 2011; Zhou and Dou 2011). A form of controlled donation after cardiac death (DCD), termed donation after brain death plus cardiac death (DBCD), is occasionally employed to circumvent the legal obstacles to allow organ procurement from pre- sumed brain dead patients (Chen 2011). Tables 1 and 2 compares the epidemiology of brain death and DBD in selected Western and Eastern countries. In summary, these data indicate that Eastern countries have been reluctant to adopt the concept of brain death into legal and medical practice. Among hospitalized patients in Kunming, China, 66 percent believed a brain dead person to be still alive (Wang et al. 2013), as did 40 percent of professionals and government officials in Guangdong (Song et al. 2009). Even among the Chinese hospital staff, only 35 percent accepted brain death (Sun, Wang, and Gao 2005). These studies suggest that the general public in the East are less likely to concur that brain death is human death. Confusion and misunderstanding about the concept may contribute to their apparent resistance. West In Taiwan, similar to Japan, brain death only applies to organ donors. In addition, brain death exams are performed for a handful of patients each year who request palliative care measures. All others are declared dead by the traditional cardiopulmonary standard (Y-J. Chiang, CEO of TORSC, pers. comm.) b Estimated based on DBD donor numbers and an acceptance rate of 64 percent for organ donation solicitations reported in Lee et al. (2009) a Estimated based on DBD donor rates. In Taiwan, similar to Japan, brain death only applies to organ donors. In addition, brain death exams are performed for a handful of patients each year who request palliative care measures. All others are declared dead by the traditional cardiopulmonary standard (Y-J. Chiang, CEO of TORSC, pers. comm.) (Lo 2012, S6). Rhetoric surrounding brain death has frequently involved comparing the philosophical and religious traditions in the two hemispheres (Table 3). relationship between the soul and the body (President’s Commission 1981). Irreversible loss of brain function, demonstrating cessation of the mind’s coexistence with the physical body, indicates death of the person. One transplant surgeon summarized brain death as “perma- nent loss of integrative functions and consciousness with- out a chance of returning to meaningful life” (Pratschke et al. 1999, 344). Consistent with this brain-centered definition of life, the majority of Europeans consider life without cognitive capacity not worth living (Demertzi et al. 2011), and many U.S. residents believe that a comatose or vegetative patient is dead (Siminoff, Burant, and Youngner 2004). Rationality and the dichotomy between body and soul have been identified as two principles of Western philos- ophy supporting brain death (Bowman and Richard 2003). The idea of the soul existing separately from the body traces back to Socrates (Devettere 1990; Ray 1991). Plato considered the soul as a pure spiritual existence temporarily imprisoned in the body. Descartes further identified the ability to think as the essence of person- hood, equating the soul with the conscious mind and the body with an organic machine (Bowman and Richard 2003; Shewmon 1998a). As the seat of rational thoughts, the brain occupies a prominent place in Western philos- ophy (Ohnuki-Tierney et al. 1994; Korein 1978). This neuro-essentialism can be expressed as “we are our brains” (Reiner 2011, 1; Roskies 2002). The rest of the physical body has been devalued (Bowman and Richard 2003). West What might explain this apparent difference in accep- tance of brain death between East and West? Could it be that the concept of brain death, originally conceived by Westerners out of their technological advances, is inher- ently incongruent with the East? As one anthropologist put it: “[A]lthough it is framed as if it were highly scientific and thus culture-free, the notion of brain death is culturally constructed” (Ohnuki-Tierney et al. 1994, 234). Many have speculated about this theory, which often appears in the form of overarching statements such as: “The concept of brain death is not fully accepted in China due to long-standing cultural traditions” (Zhang et al. 2011, 1423) and “Cultural resistance is a real issue” Public opinions further elucidate the difference in the integration of brain death into Western and Eastern societies. In a study surveying large demographically representative samples from Japan, the United States, the United Kingdom, Germany, and France, significant- ly fewer people considered brain death as an appropriate standard for human death in Japan than in the Western 217 Bioethical Inquiry (2015) 12:211–225 Bioethical Inquiry (2015) 12:211–225 Table 1 Epidemiology of brain death in select countries Country Year Confirmed brain deaths Brain deaths per million population per year Source West United States 2012 ~15,000–20,000 48–64 (UNOS 2013; Swerdlow 2004) United Kingdom 2010 992 15.9 (Murphy and Smith 2012) France 2011 3,174 48.7 (Agence de la Biomedecine 2011) Spain 2008 2,478 55.4 (Matesanz et al. 2011) Canada 2011 2,425 72.4 (Canadian Organ Replacement Register 2011) Australia 2012 ~790 34.9 (Organ and Tissue Authority 2012) East Japan 2010 32 0.25 (The Committee on Organ Transplant 2012) 1997–2012 198 0.098 PR China 2003–2009 ~200 0.019 (Zhou and Dou 2011) Taiwan 2012 ~200a 8.58 (TORSC 2012) Singapore 2007 86 18.7 (Kwek et al. 2009) Korea 2007 ~250b 4.76 (KNOS 2011; Lee et al. 2009) a Estimated based on DBD donor rates. In Taiwan, similar to Japan, brain death only applies to organ donors. In addition, brain death exams are performed for a handful of patients each year who request palliative care measures. All others are declared dead by the traditional cardiopulmonary standard (Y-J. Chiang, CEO of TORSC, pers. comm.) b Estimated based on DBD donor numbers and an acceptance rate of 64 percent for organ donation solicitations reported in Lee et al. (2009) Table 1 Epidemiology of brain death in select countries a Estimated based on DBD donor rates. West The brain as the integrator and regulator of bodily functions, one of the theoretical foundations of the initial brain death proposal, also stemmed from the master–tool Life as a synthesis of body and soul is also instilled through the monotheist Western religions. Life is defined through individuality and personhood, carried in distinct- ly brain-oriented abilities to believe, make decisions, feel, and interact with the world (Grodin 1994; Arbour, AlGhamdi, and Peters 2012). A body without the soul is no longer a person (Bowman and Richard 2003). The term “physiological decapitation” has been used to de- scribe brain death in Christian, Judaic, and Muslim Bioethical Inquiry (2015) 12:211–225 218 Table 2 Epidemiology of brain death in select countries Country Year Brain dead donors Brain dead donors as % of all deceased donors Source West United States 2011 7,071 87 % (European Committee on Organ Transplantation 2012) United Kingdom 2010 652 60 % (NHS Blood and Transplant 2012) France 2011 1,572 96 % (Agence de la Biomedecine 2011) Spain 2011 1,550 93 % (European Committee on Organ Transplantation 2012) Canada 2011 466 88 % (European Committee on Organ Transplantation 2012; Canadian Organ Replacement Register 2011) Australia 2012 277 78 % (Organ and Tissue Authority 2012) East Japan 1997 4 5 % (The Committee on Organ Transplant 2012) 2012 38 40 % 1997–2012 197 14 % PR China 2003–2009 61 <0.1 % (Zhou and Dou 2011) Taiwan 2012 193 100 %a (TORSC 2012) Singapore 2007 26 100 %b (Kwek et al. 2009) Korea 2000 52 41 % (Min et al. 2010; KNOS 2011) 2011 368 73 % a In Taiwan, only brain dead donors have been used for transplant. No DCD cases have been reported to date (Y-J. Chiang, CEO of TORSC, pers. comm.) b In 2007, Singapore’s Human Organ Transplant Act only allowed DBD. This was changed in 2009 when a revision of the law allowed DCD Table 2 Epidemiology of brain death in select countries have endorsed the concept of brain death (Bernat 2005). As early as 1957, Pope Pius XII affirmed that physicians have the ultimate authority to set the criteria for death. He vernaculars (Grodin 1994; Bernat 2005; Fins 1995; Niu 2011). With the exception of Orthodox Jews who main- tain that the soul resides in the heart, all three religions vernaculars (Grodin 1994; Bernat 2005; Fins 1995; Niu 2011). West In a Japanese documentary, the wife of a man who is presumed brain dead from an intracra- nial hemorrhage complained of feeling uncomfortable with designating the time of death as at the end of the second brain death exam. She refused the brain death exam in order to “see him off in a natural way” (Yanagida 2012). also urged against futile attempts at prolonging life in “hopeless situations” (Korein 1978). Pope John Paul II upheld the whole brain criteria for neurological death (Bresnahan and Mahler 2010). Brain death was accepted by the Chief Rabbinical Councils of Israel and America in 1986 and 1991, respectively (Dorff 2005; Grodin 1994). The Islamic Fiqh Academy permitted the decla- ration of death with irreversible loss of whole brain function in 1981. The Council of Islamic Jurisprudence officially designated brain death as death in 1986 (Arbour, AlGhamdi, and Peters 2012). Doctrines of pragmatism and utilitarianism, hall- marks of the industrial West, further provide support for brain death by giving it “great instrumental value” to the society (Fins 1995, 36). Margaret Lock, who published the seminal work comparing the reception of brain death in America and Japan, judged the reasons for establishing the brain death criteria in the Harvard Re- port as essentially pragmatic: to reduce the burden of futile medicine and to facilitate organ procurement (Lock 1996, 2002). Obsession with planning and con- trolling one’s body and life events is another prominent characteristic of modern Western society, which mani- fests in brain death as a way to control the timing and manner of death (Lock 1996; Bowman and Richard 2003). It is desirable and necessary to know when death may be declared, and time and technology affected this. Interestingly, in the recent debates surrounding brain death legislation in China, the proponents have been vocalizing traditionally Western arguments emphasizing rationality, utility, and societal benefits (Huang, Mao, and Millis 2008; Chen 2002; Ouyang 2004; Wang 2003). On the other hand, the opposition has been quoting originally Eastern beliefs and traditions. For example, Buddhist beliefs about reincarnation and warnings against mutilation of the body have been used to explain Asians’ resistance toward organ transplanta- tion. However, the prosperity of organ transplant pro- grams in China despite scarcity of brain dead donors suggest that a society’s resistance toward brain death does not necessarily correlate with its aversion to trans- plant or removal of organs. West With the exception of Orthodox Jews who main- tain that the soul resides in the heart, all three religions Table 3 Postulated differences in philosophical and religious be- liefs of East and West West East Clear body/soul separation Integrated view of life consisting of body, spirit, and nature Brain is the dominant organ and location of command and integration functions No obvious dominant body part Soul lives in the brain Spirit is distributed throughout the body Life can be explained by physical laws Life is mysterious Clear boundary between life and death Ambiguous transition from life to death Life is to be controlled and planned Life is to be awed Desire to control the mode and timing of death Desire to let death come naturally Values autonomy Values interpersonal connections and family-centered approaches Table 3 Postulated differences in philosophical and religious be- liefs of East and West Bioethical Inquiry (2015) 12:211–225 219 four decades of modernization, it is difficult to imagine that beliefs about death that have been so central to Japanese culture for the past several thousand years would vanish, even in the face of high technology medicine.” (Feldman 1988, 342) In Shintoism and Tao- ism, the native beliefs of Japan and China respectively, it is not only difficult to separate the mind from the body, human life is also intimately associated with the sur- rounding environment (Kasai 2009; Liao 2005). Shin- toism understands humans within the context of the forest, coexisting with mountains, rivers, sun, earth, plants, and animals (Kasai 2009). Taoism also advocates accepting and following the laws of nature, albeit often mysterious. Confucianism classifies longevity and nat- ural death among the “five principles of happiness,” while violent or premature death and sickness are count- ed as “extreme ferocities” (Liao 2005, 69). In this cos- mos, the physical and spiritual mingle, the boundary between life and death is blurred and often impossible to discern (Feldman 1988; Liao 2005; Kasai 2009). Death represents an ambiguous and gradual process with disintegration of both the physical and spiritual existences, accompanied by rituals of leave-taking and seeing-off. From the perspective of the nature worship- er, brain death is too specific and artificial (Feldman 1988; Kasai 2009). West Does the concept of brain death itself evoke an unsettling sense of violation? Beliefs about the importance of the physical body in the afterlife pervade Eastern religions. The body is a gift from one’s parents and ancestors who can be traced to the mythological era (Ohnuki-Tierney et al. 1994). An- nihilation of the body’s wholeness represents disrespect for the parents and desecration toward the ancestors, therefore a premier violation of the “filial duty” (Tang, Li, and Wu 2008; Chang 2003). Not being buried whole is considered a curse to be dodged and one of the most severe punishments for one’s enemy (Feldman 1988). An intact body is required for the spirit to transition upon death, to enjoy life in the underworld that resem- bles the current one in every way from bribery to bu- reaucracy, and to reincarnate into the next life (Yu and Xing 2009). Although not direct criticisms of brain death, these beliefs are thought to contribute to the rejection of brain death in Eastern societies because of East Eastern philosophy takes a more amalgamated view on life. The dichotomy between body and soul is a foreign concept to Asian societies. One scholar noted: “Despite Bioethical Inquiry (2015) 12:211–225 220 the intimate relationship between brain death and organ procurement in medical practice. East is constituted in the public domain through a web of interpersonal relationships and exchanges (Lock 2002). Confucians define the meaning of human life through a series of obligations. Filial duty has been mentioned often as an obstacle to popular acceptance of the brain death criteria in China (Ouyang 2004). The dying ought not die for themselves, but are required to keep on living to comfort the survivors. The ostensibly lifelike body with mechanically maintained vital signs represents a lingering hope that helps uphold the social connections surrounding the brain dead patient. For the survivors, accepting brain death would mean relinquishing one’s obligations. The principle of zhongsheng advocated by both Taoism and Confucian- ism emphasizes making a rigorous effort toward contin- uation of both physical and social life (Liao 2005). People’s understanding of organ donation also reflects this desire to cling to a physical existence that sustains social connections. Whereas organ donation is a gift of life benefitting the recipient in the West (Quiroga 2011), it serves as an extension of the donor’s life to families who agree to donate in the East (Zhan 2012). The brain does not occupy a special position as the dominant organ. Traditional Chinese Medicine teaches that the human body is a system of correspondence, rather than a system of causation (Ohnuki-Tierney et al. 1994). Functions of living result from interactions between all organ systems; the brain neither controls nor integrates. In Buddhism, alaya-vijnana, or the Eighth Consciousness representing one’s personal and collec- tive identity, is distributed throughout the body and not exclusively located in the brain (Bowman and Richard 2003; Keown 2010). Even in the absence of measurable brain activity, consciousness may still be dwelling in the body (Bresnahan and Mahler 2010; Keown 2010). For Asians, the definition of life reaches beyond ratio- nality. Takeshi Umehara, a prominent polytheist, criticized the rationale of brain death as “lacking the sense of awe toward life” (Kasai 2009, 36). To label physical existence without consciousness and thoughts as mere matter disre- gards the fact that insects and plants are also alive. This suggests a world of total human control, contradictory to nature worship. East Buddhism identifies three crucial ele- ments for life—vitality, heat, and sentiency (Bresnahan and Mahler 2010; Keown 2010). Vitality is the energy and driving force of life. Vitality generates work, represented by body heat. Sentiency means the way in which con- sciousness perceives and interacts with the surrounding world. It is in no way limited to the sensory arm of the nervous system and does not warrant existence of the brain. Zhu Xi, a Confucian scholar of the Song Dynasty, also noted that “[w]hen a person is about to die, heat leaves his body, which indicates that the spirit is gone” (Liao 2005, 70). The importance of body heat as a sign of life makes brain death unacceptable. Several scholars have reached the conclusion that, for Asians, “the inactive brain represented a prolongation of life rather than, as it has come to be viewed in Western countries, a prolongation of the process of dying” (Ohnuki-Tierney et al. 1994). When looking at a warm body with a beating heart on mechan- ical ventilation, Asians do not see a collection of cadaveric organs being perfused, but “the co-habitual relationship between the body and the spirit and integration of life being supported” (Kasai 2009, 39). Family members fre- quently quote heartbeat and body heat as tangible evi- dence that the brain dead patient is still alive (Hong 2007; Yu et al. 2012; Gao 2012). In contrast to the West, none of the authority figures for the Eastern religions has announced a clear opinion for or against brain death. This lack of clear religious guidance impedes further the acceptance of the brain death standard. Conclusion and Outlook for Future Research Advocates for le tion of brain death around the world recognize its Table 4 Timeline of the main events in neuroscience, cri death Neuroscience 1890 ICP increase associated with respiratory arrest preceding circulatory arrest 1900 1910 1920 EEG 1930 Cerebral angiograms 1940 1950 Coma dépassé: state of irreversible coma and apnea 1960 Proposals to equate death of the nervous system to cardiopulmonary death Syney conference Harvard criteria 1970 Brain stem death criteria 1980 Universal Declaration of Death Act Bioethical Inquiry (2015) 12:211–225 Bioethical Inquiry (2015) 12:211–225 221 individuals, especially regarding controversial issues, is often intuitive. In a survey of American families who did not consent to donate the organs of their brain dead relatives, less than 2 percent cited either religion or culture as the reason for their refusal, and 80 percent disclosed emotion as the deciding factor (Ojo et al. 2004). In a Chinese study, the possession or lack of religious faith did not correlate with respondents’ approval of the brain death standard (Yu and Xing 2009). For families who refuse to accept, or physicians who refuse to make, a brain death diagnosis, emotional or intuitive states may play a crucial role. These emotions may include disbelief, guilt, helplessness, anger, and pain, as found in a study of families coping with ICU death (Townsend 1995). What types of emotions are provoked by brain death and how people’s emotional responses differ or resemble each other in East and West are unknown. in organ transplantation and conservation of medical resources. Procurement and donor registry organizations in both Eastern and Western countries use “the gift of love” as their slogan of choice. Interviews with Swedish families of the brain dead revealed that Westerners also conjure a living person in the warm body with a beating heart and an undulating chest, although the images eventually change into that of an empty shell (Frid et al. 2007). Instead of a contrasting list of concepts as presented in Table 3, Easterners and Westerners may take into consideration a similar set of principles when faced with the dilemma of brain death, but assign them different priorities to guide their decisions (Haidt 2007). Lastly, social circumstances unique to the modern era, unforeseen by Socrates or Confucius, likely make a significant contribution to how people perceive brain death. Conclusion and Outlook for Future Research Existing discussions on the differences in attitudes toward brain death between East and West have focused on cultural heritages. In summary, neuro-essentialism and elevation of rationality and autonomy as the defining characteristics of personhood nurture the concept of brain death and its widespread acceptance in the West. Obscure boundaries between an individual human life and the rest of the universe, as well as interpreting the preserved vital signs as being alive, hinder the utility of brain death in the East. Overall, philosophical and religious backgrounds appear to reflect the degree of ease or difficulty with which societies have adapted to brain death. However, little is known about the application of these ideological disparities in real life. Ordinary individuals may not explicitly express the concepts mentioned above by medical anthropologists, and perhaps cultural influ- ences may be subtle and not consciously acknowledged. High-stakes medical decision-making by typical In addition, unlike the Western portrayal of an auton- omous self enclosed inside the brain, personhood in the individuals, especially regarding controversial iss often intuitive. In a survey of American families w not consent to donate the organs of their brain relatives, less than 2 percent cited either religion or as the reason for their refusal, and 80 percent dis emotion as the deciding factor (Ojo et al. 2004 Chinese study, the possession or lack of religiou did not correlate with respondents’ approval of th death standard (Yu and Xing 2009). For familie refuse to accept, or physicians who refuse to make, death diagnosis, emotional or intuitive states may crucial role. These emotions may include disbelief helplessness, anger, and pain, as found in a st families coping with ICU death (Townsend 1995) types of emotions are provoked by brain death an people’s emotional responses differ or resemble eac in East and West are unknown. Moreover, the philosophical arguments are ways unique to any one culture, although the represent mainstream thinking. We acknowledg variations exist within the larger cultural frame we refer to as East and West; certain subpopu may show reasoning patterns resembling their co parts in the contrasting culture. Conclusion and Outlook for Future Research One example, trust in the health care system, has been explored to explain the cautious approach to brain death in Japan (Lock 2002). Escalating physician–pa- tient conflicts in China in recent years (Bai 2012; Hu, Zhang, and Zhang 2012) may contribute to the disbelief of patients’ families in the diagnosis of brain death. The information gap between physician and patient certainly invites misunderstanding. Care providers may then avoid broaching subjects such as brain death to prevent Moreover, the philosophical arguments are not al- ways unique to any one culture, although they may represent mainstream thinking. We acknowledge that variations exist within the larger cultural frameworks we refer to as East and West; certain subpopulations may show reasoning patterns resembling their counter- parts in the contrasting culture. Advocates for legaliza- tion of brain death around the world recognize its utility Table 4 Timeline of the main events in neuroscience, critical care medicine, and transplant surgery that led to conceptualization of brain death Neuroscience Critical care Transplant 1890 ICP increase associated with respiratory arrest preceding circulatory arrest 1900 Vascular anastomosis 1910 1920 EEG Iron lung 1930 Cerebral angiograms Invasive monitoring Unsuccessful kidney transplant attempts using unrelated donors 1940 Positive pressure ventilation in response to European polio epidemic 1950 Coma dépassé: state of irreversible coma and apnea ICU built in hospitals Mechanical ventilation Successful kidney transplant between identical twins Immunosuppressive therapy 1960 Proposals to equate death of the nervous system to cardiopulmonary death Syney conference Harvard criteria Liver, lung, pancreas transplants First transplant from a heart-beating donor 1970 Brain stem death criteria 1980 Universal Declaration of Death Act 222 Bioethical Inquiry (2015) 12:211–225 Arbour, R., H.M.S. AlGhamdi, and L. Peters. 2012. Islam, brain death, and transplantation: Culture, faith, and jurisprudence. AACN Advanced Critical Care 23(4): 381–394. doi:10.1097/ NCI.0b013e3182683b1e. retaliation from patients. The contemporary social cli- mate may further eclipse the influence of philosophical and religious traditions on medical decision-making (Table 4). Bagheri, A. 2003. Criticism of “brain death” policy in Japan. Kennedy Institute of Ethics Journal 13(4): 359–372. ( ) Addressing some of these hypotheses can help eluci- date the root cause of differences in attitudes toward brain death in East and West. By understanding what obstacles lie in the journey of brain death from academia to real-life settings, future research should aim to inform policy-makers in countries where brain death remains controversial, such as China. Conclusion and Outlook for Future Research For example, if research reveals that Chinese citizens firmly believe that vitality and body heat represent life, and therefore are unlikely to see the brain dead as truly dead, legalizing brain death may evoke popular rage. However, if it shows that education and economic status significantly correlate with acceptance of brain death, propagating factual knowledge about brain death through the media or giving financial incentives may facilitate execution of the brain death law. In addition, by bringing attention to the cultural context, we hope to empower medical pro- viders to communicate about brain death to diverse patient populations with more clarity and empathy. At the institutional level, hospitals are able to consider places of potential conflict and mitigation strategies when implementing protocols for ventilation withdraw- al or organ donation upon brain death. 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https://openalex.org/W2795556739	https://europepmc.org/articles/pmc5831695?pdf=render	English	null	Locomotor training using an overground robotic exoskeleton in long-term manual wheelchair users with a chronic spinal cord injury living in the community: Lessons learned from a feasibility study in terms of recruitment, attendance, learnability, performance and safety	Journal of neuroengineering and rehabilitation	2,018	cc-by	10,526	Locomotor training using an overground robotic exoskeleton in long-term manual wheelchair users with a chronic spinal cord injury living in the community: Lessons learned from a feasibility study in terms of recruitment, attendance, learnability, performance and safety Dany H. Gagnon1,2* , Manuel J. Escalona1,2, Martin Vermette1,2, Lívia P. Carvalho3, Antony D. K Cyril Duclos1,2 and Mylène Aubertin-Leheudre3 Dany H. Gagnon1,2* , Manuel J. Escalona1,2, Martin Vermette1,2, Lívia P. Carvalho3, Antony D. Karelis3, Cyril Duclos1,2 and Mylène Aubertin-Leheudre3 Dany H. Gagnon1,2* , Manuel J. Escalona1,2, Martin Vermette1,2, Lívia P. Carvalho3, Antony D. Karelis3, Cyril Duclos1,2 and Mylène Aubertin-Leheudre3 © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. * Correspondence: dany.gagnon.2@umontreal.ca 1School of Rehabilitation, Université de Montréal, Montreal, QC, Canada 2Pathokinesiology Laboratory, Centre for Interdisciplinary Research in Rehabilitation of Greater Montreal, Centre intégré universitaire de santé et services sociaux du Centre-Sud-de-l’Île-de-Montréal, Installation Institut de réadaptation Gingras-Lindsay-de-Montréal, 6300 Avenue Darlington, Montreal, QC H3S 2J4, Canada Full list of author information is available at the end of the article Abstract Background: For individuals who sustain a complete motor spinal cord injury (SCI) and rely on a wheelchair as their primary mode of locomotion, overground robotic exoskeletons represent a promising solution to stand and walk again. Although overground robotic exoskeletons have gained tremendous attention over the past decade and are now being transferred from laboratories to clinical settings, their effects remain unclear given the paucity of scientific evidence and the absence of large-scale clinical trials. This study aims to examine the feasibility of a locomotor training program with an overground robotic exoskeleton in terms of recruitment, attendance, and drop-out rates as well as walking performance, learnability, and safety. Methods: Individuals with a SCI were invited to participate in a 6 to 8-week locomotor training program with a robotic exoskeleton encompassing 18 sessions. Selected participants underwent a comprehensive screening process and completed two familiarization sessions with the robotic exoskeleton. The outcome measures were the rate of recruitment of potential participants, the rate of attendance at training sessions, the rate of drop-outs, the ability to walk with the exoskeleton, and its progression over the program as well as the adverse events. (Continued on next page) * Correspondence: dany.gagnon.2@umontreal.ca Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 https://doi.org/10.1186/s12984-018-0354-2 Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 https://doi.org/10.1186/s12984-018-0354-2 Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 https://doi.org/10.1186/s12984-018-0354-2 RESEARCH Open Access Locomotor training using an overground robotic exoskeleton in long-term manual wheelchair users with a chronic spinal cord injury living in the community: Lessons learned from a feasibility study in terms of recruitment, attendance, learnability, performance and safety Dany H. Gagnon1,2* , Manuel J. Escalona1,2, Martin Vermette1,2, Lívia P. Carvalho3, Antony D. Karelis3, Cyril Duclos1,2 and Mylène Aubertin-Leheudre3 Open Access * Correspondence: dany.gagnon.2@umontreal.ca Keywords: Exercise, Paraplegia, Physical medicine and rehabilitation, Robotics, Therapies, Technology, Walking To date, only few studies have reported their recruitment rate or identified the personal or environmental factors that interfered in the selection of potential participants, and even fewer have reported their attendance rate when- ever a training program was offered [14–17]. Moreover, gaining a better understanding of the skill-acquisition process during overground walking with a robotic exo- skeleton, and of a safe and well-tolerated progression is es- sential for planning future larger-scale interventional trials. To date, very few studies have precisely described the trajectory of change observed over the course of the training program, especially in regard to the number of therapist required and their level of physical assistance, the type of walking aid required during each training ses- sion, and the time needed to achieve autonomous control of the exoskeleton [18]. However, many studies have re- ported training-related measures (e.g., standing time, walking time, number of steps taken during a session) or performance-based measures (e.g., walking speed- or dis- tance) that were typically measured only at the start, mid- term, or end of the intervention [15]. Based on a recent systematic review incorporating 14 case-series or quasi- experimental studies using a robotic exoskeleton as an as- sistive device [2], mostly small heterogeneous group of in- dividuals with complete and incomplete sensorimotor SCI (i.e., N ≤8 participants in 86% of the studies) who com- pleted, using different models of overground robotic exo- skeletons having various control modes (i.e., 5 different exoskeletons used with 4 different control modes), various training protocols encompassing a wide range of training sessions (range: 2 to over 100 training sessions) and fre- quencies (range: unspecified to 6 training sessions per week) offered at a single center have been investigated to date. Hence, stronger evidence continues to be needed to * Correspondence: dany.gagnon.2@umontreal.ca * Correspondence: dany.gagnon.2@umontreal.ca 1School of Rehabilitation, Université de Montréal, Montreal, QC, Canada 2Pathokinesiology Laboratory, Centre for Interdisciplinary Research in Rehabilitation of Greater Montreal, Centre intégré universitaire de santé et services sociaux du Centre-Sud-de-l’Île-de-Montréal, Installation Institut de réadaptation Gingras-Lindsay-de-Montréal, 6300 Avenue Darlington, Montreal, QC H3S 2J4, Canada Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Page 2 of 12 Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 (Continued from previous page) Results: Out of 49 individuals who expressed their interest in participating in the study, only 14 initiated the program (recruitment rate = 28.6%). Of these, 13 individuals completed the program (drop-out rate = 7.1%) and attended 17.6 ± 1.1 sessions (attendance rate = 97.9%). Their greatest standing time, walking time, and number of steps taken during a session were 64.5 ± 10.2 min, 47.2 ± 11.3 min, and 1843 ± 577 steps, respectively. During the training program, these last three parameters increased by 45.3%, 102.1%, and 248.7%, respectively. At the end of the program, when walking with the exoskeleton, most participants required one therapist (85.7%), needed stand-by or contact-guard assistance (57.1%), used forearm crutches (71.4%), and reached a walking speed of 0.25 ± 0.05 m/s. Five participants reported training-related pain or stiffness in the upper extremities during the program. One participant sustained bilateral calcaneal fractures and stopped the program. Conclusions: This study confirms that larger clinical trials investigating the effects of a locomotor training program with an overground robotic exoskeleton are feasible and relatively safe in individuals with complete motor SCI. Moreover, to optimize the recruitment rate and safety in future trials, this study now highlights the need of developing pre-training rehabilitation programs to increase passive lower extremity range of motion and standing tolerance. This study also calls for the development of clinical practice guidelines targeting fragility fracture risk assessment linked to the use of overground robotic exoskeletons. Background All potential participants were also screened by a research physiother- apist to rule out other potential standing- and walking- related exclusion criteria such as lower extremity passive range of motion limitations (hip flexion contracture ≥5°, knee flexion contracture ≥10°, and ankle dorsiflexion ≤− 5° with knee extended), moderate-to-severe lower extrem- ity spasticity (> 2 modified Ashworth score), inability to sit with hips and knees ≥90° flexion, and a standing tolerance test with full lower extremity weight-bearing of ≤30 min. Moreover, for participants to be fitted within the robotic exoskeleton, their height and pelvis width needed to range between 1.52–1.93 m and 30–46 cm, respectively, whereas the length of their thigh and lower leg segments also needed to range between 51 and 61.4 cm and 48–63.4 cm, respectively. Furthermore, a length discrepancy of no more than 1.3 and 1.9 cm was essential at the thigh and lower leg segments, respectively. Last, the participant’s body weight needed to be less than 100 kg. The main re- cruitment strategies implemented by the research team in- cluded contacting individuals with a complete motor SCI who previously participated in research projects and had agreed to be informed when a new study starts; posting flyers containing information about the study in key areas within the rehabilitation facility; advertising in the maga- zine of a non-profit organisation dedicated to the social and professional reintegration of individuals with spinal cord injury in the province of Quebec (i.e., www.moellee- piniere.com/en/our-publication/paraquad/); and educating physicians servicing individuals with SCI living in the community via the outpatient clinic at the rehabilitation facility about the study for them to refer potential partici- pants to the research team. Alternative recruitment strat- egies included potential participants who directly contacted the research team to express interest in partici- pating after having observed part of a training session dur- ing a visit to the rehabilitation facility; who saw a television show reporting on the research project (https:// www.youtube.com/watch?v=1y1c6ynYySk); who discussed with a participant involved in or who had completed the study; or who had read comments about the study or seen videos of participants engaged in the study posted on so- cial media (i.e., Facebook). The study was conducted at the Pathokinesiology Laboratory of the Centre for Inter- disciplinary Research in Rehabilitation of Greater Montreal inform the development of future evidence-based adapted physical activity or neurorehabilitation training programs to be tested and compared in larger-scale interventional trials. Background There has been a growing interest for overground ro- botic exoskeletons over the past decade [1–8]. These overground robotic exoskeletons typically provide mo- torized assistance at the hips and knees via motors while the ankles and feet are generally assisted with dynamic ankle-foot orthoses. This assistance fully or partially gen- erates and coordinates flexion and extension movements and moments at these joints to produce or assist with sit-stand transfers and overground walking. For individ- uals who are affected by sensorimotor impairments and rely on a wheelchair as their primary mode of locomo- tion, overground robotic exoskeletons figure among the most promising solutions to stand and walk although their effects and effectiveness remain unclear given the paucity of scientific evidence. In this population, overground robotic exoskeletons can be used for standing and walking in the context of adapted physical activity programs offered during rehabilitation or in the community. In fact, based on the available evidence, adapted physical activity programs incorporating standing and walking with a robotic exoskeleton could potentially alleviate the development of musculoskeletal [9], cardiore- spiratory [10–13], and endocrine-metabolic [9] secondary health conditions and complications. Hence, there is a need to develop and test such programs that integrate an overground walking component while also democratizing accessibility to the robotic exoskeleton, especially in publically-funded healthcare environments. However, be- fore doing so and assessing such programs in terms of their efficacy and effectiveness, it is crucial to gain a better understanding of the factors that could potentially inter- fere in the process of participants’ recruitment and selec- tion as well as in their attendance at the training sessions. Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 3 of 12 Page 3 of 12 ossification, rotator cuff tendinopathy), history of lower extremity fracture within the past year, unstable cardiovas- cular or autonomic system, cognitive or oral communica- tion problems, or any other conditions that could restrict their ability to train walking ability or confound in other ways the results of this study were excluded. Methods Design A single-group longitudinal prospective feasibility study. Background Alongside, stronger evidence on the cardiorespira- tory [11], musculoskeletal, balance, and cognitive require- ments during overground walking with a robotic exoskeleton, for examples, is also needed to better under- stand the underlying mechanisms of intervention effects and to select the best comparators in these future trials. The overall aim of the present study was to investigate the feasibility and safety of a new locomotor training program with a robotic exoskeleton offered to long-term manual wheelchair users with a spinal cord injury (SCI) living in the community. Specifically, the intent of the present study was to precisely determine the recruit- ment, attendance and drop-out rates, the learnability and detailed progression over the course of the training program (including the level of human assistance, the level of technical assistance, and the walking perform- ance in terms of walking time, number of steps taken, and walking speed), and the safety. These attributes of the new locomotor training program could provide valu- able information for the development of future larger- scale clinical trials investigating the effects or the effi- ciency of a locomotor training program with an over- ground robotic exoskeleton. These clinical trials are important to better understand if and how locomotor training programs with the robotic exoskeleton can alle- viate secondary health conditions and complications, maximize functional abilities, or optimize psychological well-being, social participation, and life satisfaction among long-term manual wheelchair users living in the community. Participants p A sample of 14 adults with a motor complete SCI (ASIA Impairment Scale = A or B) who use a wheelchair as their primary mode of mobility were recruited (Table 1). To be included in the study, potential participants had to be at least 18 years of age, had been discharged from an inten- sive inpatient rehabilitation program for at least 18 months, resided in a community within a 75 km radius of the rehabilitation center, and communicated in either French or English. Potential participants who had previ- ously underwent training with robotic exoskeleton for overground walking, with other nervous system damage aside from the SCI (e.g., multiple sclerosis), impaired skin integrity (e.g., pressure sores in areas in contact with the robotic exoskeleton), concomitant or secondary musculo- skeletal impairments (e.g., lower extremity heterotopic Gagnon et al. Locomotor training program Initially, participants attended two familiarization ses- sions over a one-week period that lasted about 45– 60 min per session. During these familiarization sessions, participants were properly fitted with the EKSO GT ro- botic exoskeleton before performing balance, walking- related tasks (e.g., sit-stand transfers), and walking on short distances with visual and verbal feedbacks while the certified therapist actuated each step (i.e., FirstStep mode). As the participants’ level of proficiency increased during the familiarization sessions, they learned to safely ambulate with the exoskelon at a self-selected comfort- able speed using their own walking aid (i.e., rolling walker or forearm crutches) while taking control of each step triggered via anterolateral body weight shifts (i.e., ProStep mode) under the direct supervision of a certified therapist. Following these two familiarisation sessions, participants began the six-week progressive locomotor training program administered by a certified therapist that encompassed a total of 18 training sessions (three sessions/week; 60 min/session). Depending on the level of each participant’s proficiency, on the participant’s tol- erance, and on the activities planned for the session walking speed of 1.6 m/s. The EKSO GT robotic exo- skeleton is approved by Health Canada for clinical use. walking speed of 1.6 m/s. The EKSO GT robotic exo- skeleton is approved by Health Canada for clinical use. study’s objectives and of the nature of their participation. The Research Ethics Committee of the Centre for Inter- disciplinary Research in Rehabilitation of Greater Mon- treal approved the study (CRIR-1083-0515). Participants Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 4 of 12 Table 1 Description of participants Participant # Sex Age (years) Height (m) Weight (kg) Body Mass Index (Weight/Height2) Time since SCI/D (years) Origin of SCI/D ASIA-Motor Score (/100) ASIA-Sensory Score (/224) ASIA Impairment Scale (AIS) ASIA Neurological level 1 F 26.7 1.61 61.4 23.7 2.2 Trauma 50 104 A T6 2 M 28.4 1.78 73.9 23.3 5.1 Trauma 50 108 A T6 3 M 63.1 1.85 96.0 28.0 8.3 Trauma 50 143 A T10 4 M 32.2 1.92 91.2 24.7 8.0 Trauma 50 118 A T6 5 M 42.9 1.8 66.6 20.6 14.4 Trauma 28 48 A C6 6 M 51.5 1.67 61.9 22.2 31.4 Trauma 50 140 B T6 7 M 43.8 1.8 107.2 33.1 3.4 Trauma 50 143 A T10 8 M 35.3 1.87 67.9 19.4 8.6 Trauma 50 108 A T6 9 M 38.1 1.6 64.3 25.1 6.9 Trauma 50 115 A T9 10 M 27.2 1.7 56.2 19.4 4.2 Trauma 50 104 A T4 11 F 31.1 1.6 63.7 24.9 1.0 Trauma 54 123 A T8 12 F 39.4 1.68 75.5 26.8 4.7 Trauma 50 80 A T3 13 F 51.9 1.62 58.5 22.3 5.2 Non- Trauma 50 96 A T4 14 F 30.9 1.63 48.7 18.3 0.8 Trauma 50 106 A T6 Mean 38.7 1.7 70.9 23.7 7.4 48.7 109.7 Standard deviation 10.9 0.1 16.5 3.9 7.8 6.1 25.4 AIS ASIA Impairment Scale, ASIA American Spinal Cord Injury Association, A No motor or sensory function is preserved below the neurological level, B Sensory function is preserved but no motor function below the neurological level, C Motor function is preserved below the neurological level, and more than half of the key muscles below the neurological level have a muscle grade < 3 out of 5 (manual muscle testing), D motor function is preserved below the neurological level, and at least half of the key muscles below the neurological level have a muscle grade of ≥3 out of 5, E motor and sensory function are normal AIS ASIA Impairment Scale, ASIA American Spinal Cord Injury Association, A No motor or sensory function is preserved below the neurological level, B Sensory function is preserved but no motor function below the neurological level, C Motor function is preserved below the neurological level, and more than half of the key muscles below the neurological level have a muscle grade < 3 out of 5 (manual muscle testing), D motor function is preserved below the neurological level, and at least half of the key muscles below the neurological level have a muscle grade of ≥3 out of 5, E motor and sensory function are normal AIS ASIA Impairment Scale, ASIA American Spinal Cord Injury Association, A No motor or sensory function is preserved below the neurological level, B Sensory function is preserved but no motor function below the neurological level, C Motor function is preserved below the neurological level, and more than half of the key muscles below the neurological level have a muscle grade < 3 out of 5 (manual muscle testing), D motor function is preserved below the neurological level, and at least half of the key muscles below the neurological level have a muscle grade of ≥3 out of 5, E motor and sensory function are normal Attendance Most participants (N = 11/14) completed all training ses- sions (attendance rate = 100%) whereas two participants were deprived of one (adjusted attendance rate = 94%) and four training sessions (adjusted attendance rate = 78%), re- spectively, since the program was temporarily suspended during the holiday season. Hence, the overall attendance rate was 97.9% (229 completed training sessions/234 planned training sessions). One participant was withdrawn from the study by the research team after one training ses- sion and was not accounted for in the attendance rate and learnability statistics (details provided in Adverse Events section). This was the only participant who dropped out of the study (n = 1/14; drop-out rate = 7.1%). Robotic exoskeleton The wearable robotic exoskeleton EKSO™(version 1.1) (Ekso Bionics, Richmond, CA, USA) is a ready-to-wear, battery-powered, motor driven, robotic pair of legs gen- erating motion at the hip and knee joints in a properly sequenced manner. Each joint is independently con- trolled by different sensors linked to a small, portable, computerized control system attached to the flexible trunk module that also encompass the battery. Informa- tion gathered by over 35 different sensors (e.g., acceler- ometers, speed controllers, gyroscopes, pressure sensors) feed a decisional algorithm loop allowing manual wheel- chair users with SCI to perform sit-stand transfers and walk. When walking with the robotic exoskeleton, each step is primarily commanded by combined forward and lateral bodyweight shifts toward the weight-bearing lower extremity before initiating the oscillation with the opposite lower extremity. The certified therapist can control numerous walking features (e.g., speed, step height, step length). The EKSO GT robotic exoskeleton weighs about 28 kg and can technically reach a maximal Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 5 of 12 Page 5 of 12 different reasons during this second step. Finally, out of the last 19 potential participants who underwent clinical screening and completed the familiarization sessions, five individuals with SCI refused to participate in the study during this last recruitment step. Hence, a total of 14 individuals with SCI were enrolled in the study (re- cruitment rate = 28.6%). The most common reasons for excluding potential participants were the presence of limited passive dorsiflexion range of motion at the ankle (n = 13/30; rate = 43.3%) as well as time, transportation, or accommodation constraints linked to the program re- quirements (N = 9/30; rate = 30%). The most common reason for refusing to participate was the fear of devel- oping a complication as a result of ambulating with the robotic exoskeleton system after having tried the robotic exoskeleton (n = 3/5; rate = 60%). (e.g., instructions and basic training to initiate sit-stand transfers, walking and turning with forearm crutches), the workload was periodically adjusted using walking distance, duration, and speed parameter progressions [7]. After each session, all training parameters and other relevant information (e.g. total standing time, total walk- ing time, and total number of steps) were recorded. Main outcome measures The main outcome measures are the rate of recruitment of potential participants, the drop-out rate of partici- pants enrolled into the study, the rate of attendance at training sessions, the progression in the ability to walk with the exoskeleton (i.e., standing time, walking time, number of steps taken per session, type of waking aid, number of therapists needed, level of assistance provided per session), the performance when walking with the exoskeleton at self-selected comfortable walking speed measured using the 10-m walking test (10MWT) [19] at the start (within the first 5 sessions) and end of the pro- gram, and adverse events (Table 2). Learnability and performance Learnability and performance A summary of the progression of the standing time, walk- ing time, and number steps taken per session is illustrated in Fig. 2. On average, during the locomotor training pro- gram, the standing time, the walking time, and the number of steps taken per session were 49.7 ± 12.7 min, 33.4 ± 12.5 min, and 1190 ± 561 steps, respectively. Between the start (mean of sessions 1 and 2) and the end (mean of ses- sions 17 and 18) of the locomotor training program, the standing time, walking time, and number of steps taken per session progressed by 45.3%, 102.1%, and 248.7%. The ma- jority of participants (N = 10/13) were already self-initiating their steps via lateral and anterior bodyweight shifts toward the weight bearing lower extremity (i.e., prostep mode) at the first session of the training program. Two additional participants reached this level at the second training session whereas another participant reached it at the 8th session of the training program. Most participants (N = 10/13) devel- oped the ability to ambulate with Canadian crutches after 3.5 ± 3.3 training sessions whereas the other 3 participants continued to use a rollator walker throughout the training program (Fig. 3). Most participants (N = 11/13) needed Statistical analysis Descriptive statistics (i.e., mean, standard deviation) were calculated for all demographics and clinical characteristics as well as for all outcome measures. After a Shapiro-Wilk test confirmed the normality of the walking speed mea- sures, the pre- and post-training walking speed measures were compared using a paired Student t test for repeated measures with the significance level set at p ≤0.05. These statistics were computed using SPSS statistic software ver- sion 17.0 (IBM Corporation, Armonk, New York). Recruitment The contact is made to help steady the body or help with balance (6): Stand-by Assist: The physical therapist does not touch the participant or provide any assistance, but he or she may need to be close by for safety in case the participant loses their balance or needs help to maintain safety during the task being performed (7): Modified Independence: The participant can walk with the exoskeleton without any supervision, with the help of a walker or crutches (8): Total independence: The participant can walk with the exoskeleton without supervision and the use of a walking aid Walking Aid: R.W. Rigid walker and F.C. Forearm crutches Table 2 Summary of the key outcome measures Attendance Learnability and Performance Mobility Aid and Physical Assistance Safety Subject ID # of training sessions completed (/18) Length of time spent standing upright (min/session) Length of time spent walking (min/session) Number of steps taken (steps/session) Walking Speed (m/s) First training session Last training session Use of controller (session achieved) Shoulder pain or stiffness Thumb Tendinitis (Adductor) Knee instability Pressure Drop (Request to sit) Fracture (Ankle - Bilat) Level of assistance Walking Aid Number of PT needed Level of assistance Walking Aid Number of PT needed mean (SD) [min;max] mean (SD) [min;max] mean (SD) [min;max] Start End 1 18 52(12) [19; 70] 30(9) [8;45] 1240(492) [297;2087] 0.16 0.29 3 F.C. 2 5 F.C. 1 15 ☑ – – ☑ – 2 18 49(6) [41; 62] 31(9) [13;49] 1169(412) [420;2050] 0.19 0.30 3 F.C. 2 6 F.C. 1 15 – – – ☑ – 3 18 56(13) [33;87] 30(7) [19;41] 828(260) [407;1262] 0.12 0.24 3 R.W. 2 4 F.C. 1 Not Achieved – – – – – 4 17 54(11) [29;75] 33(10) [20;54] 1096(453) [371;2148] 0.16 0.23 3 R.W. 2 5 F.C. 1 Not Achieved – – – – – 5 14 37(11) [18;51] 19(8) [5;33] 588(293) [114;1056] – 0.20 2 R.W. 2 3 R.W. 2 Not Achieved – – – ☑ – 6 18 53(9) [29;63] 40(11) [19;52] 1575(535) [738;2272] 0.16 0.27 4 R.W. 1 6 F.C. 1 12 ☑ – – – – 7 18 56(9) [34;71] 38(9) [22;54] 1226(407) [768;2101] 0.13 0.30 3 R.W. 2 4 R.W. 1 Not Achieved ☑ – ☑ – – 8 1 51- [51;51] 19- [19;19] 455 - [455;455] – – 3 R.W. 2 3 R.W. Recruitment 2 Not Achieved – – – – ☑ 9 18 45(14) [18;60] 34(16) [7;55] 1347(757) [221;2397] 0.13 0.24 3 R.W. 1 6 F.C. 1 13 – – – – – 10 18 32(10) [12;50] 21(9) [6;40] 765(386) [254;1555] 0.17 0.17 3 R.W. 2 4 R.W. 1 Not Achieved – – – ☑ – 11 18 55(7) [37;64] 41(11) [19;53] 1504(491) [601;2097] 0.16 0.29 3 F.C. 2 5 F.C. 1 12 – – – – – 12 18 57(6) [38;65] 44(9) [24;59] 1266(454) [445;2093] 0.12 0.22 3 F.C. 2 5 F.C. 1 12 ☑ ☑ – ☑ – 13 18 43(13) [17;62] 29(11) [11;45] 1057(485) [317;1702] 0.13 0.14 3 R.W. 2 4 F.C. 1 Not Achieved – – – ☑ – 14 18 55(10) [39;71] 42(12) [22;58] 1711(614) [567;2527] 0.15 0.28 3 R.W. 2 6 F.C. 1 8 – – – – – Mean 17.6a 49.7 33.4b 1190b 0.15 0.25 Standard deviation 1.1 12.7 12.5 561.4 0.02 0.05 Level of Assistance:(1): Dependent: During dependent mobility, the participant is unable to help at all. The physical therapist - or another healthcare provider - will do all of the work (2): Maximal Assist: The participant performs 25% or less of the work during mobility and the physical therapist provides the rest of the work (3): Moderate Assist: The participant performs between 25% and 75% of the work necessary to move and the physical therapist provides the rest of the work (4): Minimal Assist: The participant performs 75% of the work to move and the physical therapist provides the rest of the work (5): Contact Guard Assist: The physical therapist needs to merely have one or two hands on the participant’s body, but provides no other assistance to perform the functional task. The contact is made to help steady the body or help with balance (6): Stand-by Assist: The physical therapist does not touch the participant or provide any assistance, but he or she may need to be close by for safety in case the participant loses their balance or needs help to maintain safety during the task being performed (7): Modified Independence: The participant can walk with the exoskeleton without any supervision, with the help of a walker or crutches (8): Total independence: The participant can walk with the exoskeleton without supervision and the use of a walking aid Walking Aid: R.W. Rigid walker and F.C. Recruitment A summary of the recruitment process, along with the number of participants who successfully completed each stage of this process and reasons for excluding potential participants, are illustrated in Fig. 1. A total of 49 indi- viduals with a SCI contacted the research team by phone or via email to express their interest in participating in the research project during an overall 11-month enroll- ment period split into two phases: October to December 2015 (3 months) and May to December 2016 (8 months). In reality, the overall enrollment period per se was shorter (about 8 months) since the last participants needed to be recruited no later than November 1, 2015 and 2016, respectively. Upon completion of the pre- screening interview of potential participants over the phone (N = 49), 19 individuals with a SCI were excluded for different reasons based on the answers provided to specific screening questions during this initial step. Out of the 30 potential participants who underwent clinical pre-screening, 11 individuals with SCI were excluded for Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 6 of 12 Level of Assistance:(1): Dependent: During dependent mobility, the participant is unable to help at all. The physical therapist - or another healthcare provider - will do all of the work (2): Maximal Assist: The participant performs 25% or less of the work during mobility and the physical therapist provides the rest of the work (3): Moderate Assist: The participant performs between 25% and 75% of the work necessary to move and the physical therapist provides the rest of the work (4): Minimal Assist: The participant performs 75% of the work to move and the physical therapist provides the rest of the work (5): Contact Guard Assist: The physical therapist needs to merely have one or two hands on the participant’s body, but provides no other assistance to perform the functional task. Recruitment Forearm crutches of Assistance:(1): Dependent: During dependent mobility, the participant is unable to help at all. The physical therapist - or another healthcare provider - will do all of the work aximal Assist: The participant performs 25% or less of the work during mobility and the physical therapist provides the rest of the work oderate Assist: The participant performs between 25% and 75% of the work necessary to move and the physical therapist provides the rest of the work inimal Assist: The participant performs 75% of the work to move and the physical therapist provides the rest of the work ontact Guard Assist: The physical therapist needs to merely have one or two hands on the participant’s body, but provides no other assistance to perform the functional task. The contact is made to help steady the or help with balance and-by Assist: The physical therapist does not touch the participant or provide any assistance, but he or she may need to be close by for safety in case the participant loses their balance or needs help to maintain during the task being performed odified Independence: The participant can walk with the exoskeleton without any supervision, with the help of a walker or crutches otal independence: The participant can walk with the exoskeleton without supervision and the use of a walking aid ng Aid: R.W. Rigid walker and F.C. Forearm crutches Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 7 of 12 Fig. 1 Summary of the key milestones of the project Fig. 1 Summary of the key milestones of the project Fig. 1 Summary of the key milestones of the project moderate assistance provided by one physiotherapist at the start of the training sessions whereas the two participants needed minimal and maximal assistance, respectively (Fig. 3). Upon completion of the training sessions, one partici- pant needed moderate, four participants needed minimal, and four needed contact guard assistance provided by one physiotherapist, whereas four participants needed a physio- therapist to stand-by while walking (Fig. 3). All participants needed moderate to maximal assistance provided by one physiotherapist for all sit-stand transitions throughout the training sessions. As for the walking speed, it increased sig- nificantly (p ≤0.0001; + 66.8%) between the start (mean ± 1 SD = 0.15 ± 0.02 m/s) and end (mean ± 1 SD = 0.25 ± 0.05 m/s) of the training program. Recruitment These last results do not include the data of one participant with C6 tetraplegia as he only took a limited number of steps at a very slow pace and needed maximal assistance of the certified therapist during the first week (i.e., invalid result for the 10MWT). Fig. 2 Group mean ± 1 SD of the standing time, walking time, and number of steps measured per session Adverse events As mentioned previously, one participant was diagnosed with bilateral type I non-displaced fractures of the calca- neus after having completed the two familiarization ses- sions and the first training session. Uncertainties exist about the specific cause of the fractures. This participant was withdrawn from the study. Four participants reported exacerbation of pre-existing shoulder pain, stiffness, or discomfort whereas one participant developed soreness at the thumb over the course of the locomotor training pro- gram. This finding was unexpected as no previous study reported on upper extremity pain, stiffness, or discomfort. One participant failed to report a previously complete Fig. 2 Group mean ± 1 SD of the standing time, walking time, and number of steps measured per session Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 8 of 12 Fig. 3 Description of the level of therapist assistance required and of the walking aid used during each session Fig. 3 Description of the level of therapist assistance required and of the walking aid used during each session considering the number of potential participants (N = 49) who initially expressed their interest in participating into the proposed study and completed the different steps of the recruitment process. Moreover, considering that the study was conducted in a rehabilitation center hosting an ultra-specialized SCI rehabilitation program servicing the western part of the Province of Quebec, the fact that numerous strategies were implemented to overcome potential barriers (e.g., a dedicated research professional in charge of the recruitment, multiple re- cruitment strategies implemented, telephone pre- screening interview to minimize the number of visits, free parking, free training sessions), and that all partici- pants were allocated to the locomotor training program with the robotic exoskeleton, a higher recruitment rate was anticipated (i.e., ≥50%). Nonetheless, this recruit- ment rate is 1.7 times greater than the one reported in other feasibility studies investigating locomotor training anterior cruciate ligament tear to the research team and developed severe knee hyperextension at the 10th training session that was solved by blocking knee extension to −3° thereafter. Six participants experienced orthostatic hypotension with systolic blood pressure drops of ≥ 20 mmHg during a training session. No participant devel- oped any soft tissue or skin problem nor fell during the locomotor training program. For the three certified trainers involved in the intervention, no adverse effect was documented. Adverse events Last, a battery failure and a hip joint bearing assembly failure were directly linked to the robotic exo- skeleton itself over the course of the locomotor training program. Recruitment The recruitment rate reached in this preliminary study (28.6%) was acceptable although it remains relatively low Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 9 of 12 Page 9 of 12 commitment of the participants who engaged into the locomotor training program. This is further supported by the fact that only one participant dropped out of the program following an adverse event (i.e. calcaneus frac- ture). Hence, a completion rate of 92.9% (n = 13 partici- pants/14 participants) was reached and is greater than the 50% documented in another feasibility study propos- ing a comparable program [15]. The importance of the familiarization sessions needs to be highlighted since 5 out of the 19 participants (26.3%) decided not to engage into the locomotor training program at that time. Al- though not formally documented, these familiarization sessions allowed the research team, to some extent, to further screen potential participants who were hesitant to engage into the locomotor training program and po- tential participants to take an informed decision about their commitment based on a lived experience. Add- itionally, it supports the relevance of adopting a flexible approach when scheduling the training sessions to accom- modate all stakeholders, especially the participants. Since the training sessions involved no or very limited socialization with individuals with similar sensorimotor impairments and functional disabilities (i.e., individualized approach), aside from the interaction with one or two therapists, the commitment of participants to complete the training session and, to some extent, their acceptance of the new technology, especially with regard to its per- ceived usefulness and ease-of-use, is also established. programs with a robotic exoskeleton in individuals with complete or incomplete SCI in England (17%) [15] and Germany (12%) [18]. Only one recent multi-center study investigating a single training session with a self- stabilizing robotic exoskeleton in individuals with SCI has reached a recruitment rate near 50% (i.e., 20 partici- pants recruited among 46 screened for eligibility) [17]. Nonetheless, the recruitment rate of the present study compares relatively well to the rates reached in other feasibility studies investigating various task-specific gait- training programs offered to relatively homogeneous samples of individuals with neurological impairments (e.g., stroke = 6.7% [20], Parkinson disease = 11% [21]). Learnability and performance Learnability and performance Individuals with a complete motor SCI demonstrated a capability to quickly learn to ambulate overground with a robotic exoskeleton. Overall, the standing and walking time (including the number of steps/session) progressed at a faster rate during the first half than during the sec- ond half of the locomotor training program. Overall, the participants stood and walked at least 30 and 20 min, re- spectively, at the first training session which is recom- mended in clinical practice to anticipate beneficial effects among long-term manual wheelchair users with a spinal cord injury [23, 24]. The level of therapist assist- ance also rapidly decreased over the course of the loco- motor training program with most participants requiring no more than minimal assistance after the 8th and 9th training sessions (halfway into the program) and only contact guard or stand-by assistance by the end of the program when walking. Additionally, most participants walked with forearm crutches, with or without the use of the self-controller that allows the user to drive few basic functions of the robotic exoskeleton (e.g., initiation of the first step, continuous walking in ‘prostep’ mode, and stops), by the end of the program. Overall, the learnability trajectory, illustrated for the first time in the present study using measures systematically collected at Different strategies may need consideration to optimize recruitment rate and facilitate attendance in future clinical trials [e.g., offering training sessions during the evening and weekend; offering training sessions away from the main rehabilitation center affiliated with the project (e.g., other rehabilitation centres, community-based physical ac- tivity centers, living labs in shopping malls); adjusting the training schedule to best match participants’ availability with a minimum of two training sessions per week; pro- posing temporary housing alternatives for potential partic- ipants living further away who demonstrate an interest in participating]. Last, it is important to highlight that the re- cruitment rate may have been lower if potential partici- pants had had a chance to be allocated to an alternative experimental group undergoing a different training pro- gram or a control group with no training program. Recruitment In the present study, the most important reason (16 out of 35 potential participants = 46%) for not qualifying for the training program was due to musculoskeletal impair- ments with the leading cause being a reduced passive range of motion at the ankle, knee, or hip. For the same reason, other preliminary studies have excluded up to 77.8% of potential participants (7 out of 9 potential par- ticipants excluded) [16]. The second most important reason was linked to time constraints (7 out of 35 poten- tial participants = 20%). Contrary to other preliminary studies [16, 22], transportation did not emerge as a major barrier to the recruitment process nor the drop- out rate in the present study since only two potential participants based their decision on this criterion (2 out of 35 potential participants = 5.7%). Taking these reasons together, developing a home-based pre-training program with indirect supervision of a therapist that would target gains in passive range of motion at the lower extremity and progressive standing time prior to initiating the locomotor training program may be warranted. Attendance Yet, all these participants opted to continue the training sessions while exploring personalized solutions to alleviate or even eliminate pain over the course of the program (e.g., increased number and duration of rest periods dur- ing sessions; cushioning at the handle of the walking aid; recommendation of stretching exercises post-training; use of nonsteroidal anti-inflammatory drugs) with the certified trainer(s). Unexpectedly, no exoskeleton-related skin or soft tissue issue was observed in the present study al- though it affected up to 50% of participants in previous studies and typically leads to interruptions of the interven- tion or withdrawal of participants from the studies [15]. All the above-identified risks remain impossible to elimin- ate, warrant thoughtful consideration, and should be care- fully explained within the informed consent form along with the strategies implemented to minimize or overcome them. Finally, two events linked to the robotic exoskeleton itself (device malfunction) occurred over the course of the locomotor training program. The first event was a battery failure that required its replacement while the second The learning process also may have been facilitated by optional distinct auditory feedbacks automatically gener- ated when the participant respectively reached the lateral and forward body weight shift targets required prior to initiating steps, especially early on during the learning stage [25]. Moreover, although not formally assessed in the present study, some participants periodically filmed their performance, especially at the beginning of the study, to facilitate their learning and complement the therapist’s subjective feedback (i.e., visual feedback- induced performance improvement) [26]. Hence, in addition to the adjustability of some exoskeleton param- eters (e.g., reducing body shift amplitudes to initiate steps, reducing step height, increasing step length), nu- merous clinical strategies (e.g., reducing level of human assistance, changing walking aid) are also possible to ad- just the level of challenges during overground walking with the robotic exoskeleton as the participant’s level of proficiency improves. Maintaining a level of challenge during learning is also known to positively impact a par- ticipant’s level of motivation and attendance, both of which are crucial in the context of any clinical trial in which participants are assigned to receive an interven- tion [27]. As for the performance, the walking speed was found to increase significantly between the start and end of the training program. Attendance In fact, the mean walking speed reached in the present study (i.e., 0.25 ± 0.05 m/s) is similar to the weighted mean gait speed of 0.25 ± 0.14 m/s reported in a recent meta-analysis investigating a heterogeneous group of individuals with a complete SCI who completed, with different models of overground robotic exoskeletons, various training protocols encom- passing a wide range of training sessions [2]. Nonethe- less, reaching faster walking speed after 18 sessions may still be possible with additional training since able- bodied adults, who have completed basic training with the robotic exoskeleton, reach on average a self-selected comfortable walking speed of 0.38 ± 0.09 m/s when they were asked to avoid all voluntary muscular contraction of their lower extremities (i.e., passive walking) [28]. 35.7%) over the course of the locomotor training program. Yet, all these participants opted to continue the training sessions while exploring personalized solutions to alleviate or even eliminate pain over the course of the program (e.g., increased number and duration of rest periods dur- ing sessions; cushioning at the handle of the walking aid; recommendation of stretching exercises post-training; use of nonsteroidal anti-inflammatory drugs) with the certified trainer(s). Unexpectedly, no exoskeleton-related skin or soft tissue issue was observed in the present study al- though it affected up to 50% of participants in previous studies and typically leads to interruptions of the interven- tion or withdrawal of participants from the studies [15]. All the above-identified risks remain impossible to elimin- ate, warrant thoughtful consideration, and should be care- fully explained within the informed consent form along with the strategies implemented to minimize or overcome them. Finally, two events linked to the robotic exoskeleton itself (device malfunction) occurred over the course of the locomotor training program. The first event was a battery failure that required its replacement while the second Attendance The attendance rate reached in the present preliminary study (97.9%) was excellent. The high attendance with respect to the scheduled training sessions confirms the Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 10 of 12 Page 10 of 12 known to reach about 36% ± 15% of the bodyweight at heel strike when walking with an overground robotic exo- skeleton [29], are potential explanatory factors. This par- ticipant was withdrawn from the study and referred to the medical team until the fractures were healed. These frac- tures occurred even though the screening process was thoroughly completed by an experienced research physio- therapist and the minimal standing time tolerance (i.e., ≥30 min), recommended by the manufacturer of the exo- skeleton, was verified. Unfortunately, another preliminary study also reported a comparable fracture of the talus dur- ing a locomotor training program with another over- ground robotic exoskeleton [15] whereas a review recently suggested an overall incidence rate of bone fracture of 3.4% [3]. The fact that some studies have predominantly included individuals with recent SCI (≤1 year), a time period during which bone mineral density declines at the L/Es and distal vertebrae (i.e., infralesional osteoporosis) may not have yet stabilized at levels significantly below those of age and gender-matched able-bodied individuals [30], may explain why this risk may have been underesti- mated [16]. Further investigation will be needed to explore all potential causes to implement additional screening ele- ments for severe osteoporosis into the process (e.g., frac- ture risk stratification algorithms for adults with SCI [31], threshold for bone mineral density or architecture at the ankle and foot) and to develop solutions addressing the complex challenges linked to physical activities performed in standing position in individuals with SCI in the future. Other minor adverse events, predominantly linked to ex- acerbation of pre-existing (N = 4) or the development of new (N = 1) musculoskeletal-related non-debilitating pain at the upper extremity, were also documented (n = 5/14; 35.7%) over the course of the locomotor training program. Yet, all these participants opted to continue the training sessions while exploring personalized solutions to alleviate or even eliminate pain over the course of the program (e.g., increased number and duration of rest periods dur- ing sessions; cushioning at the handle of the walking aid; recommendation of stretching exercises post-training; use of nonsteroidal anti-inflammatory drugs) with the certified trainer(s). Attendance Unexpectedly, no exoskeleton-related skin or soft tissue issue was observed in the present study al- though it affected up to 50% of participants in previous studies and typically leads to interruptions of the interven- tion or withdrawal of participants from the studies [15]. All the above-identified risks remain impossible to elimin- ate, warrant thoughtful consideration, and should be care- fully explained within the informed consent form along each training session, compares to some extent with the ones reported using only pre- and post-intervention measures in previous study using a similar or different robotic exoskeletons [3, 16, 18, 22]. known to reach about 36% ± 15% of the bodyweight at heel strike when walking with an overground robotic exo- skeleton [29], are potential explanatory factors. This par- ticipant was withdrawn from the study and referred to the medical team until the fractures were healed. These frac- tures occurred even though the screening process was thoroughly completed by an experienced research physio- therapist and the minimal standing time tolerance (i.e., ≥30 min), recommended by the manufacturer of the exo- skeleton, was verified. Unfortunately, another preliminary study also reported a comparable fracture of the talus dur- ing a locomotor training program with another over- ground robotic exoskeleton [15] whereas a review recently suggested an overall incidence rate of bone fracture of 3.4% [3]. The fact that some studies have predominantly included individuals with recent SCI (≤1 year), a time period during which bone mineral density declines at the L/Es and distal vertebrae (i.e., infralesional osteoporosis) may not have yet stabilized at levels significantly below those of age and gender-matched able-bodied individuals [30], may explain why this risk may have been underesti- mated [16]. Further investigation will be needed to explore all potential causes to implement additional screening ele- ments for severe osteoporosis into the process (e.g., frac- ture risk stratification algorithms for adults with SCI [31], threshold for bone mineral density or architecture at the ankle and foot) and to develop solutions addressing the complex challenges linked to physical activities performed in standing position in individuals with SCI in the future. Other minor adverse events, predominantly linked to ex- acerbation of pre-existing (N = 4) or the development of new (N = 1) musculoskeletal-related non-debilitating pain at the upper extremity, were also documented (n = 5/14; 35.7%) over the course of the locomotor training program. Competing interests Th h d l h The authors declare that they have no competing interests. Funding h This project was funded largely by the Rick Hansen Institute and the Traumatology Research Consortium of the Fonds de Recherche du Québec – Santé (FRQS). The equipment and material required to complete this project were financed in part by a John R. Evans Leaders’ Fund Award from the Canadian Fund for Innovation. Availability of data and materials The datasets used and analysed during the current study are available from the corresponding author on reasonable request. Safety Among all participants, one serious adverse event oc- curred during the study. One participant sustained bilat- eral type I non-displaced fracture of the calcaneus after completing the two familiarization and the first training sessions. Although uncertainties exist about the specific cause of the fractures, both the fragility fracture risk of the calcaneus and the elevated vertical ground reaction force, Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Gagnon et al. Journal of NeuroEngineering and Rehabilitation (2018) 15:12 Page 11 of 12 Page 11 of 12 event was a mechanical problem with a hip joint bearing assembly malfunction due to a damaged bolt holding the proximal and distal joint components together. In both cases, the problems were solved within a 48-h period with the prompt assistance of the company’s customer service department and had minimal impact on the conduct of the study. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Limits of the study Limitations in the present study were the small sample size of relatively homogeneous participants recruited at a single site as well as the absence of a control group. Un- certainties about the best research design and outcome measures to adopt in future clinical trials continue. Be- cause the study only included long-term manual wheel- chair users with a chronic SCI living in the community, the generalizability of the results beyond this reference population, such as in ambulatory individuals with an in- complete SCI, requires caution. Prudence is also suggested when inferring about participants’ acceptance and satisfac- tion, particularly when addressing attendance and learn- ability, as this dimension was not reported. Hence, the findings of the present study should be considered prelim- inary, but it is anticipated that they will stimulate interest in conducting future larger-scale level I or II clinical trials investigating the efficacy or effectiveness of locomotor training programs with an overground robotic exoskeleton in long-term manual wheelchair users. Author details 1 1School of Rehabilitation, Université de Montréal, Montreal, QC, Canada. 2Pathokinesiology Laboratory, Centre for Interdisciplinary Research in Rehabilitation of Greater Montreal, Centre intégré universitaire de santé et services sociaux du Centre-Sud-de-l’Île-de-Montréal, Installation Institut de réadaptation Gingras-Lindsay-de-Montréal, 6300 Avenue Darlington, Montreal, QC H3S 2J4, Canada. 3Department of Exercise Science, Université du Québec à Montréal, Montreal, QC, Canada. Montreal, QC H3S 2J4, Canada. 3Department of Exercise Science, Université du Québec à Montréal, Montreal, QC, Canada. Authors’ contributions Design of protocol: DHG, ADK, CD, MAL. Implementation of protocol: DHG, ME, MV, CD, MAL. Data collection: DHG, ME, MV. Primary drafting of the manuscript and data-analysis: DHG, ME, MV, ADK, CD, MAL. All authors read and approved the final manuscript. Abbreviations ASIA: American Spinal Injuries Association; SCI: Spinal Cord Injury Acknowledgements D. H. Gagnon co-chairs the Initiative for the Development of New Technologies and Practices in Rehabilitation (INSPIRE) funded by the LRH Foundation and co- leads the Rehabilitation Intervention for Individuals with a SCI in the Community (RIISC) research team funded by the Ontario Neurotrauma Foundation and the Quebec Rehabilitation Research Network. M. Aubertin- Leheudre holds a junior 2 salary award from the FRQS. Conclusion This study reinforces what other pilot studies have shown and confirms that a locomotor training program with an overground robotic exoskeleton under the direct supervi- sion of a certified therapist is feasible and relatively safe in long-term manual wheelchair users with complete motor SCI. This finding is expected to stimulate interest in con- ducting future Level I and II large clinical trials investigat- ing, for example, the physical and psychological health effects or the cost-effectiveness of a locomotor training program with an overground robotic exoskeleton in this population. While doing so, strategies may need to be im- plemented to overcome potential challenges related to re- cruitment rate and minor safety issues. In fact, this study now confirms the relevance of developing pre-training re- habilitation programs to optimize passive lower extremity range of motion and standing tolerance to optimize the recruitment rate and safety, respectively. This study also calls for the development of clinical practice guidelines targeting fragility fracture risk assessment linked to the use of overground robotic exoskeletons. Received: 28 November 2017 Accepted: 15 February 2018 References 1. Esquenazi A, Talaty M, Jayaraman A. 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https://openalex.org/W3149503720	https://miun.diva-portal.org/smash/get/diva2:1547514/FULLTEXT01	English	null	Asymmetries in Ground Reaction Forces During Turns by Elite Slalom Alpine Skiers Are Not Related to Asymmetries in Muscular Strength	Frontiers in physiology	2,021	cc-by	9,614	ORIGINAL RESEARCH published: 30 March 2021 doi: 10.3389/fphys.2021.577698 Asymmetries in Ground Reaction Forces During Turns by Elite Slalom Alpine Skiers Are Not Related to Asymmetries in Muscular Strength Jan Ogrin1, Nejc Šarabon2,3, Mads Kjær Madsen4, Uwe Kersting4,5, Hans-Christer Holmberg6,7 and Matej Supej1,8* 1 Faculty of Sport, University of Ljubljana, Ljubljana, Slovenia, 2 Faculty of Health Sciences, University of Primorska, Izola, Slovenia, 3 S2P, Science to Practice, Ltd., Laboratory for Motor Control and Motor Behavior, Ljubljana, Slovenia, 4 Department of Health Science and Technology, Sport Sciences – Performance and Technology, Aalborg University, Aalborg, Denmark, 5 Institute of Biomechanics and Orthopedics, German Sport University Cologne, Cologne, Germany, 6 Department of Physiology and Pharmacology, Biomedicum C5, Karolinska Institute, Stockholm, Sweden, 7 China Institute of Sport and Health Science, Beijing Sport University, Beijing, China, 8 Swedish Winter Sports Research Centre, Mid Sweden University, Östersund, Sweden Edited by: Giuseppe D’Antona, University of Pavia, Italy The ground reaction forces (GRF) associated with competitive alpine skiing, which are relatively large, might be asymmetric during left and right turns due to asymmetries in the strength of the legs and torso and the present investigation was designed to evaluate this possibility. While skiing a symmetrical, 20-gate slalom course, the asymmetries of 9 elite alpine skiers were calculated on the basis of measurements provided by inertial motion units (IMU), a Global Navigation Satellite System and pressure insoles. In addition, specialized dynamometers were utilized to assess potential asymmetry in the strength of their legs and torso in the laboratory. In total, seven variables related to GRF were assessed on-snow and eight related to strength of the legs and torso in the laboratory. The asymmetries in these parameters between left and right turns on snow were expressed in terms of the symmetry (SI) and Jaccard indices (JI), while the asymmetries between the left and right sides of the body in the case of the laboratory measurements were expressed as the SIs. The three hypotheses to be tested were examined using multivariable regression models. Our ﬁndings resulted in rejection of all three hypotheses: The asymmetries in total GRF (H1), as well as in the GRF acting on the inside and outside legs (H2) and on the rear- and forefeet GRF (H3) during left and right turns were not associated with asymmetries in parameters related to muscular strength. Nevertheless, this group of elite slalom skiers exhibited signiﬁcant asymmetry between their right and left legs with respect to MVC during ankle ﬂexion (0.53 ± 0.06 versus 0.60 ± 0.07 Nm/kg, respectively) and hip extension (2.68 ± 0.39 versus 2.17 ± 0.26 Nm/kg), as well as with respect to the GRFs on the inside leg while skiing (66.8 ± 7.39 versus 76.0 ± 10.0 %BW). As indicated by the JI values, there were also large asymmetries related to GRF as measured by pressure insoles Reviewed by: Frédérique Hintzy, Université Savoie Mont Blanc, France Alain Steve Comtois, Université du Québec à Montréal, Canada *Correspondence: Matej Supej matej.supej@fsp.uni-lj.si Specialty section: This article was submitted to Exercise Physiology, a section of the journal Frontiers in Physiology Received: 29 June 2020 Accepted: 12 March 2021 Published: 30 March 2021 Edited by: Giuseppe D’Antona, University of Pavia, Italy INTRODUCTION Recently, Bell et al. (2014) reported that more pronounced inter-leg asymmetry (>10%) in power production during the push-oﬀphase of vertical jumping is associated with 20– 25% lower jump height, as well as less favorable changes in direction. Moreover, a systematic review exploring the relationship between asymmetries and performance describes widely discrepant ﬁndings, with negative associations in some cases (e.g., jumping and kicking), no associations in others (e.g., sprinting), and occasionally even a positive relationship (e.g., cycling) (Bishop et al., 2018; Maloney, 2019). Such relationships may be dependent on the sport under consideration, as well as the age and sex of the athletes. Alpine ski racing is an extremely complex and highly competitive sport, involving numerous physical, technical and tactical challenges. The diﬀerence between the ﬁrst and second ﬁnishers in a race is often only hundredths of a second (Hébert-Losier et al., 2014). Accordingly, small diﬀerences in a variety of factors can exert considerable inﬂuence on the successful performance of elite alpine ski racers. Although several studies have focused on determinants of sectional or instantaneous performance (Hébert-Losier et al., 2014), there is still no deeper understanding of the inﬂuence of biomechanical, anatomical variables or physical preparation (Spörri et al., 2012; Supej and Holmberg, 2019). In this context, vertical jump eﬃciency has been shown to be a good predictor of slalom performance (Strojnik and Dolenc, 2009), although at the same time Schobersberger et al. (2021) demonstrated recently that neither maximal aerobic capacity nor maximal power output were signiﬁcantly correlated to competitive performance, as indicated by FIS ranking. These ﬁndings reinforce the proposal that there is no single determinant of competitive performance in this complex sport. On-snow training, oﬀ-snow resistance training and conditioning of elite alpine skiers have received relatively little attention from sports researchers (Gilgien et al., 2018). Indeed, our search of the relevant scientiﬁc literature revealed only one study dealing with the laterality of ground reaction forces (GRF) in connection with slalom skiing (Vaverka and Vodickova, 2010) and one other case study demonstrating that an inertial motion tracker placed on the pelvis can detect lateral asymmetries during giant slalom skiing (Yu et al., 2016). More recently, Supej et al. (2020) found that asymmetries in technique and GRF were associated with asymmetries in performance. More speciﬁcally, asymmetries in GRF acting on the outside leg in combination with the shank angle inﬂuenced the asymmetries in turning radius. INTRODUCTION The risk of injury associated with alpine skiing is relatively high (Haaland et al., 2016) and recent research has focused on determining why and, at the same time, reducing this risk (Bere et al., 2014; Müller et al., 2016; Spörri et al., 2017). The handful of reports on asymmetries in alpine skiing that have appeared so far have been concerned primarily with the eﬀects of such asymmetries on injuries to the anterior cruciate ligament of the knee (Bujas et al., 2012; Jordan et al., 2015a, 2017; Steidl-Muller et al., 2018). A 10% asymmetry in the strength of the legs is generally considered to be the threshold for a safe return to alpine skiing following injury. Interestingly, healthy, uninjured younger skiers often exhibit more pronounced asymmetry in leg extension strength during their phases of rapid growth, suggesting that a diﬀerent threshold might be more appropriate for this particular subgroup (Steidl-Muller et al., 2018). However, that same investigation also found that diﬀerences between the right and left legs with respect to extension strength considerably increase the risk of injury for young ski racers. To our knowledge, kinematic and/or kinetic data have not yet been employed to search for potential links between asymmetries in muscular strength and asymmetries on-snow. This lack of research is especially disturbing since the GRFs associated with alpine skiing are large (Supej and Holmberg, 2019) and fundamental motor asymmetries may potentially exert considerable inﬂuence on both skiing performance and safety. The large GRFs associated with alpine skiing require considerable muscular strength for eﬀective turning. Consequently, lateral asymmetry by and the strength of the legs and trunk might result in diﬀerences between left and right turns and, thereby, diﬀerent patterns of GRFs. Accordingly, this study was designed to determine whether asymmetries in basic muscular strength are related to the GRFs encountered during elite slalom skiing. In this context and based on expert deconstruction of skiing as a motor task, the following three hypotheses were tested: The relationship between asymmetries with incidence and risk of injuries in connection with other sports has also been receiving attention. For both athletes and individuals who not participate in sports, inter-limb asymmetries > 15% are associated with an elevated risk of injury (Barber et al., 1990). Although it is reasonable to conclude that reducing such asymmetry can help avoid injuries, the actual eﬀect of interventions designed for this purpose remains unclear. Citation: Ogrin J, Šarabon N, Madsen MK, Kersting U, Holmberg H-C and Supej M (2021) Asymmetries in Ground Reaction Forces During Turns by Elite Slalom Alpine Skiers Are Not Related to Asymmetries in Muscular Strength. Front. Physiol. 12:577698. doi: 10.3389/fphys.2021.577698 March 2021 \| Volume 12 \| Article 577698 Frontiers in Physiology \| www.frontiersin.org 1 Ogrin et al. Asymmetries in Alpine Skiing (range: 42.7–56.0%). In conclusion, inter-limb asymmetries in GRFs during elite alpine skiing are not related to corresponding asymmetries in muscular strength. Although our elite athletes exhibited relatively small inter-limb asymmetries in strength, their asymmetries in GRF on-snow were relatively large. Keywords: biomechanics, alpine skiing, inertial suit, GNSS-global navigation satellite system, GPS-global positional system, pressure insoles, force plate Frontiers in Physiology \| www.frontiersin.org MATERIALS AND METHODS FIGURE 1 \| Setups for the skiing measurements on snow (top) and laboratory tests (bottom): (I) countermovement jump on a bilateral force platform, (II) ankle dynamometry, (III) knee dynamometry, (IV) hip dynamometry, and (V) trunk dynamometry. INTRODUCTION H1: Asymmetries in the GRFs associated with left and right turns are related to asymmetries between the left and right sides of the body with respect to at least one of the following parameters related to muscular strength: maximal March 2021 \| Volume 12 \| Article 577698 Frontiers in Physiology \| www.frontiersin.org 2 Asymmetries in Alpine Skiing Ogrin et al. force during a countermovement jump and/or the peak torque associated with maximal voluntary contraction (MVC) during knee extension and ﬂexion and/or during hip extension. Navigation Satellite System (RTK GNSS) (Figure 1), in a manner similar to previous studies (Krüger and Edelmann-Nusser, 2010; Supej, 2010). The only diﬀerence was that here a more recent inertial system MVN Biomech. 2018.2 (Xsens Technologies B.V., Enschede, Netherlands) recording data at 240 Hz and a more advanced/new generation GNSS antenna Leica Zeno GG04 plus (Leica Geosystems, Heerbrugg, Switzerland) with a sampling frequency of 20 Hz were used. Navigation Satellite System (RTK GNSS) (Figure 1), in a manner similar to previous studies (Krüger and Edelmann-Nusser, 2010; Supej, 2010). The only diﬀerence was that here a more recent inertial system MVN Biomech. 2018.2 (Xsens Technologies B.V., Enschede, Netherlands) recording data at 240 Hz and a more advanced/new generation GNSS antenna Leica Zeno GG04 plus (Leica Geosystems, Heerbrugg, Switzerland) with a sampling frequency of 20 Hz were used. H2: Asymmetries between left and right turns regarding the GRF acting on the inside and/or outside leg are related to asymmetries between the left and right sides of the body with respect to at least one of the following parameters related to muscular strength: maximal force during a countermovement jump and/or peak MVC torque during knee extension and/or ﬂexion, during hip extension and/or abduction, and/or during lateral trunk ﬂexion. The inertial sensors were embedded and placed under the skier’s racing suit, while the GNSS smart antenna (weight: 0.8 kg, height: 7.1 cm, diameter: 8.6 cm) was placed on top of a modiﬁed back-protector vest (Spine VPD 2.0, POC, Stockholm, Sweden) to ensure optimal satellite visibility in the vicinity of the shoulders and posterior neckline (Figure 1). The receiver (Conker NS6, Conker, Takeley, England) was placed in a pocket of the back-protector vest located on the front of the trunk and the two devices communicated via Bluetooth. INTRODUCTION To allow merging of the trajectories surveyed by RTK GNSS with inertial data H3: Asymmetries between left and right turns with respect to the GRF acting on the rear- and forefoot of the inside and outside legs are related to asymmetries between the left and right sides of the body regarding peak MVC torque during ankle extension and/or ﬂexion. Frontiers in Physiology \| www.frontiersin.org Laboratory Tests On the day following these skiing runs, counter-movement jump performance and maximal isometric strength of the selected muscle groups was tested. It was ensured that there was suﬃcient time for rest between the measurements in the laboratory and on-snow. Maximal isometric voluntary torque during bilateral plantar ﬂexion and dorsal ﬂexion was tested on an isometric ankle dynamometer (S2P Ltd., Ljubljana, Slovenia) adjusted to ensure proper positioning of each subject (upright trunk, 90◦ angles at the hips, knees and ankles). A metal brake above each knee and a rigid strap above each foot ensured isometric conditions. Each of the two foot pedals was linked ﬁrmly to a strain gauge (Z6FC3/200kg, Hottinger Baldwin Messtechnik GmbH, Darmstadt, Germany). For determination of maximal voluntary torque during bilateral ﬂexion and extension of the knee, the subject was seated (with 90◦and 60◦ﬂexion at the hips and knees, respectively) on an isometric knee dynamometer (S2P Ltd., Ljubljana, Slovenia) with tight straps across the pelvis, above the knees and behind the distal leg to ensure ﬁxation. This positioning of the joints was chosen to allow the knee ﬂexors/extensors to develop maximal knee torque (Brughelli and Cronin, 2007; Brughelli et al., 2010). This testing procedure has been reported to yield highly reproducible results (Sarabon et al., 2013). Measurements and Collection of Data Skiing Tests During three consecutive days, data on 9 elite male European Cup slalom skiers (age: 22.7 ± 3.4 y; height: 181.8 ± 6.9 cm; weight, 82.2 ± 5.6 kg; current SL FIS points: 24.9 ± 18.6; means ± SD), ﬁve with right and four left leg preference (assessed on the basis of leg preference in connection with testing maximal height of a single-leg jump), were collected on a groomed slope with a mean inclination of 16◦and <1◦ tilting to either side on a glacier. The snow was icy and hard and the temperature between −2 and 0◦C. The 20-gate corridor course, with two symmetrical (mirrored) slalom courses (see Supplementary Figure 1), was set up using a high-resolution geodetic global satellite navigation system (Leica Geosystems 1200, Leica Geosystems AG, Heerbrugg, Switzerland) and its accompanying “Stakeout” program to ensure a constant distance of 12 and 4-m oﬀset between the gates. The total length of the course from the start to the ﬁnish was approximately 250 m. These two courses were prepared professionally following each of the 12 individual runs on any given day. This study was pre- approved by the National Medical Ethics Committee (approval no. 0120-99/2018/5) and informed written consent to participate obtained from each subject. Each subject skied this corridor course four times and all trials were used in our calculations. The side from which the ﬁrst run was started was pre-selected randomly (and the skier informed about this assignment at the start), with subsequent starts alternating between the left and right sides. Prior to each and every run, the course was smoothed by coaches and members of the research team in order to provide nearly ideal conditions. Prior to each run, the skier was asked to perform three explosive squats, followed by three hits with the right ski to the ground, in order to synchronize the measuring instruments. The three-dimensional motion of the entire body was monitored with a full-body inertial measurement system in combination with a high-frequency Real Time Kinematics Global FIGURE 1 \| Setups for the skiing measurements on snow (top) and laboratory tests (bottom): (I) countermovement jump on a bilateral force platform, (II) ankle dynamometry, (III) knee dynamometry, (IV) hip dynamometry, and (V) trunk dynamometry. Frontiers in Physiology \| www.frontiersin.org March 2021 \| Volume 12 \| Article 577698 3 Asymmetries in Alpine Skiing Ogrin et al. Computation of Independent and Dependent Variables Independent Variables The values were calculated using the following symmetry index (SI), which is similar to the one developed by Bishop et al. (2016): SI (L, R) = 1 −\|L −R\| LR . where L represents the measurement for the left side and R the right measurement. The means of the 1-s maxima in the three successful trials (derived from the torque/time curve in the case of the isometric dynamometer and from the force/time curve for the force plate) were employed. In a similar manner, the SI was computed using the mean percentages of body weight supported by the left and right legs, obtained from the force plate signals during the quiet stances, as in the work of Sarabon and Rosker (2013). In this manner the level of fundamental motor asymmetry was derived from the following asymmetry indicating variables: The maximal strength of muscle groups acting at the hip during bilateral abduction and unilateral ﬂexion (supine position) was evaluated employing a multi-directional hip isometric dynamometer (Muscle Board, S2P Ltd., Ljubljana, Slovenia). During these tests, performed both in the prone (hip extension) and supine (hip abduction) positions, the pelvis was ﬁxated with tight non-elastic straps. The lateral trunk ﬂexors were tested in the upright position utilizing the dynamometric function of a multi-modal system for testing neuromuscular functions of the trunk (TNC system, S2P Ltd., Ljubljana, Slovenia). – the SI for the maximal force during the counter-movement jump; – the SI for the maximal voluntary contraction (MVC) associated with ﬂexion and extension of the knee and ankle; – the SI for the MVC associated with hip ﬂexion and abduction; – the SI for the MVC during lateral ﬂexion of the trunk. A high SI percentage means better symmetry between the left and right side measurement. In all cases, testing of maximal voluntary strength followed the same protocol. After the task was explained to the subject, the equipment ﬁtted and appropriate/ﬁrm ﬁxation of body segments ensured, the subject ﬁrst carried out one repetition each at approximately 50, 75, and 90% of self-estimated maximal voluntary eﬀort to become accustomed to the set-up. Thereafter, he performed three MVCs (pressing against the support with as much force as possible for 3 s), separated by 60 s to avoid fatigue and with verbal encouragement. The signals were acquired at 1,000 Hz (NI USB-6009; National Instruments Corp., Austin, TX, Measurements and Collection of Data Skiing Tests United States) and the 1-s period around the peak of the torque- time curve analyzed. The mean value of the three repetitions was used for statistical analysis. on full-body motion, the position of the antenna relative to the cervical (C7) and thoracic vertebra (T12) was measured. GRFs were monitored at a sampling frequency of 100 Hz by pressure insoles (Loadsol, Novel GmbH, Münich, Germany) that allowed separate determination of the forces on the left and right feet and fore- and rear-foot, as well as total overall force (Figure 1). To ensure appropriate synchronization, while skiing the subjects were also recorded at 50 Hz with a high-resolution JVC camcorder (GC-PX100, The Japan Victor Company Ltd., Yokohama, Japan). GRFs during bilateral counter-movement jump were determined with a bilateral force plate (2x 9260AA6, Kistler Instrumente AG, Winterthur, Switzerland). Each participant was instructed to perform a rapid counter-movement (so that the angle of the knee reached 90◦) and couple this explosively to the push-oﬀto jump as high as possible. Jumps that were not explosive, i.e., the eccentric and concentric phases were not coupled explosively and/or the heels not lifted oﬀthe force plate during the downward movement, were rejected and the trial repeated. The subject was asked to aim for maximal performance during three repetitions while adhering to the following instructions: (i) counter-movement associated with the counter-movement jump should be performed quickly and dynamically; and (ii) the hands should be on the hips at all times and the feet ﬂat and in contact with the force plate during push-oﬀ. Frontiers in Physiology \| www.frontiersin.org Dependent Variables The kinematic data supplied by the MVN Biomech were exported into Matlab R2016b (Mathworks, Natick, MA, United States) for further processing. To match their sampling frequency to that of the inertial sensors, the RTK GNSS and force data were converted to 240 Hz by cubic spline interpolation. The RTK GNSS, full- body inertial motion and video data were synchronized using the March 2021 \| Volume 12 \| Article 577698 Frontiers in Physiology \| www.frontiersin.org 4 Ogrin et al. Asymmetries in Alpine Skiing lowest point during the ﬁnal explosive squat movement. Force and kinematic data were synchronized by matching the peak foot acceleration with the maximal GRF during the three hits with the ski on the snow before the start of testing. and all right turns, respectively. The Jaccard index (JI) was then calculated from these two geometric shapes Lg and Rg as follows: JI Lg, Rg = p(Lg ∩Rg) p(Lg ∪Rg), Following synchronization, all data were ﬁltered with the Rauch-Tung-Striebel algorithm (Rauch et al., 1965), which uses two unscented Kalman ﬁlters running forward and backward in time to achieve ﬁxed-interval oﬄine smoothing of the estimated signals, as described previously (Supej, 2010). The RTK GNSS and measurements of full-body inertial motion were merged employing the GNSS data as a global reference coordinate system. These merged data was then exported into the Visual 3D v6 software (C-Motion Inc., Germantown, MD, United States), which computed the trajectory of the center-of-mass (CoM) and acceleration of the skier, as well as the trajectory of the mid- point of the ankle (arithmetic mean of the positions of the ankle joints), after which the values obtained were exported back into formats compatible with Matlab, in a manner similar to that described previously (Zorko et al., 2015). The sum of the CoM and gravitational acceleration was used to estimate the total GRFs acting on the skier (Supej and Holmberg, 2010). The variables of GRF obtained from both the kinematic system and pressure insoles had also been normalized to the overall GRF to facilitate analysis and are presented as percentages of body weight (% BW). It should be noted that measurements of forces by pressure insoles have been reported to be too inaccurate for monitoring the magnitude of the GRF (Stricker et al., 2010). Dependent Variables However, since it can be assumed that the GRF values indicated by pressure insoles are subject to approximately the same level and type of bias in the case of the left and right legs, these values can be used for calculating symmetry indices, which only reﬂect relative diﬀerences. where p represents the area enclosed by the geometric shapes (Lg ∩Rg or Lg ∪Rg). In contrast to the situation with SI, not only the mean values for JI, but also the standard deviations are utilized to calculate asymmetries between sets of data and, therefore, JI values provide a better indication of asymmetry. Moreover, there is a bijective relationship between geometrical matching of Lg and Rg and matching of the means and standard deviations of the values for these datasets, which both determine the same two-dimensional shapes. In other words, when there is a good match between the mean values and standard deviations for the two sets, there is a good geometrical match (i.e., JI is close to 1) between the two two-dimensional shapes determined by these same two sets, and vice versa. From a practical point of view, the maximal or average GFR during a slalom turn captures very few aspects of this action. Maximal GRF can often be generated by shocks or transient vibrations that have no major impact on skiing (Supej et al., 2018). Moreover, not only do the GRFs during the weight transfer and steering phases diﬀer, the magnitude of the GRF within the steering phase also varies signiﬁcantly (Supej and Holmberg, 2010; Supej et al., 2015). Therefore, averaging the GRF for the entire steering phase results in loss of important information. By determining the JI for the "steering phase," this problem can be avoided and a deeper understanding of “active” turning achieved. g g Combining SI and JI provided a measure of the level of GRF asymmetry, in particular: For all variables, mean curves and standard deviations were calculated and diagrams representing turn-cycle characteristics were created using the deﬁnitions of turn phases described by Müller et al. (1998), while the beginning and end of each turn were deﬁned according to Supej et al. (2003). For this latter purpose, a 3D terrain model of the slope was constructed and the turns analyzed were divided into initiation, steering and completion phases, as previously described in greater detail (Supej and Holmberg, 2010). Dependent Variables – the SI and JI for GRFs acting on the inside or outside of the fore-, rear- or entire foot during the steering phase (measured by the pressure insoles); – the SI and JI for the overall GRF during the steering phase (computed from the kinematic data). Frontiers in Physiology \| www.frontiersin.org Multi-Variable Regression Multi-Variable Regression To test our three hypotheses, linear multivariable regression models were applied. In the case of hypothesis H1, four independent variables and two dependent variables were included and 20 linear regression models tested. For H2 and H3, 124 and 24 models were tested, respectively. In each case, depending on the number of models, the threshold p-value obtained was subjected to Bonferroni adjustment. All models that yielded a p-value greater than the adjusted threshold were discarded, as were all models for which R2 was less than 0.7. These restrictions led to all of the models being discarded and thus to all of the hypotheses being rejected. Although none of the models was associated with a signiﬁcant p-value, Tables 3–5 present the ﬁndings with models that included a single potential predictor. Models including more than one potential predictor are not documented because of their very large total number (80). The Symmetry and Jaccard Indices (SI and JI) The mean symmetry indices (SI) for the independent variables determined in the laboratory ranged from ∼89% (for peak MVC torque during hip extension) to ∼98% (peak MVC torque during hip abduction) (Table 1), while the corresponding mean SI for the dependent variables during the steering phase of the turn while skiing ranged from ∼85% (GRFs on the rear-foot of the inside leg) to ∼98% (total GRF). The mean Jaccard Index (JI) for the dependent variables during this steering ranged from ∼43% (for the GRF on the fore-foot of the inside leg) to ∼71% (total GRF) (Table 2). The time-courses of the GRFs on the entire outside foot during left and right turns and the corresponding JI during the steering phase (ranging from 29 to 79%) are shown for all 9 skiers in Figure 1. For four of these skiers, these two variables were well matched, while the other 5 demonstrated asymmetry in GRFs on the outside leg between 29 and 59% (skiers A, C, D, E, and F in Figure 2). Of these latter 5, 4 had a right leg preference and larger mean overall GRFs during the steering phase of left turns, when the right leg was outside. Statistical Analyses All values presented are means and standard deviations. The Shapiro-Wilk test was applied to determine whether the distribution of these values was normal and, when necessary, the Box-Cox power transformation performed to achieve normality. The data on turns were examined for potential outliers (1.5 Inter-Quartile Range method of outlier detection was applied), which were excluded from analysis. For all possible combinations of the independent (at most two) and dependent variables, multivariable linear regression was performed. Initial p-values below 0.05 obtained by applying the F-test to regression models were adjusted using a Bonferroni- like correction (Curtin and Schulz, 1998). The power of diﬀerences indicated as being statistically signiﬁcant (p < 0.05) was tested using G∗Power (Faul et al., 2009) and only diﬀerences with a power greater than 0.8 were ﬁnally considered to be signiﬁcant. All statistical analyses were performed in the Matlab software (Prentice-Hall, Inc., Upper Saddle River, NJ, United States). j g The largest GRFs associated with slalom occurs during the steering phase, when the skier is actively turning, whereas during the initiation and completion phases these forces are signiﬁcantly lower (Supej and Holmberg, 2010; Supej et al., 2015). Therefore, the SI reﬂecting the degree of asymmetry in the GRF was determined during the steering phase. In addition, the Jaccard index (JI) (Jaccard, 1901) or intersection over union (IOU), a standard algorithmic estimation of the accuracy with which an object is detected by a computer vision, was calculated on the basis of the shapes of the left (Lg) and right (Rg) turns. Deﬁnition of these shapes was based on a normalized time-scale from the start to ﬁnish of the turn, with upper and lower limits one standard deviation above or below the average curve, respectively. The average curve and corresponding standard deviation for Lg and Rg was calculated from all left turns March 2021 \| Volume 12 \| Article 577698 Frontiers in Physiology \| www.frontiersin.org 5 Asymmetries in Alpine Skiing Ogrin et al. DISCUSSION The major ﬁnding is that asymmetries in GRFs during elite alpine skiing are not related to asymmetries in local muscular strength. More precisely, none of our three hypotheses was supported by the ﬁndings: none of the asymmetries between left and right TABLE 1 \| The values (means ± standard deviations, statistical comparison of the right and left legs) of the parameters measured and corresponding motor symmetry indices (independent variables). Variable Left leg/side Right leg/side p-value Cohen’s d SI (%) Countermovement jump Maximal force (N/kg) 12.2 ± 1.20 13.3 ± 1.64 0.12 0.61 94.0 ± 3.14 Maximal voluntary contraction Ankle ﬂexion (Nm/kg) 0.53 ± 0.06 0.60 ± 0.07 0.04 0.84 93.5 ± 3.74 Ankle extension (Nm/kg) 1.86 ± 0.27 1.93 ± 0.32 0.62 0.19 95.6 ± 2.60 Knee ﬂexion (Nm/kg) 1.81 ± 0.24 1.83 ± 0.16 0.84 0.07 95.6 ± 3.80 Knee extension (Nm/kg) 2.75 ± 0.47 2.69 ± 0.42 0.80 0.10 95.5 ± 2.60 Hip extension (Nm/kg) 2.68 ± 0.39 2.17 ± 0.26 0.01 1.14 89.5 ± 6.33 Hip abduction (Nm/kg) 1.77 ± 0.28 1.87 ± 0.36 0.52 0.02 97.7 ± 1.88 Trunk lateral ﬂexion (Nm/kg) 6.68 ± 1.30 7.36 ± 1.18 0.26 0.43 92.7 ± 5.60 SI, symmetry index. TABLE 2 \| The values (means ± standard deviations, statistical comparison of the right and left legs) of the ground reaction forces (GRF) measured and corresponding symmetry and Jaccard indices (dependent variables). Variable Left turn Right turn p-value Cohen’s d SI (%) JI (%) Entire foot—pressure insoles GRF outside leg (% BW) 126.2 ± 19.2 113.6 ± 21.55 0.21 0.48 92.9 ± 4.74 56.1 ± 18.9 GRF inside leg (% BW) 66.8 ± 7.39 76.0 ± 10.0 0.04 0.83 91.4 ± 5.72 56.0 ± 19.6 Fore-foot—pressure insoles GRF outside leg (% BW) 62.5 ± 20.6 58.7 ± 26.85 0.75 0.12 88.4 ± 8.35 47.2 ± 21.6 GRF inside leg (% BW) 27.4 ± 9.20 37.8 ± 17.6 0.14 0.58 85.1 ± 10.1 42.7 ± 23.2 Rear-foot—pressure insoles GRF outside leg (% BW) 63.8 ± 7.69 54.9 ± 16.6 0.17 0.54 87.8 ± 7.33 51.6 ± 14.4 GRF inside leg (% BW) 39.4 ± 9.13 38.25 ± 15.8 0.85 0.07 85.8 ± 15.2 52.9 ± 23.2 Approximation from CoM Overall GRF (% BW) 287.5 ± 26.3 283.7 ± 17.5 0.72 0.13 98.2 ± 1.11 71.3 ± 2.70 CoM, center of mass; BW, body weight; SI, symmetry index; JI, Jaccard index. DISCUSSION March 2021 \| Volume 12 \| Article 577698 Frontiers in Physiology \| www.frontiersin.org 6 Asymmetries in Alpine Skiing Ogrin et al. FIGURE 2 \| Evolution of the ground reaction forces (GRFs) on the entire outside foot during left (blue) and right (red) turns for all nine elite alpine skiers (A-I) in order to compare the Jaccard Index (JI) during the steering phase. The vertical lines indicate the start and end of the turn and the steering phase is marked with a horizontal double-arrow. FIGURE 2 \| Evolution of the ground reaction forces (GRFs) on the entire outside foot during left (blue) and right (red) turns for all nine elite alpine skiers (A-I) in order to compare the Jaccard Index (JI) during the steering phase. The vertical lines indicate the start and end of the turn and the steering phase is marked with a horizontal double-arrow. FIGURE 2 \| Evolution of the ground reaction forces (GRFs) on the entire outside foot during left (blue) and right (red) turns for all nine elite alpine skiers (A-I) in order to compare the Jaccard Index (JI) during the steering phase. The vertical lines indicate the start and end of the turn and the steering phase is marked with a horizontal double-arrow. turns in total GRF (H1) or in GRF acting on the inside and outside legs (H2) or rear- and forefoot GRF (H3) were associated with asymmetries in the parameters related to strength examined. Nevertheless, this group of elite slalom skiers exhibited signiﬁcant inter-limb asymmetries in MVC during ankle ﬂexion and hip extension, as well as in the GRFs on the inside leg while skiing. The GRFs on the skier are highest during the steering phase and considerably lower during both the initiation and completion phases (Supej and Holmberg, 2010). Therefore, it is reasonable to assume that the most pronounced asymmetries in GRF caused by asymmetries in strength will occur during the steering phase. Therefore, the values of the dependent variables were computed for this phase only. The symmetry index (SI) utilized here is symmetrical [i.e., SI(L, R) = SI(R, L)], bounded by the interval [0,1], and increases monotonically as symmetry increases. This last characteristic is a requirement for meaningful interpretation of the results of linear multivariable regression models. DISCUSSION TABLE 5 \| Triplets (p-value, coefﬁcient in the linear regression model, R2) for each pair: independent (laboratory) variable (rows) and dependent (on-snow) variable (columns) used for testing hypothesis H3. Inside leg entire foot pressure insoles measured force Outside leg entire foot pressure insoles measured force Backfoot Forefoot Backfoot Forefoot SI JI SI JI SI JI SI JI Ankle ﬂexion MVC (SI) 0.61, −0.79, 0.04 0.97, −0.09, 0.00 0.68, 0.44, 0.03 0.45, 1.80, 0.08 0.16, −1.00, 0.26 0.44, −1.14, 0.09 0.69, −0.35, 0.02 0.64, −1.04, 0.03 Ankle extension MVC (SI) 0.40, −1.90, 0.10 0.14, −4.87, 0.29 0.06, 2.61, 0.43 0.10, 2.26, 0.33 0.28, 1.17, 0.17 0.49, 1.51, 0.07 0.38, 1.09, 0.11 0.40, 2.71, 0.10 Adjusted p-value threshold was 0.0021. JI, Jaccard index; MVC, maximal voluntary contraction; SI, symmetry index.x return-to-sport proposed by Myer et al. (2006), our participants did not demonstrate levels of asymmetries as pronounced as, for example, those exhibited by skiers following reconstruction of the ACL. The recent observation that asymmetries in isometric leg extension strength constitute a signiﬁcant risk factor for traumatic injury (Steidl-Muller et al., 2018) indicates that the only potential risk factor for some of our skiers could be their SI for MVC in connection with hip extension (89.5 ± 6.3%). variables examined. In practice, this means that the mean overall GRF on the skier during left and right turns is very similar, in spite of the other diﬀerences observed. This was probably due to the use of a highly accurate RTK GNSS to ensure that the course was set symmetrically, with the chosen slope having little or no tilting to either side. However, similarity in the absolute GRF during left and right turns does not necessarily mean that the skiers’ body movements were symmetrical. A previous investigation involving the same on-snow measurements as those employed here (but no strength tests) revealed that asymmetries in the GRF acting on the entire outside leg were associated with asymmetries in turning radius, which is related to performance (Supej et al., 2020). In the present case, the asymmetries in the production of skiing force (dependent variables) turned out to be greater than fundamental asymmetries (independent variables), a ﬁnding that is at least somewhat in agreement with earlier reports that fundamental motor asymmetries of approximately 10% lowered jump height and impaired change of direction by more than 10% (Hoﬀman et al., 2007; Bell et al., 2014). DISCUSSION TABLE 3 \| Triplets (p-value, coefﬁcient in the linear regression model, R2) for each pair: independent (laboratory) variable (rows) and dependent (on-snow) variable (columns) used for testing hypothesis H1. Total GRF estimation from acceleration of CoM SI JI CMJ max force (SI) 0.71, −0.05, 0.02 0.44, 0.25, 0.08 Knee ﬂex MVC (SI) 0.78, −0.03, 0.01 0.79, 0.06, 0.01 Knee ext MVC (SI) 0.26, −0.18, 0.18 0.38, −0.34, 0.11 Hip ext MVC (SI) 0.81, −0.03, 0.01 0.17, 0.31, 0.29 Adjusted p-value threshold was 0.0026. CMJ, countermovement jump; CoM, center of mass; GRF, ground reaction forces; JI, Jaccard index; MVC, maximal voluntary contraction; SI, symmetry index. Since the absolute values for GRFs estimated using pressure insoles can deviate substantially from the true values (Lüthi et al., 2005; Stricker et al., 2010), acceleration of the CoM was also used here to compute the GRFs’ approximation. The most pronounced SI (∼98%), as well as the greatest JI (∼71%) for these absolute GRFs were relatively large compared to the other dependent March 2021 \| Volume 12 \| Article 577698 Frontiers in Physiology \| www.frontiersin.org 7 Asymmetries in Alpine Skiing Ogrin et al. TABLE 4 \| Triplets (p-value, coefﬁcient in the linear regression model, R2) for each pair: independent (laboratory) variable (rows) and dependent (on-snow) variable (columns) used for testing hypothesis H2. TABLE 4 \| Triplets (p-value, coefﬁcient in the linear regression model, R2) for each pair: independent (laboratory) variable (rows) and dependent (on-snow) variable (columns) used for testing hypothesis H2. Inside leg total force pressure insoles Outside leg total force pressure insoles SI JI SI JI CMJ max force (SI) 0.45, 0.53, 0.08 0.35, 2.20, 0.13 0.43, 0.46, 0.09 0.18, 2.93, 0.24 Knee ﬂex MVC (SI) 0.11, 0.80, 0.32 0.16, 2.48, 0.26 0.93, −0.04, 0.00 0.58, 1.00, 0.05 Knee ext MVC (SI) 0.55, −0.50, 0.05 0.47, −2.04, 0.08 0.01, −1.46, 0.66 0.06, −4.65, 0.43 Hip ext MVC (SI) 0.02, 1.25, 0.63 0.04, 3.91, 0.53 0.37, 0.43, 0.13 0.09, 2.81, 0.41 Hip abd MVC (SI) 0.31, −0.69, 0.15 0.46, 1.73, 0.29 0.27, −0.62, 0.17 0.53, −1.43, 0.06 Trunk lat ﬂex MVC (SI) 0.72, −0.15, 0.02 0.52, −0.89, 0.06 0.53, −0.21, 0.06 0.35, −1.24, 0.29 Adjusted p-value threshold was 0.0004. CMJ, countermovement jump; JI, Jaccard index; MVC, maximal voluntary contraction; SI, symmetry index. Frontiers in Physiology \| www.frontiersin.org DISCUSSION It can thus be speculated that asymmetries in strength might inﬂuence performance. Associations observed between asymmetries and performance appear to diﬀer between sports and with respect to diﬀerent measures of outcome (Bishop et al., 2018). Therefore, more speciﬁc and detailed characterization of potential relationships between asymmetries, with regards to both fundamental motor abilities and GRF during skiing, and the performance of elite alpine skiers would be of considerable value. Such an investigation should preferably involve a larger number of athletes who specialize in a variety of Interestingly, as indicated by the values provided by the asymmetry in GRF indicated by the pressure insoles, the asymmetry in GRF was statistically signiﬁcant for the inside leg only (p = 0.21 for the outside leg, Table 2). In other words, the force(s) acting on the right foot during right turns, when this leg was inside, was larger than the force(s) acting on the left leg, but this was not the case during left turns, when the right foot was outside. Although this diﬀerence might be related to the fact that ﬁve of our nine skiers exhibited right leg preference, the present ﬁndings cannot be used to fully support this possibility. Although also not statistically signiﬁcant, noticeably more maximal force was produced by the right leg during the countermovement jump (p = 0.12) (Table 1). Although vertical jump parameters were previously shown to be good predictors of slalom performance (Strojnik and Dolenc, 2009), here there was no association between performance and asymmetries associated with jumping and skiing. One speculative explanation for the lack of any such association is that the mean deviation from perfect symmetry with respect to the independent variables was no larger than ∼10%, in line with ﬁndings on elite Austrian alpine skiers (Steidl-Muller et al., 2018). Compared to the thresholds for safe March 2021 \| Volume 12 \| Article 577698 8 Asymmetries in Alpine Skiing Ogrin et al. skiing disciplines, although this is diﬃcult to arrange in the case of elite alpine skiing on-snow. and maintain conditions on the slope for them that would allow objective analyses. Another interesting observation here is that the SI for maximal power during the countermovement jump, as well as the corresponding indices for both maximal power and maximal force during the squat jump, exhibits exceptionally high symmetry (∼98%), with low standard deviations. DATA AVAILABILITY STATEMENT The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. Methodological Considerations The current investigation encompassed one of the largest sets of full-body three-dimensional kinematic data on elite alpine skiers reported to date (in total, 720 turns), collected utilizing a state-of-the-art Global Navigation Satellite System (GNSS) and capture of inertial motion sensors with pressure insoles. Such measurement equipment has been shown to be reliable for use in ﬁeld tests on alpine skiers (Krüger and Edelmann-Nusser, 2010; Supej, 2010; Spörri et al., 2015, 2016, 2018; Zorko et al., 2015; Falda-Buscaiot et al., 2017). In addition, our laboratory tests were performed with up-to-date, valid and reliable equipment. Nevertheless, a larger sample size would increase the power of the statistical tests, but it is extremely diﬃcult to assemble a larger group of elite competitors to take measurements on-snow DISCUSSION In practice, this means that the SI value for any given variable during skiing could be very high, while the corresponding JI is very low. Accordingly, the novel approach to analysis of asymmetries that we present here can provide new insights. CONCLUSION Ours is the ﬁrst examination of potential relationships between asymmetries in fundamental muscular strength and asymmetries exhibited on-snow by elite alpine skiers. Despite the well-known importance of physical ﬁtness in this context (Strojnik and Dolenc, 2009) and the association of GRF with parameters related to performance (Supej et al., 2020), our present ﬁndings do not indicate that muscular strength exerts a direct impact on GRFs. However, the asymmetries in muscular strength among the skiers involved here were probably too small to exert an observable impact on GRF asymmetries in connection with such a complex sport as slalom skiing. Thus, the potential existence of such relationships remains to be elucidated, perhaps using athletes who demonstrate more pronounced asymmetries in strength. In addition, it might be revealing to examine whether fatigue augments skiing asymmetries and, if so, whether this augmentation is more closely related to asymmetries in basic strength. From a practical point of view, the relatively large asymmetries in skiing GRF indicated by the JI values (despite much smaller asymmetries as assessed on the basis of SI) suggest that, for example, the rate of force development and duration of the maximal GRF diﬀer between left and right turns. The fact that elite skiers train extensively on snow, especially during the competition period (Gilgien et al., 2018), could contribute to the development of asymmetries in strength as the season progresses. Therefore, we suggest that testing regularly for asymmetries in combination with compensatory strength training, as required, can help avoid a potential increase in the risk of injury. DISCUSSION Similarly, the mean SI for other fundamental movements were also relatively high, in general around 95%. The small sizes of these asymmetries and their highly limited variation may have obscured other relationships between fundamental and skiing- speciﬁc asymmetries, thereby leading to the rejection of all three hypotheses. It might be useful in a future study of this nature to include skiers with more pronounced asymmetries in physical abilities, perhaps even those who have "recovered" from an ACL injury, who, despite returning fully to competition, still exhibit asymmetries in inter-limb function and strength (Jordan et al., 2015a,b). This might reveal certain underlying relationships not present among uninjured elite skiers. Nevertheless, this study did have certain limitations. The ski boot did not allow the sensors of inertial motion to be placed on the fore-foot, but since characterization of ankle mechanics was not part of our main goal, this did not aﬀect the results. As mentioned above, pressure insoles do not provide the most precise measurement of GRF during alpine skiing, giving 21 and 54% lower values than those obtained with dynamometers for the outside and inside skis, respectively (Stricker et al., 2010). At the same time, pressure insoles can monitor pressure on diﬀerent parts of the foot (e.g., the fore- and rear-foot) and the right and left legs separately. Moreover, the type and extent of bias are likely to be similar for the left and right legs, so that values provided by pressure insoles can be helpful in revealing diﬀerences between the legs, which was the primary focus here. Finally, since our measurements were performed on a moderate incline under nearly ideal conditions with respect to snow and weather, generalization of the ﬁndings is not straightforward. Future studies on slopes with diﬀerent inclines and with diﬀerent gate set-ups and snow conditions are warranted. Moreover, a comparison of left and right turns on a course that tilts to the side would be of interest, although this would require diﬀerent methodological approaches and data processing. It is noteworthy that the symmetry values in skiing (dependent) variables indicated by their Jaccard indices (JI) were, in general, much lower that what the corresponding SI suggested (Table 2). This diﬀerence can probably be explained by the fact that the JI values reﬂect time-dependent behavior during the turning cycle, whereas the SIs describe the behavior on the basis of a single (usually mean) value associated with the turn. REFERENCES Rapid hamstrings/quadriceps strength in ACL-reconstructed elite Alpine ski racers. Med. Sci. Sports Exerc. 47, 109–119. doi: 10.1249/mss.0000000000000375 Bishop, C., Turner, A., and Read, P. (2018). Eﬀects of inter-limb asymmetries on physical and sports performance: a systematic review. J. Sports Sci. 36, 1135–1144. doi: 10.1080/02640414.2017.1361894 Jordan, M. J., Aagaard, P., and Herzog, W. (2017). Asymmetry and thigh muscle coactivity in fatigued anterior cruciate ligament-reconstructed elite skiers. Med. Sci. 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JO performed data pre-processing, processing, and statistical analysis. JO, NŠ, and MS performed the data analysis and interpretation. JO, MS, NŠ, and H-CH contributed to drafting the manuscript. All authors provided feedback on and approved the ﬁnal version of the manuscript. ACKNOWLEDGMENTS We would like to thank all of the participants, their coaches, and the support team sincerely for their involvement. We also thank Alessandro Galloppini for help with the measurements and data pre-processing. The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2021.577698/full#supplementary-material The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2021.577698/full#supplementary-material FUNDING This study was supported by the Slovenian Research Agency in the framework of (1) project L5-1845: Body asymmetries as a risk factor in musculoskeletal injury development: studying REFERENCES 2020-0105 Supej, M., and Holmberg, H. C. (2019). Recent kinematic and kinetic advances in olympic alpine skiing: pyeongchang and beyond. Front. Physiol. 10:111. doi: 10.3389/fphys.2019.00111 Supej, M., Kugovnik, O., and Nemec, B. (2003). Kinematic determination of the beginning of a ski turn. Kinesiol. Slov. 9, 5–11. Spörri, J., Kröll, J., Fasel, B., Aminian, K., and Müller, E. (2016). 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The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Copyright © 2021 Ogrin, Šarabon, Madsen, Kersting, Holmberg and Supej. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Stricker, G., Scheiber, P., Lindenhofer, E., and Müller, E. (2010). 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https://openalex.org/W2526115426	https://discovery.ucl.ac.uk/id/eprint/1522318/1/Linhart_Band%20gap%20reduction%20in%20InNxSb1-x%20alloys%20VoR.pdf	English	null	Band gap reduction in InN<i>x</i>Sb1-<i>x</i> alloys: Optical absorption, k · P modeling, and density functional theory	Applied physics letters	2,016	cc-by	6,577	Band gap reduction in InNxSb1-x alloys: Optical absorption, k P modeling, and density functional theory W. M. Linhart, , M. K. Rajpalke, J. Buckeridge, P. A. E. Murgatroyd, J. J. Bomphrey, J. Alaria, C. R. A. Catlow, D. O. Scanlon, M. J. Ashwin, and T. D. Veal, Citation: Appl. Phys. Lett. 109, 132104 (2016); doi: 10.1063/1.4963836 View online: http://dx.doi.org/10.1063/1.4963836 View Table of Contents: http://aip.scitation.org/toc/apl/109/13 Published by the American Institute of Physics Band gap reduction in InNxSb1-x alloys: Optical absorption, k P modeling, and density functional theory W. M. Linhart, , M. K. Rajpalke, J. Buckeridge, P. A. E. Murgatroyd, J. J. Bomphrey, J. Alaria, C. R. A. Catlow, D. O. Scanlon, M. J. Ashwin, and T. D. Veal, Citation: Appl. Phys. Lett. 109, 132104 (2016); doi: 10.1063/1.4963836 View online: http://dx.doi.org/10.1063/1.4963836 View Table of Contents: http://aip.scitation.org/toc/apl/109/13 Published by the American Institute of Physics (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) Using infrared absorption, the room temperature band gap of InSb is found to reduce from 174 (7.1 lm) to 85 meV (14.6 lm) upon incorporation of up to 1.13% N, a reduction of 79 meV/%N. The experimentally observed band gap reduction in molecular-beam epitaxial InNSb thin ﬁlms is reproduced by a ﬁve band k P band anticrossing model incorporating a nitrogen level, EN, 0.75 eV above the valence band maximum of the host InSb and an interaction coupling matrix element between the host conduction band and the N level of b ¼ 1.80 eV. This observation is consistent with the presented results from hybrid density functional theory. V C 2016 Author(s). All article content, except where otherwise noted, is licensed under a Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/). [http://dx.doi.org/10.1063/1.4963836] The substitution of highly electronegative nitrogen for a few percent of the host anions in III-V semiconductors is known to lead to a signiﬁcant band gap reduction. Such dilute nitride III-V compounds have attracted attention in recent years due to realised device applications, including subcells in world record efﬁciency multi-junction solar cells, as in the case of GaInNAsSb,1 and potential applications such as light sources and photodetectors in the long-wavelength infrared range.2–4 The property of band gap reduction upon N incorpo- ration has already been observed in GaNAs,5 GaNP,6 GaInNAs,7 InNAs,8 and GaNSb.9,10 It has been described in terms of conduction band anticrossing (BAC), wherein a localized nitrogen level interacts with the extended host con- duction band.7 The linear combination of isolated nitrogen states model also describes this phenomenon very well.11–14 modeling relied on assumed band anticrossing parameters rather than those determined from experimental results. Photoluminescence has been observed from InNSb grown by metal-organic vapor phase epitaxy, but without a clear trend of band gap versus N content.23 In addition to band gap reduction, the Auger recombina- tion rate for InNSb alloys has been suggested to be three times lower than that of comparable band gap HgCdTe alloys due to the high electron effective mass, which makes InNSb a prom- ising material for long-wavelength light sources and photode- tectors.2,24 However, in spite of continued interest in InNSb alloys over the last 15 years, straightforward optical spectro- scopic evidence of the band gap reduction in samples of known N content has still not been reported and so reliable BAC parameters have not been determined. Band gap reduction in InNxSb1-x alloys: Optical absorption, k P modeling, and density functional theory W M Li h t, M K R j lk J B k id P A E M t d J J B h J Al i C R A C tl Citation: Appl. Phys. Lett. 109, 132104 (2016); doi: 10.1063/1.4963836 View online: http://dx.doi.org/10.1063/1.4963836 View Table of Contents: http://aip.scitation.org/toc/apl/109/13 Published by the American Institute of Physics APPLIED PHYSICS LETTERS 109, 132104 (2016) a)Electronic mail: wojciech.linhart@gmail.com b)Electronic mail: T.Veal@liverpool.ac.uk Band gap reduction in InNxSb1-x alloys: Optical absorption, k P modeling, and density functional theory W. M. Linhart,1,a) M. K. Rajpalke,2 J. Buckeridge,3 P. A. E. Murgatroyd,2 J. J. Bomphrey,4 J. Alaria,2 C. R. A. Catlow,3 D. O. Scanlon,3,5 M. J. Ashwin,4 and T. D. Veal2,b) 1Laboratory for Optical Spectroscopy of Nanostructures, Faculty of Fundamental Problems of Technology, Wrocław University of Technology, Wybrze_ze Wyspianskiego 27, Wrocław, Poland 2Stephenson Institute for Renewable Energy and Department of Physics, School of Physical Science, University of Liverpool, Liverpool L69 7ZF, United Kingdom 3University College London, Department of Chemistry, London WC1H 0AJ, United Kingdom 4Department of Chemistry, University of Warwick, Coventry CV4 7AL, United Kingdom 5Diamond Light Source Ltd., Didcot, Oxfordshire OX11 0DE, United Kingdom (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) Prior to growth, the N plasma was struck in a separate chamber with a power of 500 W and a ﬂow rate of 0.2 sccm. The InNSb epilayers were grown to a thickness of 400 nm at a ﬁxed growth rate of nominally 0.5 lm h1 using substrate temperatures, determined by pyrometer, ranging from 270 to 365 C. The surface mor- phology was investigated by atomic force microscopy (AFM) with an Asylum research MFP-3D microscope in tap- ping mode. The lattice constant of the InNSb ﬁlms was deter- mined using high resolution X-ray diffraction (XRD) using a Philips X’Pert diffractometer equipped with monochromatic Cu Ka1 X-ray source (k ¼ 0.15406 nm) with a four bounce Ge monochromator. XRD analysis was performed using the Panalytical X’pert Epitaxy application. AFM indicates the root mean square roughness values (uncorrelated with N con- tent) in the range of 0.5–3.0 nm for all samples from 5 5 lm2 images. There is no evidence from AFM measure- ments of any condensation of Sb on the ﬁlms’ surface. Transmittance measurements were performed at room tem- perature using a Bruker Vertex 70V Fourier-transform infra- red spectrometer, using a liquid nitrogen-cooled HgCdTe detector between 50 and 1200 meV. Additionally, transmis- sion measurements were made in the temperature range 4–300 K for a ﬁlm with 0.73% N content using a continuous- ﬂow He cryostat. The incorporation of N in InSb has also been investi- gated using DFT calculations employing hybrid functionals which often provide better structural data and more accurate band gaps than the local density and generalized gradient approximations.25–29 Here, the screened HSE06 (Heyd-Scuseria-Ernzerhof) hybrid density functional is used,30 as implemented in the Vienna Ab initio Simulation Package (VASP).31–33 The value of exact nonlocal exchange, a, was 31.0%, which gives a band gap of 231 meV for InSb, in good agreement with the 0 K band gap of 235 meV extrapolated from the low temperature experimen- tal data.34 The valence–core interaction was described using the projector augmented wave (PAW) approach,35 and cores of [Kr] for indium and antimony and [He] for nitrogen are used. A cutoff of 400 eV was used for all the calculations, with the Brillouin zone sampled employing a 8 8 8 Monkhorst Pack grid, which provided convergence in the total energy of 104eV. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) p The N fraction was determined for epilayers grown at temperatures between 270 and 365 C using a ﬁxed growth rate of 0.5 lm h1. Both the N fraction and the epilayer thick- ness were determined by XRD, where both the 004 peak split- ting and the Pendell€osung fringes were modeled by dynamical simulations. The epilayer peak occurs at higher Bragg angle than the substrate peak, corresponding to the InNSb layer hav- ing a smaller lattice parameter than that of InSb. The N con- tent was determined under the assumption that Vegard’s law is valid using an endpoint lattice parameter for zinc blende InN of 5.01 A˚ .41 Example measurements and simulations are shown in Fig. 1 for layers with 1.13 and 0.73% of their Sb sub- lattice substituted by N atoms. (The sample with 0.73% N was grown in a separate growth run from the others in order to ﬁll the composition gap between 0.54% and 1.1% N. By the time of the later growth run, the N plasma source aperture plate had been modiﬁed and so, to ﬁll the composition gap, the sample was grown at 275 C but at a higher growth rate of 1.75 lm h1.) The N content from XRD (for ﬁxed N plasma source conditions and ﬁxed growth rate) increases as the growth tem- perature decreases and begins to reach a plateau corresponding to x ¼ 1.14% for growth temperatures below approximately 270 C (see Fig. S1 of supplementary material). These results are similar to those of Zhang et al. who grew InNSb at between 250 and 350 C.42 The temperature dependence of the N incorporation in InNxSb1–x has been modeled using the kinetic approach of Wood et al.43 and Pan et al.,44 which has previously been applied to N incorporation in GaNSb and GaInNSb alloys.45,46 The curve shown in Fig. S1 is a very good ﬁt to the experimental data and corresponds to an energy barrier for loss of N of 1.79eV and a characteristic surface res- idence lifetime of N atoms of 5 ls. For comparison, the previ- ously reported equivalent values for GaNSb are 2.0 eV and 5 ls.45 This lower energy barrier is consistent with the g The InNSb epilayers were grown at the University of Warwick by N plasma assisted solid-source MBE on 100 nm InSb buffer layers on quarters of 51 mm diameter undoped InSb(001) substrates. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) In this letter, infrared absorption measurements of the band gap of epitaxial InNSb thin ﬁlms with nitrogen incorpo- ration of up to 1.13% are presented, analyzed using the BAC model, and compared with the results of hybrid density func- tional theory (DFT). The band gap of InNxSb1–x alloys is shown to decrease from 174 meV (7.1 lm) for x ¼ 0 to 85 meV (14.6 lm) for x ¼ 1.13%, which corresponds to a band gap reduction of 79 meV/%N. Simulations of the absorption, based on a joint density of states method, includ- ing the BAC interaction reproduce the experimental absorp- tion spectra well. The band gap reduction as a function of N content is also well reproduced by the BAC model. The BAC matrix element coupling strength is estimated to be 1.80 eV, using a nitrogen level of 0.75 eV above the valence band maximum (VBM). These ﬁndings and preliminary low temperature absorption measurements are consistent with the hybrid DFT results. Overall, these results indicate that InNSb alloys are suitable for sources and detectors operating in the 8–14 lm atmospheric transmission window and InSb has the smallest band gap of any common binary III–V semiconductor, having a value of 174 meV at room temperature, corresponding to a wavelength of 7 lm. InNSb has long been proposed as an alternative to InAsSb for use in the 8–14 lm atmospheric transmission window.15 Some evi- dence has been reported that the addition of N to form InNSb results in a dramatic reduction of the fundamental band gap.2,16–18 However, several early studies were hampered by non-optimal samples in which the N content was not well known and/or hydrogen was added to stabilize an electron cyclotron resonance N plasma source.2,16,17,19,20 In other sam- ples, grown by radio frequency N plasma source-assisted MBE, fundamental band gap reduction in the InNSb ﬁlms grown on semi-insulating GaAs was inferred from modeling of data of Burstein–Moss shifted absorption edges versus degenerate carrier concentrations.21,22 However, quantitative V C Author(s) 2016. 109, 132104-1 0003-6951/2016/109(13)/132104/5 132104-2 Linhart et al. Linhart et al. Appl. Phys. Lett. 109, 132104 (2016) spin-orbit valence band split off of 800 meV, in good agree- ment with experiment (803 meV).40 provide the BAC parameters needed for modeling for under- standing related quaternary alloys such as GaInNSb and InNSbBi and for future device design. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) Within this model, the host InSb conduc- tion band and its nonparabolicity are described by Pidgeon and Brown’s ð4 4Þ kP Hamiltonian that includes interac- tions of the InSb conduction band with the valence bands and the inﬂuence of higher lying bands.51 So this results in a ð5 5Þ Hamiltonian (or ð10 10Þ if spin degeneracy is included) to describe the InNSb. As a result of the interaction of the nitrogen level with the host conduction band, two con- duction subbands form and are denoted as Eþ and E. An example with x ¼ 1% is shown in Fig. 2. (2) where gcvðEÞ is the joint density of states, h is the incident photon energy, and Pcv are the momentum matrix elements for the optical transitions, derived from the BAC model. Here, the calculated absorption of InSb was subtracted from the cal- culated absorption spectrum of InNxSb1–x which is equivalent to ratioing the transmission data. Within this model approach, the trends observed in the experimental absorption spectra are well reproduced. The absorption onsets are sharper in the model than the measured curves as a result of lifetime broad- ening being neglected. The experimental absorption edge as a function of N content is shown by the open circles in Fig. 4. The BAC model well reproduces the trend of the band gap reduction, which is determined by the transition between the VBM and the minimum of the E– subband, which is shown by the thick solid line in Fig. 4. Within the BAC model, the adjustable parameters of the InNSb band structure are the energy of the N impurity level, EN, and the BAC coupling strength, b, in the coupling matrix element, VMN ¼ b ﬃﬃﬃx p . The N level is set to 0.75 eV above the VBM, according to the results of previ- ous tight binding calculations.50 The optimum ﬁt is then The optical properties of the InNSb epilayers as a func- tion of N content were studied using transmission measure- ments. The transmission data from each sample were divided by the transmission from a different quarter of the same InSb wafer so that the remaining signal corresponds to transmis- sion through the InNSb layer. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) N incorporation was modeled by substituting one Sb for an N in a 2 2 2 expansion of the 8 atom cubic cell (i.e., a 64 atom supercell), corresponding to an N concentration of 3.125%. This supercell size has been shown to be well converged when modeling N in other III–V materials.36,37 Geometry optimisation was deemed to have converged when the forces on all the atoms were less than 0.01 eV A˚ 1. The band structure of the supercell con- taining one N was unfolded to the primitive cell band struc- ture using the BandUP code.38,39 All the calculations included spin-orbit coupling, which, for bulk InSb gives a FIG. 1. XRD (points) of the 004 diffraction maximum from the InNSb ﬁlms grown on an InSb substrate at 275 C at growth rates of 0.5 and 1.75lm h1. The results of the simulations (solid line) indicate that the InNSb ﬁlms con- tain 1.13 and 0.73% N and their thickness are 390 6 10 nm and 400 6 10 nm, respectively. FIG. 1. XRD (points) of the 004 diffraction maximum from the InNSb ﬁlms grown on an InSb substrate at 275 C at growth rates of 0.5 and 1.75lm h1. The results of the simulations (solid line) indicate that the InNSb ﬁlms con- tain 1.13 and 0.73% N and their thickness are 390 6 10 nm and 400 6 10 nm, respectively. Appl. Phys. Lett. 109, 132104 (2016) 132104-3 Linhart et al. requirement to grow InSb at a lower temperature than GaSb for N incorporation. with x ¼ 0.54%, 0.73%, and 1.1% are shown in Fig. 3(a). The band gap red shifts with increasing N content. The absorption edge decreases in energy to 85 6 15 meV as the N content is increased to x ¼ 1.13%, corresponding to a wave- length of 14.6 lm. This corresponds to a band gap reduc- tion of 79 6 5 meV/%N. Model absorption curves are shown in Fig. 3(b). The approach of Perlin et al.52 has been used to calculate the model absorption spectra. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) Accordingly, the absorption coefﬁcient, a, is The band structure close to the C point of InNxSb1–x alloys was calculated using the kP BAC model.47–49 The dispersion of the E6 subbands can be determined by ﬁnding the eigenvalues of the 2 2 determinant EcðxÞ E b ﬃﬃﬃx p b ﬃﬃﬃx p ENðxÞ E ¼ 0; (1) (1) a E ð Þ / X c;v jPcvj2gcv E ð Þ h ; (2) where EcðxÞ ¼ Ec0 ax and ENðxÞ ¼ EN0 cx are the assumed energies of the conduction band edge and isolated N level with respect to the InSb valence band maximum (VBM). The values a ¼ 2.44 eV and c ¼ 2.47 eV are taken from ﬁtting to previous tight binding results.50 The x-depen- dence of the effective N level is due to interactions with other N-related states, such as different N pair and cluster conﬁgurations that are more likely to occur as the N content, x, increases. The value of b is determined by ﬁtting to the experimental data. Within this model, the host InSb conduc- tion band and its nonparabolicity are described by Pidgeon and Brown’s ð4 4Þ kP Hamiltonian that includes interac- tions of the InSb conduction band with the valence bands and the inﬂuence of higher lying bands.51 So this results in a ð5 5Þ Hamiltonian (or ð10 10Þ if spin degeneracy is included) to describe the InNSb. As a result of the interaction of the nitrogen level with the host conduction band, two con- duction subbands form and are denoted as Eþ and E. An example with x ¼ 1% is shown in Fig. 2. where EcðxÞ ¼ Ec0 ax and ENðxÞ ¼ EN0 cx are the assumed energies of the conduction band edge and isolated N level with respect to the InSb valence band maximum (VBM). The values a ¼ 2.44 eV and c ¼ 2.47 eV are taken from ﬁtting to previous tight binding results.50 The x-depen- dence of the effective N level is due to interactions with other N-related states, such as different N pair and cluster conﬁgurations that are more likely to occur as the N content, x, increases. The value of b is determined by ﬁtting to the experimental data. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) This ratioing of the sample data with data from an InSb substrate accounts for the decrease in Da above the band gap of the InSb—the absorp- tion of InNSb converges to that of InSb as Eþ approaches the InSb conduction band (see Fig. 2). The absorption spec- tra calculated from the transmission data for the samples sion through the InNSb layer. This ratioing of the sample data with data from an InSb substrate accounts for the decrease in Da above the band gap of the InSb—the absorp- tion of InNSb converges to that of InSb as Eþ approaches the InSb conduction band (see Fig. 2). The absorption spec- tra calculated from the transmission data for the samples FIG. 2. (a) Conduction band dispersion of InN0.01Sb0.99 close to the C point of the Brillouin zone calculated using the kP BAC model. The host conduc- tion band (dashed line) interacts with the N level (dashed-dotted line) and results in two conduction subbands E and Eþ (solid lines). FIG. 3. (a) Absorption spectra from InNxSb1-x with x ¼ 0.54% (solid line), 0.73% (dashed line), and 1.1% (dashed-dotted line) grown on InSb sub- strates. Da denotes the difference in absorption by the InNSb ﬁlm on InSb substrate and by InSb substrate alone. (b) The calculated absorption spectra within the BAC model. FIG. 3. (a) Absorption spectra from InNxSb1-x with x ¼ 0.54% (solid line), 0.73% (dashed line), and 1.1% (dashed-dotted line) grown on InSb sub- strates. Da denotes the difference in absorption by the InNSb ﬁlm on InSb substrate and by InSb substrate alone. (b) The calculated absorption spectra within the BAC model. FIG. 2. (a) Conduction band dispersion of InN0.01Sb0.99 close to the C point of the Brillouin zone calculated using the kP BAC model. The host conduc- tion band (dashed line) interacts with the N level (dashed-dotted line) and results in two conduction subbands E and Eþ (solid lines). FIG. 3. (a) Absorption spectra from InNxSb1-x with x ¼ 0.54% (solid line), 0.73% (dashed line), and 1.1% (dashed-dotted line) grown on InSb sub- strates. Da denotes the difference in absorption by the InNSb ﬁlm on InSb substrate and by InSb substrate alone. (b) The calculated absorption spectra within the BAC model. FIG. 3. (a) Absorption spectra from InNxSb1-x with x ¼ 0.54% (solid line), 0.73% (dashed line), and 1.1% (dashed-dotted line) grown on InSb sub- strates. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) See supplementary material for ﬁgures showing N con- tent from XRD versus growth temperature along with kinetic modeling and also the absorption spectra recorded at 4 K from an InSb substrate and from InNSb/InSb with 0.73% N. given by adjusting the value of b to 1.80 eV. The band gap reduction is 79 meV/%N due to downward shift of the con- duction band minimum due to the BAC interaction. The interaction strength of b ¼ 1.80 eV is similar to the value of 1.99 eV suggested by tight binding calculations.50 The BAC parameters determined here differ signiﬁcantly from those recommended in Vurgaftman and Meyer’s review34 where, largely based on the work by Murdin et al.16 but using an N content-independent N level, they suggested EN ¼ 0.65 eV and b ¼ 3.0 eV. These values correspond to 145 meV/%N band gap reduction and band gap closure for 1.25% N at room temperature. The experimental work was supported by the Engineering and Physical Sciences Research Council (EPSRC) under Grant No. EP/G004447/2. W.M.L. acknowledges support from the National Science Center (NCN) Grant No. 2014/13/D/ST3/ 01947. D.O.S. and T.D.V. acknowledge support from the Materials Design Network. J.B. and C.R.A.C. acknowledge funding from the EPSRC Grant No. EP/K016288/1. The hybrid DFT was performed on the national supercomputer ARCHER via funding from the EPSRC Grant No. EP/ L000202/1. p Results from hybrid DFT are also shown in Fig. 4 for InSb and InNSb with 3.125% N. As the DFT corresponds to 0 K, the results are compared with absorption results at 4 K for InSb and one InNSb sample with 0.73% N (see Fig. S2 of supplementary material). The DFT band gap of InNSb with 3.125% N is 23 meV. Assuming linear variation of band gap with N content, this gives a band gap reduction of 67 meV/%N. This is somewhat lower than the room temperature experi- mental value, which is probably due to the absence of N pair and cluster effects in DFT calculations using a 64 atom supercell containing only one N atom, which corresponds to a perfectly ordered array of substitutional N atoms, rather than a random distribution. The low temperature band gap dependence on N content has been calculated using kP with and without the effects of N pairs and clusters on the effec- tive N level and is shown in Fig. 4. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) Da denotes the difference in absorption by the InNSb ﬁlm on InSb substrate and by InSb substrate alone. (b) The calculated absorption spectra within the BAC model. FIG. 2. (a) Conduction band dispersion of InN0.01Sb0.99 close to the C point of the Brillouin zone calculated using the kP BAC model. The host conduc- tion band (dashed line) interacts with the N level (dashed-dotted line) and results in two conduction subbands E and Eþ (solid lines). FIG. 4. The room temperature (300 K) band gap versus N content deter- mined from the absorption spectra for the InNxSb1-x samples with 0 < x 6 1:13% (open red circles) and calculated composition dependence of the band gap of InNxSb1-x using the k P BAC model (thick solid red line). The hybrid DFT band gaps (0 K) for InSb and InNSb with 3.125% N are also shown (closed blue circles) and joined by a dotted blue line. The 4 K experimental band gaps from optical absorption of InSb and InNSb with 0.73% N are also shown (open blue squares). The thin solid blue line is the band gap variation for 4 K using the k P BAC model and the dashed blue line is from the same model but with a ﬁxed effective N level, equivalent to removing the effect of N pairs and clusters. Inset is the C to X direction band dispersions of the highest valence band (the heavy hole valence band) and the two lowest conduction bands (the E and Eþ) subbands from hybrid density functional theory (points) and the k P modeling (solid lines). 132104-4 Linhart et al. Appl. Phys. Lett. 109, 132104 (2016) 132104-4 Linhart et al. Linhart et al. bands, E and Eþ, are shown in inset in Fig. 4. The band dis- persions from k P calculations are also shown. Without sig- niﬁcantly changing the band dispersions, the value of b in the k P was adjusted from 1.80 to 1.65 eV to reproduce the DFT band gap for x ¼ 3.125% N. The k P dispersions match those from hybrid DFT for the topmost valence band and the E subband. The energy position of the Eþ band is similar for both methods, but with ﬂatter dispersion in the k P results. (Received 29 April 2016; accepted 19 September 2016; published online 29 September 2016) The band gap reduction of 79 meV/%N represents a large proportional change due to the small band gap of InSb, but is about half the absolute band gap change per %N compared with that seen in GaNAs alloys (150 meV/%). This is a conse- quence of the N level lying more than 550 meV above the CBM in InNSb compared with only 250 meV for GaNAs. The InNSb band gap reduction per %N is greater than twice that observed per %Bi in InSbBi alloys (35meV%Bi),53 but is accompanied by much greater lattice parameter change due to the greater difference between the zinc blende InN and InSb lattice parameters compared with that of the zinc blende InBi and InSb parameters. Additionally, the InNSb calculation sug- gests that at room temperature band gap closure will occur for x > 2.3%, giving a semimetallic band structure. Some circum- stantial evidence of such band gap closure has previously been reported by Veal et al.18 At low temperature (0 or 4 K), band gap closure is predicted to occur at or greater than 3% N. FIG. 4. The room temperature (300 K) band gap versus N content deter- mined from the absorption spectra for the InNxSb1-x samples with 0 < x 6 1:13% (open red circles) and calculated composition dependence of the band gap of InNxSb1-x using the k P BAC model (thick solid red line). The hybrid DFT band gaps (0 K) for InSb and InNSb with 3.125% N are also shown (closed blue circles) and joined by a dotted blue line. The 4 K experimental band gaps from optical absorption of InSb and InNSb with 0.73% N are also shown (open blue squares). The thin solid blue line is the band gap variation for 4 K using the k P BAC model and the dashed blue line is from the same model but with a ﬁxed effective N level, equivalent to removing the effect of N pairs and clusters. 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https://openalex.org/W1963707677	https://www.hal.inserm.fr/inserm-01072122/document	English	null	Acoustic Radiation Force Impulse (ARFI) and Transient Elastography (TE) for evaluation of liver fibrosis in HIV-HCV co-infected patients	BMC infectious diseases	2,014	cc-by	7,705	To cite this version: Nora Frulio, Hervé Trillaud, Paul Perez, Julien Asselineau, Marianne Vandenhende, et al.. Acoustic Radiation Force Impulse (ARFI) and Transient Elastography (TE) for evaluation of liver fibrosis in HIV-HCV co-infected patients.. BMC Infectious Diseases, 2014, 14, pp.405. 10.1186/1471-2334-14- 405. inserm-01072122 Nora Frulio, Hervé Trillaud, Paul Perez, Julien Asselineau, Marianne Vandenhende, et al.. Acoustic Radiation Force Impulse (ARFI) and Transient Elastography (TE) for evaluation of liver fibrosis in HIV-HCV co-infected patients.. BMC Infectious Diseases, 2014, 14, pp.405. 10.1186/1471-2334-14- 405. inserm-01072122 © 2014 Frulio et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. HAL Id: inserm-01072122 https://inserm.hal.science/inserm-01072122v1 Submitted on 8 Oct 2014 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Frulio et al. BMC Infectious Diseases 2014, 14:405 http://www.biomedcentral.com/1471-2334/14/405 * Correspondence: norafrulio@yahoo.fr 1Department of Diagnostic and Interventional Imaging, Saint-André hospital, CHU de Bordeaux, 1 rue Jean Burguet, 33075 Bordeaux, France Full list of author information is available at the end of the article Acoustic Radiation Force Impulse (ARFI) and Transient Elastography (TE) for evaluation of liver fibrosis in HIV-HCV co-infected patients Nora Frulio1, Hervé Trillaud1, Paul Perez2, Julien Asselineau2, Marianne Vandenhende3, Mojgan Hessamfar3, Fabrice Bonnet3, Florent Maire1, Jean Delaune3, Victor De Ledinghen4,5 and Philippe Morlat3 Abstract Background: Transient elastography (TE) is widely used for non-invasive assessment of liver fibrosis in HIV-HCV co-infected patients. TE, however, cannot determine liver morphology. Acoustic radiation force impulse (ARFI) imaging is a novel procedure enabling assessment of liver fibrosis during a conventional ultrasonographic examination. This study evaluated the correlation between liver fibrosis measurements by TE and ARFI. Methods: Each of 46 HIV-HCV patients underwent both ARFI and TE within 6 months. Patients were evaluated by the “equivalent METAVIR” scoring system, using previously established cut-off values. Agreements between the ARFI and TE scores were estimated by Kappa coefficients, with Kappa values ≥0.40, ≥0.60, and ≥0.80 defined as moderate, good and very good agreement, respectively. Results: ARFI and TE yielded "Equivalent Metavir" fibrosis scores of F1 in 26 and 31 patients, respectively; F2 in nine and seven, respectively; F3 in three and two, respectively; and F4 in eight and six, respectively. The two methods showed very good agreement in predicting overall stages [Kappa = 0.82] and for F ≥3 [Kappa = 0.80] and moderate agreement in predicting significant fibrosis F ≥2 [Kappa = 0.50]. Morphologic ultrasound analysis concomitant to ARFI detected two hepatocarcinomas. Conclusions: ARFI showed promising results in the non-invasive assessment of liver fibrosis in HIV-HCV patients, with liver fibrosis staging similar to that of TE. Moreover, ARFI can assess morphology and fibrosis during the same session. Keywords: ARFI elastography, Liver fibrosis, HIV-HCV co-infected patients, Transient elastography Keywords: ARFI elastography, Liver fibrosis, HIV-HCV co-infected patients, Transient elastography Keywords: ARFI elastography, Liver fibrosis, HIV-HCV co-infected patients, Transient elastography Background been considered the gold standard to assess activity and fibrosis in patients with chronic liver disease. Despite its diagnostic utility, liver biopsy is limited because of its in- vasiveness and cost, the risk of complications including death, its poor acceptance by patients, the lack of avail- ability of expert practitioners, and intra/inter-observer variability [8-10]. In recent years, various non-invasive methods for liver fibrosis assessment, have been devel- oped, including serum biomarkers and elastography techniques. Transient elastography (TE), real-time elas- tography (RTE), acoustic radiation force impulse im- aging (ARFI), and shear wave elastography are the most frequent elastography techniques [11-15]. Since the introduction of highly active antiretroviral ther- apy (HAART), chronic liver disease has emerged as one of the leading causes of morbidity and mortality in HIV-HCV co-infected patients [1,2]. Therefore, the management of HCV-related liver disease has become a major challenge in these patients [3]. Evaluation of liver fibrosis is considered essential to the management of patients with chronic hepatitis C, providing prognostic information and assisting in mak- ing therapeutic decisions [4-7]. To date, liver biopsy has Correspondence: norafrulio@yahoo.fr 1Department of Diagnostic and Interventional Imaging, Saint-André hospital, CHU de Bordeaux, 1 rue Jean Burguet, 33075 Bordeaux, France Full list of author information is available at the end of the article TE (FibroScan®) is currently the most extensively used elastography technique in clinical practice [16-19]. Many Page 2 of 8 Frulio et al. BMC Infectious Diseases 2014, 14:405 http://www.biomedcentral.com/1471-2334/14/405 included small nodular and irregular livers with increased echogenicity and/or a significant reduction in Doppler flow in the portal circulation on ultrasound, CT, or MRI. Quantitative ARFI imaging (Acuson S2000 Siemens) and TE were performed by two different observers, with each subject undergoing the two examinations no more than 6 months apart. The physician performing the sec- ond procedure was blinded to the results of the first procedure. included small nodular and irregular livers with increased echogenicity and/or a significant reduction in Doppler flow in the portal circulation on ultrasound, CT, or MRI. prospective studies have evaluated the diagnostic perform- ance of TE in staging liver fibrosis in patients with chronic liver diseases, including chronic viral hepatitis C [11,20-22], chronic viral hepatitis B [23], HIV–HCV co-infection [24-26], alcoholic disease [27], and non-alcoholic fatty liver disease [28]. ARFI examination ARFI imaging is a ARFI imaging is a method of quantifying the mechanical properties of tissues, without manual compression, by measuring the shear wave velocity induced by acoustic ra- diation and propagating in the tissue. This quantitative technique provides a single unidimensional measurement of tissue elasticity like TE, although the measurement area can be positioned on a two-dimensional B mode image. The region is a 1 × 0.5-cm rectangle, which can be freely moved in the two-dimensional B mode image to a max- imum depth of 8 cm from the skin plane. Measurements are expressed as m/s, indicating shear wave speed travel- ing perpendicular to the shear wave source. The principle of ARFI is described in Figure 1 [30,42,43]. Background Two meta-analyses found that TE is highly accurate for the diagnosis of cirrhosis and advanced fibro- sis [16,17], suggesting that TE may be used in place of bi- opsy in most patients with chronic hepatitis C [18,29]. Quantitative ARFI imaging (Acuson S2000 Siemens) and TE were performed by two different observers, with each subject undergoing the two examinations no more than 6 months apart. The physician performing the sec- ond procedure was blinded to the results of the first procedure. TE, however, has several drawbacks, including the re- quirement for specific equipment that performs only elas- tography, its ability to assess only the right liver, and its inability to visually determine the site of measurement. Moreover, ascites and obesity are limiting factors. ARFI imaging has many advantages over TE. The technology used for ARFI has been incorporated into a conventional ultrasound system, allowing ultrasound analysis of liver morphology at the same time. In addition, ARFI allows elastography to be performed with a flexible metering box at variable depths, enabling the exact localization of the measurement site, and the examination can also be per- formed in patients with ascites. ARFI measurements have been found to correlate well with the stage of hepatic fibro- sis [30-36]. However, to our knowledge, no study to date has investigated the potential benefits of ARFI elastography in evaluating fibrosis stage in HIV-HCV co-infected pa- tients. This study therefore assessed the agreement be- tween TE and ARFI for the evaluation of liver fibrosis in this patient population. Transient elastography (TE) TE was performed in the right lobe of the liver through the intercostal space as described [39,40]. Liver stiffness was measured in a volume approximating a cylinder 1 cm wide and 4 cm long, and at a depth between 25 and 65 mm. As suggested by the manufacturer, 10 successful acquisitions were obtained for each patient in fasting conditions. TE results were defined as very reliable (IQR/M ≤0.10), reliable (0.10 < IQR/M ≤0.30 or IQR/ M >0.30 with LSE median <7.1 kPa) and poorly reliable (IQR/M >0.30 with LSE median ≥7.1 kPa) [41]. Poorly re- liable results were excluded. The median value for each patient was expressed according to “equivalent Metavir” fi- brosis scores using previously determined cut-off values for HCV mono-infected patients of 7.1, 9.5 and 12.5 kPa for liver fibrosis scores of F ≥2, F ≥3, and F = 4, respect- ively [11]. Methods Patients The study was approved by the institutional review board of our University hospital. Between July 2009 and May 2012, HIV-HCV co-infected patients regularly followed up at the HIV out-patient Clinic of the Department of Internal Medicine and Infectious Disease of our hospital were pro- spectively enrolled. All patients provided written informed consent before inclusion. The demographic, clinical and biological information of all patients was obtained from their medical records. Fac- tors included patient age, body mass index (BMI), alcohol consumption, medications, specific antiretroviral drugs, plasma HIV-RNA levels (IU/ml), complete blood counts, absolute number of CD4+ T-cells (/mm3), plasma HCV- RNA concentrations (IU/ml), and HCV genotype. Men and women were considered heavy drinkers if they con- sumed >3 and >2 glasses of alcohol per day, respectively. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), and bilirubin concentrations were also measured. All patients underwent quantitative elastographic ex- aminations in fasting conditions with a Siemens Acuson S2000TM ultrasound system (Siemens AG, Erlangen, Germany) and an abdominal curved transducer (4C1), using the virtual touch tissue quantification system. The first step assessed liver morphology and echo- structure, looking for cirrhosis, signs of portal hyperten- sion and tumor lesions. In the second step, 10 ARFI mea- surements of the right liver through the intercostal space were performed by a single examination. To standardize these examinations, an area of liver tissue free of large blood vessels was chosen at a minimal distance of 2 cm The presence of cirrhosis was evaluated in all patients histologically and from imaging, clinical and biological results [37,38]. Imaging findings indicative of cirrhosis Frulio et al. BMC Infectious Diseases 2014, 14:405 http://www.biomedcentral.com/1471-2334/14/405 Page 3 of 8 b a Figure 1 Principle or ARFI quantitative measurement. a) Schematic of principles of ARFI imaging in virtual touch quantification mode – b) Example of a SWV measurement of the right liver. Transmission of a longitudinal acoustic pulse leads to tissue displacement, resulting in shear wave propagation away from the region of excitation. The shear wave velocity is measured within a defined region of interest (ROI) using ultrasound tracking beams laterally adjacent to the single push beam. The shear velocity is estimated in a graphically displayed ROI measuring 1 × 0.5 cm. The shear wave propagation velocity is proportional to the square root of tissue elasticity. a b b Figure 1 Principle or ARFI quantitative measurement. Methods Patients a) Schematic of principles of ARFI imaging in virtual touch quantification mode – b) Example of a SWV measurement of the right liver. Transmission of a longitudinal acoustic pulse leads to tissue displacement, resulting in shear wave propagation away from the region of excitation. The shear wave velocity is measured within a defined region of interest (ROI) using ultrasound tracking beams laterally adjacent to the single push beam. The shear velocity is estimated in a graphically displayed ROI measuring 1 × 0.5 cm. The shear wave propagation velocity is proportional to the square root of tissue elasticity. Results below the liver capsule. ARFI measurements were expressed in m/s as median (M), mean and standard deviation, and interquartile range (IQR), with the IQR/M ratio used to evalu- ate the variability of the measurements. An IQR/M < 0.3 was considered a homogeneous set of measurements. Median ARFI value was expressed according to the ‘equivalent Metavir’ score using previously determined cutt-off values for HCV mono-infected patients of 1.34, 1.55, 1.80 m/s for liver fibrosis scores of F ≥2, F ≥3, and F = 4, respectively [44]. below the liver capsule. ARFI measurements were expressed in m/s as median (M), mean and standard deviation, and interquartile range (IQR), with the IQR/M ratio used to evalu- ate the variability of the measurements. An IQR/M < 0.3 was considered a homogeneous set of measurements. Median ARFI value was expressed according to the ‘equivalent Metavir’ score using previously determined cutt-off values for HCV mono-infected patients of 1.34, 1.55, 1.80 m/s for liver fibrosis scores of F ≥2, F ≥3, and F = 4, respectively [44]. y p p Of the 86 examinations, 40 were excluded because of missing data (n = 8), too long delay (>6 months) between ARFI and TE (n = 24) or repeated examinations for the same patient (n = 8). When liver stiffness was measured repeatedly in the same patient, only the measurement with minimal delay between ARFI and TE was included in the analysis. Valid results of both ARFI and TE were obtained for 46 patients; their demographic and clinical and biological characteristics are summarized in Table 1. Evaluation of liver fibrosis The median delay between ARFI and TE in the 46 evalu- ated patients was 10 days (range, 0–25 days). Median liver stiffness on TE was 6.1 kPa (range 3.4–35.3 kPa) and me- dian shear wave velocity (SWV) on ARFI was 1.29 m/s (range 0.93–2.86 m/s). Median liver stiffnesses on TE in patients with F1, F2, F3, and F4 fibrosis stages were 5.4 kPa (range 4.1–6.1 kPa), 7.2 kPa (range, 7.1–7.8 kPa), 11.6 kPa (range, 10.8–12.3 kPa) and 24.6 kPa (range, 18.0–27.7 kPa), respectively. Median SWVs on ARFI in patients with F1, F2, F3 and F4 fibrosis stages were 1.15 m/s (range, 1.06–1.25 m/s), 1.44 m/s (range, 1.40–1.47 m/s), 1.60 m/s (range, 1.58–1.61 m/s), and 2.25 m/s (range, 2.12–2.52 m/ s), respectively. Figure 2 shows a scatterplot of the rela- tionship between median results on TE and ARFI. Statistical analysis Of the 46 patients, 28 did not receive any treatment for HCV, whereas the other 18 were treated with pegin- terferon and ribavirin (SOC). At the time of ARFI, eight had achieved a sustained virologic response, whereas the other 10 had failed treatment. No patient received any direct-acting antiviral agents (DAAs). Moreover, all 46 patients had received highly active antiretroviral therapy (HAART), including 28 who received third-generation protease inhibitors (PIs), three who received integrase inhibitors (INIs), eight who received non-nucleoside re- verse transcriptase inhibitors (NNRTIs), and seven who received other treatment combinations (Table 1). Quantitative characteristics were expressed as mean (stand- ard deviation), median (first-third quartile) and range, and compared using non-parametric Wilcoxon statistics. Quali- tative characteristics were expressed as frequencies and proportions, and compared using Fisher’s exact test. Correlations between TE and ARFI measurements were estimated using Spearman’s correlation coefficient and its two-sided 95% confidence interval (CI), as calculated with Fisher's z transformation. Agreements between “equivalent Metavir” fibrosis stages on ARFI and TE were assessed by simple Kappa coeffi- cients for predictions of F ≥3 or F ≥2, and F = 4, and weighted Kappa coefficients for predictions of F1 to F4 Metavir scores; two-sided 95% CIs were also estimated. Kappa values ≥0.60 and ≥0.80 were considered indicative of good and very good agreement, respectively. Additional analysis was performed in patients infected with HCV ge- notypes 1 and 4, who usually have a poorer response than patients infected with genotypes 2 and 3. Ultrasound analysis allowed the detection of focal liver le- sions in five patients, including two with hepatocellular car- cinoma (HCC) and three with benign liver lesions. The two HCCs presented with arterial hypervascularity and wash- out in venous and delayed phase on dynamic enhanced MRI [45]. Their diagnosis was confirmed by histologic examination of biopsy specimens obtained just before liver nodule radiofrequency. The three benign liver lesions were diagnosed based on their tumor-specific vascularity patterns in the arterial, portal, and late phases on dynamic enhanced MRI, as described [46-48]. Imaging features were typical, with liver biopsies not required to confirm their diagnosis. Quantitative and qualitative variables associated with discordances between ARFI and TE staging were assessed by the Wilcoxon test and Fisher’s exact test, respectively. Only univariate analyses were performed because of the small number of subjects. Frulio et al. Comparison of equivalent Metavir fibrosis scores on TE and ARFI Spearman correlation analysis showed that the correlation coefficient between ARFI and TE measurements was 0.76 [95% CI, 0.61–0.86]. Table 2 shows the agreement between TE and ARFI measurements, as determined by equivalent Metavir fibrosis scores. Overall agreement between the two methods was very good [concordance 69.6%, weighted Kappa = 0.82; 95% CI = 0.70–0.95]. Agreement was also very good for predicting severe fibrosis (≥F3) [concord- ance 93.5%, Kappa = 0.80, 95% CI = 0.59–1.00], and was moderate for predicting significant fibrosis (≥F2) [con- cordance 76.1%, Kappa = 0.5, 95% CI = 0.25–0.75]. Statistical analysis BMC Infectious Diseases 2014, 14:405 http://www.biomedcentral.com/1471-2334/14/405 Page 4 of 8 Page 4 of 8 Table 1 Patient demographic and clinical characteristics Demographics Age (y) Median (Q1; Q3) 48 (45; 51) Gender n (%) Female 14 (30.4) Male 32 (69.6) BMI Median (Q1; Q3) 22.4 (19.8; 24.3) Overweight (BMI > =25) n (%) 8 (17.4) Chronic alcohol abuse n (%) 15 (34.1) Cirrhosis n (%) 6 (13) Child Pugh stage n 6 A n (%) 6 (100) B, C, D n 0 HIV treatment Protease inhibitors n (%) 28 (60.9) Integrase inhibitors n (%) 3 (6.5) Non-nucleoside RT inhibitors n (%) 8 (17.4) Other treatment n (%) 7 (15.2) HCV treatment None, n (%) 28 (60.9) Previous treatment failure n (%) 10 (21.7) Previous sustained virologic response n (%) 8 (17.4) Virology HCV genotype n (%) 1-1a-1b 24 (59) 2-3-3a 9 (22) 4 41b-4a 8 (19) ND 5 (10) HCV RNA > 15 IU/ml n (%) 38 (83) HCV RNA IU/ml (Q1; Q3) 1116144 (391800; 3680000) Undetectable HIV RNA n (%) 40 (87) CD4/mm3 (Q1; Q3) 576 (377; 833) Liver blood test Delay between ARFI and blood test days (Q1; Q3) 0 (0; 1) AST or ALT ≥2N n (%) 7 (15.6) GGT and/or ALP ≥2N n (%) 15 (33.3) Bilirubin ≥2N n (%) 10 (22.2) US liver characteristics Steatosis n (%) 3 (6.5) Cirrhosis in US n (%) 6 (13) US portal hypertension signs n (%) 2 (4.3) US hepatomegaly n (%) 14 (30.4) US focal liver lesion n (%) 5 (10.9) Table 1 Patient demographic and clinical characteristics h Table 1 Patient demographic and clinical characteristics (Continued) ARFI characteristics Median (Q1; Q3) (m/s) 1.29 (1.15; 1.55) Min; Max 0.93; 2.86 TE characteristics Median (Q1; Q3) (kPa) 6.1 (4.6; 7.2) Min; Max 3.4; 35.3 Table 1 Patient demographic and clinical characteristics (Continued) Evaluation of liver fibrosis Evaluation of liver fibrosis Liver blood test Of the 46 patients, 14 (30%) showed discordant Metavir scores between the two methods, 11 in predicting F ≥2 and three in predicting F ≥3. ARFI yielded a higher fibro- sis score than TE for 11 of these patients. As the discord- ance was more important for predicting F ≥2, factors possibly associated with these discordances were analyzed, including age; sex; BMI; alcohol consumption; plasma HCV-RNA levels; absolute CD4+ (/mm3) cells; HCV genotype; increased serum AST, ALT, ALP, and GGT (>2N) and bilirubin concentrations; steatosis; cirrhosis; HIV and HCV treatment; and response to HCV treat- ment. Only age was associated with the discordance be- tween the two methods (p = 0.047) with older patients in the discordant group. Discordance also tended to be Frulio et al. BMC Infectious Diseases 2014, 14:405 http://www.biomedcentral.com/1471-2334/14/405 Frulio et al. BMC Infectious Diseases 2014, 14:405 http://www.biomedcentral.com/1471-2334/14/405 Page 5 of 8 Figure 2 Scatterplot of the relationship between median ARFI (m/s) and TE (kPa) measurements. Figure 2 Scatterplot of the relationship between median ARFI (m/s) and TE (kPa) measurements. HIV and HCV, is of paramount importance for patient management. The presence of significant fibrosis is an indication for treatment, regardless of HCV genotype, whereas the presence of cirrhosis is an indication for ini- tiating a surveillance program for the early detection of HCC and for esophageal varices. associated directly with hepatomegaly and inversely with overweight, cytolysis, and cholestasis. No significant differ- ence was found with HIV (p=0.22) and HCV(p=0.55) treatment. Similar results were observed in the subgroup of pa- tients infected with HCV genotypes 1 and 4. Table 3 shows the agreement between TE and ARFI measure- ments, as determined by equivalent Metavir fibrosis stage classification, in these patients. Agreement between the two methods was very good overall [concordance 65.6%, weighted Kappa = 0.84; 95% CI = 0.71–0.96], very good in predicting severe (F ≥3) fibrosis [concordance 93.8%, Kappa = 0.83, 95% CI = 0.61–1.00], and moderate in pre- dicting significant fibrosis (F ≥2) fibrosis [concordance 75%, Kappa = 0.51, 95% CI = 0.22–0.79]. To our knowledge, no study to date has used ARFI to evaluate liver fibrosis in HIV-HCV co-infected patients or compared the results of ARFI and TE in this popula- tion. This pilot study, found that these two methods yielded similar results. ARFI and TE showed very good agreement in predicting overall fibrosis stage and stage F ≥3. Liver blood test According to the literature, the two methods also showed similar results in mono-infected patients and in patients with other types of chronic liver disease. A meta- analysis of nine studies showed that ARFI imaging had ex- cellent diagnostic accuracy for the staging of liver fibrosis in various chronic liver diseases; compared with liver bi- opsy, ARFI was highly accurate in the diagnosis of fibrosis Discussion h The correct evaluation of liver fibrosis in patients with chronic liver disease, especially in those co-infected with Table 2 Agreement between TE and ARFI fibrosis stage classification according to “equivalent Metavir” fibrosis scores ARFI F1 F2 F3 F4 TOTAL TE F1 23 6 2 0 31 F2 3 3 1 0 7 F3 0 0 0 2 2 F4 0 0 0 6 6 TOTAL 26 9 3 8 46 Observed agreement: 69.6% 95% CI = [54.2–82.3]. Weighted Kappa coefficient: 0.82 95% CI = [0.70–0.95]. Table 3 Agreement between TE and ARFI fibrosis stage classification according to “equivalent Metavir” fibrosis stage in patients infected with HCV genotypes 1 and 4 ARFI F1 F2 F3 F4 TOTAL TE F1 13 5 1 0 19 F2 2 3 1 0 6 F3 0 0 0 2 2 F4 0 0 0 5 5 TOTAL 15 8 2 7 32 Overall proportion of agreement: 65.6% 95% CI [46.8–81.4]. Kappa: 0.84 95% CI [0.71–0.96]. Table 3 Agreement between TE and ARFI fibrosis stage classification according to “equivalent Metavir” fibrosis stage in patients infected with HCV genotypes 1 and 4 Table 2 Agreement between TE and ARFI fibrosis stage classification according to “equivalent Metavir” fibrosis scores Table 3 Agreement between TE and ARFI fibrosis stage classification according to “equivalent Metavir” fibrosis stage in patients infected with HCV genotypes 1 and 4 Table 3 Agreement between TE and ARFI fibrosis stage classification according to “equivalent Metavir” fibrosis stage in patients infected with HCV genotypes 1 and 4 ARFI F1 F2 F3 F4 TOTAL TE F1 13 5 1 0 19 F2 2 3 1 0 6 F3 0 0 0 2 2 F4 0 0 0 5 5 TOTAL 15 8 2 7 32 Overall proportion of agreement: 65.6% 95% CI [46.8–81.4]. Kappa: 0.84 95% CI [0.71–0.96]. Page 6 of 8 Frulio et al. BMC Infectious Diseases 2014, 14:405 http://www.biomedcentral.com/1471-2334/14/405 stage F ≥3 (AUROC = 0.91) and for the diagnosis of liver cirrhosis (AUROC = 0.93) [44]. Moreover, an international multicenter study of 911 HCV mono-infected patients found that ARFI was highly accurate in the diagnosis of fi- brosis stage F ≥3 (AUROC = 0.83) [49]. with biopsy results. Moreover, when a discrepancy was ob- served between the two techniques, it was not possible to conclude whether TE or ARFI was more correct. Discussion h These discrepancies may be linked to the cut-off values used, as these are not the same in all published studies. The TE cut-off values we used [11] yielded similar results as other cut-off values from a recent meta-analysis [17] (data not shown). However, although meta-analyses have found TE to be reliable in assessing liver fibrosis stage, irrespective of etiology, most studies were performed in western patients with HCV mono-infection. Thus, applying these cut-off values to patients co-infected with HIV and HCV may re- sult in discrepancies. To date, however, no ARFI cut-off values have been determined for co-infected patients. We observed a moderate agreement between the two techniques for predicting F ≥2. Earlier studies also showed that TE or ARFI was more accurate for predicting extreme stages (F1 or F4) than moderate (F2) fibrosis. For example, the AUROCs for ARFI were higher for predicting F=4 (0.84;0.95;0.91) than F≥2 (0.79;0.77;0.82) [30,33,49]. In addition, agreement between the TE and ARFI scores was determined in patients infected by genotype 1 and 4 who usually have poorer response than patients infected with genotype 2 and 3. Similarly, there was no difference in concordance level between patients with genotypes 1 and 4 and those with other genotypes. The TE and ARFI methods showed very good agreement in predicting overall fibrosis stage and F ≥3 in patients with genotypes 1 and 4. Finally, although liver biopsy has been considered the gold standard for the assessment of liver fibrosis, we elected to use non-invasive methods for comparison [12]. Fibrosis is heterogeneously distributed throughout the liver, whereas a biopsy evaluates only 1/50000 of the total volume of the liver. The likelihood of misdiagnosing a fragment 2.5 cm in length has been estimated to be as high as 25%, with the optimal size of a biopsy sample be- ing 4 cm, which is difficult to obtain in daily practice [10]. Moreover, liver biopsy is an invasive and costly method, with high risks of complications and intra-/inter-observer variability. Thus, liver biopsy is increasingly replaced by non-invasive methods, especially TE. Since TE is consid- ered a reliable diagnostic modality in clinical practice, we compared the results of ARFI with those of TE. This study included a small number of patients (n = 46). AASLD: American Association for the Study of Liver Diseases; ALP: Alkaline phosphatase; ALT: Alanine aminotransferase; ARFI: Acoustic radiation force impulse; AST: Aspartate aminotransferase; AUROC: Area under the receiver operating characteristic curve; BMI: Body mass index; CT: Computed tomography; DAA: Direct-acting antiviral agents; GGT: γ-glutamyl transferase; HAART: Highly active antiretroviral therapy; HCC: Hepatocellular carcinoma; HCV: Hepatitis C virus; HIV: Human immunodeficiency virus; INIs: Integrase Consent and ethics committee Written informed consent was obtained from all patients for publication of this paper; copies are available for re- view by the Editor of this journal. This study and its publication were approved by our local ethics committee: CPP Sud-Ouest et Outre Mer III Bordeaux. TE and ARFI showed a discrepancy in predicting fi- brosis score in 30% of patients, especially in predicting F ≥2. Apart from age, no factor was clearly associated with these discrepancies. However, the study size was not powered for this aim. Liver biopsy should have been performed in patients with discordance, but was not ex- cept in one patient, in whom ARFI, but not TE, agreed Conclusions h l This pilot study showed good agreement between TE and ARFI in evaluating liver fibrosis in HIV-HCV co- infected patients, suggesting that ARFI may replace TE in this population. In addition to being a new and prom- ising sonography-based method for non-invasive assess- ment of liver fibrosis, ARFI is cost effective, allowing routine ultrasound examination and fibrosis assessment during the same session. Discussion h However, it was designed as a pilot study, with intraclass correlation coefficients precise enough to draw conclusions by comparing the lower limit of the 95% CIs with 0.6, deemed to represent the lower limit of good agreement. ARFI has several advantages compared with TE. TE has been reported to have an average failure rate of 3.1% and to be highly dependent on BMI. Moreover, TE measure- ments were estimated to be unreliable in 15.8% of patients in a very large study [19]. We found that ARFI examina- tions were reliable in all patients, with no measurement limitations due to overweight or the presence of ascites. ARFI measurement depth can be adapted according to the distance between the skin and liver capsule. In addition, ARFI is an ultrasound-based elastography method, inte- grated into a routine ultrasound machine, allowing simul- taneous morphological and Doppler analysis of the liver, screening for focal liver lesions and evaluation of liver fi- brosis. Moreover, the presence of B mode allows ARFI measurements to be localized to liver segments far from the capsule and blood vessels. Ultrasound morphological analysis of our patients identified cirrhosis in six patients and focal liver lesions in five, including two HCCs. More- over, the presence of HCC did not influence fibrosis evalu- ation because ARFI measurements in non-tumoral liver were performed at a distance (>3 cm) from the tumor. In- deed, the visual control in B mode allowed the ROI to be positioned to avoid specific areas, including tumors. Competing interests chronic hepatitis C. Hepatology 2003, 38:1449–145 p g The authors state that they have no competing interests. 11. Castera L, Vergniol J, Foucher J, Le Bail B, Chanteloup E, Haaser M, Darriet M, Couzigou P, De Ledinghen V: Prospective comparison of transient elastography, Fibrotest, APRI, and liver biopsy for the assessment of fibrosis in chronic hepatitis C. Gastroenterology 2005, 128:343–350. Received: 27 January 2014 Accepted: 15 July 2014 Published: 21 July 2014 20. 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Author details 1 1Department of Diagnostic and Interventional Imaging, Saint-André hospital, CHU de Bordeaux, 1 rue Jean Burguet, 33075 Bordeaux, France. 2Unité de Soutien Méthodologique à la Recherche Clinique (USMR), CHU Bordeaux Pôle de Santé Publique, place Amélie Rabat-Leon, 33000 Bordeaux, France. 3Department of internal medicine and infectious diseases, Saint-André hospital, CHU de Bordeaux, 1 rue Jean Burguet, 33075 Bordeaux, France. 4INSERM U1053, Bordeaux university, Bordeaux, France. 5Centre 17. Friedrich-Rust M, Ong MF, Martens S, Sarrazin C, Bojunga J, Zeuzem S, Herrmann E: Performance of transient elastography for the staging of liver fibrosis: a meta-analysis. Gastroenterology 2008, 134:960–974. 18. Jung KS, Kim SU: Clinical applications of transient elastography. Clin Mol Hepatol 2012, 18:163–173. d’investigation de la fibrose hépatique, Haut-Lévêque hospital, CHU de Bordeaux, 1 Avenue de Magellan, 33604 Pessac, France. 19. Castera L, Foucher J, Bernard PH, Carvalho F, Allaix D, Merrouche W, Couzigou P, de Ledinghen V: Pitfalls of liver stiffness measurement: a 5-year prospective study of 13,369 examinations. Hepatology 2010, 51:828–835. Received: 27 January 2014 Accepted: 15 July 2014 Published: 21 July 2014 Acknowledgements h k 15. Sporea I, Sirli R, Bota S, Fierbinteanu-Braticevici C, Petrisor A, Badea R, Lupsor M, Popescu A, Danila M: Is ARFI elastography reliable for predicting fibrosis severity in chronic HCV hepatitis? World J Radiol 2011, 3:188–193. We thank Dr. Denis Lacoste and Dr. Noëlle Bernard for referring patients, Dr. Sabrina Caldatto for extracting data, and Dr. Cécile Salut, Dr. Mounir Bouzgarrou and Dr P Balageas for their valuable help with imaging methods. We thank Dr. Denis Lacoste and Dr. Noëlle Bernard for referring patients, Dr. Sabrina Caldatto for extracting data, and Dr. Cécile Salut, Dr. Mounir Bouzgarrou and Dr P Balageas for their valuable help with imaging methods. 16. 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Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: • Convenient online submission • Thorough peer review • No space constraints or color ﬁgure charges • Immediate publication on acceptance • Inclusion in PubMed, CAS, Scopus and Google Scholar • Research which is freely available for redistribution Submit your manuscript at www.biomedcentral.com/submit Submit your next manuscript to BioMed Central and take full advantage of: Submit your next manuscript to BioMed Central and take full advantage of: 42. 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https://openalex.org/W4313188751	https://zenodo.org/records/7541912/files/Effectiveness%20of%20ecologically%20safe%20disinfectants%20against%20Pseudomonas%20aeruginosa%20and%20the%20poultry%20main%20bacteriosis%20pathogens.pdf	English	null	Effectiveness of ecologically safe disinfectants against Pseudomonas aeruginosa and the poultry main bacteriosis pathogens	ScienceRise. Biological science	2,022	cc-by	3,534	How to cite: How to cite: Berezovskiy, A., Fotina, T., Vashchyk, Y., Berezhnyi, D., Morozenko, D. (2022). Effectiveness of ecologically safe disinfectants against Pseudomo- nas aeruginosa and the poultry main bacteriosis pathogens. ScienceRise: Biological Science, 3 (32), 00-00. doi: http://doi.org/10.15587/2519- 8025.2022.266239 © The Author(s) 2022 © The Author(s) 2022 This is an open access article under the Creative Commons CC BY license hydrate © The Author(s) 2022 Thi i © The Author(s) 2022 This is an open access article under the Creative Commons CC BY license hydrate Andriy Berezovskiy, Tetiana Fotina, Yevheniia Vashchyk, Dmytro Berezhnyi, Dmytro Morozenko Andriy Berezovskiy, Tetiana Fotina, Yevheniia Vashchyk, Dmytro Berezhnyi, Dmytro Morozenko The aim: study of the effectiveness of environmentally safe disinfectants against P. aeruginosa and p main bacteriosis of poultry at test facilities. Materials and methods. To study the antimicrobial action of the investigated disinfectants against a mixture of epizoot- ic cultures of E. coli, P. aeruginosa, S. aureus, S. typhimurium, isolated from poultry, bacteriological studies were car- ried out on test objects: galvanized iron, wooden bars (painted and unpainted), red brick, cutouts from plaster, size 10×10 cm. Results. The working solutions of the new disinfectant "Dezsan" were studied in comparison with the control agents: "Virocid" and "Bi-dez" at a concentration of 0.01; 0.1; 0.25; 0.5; 1; 1.5 % in relation to suspension cultures of E. coli, P. aeruginosa, S. aureus, S. typhimurium. In this case, it was established, that the "Dezsan" agent showed an antimicrobial effect on rough test objects after exposure for 3 hours at a concentration of 0.1 %, and at a concentration of 0.25 % - for 1 hour. On smooth surfaces, the agent neutralized bacterial cultures at a concentration of 0.1 % after exposure for 1 hour. The preparation "Bi-dez" at a concentration of 0.25 % was effective on smooth surfaces after exposure for 1 hour, on rough surfaces (brick, plaster) - at a concentration of 0.5 % after exposure for 3 hours or more. The working solution of 1 % concentration neutralized bacterial cultures on all types of surfaces after exposure for 1 hour or more. "Virocid" agent after exposure for 1 hour neutralized bacterial cultures on smooth surfaces in concentrations of 0.25 % and higher; on rough surfaces, the growth of cultures was not detected when using a 0.5 % solution. Conclusions. Environmentally safe disinfectants "Dezsan" and "Shumerske sryblo" compared to the control ones ("Bi Dez" and "Virotsid") show an active antimicrobial effect at a concentration of 0.25 % and 3 %, respectively, against the suspension of epizootic cultures of E. coli, P. aeruginosa, S. aureus and S. typhimurium on different types of production surfaces, which justifies the feasibility of their use based on the principle of rotation of disinfectants for the prevention of bacterial pseudomonosis of poultry ords: poultry, pseudomonosis, E. coli, P. aeruginosa, S. aureus, S. typhimurium, effectiveness, prevention №3(32)2022 №3(32)2022 Scientific Journal «ScienceRise: Biological Science» UDC 636.5:614.4 DOI: 10.15587/2519-8025.2022.266239 Veterinary research 2. Materials and methods of isolation of P. aeruginosa and E. coli was 3 times higher than the cases of coccal microflora isolation and 3.15 times higher than the frequency of other causative agents of poultry bacteriosis. To study the antimicrobial action of the investigated disinfectants against a mixture of epizootic cultures of E. coli, P. aeruginosa, S. aureus, S. typhimurium, isolated from pat material from poultry, bacteriological studies were car- ried out on test objects in accordance with the methodologi- cal recommendations "Use of the latest means and methods of rehabilitation of poultry facilities and control of their ef- fectiveness", 2007. The test objects were: galvanized iron, wooden bars (painted and unpainted), red brick and plaster cutouts, measuring 10×10 cm. Before applying the cultures, the test objects were subjected to heat treatment for the pur- pose of sanitation. The research was conducted in a bacteri- ological box. Using a sterile pipette, a mixture of 1 billion suspended microbial cells of bacterial cultures of E. coli, P. aeruginosa, S. aureus, S. typhimurium in an isotonic solu- tion was applied to test objects in enameled cuvettes. An hour later, using a sprayer, aerosols of disinfectant solutions were sprayed at the rate of 10 cm3 per 10 cm2 for exposure from 1 hour up to 1 day Working solutions of disinfectants "Dezsan", "Virocid" and "Bi-dez" at a concentration of 0.01 were studied; 0.1; 0.25; 0.5; 1; 1.5 %. Control test objects were irrigated with sterile distilled water. Our results and the data of other researchers re- garding the isolation of bacterial flora from poultry farms differed somewhat, which can be explained by climatic features, different methods and focus of research. A higher percentage of P. aeruginosa isolation confirms the effectiveness of our proposed medium, which was used to isolate the pathogen from pat material and facilities of poultry farms. Thus, according to the results of studies by Stegnii B.T., Gliebova K.V., Petrenchuk E.P. et al. [6], the percentage of productive poultry, affected by salmo- nella, is 7.7 % of the total number of poultry examined. The share of pathogenic E. coli cultures accounts for 14.7 % of the number of isolated pathogens. The frequency of isolation of Enterobacter, Citrobacter, and Proteus cul- tures is 3.9 %, 15.6 %, and 8.2 %, respectively. The per- centage of poultry, infected with representatives of the Staphylococcus, Pseudomonas, Neisseria and Ornitho- bacterium families, is not significant [7]. 2. Materials and methods Disinfection measures, aimed at destroying path- ogens in the environment and preventing their penetra- tion into the bird's body, are an integral part of the effec- tive fight against bacterial infections. Effectively carried out disinfection measures in premises for growing poul- try, hatcheries allow to prevent the spread of pathogens and the occurrence of epizootic outbreaks. Long-term use of disinfectants of the same chemical group causes the development of microflora resistance to antimicrobial and disinfectant drugs. There is a need to constantly search for new effective antimicrobial substances. The environmental aspect is extremely important when choosing disinfectants. Effective, but aggressive sub- stances (formalin, caustic soda, chlorine-containing dis- infectants, etc.) are recognized by the world as ecologi- cally dangerous, have an irritating and carcinogenic ef- fect, so they are abandoned in many countries [8, 9]. The composition of the "Bi-dez" product: 100 cm3 of the drug contains active substances (g): polyhexa- methyleneguanidine hydrochloride – 6.5; dodecyldipro- pylene triamine – 6.5. Excipients: glutamic acid, co- coamidopropyl betaine, demineralized water (NVF BROVAFARMA LLC, Ukraine). The composition of "Dezsan": 100 cm3 of the drug contains active substances: alkyldimethylbenzylammonium chloride – 4.8 %, octyldecyldimethylammonium chloride – 3.6 %, dioctyldimethylammonium chloride – 1.44 %, didecyldimethylammonium chloride – 2.16 %, glutaralde- hyde – 10 % (Brovafarma Ltd., Ukraine). "Virocid" contains a composition of two quater- nary ammonium compounds: alkyldimethylben- zylammonium chloride – 17.06 %, didecyldime- thylammonium chloride – 7.8 %), glutaraldehyde – 10.7 %, isopropanol – 14.6 %, turpentine derivative – 2.0 % ("CID LINES NV/SA", Belgium). Epizootic well-being in the economy directly de- pends on timely and regular disinfection with effective disinfectants. The competitiveness of modern poultry en- terprises is determined by many factors, including the quality of disinfection measures. The main purpose of disinfection is not only to improve the health of the live- stock industry, but also to prevent infectious diseases in healthy farms. Therefore, preventive disinfection is gain- ing more and more popularity as a combination of disin- fection measures, carried out in the absence of infectious diseases, its purpose is to prevent the occurrence and spread of infections [10–12]. 1. Introduction conditions of intensification of production, the effect of factors, contributing to the disruption of normal micro- flora in farm poultry, is noted. Non-observance of veteri- nary and sanitary norms and zoohygiene requirements causes a violation of the balance between normal and conditionally pathogenic microflora of the gastrointesti- nal tract, which, against the background of constant stress and a decrease in the natural resistance of the body of birds, leads to an increase in the pathogenic and viru- lent properties of microorganisms and causes the devel- opment of an infectious disease [5]. Poultry farming, as a branch of animal husbandry, is progressive and constantly improving. This especially applies to the prevention of bacterial diseases [1, 2]. It differs from other branches of animal husbandry in its high reproduction rate and prematurity, which makes it the main source of providing the population with proteins of animal origin [3]. The use of modern technologies allows in broiler production to reduce the period of poultry fattening to 35 days, to ensure an average daily gain of more than 50 grams for feed conversion of 1.75 kg, and to obtain more than 230 kg of broiler meat in slaughter weight per laying hen of the parent flock; in egg production, more than 340 eggs can be obtained for an average annual lay- ing hen with feed conversion of 1.17 kg [4]. However, in The aim of the research study of the effective- ness of environmentally safe modern disinfectants against P. aeruginosa and pathogens of the main bacteri- osis of poultry at test facilities. Veterinary research Veterinary research 9 Scientific Journal «ScienceRise: Biological Science» №3(32)2022 2. Materials and methods To determine the quality of disinfection with ster- ile cotton swabs that were previously immersed in tubes with MPB, washings were taken from the surfaces of the test objects and again placed in tubes with MPB, which were incubated in a thermostat at a temperature of + 38 oC, followed by recording the growth of the culture after 12, 24 and 48 hours. The presence of the develop- ment of signs of culture growth (turbidity, change in the color of MPB, the formation of a film on the surface and sediment at the bottom of the test tube) indicated the ab- sence of antimicrobial action of the studied disinfectant. The working solutions of the new disinfectant "Dezsan" were studied in comparison with the control agents: "Virocid" and "Bi-dez" at a concentration of 0.01; 0.1; 0.25; 0.5; 1; 1.5 % in relation to suspension cultures of E. coli, P. aeruginosa, S. aureus, S. typhimurium. In this case, it was established, that the "Dezsan" agent showed an antimicrobial effect on rough test objects after exposure for 3 hours at a concentration of 0.1 %, and at a concentration of 0.25 % – after 1 hour. On smooth sur- faces, the agent neutralized bacterial cultures at a concen- tration of 0.1 % after exposure for 1 hour. The results of the study are presented in Table 1. 3. Research results typhimurium on various test objects Test objects Exposure, hours Growth of bacterial cultures on test objects / concentration of disinfection solution, % "Dezsan" "Bides" "Virocide" 0.01 0.1 0.25 0.5 1 1.5 0.01 0.1 0.25 0.5 1 1.5 0.01 0.1 0.25 0.5 1 1.5 Galvanized iron 1 + – – – – – + + – – – – + + – – – – 3 + – – – – – + – – – – – + – – – – – 24 + – – – – – + – – – – – + – – – – – Painted wood 1 + – – – – – + + – – – – + + – – – – 3 + – – – – – + – – – – – + – – – – – 24 + – – – – – + – – – – – + – – – – – Unpainted wood 1 + – – – – – + + + – – – + + – – – – 3 + – – – – – + + – – – – + + – – – – 24 + – – – – – + – – – – – + – – – – – Brick 1 + + – – – – + + + + – – + + + – – – 3 + – – – – – + + + – – – + + + – – – 24 + – – – – – + + – – – – + – – – – – Plaster 1 + + – – – – + + + + – – + + + – – – 3 + – – – – – + + + – – – + + + – – – 24 + – – – – – + + – – – – + – – – – – Note: "+" – presence of growth, "–" – absence of growth The preparation "Bi-dez" at a concentration of 0.25 % was effective on smooth surfaces after exposure for 1 hour, on rough surfaces (brick, plaster) – at a con- centration of 0.5 % after exposure for 3 hours or more. 3. Research results According to the results of our monitoring bacte- riological studies of pathological material and washings from the production surfaces of poultry premises, on av- erage, in all farms of different technological direction, escherichia prevailed – 37.58 % and Pseudomonas aeru- ginosa – 22.98 %, coccus microflora was detected in 20.23 % of cases. The number of Proteus, Campylobac- ter, Enterobacter, Citrobacter, Klebsiella, Yersinia, and Clostridium cultures was 19.21 %. Thus, the frequency 10 10 Scientific Journal «ScienceRise: Biological Science» №3(32)2022 Table 1 Antimicrobial effect of modern disinfectants on suspended cultures of E. coli, P. aeruginosa, S. aureus, S. 3. Research results A working solution of 1 % concentration neutralized bacte- rial cultures on all types of surfaces after exposure for 1 hour or more. The "Virocid" agent after exposure for 1 hour neutralized bacterial cultures on smooth surfaces in concentrations of 0.25 % and higher; on rough surfaces the growth of cultures was not detected when using a 0.5 % solution. painted and unpainted wood, brick and plaster. There- fore, the effectiveness of the tested disinfectants on other materials cannot be guaranteed. Prospects for further research. A promising di- rection of research is the study of other concentrations of disinfectants for further use in poultry farming for dis- ease prevention. Financing Financing The study was performed without financial support. 4. Conclusions Environmentally safe disinfectants "Dezsan" and "Shumerske sryblo" compared to the control ones ("Bi Dez" and "Virotsid") show an active antimicrobial effect at a concentration of 0.25 % and 3 %, respectively, against the suspension of epizootic cultures of E. coli, P. aeruginosa, S. aureus and S. typhimurium on different types of production surfaces, which justifies the feasibil- ity of their use based on the principle of rotation of disin- fectants for the prevention of bacterial pseudomonosis of poultry. Thus, the studied disinfectant "Dezsan" and the control "Bi-dez" and "Virocid" on all types of surfaces in 3 hours of exposure showed an antimicrobial effect against the suspension of epizootic cultures of E. coli, P. aeruginosa, S. aureus and S. typhimurium, isolated from the pat material from the bird, in a concentration of 0.1 %, 0.5 % and 0.5 %, and after exposure for 1 hour – in 0.25 %, 1 % and 0.5 %, respectively. In comparison with modern studies of other au- thors, it should be noted, that standard disinfection schemes in poultry farming do not give results in terms of complete destruction of pathogens. This is caused by various factors, among which the leading role belongs to antibiotic resistance, in particular, Salmonella [13]. Peracetic acid, hydrogen peroxide, and formaldehyde proved to be the best disinfectants for poultry rooms, but their effectiveness was not ideal [14]. According to foreign authors, the selection and testing of disinfection is an important problem [15], which determines the relevance and necessity of con- ducting our research. Conflict of interests The authors declare that they have no conflict of interest in relation to this study, including financial, per- sonal, authorship, or any other, that could affect the study and its results, presented in this article. Veterinary research Acknowledgments The authors express their gratitude to the limited liability company "Brovafarma" (Ukraine) for their sup- port in conducting this research and providing disinfect- ants for testing. Research limitations. The research was conduct- ed exclusively on such test objects as galvanized iron, Veterinary research 11 Scientific Journal «ScienceRise: Biological Science» №3(32)2022 №3(32)2022 References References 1. Jeni, R. E., Dittoe, D. K., Olson, E. G., Lourenco, J., Corcionivoschi, N., Ricke, S. C.et al. (2021). Probiotics and potential appli- cations for alternative poultry production systems. Poultry Science, 100 (7), 101156. doi: https://doi.org/10.1016/j.psj.2021.101156 References 1. Jeni, R. E., Dittoe, D. K., Olson, E. G., Lourenco, J., Corcionivoschi, N., Ricke, S. C.et al. (2021). Probiotics and potential appli- cations for alternative poultry production systems. Poultry Science, 100 (7), 101156. doi: https://doi.org/10.1016/j.psj.2021.101156 2. 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Portico. https://doi.org/10.1111/zph.12830 Received date 04.08.2022 Accepted date 22.09.2022 Published date 30.09.2022 Andriy Berezovskiy, Doctor of Veterinary Sciences, Professor, Department of Veterinary Expertise, Microbiology, Zoohygiene and Safety and Quality of Livestock Products, Sumy National Agrarian University, Gerasima Kodratieva str., 160, Sumy, Ukraine, 40000 Tetiana Fotina, Doctor of Veterinary Sciences, Professor, Head of Department, Department of Veterinary Expertise, Microbiology, Zoohygiene and Safety and Quality of Livestock Products, Sumy National Agrarian University, Gerasima Kodratieva str., 160, Sumy, Ukraine, 40000 Yevheniia Vashchyk, Doctor of Veterinary Sciences, Associate Professor, Department of Veterinary Medicine and Pharmacy, National University of Pharmacy Pushkinska str., 53, Kharkiv, Ukraine, 61002 Dmytro Berezhnyi, PhD, Associate Professor, Department of Veterinary Medicine and Pharmacy, National University of Pharmacy, Pushkinska str., 53, Kharkiv, Ukraine, 61002 Dmytro Morozenko, Doctor of Veterinary Sciences, Senior Researcher, Head of Department, Department of Veterinary Medicine and Pharmacy, National University of Pharmacy, Pushkinska str., 53, Kharkiv, Ukraine, 61002 Corresponding author: Yevheniia Vashchyk, e-mail: yevgeniavashik@gmail.com 12 12
https://openalex.org/W2740437078	https://www.scielo.br/j/cadsc/a/sRQkf7ZByGBnDwvg9xVrsLz/?lang=pt&format=pdf	Portuguese	null	Prevalência de doenças musculoesqueléticas autorreferidas segundo variáveis demográficas e de saúde: estudo transversal de idosos de Goiânia/GO	Cadernos Saúde Coletiva	2,017	cc-by	4,871	DOI: 10.1590/1414-462X201700010274 DOI: 10.1590/1414-462X201700010274 Artigo Original Resumo Objetivo: Estimar a prevalência de doenças musculoesqueléticas autorreferidas por idosos segundo variáveis demográficas, dor, autoavaliação de saúde e quedas. Métodos: Estudo transversal, de base populacional, com amostra probabilística de 934 idosos residentes em Goiânia, em Goiás. Aplicou-se um questionário padronizado e semiestruturado. Considerou-se doença musculoesquelética autorreferida pelo idoso como variável desfecho, enquanto as variáveis de exposição foram demográficas, autoavaliação de saúde, quedas, dor e atividade física. Para análise, foram utilizados os testes qui-quadrado ou Fisher e regressão de Poisson (valor de p<0,05). Resultados: Dos 934 idosos, 62,2% eram mulheres, com média de 71,4 anos (±8,3). A prevalência de doenças musculoesqueléticas foi de 39,1% (IC95% 35,9-42,3), sendo as mais frequentes: osteoporose (24,6%), artrose (2,9%), reumatismo (1,2%) e artrite (0,6%). A prevalência foi superior nas mulheres (44,7%; p=0,000); faixa etária ≥80 anos (44,7%; p=0,002); autoavaliação de saúde ruim (55,9%; p=0,000); relato de dor (43,4%; p=0,001). Não houve associação entre doenças musculoesqueléticas e quedas (p=0,671) e sedentarismo (p=0,167). Conclusão: Observaram-se elevada prevalência de doenças musculoesqueléticas nos idosos de elevada faixa etária e associação com autoavaliação de saúde ruim e relato de dor, o que sugere intervenções no controle das condições de saúde gerais. P l h d l lé i id d i vras-chave: doenças musculoesqueléticas; idoso; estudos transversais. Prevalence of self-reported musculoskeletal diseases by demographic and health variables: cross-sectional study of elderly of Goiânia/GO Prevalence of self-reported musculoskeletal diseases by demographic and health variables: cross-sectional study of elderly of Goiânia/GO Anna Cássia Fernandes Melo1, Adélia Yaeko Kyosen Nakatani2, Lilian Varanda Pereira3, Ruth Losada de Menezes4, Valéria Pagotto5 ▄ ▄METODOLOGIA Trata-se de um estudo de delineamento transversal, de base populacional, inserido na pesquisa matriz “Situação de saúde da população idosa do município de Goiânia-GO”, vinculado à Rede de Vigilância à Saúde do Idoso no Estado de Goiás (REVISI). Os detalhes metodológicos e de cálculo amostral já foram descritos em publicações prévias17,18. Essas enfermidades, conhecidas como doenças musculoesqueléticas (DME), são a principal causa de incapacidade relacionada à diminuição da mobilidade, principalmente, na faixa etária 65 anos ou mais. Elas podem comprometer a realização de atividades da vida diária (AVD)3, bem como, eventualmente, ocasionar dependência e institucionalização, influenciar na autopercepção de saúde e qualidade de vida4-6, e implicar em aumento de custos para os serviços7,8. Entre as DME, a osteoporose é a mais prevalente mundialmente7,9, apresentando como principais complicações fragilidade óssea, fratura e incapacidade10. Resumidamente, a amostra foi calculada considerando-se os seguintes parâmetros: população de idosos de Goiânia (7% do total da população, que era de 1.249.645, tendo como ano‑base 2007); frequência esperada de 30% para todos os eventos do inquérito; nível de confiança de 95%; nível de significância de 5%; frequência esperada de 30%; precisão absoluta de 5%; efeito de delineamento de 1,8. Com isso, obteve-se um valor amostral de 823 idosos. A esse cálculo foram acrescidos 11% para possíveis perdas, resultando em 934 idosos, conferindo poder amostral suficiente para as análises do presente estudo. Poucos estudos analisaram a prevalência de DME no Brasil. Estudo de revisão sistemática mostrou que as DME mais prevalentes foram artrite ou reumatismo (24,2-37,5%), osteoartrose (16,6‑39,6%), tendinite (9,2%), bursite (3,8%), e outras (10,7%)11. Os fatores que se associam à ocorrência de DME podem ser de caráter intrínseco, como sexo feminino, idade avançada, origem asiática, cor branca, deficiência hormonal, presença de comorbidades, fatores genéticos, fragilidade ou histórico de fratura prévia3, fraqueza de membros inferiores, déficit de equilíbrio, diminuição da força de preensão e polifarmácia12, e extrínsecos, que são relacionados ao estilo de vida, como baixo peso corpóreo, sedentarismo, tempo de exposição ao tabaco, uso abusivo de álcool e deficiência de vitamina D3,12. A dor também está associada às DME, constituindo-se a principal queixa das pessoas com DME crônicas, promovendo impacto na qualidade de vida, independência e participação social do idoso11. i Para seleção dos idosos, foi realizada amostragem por conglomerados. A área geográfica do estudo foi definida a partir dos Setores Censitários (SC) estabelecidos pelo Instituto Brasileiro de Geografia e Estatística (IBGE). Abstract Objective: Estimate the prevalence of self-reported musculoskeletal disorders in elderlies according to demographic variables, pain, self-assessment of health and falls. Methods: Cross-sectional and population-based study with a probabilistic sample of 934 seniors living in Goiania, GO. It was applied a standardized and semi-structured questionnaire. The outcome variable was self-reported musculoskeletal disorders by elderly and exposure were demographic, self-assessment health, falls, pain and physical activity. 1Hospital Araújo Jorge, Associação de Combate ao Câncer em Goiás (ACCG) - Goiânia (GO), Brasil. 2Pontifícia Universidade Católica de Goiás (PUC Goiás) - Goiânia (GO), Brasil. 3Universidade Federal de Goiás (UFG) - Goiânia (GO), Brasil. 4Universidade de Brasília (UnB) - Brasília (DF), Brasil. 5Faculdade de Enfermagem, Universidade Federal de Goiás (UFG) - Goiânia (GO), Brasil. Trabalho realizado em setores censitários urbanos do município de Goiânia - Goiânia (GO), Brasil. Endereço para correspondência: Anna Cássia Fernandes Melo – Hospital Araújo Jorge, Associação de Combate ao Câncer em Goiás (ACCG), Rua 227, Qd-68, s/n, Setor Leste Universitário – CEP: 74605-080 – Goiânia (GO), Brasil – Email: anna_melo_15@hotmail.com Fonte de financiamento: Fundação de Amparo à Pesquisa no Estado de Goiás – FAPEG e auxilio logístico da Secretaria Municipal de Saúde de Goiânia. Conflito de interesses: nada a declarar. p j g ç ( ) 2Pontifícia Universidade Católica de Goiás (PUC Goiás) - Goiânia (GO), Brasil. 3Universidade Federal de Goiás (UFG) - Goiânia (GO), Brasil. 4Universidade de Brasília (UnB) - Brasília (DF), Brasil. 5Faculdade de Enfermagem, Universidade Federal de Goiás (UFG) - Goiânia (GO), Brasil. Cad. Saúde Colet., 2017, Rio de Janeiro, 25 (2): 138-143 138 Doenças musculoesqueléticas, dor e autoavaliação de saúde em idosos For analysis, we used the Chi-square test or Fisher and Poisson regression (value of p<0.05). Results: We evaluated 934 elderly, 62.2% were women, average of 71.4 years (±8.3). The prevalence of musculoskeletal disorders was 39.1% (95% CI -42.3 35.9), being the most frequent: osteoporosis (24.6%), arthritis (2.9%), rheumatism (1.2%) and arthritis (0.6%). The prevalence was higher in women (44.7%; p=0.000); age ≥ 80 years (44.7%; p=0.002); self-assessment of bad health (55.9%; p=0.000); report of pain (43.4%; p=0.001). There was no association between musculoskeletal disorders and falls (p=0.671) and sedentary (p=0.167). Conclusion: We observed a high prevalence of musculoskeletal disorders in the elderly with high age group which suggests interventions in the control of the general health conditions and pain. Keywords: musculoskeletal diseases; aged; cross-sectional studies. ▄ ▄INTRODUÇÃO as consequências das DME para a saúde do idoso, o objetivo deste estudo foi estimar a prevalência de DME autorreferidas por idosos segundo variáveis demográficas, dor, autoavaliação de saúde e quedas. O envelhecimento humano contribui para a ocorrência de diminuição de massa óssea, dano às estruturas cartilaginosas, redução da elasticidade dos ligamentos, perda de força muscular e infiltração gordurosa nos tecidos, diminuindo a capacidade destes em manter suas funções normais e podendo levar a doenças como osteoporose, sarcopenia, fratura por trauma leve, osteoartrite e artrite inflamatória1-3. ▄ ▄METODOLOGIA Para a delimitação dos SC, utilizou-se o mapa urbano básico digital de Goiânia, fornecido pela COMDATA (instituição municipal responsável pela construção da malha digital da cidade). Identificaram‑se 1.068 SC no município, dos quais 912 eram estritamente urbanos. A média de indivíduos por setor era de 980 pessoas. Considerando 7% de idosos na população de Goiânia, estimaram‑se 16,3 idosos por SC. Dividindo-se o total da amostra (n=934) pelo número de idosos estimados por SC (17,0), calculou-se que seriam necessários 55 SC para a coleta dos dados. Sorteou-se um setor a mais (56) por meio de tabela de números aleatórios criada em sistema eletrônico de randomização. Nesses setores, foram sorteados o quarteirão e a esquina para o início da coleta. A partir da esquina sorteada, a primeira residência foi visitada. Caso não houvesse idoso, a entrevistadora deslocava-se para o próximo domicílio até identificá-lo. Na ocorrência de mais de um idoso residindo no domicílio, todos foram entrevistados. i Para seleção dos idosos, foi realizada amostragem por conglomerados. A área geográfica do estudo foi definida a partir dos Setores Censitários (SC) estabelecidos pelo Instituto Brasileiro de Geografia e Estatística (IBGE). Para a delimitação dos SC, utilizou-se o mapa urbano básico digital de Goiânia, fornecido pela COMDATA (instituição municipal responsável pela construção da malha digital da cidade). Identificaram‑se 1.068 SC no município, dos quais 912 eram estritamente urbanos. A média de indivíduos por setor era de 980 pessoas. Considerando o aumento da expectativa de vida na atualidade e nos próximos anos no Brasil, as DME têm tendência crescente, pois os idosos constituem o grupo com maior prevalência dessas doenças13,14, o que pode levar ao aumento da morbimortalidade, restrição de mobilidade, incapacidade funcional, isolamento social e aumento do risco de quedas15,16. Considerando que poucos estudos analisaram essa temática no Brasil, e dadas Cad. Saúde Colet., 2017, Rio de Janeiro, 25 (2): 138-143 139 Anna Cássia Fernandes Melo, Adélia Yaeko Kyosen Nakatani, Lilian Varanda Pereira, Ruth Losada de Menezes, Valéria Pagotto Quando o número de domicílios residenciais do setor não era suficiente para completar a amostra, outro SC já estava sorteado; então o entrevistador prosseguia até aproximar ou completar o total estimado de idosos. Foram incluídos: indivíduos que possuíam idade igual ou superior a 60 anos, que residiam na área urbana de Goiânia e que eram moradores da residência da entrevista. Foram excluídos os idosos que não responderam à pergunta sobre DME. ▄ ▄RESULTADOS Foram entrevistados 934 idosos, sendo 62,2% (n=581) do sexo feminino, 48,1% na faixa etária de 60-69 anos, com idade média de 71,4 anos (±8,3), 57,9% com renda inferior a um salário mínimo, 48,2% com nível de escolaridade na fase do ensino primário, e 49,1% casados. Os dados foram coletados entre os meses de dezembro de 2009 e abril de 2010 por entrevistadores previamente treinados para a aplicação do questionário, após a aplicação do teste‑piloto que foi realizado nos meses de outubro e novembro de 2009 com população de 50 idosos de um SC não sorteado para a coleta dos dados. A prevalência de DME foi de 39,1% (IC95% 35,9-42,3) (Figura 1). As principais doenças autorreferidas foram: osteoporose (24,6%), artrose (2,9%), reumatismo (1,2%) e artrite (0,6%). Do total, 9,8% das pessoas não souberam relatar o tipo de DME que referiram ter (Tabela 1). Os instrumentos foram preenchidos e entregues em uma secretaria central para conferência e identificação de possível inconsistência dos dados. Quando alguma incongruência de dados era identificada, o entrevistador retornava ao domicílio e corrigia os registros antes de seu envio ao banco de dados eletrônico. O controle de qualidade dos dados foi realizado por pesquisadores e técnicos treinados que supervisionaram todos os questionários aplicados logo após a entrevista e antes de sua inserção no banco de dados. Já o fomento de dados no sistema foi feito por pares. Após o término do processo de digitação dos dados, o banco foi integralmente conferido para reavaliação de possíveis incongruências que poderiam ter persistido. Observou-se maior prevalência de DME entre as mulheres (49,6%). A probabilidade de apresentar DME foi 2,3 vezes maior entre as mulheres do que nos homens (RP=2,3; IC95% 1,8-2,8). Em relação à faixa etária, a prevalência foi superior nos idosos acima de 70 anos (superior a 40%), e as medidas de efeito demonstraram que a prevalência de DME nas faixas etárias mais altas pode ser 13% maior em relação à faixa etária mais jovem (RP=1,3; IC95% 1,1-1,7) (Tabela 2). Analisando a prevalência conforme as variáveis de exposição (Tabela 3), observa-se que a prevalência de DME foi superior em idosos que autoavaliaram sua saúde como ruim (55,9%), A variável desfecho deste estudo foi DME autorreferida, avaliada por meio da seguinte pergunta: “Quais doenças o médico já disse que o(a) Sr.(a) têm?”. ▄ ▄METODOLOGIA conforme as normas nacionais em ética em pesquisa vigente sobre pesquisas envolvendo seres humanos. Os participantes consentiram em participar mediante assinatura do Termo de Consentimento Livre e Esclarecido (TCLE). ▄ ▄RESULTADOS Prevalência de doenças musculoesqueléticas (DME) e razão de prevalência (RP) conforme sexo e faixa etária em idosos no município de Goiânia, Goiás, em 2010 (n=934) Variáveis N (%) Prevalência de DME (%) RP (IC%) p* Sexo 0,000 Feminino 581(62,2) 49,7 2,3 (1,8-2,8) Masculino 353(37,8) 21,6 1,0 Faixa etária (1) 0,002 60-69 anos 451(48,3) 33,2 1,0 70-79 anos 310(33,2) 44,5 1,3 (1,1-1,6) ≥80 anos 173(18,5) 44,8 1,3 (1,1-1,7) Qui-quadrado musculoesqueléticas (DME) e razão de prevalência (RP) conforme sexo e faixa etária em idosos no município 34) s musculoesqueléticas (DME) e razão de prevalência (RP) conforme condições de saúde em idosos no 2010 ( 934) Tabela 3. Prevalência de doenças musculoesqueléticas (DME) e razão de prevalência (RP) conforme condições de saúde em idosos no município de Goiânia, Goiás, em 2010 (n=934) Variáveis N (%) Prevalência de DME (%) RP (IC%) p Autoavaliação de saúde 0,000 Muito bom/bom 396(44,5) 30,8 1,00 Regular 391(44) 44,0 1,4 (1,2-1,7) Ruim/muito ruim 102(11,5) 55,9 1,8 (1,4-2,3) Quedas no último ano 0,671 Sim 318(34,8) 39,3 1,0 (0,9-1,2) Não 595(65,2) 37,9 1,0 Dor 0,001 Sim 543(59,4) 43,4 1,3 (1,1-1,6) Não 371(40,6) 32,6 1,0 Atividade física 0,167 Sim 287(31,2) 35,5 1,0 Não 632(68,8) 40,4 1,1 (0,9-1,4) *Qui-quadrado Um estudo feito em São Paulo22 evidenciou prevalência de 21,3% de osteoporose em mulheres menopausadas com idade superior a 50 anos. Recentemente, uma pesquisa23 em São Paulo com idosos com idade de 80 anos ou mais mostrou prevalência de artrite/artrose de 26 e 33%, alguma doença na coluna com 12 e 19%, e osteopenia/osteoporose de 71 e 49% em homens e mulheres, respectivamente. que referiram dor (43,4%), quedas (39,3%) e naqueles que não praticavam atividade física (40,4%). Foram associadas às DME: autoavaliação do estado de saúde regular (RP=1,4; IC95% 1,2‑1,7) e ruim/muito ruim (RP=1,8; IC95% 1,4-2,3), além de relato de dor (RP=1,3; IC95% 1,1-1,6). que referiram dor (43,4%), quedas (39,3%) e naqueles que não praticavam atividade física (40,4%). Foram associadas às DME: autoavaliação do estado de saúde regular (RP=1,4; IC95% 1,2‑1,7) e ruim/muito ruim (RP=1,8; IC95% 1,4-2,3), além de relato de dor (RP=1,3; IC95% 1,1-1,6). ▄ ▄RESULTADOS Todas as respostas referentes às DME foram agrupadas de acordo com a Classificação Internacional de Doenças (CID) em uma única variável dependente: DME.i Figura 1. Prevalência de doenças musculoesqueléticas conforme sexo em idosos do município de Goiânia, Goiás, em 2010 (n=934) As variáveis de exposição foram demográficas: (sexo e faixa etária), quedas, dor autorreferida e autoavaliação do estado de saúde. A ocorrência de quedas foi avaliada por meio da pergunta: “O(a) Sr.(a) caiu no último ano?”. Para autoavaliar sua saúde, os idosos responderam à seguinte questão: “O que o(a) Sr.(a) acha do seu estado de saúde no último mês?”. As opções de resposta foram categorizadas em: bom/muito bom; regular; ruim/muito ruim. A dor existente há seis meses ou mais foi avaliada pelo autorrelato. Os dados foram digitados no programa Excel for Windows 2003-2007 e analisados no software Stata, versão 11.0. As variáveis foram primeiramente analisadas de forma descritiva por meio de frequência absoluta, relativa, média e desvio-padrão. Para a análise de associação entre as variáveis independentes e o desfecho, foi utilizado o teste qui quadrado ou Fisher, adotando‑se nível de significância de 5%. A magnitude da associação foi estimada pela razão de prevalência (RP), considerando-se nível de significância de 5% (p<0,05). Figura 1. Prevalência de doenças musculoesqueléticas conforme sexo em idosos do município de Goiânia, Goiás, em 2010 (n=934) Tabela 1. Prevalência de doenças musculoesqueléticas (DME) autorreferidas por idosos do município de Goiânia, Goiás, em 2010 (n=934) ( ) DME n (%) IC95% Osteoporose 229 (24,6) 21,8-27,5 Artrose 25 (2,7) 1,7-3,9 Reumatismo 11(1,2) 0,5-2,1 Artrite 6 (0,6) 0,2-1,4 Outros 94 (9,8) 8,2-12,2 A pesquisa foi aprovada pelo Comitê de Ética em Pesquisa da Universidade Federal de Goiás sob o protocolo n° 050/2009, Cad. Saúde Colet., 2017, Rio de Janeiro, 25 (2): 138-143 140 Doenças musculoesqueléticas, dor e autoavaliação de saúde em idosos Tabela 2. ▄ ▄DISCUSSÃO A maior predominância de DME em mulheres neste estudo se justifica, primeiramente, ao fato da maior expectativa de vida para o sexo feminino, o que faz com que apresentem maior prevalência de doenças em relação aos homens7. Estudos prévios realizados no Brasil22,24,25, no Paquistão26 e no Irã27 mostraram que a prevalência de osteoporose nas mulheres é quase o dobro em relação aos homens. Outra justificativa é porque as mulheres apresentam maior velocidade de perda óssea após a menopausa devido à diminuição hormonal significativa nos níveis de estrogênio9. A prevalência total de DME nos idosos deste estudo foi elevada (39,1%), com destaque para osteoporose (24,6%). Prevalência inferior foi observada no Canadá4, com 18,5% de idosos com artrite, 20,5% com problemas na coluna e 30% referindo alguma DME em geral. Quanto à prevalência de osteoporose, resultados inferiores foram encontrados na China7 (15,7%) e na Noruega19 (19,6%), mas semelhantes em Portugal8 (25,3%). Já no Brasil20, a Pesquisa Nacional por amostra de Domicílio (PNAD) identificou prevalência decrescente de artrite, com 37,5, 27,3 e 24,2%, respectivamente, nos anos de 1998, 2003 e 2008. Houve uma maior prevalência de DME na faixa etária mais idosa. Esse achado era esperado porque os tecidos musculoesqueléticos apresentam, com o envelhecimento, crescente fragilidade óssea, dano às estruturas cartilaginosas, redução da elasticidade dos ligamentos, perda de força muscular e infiltração gordurosa nos tecidos, condições que em faixas etárias avançadas estão Outro estudo nacional11 apontou estimativas de prevalência de DME de 9 a 40% na população idosa. Já em uma pesquisa de base populacional realizada no Rio Grande do Sul21, a artrite foi relatada por 43,1% dos idosos. Observa-se elevada prevalência de DME em estudos brasileiros quando comparados aos internacionais. Cad. Saúde Colet., 2017, Rio de Janeiro, 25 (2): 138-143 141 Anna Cássia Fernandes Melo, Adélia Yaeko Kyosen Nakatani, Lilian Varanda Pereira, Ruth Losada de Menezes, Valéria Pagotto intensificadas e podem levar à DME1-3,12. Entretanto, um estudo de base populacional realizado em Portugal8 evidenciou uma associação entre reumatismo e DME maior em idosos jovens, entre 60 e 70 anos, sugerindo que há uma tendência de aumento de DME já nessa faixa etária. fatores, sexo feminino (RP=2,4; 1,5-3,9), idade maior ou igual a 80 anos (RP=2,5; 1,6-3,9), reumatismo/artrite/artrose (RP=1,6; 1-2,5) e osteoporose (RP=1,7; 1,2-2,5) são preditores de queda. ▄ ▄DISCUSSÃO Embora neste estudo não tenha sido significativa a associação com presença de DME, algumas pesquisas6,34 apontam a atividade física como fator protetor tanto na prevenção quanto na morbidade provocada por DME, principalmente incapacidade e dor. Em um estudo prospectivo envolvendo idosos com alguma DME6, exercícios de fisioterapia implicaram na redução de dor autorrelatada e melhora gradual do desempenho. A presença de DME foi associada a uma autoavaliação de saúde ruim/muito ruim. A autoavaliação do estado de saúde é um indicador subjetivo da percepção do indivíduo sobre a própria saúde, que expressa aspectos físicos, emocionais, de bem-estar e de satisfação com a própria vida28. Essa associação foi também encontrada em outro estudo desenvolvido em Goiânia com idosos usuários do SUS29. Tanto a presença de uma DME, como os seus sintomas podem trazer limitações e falta de autonomia para o idoso executar suas tarefas, o que, por consequência, pode gerar uma percepção negativa de sua saúde geral29,30. Pode-se destacar como limitações deste estudo o tipo de delineamento transversal, que impossibilita inferir causalidade, não sendo possível afirmar em qual momento do tempo ocorreram a exposição e o desfecho. Outra limitação refere-se à natureza autorreferida da presença de DME. Entretanto, os entrevistadores foram treinados para garantir a qualidade e a fidedignidade das informações aqui apresentadas. A associação entre dor e DME é um evento esperado, uma vez que a dor é uma das principais queixas das pessoas com essas doenças, o que é coerente em estudos prévios11,12. Um trabalho prospectivo observacional31 identificou que a dor foi proporcionalmente aumentada conforme a idade e a perda da mobilidade por DME autorrelatadas. Neste estudo, não foi abordado o controle da dor nem a adesão a medicamentos, porém, outra pesquisa com idosos em um centro de tratamento medicamentoso para dor crônica32 identificou que a maioria dos agentes causadores da dor crônica era as DME. Além disso, outro estudo33 revelou ser frequente na população idosa com alguma DME a automedicação para o controle dos sintomas. Os resultados desta pesquisa demonstraram elevada prevalência de idosas com DME, sendo a osteoporose a mais prevalente. O autorrelato de dor e a autoavaliação negativa da saúde foram associadas à ocorrência de DME e sinalizam a necessidade de monitoramento dessas condições, uma vez que podem interferir na limitação funcional do idoso devido à diminuição da mobilidade. ▄ ▄AGRADECIMENTOS Os autores gostariam de agradecer o apoio financeiro da Fundação de Amparo à Pesquisa no Estado de Goiás (FAPEG) e o auxílio logístico da Secretaria Municipal de Saúde (SMS), sem os quais não seria possível dar prosseguimento a este estudo. ▄ ▄DISCUSSÃO Considerando o aumento da expectativa de vida na atualidade e que as DME são prevalentes, recomenda-se que ações de prevenção sejam reforçadas na atenção primária à saúde, nível de atenção em que os idosos têm maior acesso aos profissionais de saúde. Espera-se também que estes dados contribuam para subsidiar políticas e ações de saúde locais e possam ser utilizados na implantação de projetos e no aumento de informação para adoção de atitudes preventivas. As DME associadas à dor também podem interferir na locomoção desses idosos, propiciando desequilíbrio, restrição de mobilidade, e, consequentemente, aumentando o risco de queda12. Esse processo é atribuído às perdas progressivas de equilíbrio e de alterações na massa muscular e óssea, que ocorrem com o processo de envelhecimento. Um estudo de base populacional2 avaliou a ocorrência de quedas em 1.520 idosos por 12 meses. Embora a prevalência geral tenha sido menor do que neste estudo (6,5%), evidenciou-se que, entre outros 6. Fritz JM, Hunter SJ, Tracy DM, Brennan GP. Utilization and clinical outcomes of outpatient physical therapy for medicare beneficiaries with musculoskeletal conditions. Phys Ther. 2011;91(3):330-45. PMid:21233306. http://dx.doi.org/10.2522/ptj.20090290. Cad. Saúde Colet., 2017, Rio de Janeiro, 25 (2): 138-143 142 5. Slater M, Perruccio AV, Badley EM. 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EpiReumaPt: the study of rheumatic and musculoskeletal diseases Cad. Saúde Colet., 2017, Rio de Janeiro, 25 (2): 138-143 142 Doenças musculoesqueléticas, dor e autoavaliação de saúde em idosos in Portugal: a detailed view of the methodology. Acta Reumatol Port. 2015;40(2):110-24. PMid:26219965. in Portugal: a detailed view of the methodology. Acta Reumatol Port. 2015;40(2):110-24. PMid:26219965. 22. Baccaro LF, Machado VSS, Costa-Paiva L, Sousa MH, Osis MJ, Pinto- Neto AM. ▄ ▄REFERÊNCIAS Quedas intra-hospitalares na Santa Casa de Belo Horizonte MG são adequadamente relatadas? Fisioterapia e Pesquisa. 2011;18(1):72-8. http://dx.doi.org/10.1590/S1809- 29502011000100013. 29. Pagotto V, Nakatani AYK, Silveira EA. Fatores associados à autoavaliação de saúde ruim em idosos usuários do Sistema Único de Saúde. Cad Saude Publica. 2011;27(8):1593-602. PMid:21877007. http://dx.doi.org/10.1590/ S0102-311X2011000800014. 16. Tomita Y, Arima K, Kanagae M, Okabe T, Mizukami S, Nishimura T, et al. 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Pimentel WRT, Pagotto V, Nakatani AYK, Pereira LV, Menezes RL. Quedas e qualidade de vida: associação com aspectos emocionais em idosos comunitários. Geriatr Gerontol Aging. 2015;9(2):42-8. http://dx.doi. org/10.5327/Z2447-2115201500020002. 32. Markotic F, Cerni Obrdalj E, Zalihic A, Pehar R, Hadziosmanovic Z, Pivic G, et al. Adherence to pharmacological treatment of chronic nonmalignant pain in individuals aged 65 and older. Pain Med. 2013;14(2):247-56. PMid:23368967. http://dx.doi.org/10.1111/pme.12035. 19. Leiknes KA, Finset A, Moum T. Commonalities and differences between the diagnostic groups: Current somatoform disorders, anxiety and/or depression, and musculoskeletal disorders. J Psychosom Res. 2010;68(5):439- 46. PMid:20403502. http://dx.doi.org/10.1016/j.jpsychores.2010.02.003. 33. Driban JB, Boehret SA, Balasubramanian E, Cattano NM, Glutting J, Sitler MR. Medication and supplement use for managing joint symptoms among patients with knee and hip osteoarthritis: a cross-sectional study. BMC Musculoskelet Disord. 2012;13(1):47. PMid:22458305. http://dx.doi. org/10.1186/1471-2474-13-47. 20. Lima-Costa MF, Matos DL, Camargos VP, Macinko J. Tendências em dez anos das condições de saúde de idosos brasileiros: evidências da Pesquisa Nacional por Amostra de Domicílios (1998, 2003, 2008). Cien Saude Colet. 2011;16(9):3689-96. PMid:21987313. http://dx.doi.org/10.1590/ S1413-81232011001000006. 34. Cawthon PM, Blackwell TL, Cauley JA, Ensrud KE, Dam T, Harrison SL, et al. Objective assessment of activity, energy expenditure, and functional limitations in older men: the osteoporotic fractures in men study. J Gerontol A Biol Sci Med Sci. 2013;68(12):1518-24. PMid:23682162. http://dx.doi. org/10.1093/gerona/glt054. 21. ▄ ▄REFERÊNCIAS Blay SL, Fillenbaum GG, Andreoli SB, Gastal FL. Prevalence and concomitants of arthritis in the elderly in Rio Grande do Sul, Brazil. PLoS One. 2012;7(9):1- 7. PMid:23028995. http://dx.doi.org/10.1371/journal.pone.0045418. Recebido em: Nov. 01, 2016 Aprovado em: Mar. 30, 2017 Recebido em: Nov. 01, 2016 Aprovado em: Mar. 30, 2017 Cad. Saúde Colet., 2017, Rio de Janeiro, 25 (2): 138-143 143
W3169377977.txt	https://quantum-journal.org/papers/q-2021-06-04-466/pdf/	en		Analyzing the barren plateau phenomenon in training quantum neural networks with the ZX-calculus	Quantum	2,021	cc-by	19,013	Analyzing the barren plateau phenomenon in training quantum neural networks with the ZX-calculus Chen Zhao1,2 and Xiao-Shan Gao1,2 1 Academy arXiv:2102.01828v2 [quant-ph] 30 May 2021 2 University of Mathematics and Systems Science, Chinese Academy of Sciences of Chinese Academy of Sciences In this paper, we propose a general scheme to analyze the gradient vanishing phenomenon, also known as the barren plateau phenomenon, in training quantum neural networks with the ZX-calculus. More precisely, we extend the barren plateaus theorem from unitary 2-design circuits to any parameterized quantum circuits under certain reasonable assumptions. The main technical contribution of this paper is representing certain integrations as ZX-diagrams and computing them with the ZX-calculus. The method is used to analyze four concrete quantum neural networks with different structures. It is shown that, for the hardware efficient ansatz and the MPS-inspired ansatz, there exist barren plateaus, while for the QCNN ansatz and the tree tensor network ansatz, there exists no barren plateau. 1 Introduction In recent years, hybrid quantum-classical algorithms are widely used in quantum chemistry [1–4], combinatorial optimization [5, 6], and quantum machine learning [7–12]. In these hybrid quantumclassical algorithms, the goal is usually training parameterized quantum circuits (PQCs) with classical optimizers. The PQC will be applied to an initial state and then the state will be measured on a quantum device. The classical optimizer will update the parameters of the PQC according to the measurement results. As the PQC can be run on noisy intermediate-scale quantum (NISQ [13]) devices, these algorithms are regarded as near-term practical quantum algorithms with potential quantum advantages. There exist many methods to train PQCs. Some of these are gradient-based [14–17] and some are not [17, 18]. In quantum machine learning, gradient-based methods are widely used. When using gradient-based methods to train PQCs, one may suffer from the barren plateau (BP) phenomenon which was first studied in [19]. The BP phenomenon is that the gradient of parameters of the PQC will vanish exponentially in terms of the system size. It was proved that if the PQCs form unitary 2-designs, then the BP phenomenon exists [19]. This result has been extended to the case when the PQCs form approximately 2-designs in [20]. The BP phenomenon in PQCs of various structures has been proposed. For PQCs with a brick-like structure, if the PQC has locally 2-design, then the existence of BPs depends on the depth of the circuit and the cost-function [21]. Let n be the number of qubits of the PQC. For poly(n)-depth PQCs with a brick-like form, there always exist BPs. For log(n)-depth PQCs with a brick-like form, if the cost-function is global, there exist BPs. Otherwise, there exists no BP when the cost-function is local. Too much entanglement will induce BPs [22, 23]. The BP phenomenon in dissipative quantum neural networks has been studied in [24]. And the noise from quantum hardware also causes BPs, which are called noiseinduced BPs [25]. Several methods to avoid BPs have been proposed [20, 23, 26, 27]. Chen Zhao: zhaochen17@mails.ucas.ac.cn Xiao-Shan Gao: xgao@mmrc.iss.ac.cn Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 1 The above results about the BP phenomenon are obtained under certain assumptions of unitary 2-design and it is still difficult to analyze the BP phenomenon for PQCs besides those containing t-design parts. In this paper, we develop a general scheme to analyze whether there exist BP phenomena when training a concrete PQC. We focus on BP phenomena induced by the structure of PQCs and noise-induced BPs are not considered in this paper. The most important tool used in this paper is the ZX-calculus, a graphical language for describing and reasoning about quantum processes. The ZX-calculus was developed by Coecke and Duncan in [28, 29], which has various applications including quantum circuit synthesis [30–33], measurement-based quantum computing [34, 35], quantum error correction [36, 37], condensed matter physics [38], quantum machine learning [39], and quantum natural language processing [40]. In the ZX-calculus, the objects under consideration are ZX-diagrams, which consist of two kinds of tensors: Z-spiders and X-spiders. A ZX-diagram can be rewritten with ZX-calculus rules. Moreover, every quantum circuit can be converted into a ZX-diagram. ~ with Let θ~ = (θ1 , . . . , θm ) be a set of parameters. To analyze the gradient of a PQC U (θ) respect to a Hamiltonian H, we need to estimate the following expectation and the variance ∂ hHi ∂ hHi , Var (1) E ∂θj ∂θj where hHi is defined in Eq. (3). It will be shown that the expectation in Eq. (1) is always zero. The PQC is said to have barren plateaus if the variance in Eq. (1) vanishes exponentially in terms of the size of the PQC. The PQC is said to have no barren plateau or trainable if the variance in Eq. (1) vanishes polynomially in terms of the size of the PQC. To estimate the expectation and variance in Eq. (1), we first represent them as ZX-diagrams. Since the expectation and the variance are integrations, the main technical contribution of this paper is representing these integrations as ZX-diagrams and computing them with the ZX-calculus when the PQC satisfies Assumption 1. More precisely, with the rewriting rules in the ZX-calculus, we prove that Eq. (1) is equal to the contraction of a tensor network with a similar structure as the PQC. Hence, the existence of BPs is totally characterized by the scaling property of the tensor network. We use these techniques to analyze whether there exist BP phenomena in the hardware-efficient ansatz [2], the QCNN ansatz [41], the tree tensor network ansatz [42], and the MPS-inspired ansatz [43]. We show that there exist BPs in hardware-efficient ansatz and MPS-inspired ansatz, and there exists no BP in the QCNN ansatz and the tree tensor network ansatz. This paper is organized as follows. A brief introduction to the PQC, the BP phenomenon, and the ZX-calculus will be given in Section 2. We will prove the main result that characterizes Eq. (1) in Section 3. And the analysis of four concrete PQCs is given in Section 4. 2 Preliminary 2.1 Hybrid quantum-classical algorithms In a hybrid quantum-classical algorithm, there will be an ansatz, which is a PQC of the form ~ = U (θ) M Y [Uj (θj ) · Vj ]. (2) j=1 In (2), Uj (θj ), j = 1, . . . , M are parameterized gates, such as the rotational gates RX , RY , RZ ; and Vj are non-parameterized gates, such as the Hadamard gate H and the CNOT gate. The PQC will be applied to an initial state ρ0 and then the state will be measured. The above procedure, which is the quantum part of the algorithm, will be run on quantum processors. Meanwhile, there will be a classical part that consists of classical processors to optimize the parameters of the PQC ~ will be estimated in the classical part based on the in the quantum part. A cost-function L(θ) Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 2 measurement results. Usually, the expectation ~ ~ hHi = Tr ρ0 U † (θ)HU (θ) (3) of a given Hamiltonian H will be regarded as the cost-function in many tasks. Quantum processor ρ0 ~ U (θ) Classical processor ~ L(θ) H measure Optimizer Update parameters Figure 1: The hybrid quantum-classical algorithm As demonstrated in Figure 1, the quantum part runs the PQC and obtains the measurement results and the classical part estimates the cost-function and updates the parameters. After several iterations, the cost-function may converge and be optimized. Then the training will be stopped. This is the main idea of the hybrid quantum-classical algorithm. 2.2 Barren plateau phenomenon When the parameterized gates are of the form θ Uj (θj ) = e−i 2 Hj , where Hj satisfies Hj2 = I, the gradient ∂hHi ∂θj can be estimated by the parameter shifting rule without changing the structure of the PQC [14]. Once we obtain the gradient, we can use gradientbased optimization methods, such as gradient descent, to optimize the parameters. Ideally, if the gradient does not vanish too fast as the size of the PQC grows, then the gradient could be estimated efficiently and the PQC could be trained easily. However, the BP phenomenon tells us that in many cases, the gradient vanishes exponentially as the system size grows up. When this happens, the PQC is difficult to be trained. The first rigorous proof of the BP phenomenon is shown below. ~ = V (θM , . . . , θj+1 )U (θj )W (θj−1 , . . . , θ1 ) and a HamilTheorem 1 ([19]). Consider a PQC U (θ) tonian h H. iThe expectation of gradient is 0 if V and W are 1-design. And the variance of gradient Var ∂hHi vanishes exponentially in terms of the number of qubits if V or W is 2-design. ∂θj Hence, when designing the ansatz PQC for a hybrid quantum-classical algorithm, we should analyze whether there exist BP phenomena in it to ensure that it is trainable. 2.3 The ZX-calculus We provide a brief introduction to the ZX-calculus. For more details, please refer to [44, 45]. In the ZX-calculus, quantum states and their transformations are represented as ZX-diagrams which consist of two kinds of tensors: Z-spiders and X-spiders. A Z-spider is denoted as a green node, and an X-spider is denoted as a red node. They can be written explicitly in the Dirac Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 3 notation as follows. ( ) .. . m θ .. . n := \|0 . . . 0i h0 . . . 0\| +eiθ \|1 . . . 1i h1 . . . 1\| \| {z } \| {z } \| {z } \| {z } n ( n (4) m ) .. . m m θ .. . n := \|+ · · · +i h+ · · · +\| +eiθ \|− · · · −i h− · · · −\| \| {z } \| {z } \| {z } \| {z } n m n m For a spider, the edges on the left-hand side are called input and the edges on the right-hand side are called output. The angle θ is called the phase of the spider. For simplicity, we will omit the phase when it is zero. Spiders can be connected with wires. Hence, ZX-diagrams can be regarded as tensor networks generated with Z-spiders and X-spiders. For example, we can use ZX-diagrams to represent the following quantum states and quantum gates. = \|0i + \|1i = √ θ = RZ (θ) θ = RX (θ) π 2 := 2 \|+i √ = \|+i + \|−i = 2 \|0i = = π 2 √1 2 √1 2 π 2 = H (5) + Z Here we introduce a new notation, the yellow box, to represent the Hadamard gate 1 1 1 H=√ . 2 1 −1 Since the gates set {RZ , RX , H, CNOT} is universal for quantum computing, in principle, one can convert every quantum circuit to a ZX-diagram with the equations in Eq. (5). = α ... = ... ... α ... α+β ... (h) ... = β ... (i1) (f ) ... ... α ... ... Moreover, the ZX-calculus is a powerful tool for reasoning. There are several rewriting rules in the ZX-calculus with which one can rewrite a ZX-diagram to another equivalent form. Figure 2 gives some basic rewriting rules1 in the ZX-calculus. Here, two ZX-diagrams A, B are said to be equivalent if and only if there exists a non-zero constant c ∈ C, such that [A] = c · [B], where [A] is the matrix corresponding to the ZX-diagram A. (i2) = −α α (c) (b) = = ... = ... α ... π ... π (π) π Figure 2: Some basic rewriting rules in the ZX-calculus. Here, “. . . ” means 0 or more (figure from [30]). Note that the ZX-calculus is universal. It means that any linear transformations can be represented as ZX-diagrams. Moreover, the rules in Figure 2 are complete for the stabilizer quantum mechanics where phases can only be multiples of π2 [46, 47]. That is, if two ZX-diagrams are equivalent, then there exists a set of rewriting rules in Figure 2 that rewrites one into another. 1 (f ) is for the “fusion” rule; (h) is for the “Hadamard color changing” rule; (i1) and (i2) are “identity” rules; (π) is for the “π-copy” rule; (c) is for the “copy” rule; (b) is for the “bi-algebra” rule. Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 4 There are also completeness results for the Clifford+T quantum mechanics, where phases can be multiples of π4 , and for arbitrary ZX-diagrams [48–52]. In this paper, we will focus on a canonical form of the ZX-diagram, the graph-like ZX-diagram which is defined in [30]. Definition 1 ([30]). A ZX-diagram is called graph-like if 1. All spiders are Z-spiders. 2. Z-spiders are only connected via Hadamard edges. 3. There exist no parallel Hadamard edges or self-loops. 4. Every input or output is connected to a Z-spider and every Z-spider is connected to at most one input or output. Two spiders being connected via a Hadamard edge means that they are connected with a Hadamard box. Alternatively, we will also use the dashed blue edge to represent a Hadamard edge. .. . α β .. . := .. . α β .. . All X-spiders can be rewritten to Z-spiders by using the rule (h) in Figure 2. Connected Hadamard boxes can be canceled with the rule (i2) and normal edges can be canceled with the rule (f ). Furthermore, parallel Hadamard edges and self-loops can be canceled with rules2 in Figure 3. Hence, every ZX-diagram is equivalent to a graph-like ZX-diagram [30]. .. . α β .. . (hopf ) = .. . α β .. . (hsl) α ... = (sl) α+π ... α ... = α ... Figure 3: Rules for canceling parallel edges and self-loops [30]. 3 Analyzing the BP phenomenon with the ZX-calculus In this section, we will show how to analyze the BP phenomenon with the ZX-calculus. More precisely, we will show how to estimate the expectation and the variance of the gradient of the cost function of a PQC with respect to a Hamiltonian with the ZX-calculus. The main technique we used is to compute integration over unitarians with the ZX-calculus. Scalars are ignored in the rules in Section 2.3. However, to consider the BP phenomenon, the scalar is necessary. Hence, by using the definition of the Z-spider and the X-spider in Eq. 4, we can obtain the precise rules with scalars in Figure 4. In this paper, we consider PQCs under the following assumptions. ~ satisfies Assumption 1. The PQC U (θ) 1. Each gate in U is one of {RX , RZ , H, CNOT}. 2. The parameters in θ~ = (θ1 , . . . , θm ) are independent uniform random variables in the interval [−π, π]. 2 (hopf ) is for the “Hopf” rule; (sl) is for the “self-loop canceling” rule; (hsl) is for the “Hadamard self-loop canceling” rule. Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 5 = ... α ... ... ... α+β α ... ... ... = β = (h) ... ... (i1) (f ) ... ... α (i2) = −α ) 2n−1 α ... = eiα ... α ... π √ ) (c) = n n ... π (π) π (b) = √ .. . 2 α (sl) = α ... .. . β (hopf ) = (hsl) = α α ... ... 1 2 .. . α β .. . √1 α + π 2 ... Figure 4: Rewriting rules with scalars. We remark that in the case that a quantum circuit contains gates not satisfying Assumption 1, if one can represent these gates as composition of gates which satisfy Assumption 1, then the results of this paper still hold. For example, we will first represent RY using RZ and RX in Section 4.2. 3.1 Representing gradients as ZX-diagrams ~ of n-qubits and a Hamiltonian H. We assume that the input state is pure. Consider a PQC U (θ) Without loss of generality, we can also assume that we apply this PQC to an initial state \|0i. Then the expectation of H can be expressed as ~ ~ \|0i . hHi = h0\| U † (θ)HU (θ) (6) ~ to a parameterized graph-like ZXAs shown in Section 2.3, we can convert the PQC U (θ) ~ diagram GU (θ) with Eq. (5). Suppose that ~ = c · [GU (θ)] ~ U (θ) for a constant c, then hHi can also be expressed as a ZX-diagram as demonstrated in the following ~ is under the Assumption 1. equation. Here, U (θ) hHi = \|c\|2 2n · .. . ... ... .. . . . . .. . ... ~ GU (θ) H .. . ... ... .. . ... .. . .. . (7) ~ GU † (−θ) We remark that in the general case, the input state can be a mixed state ρ. Because the ZX-calculus is universal, we can represent ρ as a ZX-diagram Dρ . Then by replacing the X-spiders representing zero states on the left- and right-hand sides in Eq. (7) with Dρ , we can still obtain a ZX-diagram representing the expectation hHi = Tr(ρU † HU ). And results in this paper still hold in this case. If we expand the spider by the definition of the Z-spider, we can prove that the gradient can be represented as a ZX-diagram. Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. ∂hHi ∂θj 6 Theorem 2. The gradient can be represented as the following equation. n 2 \|c\|2 · ∂hHi ∂θj = ∂ ∂θj .. . = .. . .. . .. . ... ... ... ... .. . . . . θj ... ... .. .. . . ... ... ... ... .. . . .. ... ... .. .. . . θj + .. . .. . H .. . .. . H π 2 ... ... ... ... .. . . . . −θj ... ... .. .. . . ... ... ... ... .. . . .. ... ... .. .. . . −θj − π .. . .. . .. . .. . π 2 Proof. We expand the corresponding Z-spiders according to the definition. There will be four terms on the left-hand side. Two of these terms are constants, and thus they will become 0 after taking the derivative. According to the definition of the X-spider, we can obtain the ZX-diagram on the right-hand side. The complete proof is given in Appendix B. This theorem also gives a graphical proof of the parameter-shift rule in [14]. We will demonstrate it with the following example. Consider the following ansatz. We first can convert it to an equivalent ZX-diagram. \|0i RX (θ1 ) √1 2 √1 2 RZ (θ3 ) = \|0i RX (θ2 ) + RZ (θ4 ) θ1 θ3 θ2 θ4 (f ), (h) = √1 θ1 2 θ3 √1 θ2 2 θ4 And the expectation hHi of a Hamiltonian H can be represented as the following ZX-diagram. \|0i RX (θ1 ) RZ (θ3 ) hHi = RZ (−θ3 ) h0\| RX (−θ1 ) = H \|0i RX (θ2 ) + RZ (θ4 ) RZ (−θ4 ) + h0\| RX (−θ2 ) √1 θ1 2 θ3 √1 θ1 2 θ4 −θ3 1 −θ1 √ −θ4 1 −θ2 √ H 2 2 Then by Theorem 2, we can obtain the gradient directly. π ∂ hHi = ∂θ1 Accepted in √1 θ1 + 2 √1 2 θ2 π 2 θ3 −θ3 −θ1 − −θ4 −θ2 π 2 H θ4 Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. √1 2 √1 2 7 Then we can use the definition of the X-spider to obtain the parameter-shift rule as shown below. π √1 θ1 + 2 √1 2 π 2 θ3 −θ3 −θ1 − −θ4 −θ2 √1 2 π 2 H θ2 θ4 √1 2 \|+i h+\| − \|−i h−\| = = √1 θ1 + 2 √1 2 θ4 √1 2 π 2 √1 2 −θ3 −θ1 − −θ4 −θ2 √1 2 π 2 √1 2 √1 2 θ3 θ2 π 2 √1 2 π −θ3 −θ1 − −θ4 −θ2 √1 2 π 2 H θ4 √1 θ1 + 2 (f ) 1 = 2 = θ2 θ3 H θ2 √1 θ1 + 2 √1 2 π 2 θ3 √1 2 −θ3 −θ1 − −θ4 −θ2 H θ4 π 2 √1 2 √1 2 − √1 θ1 + 2 √1 2 π 2 √1 2 θ3 θ4 θ2 −θ3 −θ1 − −θ4 −θ2 √1 2 π 2 H θ2 √1 θ1 − 2 − √1 π 2 π 2 θ3 √1 2 −θ3 −θ1 + −θ4 −θ2 H θ4 π 2 √1 2 √1 2 1 hHiθ1 ,+ − hHiθ1 ,− 2 Here hHiθ1 ,± means replacing the parameter θ1 with θ1 ± π 2 in the ansatz. To analyze the BP phenomenon, we need to compute the expectation Z ∂ hHi ~ ~ ∂ hHi dθ, E( ) = p(θ) ∂θj ∂θj ~ θ (8) and the variance Var( 2 Z 2 ∂ hHi ∂ hHi ∂ hHi ~ ∂ hHi ) − E( ) = E( ) = p(θ) ∂θj ∂θj ∂θj ∂θj ~ θ 2 2 ∂ hHi dθ~ − E( ) , ∂θj (9) ~ is the probability of the parameters θ. ~ for j = 1, . . . , m. Here p(θ) By Assumption 1, Eq. (8) and Eq. (9) can be written as Z Z ∂ hHi ∂ hHi 1 E( ··· )= dθ1 . . . dθm , ∂θj (2π)m θ1 θm ∂θj and ∂ hHi 1 Var( )= ∂θj (2π)m Z Z ··· θ1 θm ∂ hHi ∂θj 2 ∂ hHi dθ1 . . . dθm − E( ) ∂θj (10) 2 , (11) for j = 1, . . . , m. We will compute the expectation and variance of the gradients in the next two sections. 3.2 The expectation of gradients In this section, we will compute the expectation in Eq. (10). As shown in Theorem 2, the integration Z ∂ hHi 1 dθk , 2π θk ∂θj for k = 1, . . . , m, is also an integration of a ZX-diagram over its parameter θk . With the following lemma, the integration can be represented as a ZX-diagram again. Lemma 1. The following equation holds. Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 8 1 2π Z m .. . α α ... ... .. n . . n .. . . . . .. −α ... ... .. n . . n .. . . . . .. .. m dα = m .. . . .. m . Proof. Expand the Z-spiders according to its definition. There will be four terms on the left-hand side. By using Z π 1 eiα dα = 0, 2π −π only two terms left. We will use the definition of the Z-spider again to obtain the ZX-diagram on the right-hand side. The complete proof is given in Appendix C. With this lemma, we give a graphical proof of the following theorem, which is also proved in [20]. Theorem 3. Under Assumption 1, the integration Z ∂ hHi 1 dθj = 0. 2π θj ∂θj Proof. Using the relation in Lemma 1 on the ZX-diagram in Theorem 2, we have n 2 1 · \|c\|2 2π Z θj ∂ hHi dθj = ∂θj .. . .. . ... ... ... ... .. . . .. ... ... .. .. . . .. . H .. . ... ... ... ... .. . . .. ... ... .. .. . . ... ... ... ... .. . . .. ... ... .. .. . . ... ... ... ... .. . . .. ... ... .. .. . . .. . .. . .. . .. . .. . .. . π = .. . .. . ... ... ... ... .. . . .. ... ... .. .. . . .. . H .. . π = .. . .. . ... ... ... ... .. . . .. ... ... .. .. . . .. . H .. . 1 π 2 = 0. As a corollary, the expectation of the gradient in Eq. (10) is zero. Corollary 1. Under Assumption 1, the expectation Z Z ∂ hHi 1 ∂ hHi E( )= · · · dθ1 . . . dθm = 0, for j = 1, . . . , m. ∂θj (2π)m θ1 θm ∂θj Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 9 3.3 The variance of gradients In this section, we will compute the variance in Eq. (9). Because the gradient ∂hHi ∂θj is a real number and the expectation is 0, by Eq. (9), the variance 2 ∂hHi is the expectation of ∂θj , which can be represented as follows by Theorem 2. .. . 4n \|c\|2 · h ∂hHi ∂θj i2 .. . ... ... ... ... .. . . .. θj + = ... ... .. .. . . ... .. . .. . ... ... ... ... π 2 ... .. . ... ... .. . H π 2 −θj − .. . .. . ... ... ... ... .. . . .. ... ... .. .. . . π −θj − π θj + .. . H .. . π 2 (12) π 2 ... .. . .. . .. . ... ... ... ... ... .. . ... ... .. . .. . Similar to Lemma 1, we can prove the following lemma. Lemma 2. The following equation holds. 1 2π Z m ... α α m .. . −α ... ... .. n . . n .. . . . . .. ... ... .. n . . n .. . . . . .. −α .. m . α .. m . m .. . ... ... ... n . . . n ... ... m .. . ... ... .. n . . n .. . . . . .. .. m . dα = m .. . ... ... ... n . . . n ... ... m .. . ... ... .. n . . n .. . . . . .. + .. m . m .. . .. m . + π ... ... .. n . . n .. . . . . .. .. m . π .. m . m .. . ... ... .. n . . n .. . . . . .. .. m . Proof. We expand all Z-spiders and there will be 16 terms on the left-hand side. Again, using the relation Z π 1 eiα dα = 0, 2π −π 6 terms was left. We can use the definition of the Z-spider again to obtain the right-hand side. The complete proof is given in Appendix D. There exist three terms after integration. Hence, computing the variance of gradients is much more complicated than computing the expectation. We denote the three ZX-diagrams in Lemma 2 as T1 = , T2 = π , T3 = π . (13) And we introduce a new notation VUa1 ,...,am , aj ∈ {T1 , T2 , T3 }, Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 10 to represent the following ZX-diagram. the spider corresponding to θm the spider corresponding to θ1 .. . ... ... ... ... .. . . .. .. . ... ... ... ... .. .. .. . . . ... ... .. .. . . .. . .. . GU VUa1 ,...,am ... .. . .. . ... ... ... ... .. . ... .. .. . . ... ... .. . ... ... .. .. . . .. . .. . (14) .. . ... am .. . ... ... ... ... .. .. .. . . . .. . a1 = .. . ... .. . ... ... ... ... .. . H ... ... ... ... .. . . .. .. . H .. . ... ... ... ... ... .. .. . . ... ... ... ... ... .. . ... ... .. . .. . Here, U (θ1 , . . . , θm ) is a PQC with m parameters and GU is the graph-like ZX-diagram corresponding to U . With this notation, we have the following theorem. Theorem 4. Under Assumption 1, the following equation holds. Var ∂ hHi ∂θj = \|c\|2 · 4n X a ,...,aj−1 ,T2 ,aj+1 ,...,am VU 1 . ak ∈{T1 ,T2 ,T3 }, k6=j Proof. By Eq. (12) and Lemma 2, we have the following equation. .. . 4n 1 · 2 \|c\| 2π Z θj ∂ hHi ∂θj ... ... .. .. . . .. . H .. . ... ... ... ... .. . . .. ... ... .. .. . . . . .. .. ... ... ... ... ... . . . ... ... .. . .. . .. . .. . π 2 dθj = X aj aj ∈{T1 ,T2 ,T3 } π .. . Accepted in .. . ... ... ... ... .. . . .. .. . . . .. .. ... ... ... ... ... . . . ... ... .. . Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. H .. . 11 And by the following equations, ... ... ... ... ... ... π π π = = 1 4 =0 π ... ... ... ... ... ... π π ... ... ... ... π ... ... π = π = π π π π ... ... ... ... ... ... π ... ... ... ... π ... π π π π π π π ... = π π ... π ... = ... ... π ... ... ... ... π π = π = π 1 4 =0 π π ... ... ... π ... ... π ... we have .. . 4n 1 · \|c\|2 2π Z θj ∂ hHi ∂θj ... ... ... ... .. . . .. .. . ... ... .. .. . . .. . H .. . ... ... ... ... .. . . .. ... ... .. .. . . . . .. .. ... ... ... ... ... . . . ... ... .. . .. . .. . .. . 2 dθj = π .. . . . .. .. ... ... ... ... .. . ... . . . ... ... Then, by the definition of VUa1 ,...,am , we obtain X ∂ hHi \|c\|2 Var = n · ∂θj 4 .. . H .. . a ,...,aj−1 ,T2 ,aj+1 ,...,am VU 1 . ak ∈{T1 ,T2 ,T3 }, k6=j Hence, to compute the variance, we need to sum over 3m−1 terms of the tensor a ,...,aj−1 ,T2 ,aj+1 ,...,am VU 1 . It seems inaccessible when m is large. But in many cases, we have simple ways to compute this sum. Recall that we have converted quantum circuits to graph-like ZX-diagrams. Hence, spiders are connected with Hadamard edges. Let us consider two spiders Wj , Wk corresponding to the parameters θj , θk in GU . Suppose that Wj and Wk are connected with a Hadamard edge. Then by the following lemma, the Hadamard edge can be removed after integration over θj and θk . Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 12 Lemma 3. The following equation holds. ... 1 (2π)2 ... ... ... ... Z Z θj θk −θk −θj θj −θj −θk θk θj ... ... ... ... θk ... ... dθj dθk = P aj ,ak ∈{T1 ,T2 ,T3 } ... Maj ,ak · aj ... Here Maj ,ak is the element on the aj -th row and  1 1 1 M= 4 1 ... ... ... ak ... ... ... ... ak -th column in the following 3 × 3 matrix  1 1 1 −1 −1 1 Proof. By Lemma 2, there will be 9 terms on the left-hand side. We can use rewriting rules in Figure 4 on each term to remove Hadamard edges to obtain the form on the right-hand side. The complete proof is given in Appendix E. Applying this lemma to the variance recursively, we can remove all the Hadamard edges connecting two parameterized spiders. And the big tensor a ,...,aj−1 ,T2 ,aj+1 ,...,am VU 1 will be broken into smaller tensors that are connected with M . It is a new tensor network whose structure is similar to GU . To compute the variance, the only thing we need to do is contracting this new tensor network. Figure 5 demonstrates the above procedure for the case that all spiders in GU are parameterized and are connected with Hadamard edges. The tensor I˜a1 ,...,ak is related to GU = α1 β1 α2 β2 .. . .. . αk βl ... ... ... γ1 ... ... ... .. . . . . . . .. . . .. .. . . ... ... γ2 .. . γm θ By lemma 3 Var( ∂hHi ∂θ ) = = P VUa1 ,...,ak ,b1 ,...,bl ,...,T2 ,...,c1 ,...,cm 2 ... I˜a1 ,...,aj 2 .. . .. . ... ... ... ... ... .. . . . . . . .. . . .. .. . . ... ... .. . H̃c1 ,...,cm 2 2 P2 = where .. . = M .. . P2 .. . = the projection copy tensor Figure 5: Computing the variance with the tensor network Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 13 the input state, while the tensor H̃c1 ,...,cm is related to the Hamiltonian H. And P2 is a projection that has only one non-zero entry. That is P2 (2, 2, . . . , 2) = 1. Also, note that there is a scalar 2 for each internal copy tensor. This scalar comes from the following equation. = = π (15) =2 π In Appendix A, a simple example is given to illustrate the techniques introduced in this paper. In conclusion, computing the variance of gradients is reduced to contracting a tensor network corresponding to the circuit. In the next section, we will use these techniques to analyze the BP phenomenon for several for concrete PQCs. 4 Analyzing the BP phenomenon for four PQCs In this section, we will analyze the BP phenomenon for four PQCs with the techniques introduced in Section 3. 4.1 Hardware-efficient ansatz Consider a hardware-efficient ansatz [2] PQC of the following form. \|0i RX RZ \|0i RX RZ \|0i RX .. .. . . RZ + .. . .. . \|0i RX RZ \|0i RX RZ + + ... ... + RZ RX RZ RZ RX RZ RZ RX RZ + .. . .. . .. . RZ RX RZ RZ RX RZ + + ... ... + RZ RX RZ RX RZ RX .. . .. . RZ RX RZ RX (16) ×L The first step is converting it to a graph-like ZX-diagram. Suppose the circuit is of n-qubits. By the conversion rules in Eq. (5) and rewriting rules in Figure 4, we obtain a graph-like ZXdiagram where most spiders are parameterized. √1 . . . 2 √ √1 . . . 2 √1 . . . 2 .. . ... ... ... √ ... ... .. . √1 . . . 2 ... √1 . . . 2 ... ... 2 ... .. . ... 2 .. ... √ . √ .. . 2 ... .. . ... 2 ... ... ×(L + 1) Here, the Z-spider with “. . . ” represents a Z-spider with a parameter. Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 14 Then we are going to represent the variance of gradients as a tensor network. By Lemma 3, we can remove most Hadamard edges. Then the remaining part consists of ... β ... γ α . By similar techniques used in Lemma 3, we can prove the following lemma. Lemma 4. The following equation holds. θ2 1 (2π)3 Z θ1 θ3 θ1 ,θ2 ,θ3 −θ1 −θ3 −θ2 ... ... ... ... ... ... ... ... −θ2 −θ3 −θ1 θ3 θ1 dθ1 dθ2 dθ3 = P ETa1 ,a2 ,a3 · a1 ,a2 ,a3 ∈{T1 ,T2 ,T3 } ... ... ... a1 θ2 Here ETa1 ,a2 ,a3 is an element in the following 3 × 3 × 3    1 0 0 0 1 1 1 0 1 0 , ET [2, ·, ·] = 1 0 ET [1, ·, ·] = 8 8 0 0 1 0 0 ... a2 a3 ... ... ... ... tensor   0 0 1 0 , ET [3, ·, ·] = 0 8 0 1 0 0 0  1 0 . 0 Proof. Refer to Appendix F. Then we can construct a tensor network that is similar to Figure 5 as follows. 1 4 1 4 1 4 1 4 1 4 2 2 2 .. . 2 2 2 M M M 2 2 .. . M M 4 ET 4 ET 2 2 M M 4 ET .. . 2 2 .. M 2 2 2 4 ET 4 ET M 2 M 2 M 2 M 2 M 2 2 4 ET 4 ET 2 M .. .. . . 4 ET 2 M M M 2 M 2 2 4 ET .. . .. . .. . . 2 2 2 4 ET ×L H̃c1 ,...,cn (17) M M 2 And if we want to compute the variance Var( ∂hHi ∂θj ), we can just simply replace the copy tensor corresponding to θj in the above tensor network with the projection P2 . By now we have represented the variance as a tensor network. And now we are going to analyze the scaling property of this tensor network when the number of qubits n and the depth of the circuit grow up. If we denote 2 = EM M 2 M 2 , 4 ET then a layer can be represented as M EM EM LT = ... .. . . ... EM EM E Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 15 And the whole tensor network in Eq. (17) is 1 2 M ... M −1 1 2 M −1 ... M 1 2 M −1 ... .. . LT M .. . .. LT . .. . H̃c1 ,...,cn . 1 2 M −1 ... M 1 2 M −1 ... M 1 2 M −1 ... M L+1 Hence, the variance will be 1 2 1 2 1 2 Var ∂hHi ∂θj ... ... M −1 M −1 ... ... M M −1 ... ... M .. . = LT .. . .. LT . M P2 M −1 .. . .. . LT .. . M .. LT . .. . ... M ... ... M ... ... M 1 2 M −1 ... 1 2 M −1 1 2 −1 M .. . .. . L1 H̃c1 ,...,cn . (18) L2 corresponding to θj We can prove that only two eigenvalues of the matrix LT are 1 and the norms of other eigenvalues are less than 1 (for the complete proof, please refer to Appendix G). Moreover, the eigenspace corresponding to the eigenvalue 1 is generated with two vectors     1 1 E1 = span{v1,2 ⊗ · · · ⊗ v1,2 , v1,3 ⊗ · · · ⊗ v1,3 }, v1,2 = 1 , v1,3 = 0 . (19) 0 1 Hence, LT d will converge to PE1 , the projection to the eigenspace E1 , exponentially, as d → ∞. If we replace LT L1 and LT L2 with the projection PE1 , then the Eq. (18) will become limL1 ,L2 →∞ Var ∂hHi ∂θj 1 2 M M −1 1 2 M −1 M 1 2 −1 M .. . = .. . .. . PE 1 M P2 M −1 .. . .. . M PE 1 .. . 1 2 M −1 1 2 M −1 M 1 2 M −1 M H̃c1 ,...,cn . (20) M corresponding to θj This term is 1 Tr(H 2 ), (21) 4n which is exponentially small. That is, the number of qubits n determines an exponential small limitation of the variance in Eq. (21) when the number of layers L → ∞. And the number of 4· Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 16 layers how the variance will be close to the limitation. More precisely, the variance L determines ∂hHi Var ∂θ is exponentially (in L) close to the exponential small (in n) value Eq. (21). Thus, there exist BPs in the hardware-efficient ansatz. Theorem 5. The variance of gradients in the hardware-efficient ansatz defined in Eq. (16) vanishes exponentially as the number of qubits n and the number of layers L grow up. Note that the above analysis can be generalized to any hardware-efficient ansätze if the entangler connects all of the qubits. 4.2 Tree tensor network ansatz The tree tensor network is a special kind of tensor network with tree structures. The quantum analog of the tree tensor network was developed in [42]. In [53], it was proved that the sum of the variance n X ∂ hHi Var ∂θj j=1 will not vanish exponentially. In this section, we will prove that not only the sum of the variance but also the variance of each parameter vanishes polynomially. Consider the tree tensor network ansatz with n-qubit of the following form. RY RY + RY + RY + RY + RY RY RY + RY + RY (22) RY RY RY RY RY + Measure To analyze the BP phenomenon of this ansatz, we first use the gate decomposition π π RY (θ) = RZ ( )RX (θ)RZ (− ) 2 2 to convert the PQC to a ZX-diagram as follows. ... −π 2 π 2 ... −π 2 π 2 ... −π 2 π 2 √ ... −π 2 π 2 ... −π 2 π 2 −π 2 ... π 2 ... −π 2 π 2 ... −π 2 Accepted in √ √ √ π 2 2 ... −π 2 π 2 √ 2 ... −π 2 π 2 ... −π 2 π 2 2 ... −π 2 π 2 2 √ 2 ... −π 2 π 2 √ 2 ... −π 2 π 2 2 Measure Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 17 The X-spiders with phase “. . . ” are spiders with parameters. And the ZX-diagram can be rewritten to a graph-like ZX-diagram as follows. −π 2 ... −π 2 ... −π 2 ... −π 2 ... −π 2 ... −π 2 ... −π 2 ... −π 2 ... π 2 π 2 −π 2 ... −π 2 ... −π 2 ... −π 2 ... π 2 π 2 π 2 π 2 −π 2 ... −π 2 ... π 2 π 2 π 2 π 2 π 2 π 2 π 2 π 2 −π 2 π 2 ... Measure By using the rewriting rule (lc) in [30], we can remove the spiders with phases ± π2 . −π 2 ... −π 2 ... −π 2 ... −π 2 ... −π 2 ... −π 2 ... −π 2 ... −π 2 ... π 2 ... π 2 π 2 ... π 2 ... π 2 ... π 2 π 2 ... ... π 2 ... Measure Now we are going to construct a tensor network from this graph-like ZX-diagram to represent the variance. By Eq. (12), the building block of the variance ... α ... β γ ... ... −β −γ ... ... −α ∂hHi 2 ∂θj is −α ... −γ −β ... γ β ... α ... . We can prove that (for the complete proof, please refer to Appendix G), after integration over the parameters α, β, γ, the building block will become 1 (2π)3 Z ... α ... β γ ... −γ −α ... −β ... ... ... dαdβdγ = α,β,γ ... −β ... −α −γ ... Here, TTTN is a 3 × 3 × 3  1 0 1  0 1 TTTN [1, ·, ·] = 16 1 0 Accepted in γ β ... α ... ... a,b,c a,b,c∈T1 ,T2 ,T3 TTTN P tensor defined as follows.   1 1 1 0 0 , TTTN [2, ·, ·] = 16 1 −1 ... ...  0 −1 1 0 , 0 1 Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. ... a b ... ... (23) c ... ...  1 1  0 TTTN [3, ·, ·] = 16 1 0 1 0  1 0 . 1 18 Hence, the variance of ∂hHi ∂θj can be obtained by replacing one of the copy tensors with the projection P2 in the following tensor network. 4 2 4 2 4 2 . (24) I˜ a,b,c TTTN = a 4 2 c b 4 2 4 2 4 H Now let us analyze the scaling property of this tensor network. Since the Hamiltonian H is a 1-qubit Hermitian operator, it can be expressed as kj ∈ R. H = k0 I + k1 X + k2 Y + k3 Z, Then       1 0 1 H̃ = 2k02 0 + 2(k12 + k32 ) 1 + 2k22  0  . 1 0 −1 We denote   0 v2 = 1 , 0 v1,3   1 = 0 , 1  1 =  0 . −1  − v1,3 Note that the building block of this tensor network is 8 · TTTN . By the definition of TTTN , we have v1,3 = = , v1,3 v1,3 v2 v1,3 1 2 v2 + v2 v2 , v2 = − v1,3 . v1,3 (25) With the above equations, we can compute the variance simply. For example, consider the following variance. 4 2 Var ∂hHi ∂θ P2 = 4 2 4 2 . I˜ 4 2 4 2 4 2 4 − H̃ = 2k02 v1,3 + 2(k12 + k32 )v2 + 2k22 v1,3 It is a linear function of H̃. Hence, we can analyze each term of H̃ individually. Since P2 v1,3 = 0, the first term 2k02 v1,3 in H̃ will become 0. Now let us consider the second term 2(k12 + k32 )v2 . With Eq. (25), we can expand the variance Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 19 as follows. 4 2 4 2 P2 P2 4 2 4 2 4 2 I˜ I˜ = 4 2 4 2 4 2 4 2 4 4 2 2v2 4 2 4 2 I˜ 1 (v1,3 2 4 2 + v2 ) = 1 2 v2 v2 4 2 4 2 4 2 4 2 I˜ 4 2 4 2 1 (v1,3 2 4 2 v2 4 2 + v2 ) P2 4 2 1 (v1,3 2 v2 + v2 ) v2 4 2 1 2 4 2 P2 4 2 = 4 2 4 2 P2 = 4 2 v2 I˜ = 1 22 v2 4 2 4 2 . I˜ 4 2 4 2 4 2 v2 4 2 v2 Expanding it recursively, the variance can be represented as a summation of terms of the following form, u1 u2 ˜ 1 , . . . , un ) = I(u I˜ .. . , where uj ∈ {v2 , v1,3 }. (26) un ˜ each of the term I(u ˜ 1 , . . . , un ) ≥ 0. Hence, we can obtain a lower And by the definition of I, Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 20 bound, 4 2 1 (v1,3 2 4 2 + v2 ) v2 1 22 v2 v2 I˜ ≥ 1 22 v2 I˜ 4 2 4 2 4 2 ≥ 1 23 1 v 2 1,3 4 2 4 2 v2 v1,3 v1,3 v1,3 v1,3 v2 v2 v2 I˜ ≥ 4 2 1 25 v2 I˜ 4 2 v2 . v1,3 1 v 2 1,3 v1,3 v1,3 4 2 v2 v2 − Similarly, we have a lower bound for the term v1,3 . For the general case of n-qubit, we can prove that it has a lower bound. Theorem 6. For tree tensor network ansatz shown in (22), if H = k0 I + k1 X + k2 Y + k3 Z, then we have Var ∂ hHi ∂θ ≥ k12 + k32 ˜ k22 ˜ I(u , . . . , u ) + I(w1 , . . . , wn ), 1 n n2 n2 (27) − ˜ 1 , . . . , un ) is defined in Eq. (26). And I˜ is a 3n dimenfor some uj , wj ∈ {v1,3 , v2 , v1,3 }. Here I(u sional tensor which only depends on the input state. If the input state is ρ, then I˜ is defined as follows. −π 2 I Ta1 −π 2 π 2 Ta2 π 2 −π 2 .. . .. . .. . = π 2 π 2 Accepted in ρ π 2 −π 2 ˜a1 ,...,an π 2 .. . ρ .. . −π 2 , where aj ∈ {1, 2, 3}. Tan −π 2 Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 21 Proof. See Appendix G.2. ˜ 1 , . . . , un ) only depends on the input state. If Note that I(u ˜ 1 , . . . , un ) ∈ Ω( I(u 1 1 ˜ 1 , . . . , wn ) ∈ Ω( ) or I(w ), poly(n) poly(n) there exist no BP in the tree tensor network ansatz. 4.3 QCNN QCNN was developed in [41]. It was proved that there exists no BP in the QCNN ansatz if the subblocks form unitary 2-design [54]. In this section, we will use the ZX-calculus to analyze the BP phenomenon in a QCNN ansatz without the assumption of unitary 2-design. Consider a QCNN ansatz as follows. RX RZ RX RZ RX RZ RX RZ RX RZ RX RZ RX RZ RX RZ + + + + RX RZ RX RZ RX RZ RX RZ RX RZ RX RZ RX RZ RX RZ + + + RX RZ RX RZ RX RZ RX RZ RX RZ RX RZ + RX RZ + RX RZ + RX RZ + RX RZ + + RX RZ RX RZ RX RZ RX RZ + RX RZ RX RZ + RX RZ + RX RZ (28) RX + RZ Measure It can be represented as the following ZX-diagram. ... ... ... ... ... ... ... ... √ ... ... ... ... ... ... ... √ ... ... ... ... ... ... ... √ ... ... √ ... ... √ ... ... ... ... √ ... 2 ... ... ... ... √ ... ... ... ... ... 2 ... ... ... ... ... ... 2 2 ... √ ... √ 2 ... 2 2 ... ... 2 2 ... ... ... ... 2 2 ... ... √ √ 2 ... √ √ 2 ... ... ... ... √ ... √ ... ... √ 2 √ 2 2 2 ... ... ... ... ... ... ... ... Measure where the Z-spiders with “. . . ” are parameterized. Note that this is a graph-like ZX-diagram whose spiders are all parameterized. Hence, by using Lemma 3, the variance can be obtained by replacing one of the copy tensors with the projection P2 in the following tensor network. 2 2 4 2 2 2 4 I˜ 2 2 2 4 2 2 2 Accepted in 4 2 2 2 2 2 4 2 2 2 4 2 2 2 2 2 4 2 2 2 2 4 2 2 2 4 2 2 2 2 2 4 2 2 2 4 2 v1,3 2 v1,3 2 2 4 2 2 2 2 2 2 2 2 v1,3 4 2 2 v1,3 4 v1,3 v1,3 2 2 2 2 2 4 2 v1,3 4 4 2 2 Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 2 2 2 H̃ 22 Now we are going to analyze the scaling property of this tensor network. If we denote 2 = TQCNN 2 4 2 2 , then we have the following equations 2 2 2 4 2 v1,3 2 v1,3 v1,3 2 , = 4 2 v1,3 1 − v 2 1,3 v1,3 + 12 v2 = 2 1 v 4 1,3 v2 + 34 v2 2 , 2 2 4 2 v1,3 v1,3 . = − v1,3 1 − v 2 1,3 (29) + 21 v2 By using Eq. (29), we can expand the variance as a sum of terms of the following form. u1 u2 ˜ 1 , . . . , un ) = I(u I˜ − .. , where uj ∈ {v2 , v1,3 , v1,3 }. . un Each of these terms is non-negative. Hence, similar to the analysis of tree tensor network ansatz, we can prove that the variance of gradients in the QCNN ansatz has a lower bound, since the QCNN ansatz is of O(log(n))-depth. Theorem 7. For the QCNN ansatz shown in (28), if H = k0 I + k1 X + k2 Y + k3 Z, then we have Var ∂ hHi ∂θ ≥ k22 + k32 ˜ k12 ˜ I(u , . . . , u ) + I(w1 , . . . , wn ), 1 n n9 n9 (30) − for some uj , wj ∈ {v1,3 , v2 , v1,3 }. Here I˜ is a 3n dimensional tensor which only depends on the input state ρ. .. . ρ .. . Ta1 Ta2 I˜a1 ,...,an = .. . .. . ρ .. . , where aj ∈ {1, 2, 3}. Tan Proof. See Appendix G.3. Hence, if provided ˜ 1 , . . . , un ) ∈ Ω( I(u 1 1 ˜ 1 , . . . , wn ) ∈ Ω( ) or I(w ), poly(n) poly(n) then there exists no BP in the QCNN ansatz. Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 23 4.4 MPS-inspired ansatz The matrix product state (MPS) is a special structure of tensor networks, which is widely used in quantum physics and machine learning [55, 56]. There are also PQCs with a similar structure as MPS, and we call it MPS-inspired ansatz. It has been shown that MPS-inspired ansatz can be implemented efficiently in quantum computers with a small number of qubits [43]. We will analyze the BP phenomenon in MPS-inspired ansatz in this section. Let us consider the following MPS-inspired ansatz \|0i RX RZ \|0i RX RZ + \|0i RX RZ RX RZ RX RZ .. . RX + .. RZ . .. . + \|0i RX RZ RX RZ + \|0i RX RZ RX RZ RX RZ , + RX RZ RX RZ (31) and the Hamiltonian H = I ⊗ I · · · ⊗ I ⊗ X. We will prove that the variance Var ∂hHi is ∂θ1 exponentially small. Here θ1 is the parameter of the first RX gate applying on the first qubit. Firstly, we convert the PQC into a ZX-diagram as follows. √1 . . . 2 √ √1 . . . 2 ... √1 . . . 2 ... ... ... ... ... √ 2 ... ... ... 2 .. . .. .. . ... .. . .. . . ... √1 . . . 2 ... √1 . . . 2 √ ... ... ... ... ... 2 ... √ ... ... 2 ... ... This is a graph-like ZX-diagram whose spiders are all parameterized. We can use Lemma 3 to represent the variance as the following tensor network. 1 4 2 2 P2 12 2 4 2 4 Var ∂hHi ∂θ1 1 4 = 2 2 2 2 2 2 4 2 .. . 2 .. . .. v1,3 2 v1,3 .. . . 2 2 1 4 2 .. . 2 4 12 2 2 2 2 2 2 4 2 v1,3 2 2 4 v1,3 v2 By using 2M v2 = and 2 4 Accepted in 2 v1,3 = v2 1 2 1 − v2 + v1,3 , 2 + − v1,3 2 − v1,3 v1,3 , v2 4 2 2M v1,3 = v1,3 , v1,3 = − v1,3 2 − v1,3 , v2 Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 4 2 v1,3 v1,3 = v1,3 , v1,3 24 we can simplify the variance as 1 4 1 4 Var ∂hHi ∂θ1 = 1 4 1 4 1 4 2 P2 2 2 2 2 2 .. . 3 − v 4 1,3 v2 v2 .. . 2 2 2 2 = 1 22n−1 (32) v2 v2 It is exponential in the number of qubits n. Hence, there exist BPs in the MPS-inspired ansatz. 5 Discussion We developed powerful techniques to analyze the BP phenomenon for quantum neural networks training with the ZX-calculus. The quantum neural networks under consideration are PQCs under certain reasonable assumptions and the cost function is the expectation hHi of the PQC with respect to a given Hamiltonian H. The basic idea of the method is to represent the PQC, the cost function hHi, and the gradients ∂hHi ∂θj as ZX-diagrams. And then computing the expectation and the variance of the gradient of hHi becomes computing the integration of certain ZX-diagrams. We show that these integrations are sums of ZX-diagrams that can be computed explicitly in many cases. As future works, it would be desirable to use the completeness of ZX-calculus to represent these sums by ZX-diagrams, or more generally, diagrams in other complete graphical calculi. In principle, these techniques can be used to any given ansatz under Assumption 1. We remark that these techniques can be used to analyze the BP phenomenon for PQCs which contain t-design sub-blocks, for example, the PQCs considered in [21, 54]. Because the t-design sub-blocks can be replaced with concrete t-design PQCs and then the techniques proposed in this paper can be applied. Techniques introduced in this paper can be used in more cases including circuits with global cost functions and circuits of any depth. To analyze a PQC with a global cost function, one can represent the global Hamiltonian as a ZX-diagram and then the method introduced in this paper can be used. As shown in Section 4.1, the BP phenomenon for the hardware-efficient ansatz has been analyzed when the depth is O(poly(n)). In conclusion, we extend the BP theorem from unitary 2-design circuits to any parameterized quantum circuits under Assumption 1. Using the techniques proposed in this paper, we analyzed four kinds of ansätze, including the hardware-efficient ansatz, the tree tensor network ansatz, the QCNN ansatz, and the MPS-inspired ansatz. It is shown that there exist BPs in the hardware-efficient-ansatz and the MPS-inspired ansatz, while there exists no BP in the tree tensor network ansatz and the QCNN ansatz. Acknowledgment. This work is partially supported by a NSFC grant No.11688101 and by a NKRDP grant No.2018YFA0306702. We want to thank the anonymous referees for their valuable suggestions. References [1] Alberto Peruzzo, Jarrod McClean, Peter Shadbolt, Man-Hong Yung, Xiao-Qi Zhou, Peter J. Love, Alán Aspuru-Guzik, and Jeremy L. O’Brien. A variational eigenvalue solver on a photonic quantum processor. Nature Communications, 5(1):4213, Jul 2014. ISSN 2041-1723. DOI: 10.1038/ncomms5213. [2] Abhinav Kandala, Antonio Mezzacapo, Kristan Temme, Maika Takita, Markus Brink, Jerry M. Chow, and Jay M. Gambetta. Hardware-efficient variational quantum eigensolver for Accepted in Quantum 2021-05-28, click title to verify. 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Recall that the PQC considered is \|0i RX (θ1 ) RZ (θ3 ) = \|0i Accepted in RX (θ2 ) + RZ (θ4 ) √1 2 √1 2 θ1 θ3 θ2 θ4 (f ), (h) = Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. √1 θ1 2 θ3 √1 θ2 2 θ4 28 and the gradient with respect to θ1 can be represented as π √1 θ1 + 2 ∂ hHi = ∂θ1 √1 2 π 2 θ3 −θ3 −θ1 − −θ4 −θ2 π 2 H θ2 θ4 √1 2 . √1 2 By Lemma 1, the expectation of this gradient is zero. The variance of the gradient is the following integration. π π 2 θ1 + θ3 −θ3 −θ1 − −θ4 −θ2 π 2 H Var ∂ hHi ∂θ1 1 = 16(2π)4 Z Z Z Z θ1 θ2 θ3 θ2 θ4 −θ2 θ4 −θ4 θ4 θ2 θ3 θ1 + dθ1 dθ2 dθ3 dθ4 . H −θ1 − π 2 −θ3 π 2 π Now we choose the Hamiltonian H = X ⊗ X. Then by Lemma 2 and Theorem 4, we have π π π π 16 · Var ∂ hHi ∂θ1 = X a1 a2 π a3 X = a4 a1 ,a2 ,a3 ,a4 ∈{T1 ,T2 ,T3 } a2 π a3 a4 . a2 ,a3 ,a4 ∈{T1 ,T2 ,T3 } π π π π π Using Lemma 3 recursively, we can break the large ZX-diagram into small parts as follows. π π π 16 · Var ∂ hHi ∂θ1 X = a2 π π a3 X MT2 ,a3 · a2 ,a3 ,a4 ∈{T1 ,T2 ,T3 } = a4 a2 ,a3 ,a4 ∈{T1 ,T2 ,T3 } a2 π π a3 π π π π π π = X MT2 ,a3 · Ma2 ,a3 · a2 π π a3 a4 a2 ,a3 ,a4 ∈{T1 ,T2 ,T3 } = X MT2 ,a3 a2 ,a3 ,a4 ∈{T1 ,T2 ,T3 } · Ma2 ,a3 · Ma2 ,a4 · a2 π π MT2 ,a3 · Ma2 ,a3 · Ma2 ,a4 · a2 ,a3 ,a4 ∈{T1 ,T2 ,T3 } Accepted in a4 π π X a3 π π = a4 π · a2 · a3 π π · a4 . π Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 29 We denote I˜a1 ,a2 = · a1 , a2 π H̃a3 ,a4 = π · a3 a4 π . π Then the variance can be represented as the following tensor network. 16 · Var ∂ hHi ∂θ1 X = P2 MT2 ,a3 · Ma2 ,a3 · Ma2 ,a4 · I˜T2 ,a2 · H̃a3 ,a4 = I˜ H̃ . a2 ,a3 ,a4 ∈{T1 ,T2 ,T3 } By contracting this tensor network, we finally obtain ∂ hHi 1 3 Var = · . ∂θ1 16 4 B Proof of theorem 2 Theorem 2. The gradient can be represented as the following equation. n 2 \|c\|2 · ∂hHi ∂θj = ∂ ∂θj = .. . .. . .. . .. . ... ... ... ... .. . . . . θj ... ... .. .. . . ... ... ... ... .. . . .. ... ... .. .. . . θj + .. . .. . π 2 H H .. . .. . ... ... ... ... .. . . . . −θj ... ... .. .. . . ... ... ... ... .. . . .. ... ... .. .. . . π −θj − .. . .. . .. . .. . π 2 Proof. Consider the spiders corresponding to θj . We expand the spiders as follows. m .. . θj ... ... .. n . . n .. . . . . .. −θj ⊗n ⊗m . . . ⊗m ⊗n ⊗n ⊗m . . . ⊗m ⊗n \|0i h0\| \|0i h0\| \|0i h0\| +eiθj \|1i h1\| .. m = −iθj \|0i⊗n h0\|⊗m . . . \|1i⊗m h1\|⊗n + \|1i⊗n h1\|⊗m . . . \|1i⊗m h1\|⊗n . +e . Taking the partial derivative of θj on the two sides, we obtain ∂ ∂θj m .. . θj ... ... .. n . . n .. . . . . .. −θj .. m = ieiθj \|1i⊗n h1\|⊗m . . . \|0i⊗n h0\|⊗m −ie−iθj \|0i⊗n h0\|⊗m . . . \|1i⊗n h1\|⊗m . = ei(θj + π2 )\|1i⊗n h1\|⊗m . . . \|0i⊗n h0\|⊗m +e−i(θj + π2 ) \|0i⊗n h0\|⊗m . . . \|1i⊗n h1\|⊗m ... = m . θj + π2 . n ... . n . −θj − π2 . m .. .. . .. .. . . .. . π C Proof of lemma 1 Lemma 1. The following equation holds. Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 30 1 2π ... ... .. n . . n .. . . . . .. Z α m .. . α ... ... .. n . . n .. . . . . .. .. m dα = m .. . . −α .. m . Proof. We expand the spiders as follows. m .. . α ... ... n . . n .. ... . . . .. −α ⊗n ⊗m . . . ⊗m ⊗n ⊗n ⊗m . . . ⊗m ⊗n \|0i h0\| \|0i h0\| + eiα\|1i h1\| \|0i h0\| .. m = ⊗n ⊗m . . . ⊗m ⊗n −iα \|0i⊗n h0\|⊗m . . . \|1i⊗m h1\|⊗n + \|1i h1\| \|1i h1\| . +e . By Z π eikα dα = 0, k = ±1, −π we have 1 2π R α dα m .. . ... ... .. n . . n .. . . . . .. α −α .. m = \|0i⊗n h0\|⊗m . . . \|0i⊗m h0\|⊗n + \|1i⊗n h1\|⊗m . . . \|1i⊗m h1\|⊗n . ... ... = m . n . .. m . . . n .. .. .. . . . . .. D Proof of lemma 2 Lemma 2. The following equation holds. 1 2π Z m ... α α m .. . −α ... ... ... n . . . n ... ... ... ... .. n . . n .. . . . . .. −α .. m . α .. m . m .. . ... ... .. n . . n .. . . . . .. m .. . ... ... ... n . . . n ... ... .. m . dα = m .. . ... ... .. n . . n .. . . . . .. m .. . ... ... ... n . . . n ... ... + .. m . ... ... .. n . . n .. . . . . .. m .. . .. m . + π .. m . π .. m . ... ... .. n . . n .. . . . . .. m .. . .. m . Proof. We expand each spider on the left-hand side of the equation as follows. m .. . α m .. . −α ... ... .. n . . n . . . .. ... ... .. n . . n . . . .. .. . −α .. . α .. m . +e−iα = +eiα .. m . + ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| ⊗ iα ⊗n ⊗m ⊗m ⊗n +e \|0i h0\| . . . \|0i h0\| ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| ⊗ + ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|0i h0\| ⊗m ⊗n ⊗n ⊗m . . . \|0i h0\| ⊗ i2α \|0i h0\| ⊗n ⊗m ⊗m ⊗n +e \|0i h0\| . . . \|1i h1\| ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| ⊗ iα ⊗n ⊗m ⊗m ⊗n +e \|1i h1\| . . . \|1i h1\| Since Z ⊗n ⊗m ⊗m ⊗n ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|0i h0\| ⊗ −iα \|0i h0\| . . . \|1i h1\| ⊗ + ⊗n ⊗m ⊗m ⊗n +e ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| \|0i h0\| . . . \|0i h0\| ⊗n ⊗m . . . ⊗n ⊗m . . . ⊗m ⊗n ⊗m ⊗n \|1i h1\| \|0i h0\| ⊗ −i2α \|0i h0\| \|1i h1\| ⊗ −iα ⊗n ⊗m ⊗m ⊗n +e ⊗n ⊗m ⊗m ⊗n +e \|1i h1\| . . . \|0i h0\| \|1i h1\| . . . \|0i h0\| ⊗m ⊗n ⊗m ⊗n ⊗n ⊗m . . . ⊗n ⊗m . . . \|0i h0\| ⊗ \|1i h1\| ⊗ iα \|1i h1\| \|0i h0\| + ⊗n ⊗m ⊗m ⊗n ⊗n ⊗m ⊗m ⊗n +e \|0i h0\| . . . \|1i h1\| \|0i h0\| . . . \|1i h1\| ⊗n ⊗m ⊗m ⊗n ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|0i h0\| ⊗ −iα \|0i h0\| . . . \|1i h1\| ⊗ + ⊗n ⊗m ⊗m ⊗n +e ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|1i h1\| \|1i h1\| . . . \|1i h1\| ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|1i h1\| ⊗ ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|1i h1\| ⊗ ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|0i h0\| ⊗m ⊗n ⊗n ⊗m . . . \|1i h1\| ⊗ \|1i h1\| ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|1i h1\| ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|1i h1\| ⊗ ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|1i h1\| 2π eikα dα = 0, k = ±1, ±2, 0 we integrate over α on each side and obtain 1 2π R m .. . α α m .. . −α ... ... .. n . . n . . . .. ... ... .. n . . n . . . .. .. . −α .. . α .. m . dα = .. m . ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| ⊗ ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| ⊗n ⊗m . . . ⊗m ⊗n \|1i h1\| \|1i h1\| ⊗ + ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|1i h1\| m .. . ... ... ... n . . . n ... ... m .. . ... ... ... n . . . n ... ... .. m . = Accepted in + ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|0i h0\| ⊗ ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|0i h0\| ⊗n ⊗m . . . ⊗m ⊗n \|0i h0\| \|1i h1\| ⊗ + ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|1i h1\| m .. . ... ... ... n . . . n ... ... m .. . ... ... ... n . . . n ... ... + .. m . + m .. . .. m . + π ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| ⊗ ⊗n ⊗m ⊗m ⊗n \|1i h1\| . . . \|1i h1\| ⊗n ⊗m . . . ⊗m ⊗n \|1i h1\| \|1i h1\| ⊗ + ⊗n ⊗m ⊗m ⊗n \|0i h0\| . . . \|0i h0\| ... ... .. n . . n .. . . . . .. .. m . π . .. m . Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. m .. . ... ... .. n . . n .. . . . . .. .. m . 31 E Proof of lemma 3 Lemma 3. The following equation holds. ... 1 (2π)2 ... ... ... ... Z Z θj θk −θk −θj θj −θj −θk θk θj ... ... ... ... θk ... ... dθj dθk = P aj ,ak ∈{T1 ,T2 ,T3 } ... Maj ,ak · ... ... aj ... ... ak ... ... ... ... column in the following 3 × 3 matrix  1 −1 1 Here Maj ,ak is the element on the aj -th row and ak -th  1 1 1 1 1 M= 4 1 −1 Proof. By Lemma 2, we have ... 1 (2π)2 ... ... ... ... Z Z θj θk −θk −θj θj −θj −θk θk θj ... ... ... ... θk ... ... dθj dθk = P ... ... aj aj ,ak ∈{T1 ,T2 ,T3 } ... ... ... . ak ... ... ... ... Hence, it is sufficient to prove ... ... ... aj ... ... ... ... ak ... ... ... ... = Maj ,ak aj ... ... ... ... ... ... ak ... ... ... for aj , aj ∈ {T1 , T2 , T3 }. For aj = ak = T1 , we have ... ... ... ... ... (f ) = (hopf ) 1 = 4 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... (f ) = ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... (33) 1 4 . ... ... ... ... ... For (aj , ak ) ∈ / {(T2 , T3 ), (T3 , T2 )}, the proof is almost the same as that of Eq. (33). Now, let us consider the case when (aj , ak ) = (T2 , T3 ). We can use the rules in Figure 4 as Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 32 follows. ... ... ... ... π ... ... (f ) = π ... ... π π ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... π ... (π), (f ) = ... π π π ... ... (h), (i2) (f ), = ... π ... π π ... ... π ... ... ... ... ... ... ... ... π ... π ... ... (hopf ) = ... 1 4 ... π π ... π ... ... ... ... ... ... (π) = − 14 ... π ... π ... π ... ... π π ... ... ... ... π ... (f ) = ... ... (f ) = ... − 41 ... ... π ... ... ... π . π ... ... ... ... ... ... ... ... F Proof of lemma 4 Lemma 4. The following equation holds. θ2 1 (2π)3 Z θ1 θ3 θ1 ,θ2 ,θ3 −θ1 −θ3 −θ2 ... ... ... ... ... ... ... ... −θ2 −θ3 −θ1 θ3 θ1 dθ1 dθ2 dθ3 = P a1 ,a2 ,a3 ∈{T1 ,T2 ,T3 } θ2 Here ETa1 ,a2 ,a3 is an element in the following 3 × 3 × 3 tensor     1 0 0 0 1 0 1 1 0 1 0 , ET [2, ·, ·] = 1 0 0 , ET [1, ·, ·] = 8 8 0 0 1 0 0 0 Accepted in ETa1 ,a2 ,a3 · ... ... ... ... a1 a2 ... ... ... ...  0 1 0 ET [3, ·, ·] = 8 1 Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. a3 0 0 0  1 0 . 0 33 Proof. By Lemma 2, it is sufficient to prove that ... ... ... ... ... ... ... ... ... ... ... ... a1 a2 = ETa1 ,a2 ,a3 · a3 a1 a2 = 2ETa1 ,a2 ,a3 · a3 a2 a3 ... ... ... ... ... ... ... ... ... ... ... ... for a1 , a2 , a3 ∈ {T1 , T2 , T3 }. We first consider the case when a1 = T1 . Since a1 = T1 , we have ... ... ... ... ... ... ... ... a2 (h), (f ) = (f ) = a3 a2 ... ... ... ... ... ... ... ... ... ... ... ... ... a2 (b) 2 = ... ... ... ... ... ... ... ... a3 ... (h), (f ) = 2 a3 ... a2 a3 ... ... ... ... ... a2 a3 ... ... ... ... . Now, if a2 = T1 , then we have ... ... ... ... ... ... ... ... ... ... ... ... 2 Accepted in a3 (f ), (hopf ) 1 = 4 a3 (i1), (i2) = 1 4 a3 ... ... ... ... ... ... ... ... ... ... ... ... Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. . 34 Hence, ... ... ... ... ... ... ... ... (f ) = 1 4 , ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... 1 4 π (f ), (h) 1 = 4 (hopf ) 1 = 4 π ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... π ... ... ... ... 1 8 1 0 (f ), (h) 1 = 4 (hopf ) 1 = 4 π = 0, π ... 1 4 That is ET [1, 1, ·] = 1 4 = 0. π ... ... ... ... ... ... ... ... 0 . If a2 = T2 , then 2 ... ... ... ... ... ... ... ... ... ... ... ... a3 π ... ... ... ... ... ... ... ... (f ), (hopf ) = 1 2 ... (h), (f ) = ... ... π ... ... ... ... π ... 1 2 ... a3 π Accepted in a3 π π ... a3 (i1), (i2) 1 = 2 ... ... ... ... ... π π (π), (f ) 1 = 2 a3 π (f ), (hopf ), (π) = 1 4 ... a3 π π ... ... ... ... ... ... ... ... ... ... ... ... Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 35 Hence, ... ... ... ... π ... 1 4 π ... ... (f ), (hopf ) 1 = 8 π ... ... ... ... ... ... 1 4 = 0, π ... ... ... (h) = π 1 4 ... π (f ), (π) 1 = 4 π ... ... ... ... ... ... ... ... ... ... ... ... ... ... π ... π ... ... π π 1 4 ... ... ... ... ... π ... ... ... π ... (f ), (h) = π 1 4 π ... π ... ... ... ... ... π ... ... (hopf ) = π 1 4 ... π ... ... ... ... ... ... ... ... ... ... ... 1 8 = 0. π ... That is ET [1, 2, ·] = , π 1 0 . 0 If a2 = T3 , then 2 ... ... ... ... ... ... ... ... ... ... ... ... π π ... ... ... ... ... ... ... ... (π), (f ) = Accepted in (f ), (hopf ) = a3 π π π π a3 (h), (f ) = (i1), (i2) = a3 π ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... π a3 π a3 π π (f ), (hopf ), (π) = 1 4 π π a3 ... ... ... ... ... ... ... ... ... ... ... ... Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. π 36 Hence. 1 4 1 4 1 4 That is ET [1, 3, ·] = 1 8 0 π ... ... ... ... ... ... ... ... π π π ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... π (f ), (hopf ) 1 = 4 π π π π ... ... ... ... ... ... ... ... ... ... π 0 π ... π π (f ), (hopf ) 1 = 4 π π (h), (f ), (π) = π 1 4 π = 0, π = 0, ... ... ... ... ... ... π π . ... ... ... ... ... ... ... ... 1 . By now, we have proved that  1 1 ET [1, ·, ·] = 0 8 0 0 1 0  0 0 . 1 (34) Now, let us consider the case when a1 = T2 . Since a1 = T2 , we have ... ... ... ... ... ... ... ... a2 π (h), (f ) = (f ) = a3 a2 π a3 ... ... ... ... ... ... ... ... ... ... ... ... ... a2 ... ... (b) 2 = a3 π ... ... ... ... ... ... a2 ... a3 π ... ... ... ... π ... (h), (f ) = 2 Accepted in ... a2 a3 ... ... ... ... . Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 37 Now, if a2 = T1 , then we have ... ... ... π ... ... ... ... π ... 2 π ... a3 (f ), (hopf ) = ... 1 4 ... ... (i1), (i2) 1 = 4 a3 a3 ... ... ... ... ... ... ... ... ... ... ... ... Hence, ... ... ... ... ... π (f ), (hopf ) = 1 4 = 0, ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... π 1 4 (h) = π 1 4 (f ), (π) = π ... ... ... ... ... ... ... ... 1 4 ... ... ... π , π ... ... ... ... ... π ... ... ... ... ... ... 0 ... (f ), (hopf ) = π That is ET [1, 1, ·] = ... ... π ... 1 8 ... 1 8 ... 1 4 ... π ... 1 ... 1 8 = 0. π ... ... ... ... 0 . If a2 = T2 , then ... ... ... π ... 2 ... a3 ... ... ... ... ... ... π ... (f ), (hopf ) = 1 2 π ... ... ... a3 π ... ... 1 2 ... a3 (i1), (i2) 1 = 2 ... a3 π ... ... ... ... ... ... ... π ... ... (h), (f ) = 12 a3 ... ... π (f ), (hopf ) = ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... a3 (f ), (π) 1 = 4 a3 ... ... ... ... π ... ... (i1), (i2) 1 = 4 1 4 ... π π ... π π ... (π) = ... π π ... π a3 π ... ... ... ... ... ... ... ... π Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 38 Hence, ... ... ... ... ... ... ... ... 1 4 1 4 That is ET [1, 2, ·] = , π ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... π (f ), (h) 1 = 4 π ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... π 1 1 4 ... π 1 4 1 8 (f ) = π π (f ), (h) 1 = 4 π ... ... ... ... ... ... ... ... π (hopf ) = 0, π (hopf ) = 0. 0 . 0 If a2 = T3 , then ... ... ... π ... 2 ... ... (f ), (hopf ) = a3 π Accepted in ... ... ... ... ... ... ... ... ... ... ... ... ... ... (f ), (hopf ) = a3 ... ... ... ... π π 1 4 a3 π π ... ... ... ... ... ... π π π π ... a3 π ... (π) = π π ... ... a3 π π π ... π ... ... (h), (f ) = ... π ... ... ... ... ... π (f ), (π) = π Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 1 4 π π ... a3 ... ... ... ... π 39 Hence. ... ... ... π ... 1 4 π ... ... π π (f ), (hopf ) 1 = 32 π π ... ... ... ... ... ... ... ... ... ... ... ... ... π π π π (f ), (hopf ) 1 = 16 π That is ET [1, 3, ·] = 1 8 0 0 = 0, π = 0, π = 0. ... π π ... ... ... ... ... ... ... ... ... ... ... ... ... ... π π π π π ... 1 4 ... ... ... 1 4 ... π π π π π (f ), (hopf ) 1 = 8 π ... π π ... ... ... ... ... ... ... ... 0 . By now, we have proved that  0 1 ET [2, ·, ·] = 1 8 0  0 0 . 0 1 0 0 (35) Let us consider the case when a1 = T3 . When a1 = T3 , we have ... ... ... ... ... ... ... ... a2 π (h), (f ) = (f ) = a3 ... ... ... ... ... ... ... ... ... ... ... ... ... ... ... a2 π (b) = a3 ... ... ... ... ... ... Accepted in π a3 ... ... (h), (f ) = 2 a2 π 2 a2 π a3 ... ... ... ... ... a2 a3 ... ... ... ... . Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 40 According to the symmetry, we can use the result of the  0 0 1 0 0 ET [3, ·, ·] = 8 1 0 case when a1 = T2 . And we obtain  1 0 . (36) 0 G Analysis of PQCs in section 4 G.1 Hardware-efficient ansatz We will prove some properties of LT , which are used in the analysis in Section 4.1. Theorem 8. Suppose that λ1 , . . . , λ3n are eigenvalues of LT . And \|λ1 \| ≥ \|λ2 \| ≥ · · · ≥ \|λ3n \| . Then we have λ1 = λ2 = 1, \|λj \| < 1 for j > 2. Proof. By definition of EM , we can compute that 3 1 1 4 1  41  4   EM =        4 3 4 − 14 4 − 14 3 4 1 4 3 4 − 41  3 4 1 4 1 4 0 0 0 1 4 − 14 3 4 0 0 0       .    3 4 1 (37) 4 1 4 By computation, EM can be diagonalized. Four of its eigenvalues are 1 and other eigenvalues are in the interval (−1, 1). Moreover, the eigenspace of the eigenvalue 1 is span {v1 ⊗ v1,2 , v1 ⊗ v1,3 , v1,2 ⊗ v1,2 , v1,3 ⊗ v1,3 } , where (38)       1 1 1 v1 = 0 , v1,2 = 1 , v1,3 = 0 . 0 0 1 LT is an operator on the tensor product of n R3 . We denote the operator EM on the i-th and j-th R3 as EMi,j . Then the eigenspace of LT corresponding to the eigenvalue 1 is the intersection of the eigenspaces corresponding to the eigenvalue 1 of EM1,2 , EM2,3 , . . . , EMn−1,n , EMn,1 . Hence, by Eq. (38), we have E1 = span {v1,2 ⊗ · · · ⊗ v1,2 , v1,3 ⊗ · · · ⊗ v1,3 } . Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 41 G.2 Tree tensor network ansatz In Section 4.2, we used Eq. (23). Here we will prove this equation. Proof of Eq. (23). By Lemma 2, it suffices to prove that ... ... ... a b ... ... ... ... ... ... ... ... a,b,c TTTN = c ... a ... ... ... ... ... b . c ... ... ... Similar to the proof of Lemma 4, we first consider the case when a = T1 . When a = T1 , we have ... ... ... ... ... ... b c ... ... ... ... ... (f ), (hopf ) 1 = 4 ... ... ... ... ... ... b c ... ... ... ... (i1), (i2) 1 = 4 ... ... ... ... ... ... ... b . c ... ... Now, a is disconnected with b and c. Hence, we can consider b and c separately. If b = T1 , then ... ... 1 4 ... c ... ... ... (f ), (hopf ) 1 = 16 ... ... ... ... c ... ... (i1), (i2) 1 = 16 ... ... ... ... c ... ... Hence, ... ... = , ... ... ... ... ... (f ) = π (hopf ) = π ... ... ... = 0, π ... ... (f ) = π π ... . ... That it TTTN [1, 1, ·] = 1 1 16 1 . 0 If b = T2 , then ... ... 1 4 π ... ... c ... ... (π) = ... π π ... π ... 1 4 ... c ... ... (h), (f ) 1 = 4 ... ... π π ... π π ... ... c ... π ... (f ), (hopf ) 1 = 16 ... π π ... ... c π π ... ... π ... (π), (f ) 1 = 16 ... ... ... π ... c ... π Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 42 Hence, π π ... ... (f ), (hopf ) = = 0, ... ... π π π ... ... (f ), (h), (π) = π , π ... ... π π π ... ... (f ), (hopf ) = π = 0. ... ... π π That it TTTN [1, 2, ·] = 1 0 16 1 0 . If b = T3 , then ... 1 4 ... ... ... c π ... ... (f ), (hopf ) 1 = 16 ... ... c π ... ... ... (i1), (i2) 1 = 16 ... ... ... ... ... c π ... ... Hence, ... ... = ... ... π ... (f ) = ... ... ... (hopf ) = π π ... ... π , ... = 0, ... ... (f ) = π . ... That it 1 1 16 0 1 .  1 1  0 TTTN [1, ·, ·] = 16 1 0 1 0  1 0 . 1 TTTN [1, 3, ·] = By now, we have proved that Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. (39) 43 Now, let us consider the case when a = T2 . When a = T2 , we have ... ... ... ... b π ... ... ... π ... π ... ... (π) = c ... b ... ... ... c ... ... ... π ... ... π ... ... (f ), (hopf ) 1 = 4 ... ... ... ... ... ... ... ... ... ... ... π ... ... π ... ... π ... b π (π) = c π b π π ... π ... ... ... ... ... b ... ... π 1 4 ... ... ... (h), (f ) = π . c ... ... π c Now, a is disconnected with b and c. Hence, we can consider b and c individually. If b = T1 , then π ... π ... 1 4 (h) = c ... ... ... ... ... ... ... 1 4 ... ... (f ), (hopf ) = c ... ... π π π ... π π ... ... 1 4 ... c ... ... π ... π ... (π) = c ... π ... (i1), (f ), (h) = ... 1 4 ... ... π 1 16 ... ... (i1), (i2) = c ... ... π ... π ... π ... ... 1 16 c ... ... Hence, π ... ... (f ) = 1 16 π ... π ... 1 16 ... (f ), (h) 1 = 16 π ... π ... π (hopf ) = 0, ... ... π (f ), (π) 1 = − 16 π π , ... π 1 16 1 16 ... π π π ... (f ), (π) 1 = − 16 π ... That it TTTN [2, 1, ·] = . ... 1 1 16 0 −1 . If b = T2 , then π ... ... 1 4 c π ... ... ... ... (h), (π) 1 = 4 ... π π ... c π ... π ... ... (f ), (π) 1 = 4 π ... ... ... π ... ... ... (π), (f ) = π π π ... ... c π π π π ... ... ... π π ... (i1), (f ), (h) = ... π π π ... 1 4 π c π π ... ... ... (f ), (hopf ) = ... ... π π 1 16 π c π π ... ... ... 1 16 ... c π π ... ... π Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 44 Hence, π π π ... ... (f ), (hopf ) 1 = 16 1 16 π = 0, ... ... π π π ... π 1 16 ... (f ), (π) = π 1 16 ... π , π ... π π π ... π 1 16 (f ), (hopf ) 1 = 16 π ... π ... π = 0. π ... π π π That it 1 0 16 TTTN [2, 2, ·] = 0 .ow 1 If b = T3 , then π ... π ... 1 4 ... (h) = c π ... ... ... ... ... 1 4 ... π 1 4 ... ... π 1 4 π ... π ... ... c π ... π ... π ... ... ... (f ), (hopf ) = c π π ... (f ), (π) 1 = − 16 ... π ... (π) = c π ... π ... (i1), (f ), (h) = ... π ... ... π ... ... ... π ... π 1 16 c π π ... ... (i1), (i2) = ... π ... ... π 1 16 π ... π ... c π π ... ... c π π ... ... Hence, π ... 1 − 16 π ... π ... 1 − 16 π ... π ... π , ... ... (f ), (h) 1 = − 16 π 1 − 16 ... (f ) 1 = − 16 (hopf ) = 0, ... ... (f ), (π) 1 = 16 π π π ... That it TTTN [2, 3, ·] = ... π π π (f ), (π) 1 = 16 ... 1 −1 16 π . ... 0 1 . By now, we have proved that  1 1  0 TTTN [2, ·, ·] = 16 −1 Accepted in  0 −1 1 0 . 0 1 Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. (40) 45 Now, let us consider the case when a = T3 . When a = T3 , we have ... ... ... ... π ... b ... ... ... ... (f ), (hopf ) 1 = 4 c ... ... ... ... ... ... ... π b ... ... (i1), (i2) 1 = 4 c ... Now, a is disconnected with b and c. And the parts of b Hence, we have  1 0 1  0 1 TTTN [3, ·, ·] = 16 1 0 ... ... ... ... ... ... ... π b ... . c ... ... ... and c are the same as that of a = T1 .  1 0 . 1 (41) Now we are going to prove that there is a lower bound for the variance of gradients in the tree tensor network ansatz. Theorem 6. For tree tensor network ansatz shown in (22), if H = k0 I + k1 X + k2 Y + k3 Z, then we have Var ∂ hHi ∂θ ≥ k12 + k32 ˜ k2 ˜ I(u1 , . . . , un ) + 22 I(w 1 , . . . , wn ), 2 n n (27) − ˜ 1 , . . . , un ) is defined in Eq. (26). And I˜ is a 3n dimenfor some uj , wj ∈ {v1,3 , v2 , v1,3 }. Here I(u sional tensor which only depends on the input state. If the input state is ρ, then I˜ is defined as follows. −π 2 π 2 ρ π 2 Ta1 −π 2 π 2 Ta2 π 2 −π 2 .. . −π 2 .. . .. . I˜a1 ,...,an = π 2 π 2 .. . ρ .. . −π 2 , where aj ∈ {1, 2, 3}. Tan −π 2 Proof. We first prove that ˜ 1 , . . . , un ) ≥ 0, I(u where Accepted in   0 v2 = 1 , 0 v1,3 − uj ∈ v2 , v1,3 , v1,3 ,   1 = 0 , 1  − v1,3  1 =  0 . −1 Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 46 − Note that if u1 is v1,3 or v1,3 , then −π 2 −π 2 π 2 .. . ρ .. . −π 2 −π 2 π 2 T1 + T3 −π 2 π 2 ... ˜ 1,3 , . . . ) = I(v π 2 π 2 π 2 .. . −π 2 .. . ρ .. . −π 2 π 2 ρ π 2 T1 − T3 −π 2 π 2 ... π 2 −π 2 .. . .. . .. . ˜ − ,...) = , I(v 1,3 π 2 ... −π 2 .. . π 2 ρ .. . −π 2 . ... −π 2 By T1 + T3 = + = \|00i h00\| + \|10i h10\| + \|01i h01\| + \|11i h11\| , π = (\|0i h0\| + \|1i h1\|) ⊗ (\|0i h0\| + \|1i h1\|) = T1 − T3 = + , = \|00i h00\| − \|10i h10\| − \|01i h01\| + \|11i h11\| π π = (\|0i h0\| − \|1i h1\|) ⊗ (\|0i h0\| − \|1i h1\|) = \|0i h1\| T2 = = π π , \|1i h0\| + \|0i h1\| \|1i h0\| , ˜ 1 , . . . , un ) as a sum of squares. Thus, we have proved that we can expand I(u ˜ 1 , . . . , un ) ≥ 0. I(u Now let us consider the lower bound of Var ∂hHi . By the graph-like ZX-diagram just ob∂θ   H̃1 tained, we have H̃ = H̃2  which is defined as follows. H̃3 π 2 H̃j = H −π 2 , for j = 1, 2, 3. Tj −π 2 H π 2 Suppose that H = k0 I + k1 X + k2 Y + k3 Z. Then we can obtain − H̃ = 2k02 v1,3 + 2(k12 + k32 )v2 + 2k22 v1,3 . (42) And by the definition of TTTN , we can obtain Eq. (25). Hence, we can expand the variance as X ∂ hHi ˜ 1 , . . . , un ), a(u1 , . . . , un ) · I(u Var = ∂θ − uj ∈{v2 ,v1,3 ,v1,3 } Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 47 where a(u1 , . . . , un ) is a non-negative number. We will prove that there exists one choise of (u1 , . . . , un ) such that 1 a(u1 , . . . , un ) ∈ Ω( ). poly(n) When we use Eq. (25) to expand the variance, the only case that will cause a coefficient < 1 is the case when we use the second equation in Eq. (25). Hence, the coefficients a(u1 , . . . , un ) only depend on the number of times that we use the second equation. And it depends on the location of θ. The worst case is that θ is the parameter of the first gate applying to the first qubit. In this case, we need to use the second equation in Eq. (25) for 2 log(n) − 1 times. That means we have a lower bound for the variance k22 ˜ k2 + k2 ˜ ∂ hHi ≥ 1 2 3 I(u Var 1 , . . . , un ) + 2 I(w1 , . . . , wn ), ∂θ n n − for some uj , wj ∈ {v1,3 , v2 , v1,3 }. Note that I(u1 , . . . , un ) only depends on the input state ρ. If ˜ 1 , . . . , un ) ∈ Ω( I(u 1 1 ˜ 1 , . . . , wn ) ∈ Ω( ) or I(w ), poly(n) poly(n) then there exists no BP in the tree tensor network ansatz. G.3 QCNN Theorem 7. For the QCNN ansatz shown in (28), if H = k0 I + k1 X + k2 Y + k3 Z, then we have k22 + k32 ˜ k2 ˜ I(u1 , . . . , un ) + 19 I(w (30) 1 , . . . , wn ), 9 n n − for some uj , wj ∈ {v1,3 , v2 , v1,3 }. Here I˜ is a 3n dimensional tensor which only depends on the input state ρ. Var ∂ hHi ∂θ ≥ .. . ρ .. . Ta1 Ta2 I˜a1 ,...,an = .. . .. . ρ .. . , where aj ∈ {1, 2, 3}. Tan Proof. The proof is similar to that of the tree tensor network ansatz (Theorem 6), so we will only give a sketch of the proof. We also have ˜ 1 , . . . , un ) ≥ 0, I(u − uj ∈ {v1,3 , v2 , v1,3 }. From the graph-like ZX-diagram, we have H H̃j = Tj , for j = 1, 2, 3. H Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 48 Hence, − H̃ = 2k02 v1,3 + 2(k12 + k22 )v2 + 2k32 v1,3 . We will analyze each term of H̃ in the variance. Using Eq. (29), the term 2k02 v1,3 will become 0 by P2 v1,3 = 0. The terms 2(k12 + k22 )v2 and − 2k32 v1,3 will generate terms containing v2 after expanding using Eq. (29). And each time after generating terms containing v2 , a coefficient ≥ 81 will be multiplied to the variance. Hence, if we want to bring v2 to P2 , we need to generate terms containing v2 for l times, where l is a path from the location of v2 to the location of P2 . By the structure of the QCNN ansatz, we have l ≥ 3 log(n). It will generated a coefficient ≥ 1 . 83 log(n) Hence we have Var ∂ hHi ∂θ ≥ k12 ˜ k22 + k32 ˜ I(u , . . . , u ) + I(w1 , . . . , wn ), 1 n n9 n9 − for some uj , wj ∈ {v1,3 , v2 , v1,3 }. Accepted in Quantum 2021-05-28, click title to verify. Published under CC-BY 4.0. 49
https://openalex.org/W2152010581	https://lirias.kuleuven.be/retrieve/534036	English	null	Delay and secrecy: does industry sponsorship jeopardize disclosure of academic research?	Industrial and corporate change	2,014	public-domain	7,589	Faculty of Business and Economics Delay and secrecy: Does industry sponsorship jeopardize disclosure of academic research? Dirk Czarnitzki, Christophe Grimpe and Andrew A. Toole OR 1101 Non-technical summary It has almost become conventional wisdom that the viability of modern open science norms and practices depends on public disclosure of new knowledge, methods, and materials. At the same time, long-run trends suggest a broad shift is taking place in the institutional financing structure that supports academic research. According to data compiled by the OECD, industry sources are financing a growing share of academic research while “core” public funding is generally shrinking. This ongoing shift from public to private sponsorship is a cause for concern because these sponsorship relationships are fundamentally different. Available evidence suggests that industry financing does not simply replace dwindling public money, but imposes additional restrictions on academic researchers. In particular, industry sponsors frequently limit disclosure of research findings, methods, or materials by delaying or banning public release. Hence, in this paper we examine the relationship between industry sponsorship and restrictions on disclosure using individual-level data on German academic researchers. Our results show a strong positive relationship between the degree of publication restrictions and the share of industry sponsorship. The percentage of respondents who reported higher secrecy (partial or full) is significantly larger for industry sponsored researchers than it is for researchers with other extramural sponsors, 41% and 7% respectively. Holding other factors constant, a 10% increase in a researcher’s share of industry sponsorship increases the probability that he or she will experience publication delay or secrecy by 4.4 percentage points. In this respect, our results shed light on an important challenge facing policymakers. Understanding the trade-off between public and private sponsorship of academic research involves gauging the impact of disclosure restrictions on the quantity, quality, and evolution of academic research to better understand how these restrictions may ultimately influence innovation and economic growth. Delay and secrecy: Does industry sponsorship jeopardize disclosure of academic research? Dirk Czarnitzkia,b,c, Christoph Grimped, and Andrew A. Toolec,e a) K.U.Leuven, Dept. of Managerial Economics Strategy and Innovation, Leuven (Belgium) b) Centre for R&D Monitoring (ECOOM) at K.U.Leuven c) Centre for European Economic Research (ZEW), Mannheim (Germany) d) Copenhagen Business School, Copenhagen (Denmark) e) U.S. Department of Agriculture, Economic Research Service, Washington DC (United States) a) K.U.Leuven, Dept. of Managerial Economics Strategy and Innovation, Leuven (Belgium) b) Centre for R&D Monitoring (ECOOM) at K.U.Leuven c) Centre for European Economic Research (ZEW), Mannheim (Germany) d) Copenhagen Business School, Copenhagen (Denmark) e) U.S. Department of Agriculture, Economic Research Service, Washington DC (United States) a) K.U.Leuven, Dept. of Managerial Economics Strategy and Innovation, Leuven (Belgium) b) Centre for R&D Monitoring (ECOOM) at K.U.Leuven c) Centre for European Economic Research (ZEW), Mannheim (Germany) d) Copenhagen Business School, Copenhagen (Denmark) e) U.S. Department of Agriculture, Economic Research Service, Washington DC (United States) January 2011 Abstract The viability of modern open science norms and practices depend on public disclosure of new knowledge, methods, and materials. Aggregate data from the OECD show a broad shift in the institutional financing structure that supports academic research from public to private sponsorship. This paper examines the relationship between industry sponsorship and restrictions on disclosure using individual-level data on German academic researchers. Accounting for self- selection into extramural sponsorship, our evidence strongly supports the perspective that industry sponsorship jeopardizes public disclosure of academic research. Keywords: open science, research funding, industry sponsorship, disclosure, secrecy JEL-Classification: O31; O32; L33 Keywords: open science, research funding, industry sponsorship, disclosure, secrecy JEL-Classification: O31; O32; L33 Contact details: Dirk Czarnitzki, K.U.Leuven, Dept. of Managerial Economics, Strategy and Innovation Naamsestraat 69, 3000 Leuven, Belgium Phone: +32 16 326 906, Fax: +32 16 326 732, E-Mail: dirk.czarnitzki@econ.kuleuven.be Christoph Grimpe, Copenhagen Business School, Dept. of Innovation and Organizational Economics, Kilevej 14A, 2000 Frederiksberg, Denmark Phone: +45 3815 2530, Fax: +45 3815 2540, E-Mail: christoph@cbs.dk Andrew A. Toole, USDA, Economic Research Service 1800 M Street NW, Washington DC 20036, USA Phone: +1 202 694 5208, Fax: +1 202 694 5776, E-Mail: atoole@ers.usda.gov Andrew A. Toole, USDA, Economic Research Service 1800 M Street NW, Washington DC 20036, USA Phone: +1 202 694 5208, Fax: +1 202 694 5776, E-Mail: atoole@ers.usda.gov 1 OECD data show the share of industry sponsorship has grown in all countries since 1980, although this share is still relatively small. General university funds (“core” funds) as a share of civilian government budget appropriations fell from 26% in 1995 to 23% in 2007 (OECD, Main Science and Technology Indicators, 2010). Acknowledgements and disclaimer: g Czarnitzki gratefully acknowledges funding from the Flemish Science Foundation (FWO grant: G.0501.11). The views expressed in this article are the authors’ and do not necessarily represent the views of the U.S. Department of Agriculture or the Economic Research Service. Czarnitzki gratefully acknowledges funding from the Flemish Science Foundation (FWO grant: G.0501.11). The views expressed in this article are the authors’ and do not necessarily represent the views of the U.S. Department of Agriculture or the Economic Research Service. 1 Introduction Long-run trends suggest a broad shift is taking place in the institutional financing structure that supports academic research. According to data compiled by the OECD, industry sources are financing a growing share of academic research while “core” public funding is generally shrinking.1 This ongoing shift from public to private sponsorship is a cause for concern because these sponsorship relationships are fundamentally different. Available evidence suggests that industry financing does not simply replace dwindling public money, but imposes additional restrictions on academic researchers. In particular, industry sponsors frequently limit disclosure of research findings, methods, or materials by delaying or banning public release (Blumenthal et al. 1996, Cohen et al. 1998, Thursby and Thursby 2007). Recent economic research highlights why public disclosure of academic research is important. Disclosure permits the “stock of public knowledge” to be cumulative, accessible, and reliable. It limits duplication of research efforts, allows new knowledge to be replicated and verified by professional peers, and permits access and use by other researchers which enhances opportunities for complementary research (Dasgupta and David 1994). In recent work, Murray et al. (2009) found that greater access to ideas and materials in academic research not only increased incentives for direct follow-on research, but led to an increase in the diversity of research by increasing the number of experimental research lines. Mukherjee and Stern (2009), who examined the theoretical conditions supporting “open science” versus “secrecy”, stressed that maintaining and growing the stock of public knowledge requires a limit on the private financial returns obtained through secrecy. 1 This paper examines the relationship between industry sponsorship and restrictions on disclosure using individual-level data on German academic researchers. Germany is an apt setting for examining this relationship. It has a strong tradition of public financial support for academic research and, among advanced economies, Germany experienced the most dramatic growth in its share of industry sponsorship, a 13.4 percentage point increase from 1995 to 2007 (OECD 2010). German academic researchers were surveyed about the degree of disclosure restrictions on publications experienced during sponsored research projects. To examine if industry sponsorship jeopardizes disclosure of academic research, we modeled the degree of restrictiveness (i.e. delay, partial secrecy, and complete secrecy) as a function of the researcher’s budget share financed by industry. 1 Introduction Using the share of industry sponsorship mirrors the shift in institutional financing highlighted by the OECD data and advances the literature beyond the use of simple indicator variable formulations. Our models also take into account non-industry extramural sponsorship, personal characteristics, research characteristics, institutional affiliations, and scientific fields of study. Both the descriptive and regression results show a strong positive relationship between the degree of publication restrictions and the share of industry sponsorship. The percentage of respondents who reported higher secrecy (partial or full) is significantly larger for industry sponsored researchers than it is for researchers with other extramural sponsors, 41% and 7% respectively. Holding other factors constant, a 10% increase in a researcher’s share of industry sponsorship increases the probability that he or she will experience publication delay or secrecy by 4.4 percentage points, which is a 17.4% increase in the predicted probability that an “average” researcher will experience publication delay or secrecy. This result is robust to the possibility that researchers self-select into extramural sponsorship and to the possibility that the share of industry sponsorship is endogenous due to unobserved variables. Based on our analysis, the shift from public to private sponsorship 2 2 seen in the OECD aggregate data reflect changes in the microeconomic environment shaping incentives for disclosure by academic researchers. On average, academic researchers are willing to restrict disclosure in exchange for financial support by industry sponsors. Our results shed light on an important challenge facing policymakers. Understanding the trade-off between public and private sponsorship of academic research involves gauging the impact of disclosure restrictions on the quantity, quality, and evolution of academic research to better understand how these restrictions may ultimately influence innovation and economic growth. The rest of the paper is organized as follows. The next section summarizes the current literature on sponsorship of academic research. The researcher-level data, estimation issues and methods are discussed in section 3. The results and concluding remarks appear in sections 4 and 5, respectively. p y p practice the “priority” reward system and support the professional ethos associated with the community of academic scientists as articulated by sociologist Robert K. Merton (see, for instance, Merton 1973, Dasgupta and David 1994, Stephan 1996). David (2004) highlights the norms of “universalism” (open entry and discourse) and “communism” (full and open disclosure) as particularly relevant to openness. 2 Open science is broadly associated with universities and other not-for-profit research institutions th y p p 4 We did not find any studies that systematically analyze the contractual terms of scientific or military- oriented contracts from state sponsors or private foundations. Cohen at al. (1998) reported that 53 percent of university-industry research centers allowed firms to impose publication delays and 35 percent allowed firms to impose secrecy through the deletion of information before publishing. For a sample of 130 French public labs that have 875 industrial partners, Goddard and Isabelle (2006) reported that 55% allowed contract provisions to delay publication and 53% allowed contract provisions to suppress information from publication. 3 Gans et al. (2010) use a theoretical framework to examine the “regimes” of disclosure under private industry sponsorship. 2 Sponsorship of Academic Research More than a simple transfer of funds, sponsorship of academic research involves contractual relationships that often specify the nature, ownership, and control rights for research findings, methods, or materials. While these contracts are necessarily “incomplete” due to a number of informational problems, they reflect negotiated outcomes between sponsors and researchers that can have far reaching implications for the organization and conduct of academic research. Historically, as argued by David (2004), sponsorship relationships helped to transform the norms, incentives, and organizational structures of scientific inquiry from a system dominated by secrecy to a modern “open science” system characterized by rapid public disclosure of new knowledge.2 Relative to a secrecy system, open science is 3 3 considered to be an efficient and welfare enhancing system for the production of a cumulative, accessible, and reliable stock of public scientific and technical knowledge (Dasgupta and David 1994, Mukherjee and Stern 2008). As history suggests, the objectives and institutional reward systems practiced by different sponsors may influence the norms, incentives, and organizational structures of academic research differently. “Public” sponsors such as science-oriented state agencies or private foundations focus on advancing public knowledge. These institutions expect sponsored research to result in new knowledge that is publicly disclosed through various channels including publication. In fact, public support often depends on a satisfactory performance as indicated by a researcher’s publication output. Advancing public knowledge through disclosure is consistent with the “priority” reward system and reinforces open science norms and behaviours. In contrast, “private” sponsors such as military-oriented state agencies or private industry focus on extracting rents from new knowledge by restricting public disclosure.3 Advocating restrictions on disclosure is likely to have a corrosive effect on open science. One approach to learning about sponsorship relationships is to ask the sponsors. We found two published studies that surveyed private firms about the characteristics of sponsored research contracts.4 Based on survey responses from 210 life science companies, Blumenthal et al. (1996) found evidence of both publication delay and secrecy (nondisclosure) restrictions on information resulting from academic research. For instance, fifty-eight percent of the companies typically required researchers to keep information confidential for 4 more than six months. Using survey responses from 112 firms engaged in university licensing, Thursby and Thursby (2007) reported that ninety percent of the university contracts included publication delay clauses. 5 Using an industry funding indicator, Walsh et al. (2007) found no relationship between industry funding and compliance with requests for research inputs among biomedical researchers performing genomic and proteomic-related research. 6 Major research institutions in Germany are not only universities but other public research institutions that have many branches in a variety of different scientific disciplines. For instance, the Fraunhofer Society has 59 institutes in Germany with about 17,000 employees, the Max-Planck Society has 76 institutes with about 12,000 employees. The Leibniz Association employs 16,100 people in 86 research centres.The Helmholtz Association has about 30,000 employees in 16 research centres. University professors are frequently heads of research groups at these institutions, i.e. they have a university affiliation but are typically on leave full-time when working with the research institutes. 2 Sponsorship of Academic Research A more direct approach, which is followed in this paper, is to ask academic researchers about any disclosure restrictions they experienced when undertaking extramurally sponsored research. We found six studies that used researcher-level survey data to shed light on the relationship between industry sponsorship and disclosure. In five of these studies, Blumenthal and colleagues described the results of three separate life science faculty surveys conducted between 1985 and 2000 (Blumenthal et al.1986, 1996a,b , 1997, 2006; Campbell et al. 2002). Their findings show that researchers with at least one industry sponsored project are more likely to report industry ownership of research results, pre-publication review, publication delays, and secrecy to protect proprietary information.5 Taking a slightly different perspective, Hong and Walsh (2009) ask researchers how “safe” they feel about discussing their current work with non-collaborating colleagues. For their full sample, academic researchers with at least one industry sponsored project were more likely to feel “unsafe” (interpreted as being more secretive). Our analysis extends this mostly descriptive literature in three ways. First, we expand the scope of evidence by analyzing academic researchers who work outside the United States in a broader set of scientific fields and institutional settings. All of the prior work analyzing how industry sponsorship influences disclosure looked at U.S. researchers working in a handful of scientific fields at American universities. Dasgupta and David (1994) highlighted how alternative institutional settings may influence a researcher’s choice about disclosure. Second, to account for heterogeneity in the degree of industry sponsorship, our analysis uses the budget share of industry support instead of a simple indicator variable formulation. 5 5 Third, our empirical analysis is the first to address potential self-selection by academic researchers into extramural sponsorship and to use an instrumental variables method to account for unobserved factors. Third, our empirical analysis is the first to address potential self-selection by academic researchers into extramural sponsorship and to use an instrumental variables method to account for unobserved factors. Major research institutions in Germany are not only universities but other public research institutions Major research institutions in Germany are not only universities but other public research institution ave many branches in a variety of different scientific disciplines. For instance, the Fraunhofer Society ha i i G i h b 17 000 l h M Pl k S i h 76 i i i h b 3.1 Data To analyze the relationship between industry sponsorship and disclosure restrictions on publications we used a researcher-level database. In 2008, the Centre for European Economic Research (ZEW) undertook an online survey of German academic researchers. The survey population was defined to include German researchers who held a Ph.D. degree and worked at either a university or a not-for-profit research institution. Information on university affiliated researchers was collected from a register of German professors (“Hochschullehrerverzeichnis”) which excludes universities of applied sciences that focus on teaching. For Germany’s largest not-for-profit research institutions (Fraunhofer Society, Max Planck Society, Helmholtz Association, Leibniz Association), information on affiliated researchers was collected using internet searches.6 In total, the survey led to 1,404 valid responses. After dropping observations with missing values in the variables of interest for this study, we end up with a final sample of 1,060 observations. The dependent variable is drawn from a question that asked respondents to indicate the degree of disclosure restrictions on publications resulting from extramural sponsorship. It 6 6 asked: “Has the funding of your research by public or private extramural sponsors resulted in: (a) a complete ban on publishing research; (b) a partial ban on publishing research; or (c) a delay in publishing research due to contractual agreements.” Respondents could check one of three boxes for each outcome indicating “yes”, “no”, or “not relevant”. For the empirical analysis, we coded two alternative dependent variables based on this question. The first is a dummy variable indicating the respondent experienced any delay or secrecy due to extramural funding. This binary variable takes the value of one if the respondent indicated “yes” to any of the outcomes. The second is an ordered variable with four categories indicating higher levels of secrecy: no delay or ban, delay of publications, partial ban on publication, and total ban on publication. The main explanatory variable in the analysis is the share of the researcher’s extramural budget funded by private industry sponsors. This variable was constructed using two survey questions. The first question asked the researcher to report his or her total extramural budget over the five year period from 2002 to 2006. Conditional on having extramural funding, the second question asked the researcher to provide the source (as a percentage) of his or her total extramural budget over the five year period. The share of industry sponsorship is the proportion of researcher’s budget funded by private sector organizations. 7 Define Y0i as the non-disclosure (or secrecy) outcome for academic researcher i in the state of not receiving extramural funding and define Di as the funding indicator: Di=1 when funded and Di=0 when not funded. Selection bias is positive when those who actually received funding value non-disclosure more in the unfunded state: 0 0 [ \| 1] [ \| 0 ]0 i i i i E Y D E Y D = − = > . 3.1 Data We used a number of other variables collected through the survey as controls or instrumental variables in the empirical analysis. These variables are grouped into four categories: research characteristics, personal characteristics, institutional affiliations, and scientific fields of study. Research characteristics relate to the individual’s position at the research institution, his or her total extramural funding, publications, patent applications, and his or her opinion about the peer review process. Personal characteristics include the individual’s age and gender, and whether he or she is tenured. Institutional affiliations cover 7 7 universities, the four major not-for-profit public research institutions mentioned above, and a catchall group for all other affiliations. Further, the researchers were grouped into four broad scientific fields specified as life sciences, natural sciences, engineering, and social sciences. 8 In addition to being statistically valid, other research on academic sharing behaviors and attitudes toward cooperation with private firms do not find gender to be significant (Haeussler 2011, Audretsch et al. 2010). Haeussler (2011) finds that a researcher’s age decreases the percent of requested information that is shared. 3.2 Methods In the ideal case, we would use an experimental design to identify the causal effect of industry sponsorship on disclosure choices by academic researchers. For instance, one might randomly assign industry sponsorship to academic researchers, allow for negotiation and research, and observe changes in disclosure. This type of experiment would eliminate any bias due to self-section by academic researchers into funding. Our survey data, however, were not collected using a randomized experimental design. With our data, we do not observe the choice by an academic researcher to seek or accept extramural sponsorship and this suggests simple Probit and ordered Probit estimators could be biased by self-section. Academic researchers who received extramural funding are probably different from those who did not receive funding. For instance, researchers who are less concerned about disclosure or perform more “applied” research may be more willing to accept extramural sponsorship that imposes disclosure restrictions.7 This would lead to an upward bias. To address this possibility, our empirical analysis includes Probit and ordered Probit models accounting for selection into funding. In these models, the academic researcher’s age and gender serve as exclusion restrictions that predict the receipt of extramural funding, but do not influence the researcher’s disclosure outcome. These exclusion restrictions are supported 8 8 statistically. Neither gender nor age significantly influence disclosure restrictions on publications once other factors are held constant.8 While our survey instrument provides fairly rich researcher-level information, we do not observe the researcher’s perception of scientific competition within his or her field. Current studies find that greater scientific competition is associated with greater secrecy (Hong and Walsh 2009, Haeussler 2011). Relevant for this analysis, however, is the relationship between scientific competition and extramural sponsorship. Scientific competition may either increase or decrease the attractiveness of extramural sponsorship. On the one hand, researchers feeling intense competition for priority may be less willing to accept third party disclosure restrictions. On the other hand, extramural sponsorship may provide financial resources that help the researcher get work done faster. The direction of potential bias could go either way. In our analysis, we included scientific field dummy variables to capture differences in the level of scientific competition across fields. Standard descriptive statistics for the variables used in the regression models are reported in Table 1. 9 Table 1: Regression descriptive statistics Table 1: Regression descriptive statistics Data used in selection equation (N = 1,060) Dependent variable Mean Std. Dev. 3.2 Methods Min Max Any extramural sponsorship 0.808 0.394 0 1 Research Characteristics Research group leader 0.722 0.448 0 1 Journal publications 21.420 26.920 0 178 Patent applications 0.749 2.101 0 24 Personal Characteristics Tenure 0.842 0.364 0 1 Female 0.148 0.355 0 1 Age 49.531 8.225 28 74 Institutions University 0.586 0.493 0 1 Fraunhofer Society 0.051 0.220 0 1 Max Planck Society 0.085 0.279 0 1 Helmholtz Association 0.165 0.371 0 1 Leibniz Association 0.070 0.255 0 1 Other Institution 0.087 0.282 0 1 Science Fields Life sciences 0.305 0.461 0 1 Natural sciences 0.292 0.455 0 1 Engineering 0.192 0.394 0 1 Social sciences 0.211 0.408 0 1 Data used in withholding regressions (N = 856) Dependent variable Mean Std. Dev. Min Max Delay or ban (ordered variable) 0.560 0.957 0 3 Delay or ban (dummy variable) 0.289 0.453 0 1 Research Characteristics Industry share 0.111 0.212 0 1 Total budget (million Euro) 1.695 3.738 0.001 75 Research group leader 0.792 0.406 0 1 Journal publications 23.557 28.125 0 176 Patent applications 0.836 2.215 0 24 Personal Characteristics Tenure 0.864 0.342 0 1 Institutions University 0.613 0.487 0 1 Fraunhofer Society 0.051 0.221 0 1 Max Planck Society 0.074 0.261 0 1 Helmholtz Association 0.152 0.359 0 1 Leibniz Association 0.065 0.247 0 1 Other Institution 0.084 0.278 0 1 Science Fields Life sciences 0.299 0.458 0 1 Natural sciences 0.292 0.455 0 1 Engineering 0.210 0.408 0 1 Social sciences 0.199 0.399 0 1 10 4.1 Main results Table 2 presents descriptive statistics for the full sample of German researchers and for subsamples broken out by extramural funding. Most respondents indicated some extramural sponsorship (81%) with nearly one third having industry sponsorship. On average, researchers with industry sponsorship had larger research budgets, published more in journals, and applied for more patents. Personal characteristics of researchers were similar except for a significant drop in the proportion of females for the group of industry sponsored researchers. A greater proportion of university and Fraunhofer affiliated researchers reported industry sponsorship while the proportion of industry sponsored researchers is quite small for affiliates of the Max Planck Society, which is strongly oriented toward basic research. Among the science fields, industry sponsorship was greatest in engineering. Next we examined the average values of the covariates for different levels of restriction (no delay or secrecy, delay, partial or full secrecy) grouped by extramural, industry, and non-industry sponsorship as shown in Table 3. Out of the 341 respondents that reported some industry sponsorship, 50% reported no delay or secrecy on publications, 9% reported a delay, and 41% reported a partial or full secrecy on publications. The percentage of respondents who reported the higher secrecy (partial or full) is significantly (at the 1% level) larger for industry sponsored researchers than it is for researchers with non-industry sponsorship, 41% and 7% respectively. The positive association between industry share and level of secrecy is already evident in Table 3. As one looks across the columns from no restrictions (no delay/ban) to higher secrecy (partial/full), researchers reported larger industry sponsorship shares. Higher secrecy was reported more frequently by researchers affiliated with applied public research organizations such as the Fraunhofer Society and Helmholtz 11 11 Association. Among the science fields, the proportion who reported partial or total secrecy on publishing is greatest in engineering. 4.1 Main results Table 2: Sample averages for all covariates by extramural sponsorship All No External Any External Industry Respondents Funding Funding Funding Total Observations (% of all obs) 1060 204 (19%) 856 (81%) 341 (32%) Report a delay or ban of research - 0.289 0.504 Research Characteristics Industry share - - 0.111 0.279 Total budget (million Euro) - - 1.695 2.163 Research group leader 0.722 0.426 0.792 0.862 Journal publications 21.42 12.451 23.557 26.595 Patent applications 0.749 0.382 0.836 1.537 Personal Characteristics Tenure 0.842 0.75 0.864 0.918 Female 0.148 0.176 0.141 0.088 Age 49.5 50.4 49.3 50.2 Institutions University 0.586 0.471 0.613 0.642 Fraunhofer Society 0.051 0.049 0.051 0.117 Max Planck Society 0.085 0.132 0.074 0.035 Helmholtz Association 0.165 0.221 0.152 0.106 Leibniz Association 0.07 0.088 0.065 0.053 Other Institution 0.087 0.098 0.084 0.073 Science Fields Life sciences 0.305 0.328 0.299 0.279 Natural sciences 0.292 0.294 0.292 0.214 Engineering 0.192 0.113 0.21 0.378 Social sciences 0.211 0.265 0.199 0.129 Table 2: Sample averages for all covariates by extramural sponsorship All No External Any Table 2: Sample averages for all covariates by extramural sponsorship 12 Table 3: Sample averages by level of publication restriction and type of extramural sponsorship Any External Funding (N=856) Industry Funding (N=341) Non-Industry Funding (N=515) No Delay or Ban Delay Partial or Full Ban No Delay or Ban Delay Partial or Full Ban No Delay or Ban Delay Partial or Full Ban Total Observations (%) 609 (71%) 69 (8%) 178 (21%) 169 (50%) 32 (9%) 140 (41%) 440 (85%) 37 (7%) 38 (7%) Research Characteristics Industry share 0.067 0.116 0.26 0.241 0.25 0.331 - - - Total budget (million Euro) 1.517 1.633 2.327 1.993 1.938 2.42 1.334 1.370 1.982 Research group leader 0.788 0.841 0.787 0.882 0.938 0.821 0.752 0.757 0.658 Journal publications 25.504 27.507 15.365 34.64 34.906 14.979 21.993 21.108 16.789 Patent applications 0.473 1.551 1.803 0.941 2.031 2.143 0.293 1.135 0.553 Personal Characteristics Tenure 0.856 0.884 0.888 0.911 0.969 0.914 0.834 0.811 0.789 Female 0.151 0.159 0.101 0.089 0.1875 0.064 0.175 0.135 0.237 Age 49.2 50 49.2 50.4 51.2 49.7 48.9 48.9 47.3 Institutions University 0.65 0.609 0.489 0.769 0.688 0.479 0.605 0.541 0.526 Fraunhofer Society 0.016 0.058 0.169 0.041 0.094 0.214 0.007 0.027 0 Max Planck Society 0.094 0.029 0.022 0.053 0.031 0.014 0.109 0.027 0.053 Helmholtz Association 0.146 0.13 0.180 0.065 0.063 0.164 0.177 0.189 0.237 Leibniz Association 0.062 0.072 0.073 0.036 0.094 0.064 0.073 0.054 0.105 Other Institution 0.076 0.116 0.101 0.059 0.063 0.093 0.082 0.162 0.132 Science Fields Life sciences 0.332 0.333 0.174 0.385 0.344 0.136 0.311 0.324 0.316 Natural sciences 0.322 0.304 0.185 0.213 0.25 0.207 0.364 0.351 0.105 Engineering 0.13 0.116 0.522 0.266 0.219 0.55 0.077 0.027 0.421 Social sciences 0.217 0.246 0.118 0.136 0.397 0.107 0.248 0.297 0.158 ages by level of publication restriction and type of extramural sponsorship Non-Industry Funding (N=515) 13 Our regression analysis begins by considering a binary outcome that indicates whether a researcher who was supported by an extramural sponsor experienced any type of publication delay or secrecy. 4.1 Main results Model A in Table 4 shows the results of a basic Probit regression that ignores selection into extramural funding. Model B controls for self-selection into extramural funding (see e.g. Wooldridge, 2002: 570, for technical details). Holding the size of the researcher’s extramural budget constant (as well as other factors), the share of industry sponsorship significantly increases the probability of publication delay or secrecy in both models. The correlation across equations in Model B, reported at the bottom of the table as rho, is negative and highly significant which indicates self-selection into extramural funding is important. Controlling for self-selection, the coefficient estimate on industry share is 18% smaller, but still highly significant. From Model B, a 10% increase in the industry share increases the probability that he or she will experience publication delay or secrecy by 4.4 percentage points. At the mean values of the covariates, the predicted probability of experiencing delay or secrecy increases by 17.4% from 0.253 to 0.297. This result supports concerns that industry sponsorship undermines the norms and practices of open science and jeopardizes the cumulative nature and reliability of public scientific and technical knowledge. A larger total extramural budget is also associated with greater publication restrictions. It is also informative to examine how other covariates influence publication delay or secrecy when the effects of industry share and extramural budget size are held constant. From Model B, research characteristics and institutional affiliations matter even after controlling for selection. A researcher who is a group leader or had more journal publications is less likely to experience delay or secrecy restrictions. Group leaders and productive researchers are likely to value disclosure more and possess more bargaining power with extramural sponsors. This is 14 consistent with Audretsch et al. (2010) who found group leadership to be associated with more cooperation experience and planned cooperation with private companies. consistent with Audretsch et al. (2010) who found group leadership to be associated with more cooperation experience and planned cooperation with private companies. Researchers who submit more patent applications are more likely to restrict publications either through delay or secrecy. Relative to university researchers, those affiliated with the Fraunhofer Society, Helmholtz and Leibniz Associations are more likely to experience publication delay or secrecy. 4.1 Main results Given our data, we cannot distinguish between an institutional “management” effect, reflecting the strength of the technology transfer capabilities at these institutions, versus an institutional “focus” effect, reflecting the relatively applied orientation of research at these institutions. As described in Section 2, most of the literature has focused on researchers in the life sciences and its subfields. Looking at Model A, our results indicate that the life sciences are not significantly more likely to experience delays or secrecy on publications relative to the base group of social scientists. Only engineering researchers are more likely to experience these publication restrictions. Interestingly, after controlling for selection into extramural sponsorship in Model B, engineering researchers are no longer significantly different from social scientists. 15 Table 4: Probit of Withholding Research (binary outcome) Model A Model B Variable Probit (no selection) Probit with Selection Second stage Probit with Selection First stage Industry share 1.427 * 1.168 * (0.235) (0.210) Total external funding 0.152 * 0.111 * (0.039) (0.033) Female 0.011 (0.116) Age 0.119 (0.053) Age-squared -0.001 * (0.0005) Research group leader 0.077 -0.352 * 0.736 * (0.137) (0.127) (0.106) Tenure -0.036 -0.091 0.209 (0.153) (0.126) (0.148) Journal publications -0.006 * -0.007 * 0.007 * (0.002) (0.002) (0.003) Patent applications 0.098 * 0.090 *** 0.057 * (0.023) (0.022) (0.031) Fraunhofer Society 0.595 0.542 -0.284 (0.247) (0.224) (0.233) Max Planck Society -0.380 -0.089 -0.334 ** (0.245) (0.202) (0.169) Helmholtz Association 0.221 0.238 * -0.236 * (0.151) (0.131) (0.140) Leibniz Association 0.423 0.368 -0.201 (0.200) (0.173) (0.185) Other institution 0.301 * 0.227 -0.006 (0.177) (0.157) (0.173) Life sciences -0.175 -0.130 -0.038 (0.155) (0.131) (0.139) Natural sciences -0.158 -0.201 0.146 (0.162) (0.137) (0.144) Engineering 0.390 0.181 0.448 * (0.161) (0.144) (0.164) Intercept -0.795 *** 0.042 -2.354 * (0.206) (0.190) (1.284) Log-Likelihood -415.788 -854.607 Equation corr (rho) - -0.916* # Observations 856 1060 Note: Standard errors in parentheses. * (*,) indicate a significance level of 1% (5%, 10%) Reference: male, non group leader, untenured, university, social scientist. Table 4: Probit of Withholding Research (binary outcome) Model A 16 Given the structure of the survey question, it is perhaps more natural to specify an ordered dependent variable with four categories (no delay or secrecy, delay of publications, partial secrecy on publication, and total secrecy on publication) indicating increasing levels of secrecy. 4.1 Main results Model A in Table 5 reports the ordered Probit results without accounting for selection. The signs and statistical significance of the explanatory variables are nearly identical to those presented in Table 4 Model A. When self-selection is taken into account (see Miranda and Rabe-Hesketh, 2006, for econometric details of the ordered Probit model with selection), as shown in Model B of Table 5, the results from the binary Probit analysis continue to hold. Once again, controlling for self-selection reduces the coefficient estimate on industry share, but it remains highly significant. 17 17 Table 5: Ordered Probit of Withholding Research Model A Model B Variable Ordered Probit (no selection) Ordered Probit with Selection Second stage Ordered Probit with Selection First stage Industry share 1.567 * 1.342 * (0.209) (0.189) Total external funding 0.153 * 0.129 * (0.037) (0.030) Female 0.006 (0.118) Age 0.134 (0.054) Age-squared -0.002 * (0.0005) Research group leader 0.030 -0.348 * 0.718 * (0.127) (0.126) (0.107) Tenure -0.069 -0.126 0.205 (0.144) (0.125) (0.147) Journal publications -0.006 * -0.007 * 0.008 * (0.002) (0.002) (0.003) Patent applications 0.065 * 0.053 * 0.043 (0.019) (0.018) (0.032) Fraunhofer Society 0.678 * 0.589 * -0.273 (0.202) (0.191) (0.230) Max Planck Society -0.380 -0.147 -0.354 (0.239) (0.205) (0.169) Helmholtz Association 0.251 * 0.261 ** -0.264 * (0.140) (0.126) (0.141) Leibniz Association 0.393 0.350 -0.221 (0.186) (0.168) (0.186) Other institution 0.287 * 0.210 -0.051 (0.164) (0.151) (0.171) Life sciences -0.131 -0.103 -0.034 (0.147) (0.130) (0.138) Natural sciences -0.169 -0.209 0.154 (0.154) (0.136) (0.144) Engineering 0.538 * 0.305 0.482 * (0.149) (0.143) (0.163) Intercept -2.665 (1.311) Log-Likelihood -642.906 -1,082.116 Equation corr (rho) - -0.834* # Observations 856 1060 Note: Standard errors in parentheses. * (,) indicate a significance level of 1% (5%, 10%). Reference: male, non group leader, untenured, university, social scientist Table 5: Ordered Probit of Withholding Research 18 4.2 Robustness test 4.2 Robustness test As a further check on the robustness of our main results, we implemented an instrumental variables (IV) approach to account for any endogeneity of industry budget share using an IV Probit model. A valid IV for industry share must be relevant (highly correlated with industry share) and exogenous (independent of unobserved factors influencing the degree of disclosure restrictions on publications). One survey question asked respondents “How do you evaluate the transparency of the referees’ decisions regarding your proposal to the 6th European Union Framework program?” Respondents had five choices on a Likert scale ranging from 1 for “very low” to 5 for “very high”. This question provides a relevant IV because those researchers who indicated a poor opinion of the EU proposal review process are more likely to consider industry as an alternative extramural sponsor. At the same time, a researcher’s opinion about referee reports is arguably independent of factors that influence his or her choice about disclosure. As a second IV, we used the researcher’s gender. These IVs were correlated with industry share in the first stage regression with an F-statistic of 5.3 and passed the over-identification test for exogeneity. The models used fewer observations (542) than the models reported above because responses about the refereeing outcomes were conditional on having applied to the 6th EU Framework program. The first regression reported in Table 6 is a standard binary Probit. The results are quite similar to earlier results except for the science fields. Researchers in the natural sciences are now significantly less likely to report publication restrictions than the base group of social scientists and engineers are no longer significantly different. When an IV Probit method is used as shown in Model B, the coefficient on industry share remains positive and significant. At the same time most of the research characteristics and institutional affiliations are no longer 19 significantly related to experiencing publication restrictions. This suggests the effect of industry sponsorship was partially absorbed by research characteristics and institutional affiliation covariates in earlier models. The IV Probit model also reveals more heterogeneity across science fields with researchers in both life sciences and natural sciences being significantly less likely to experience publication restrictions relative to social scientists. 4.2 Robustness test 20 20 Table 6: Probit of Withholding Research (binary outcome, all sponsors) Table 6: Probit of Withholding Research (binary outcome, all sponsors) Model A Model B Variable Probit IV Probit Industry share 1.815 4.620 (0.393) (2.182) Total external funding 0.137 * 0.152 * (0.050) (0.047) Research group leader 0.031 -0.094 (0.172) (0.196) Tenure 0.020 -0.081 (0.198) (0.206) Journal publications -0.005 ** -0.005 * (0.002) (0.002) Patent applications 0.081 * 0.044 (0.026) (0.047) Fraunhofer Society 0.671 0.036 (0.282) (0.661) Max Planck Society -0.396 -0.373 (0.307) (0.291) Helmholtz Association 0.309 * 0.290 (0.181) (0.178) Leibniz Association 0.670 *** 0.556 * (0.242) (0.285) Other institution 0.320 0.212 (0.211) (0.236) Life sciences -0.299 -0.359 * (0.205) (0.195) Natural sciences -0.447 -0.458 (0.213) (0.205) Engineering 0.027 0.430 (0.212) (0.435) Intercept -0.570 -0.388 (0.268) (0.333) Log-Likelihood -285.914 -35.757 # Observations 542 542 Note: Standard errors in parentheses. * (*,) indicate a significance level of 1% (5%, 10% Reference: male, non group leader, untenured, university, social scientist 5 Conclusion Using data obtained from individual academic researchers, our evidence strongly supports the perspective that industry sponsorship jeopardizes public disclosure of academic research. Firms expect proprietary benefits from their sponsorship relationships and realizing these benefits often 21 requires disclosure restrictions that academic researchers would not otherwise impose. Academic researchers who accepted industry sponsorship reported significantly more disclosure delays and greater secrecy. Importantly, both the descriptive statistics and the regression models indicate these delay and secrecy outcomes increase as the share of industry sponsorship grows. While we cannot unequivocally state that this association is causal, our empirical analysis offered significant advances in this direction. Selection models accounted for self-selection by academic researchers into extramural research funding and IV regression methods addressed concerns about confounding omitted characteristics or potential measurement error. Our microeconomic evidence provides a lens for interpreting the on-going aggregate shift in institutional financing that supports academic research. As revealed in Figure 1, country-level OECD data show the share of industry sponsorship is generally rising, although not universally or monotonically. Among OECD countries, Germany has the largest share of industry sponsorship, about 25% in 2007. Our researcher-level evidence from Germany suggests the aggregate shift does not simply represent the substitution of private money for public, but involves a real change in when or if academic research findings, methods, and materials are publicly disclosed. This interpretation is consistent with prior research that examined researchers working in a handful of science fields at American universities. It appears the adverse effect of industry sponsorship on disclosure of academic research is not country specific or university specific, although more research is clearly needed to understand how country-level and institution-level characteristics influence the relationship between sponsorship and disclosure. 22 Figure 1: Percentage of higher education and government R&D financed by industry 1981, 1995, 2007 Source: OECD, Main Science and Technology Indicators 2010 0 5 10 15 20 25 30 Germany France United Kingdom Italy Spain EU-27 EU-15 Australia Canada United States Total OECD 1981 1995 2007 Source: OECD, Main Science and Technology Indicators 2010 23 The challenge facing policymakers is to gauge the impact of disclosure restrictions on the quantity, quality, and evolution of academic research to better understand how these restrictions may ultimately influence innovation and economic growth. This is a significant challenge and our study only lays the groundwork for more research. Before policy recommendations can be made numerous follow-on questions must be answered. 5 Conclusion For instance: What is the quantity and nature of information delayed or withheld? How do these disclosure restrictions affect the access costs, fidelity, and use of ideas that compose the stock of public scientific and technical knowledge? How important is the information delayed or withheld for private returns? What are the net social costs or benefits of disclosure restrictions? At this stage of the research, policymakers should at least be aware that academic researchers are accepting disclosure restrictions in exchange for financial support by industrial sponsors. If, as David (1994) argued, sponsorship relationships played an important role in the emergence of open science, it is only 23 logical that sponsorship relationships are influential enough to undermine open science norms and practices. logical that sponsorship relationships are influential enough to undermine open science norms and practices. References Audretsch, D.B., W. Bönte, and S. Krabel (2010), Why do scientists in public research institutions cooperate with private firms?, DRUID Working Paper No. 10-27, Copenhagen. Blumenthal, D., E.G. Campbell, M.S. Anderson, N. Causino, and K. Seashore-Louis (1997), Withholding research results in academic life science, Journal of the American Medical Association 277(15), 1224-1228. Blumenthal, D., E.G. Campbell, N. Causino, and K. Seashore-Louis (1996), Participation of life- science faculty in research relationships with industry, The New England Journal of Medicine 335(23), 1734-1739. Blumenthal, D., E.G. Campbell, M. Gokhale, R. Yucel, B. Clarridge, S. Hilgartner, and N.A. Holtzman (2006), Data withholding in genetics and other life sciences: Prevalences and practices, Academic Medicine 81(2), 137-145. Blumenthal, D., N. Causino, E.G. Campbell, and K. Seashore-Louis (1996), Relationships between academic institutions and industry in the life sciences – an industry survey, The New England Journal of Medicine 334(6), 368-373. Blumenthal, D., M. Gluck, K. Seashore-Louis, M.A. Stoto, and D. Wise (1986), University- industry research relationships in biotechnology: Implications for the university, Science Magazine 232(4756), 1361-1366. 24 Campbell, E.G., J.S. Weissman, N. Causino, and D. Blumenthal (2000), Data withholding in academic medicine: characteristics of faculty denied access to research results and biomaterials, Research Policy 29(2) 303-312. Dasgupta, P., and P.A. David (1994), Toward a new economics of science, Research Policy 23(5), 487-521. David, P.A. (2004), Understanding the emergence of ‘open science’ institutions: functionalist economics in historical context, Industrial and Corporate Change 13(4), 571-589. Cohen, W.M., R. Florida, L. Randazzese, and J. Walsh (1998), Industry and the academy: Uneasy partners in the cause of technical advance, in: R.G. Noll (ed.), Challenges to Research Universities, Washington, DC: Brookings Institution Press. Gans, J.A., F.E. Murray, and S. Stern (2010), Contracting over disclosure of scientific knowledge: Intellectual property and academic freedom, mimeo, Cambridge. Geuna, A. (2001), The changing rationale for European university research funding: Are there negative unintended consequences?, Journal of Economic Issues 35(3), 607-630. Goddard, J.G., and M. Isabelle (2006), Managing intellectual assets within knowledge-based partnerships: Insights from a survey of public laboratories collaborating with industry, IMRT Working paper 2006/03, downloaded on 12/05/2010 from http://papers.ssrn.com/sol3/papers.cfm?abstract_id=1340209. Haeussler, C. (2011), Information-sharing in academia and the industry: A comparative study, Research Policy 40(1), 105-122. Hong, W., and J.P. Walsh (2009), For money or glory?: Commercialization, competition and secrecy in the entrepreneurial university, Sociological Quarterly 50(1), 145-171. 25 Merton, R.K. References (1973), The sociology of science: theoretical and empirical investigations, Chicago: University of Chicago Press., Miranda, A., and S. Rabe-Hesketh (2006), Maximum likelihood estimation of endogenous switching and sample selection models for binary, ordinal, and count variables, The Stata Journal 6(3), 285–308. Mukherjee, A., and S. Stern (2009), Disclosure or secrecy? The dynamics of open science, International Journal of Industrial Organization 27(3), 449-462. Murray, F., P. Aghion, M. Dewatripont, J. Kolev, and S. Stern, (2009), Of mice and academics: Examining the effect of openness on innovation, NBER Working paper No. 14819, Cambridge. Stephan, P.E. (1996), The economics of science, Journal of Economic Literature 34(3), 1199- 1235. Thursby, J.G., and M.C. Thursby (2007), University licensing, Oxford Review of Economic Policy 23(4), 620-639. Walsh. J.P., W.M. Cohen, and C. Cho (2007), Where excludability matters: Material versus intellectual property in academic biomedical research, Research Policy 36, 1184-1203. Wooldridge, J.M. (2002), Econometric analysis of cross section and panel data, Cambridge: MIT Press. Merton, R.K. (1973), The sociology of science: theoretical and empirical investigations, Chicago: University of Chicago Press., Merton, R.K. (1973), The sociology of science: theoretical and empirical investigations, Chicago: University of Chicago Press., Miranda, A., and S. Rabe-Hesketh (2006), Maximum likelihood estimation of endogenous switching and sample selection models for binary, ordinal, and count variables, The Stata Journal 6(3), 285–308. Mukherjee, A., and S. Stern (2009), Disclosure or secrecy? The dynamics of open science, International Journal of Industrial Organization 27(3), 449-462. Murray, F., P. Aghion, M. Dewatripont, J. Kolev, and S. Stern, (2009), Of mice and academics: Examining the effect of openness on innovation, NBER Working paper No. 14819, Cambridge. Stephan, P.E. (1996), The economics of science, Journal of Economic Literature 34(3), 1199- 1235. Wooldridge, J.M. (2002), Econometric analysis of cross section and panel data, Cambridge: MIT Press. 26
https://openalex.org/W4299960375	https://zenodo.org/record/6116562/files/54-55.pdf	English	null	Precipitate based Selective lon Sensitive Membrane Electrodes for Tripositive Chromium and Aluminium	Zenodo (CERN European Organization for Nuclear Research)	1,989	cc-by	1,890	References Green coloured chromium(m) dithiozonate and ash coloured aluminium(m) dithiozenate precipitates were obtained by adding in dilute solutions of the salts of the respective metal ions, an ammonical solution of dithiozone 1 • After washing and drying the precipitate, the master membrane with each precipitate was prepared by mixing the precipitate (100 mg) with epoxy resin (400 mg} 8 • 1. 1. SJCI.BIN, Ohsm. Bev., 1965, 65, 153. ~. 0 S C A d A S J , ~. P. 0. STJCWART and A. L. PoRTS, J, Oh~~m. Soc., Dalton Tf"aAS., 19'12, 1661. B 0 B. fl[, PASQUAI.Io F. MARCHJI:"l'TJ, 0. FLOR.IANI and 8. MSB.I.IJfO,J. Chem. Soc., Dal~on Trans., 19'17,139. J l l Oh~~m., 4, C. E. 1\IANNIX and A. P, ZrPP, J, lnMg. Nucl. Oh~~m., 19'19, •U,69. , 6. T. G. F. HUDSON, "Vanadium Toxicology and Biological Slgnlflcance", Elsevier, New York, 196,. g 6. K. M. PUllOBIT and D. V. R. RAo,J.Indtan Ohem. Soc., 1980,57, 863. Tl'I O d A dl J Oh S Preparation of electrode8 : A small portion from each membrane was cut out and fixed at one end of the glass tube (o.d. 0.8 em and i.d. 0.5 em} with the help of Araldite. These tubes were filled with 0.1 M solution of CrCI 8 and Al(N08 ) 8 , respec· tively. A saturated calomel electrode was inserted for electrical contact. A separate saturated calomel electrode was used as external reference electrode. The electrode systems can be represented as '1. K. Tl'I. PUROHIT and D. V. R. RAo, Indlan J. Ohem., Sect. .-1,1988, :u, 620. India1~ S~t. , , , B. S.liiSHRA and K. M. PUROHIT, India1~ J, Ohsm., S~t. A, 1988, 27. '18. , 9. T. MOEI.LKB., lnorg. 8-ynth., 196'7, 5, 118. J , 9. T. MOEI.LKB., lnorg. 8-ynth., 196'7, 5, 118. 10 l d g 10. R. 0. AGGARWAl., R. BAI.A and R. L. PRASAD, Indian J, Okem., S~t • .A, 1983, 22, 668. , , 11. A. I. VoGIUn .. A Text Book of Quantitative Inorganic Analysis", 8rd. ed., Longmans, London, 1962. 12 J G CA C d Ch1 1911 81 12. W. J. GJCARY, Coord. Ch1m. Rev., 1911,7, 81. 18 B N FIGGIS d LBWIS "M d C di 12. W. J. GJCARY, Coord. Ch1m. Rev., 1911,7, 81. ••Present address: Oooservatlon Laboratory, Allahabad Maseum, Allahabad. Aeknowledgement The authors are thankful to Prof. D. V. Ramana Rao for his keen interest in this work and to Prof. Somnath Misra, Principal, Regional Engineering College, Rourkela for facilities. The authors extend their thanks to the Director, C.D.R.I, Lucknow, for recording ir spectra. References 18 B N FIGGIS d J LBWIS "M d C di ti SCE I 0.1 M MB+ I membrane I sample 1 SCE where, M=Crs-t- or Al 8 +. Before taking measure- ment the membrane of each electrode system was kept immersed for 6 days in 0.1 M solution of the respective ions. 18. B. N. FIGGIS and ;J, LBWIS, "Modern Coordination Ohem1sU,.". Intersoienoe, New York, 196'1, p. 406. 18. B. N. FIGGIS and ;J, LBWIS, "Modern Coordination Oh 1 U " I t i N Y k 196'1 p 406 8. B. N. FIGGIS and ;J, LBWIS, "Modern C Oh 1 U " I i N Y k 196'1 14. K. NAKAMOTO, "Infrared and Raman Spectra of Inorganlo and Coordination Compounds", Srd. ed,, Wiley, New York,19'18, p. 24'7. , , p 16. R. o. OBAB.I.BS, M:. FRJCISKR, R. FRI!CDJIL, L. E. HILLAND and W. D. :JOHNSTON, BpectNoh-im • .Acta, 1966,8, 1. an~ Ohemlcal Laboratories, Allahabad University, Allahabad-211 002 Manuscr-ipt received 8 .l.ugUBt 1988, accepted 20 S1ptembsr 1988 The observed data are in conformity with a distorted octahedral structureu-u for vana- dium(tv). NO commercial ion selective electrode for Criii and Al111 ions is available. Both the ions are usually determined by indirect potentiometry in which a different electrode is used, e.g. copper(n) ISEs are being used 1 for the determination of AIIII. In this paper we report the electrodes for these tri· posrtive ions which are based on the dithiozone precipitate of the respective ion as an electroactive material. N J. INDIAN CHBM. SOC., VOL, 66, JANUARY 1989 20. 1. J. R. FRANSTO DA SIX. VA and R. WOOTON, 0hB111· Oommuft., 1969, 421. hcs modes, respectively, suggesting N-bonded thiocyanate ion u. The intense band at - 2 000 cm-1 and a weaker band at - 1 280 cm-1 due to asymmetric and symmetric stretching respectively of azide ion were observed, indicating its terminal coordination18• The 950 cm-1 band was attributed to vv. 0 18• The coordination of thiocyanate and azide ion in the sixth coordination position lowers vv-o from - 990 cm-1 in VO(acac) 1 , VO(ox) 1 and VO(pyca) 1 to ,.... 950 cm-1 in the present complexes. Lowering of the vv-o suggests coordination of thio- cyanate and azide ion trans to V =0 group in the compJex.es10• f , , 22 21. H. 1. 8TOKI.OSA, 1. R WASOON and B. J. Ma00B.:YIC, Inorg. Oh~~m., 1974, 13, 59\l, J 22. g R. K. AGARWAl. and G. BINGB, J, Indian Ohem. Soo., 1986, 63,926. , , \13, B. SINGH, Indian J. Ohsm., Beet. A, 1987, 26, 960. , , \13, B. SINGH, Indian J. Ohsm., Beet. A, 1987, 26, 960. Membrane Electrodes for Tripositive Chromium and Aluminium RAJBSH CHANDRA MISRA** and M.C.CHATTOPADHYAYA* Electronic spectra of the complexes showed two bands, one transition is around 12 000 cm-1 due to d"7~d""' dn. (v1 ) and the other around 16 000 cm-1 due to d".,-d .... _.,, (v1 ). and M.C.CHATTOPADHYAYA* Ohemlcal Laboratories, Allahabad University, Allahabad-211 002 Manuscr-ipt received 8 .l.ugUBt 1988, accepted 20 S1ptembsr 1988 J. INDIAN CHBM. SOC., VOL, 66, JANUARY 1989 J. INDIAN CHBM. SOC., VOL, 66, JANUARY 1989 Results and Discussion A sharp rise in the titration curve was observed to occur near the end-point (Fig. 1). solution and after every addition of 0 1 ml Na 11HPO., solution, the electrode potential was noted. The potential values were plotted against the volume of Na 11HPO., solution consumed in titration. A sharp rise in the titration curve was observed to occur near the end-point (Fig. 1). was varied in the range from l x I0-1 to 1 x IO-' M and the potential was recorded with respective ion-selective electrode with the help of a Philips PR 9405 M pH meter at room temperature (23 ± t•). The response of each electrode was plotted against the concentration of the respective ion in a semilog graph paper. In case of Criii electrode a linear response was observed down to the concentration of 1 x w-e M and for the AJIII electrode the value was 5 x to-or M. Other properties like response time, pH range, age of the membrane of all the three electrodes w.as determined (Table I). The selectivity coefficients were determined by the mixed solution method .. (Table 2). Acknowledgement Authors are thankful to State Council of Science and Technology, Lucknow, for financial assistance. Results and Discussion , , 16. G. W. A. FowUS, B. W. MATBJCWS an~ R. A. WAJ'/l'ON, J, Ohem. Soc. (d), 1968, 1108. 1 L O J Oh~m. J 1105 17. 1. L. BURloUUS'UR, OoorcJ. Oh~m. Rev., 1966, J, 1105; 1966, 3, 8115. K 0 J I di Oh~m. Two separate sets of solution of CrCI 8 and Al(N08 ) 8 were prepared in which the concentration 18. K. 0. D.&SB and Oa. K. 0. MOBAPATRA, J, Indiata Oh~m. Soc., 1979, 56, 84. A L , 19. A, N. 8PKCA, N, M. KARAYANIS and L, L. PY'tJ.JCWSKI, Inorg. Ohtm. AcltZ, 1914, t, 8'7. S4 NOTBS Lower detecti limit Bl. Electrode no. M 1. Oriii IBE 1 x to- ll· Ailii ISE li X 1(1 81. ,Electrode no, Oe•+ La I+ 1. Oriii ISE 0.010 0.005 2. -Aliii ISE o.ooli 0.001 was varied in the range from l x I0 and the potential was recorded ion-selective electrode with the h PR 9405 M pH meter at room temp The response of each electrode wa the concentration of the respective graph paper. In case of Criii el response was observed down to t of 1 x w-e M and for the AJIII ele was 5 x to-or M. Other properties time, pH range, age of the mem three electrodes w.as determined selectivity coefficients were determin solution method .. (Table 2). Titration of crm. Aim ion agains Both Crill and Al111 give precipit phosphate ion and precipitation these species can be monitered sensor•· 11 • For the titration of th ions against phosphate ion, stand the salts of Criii and AJill (0.01 M) (0.01 M) were prepared. The sol diluted to 20 ml by double-distille electrode potential measured with ISE. The solution was then titrated a 1 0 _/,... __ .~._.___....t __ _,__ ...!-- I I J 4 ~ IK>I.UME OF "";P04 SOlUTION f m ml I - l'ls. 1. Plots of potential asalnst volume of N TABI,. 1 Lower Slope of detection response limit ourve Bl. Electrode no. M mV 1. Oriii IBE 1 x to-• 20 ll· Ailii ISE li X 1(17 20 Response Working Life of time pH range electrode 8 month 15 2.8-7.6 8 10 8.2-10.8 10 81 Electrode Ions solution and after every addition of 0 1 ml Na 11HPO., solution, the electrode potential was noted. The potential values were plotted against the volume of Na 11HPO., solution consumed in titration. Refereaces 1. L. BUC'IlA and M. BUCBAR:ItJt. Anal. Lilt., 1978, 3, 618. 51. A. I. VoG:ItL. "Quantitative Inorganic Analysis", E.L.B.S., London, 1969. 8. R. 0. MrSRA and ll!. 0. OSA'l''l'OPADHVAYA, IndiafiJ, Ohem •• Beet. A, 1988, 17, 1011. 4.. G 'J. MOODY and 'J. D. R. THOMAS, Talanta. 1971, 18, 1261. IS. R. P. Bucx:, Aflal. Okem.,1966, 38, 978. 6. W. 8. BALIG, Int. Laboratorr, 1984, 14, llO. Titration of crm. Aim ion against phosphate ion : Both Crill and Al111 give precipitate wtth ortho- phosphate ion and precipitation titration between these species can be monitered with a suitable sensor•· 11 • For the titration of the above metal ions against phosphate ion, standard solutions of the salts of Criii and AJill (0.01 M) and Na 11HPO., • (0.01 M) were prepared. The solution (5 ml} was diluted to 20 ml by double-distilled water and tts electrode potential measured with the respective ISE. The solution was then titrated against NasHPO., , , , 4.. G 'J. MOODY and 'J. D. R. THOMAS, Talanta. 1971, 18, 1261. 1 0 _/,... __ .~._.___....t __ _,__ ...!--~-.....______, I I J 4 ~ 6 18 IK>I.UME OF "";P04 SOlUTION f m ml I - l'ls. 1. Plots of potential asalnst volume of Na8HP04 solution. 1 0 _/,... __ .~._.___....t __ _,__ ...!--~-.....______, I I J 4 ~ 6 18 IK>I.UME OF "";P04 SOlUTION f m ml I - Photocycllsatlon of trans-Stilbene N. SAHOo• and R. J, GALAGALI Department of Ohemlstry, Banaraa Hindu University, Varanasi.Siln 005 Manuscri.l't rec11Ved l7March 1988, revssed Htl August 1988, acc'.l't•d sa B•ptrmber 1988 THE tran~cis isomerisation of stilbene with photo- sensitisers take place by the transfer of energy of sensitisers to stilbene during excitation under the uv irradiation1 • The photocyclisation and photoiso- merisation of trans-stilbene has been discussed under nitrogen laser. The laser irradiation diminishes the time period of irradiation and gives maximum yield than that of the normal uv irradiation. l'ls. 1. Plots of potential asalnst volume of Na8HP04 solution. l'ls.
https://openalex.org/W2918562721	https://upcommons.upc.edu/bitstream/2117/172848/1/Energy%20conv%202019.pdf	English	null	Techno-economic and exergy analysis of polygeneration plant for power and DME production with the integration of chemical looping CO2/H2O splitting	Energy conversion and management	2,019	public-domain	21,395	Abstract: In the present study we propose a novel gas feed polygeneration process for the dimethyl ether (DME) and power production and carbon capture (CCS). The process consists of chemical looping CO2/H2O dissociation (CL) unit to produce syngas (CO and H2 with methane reduction step in redox cycle) from exhaust gases for DME production in a packed bed reactor. Except for the chemical looping CO2/H2O dissociation unit (consist of two interconnected reactors) which is under development stage, the process is considered based on already existing industrial components. The aim of the present work is to assess the process based on energy efficiency and the economic performance of the integrated chemical looping unit for industrial scale DME production plant. It was found that the price of the DME produced was 34 $/GJ (DME LHV). The beneficial integration proposed resulted in a production of 103 MWe and 2.14 kg/s of DME with an energy and exergetic efficiency of about 50% and 44%, respectively. A discounted cash flow analysis was performed to evaluate the profitability of the process. In order to have a positive NPV, a selling price of electricity and DME higher than the current market was required. Finally, an exergo-economic analysis was executed to detect which are the main causes of the low economic performance. An exergo economic factor of 0.37 of the overall plant was found, suggesting that an improvement in the efficiency of the equipment is needed. The potential of CO2/H2O dissociation unleashes in liquid hydrocarbon and fuel production strategies, which are hindered by only-thermal reduction step in the existing approach for solar thermochemical syngas production. The analysis also provides information on the main economic drives associated with high capital investment in the process plant with individual sub-systems. The energy, exergy and economic analysis have proved to show the scope of improvements over the conventional method of manufacturing DME considered 100% carbon capture. The analysis also reflects the strong potential of chemical looping syngas production pathways to integrate with polygeneration system to increase the overall efficiency with reduced cost of carbon capture. The baseline simulation of the polygeneration plant proposes a methodology for assessment of the impact on the overall efficiency, economic performance and water for advances system designs. Keywords: DME production, CO2/H2O splitting, Chemical looping, Polygeneration, Oxyfuel combustion cycle, Exergo-economic, Carbon Capture and Utilization, Exergy analysis. 1. Introduction Climate change presents a global risk to society. Among the probable causes of climate change, the emission of greenhouse gases (GHG), primarily from anthropogenic activities, is the primary attribute [1]. Of the several GHGs that are emitted, carbon dioxide is of the highest concern due to its highest volume rate of emission and persistence in the atmosphere [1,2]. With this focus, the 21st Conference on the Climate Change in Paris, resulting in the so-called Paris Agreement of December 2015, has achieved significant milestones, with 195 of 197 participants agreeing “to strengthen the global response to the threat of climate change, […]. The largest part of the 49 Gt of CO2 emitted in 2010 comes from the combustion of fossil fuel (31 Gt). The 65% of this 31 Gt of CO2 accounts for energy production [1]. However, without drastic measures, proceeding with the current pace of emissions, it is expected that the limits of the Paris agreement will be overshoot by 2050 [4]. Among the multiple pathways proposed for reduction in the anthropogenic emissions of CO2, the following are the most discussed: i) reduction in global energy consumption, ii) increase in efficiency in industrial and domestic sector iii) increase in the use of renewable energy sources, iv) increase in the use of nuclear power, v) switching to less carbon-intensive fuels, vi) enhance CO2 uptake in biomass, vii) CO2 capture and storage (CCS), and viii) CO2 capture and utilization (CCU) among others. Of the several possible alternatives, Carbon capture and storage (CCS) technologies have been studied to provide considerable benefits towards decreasing the anthropogenic emissions [5–7]. Indeed the significance of CCS in a low carbon economy has been predicted to be a solution by the scientific community [8]. Nevertheless, unlike all other pathways, CCS suffers from its own drawbacks including the need for compression of CO2 to high pressures of usually 110 bars, complex transportation mechanism of the compressed CO2, large storage space requirement, a high-efficiency penalty on the power plant among others [9–11]. Therefore, an alternate pathway, to recycle and reuse the captured CO2 is via innovative processes integrated in chemical formation for subsequent use, which has received much attention [12–14]. In this regard, polygeneration systems, combining a number of utilities in a single system in an efficient way, to produce multiple products (power, chemical, fuel etc.) has received particular interest in recent years [15]. 1. Introduction This not only results in a significant gain in efficiency due to a tight integration and synergy between different processes but also, it allows the system to achieve a higher flexibility in operation, varying proportionally the products according to the fluctuating market price [16]. Multiple configurations of polygeneration systems have been reported in the literature. Farhat et al. [17] proposed a coal-fed polygeneration plant for power and fertilizer production, and further studied different flexible operations to evaluate the economic competitiveness of the plant. Li et al. [18] modelled a polygeneration plant with CO2 capture for production of power and synthetic natural gas, the proposed arrangement achieved a lower life cycle and GHG emission with respect to the starting ultra-supercritical coal power plant. Bose et al. [12] introduce a cost-effective production of urea and power combined with CCS using coal gasification. One intriguing approach to recycle exhaust gas within the power plant and hence produce chemicals in addition to power is through the chemical looping (CL) technology [19,20]. Chemical looping technology for CO2/H2O splitting usually utilizes metallic oxygen carriers to convert the thermo- chemical energy into chemical energy [21]. Both thermally driven cycles (by concentrated solar power) or by fuel reduction (by methane) are feasible alternatives [22]. Methane-driven cycles, however, besides being able to operate at low-temperature, [22,23] are also able to operate round the clock, unlike solar driven cycles. A basic schematic of the methane driven chemical looping CO2/H2O splitting cycle is shown in Figure 1. MeO MeO2-δ δCO2 δCO Methane Reduction (MR) Oxidation (WS and CDS) TRED TOXD δH2O δH2 δCH4 δCO 2δH2 Figure 1 Conceptual scheme of the chemical looping syngas production through methane reduction and corresponding splitting of water and carbon dioxide, usually present in waste gas (CO2 and H2O) from industrial applications Figure 1 Conceptual scheme of the chemical looping syngas production through methane reduction and corresponding splitting of water and carbon dioxide, usually present in waste gas (CO2 and H2O) from industrial applications Of the multiple metal oxide redox pairs studied in this regard [23–25], cerium (IV) oxide (CeO2) has been considered to be one of the most promising for the present application due to its strong ability to undergo cyclic redox reactions while retaining its chemical and structural properties together with high resilience to mechanical stress and agglomeration [26]. 1. Introduction Ceria reduction, even though non- stoichiometric, tends towards stoichiometry with methane reduction at temperatures beyond 1400oC. Accordingly, the CeO2/Ce2O3 redox pair with reduction of CeO2 in the presence of methane, and subsequent oxidation with CO2/H2O can be described by the equations (1-3). 2 4 2 3 2 Methane reduction: 2CeO + CH Ce O + CO + 2H  (1) 2 3 2 2 2 Water-splitting(WS): Ce O + H O 2CeO + H  (2) 2 2 3 2 2 CO -splitting (CDS): Ce O + CO 2CeO + CO  (3) (1) (2) 2 3 2 2 g (CDS): Ce O + CO 2CeO + CO  (3) (3) By optimally combining the composition of the exhaust gas (a mixture of water and CO2), and by controlling the temperature of reaction, the desired composition of syngas can be obtained, which can subsequently be utilized to produce multiple products through industrial processes [27–29]. Of the most interesting options, the production of synthetic fuels, able to generate electrical power in high efficient devices such as fuel cells or direct implementation in automotive applications is an interesting alternative [30,31]. Dimethyl Ether (DME) is one of the strongest candidates as a synthetic By optimally combining the composition of the exhaust gas (a mixture of water and CO2), and by controlling the temperature of reaction, the desired composition of syngas can be obtained, which can subsequently be utilized to produce multiple products through industrial processes [27–29]. Of the most interesting options, the production of synthetic fuels, able to generate electrical power in high efficient devices such as fuel cells or direct implementation in automotive applications is an interesting alternative [30,31]. Dimethyl Ether (DME) is one of the strongest candidates as a synthetic fuel due to its similarity with diesel, while thanks to its physical properties and chemical structure – produces low NOx, limited HC and almost no SOx and particulate subject to combustion [30]. However, a lower LHV than conventional diesel and requirement of pressurization to maintain liquid state like LPG are some drawbacks. Nevertheless, considering the future potential for synthetic fuels, especially in relation to closing the carbon loop, a polygeneration system, aiming to produce power and DME through the integration of chemical looping syngas production is here proposed and thoroughly investigated. 1. Introduction In this prospect, a natural gas (NG) driven cycle has been selected due to the possible integration with the metal reduction by methane, which can be easily replaced by biomethane in future, thereby increasing the sustainability of such a plant in future. A detailed energy analysis, techno-economic and exergo- economic studies have been carried out on the proposed scheme and are reported subsequently in the paper. 2. Process and plant description: The proposed plant is an oxyfuel natural gas feed combined cycle power plant integrated with a chemical looping CO2/H2O splitting (CL) unit for power and fuel generation with carbon capture (OXYF-CL-PFG). Figure 2Error! No s'ha trobat l'origen de la referència. describes the process flow concept. The natural gas supplied from the gas grid is sent to the chemical looping splitting unit (CL) where it is converted into a hydrogen-rich syngas by the simultaneous reduction of ceria. The produced syngas is sent to an oxyfuel unit where it is burnt with a pure stream of oxygen coming from an air separation unit (ASU). The hot exhaust gases composed mainly by H2O and CO2 are firstly expanded in a gas turbine and then sent in a heat recovery steam generation unit (HRSG). Here, the surplus heat is exploited to produce superheated steam for power production. Finally, the water and carbon monoxide are separated by a water condenser. The large part of the separated CO2 is sequestrated for storage, while another fraction together with steam is sent to the CL unit. In the CL unit, both H2O and CO2 are dissociated in oxidation reactor by the reduced ceria coming from the reduction reactor. The produced syngas from the oxidation reactor is sent for the DME production process. Finally, the produced fuel is separated from the other diluted product in a distillation unit (or clean up unit). In the following sub-section, the methodology adopted for the presented work and more details on each unit and their integration are described. Figure 2: General plant concept Figure 2: General plant concept Figure 2: General plant concept 2.1 Simulation Methodology The polygeneration gas to power and fuel plant integrated with a chemical looping CO2/H2O dissociation unit has been modelled by combining mass and energy balance equations. The hypothesis of chemical equilibrium has been applied to all the components of the layout with exception of the DME reactor, for which a kinetic approach has been used. Simulations were performed using the commercial software Aspen Plus v8.8. The material streams used in the model involve conventional and solid components. The Peng-Robinson-Boston-Mathias (PR-BM) property method was used for the conventional components [32][33]. This method uses the Peng-Robinson cubic equation of state combined with the Boston-Mathias alpha function for all the thermodynamic properties [32] as this approach was recommended for hydrocarbon processing applications such as gas processing, refinery, and petrochemical processes [34]. The oxygen carrier (CeO2/Ce3O4) used for the chemical looping simulation were entered as conventional solid components. For this type of streams, the Barin equation was used [35] [36]. The RGibbs reactor blocks were used for modelling the oxidation reactor, the reduction reactor and the combustor of the oxyfuel unit. The distillation unit and the air separation unit were modelled using the RadFrac column. The DME reactor was simulated with an RPlug reactor combined with a Langmuir-Hinshelwood Hougen-Watson (LHHW) kinetic model. During the simulation of this component, the Soave-Redlich-Kwong (SRK) EOS was utilized. The Redlich Kwong (RK) EOS is usually applied to binary components [37]. Graaf et al. [38] demonstrated that the chemical equilibrium of the methanol reaction and WGS reaction can be well described at high-pressure by using the SRK-EOS. The main hypotheses used in the modelling phase are reported in Table 1. Figure 3 shows the detailed layout of the plant. Table 1: main assumptions and hypothesis used in the process simulation. Natural gas Composition (std.vol%): 93.1% CH4, 3.2% C2H6, 1.6% N2, 1.1% C3H8, 1.0% CO2 LHV=47.1 MJ/kg Oxidation and reduction reactors Model: RGIBBS, no heat losses Combustor Model: RGIBBS ΔP=0.2 bar, no heat losses Compressors, pumps and turbines ηis,comp=0.9, ηmech,comp=0.98, ηis,pump=0.9, ηdriver,pump=0.90, ηis,turb=0.9, ηmech,turb=0.98 Oxygen carrier Solid ceria: CeO2/Ce2O3, diameter=100 μm Temperature drop of 20°C during ceria recycling from OXI to RED DME reactor Model: RPlug multitube reactor, Operation: T=250°C P=50 bar Heat exchangers ΔTmin=10°C Distillation unit Model: RadFrac, Reboiler type: Kettle CLN-CO2 CLN-DME CLN-MeOH P=10 bar P=9 bar P=2 bar Table 1: main assumptions and hypothesis used in the process simulation. 2.1 Simulation Methodology Table 1: main assumptions and hypothesis used in the process simulation. Figure 3: Detailed plant layout for power and DME production from natural gas utilizing the CL unit Figure 3: Detailed plant layout for power and DME production from natural gas utilizing the CL unit 2.2 Chemical looping syngas production unit (CL unit) The chemical looping unit consist of two interconnected reactors operating at 2 bar: the reduction reactor (RED) and oxidation reactor (OXI). The circulating oxygen carrier is ceria oxide (CeO2/Ce2O3). The natural gas taken from the grid at 70 bars (stream 2) [39], is heated up at 290°C and expanded to 2 bar via the turbo-expander (TURBOEXP). The preheating is necessary to prevent an outlet temperature (stream 4) from the turbo-expander lower than 0°C. After the expansion, the natural gas is heated to 890°C (stream 5) and fed to the reduction reactor (RED). Here the ceria oxide CeO2 (stream 9), whit an inlet ratio CeO2:NG=1:0.7, is completely reduced with natural gas, producing a syngas composed by H2 and CO in a 2:1 ratio (reaction 1). The reaction is endothermic and so requires an external heat source. The high temperature from oxyfuel combustion is usually controlled by CO2 recycling. A part of this heat has been proposed to be used for the endothermic reduction in RED. For this scope, a heat integrated combustion was proposed utilizing an annular combustion chamber [40]. The hot syngas (stream 6) exits the reduction reactor at 900°C and it is separated from the solid (stream 7) by a cyclone (CYC-1), cooled and sent to the oxyfuel unit. The reduced ceria is fed into oxidation reactor where it is then oxidized (reactions 2 and 3) by a mixture coming from the oxyfuel unit of 60% H2O-40% CO2 (stream 40 and 46) to have at the outlet the ideal H2:CO (1:1) for DME production as described in section 3. In order to achieve a full oxidation of Ce2O3 a 60% excess of water and carbon dioxide mixture is required. Before the oxidation, both water and carbon monoxide are compressed at the 2 bar chemical unit operation with a pump (PUMP-1) and a compressor (COMP-4) and heated up at 500°C. Since the reactions in the oxidation reactor are exothermic, the outlet temperature of the reactor goes to 1322°C. The hot syngas ( Table 5 Plant results with selected parameters. Table 5 Plant results with selected parameters. 2.2 Chemical looping syngas production unit (CL unit) NG feed 25.2 ton/h WGROSS 167.61 MW WNET 102.90 MW ηTOT 50.21% WCOMP-1 3.76 MW WCOMP-2 10.67 MW WCOMP-3 28.29 MW WASU 19.34 MW WGT 114.42 MW WST1 44.30 MW WST2 2.96 MW WTURBEXP 4.37 MW ṁDME 2.14 kg/s ṁMeOH 0.03 kg/s CO2,REC 0.85 CO2,DME 0.034 Table 6: Thermodynamics properties and composition of selected streams. Table 6: Thermodynamics properties and composition of selected streams. stream 28 10 15 16 17 20 31 37 38 43 47 7 9 T (°C) 900 1322 200 250 46 40 1377 80 40 40 46 900 1322 P (bar) 2 2 5 5 1 9 26 2 1 10 2 2 Mole flow (kmol/h) 1 0.47 0.34 0.15 0.05 0.04 3.7 2.44 0.19 0.28 0.09 0.59 0.29 Molar fraction O2 0 0 0 0 0 0 0 0 0 0 0 0 0 H2 0.57 0.32 0.44 0.06 0 0 0 0 0 0 0.01 0 0 H2O 0 0.28 0 0.01 0.03 0 0.22 0 0 0.99 0 0 0 CO2 0 0.09 0.13 0.57 0 0 0.77 0.99 0.99 0 0.93 0 0 CO 0.3 0.31 0.43 0.05 0 0 0 0.01 0 0 0.03 0 0 CH4 0.12 0 0 0 0 0 0 0 0 0 0 0 0 MeOH 0 0 0 0.01 0.02 0 0 0 0 0 0 0 0 DME 0 0 0 0.3 0.94 1 0 0 0 0 0.02 0 0 CeO2 0 0 0 0 0 0 0 0 0 0 0 1 0 Ce2O3 0 0 0 0 0 0 0 0 0 0 0 0 1 for the final composition) is separated from the oxidized ceria by the cyclone separator (CYC-2), cooled down (stream 10, 10a, 10b, 11) and sent to the DME unit, while the solid stream is re-circulated back for a new reduction cycle (stream 9). cooled down (stream 10, 10a, 10b, 11) and sent to the DME unit, while the solid stream is re-circulated back for a new reduction cycle (stream 9). 2.3 Air separation unit (ASU): The air separation unit consists of a cryogenic distillation unit able to produce 99.99% pure O2. The air is separated in two thermally interconnected distillation columns, HP-COL and LP-COL, which work at 5 and 1.2 bar respectively. The overall refrigeration is driven by the expansion from high pressure (30 bar) of the compressed air (stream 6-C and 7-C) through the VALVE-2 and the TURBOEXP-2C. The inlet air (1-C) is compressed at 6.3 bar by the compressor COMP-1C and separated in two streams (4-C and 8-C) by the splitter SPLIT-1C. The stream 8-C is cooled down in the exchanger HX-2C by the cold products of the LP-COL and is sent to the HP-COL. The HP-COL is a 40 stages distillation column which produce as top product a gaseous rich-in N2 (stream 12-C) and as a bottom product a liquid rich-in O2 stream (stream 10-C). The latest stream is further cooled down through Joule-Thomson effect in the valve VALVE 1-C and fed in the 56 stages low pressure column. The low temperature air streams 15-C and 17-C, together with the rich-in O2 liquid stream provides the necessary refrigeration in the LP-COL to obtain as a top product a pure N2 stream (20- C) while as product from the bottom condenser the pure O2 stream (18-C). The latest stream is pumped by the PUMP-1C at the operational condition of the combustor in the oxyfuel unit and heated in the HX-2C. The ASU block can be seen in Figure 4 in supplementary file and stream data is listed in table S1. Figure 4: Detailed layout of the air separation unit Figure 4: Detailed layout of the air separation unit 2.4 Dimethyl ether production (DME) unit: The syngas produced in the oxidation reactor (OXI) is used for DME synthesis within a catalytic reactor. Before the syngas is fed to the DME reactor, it undergoes water condensation (COND-1) and is compressed by a three-stage compressor at 50 bar (COMP-1). The DME reactor is a fixed bed reactor which is kept at the constant temperature of 250°C by a water-jacket cooler used for saturated steam generation at 2 bar (stream 44) for the oxidation (OXI) reactor. 2.4 Oxyfuel combustion unit (COMB) In this unit, combustion is performed with oxygen instead of air. This eliminates the presence of nitrogen in the exhaust gases that affect the subsequent separation process. Another advantage is a substantial reduction in thermal NOx since the absence of nitrogen [41]. Therefore, a highly pure stream of CO2 can be obtained after water condensation from the flue gas. The oxyfuel unit consists of a combustor (COMB), where the syngas from the reduction (stream 30) and the non-condensable gases from the clean-up unit (stream 49) are burnt with a 5% excess oxygen stream derived from the air separation unit (ASU) (stream 1). Stream 36 represents the part of the captured CO2 which is re-circulated to the combustor. The recirculation ratio was set such as the total combustion heat was sufficient to have a combustor outlet syngas temperature of 1377°C and the required heat to carry on the reduction of the ceria in the reduction (RED) reactor. The CO2 and syngas streams are compressed to 26 bar with two two-stage compressors (COMP-2 and COMP-3). The flue gas is firstly expanded in a two-stage gas turbine GT (26 bar to 5 bar and 5 bar to 1.05 bar) and then sent to heat recovery steam generator (HRSG1). Finally, the CO2 is separated from the water in a condenser (COND-2) and split into three parts. One part is recirculated to the combustor (stream 36), one is sent for sequestration (stream 35) and the last part (stream 38) is sent to the oxidation reactor for dissociation (OXI). Table 2 Distillation unit operation parameters. Table 2 Distillation unit operation parameters. Column Treb [°C] Qreb [MW] Tcond [°C] Qcond [MW] Stages Feed-in stage Purity of the product [%] CO2-CLN 45.87 1.12 -40.83 -0.64 25 10 - DME-CLN 150.99 0.93 42.57 -0.55 30 24 99.1 MeOH-CLN 101.53 0.03 66.36 -0.05 24 18 94.1 2.6 Steam cycle A heat recovery steam generator unit has been utilized to recover the extra heat generated within the polygeneration system. The turbines and the HSRG were modelled as simple units, without reheating or multi-pressure systems. The primary objective of the present study being to understand the potential of the net benefits from polygeneration by the integration of a chemical looping unit in a conventional oxyfuel plant, the optimization of the system was not further considered. The presented layout is composed of two different HRSG units: HRSG1 and HRSG2. The HRSG1 uses the heat of the flue gases from the oxyfuel unit (stream 32) to produce 125582 kg/h of super-heated steam at 150 bar and 550°C (stream 5A). While the HRSG2 uses the extra heat from the chemical looping unit to produce 8305 kg/h of steam at the same condition of stream 5A (stream 5B). Each steam stream is respectively expanded in steam turbine ST and ST2. The reason for the choice of two HRSG connected to two different steam cycle is to ensure flexible operation by minimizing the impact of DME and power over each other. 2.5 DME distillation unit Since a not pure DME stream is generated at the outlet of the DME reactor, a separation unit is hence necessary to obtain pure dimethyl ether. The separation unit is composed of a cooling unit and a gas- liquid separation unit. A cooling unit, represented in the layout by a vapour-liquid separator is used to produce a chilled syngas at -40°C resulting in a liquid stream of DME with dissolved CO2 and MeOH (stream 17) and a gas stream of incondensable gases, namely, H2, CO, undissolved CO2 and traces of other diluents (steam 47). The gas stream is re-circulated into the oxyfuel unit and burnt, while the liquid stream is further processed in the gas-liquid separation unit. The gas separation unit is composed of three different distillation columns: CO2-CLN, DME-CLN and MEOH-CLN (Table 2). The first one is used to separate the dissolved CO2, the second to produce a pure 99.99% DME and the last one to separate the methanol from the water. Before each column it is placed a valve to adjust the pressure to the optimal value and a heat exchanger in order to have at the inlet a 50% vapour inlet stream [157]. The number of stages used in the distillation columns was estimated increasing them until a certain change in composition was detected [158]. 3 Synthesis of DME There are two overall reactions to synthesize the syngas to DME route, represented by equation (8) and (9): 2 3 3 2 298 ;0:1 3 3 - 246.0 / K MPa H CO CH OCH CO H kJ mol      (8) 2 3 3 2 298 ;0:1 3 3 - 246.0 / K MPa H CO CH OCH CO H kJ mol      (8) 2 3 3 2 298 ;0:1 2 4 - 205.0 / K MPa CO H CH OCH H O H kJ mol      (9) (8) 2 3 3 2 298 ;0:1 2 4 - 205.0 / K MPa CO H CH OCH H O H kJ mol      (9) 2 3 3 2 298 ;0:1 2 4 - 205.0 / K MPa CO H CH OCH H O H kJ mol      (9) (9) The reaction (8) synthesizes the DME production in three steps (equation 4,6,7), while when WGS reaction does not take place, the overall DME formation is described by reaction (9) (reaction 4 plus reaction 7) [46]. Since both overall reactions are exothermic and generate two molecules of products from six molecules of reactants, according to the Le Châtelier principle [47], conversion is favoured working at high pressure and low temperature. 3 Synthesis of DME 2 3 298 ;0:1 2 - 90.4 / K MPa CO H CH OH H kJ mol     (4) 2 2 3 2 298 ;0:1 3 - 49.2 / K MPa CO H CH OH H O H kJ mol      (5) 2 2 2 298 ;0:1 - 41.2 / K MPa CO H O CO H H kJ mol      (6) 3 3 3 2 298 ;0:1 2 - 24.0 / K MPa CH OH CH OCH H O H kJ mol     (7) 2 3 298 ;0:1 2 - 90.4 / K MPa CO H CH OH H kJ mol     (4) 2 2 3 2 298 ;0:1 3 - 49.2 / K MPa CO H CH OH H O H kJ mol      (5) 2 2 2 298 ;0:1 - 41.2 / K MPa CO H O CO H H kJ mol      (6) 3 3 3 2 298 ;0:1 2 - 24.0 / K MPa CH OH CH OCH H O H kJ mol     (7) 3 298 ;0:1 - 90.4 / K MPa CH OH H kJ mol   (4) (4) 2 3 2 298 ;0:1 - 49.2 / K MPa CH OH H O H kJ mol     (5) (5) (6) 3 3 2 298 ;0:1 - 24.0 / K MPa CH OCH H O H kJ mol    (7) (7) A combination of either reaction (4), (6) and (7) or (5), (6) and (7) permits to fully describe the process thermodynamically. 3 Synthesis of DME Dimethyl ether (DME) is produced from methanol dehydration using a specific catalyst. The process can be realized in two steps (methanol and DME are produced in two different reactors) or in a single step adopting a dual catalyst. The disadvantage of the two-step process is that syngas conversion to methanol is significantly limited by equilibrium and thermodynamic constraints [42]. Consequently, the further conversion of methanol to DME in the single step process shifts the equilibrium toward more methanol production. For that reason, the direct DME synthesis is thermodynamically and economically preferable than the two steps process [43–45]. The overall process can be described by three main reactions: i) syngas conversion to methanol, ii) water gas shift and, iii) methanol dehydration. economically preferable than the two steps process [43–45]. The overall process can be described by three main reactions: i) syngas conversion to methanol, ii) water gas shift and, iii) methanol dehydration. 3.1 Kinetic Model:   3 2 2 2 2 2 2 2 2 2 2 1 3 ,1 3 2 3 4 1 1 1 CH OH H O H CO eq CO H CO hydrogenation H O H H O H p p k p p K p p r p k k p k p p                                  (10) 2 2 2 2 2 2 2 2 5 ,2 2 3 4 1 1 1 CO H O CO eq CO H RWGS H O H H O H p p k p K p p r p k k p k p p                   (11)   3 3 2 3 3 2 2 2 2 6 ,3 4 1 2 DME CH OH CH OH H O eq MeOH dehydration CH OH CH OH H O H O C k K c C K r K C K C                         (12)   3 2 2 2 2 2 2 2 2 2 2 1 3 ,1 3 2 3 4 1 1 1 CH OH H O H CO eq CO H CO hydrogenation H O H H O H p p k p p K p p r p k k p k p p                                  2 2 2 2 2 2 2 2 5 ,2 2 3 4 1 1 1 CO H O CO eq CO H RWGS H O H H O H p p k p K p p r p k k p k p p                     3 3 2 3 3 2 2 2 2 6 ,3 4 1 2 DME CH OH CH OH H O eq MeOH dehydration CH OH CH OH H O H O C k K c C K r K C K C                         (10) 2 CO hydrogenation r (11) (12) MeOH dehydration r Reaction rates of equation (10), (11), (12) are expressed in kmol/kgcat s, p is the partial pressure of the gases in Pa and C the concentration expressed in kmol/m3. 3.1 Kinetic Model: A kinetic model was implemented in order to simulate catalyst operation in Aspen Plus. The model well simulates the presence of the dual catalyst: Cu/ZnO/Al2O3 operates for the methanol synthesis while the acidic component γ-Al2O3 catalyse the methanol dehydration. It is reported [42] that the best loading catalyst ratio is 1:2 (Cu/ZnO/Al2O3: γ-Al2O3). Because DME synthesis is governed by catalytic reactions of syngas on the surface of the catalyst, Langmuir-Hinshelwood-Hougen-Watson (LHHW) kinetic model was used based on three simultaneous reactions (Eqs.5,6,7). It was assumed that each CO, CO2, H2 and H2O compounds are adsorbed at an active site on the catalyst surface [37]. The rate for each reaction is given by equations(10-12) [42,48,49]. 3.1 Kinetic Model: The equilibrium constant (Ki) and constant rate (ki) values used to determine the reaction rates are shown in table 3. Table 3: kinetic parameters. A unit B unit k1 1.07×10-13 (kmol/(kg-sPa2)) 36,696 (J/mol) k2 3450 - 0 (J/mol) k30.5 1.578×10-3 Pa-0.5 17,197 (J/mol) k4 6.62×10-16 Pa-1 124,119 (J/mol) k5 122 (kmol/(kg s Pa)) -94,765 (J/mol) k6 1.486×1011 (kmol/(kg s)) -143,666 (J/mol) KCH3OH 5.39×10-4 m3/kmol 70,560.92 (J/mol) KH2O 8.47×10-2 m3/kmol 42,151.98 (J/mol) Table 3: kinetic parameters. These parameters refer to the Arrhenius equation shown by equation (13): These parameters refer to the Arrhenius equation shown by equation (13): exp i i i B k A RT        exp i i i B k A RT        (13) (13) Where B represents either the activation energy or the reaction enthalpy or a combination of both [48]. This is because rate constant in LHHW kinetic mechanism is a combination of rate constants and equilibrium constants. Other equilibrium constant were obtained by the following equations [38,48,50]: , 0 1 1 3066 log 10.592 eq K T   (14) 2 10 2073 log 2.029 (1/ ) eq K T   (15) ,3 3220 ln 1.7 eq K T   (16) , 0 1 1 3066 log 10.592 eq K T   (14) , 0 1 1 3066 log 10.592 eq K T   (14) 2 10 2073 log 2.029 (1/ ) eq K T   (15) ,3 3220 ln 1.7 eq K T   (16) 2 10 2073 log 2.029 (1/ ) eq K T   (15) (15) ,3 3220 ln 1.7 eq K T   (16) (16) The described model was used to perform a sensitivity analysis of methanol and DME yield using the equations (17) and (18) varying the composition of the inlet stream, H2:CO ratio, and the amount of the diluent H2O and CO2. The described model was used to perform a sensitivity analysis of methanol and DME yield using the equations (17) and (18) varying the composition of the inlet stream, H2:CO ratio, and the amount of the diluent H2O and CO2. These parameters refer to the Arrhenius equation shown by equation (13): 3.1 Kinetic Model:   2 out yield in DME DME CO CO   (17)   2 out yield in MeOH MeOH CO CO   (18)   2 out yield in DME DME CO CO     2 out yield in MeOH MeOH CO CO   (17) (18)   2 out in MeOH CO CO   (18 Where DMEout and MeOHout are the DME and methanol molar flow at the outlet of the reactor (stream 15) and CO and CO2 the molar flow at the inlet (stream 14). Where DMEout and MeOHout are the DME and methanol molar flow at the outlet of the reactor (stream 15) and CO and CO2 the molar flow at the inlet (stream 14). Where DMEout and MeOHout are the DME and methanol molar flow at the outlet of the reactor (stream 15) and CO and CO2 the molar flow at the inlet (stream 14). As shown in Figure 5 the highest DME yield is obtained sending a syngas with an H2:CO ratio equal to 1:1, this means that the overall reaction is the reaction (8). The main advantage of reaction 8 is that all the hydrogen is consumed in the DME production, while in reaction 9 one third is wasted in water production. Another positive effect of feeding an equimolar H2:CO is that the main byproduct of the reaction is the CO2 which can be easily separated from the DME and MeOH in the separation unit. With increasing the CO2 content at the inlet feed, the DME yield decreases. This is attributed mainly due to two factors. As reported by [51], the methanol synthesis is retarded with the increase of CO2 content. CO2 molecules are absorbed by the methanol catalyst and occupy quicker than CO and H2 the active sites, affecting the MeOH production and consequently also the DME synthesis as shown in Figure 6a [52]. Moreover, with a high CO2 the beneficial effect of the WGS is neglected. The water formed in reaction (5) and (7) is removed by WGS producing hydrogen which kinetically advances the methanol production. Therefore, the higher CO2 favours the reverse-water gas shift that reduces the hydrogen content and produces more water. The effect of higher water content at the inlet is even worse than CO2 and it can be seen in Figure 6.b. 3.1 Kinetic Model: The high water percentage shifts towards the reactants the methanol dehydration, increasing the MeOH yield and reducing at the same time the DME yield. With a water percentage higher than 20, also methanol production is penalized. In addition, it is important to reduce the water content, since it tends to deposit near the catalyst enhancing the catalyst degradation [46]. Therefore, to increase the DME production it is necessary to have at the inlet of the DME reactor a syngas composed by an equimolar H2-CO mixture, reduce the CO2 percentage (molar fraction) in the 0-5% range and remove as much as possible the water content. Figure 5: Influence of the H2/CO ratio on the equilibrium synthesis of DME at T=250°C and p=50 bar. Figure 5: Influence of the H2/CO ratio on the equilibrium synthesis of DME at T=250°C and p=50 bar. Figure 5: Influence of the H2/CO ratio on the equilibrium synthesis of DME at T=250°C and p=50 bar Figure 6: a) Influence of the CO2 and b) H2O on the equilibrium synthesis of DME at T=250°C and p=50 bar. Figure 6: a) Influence of the CO2 and b) H2O on the equilibrium synthesis of DME at T=250°C and p=50 bar. 3.2 DME reactor design: The DME reactor was considered as a multitube fixed bed reactor. Each tube contains the double catalyst (physically mixed) with a bed voidage of 0.45. The total density of the catalyst particles is an average of the density of the two catalyst, Cu/ZnO/Al2O3 and γ-Al2O3, used in the 1:2 optimal ratio. The number of tubes and their diameter was fixed while the length was varied in order to maximize the DME yield. As shown in Figure 7 the maximum yield can be achieved with a reactor length of minimum 10 m, for this reason this value was selected in the design phase. The parameters used for the DME reactor are listed in table 4. Figure 7 DME yield from DME reactor varying reactor length (L). Figure 7 DME yield from DME reactor varying reactor length (L). Table 4 Fixed parameters for DME reactor design. Table 4 Fixed parameters for DME reactor design. N° tubes Diamater [m] Bed voidage ρ Cu/ZnO/Al2O3 [kg/m3] ρ γ-Al2O3 [kg/m3] ρ average [kg/m3] 5000 0.02 0.45 1200 1470 1380 4. Performance results 4.1 Effect of operating conditions 4.1 Effect of operating conditions A sensitivity analysis of the most affecting parameters (such as operating pressure of chemical looping CO2/H2O splitting unit, outlet temperature of reduction reactor, CO2/H2O composition in the oxidation reactor of the CL unit, turbine in temperature) was performed to maximize the global efficiency of the plant (Eq. 19) and the DME production. DME DME MEOH MEOH NE c N NG T G m LHV m LHV W m LHV        (19) (19) N NG G m LHV  (19) where: mDME ,mMeOH represent the DME and MeOH produced (kg/s), LHVDME and LHVMeOH the lower heat value of DME, MeOH and natural gas (MJ/kg), WNET the net power produced inside the plant (MW) and mNG the natural gas feed into the plant (kg/s) Figure 8 shows the effect of varying the pressure of the chemical looping unit where both oxidation and reduction reactor works at the same pressure. An efficiency gain is obtained by increasing the pressure operation of the CL unit, from 49.4% at 1 bar to 51.1% at 5 bar. This can be attributed to the fact that a significant saving of the auxiliary power compression (WCOMP,tot) can be obtained by reducing the pressure ratio of syngas compression. However, with a further increment of the pressure, the efficiency drops down to 43.6%. Based on the Le Châtelier principle, it can be understood that the reaction in the RED reactor is not thermodynamically favoured at high pressure since the reduction reaction has three moles of reactants and four of products.It can be seen that after 6 bar the amount of reduced ceria at the outlet of the reactor decreases. This results in a drop in power production and overall efficiency. Figure 8: Influence of chemical looping unit pressure on efficiency η, WNET, Ce2O3 outlet from RED and WCOMP,tot (=WCOMP-1+ WCOMP-2+ WCOMP-3). Figure 8: Influence of chemical looping unit pressure on efficiency η, WNET, Ce2O3 outlet from RED and WCOMP,tot (=WCOMP-1+ WCOMP-2+ WCOMP-3). Figure 8: Influence of chemical looping unit pressure on efficiency η, WNET, Ce2O3 outlet from RED and WCOMP,tot (=WCOMP-1+ WCOMP-2+ WCOMP-3). Another fundamental parameter is the metal oxide outlet temperature from the reduction reactor. It is found that below 900°C there is no complete metal oxide conversion. Since the analysis was performed considering the complete reduction of ceria, all the analysis was conducted above a reduction temperature of 900°C. 4.1 Effect of operating conditions A higher metal oxide temperature at the outlet of the reactor, inherently demands more heat supply. Since this heat is derived from the heat of combustion, to have more reduced metal oxide temperature, less CO2 needs to be recirculated. This, even though results in a corresponding drop in the power spent for recycling CO2 (WCOMP-3), also implies a lower mass flow through GT producing less power as shown in Figure 9. This is nevertheless partially counterbalanced by the increase in the power produced by the ST due to the higher temperature of the GT outlet. However, since the gas turbine generates more power than the steam turbine (about 2.5 times), the net power decreases. Eventually this also restricts the effective operation of the OXI. Since both the splitting reactions are exothermic, by principle this requires the reactions to takes place at lower temperature. More so, for the water splitting reaction which shows a higher exothermicity than CO2 with Ce2O3, this would result in an even slower reaction. The effect of this is evident in Figure 9, in which a significant drop in the DME production can be seen beyond 1000°C (from 2.14 kg/s for 900°C to 2.13 kg/s at 1000°C and to 1.99 Kg/s at 1100°C) due to a deviation from the ideal H2-CO ratio and an increase in CO2 in the produced syngas (Figure 10). Being a polygeneration power plant, the production of DME place a key role in the overall plant effectiveness as well as significantly its efficiency. This is observed in Figure 9, where the higher drop in the DME production than the corresponding drop in the GT power output influences a sharp drop in the efficiency. To be more specific a relative 10.5% of efficiency drop is observed between 1000°C and 1300°C, corresponding to a relative drop in DME production of 24% and a relative net power output drop of 2%. Figure 9: influence of the metal oxide outlet temperature of RED on efficiency η, WNET, WST1, WGT and WST2, QRED, WCOMP-3. Figure 9: influence of the metal oxide outlet temperature of RED on efficiency η, WNET, WST1, WGT and WST2, QRED, WCOMP-3. Figure 10 shown the effect of the metal inlet temperature of the OXI. Due to the higher exothermicity, water splitting is favoured at lower temperature with respect of CO2 splitting. 4.1 Effect of operating conditions Hence, in order to have the ideal 1:1 H2/CO ratio for DME production is necessary to send as inlet of the oxidation reactor a rich-in H2O mixture. With the increase of the metal oxide temperature, the water splitting is further penalized and consequently an higher H2O content is necessary at the inlet. As shown in Figure 10a the amount of water needed ranged from 60% to 74% with a metal oxide temperature inlet from 900°C to 1300°C. In addition, as already explained in section Error! No s'ha trobat l'origen de la referència., in order to enhance DME production dilution product as to be avoided. As shown in Figure 10.b, even if it might be possible to produce the ideal composition of syngas for whatever metal oxide temperature inlet, at higher temperatures the CO2 content increases. For this reason is suitable to work with low ceria inlet temperature (900-1000°C). Figure 10: Effect of the initial gas mixture composition fed into OXI on a) final syngas H2/CO ratio, b) CO2 content in the syngas after water removal Figure 10: Effect of the initial gas mixture composition fed into OXI on a) final syngas H2/CO ratio, b) CO2 content in the syngas after water removal Figure 11 describes the effect of the variation on the gas mixture composition at the inlet of the OXI on plant performance. The maximum efficiency of 50% is achieved with an OXI inlet mixture of 60% of H2O, following the above discussion. It can be seen at that point H2 and CO curve coincides gives H2/CO ratio of 1. Figure 11: Influence of the as mixture composition into the inlet of OXI on the plant performance b considering a metal outlet temperature from the RED equal to 900°C. Figure 11: Influence of the as mixture composition into the inlet of OXI on the plant performance b considering a metal outlet temperature from the RED equal to 900°C. Following the above discussions, increasing the water content of the gas mixture feed into OXI, increases the temperature of the outlet metal oxide from the oxidation reactor (Figure 12.a). In the proposed OXYF-CL-PFG plant layout, the oxidized ceria is recirculated back to RED without intermediate heat recuperation. Hence, a higher ceria outlet temperature from OXI results in a higher metal oxide inlet temperature into the RED. This reduces the heat requirement for the reduction reaction. 4.1 Effect of operating conditions This, in term, increases the CO2 recirculation into the GT and hence, the power output from the same. Thus, an increment in the net power production is achieved (Figure 12.d). However, in order to have a higher temperature, the inlet mixture to OXI has to diverge from the ideal to obtain the 1:1 ratio of H2/CO. The drop in DME production due to the non-optimal syngas composition leads to an overall efficiency decrement (Figure 12.b-c). Furthermore, as can be seen from Figure 12.d working with the ideal gas mixture for DME production, the net power is reduced. As described in section 3.2, to an inlet syngas to the DME reactor which diverges from the 1:1 H2-CO ratio, correspond unreacted hydrogen and carbon monoxide. Since after distillation the unreacted species are recirculated in the combustor, more power is generated. Figure 12: effect of the gas mixture inlet composition and metal oxide inlet temperature (TOC,OXI inlet) on the temperature of the metal oxide outlet Figure 12: effect of the gas mixture inlet composition and metal oxide inlet temperature (TOC,OXI inlet) on the temperature of the metal oxide outlet Finally, the impact of the gas turbine inlet temperature (TIT) was analysed. Efficiency and net power produced are positively influenced by TIT (Figure 13). Nevertheless, the output from the GT drops. This is due to the lower recirculation of CO2 inside the combustor and so a lower gas volume being expanded within the GT. This loss is partially recovered by the lower compression work for the recirculated CO2 in COMP-2. Also, the produced by the steam turbine ST1 increases due to a higher exhaust temperature from the GT. Hence, corresponding to a TIT of 1100°C and efficiency of 47.6% was obtained, which increases to 50.7% for a TIT of 1450°C. 4.1 Effect of operating conditions Figure 13 Effect of the gas turbine inlet temperature TIT on the efficiency of the plant (η), power produced by the steam turbine (WST), by the gas turbine GT (WGT) and the power absorbed by the COMP-3 (WCOMP-3) Figure 13 Effect of the gas turbine inlet temperature TIT on the efficiency of the plant (η), power produced by the steam turbine (WST), by the gas turbine GT (WGT) and the power absorbed by the COMP-3 (WCOMP-3) Based on the sensitivity analysis the following operation parameters were chosen:  chemical looping operation pressure of 2 bar;  chemical looping operation pressure of 2 bar;  40% CO2 and 60% H2O feed in the oxidation reactor with an excess of 60% with respect to the stochiometric value based on the Ce2O3 inlet to OXI;  reduction reactor temperature outlet equal to 900°C;  reduction reactor temperature outlet equal to 900°C;  TIT of 1377oC.  TIT of 1377oC. The plant performance based on the listed parameters are summarized in table 5. Table 6 gives the composition and main thermodynamics parameters of selected streams. The plant produces 102.90 MWe, 2.15 kg/s of DME and 0.03 kg/s of methanol with a total efficiency of 50.21% and a DME yield of 24.9% (as per eq.19. The highest power consumption is represented by the COMP-3 for the recycle of the CO2 followed by the compression work in the ASU, which account, respectively, for the 17% and 11.5% of the gross power generated. The inlet stream to the DME reactor (stream 14) has the ideal CO:H2 ratio, while the CO2 content is 13%. However, it can be seen in Figure 10b, that minimum CO2 percentage which can be achieved from the oxidation reactor is near 6%, even though not producing the equimolar mixture of H2/CO. So the actual plant configuration allows producing a syngas with a composition which diverges from the ideal one. A solution might be to propose two distinct oxidation reactors, one for the CO2 dissociation and one for the water dissociation. However, this might lead to two different oxidized metal temperatures, complicating the system design and operations. An encouraging result is that the proposed Oxyfuel-NGCC cycle with the chemical looping and DME units permits to cut the efficiency penalty of CCS. 1 considering 0.09 kWh/Nm3 energy requirement for CO2 compression [85]. 4.1 Effect of operating conditions In particular, compared with results from literature [53], it is possible to achieve a gain of 4 percentage points with respect to a stand-alone Oxyfuel- NGCC process1. The total CO2 produced and captured is equal to 3.34 million tons per year out of which 3.4% is embedded in the DME (CO2,DME). The recirculation streams of CO2 (CO2,REC) in the combustor accounts for the 85% of the mole per second produced of CO2. while the one sent into OXI for dissociation is 7%. Table 5 Plant results with selected parameters. Table 5 Plant results with selected parameters. NG feed 25.2 ton/h WGROSS 167.61 MW WNET 102.90 MW ηTOT 50.21% WCOMP-1 3.76 MW WCOMP-2 10.67 MW WCOMP-3 28.29 MW WASU 19.34 MW WGT 114.42 MW WST1 44.30 MW WST2 2.96 MW WTURBEXP 4.37 MW 1 considering 0.09 kWh/Nm3 energy requirement for CO2 compression [85]. 1 considering 0.09 kWh/Nm3 energy requirement for CO2 compression [85]. 1 considering 0.09 kWh/Nm3 energy requirement for CO2 compression [85]. ṁDME 2.14 kg/s ṁMeOH 0.03 kg/s CO2,REC 0.85 CO2,DME 0.034 Table 6: Thermodynamics properties and composition of selected streams. stream 28 10 15 16 17 20 31 37 38 43 47 7 9 T (°C) 900 1322 200 250 46 40 1377 80 40 40 46 900 1322 P (bar) 2 2 5 5 1 9 26 2 1 10 2 2 Mole flow (kmol/h) 1 0.47 0.34 0.15 0.05 0.04 3.7 2.44 0.19 0.28 0.09 0.59 0.29 Molar fraction O2 0 0 0 0 0 0 0 0 0 0 0 0 0 H2 0.57 0.32 0.44 0.06 0 0 0 0 0 0 0.01 0 0 H2O 0 0.28 0 0.01 0.03 0 0.22 0 0 0.99 0 0 0 CO2 0 0.09 0.13 0.57 0 0 0.77 0.99 0.99 0 0.93 0 0 CO 0.3 0.31 0.43 0.05 0 0 0 0.01 0 0 0.03 0 0 CH4 0.12 0 0 0 0 0 0 0 0 0 0 0 0 MeOH 0 0 0 0.01 0.02 0 0 0 0 0 0 0 0 DME 0 0 0 0.3 0.94 1 0 0 0 0 0.02 0 0 CeO2 0 0 0 0 0 0 0 0 0 0 0 1 0 Ce2O3 0 0 0 0 0 0 0 0 0 0 0 0 1 5. Heat integration in the plant Table 6: Thermodynamics properties and composition of selected streams. 4.1 Effect of operating conditions The pinch point analysis was performed to evaluate the thermal integration of the whole plant [54]. The highest temperature found is 1322°C and correspond to the oxidation reactor outlet, while the lowest is -40°C and correspond to the DME condensation temperature. The Figure 15of the hot and cold composite curve shows that a good thermal integration can be obtained between cold and hot utilities, without the use of an external heat source. Starting from the hotter utilities, the profile can be interpreted as follow:  The steep section , from 550° to 900°C for the cold utilities curve represents the last part of natural gas preheating before the reduction in RED (stream 5) driven by the NGPHX1 (HX-1 and HX-8) and NGPHX1 (HX-2 and HX-8b);  The cold utilities curve from 50°C to 550°C represents the steam generation ( stream 1A-7A and 2B-7B) driven by the exhaust gas (stream 32) and the hot syngas from the oxidation (stream 10). It also represents the CO2-H2O preheating before the dissociation (stream 39 and 45) driven by stream 10a and 10b in the CO2,PHX (HX-5 and HX-19) and H2OPHX (HX-4 and HX-12); (stream 10). It also represents the CO2-H2O preheating before the dissociation (stream 39 and 45) driven by stream 10a and 10b in the CO2,PHX (HX-5 and HX-19) and H2OPHX (HX-4 and HX-12);  The last part is mostly related to the distillation unit and the condensation up to a temperature of -40°C of the DME; The last part of the hot utilities corresponds to the distillation of DME. As can be observed a good heat integration among hot and cold utilities has been achieved as per the current model. Therefore, the scope for a further increment in the efficiency of the power plant through optimized heat integration is limited. As can be observed a good heat integration among hot and cold utilities has been achieved as per the current model. Therefore, the scope for a further increment in the efficiency of the power plant through optimized heat integration is limited. Hot utilities Cold utilities Figure 14: Pinch point analysis, hot and cold composite curve Figure 14: Pinch point analysis, hot and cold composite curve 6. Exergy analysis Exergy of a steady stream of matter is defined as the maximum amount of work obtainable when the stream is brought from its initial state to the dead state by processing during in which the stream may interact only with the environment [55]. Exergy can be divided into different components: kinetic exergy Ek, potential exergy Ep, physical exergy Eph and chemical exergy Ech. k p ph ch E E E E E     (20) (20) k p ph ch E E E E E     In the presented exergy analysis the physical and chemical exergy are considered. The physical exergy is defined as the maximum work achievable from a system that from its initial state is brought to the environmental state with only thermal and mechanical interaction with the environment. While the chemical exergy is the maximum work obtainable from a system that is brought from the environmental state to the dead state involving heat transfer and exchange of substances only with the environment. The two types of exergy are given by equation (21) and (22) [56]. In particular, for a mixture, the total chemical exergy Ech,tot is made by two contributes: the chemical exergy of the single i-th component E0,i and the work obtainable from a reversible isothermal expansion at T0 from the partial pressure p00 of the i-th component and environment pressure p0. 6. Exergy analysis     0, 0 0, ph i i i i i i x h h T E s s           (21) 0 , 0, 0 00 ln ch tot i i i E p E x R T p            (22)     0, 0 0, ph i i i i i i x h h T E s s           0 , 0, 0 00 ln ch tot i i i E p E x R T p            (21) (22) where: i) xi is the mass fraction of the i-th component in the mixture, ii) hi is the mass enthalpy of the i-th component, iii) h0,i is the mass enthalpy of the i-th component at the environment state, iv) is the temperature of the environment state, v) si is the mass entropy of the i-th component, vi) s0,i is the mass entropy of the i-th component at the environment state, vii) γ activity coefficient for the i-th component and viii) R is the ideal gas constant. The advantage of an exergy analysis is that it is based on the second principle of thermodynamics, thus permits to evaluate the so-called ‘‘destroyed’’ exergy Idest.. Destroyed exergy represents the real loss in the quality of energy that cannot be identified by means of a simple energy balance because the conservation of energy will always apply. The following equation represents an exergy balance of a general device in steady state condition: the conservation of energy will always apply. 6. Exergy analysis The following equation represents an exergy balance of a general device in steady state condition: 0 , , ) ( 1 out i in i i i dest i i i i T E E W Q I T               (23) (23) Where: i) the members at the left side represents the exergy contribution of the , in i E inlet and , out i E outlet mass flows, ii) i W the first member at right side represents the absorbed/produced work by the device, iii) the second member is the heat exchanged contribution, it represents the work obtainable from the heat flux Q operating with a Carnot machine and iv) Idest is the irreversibility generated. In order to estimate the exergy efficiency (or efficiency of the second principle) of a system is necessary to define which are the material consumed during the process (Fuel) and which are the final products of the process (Products). The exergy efficiency is shown by the following equation: p ex f E E   (24) (24) Where Ep represents the exergy of the product streams and Ef the exergy of the resource streams. However, the only exergy efficiency does not give a complete framework of the plant or subsystem. For this reason, additional exergetic factor was adopted [57]:  Relative irreversibilities: , , i destroyed i tot destroyed I I  (25)  Fuel depletion rate: , , i destroyed i f plant E I  (26)  Productivity lack: , , i destroyed i p plant E I  (27) - Exergetic factor: , , f i i f plant E E  (28)  Relative irreversibilities: , , i destroyed i tot destroyed I I  (25)  Fuel depletion rate: , , i destroyed i f plant E I  (26)  Relative irreversibilities: , , i destroyed i tot destroyed I I  (25) (25)  Fuel depletion rate: , , i destroyed i f plant E I  (26) (27) (28) Before a reference state was selected (Table 7). For the environmental state, a pressure (P0) of 1 atm and a temperature (T0) of 20°C were selected, while for the dead state the reference environment of Szargut [58] was chosen. 6. Exergy analysis Regarding Ce2O3, there are no works in literature where it is a reference substance and so there are no data about its chemical energy. In fact, in most of the works, Ce and CeO2 are used as a reference substance, since they are the most abundant form of ceria in the heart. However, Ce2O3 chemical exergy can be calculated starting from the known exergy of Ce and O2 (Eq.29 and Eq.30): (29) 2 3 2 Ce O 2 3 0 0 0 0 Ce O Ce O Ex 2Ex 1.5Ex G    (30) 0 0 0 2Ex 1 5Ex G    (30) 2 Ce O 2 3 0 0 0 Ce O 2Ex 1.5Ex G   (30 where: i)E0Ce, E0Ce2O3 and E0O2 are the standard chemical exergy of Ce, Ce2O3 and O2, respectively, ii) ΔG0Ce2O3 represents the Gibbs free energy for the formation of Ce2O3 from the Ce-O2 reaction (Eq.30) where: i)E0Ce, E0Ce2O3 and E0O2 are the standard chemical exergy of Ce, Ce2O3 and O2, respectively, ii) ΔG0Ce2O3 represents the Gibbs free energy for the formation of Ce2O3 from the Ce-O2 reaction (Eq.30) Table 7: environment state and dead state data and assumptions. Environmental state: P0=1 atm T0=20°C Dead State Chemical exergy Ech (j/mol) H2 CO CO2 H2Ovap H2Oliq N2 CH4 O2 CeO2 Ce2O3 DME [30] MeOH [30] 236090 275100 19203 9181 870 696 853356 3837 33800 384702 1414500 715520 Since in the proposed layout there are several chemical reactions, which change the composition of the gaseous streams, the first step was to evaluate the reference chemical exergy of the multiple mixture streams using the dead state of the reference elements (eq.30). The results are shown in Table 8. Table 8 specific chemical exergy of the gas mixture streams. Stream 31 28 10 15 13 47 ech,i [kJ/kg] 388.68 27,109.72 7,391.26 11,287.08 11,919.06 6,225.07 Table 8 specific chemical exergy of the gas mixture streams. A similar sensitivity analysis to the one previously reported has been performed to assess the exergetic performance of the plant (eq 31) and the irreversibilities generated during the process (eq 32). As can A similar sensitivity analysis to the one previously reported has been performed to assess the exergetic performance of the plant (eq 31) and the irreversibilities generated during the process (eq 32). 6. Exergy analysis Figure 15: exergetic efficiency and total irreversibilities generated varying a) the operating pressure of the CL unit, b)the temperature of the inlet metal into the reduction reactor (TMET,in) c) the temperature inlet into the gas turbine (TIT) Figure 15: exergetic efficiency and total irreversibilities generated varying a) the operating pressure of the CL unit, b)the temperature of the inlet metal into the reduction reactor (TMET,in) c) the temperature inlet into the gas turbine (TIT) Figure 16: Effect of the gas mixture composition feed into the OXI on the exergetic efficiency ηex and irreversibilities Idest Figure 16: Effect of the gas mixture composition feed into the OXI on the exergetic efficiency ηex and irreversibilities Idest Finally, a detailed exergy analysis of the main components of the layout operating at the conditions described was performed. Table 9 reports the exergy values of the main streams. Finally, a detailed exergy analysis of the main components of the layout operating at the conditions described was performed. Table 9 reports the exergy values of the main streams. Finally, a detailed exergy analysis of the main components of the layout operating at the conditions described was performed. Table 9 reports the exergy values of the main streams. 6. Exergy analysis As can be clearly observed, due to both electricity and heat self-sufficiency of the system, the input fuel, namely natural gas contributes entirely (100% of the total exergy input) to the net exergy input to the system. The product instead is the total DME, MeOH and the net power produced by the plant. be clearly observed, due to both electricity and heat self-sufficiency of the system, the input fuel, namely natural gas contributes entirely (100% of the total exergy input) to the net exergy input to the system. The product instead is the total DME, MeOH and the net power produced by the plant. 4 DME DME MEOH MEOH NET ex CH NG E m W E m E m        (31) 4 , tot dest CH DME DME MEOH M G EOH NET N E E I m m E m W        (32) DME MEOH MEOH NET E W E m E     (3 (31) 4 CH NG m E   ( ) 4 , tot dest CH DME DME MEOH M G EOH NET N E E I m m E m W        (32) 4 CH NG m E  ( 4 CH DME DME MEOH M G EOH NET N E E m E m W       (32 4 , tot dest CH DME DME MEOH M G EOH NET N E E I m m E m W        (32) As expected, since the efficiency is related to only natural gas, DME, MeOH and net power production, the exergy efficiency trend is specular to the thermodynamics efficiency previously described. For this reasons, the same explanation of the plot in section 4.1 can be adopted for the following figure 15 and 16. 6. Exergy analysis Table 9 Chemical, physical and total exergry of the plant streams State Specific Physical Exergy [kJ/kg] Specific Chemical Exergy [kJ/kg] Specific Exergy [kJ/kg] Exergy Flow [MW] State Specific Physical Exergy [kJ/kg] Specific Chemical Exergy [kJ/kg] Specific Exergy [kJ/kg] Exergy Flow [MW] 0 - - - - 31 1.375,23 387,84 1.763,06 246,88 1 247,15 119,91 367,05 7,98 32 515,30 387,84 903,14 126,47 1a 14,72 24,86 39,58 2,83 33 37,04 387,84 424,87 59,50 2 584,21 53.201,75 53.785,96 376,50 34 49,60 436,43 486,04 57,23 3 806,40 53.201,75 54.008,14 378,06 35 49,60 436,43 486,04 4,84 4 97,86 53.201,75 53.299,61 373,10 36 40,96 436,43 477,40 47,78 5 1.851,99 53.201,75 55.053,73 385,38 37 218,95 436,43 655,38 69,78 6 1.542,39 27.109,67 28.652,06 335,37 38 49,60 436,43 486,04 4,02 7 718,95 1.172,02 1.890,97 182,52 39 94,68 436,43 531,11 4,39 9 599,54 195,24 794,78 80,45 40 294,88 436,43 731,31 6,04 10 1.772,70 7.416,35 9.189,05 79,42 41 3,18 48,33 51,52 0,76 10a 1.063,98 7.416,35 8.480,33 73,29 42 3,18 48,33 51,52 0,50 10b 546,08 7.416,35 7.962,43 68,81 43 3,18 48,33 51,52 0,12 11 313,68 7.416,35 7.730,03 66,81 44 3,11 48,33 51,44 0,12 12 3,21 48,33 51,54 0,12 45 497,88 48,33 546,21 1,27 13 90,35 11.914,03 12.004,38 74,87 46 1.022,64 48,33 1.070,97 2,48 Table 9 Chemical, physical and total exergry of the plant streams 14 579,24 11.914,03 12.493,27 77,92 47 621,44 6.044,21 6.665,65 0,75 15 291,85 11.273,25 11.565,10 72,13 48 607,94 6.044,21 6.652,15 0,75 16 198,40 11.273,25 11.471,65 71,55 49 505,90 6.044,21 6.550,11 0,74 17 171,76 11.246,12 11.417,87 69,93 1-A 19,82 48,33 68,15 2,36 18 164,88 11.246,12 11.411,00 69,89 2-A 19,45 48,33 67,78 2,35 19 127,61 11.246,12 11.373,73 69,66 3-A 630,56 48,33 678,89 23,51 20 133,68 436,43 570,11 2,23 4-A 1.161,85 48,33 1.210,18 41,90 21 81,93 30.292,33 30.374,25 67,05 5-A 1.552,72 48,33 1.601,06 55,43 22 95,62 30.292,33 30.387,95 67,08 6-A 109,18 48,33 157,51 5,45 23 109,35 30.750,00 30.859,35 66,67 7-A 2,06 48,33 50,40 1,74 24 100,82 9.216,04 9.316,86 0,44 1-B 19,82 48,33 68,15 0,16 25 66,65 9.216,04 9.282,69 0,44 2-B 20,74 48,33 69,07 0,16 26 4.638,21 21.603,97 26.242,18 0,53 3-B 630,56 48,33 678,89 1,57 27 43,82 48,33 92,15 0,00 4-B 1.161,85 48,33 1.210,18 2,80 28 357,80 27.109,67 27.467,47 321,51 5-B 1.552,72 48,33 1.601,06 3,71 28b 189,96 27.109,67 27.299,62 319,54 6-B 109,18 48,33 157,51 0,36 29 145,98 27.109,67 27.255,65 319,03 7-B 2,06 48,33 50,40 0,12 30 868,38 27.109,67 27.978,05 327,48 Figure 17: Total irreversibilities distribution. 6. Exergy analysis 3,9% 4,2% 46,3% 14,3% 1,7% 0,2% 0,1% 2,9% 1,0% 2,7% 6,3% 2,6% 2,2% 1,7% 0,3% 2,1% 0,3% 1,7% 0,1% 0,0% 5,5% ASU TURBOEXP RED + COMB OXI HRSG2 ST2 STEAM-COND2 COMP-3 COMP-2 GT HRSG ST COND-2 STEAM-COND1 COMP-1 DME Reactor VLS CLN-CO2 CLN-DME CLN-MeOH others 14 579,24 11.914,03 12.493,27 77,92 47 621,44 6.044,21 6.665,65 0,75 15 291,85 11.273,25 11.565,10 72,13 48 607,94 6.044,21 6.652,15 0,75 16 198,40 11.273,25 11.471,65 71,55 49 505,90 6.044,21 6.550,11 0,74 17 171,76 11.246,12 11.417,87 69,93 1-A 19,82 48,33 68,15 2,36 18 164,88 11.246,12 11.411,00 69,89 2-A 19,45 48,33 67,78 2,35 19 127,61 11.246,12 11.373,73 69,66 3-A 630,56 48,33 678,89 23,51 20 133,68 436,43 570,11 2,23 4-A 1.161,85 48,33 1.210,18 41,90 21 81,93 30.292,33 30.374,25 67,05 5-A 1.552,72 48,33 1.601,06 55,43 22 95,62 30.292,33 30.387,95 67,08 6-A 109,18 48,33 157,51 5,45 23 109,35 30.750,00 30.859,35 66,67 7-A 2,06 48,33 50,40 1,74 24 100,82 9.216,04 9.316,86 0,44 1-B 19,82 48,33 68,15 0,16 25 66,65 9.216,04 9.282,69 0,44 2-B 20,74 48,33 69,07 0,16 26 4.638,21 21.603,97 26.242,18 0,53 3-B 630,56 48,33 678,89 1,57 27 43,82 48,33 92,15 0,00 4-B 1.161,85 48,33 1.210,18 2,80 28 357,80 27.109,67 27.467,47 321,51 5-B 1.552,72 48,33 1.601,06 3,71 28b 189,96 27.109,67 27.299,62 319,54 6-B 109,18 48,33 157,51 0,36 29 145,98 27.109,67 27.255,65 319,03 7-B 2,06 48,33 50,40 0,12 30 868,38 27.109,67 27.978,05 327,48 3,9% 4,2% 46,3% 14,3% 1,7% 0,2% 0,1% 2,9% 1,0% 2,7% 6,3% 2,6% 2,2% 1,7% 0,3% 2,1% 0,3% 1,7% 0,1% 0,0% 5,5% ASU TURBOEXP RED + COMB OXI HRSG2 ST2 STEAM-COND2 COMP-3 COMP-2 GT HRSG ST COND-2 STEAM-COND1 COMP-1 DME Reactor VLS CLN-CO2 CLN-DME CLN-MeOH others Figure 17: Total irreversibilities distribution. Figure 17: Total irreversibilities distribution. The global exergy efficiency of the plant is 45.0%, five points lower than the calculated thermal efficiency. The total irreversibilities generated are 202.72 MW with an overall fuel depletion rate (θ) of 53.84%.All the components present an exergetic efficiency over the 80%, with the exception of the air separation unit (55.9%) and the two condensers for the steam cycle (32%). However, the contribution of COND-A, COND-B and ASU to the overall efficiency is marginal since their relative irreversibilities χi don’t exceed the 3.9% (Tabe 10). The exergy efficiency of the RED+COMB results in 88.1%. Although this value is not extremely low, more than half of the 202.72 MW total irreversibities are located in this component. 6. Exergy analysis As shown in Table 10, the COMB+RED exergetic factor ψ results in 231.3%, so the irreversibilities are not due to the efficiency but are mainly proportionally correlated to the high exergy of the inlet streams. In fact, the exergy inlet of the COMB+RED ranks first among the components (870 MW), the second is the turbo-expander inlet (378 MW). Moreover it is worth mentioning that, thanks to the hypothesis of not heat losses inside the combustor and reduction reactor, the main contributor to the exergy losses are of chemical form. In fact the exergy efficiency of the COMB+RED, considering only the chemical exergy of the inlet and outlet streams, results in 70%. The oxidation reactor is the second ranked component for the relative irreversibilities parameter (14.4%) even if the exergy efficiency (83.4%) results in to be lower than the one of the RED+COMB. This is due to the lower exergy factor (50.7%). The other irreversibilities are mostly located in the HRSG1 (13.99 MW) and in the compression process (9.3 MW). The DME reactor jacketing for saturated steam production allows to increase the exergy efficiency of the component of 2.2%. Table 10 Results from the exergetic analysis of the main components. Componet Exergy balance eq.1 Irr [MW] ηex,i [%] θi [%] ψi [%] ξi [%] ASU 𝐸0 + 𝑊𝐴𝑆𝑈= 𝐸1 + 𝐸1𝑏+ 𝐼𝐴𝑆𝑈 8,53 55,91 2,26 5,14 5,03 TURBOEXP 𝐸3 = 𝑊𝑇𝑈𝑅𝐵𝑂𝐸𝑋𝑃+ 𝐼𝑇𝑈𝑅𝐵𝑂𝐸𝑋𝑃 0,59 99,84 0,16 100,41 0,35 Table 10 Results from the exergetic analysis of the main components. Componet Exergy balance eq.1 Irr [MW] ηex,i [%] θi [%] ψi [%] ξi [%] ASU 𝐸0 + 𝑊𝐴𝑆𝑈= 𝐸1 + 𝐸1𝑏+ 𝐼𝐴𝑆𝑈 8,53 55,91 2,26 5,14 5,03 TURBOEXP 𝐸3 = 𝑊𝑇𝑈𝑅𝐵𝑂𝐸𝑋𝑃+ 𝐼𝑇𝑈𝑅𝐵𝑂𝐸𝑋𝑃 0,59 99,84 0,16 100,41 0,35 Table 10 Results from the exergetic analysis of the main components. 6. Exergy analysis 1 The left-side of the equation represents the fuel of the component, while the right-side represent the product and the irreversibilities of the component. 2 Q* represents the exergy obtainable using the heat of the selected stream 2 Q* represents the exergy obtainable using the heat of the selected stream 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐶𝑂2= ∗ 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐶𝑂2 × (1 − 𝑇𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐶𝑂2 𝑇0 ) , 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐶𝑂2 ∗ = 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐶𝑂2 × (1 − 𝑇0 𝑇𝑅𝐸𝐵,𝐶𝐿𝑁−𝐶𝑂2) 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐷𝑀𝐸= ∗ 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐷𝑀𝐸× (1 − 𝑇0 𝑇𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐷𝑀𝐸) , 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐷𝑀𝐸 ∗ = 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐷𝑀𝐸× (1 − 𝑇0 𝑇𝑅𝐸𝐵,𝐶𝐿𝑁−𝐷𝑀𝐸) 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻 ∗ = 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻× (1 − 𝑇0 𝑇𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻), 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻 ∗ = 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻× (1 − 𝑇0 𝑇𝑅𝐸𝐵,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻) 8 2 Economic anal sis 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐶𝑂2= ∗ 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐶𝑂2 × (1 − 𝑇𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐶𝑂2 𝑇0 ) , 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐶𝑂2 ∗ = 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐶𝑂2 × (1 − 𝑇0 𝑇𝑅𝐸𝐵,𝐶𝐿𝑁−𝐶𝑂2) 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐷𝑀𝐸= ∗ 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐷𝑀𝐸× (1 − 𝑇0 𝑇𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐷𝑀𝐸) , 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐷𝑀𝐸 ∗ = 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐷𝑀𝐸× (1 − 𝑇0 𝑇𝑅𝐸𝐵,𝐶𝐿𝑁−𝐷𝑀𝐸) 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻 ∗ = 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻× (1 − 𝑇0 𝑇𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻), 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻 ∗ = 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻× (1 − 𝑇0 𝑇𝑅𝐸𝐵,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻) 6. Exergy analysis RED + COMB 𝐸9𝑎+ 𝐸5 + 𝐸30 + 𝐸1 + 𝐸49 + 𝐸37 = 𝐸31 + 𝐸28 + 𝐸7 + 𝐼𝑅𝐸𝐷+𝐶𝑂𝑀𝐵 103,83 88,08 27,58 231,29 61,23 OXI 𝐸7 + 𝐸46 + 𝐸40 = 𝐸10 + 𝐸9𝑏+ 𝐼𝑂𝑋𝐼 29,47 84,57 7,83 50,74 17,38 HRSG2 𝐸10 + 𝐸2−𝐵= 𝐸10𝑎+ 𝐸5−𝐵+ 𝐼𝐻𝑅𝑆𝐺−2 3,68 97,49 0,98 38,88 2,17 ST2 𝐸5−𝐵= 𝐸6−𝐵+ 𝑊𝑆𝑇2 + 𝐼𝑆𝑇2 0,38 89,77 0,10 0,99 0,22 COND-B 𝐸6−𝐵= 𝐸7−𝐵+ 𝐼𝐶𝑂𝑁𝐷−𝐵 0,25 32,00 0,07 0,10 0,15 COMP-4 𝐸38 + 𝑊𝐶𝑂𝑀𝑃−4 = 𝐸39 + 𝐼𝐶𝑂𝑀𝑃−4 0,08 98,262 0,021 1,186 0,046 COMP-3 𝐸49 + 𝑊𝐶𝑂𝑀𝑃−3 = 𝐸37 + 𝐼𝐶𝑂𝑀𝑃−3 6,29 91,73 1,67 20,21 3,71 COMP-2 𝐸29 + 𝑊𝐶𝑂𝑀𝑃−2 = 𝐸30 + 𝐼𝐶𝑂𝑀𝑃−2 2,21 99,33 0,59 87,57 1,30 GT 𝐸31 = 𝑊𝐺𝑇+ 𝐸32 + 𝐼𝐺𝑇 6,00 97,57 1,59 65,57 3,54 HRSG1 𝐸32 + 𝐸2−𝐴= 𝐸33 + 𝐸5−𝐴+ 𝐼𝐻𝑅𝑆𝐺−1 12,88 90,00 3,42 34,21 7,60 ST 𝐸5−𝐴= 𝐸6−𝐴+ 𝑊𝑆𝑇+ 𝐼𝑆𝑇 5,68 89,75 1,51 14,72 3,35 COND-2 𝐸33 = 𝐸34 + 𝐸41 + 𝐼𝐶𝑂𝑁𝐷−2 0,88 98,51 0,23 15,80 0,52 COND-A 𝐸6−𝐴= 𝐸7−𝐴+ 𝐼𝐶𝑂𝑁𝐷−𝐴 3,71 32,00 0,99 1,45 2,19 COMP-1 𝐸13 + 𝑊𝐶𝑂𝑀𝑃−1 = 𝐸14 + 𝐼𝐶𝑂𝑀𝑃−1 0,71 99,10 0,19 20,88 0,42 DME Reactor 𝐸14 + 𝐸44 = 𝐸15 + 𝐸45 + 𝐼𝐷𝑀𝐸 𝑟𝑒𝑎𝑐𝑡𝑜𝑟 4,64 94,05 1,23 20,73 2,74 VLS 𝐸15 = 𝐸47 + 𝐸17 + 𝐼𝑉𝐿𝑆 1,45 97,99 0,39 19,16 0,86 CLN-CO2 𝐸19 + 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐶𝑂2 ∗ + 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐶𝑂2 ∗ 2 = 𝐸20 + 𝐸21 + 𝐼𝐶𝐿𝑁−𝐶𝑂2 0,60 99,15 0,16 18,56 0,35 CLN-DME 𝐸22 + 𝑄𝐶𝑂𝑁,𝐶𝐿𝑁−𝐷𝑀𝐸 ∗ + 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐷𝑀𝐸 ∗ 2 = 𝐸22 + 𝐸23 + 𝐼𝑅𝐸𝐵−𝐷𝑀𝐸 0,30 99,56 0,08 17,90 0,18 CLN-MeOH 𝐸25 + 𝑄𝐶𝑂𝑁,𝐶𝐿𝑁−𝑀𝑒𝑂 ∗ + 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻 ∗ = 𝐸22 + 𝐸23 + 𝐼𝑅𝐸𝐵−𝑀𝑒𝑂𝐻 2 0,15 77,39 0,04 0,18 0,09 RED + COMB 𝐸9𝑎+ 𝐸5 + 𝐸30 + 𝐸1 + 𝐸49 + 𝐸37 = 𝐸31 𝐸28 + 𝐸7 + 𝐼𝑅𝐸𝐷+𝐶𝑂𝑀𝐵 OXI 𝐸7 + 𝐸46 + 𝐸40 = 𝐸10 + 𝐸9𝑏+ 𝐼𝑂𝑋𝐼 HRSG2 𝐸10 + 𝐸2−𝐵= 𝐸10𝑎+ 𝐸5−𝐵+ 𝐼𝐻𝑅𝑆𝐺−2 ST2 𝐸5−𝐵= 𝐸6−𝐵+ 𝑊𝑆𝑇2 + 𝐼𝑆𝑇2 COND-B 𝐸6−𝐵= 𝐸7−𝐵+ 𝐼𝐶𝑂𝑁𝐷−𝐵 COMP-4 𝐸38 + 𝑊𝐶𝑂𝑀𝑃−4 = 𝐸39 + 𝐼𝐶𝑂𝑀𝑃−4 COMP-3 𝐸49 + 𝑊𝐶𝑂𝑀𝑃−3 = 𝐸37 + 𝐼𝐶𝑂𝑀𝑃−3 COMP-2 𝐸29 + 𝑊𝐶𝑂𝑀𝑃−2 = 𝐸30 + 𝐼𝐶𝑂𝑀𝑃−2 GT 𝐸31 = 𝑊𝐺𝑇+ 𝐸32 + 𝐼𝐺𝑇 HRSG1 𝐸32 + 𝐸2−𝐴= 𝐸33 + 𝐸5−𝐴+ 𝐼𝐻𝑅𝑆𝐺−1 ST 𝐸5−𝐴= 𝐸6−𝐴+ 𝑊𝑆𝑇+ 𝐼𝑆𝑇 COND-2 𝐸33 = 𝐸34 + 𝐸41 + 𝐼𝐶𝑂𝑁𝐷−2 COND-A 𝐸6−𝐴= 𝐸7−𝐴+ 𝐼𝐶𝑂𝑁𝐷−𝐴 COMP-1 𝐸13 + 𝑊𝐶𝑂𝑀𝑃−1 = 𝐸14 + 𝐼𝐶𝑂𝑀𝑃−1 DME Reactor 𝐸14 + 𝐸44 = 𝐸15 + 𝐸45 + 𝐼𝐷𝑀𝐸 𝑟𝑒𝑎𝑐𝑡𝑜𝑟 VLS 𝐸15 = 𝐸47 + 𝐸17 + 𝐼𝑉𝐿𝑆 CLN-CO2 𝐸19 + 𝑄𝐶𝑂𝑁𝐷,𝐶𝐿𝑁−𝐶𝑂2 ∗ + 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐶𝑂2 ∗ 2 = 𝐸20 + 𝐸21 + 𝐼𝐶𝐿𝑁−𝐶𝑂2 CLN-DME 𝐸22 + 𝑄𝐶𝑂𝑁,𝐶𝐿𝑁−𝐷𝑀𝐸 ∗ + 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝐷𝑀𝐸 ∗ 2 = 𝐸22 + 𝐸23 + 𝐼𝑅𝐸𝐵−𝐷𝑀𝐸 CLN-MeOH 𝐸25 + 𝑄𝐶𝑂𝑁,𝐶𝐿𝑁−𝑀𝑒𝑂 ∗ + 𝑄𝑅𝐸𝐵,𝐶𝐿𝑁−𝑀𝑒𝑂𝐻 ∗ = 𝐸22 + 𝐸23 + 𝐼𝑅𝐸𝐵−𝑀𝑒𝑂𝐻 2 NGPHX1 𝐸2 + 𝐸28 = 𝐸3 + 𝐸28𝑏+ 𝐼𝑁𝐺𝑃𝐻𝑋1 0,41 99,94 0,11 185,39 0,24 NGPHX2 𝐸4 + 𝐸28𝑏= 𝐸5 + 𝐸29 + 𝐼𝑁𝐺𝑃𝐻𝑋2 1,59 99,78 0,42 188,17 0,94 CO2PHX 𝐸10𝑎+ 𝐸39 = 𝐸10𝑏+ 𝐸40 + 𝐼𝐶𝑂2𝑃𝐻𝑋 0,38 99,48 0,10 19,34 0,23 H2OPHX 𝐸10𝑏+ 𝐸45 = 𝐸11 + 𝐸46 + 𝐼𝐻2𝑂𝑃𝐻𝑋 3,26 95,63 0,87 19,80 1,92 1 The left-side of the equation represents the fuel of the component, while the right-side represent the product and the irreversibilities of the component. 8.2 Economic analysis An economic assessment was performed to calculate the capital cost of investment for the construction of the proposed plant. The National Energy Technology Laboratory (NETL) guidelines for techno-economic analysis for power plants was adopted [59,60]. This methodology defines capital cost at five levels: bare erected cost (BEC), engineering, Procurement and Construction Cost (EPCC), total project cost (TPC), total overnight cost (TOC) and total as-spent cost (TASC). In the current study, the TOC was considered for the capital investment expenditure. The first four items are “overnight cost” and are expressed in base year dollar that is the first year of capital expenditure. While, TASC is specified in mixed, current-year dollar over the all capital expenditure period. The Bare Erected Cost (BEC) comprises the cost of the equipment, facilities and infrastructure, and labour required for its installation. The equipment cost estimation was done using the scaling factor exponent n, equation 29 [61]: (33) n equ equ,0 0 C =C (A/A ) All the estimated equipment costs were converted to year 2017 dollar using the chemical engineering plant cost index (CEPCI, Table 12Error! No s'ha trobat l'origen de la referència.): equ,actual equ CEPCI 2017 C = C CEPCI at the time of original cost (34) The cost of the cooling tower system was included in the cost of the four condensers (COND-1, COND-2, HRSG1 and HRSG2 condenser). The overall cost was subdivided between the four components proportionally to the calculated rejected heat. The cost of the two condensers (COND-A and COND-B) of the two HRSG were included in the HRSG investment cost.The most expensive equipment is the ASU, followed by the GT. The RED+COMB unit accounts for 5.2% of the total expenditure. The individual contribution of the respective equipment to the total overnight cost is shown in Figure 18. Table 11 Equipment cost Equipment Scaling Parameter A Cost Year Cequ,0 [M$] Cequ,actual [M$] GT [39] GT Net Power [MW] 2008 $60.99 $25.70 HRSG, ducting and stack [62] ST Net Power [MW] 2003 $6.10 $4.55 ST and ST2, generator and auxiliaries [39] ST Gross Power [MW] 2008 $41.60 $15.82 Cooling Water System and Balance of Plant [39] Q rejected [MW] 2008 $61.23 $24.84 COMP-3 [39] Compressor Power [MW] 2008 $9.95 $16.77 COMP-2 [63] Compressor Power [MW] 2003 $4.83 $5.02 COMP-1 [63] Compressor Power [MW] 2003 $4.83 $2.50 ASU [64] Oxygen Production [ktO2/day] 2008 $62.96 $117.65 CLC and Oxy Reactor [65] Sized According to CLC Plant, 150MW 2016 $48.72 $14.55 TURBEXP [66] MW Power Produced 2008 $0.73 $1.32 DME reactor and BOP [63] Inlet gas [kmol/s] 2007 $21.00 $5.16 DME cooling system [63] Electrical power [MW] 2007 $1.70 $0.73 Clean-Up unit [63] inlet DME [kg/s] 2007 $28.40 $13.33 HRSG2, ducting and stack [62] ST Net Power [MW] 2003 $6.10 $13.23 COMP-4 [39] Compressor Power [MW] 2008 $9.95 $0.74 Exchangers [63] Heat exchanged [MWth] 2007 $52.00 $1.32 The bare erected cost (BEC) of each equipment was given summing to the equipment cost all the installation costs shown in Table 13: The bare erected cost (BEC) of each equipment was given summing to the equipment cost all the installation costs shown in Table 13: equ,actual BEC = C + Installation Cost (35) (35) The engineering, Procurement and Construction Cost (EPCC) comprises the BEC plus all the cost of all services provided by the engineering, procurement and construction contractor (eq. 36). These items include detailed design, contractor permitting and project management costs. EPCC = BEC + INDIRECT COST (36) (36) The total project cost (TPC) takes into account the EPCC plus the contingencies cost (eq. 37). equ,actual equ CEPCI 2017 C = C CEPCI at the time of original cost Contingencies are added to account for unknown costs that are omitted or unheralded due to lack of complete project definition or uncertainties with the development status of a technology. In the present case, since the proposed plant is based on a novel technologies arrangement a high contingency cost was selected (table 11). TPC = EPCC + CONTINGENCIES (37) TPC = EPCC + CONTINGENCIES (37) The total overnight cost (TOC) comprises the TPC plus other overnight costs (table 11), owner's cost included (i.e pre-production, inventory capital, land, financing), it was calculated as: TOC=TPC + OWNER'S COST (38) The total overnight cost (TOC) comprises the TPC plus other overnight costs (table 11), owner's cost included (i.e pre-production, inventory capital, land, financing), it was calculated as: The total overnight cost (TOC) comprises the TPC plus other overnight costs (table 11), owner's cost included (i.e pre-production, inventory capital, land, financing), it was calculated as: included (i.e pre production, inventory capital, land, financing), it was calculated as: TOC=TPC + OWNER'S COST (38) TOC=TPC + OWNER'S COST (38) TOC=TPC + OWNER'S COST (38) (38) The total overnight cost (TOC) of the plant resulted in 537.45 M$. In Figure 18 are shown the contribution to the total overnight cost of the different equipment. The most expensive equipment resulted in the ASU, followed by the GT. The RED+COMB unit accounts for 5.2% of the total expenditure. For evaluation of the economic analysis listed assumption in table 13, 14 and 15 for CAPEX estimation, OPEX estimation and economic assumption. expenditure. For evaluation of the economic analysis listed assumption in table 13, 14 and 15 for CAPEX estimation, OPEX estimation and economic assumption. Table 12 CEPCI Index Year CEPCI index 2017 572.7 2016 585.7 2008 575.4 2007 525 2006 499.6 2003 402 Table 13: main assumption in CAPEX estimation. Installation Cost [36] Accessory Electrical Plant 16,93 M$ FO Supply System and Natural Gas Supply System 10,04 M$ Erection, Steel Structures and Painting 49% of equipment cost Piping 9% of equipment cost Indirect cost [36] Yard improvement 2% BEC Services facilities 2% BEC Engineering/consulting costs 5% BEC Building 4% BEC Miscellaneous 2% BEC Owner’s cost 5% EPC Contingency [60] 30% EPC Table 13: main assumption in CAPEX estimation. Table 14: main assumption in OPEX estimation. Variable costs [36] Process Water 7.41 $/m3 Make-up water 0.41 $/m3 Fuel cost [67] 0.04 $/kWh Ceria oxide cost ([68]) 49 $/kg Yearly Ceria oxide make-up 30% of the total Fixed costs [36] Property, Taxes and insurance 2% TOC Maintenance cost 2.5% TOC Labour cost 1% TOC Figure 18: contribution of the components to the TOC. 22,7% 2,9% 9,7% 14,8% 4,6% 0,8% 5,2% 3,4% 0,6% 0,6% 1,4% 3,0% 0,4% 7,6% 8,6% 11,0% 0,6% 1,2% 0,9% ASU COMP-2 COMP-3 GT COND-2 TURBEXP RED+COMB OXI COMP-4 COND-1 COMP-1 DME reactor Vapour-Liquid Separator CLEAN-UP ST HRSG ST2 HRSG2 Exchangers 22,7% 2,9% 9,7% 14,8% 4,6% 0,8% 5,2% 3,4% 0,6% 0,6% 1,4% 3,0% 0,4% 7,6% 8,6% 11,0% 0,6% 1,2% 0,9% Figure 18: contribution of the components to the TOC. Table 15: economic assumptions. Life of the system (t) 30 years Discount rate (i) [41] 10% Capacity factor (CF) 85% Debt 60% Equity 40 % Cost of debt 2.4 % Inflation 1.5% Project financing 10 years TASC 7.8% TOC Construction 3 years Table 15: economic assumptions. Table 15: economic assumptions. Finally, to evaluate the profitability of the plant during its lifetime a discounted cash flow analysis (DCF) was adopted. DCF is based on the concept of the time value of money, all the future cash flows are estimated and discounted by a discounted factor (i) (Table 15), obtaining their present value [69]. The sum of the all discounted cash flows, both positive (revenues) and negative (operation cost, Table 14), gives the net present values (NPV). Finally, to evaluate the profitability of the plant during its lifetime a discounted cash flow analysis (DCF) was adopted. expenditure. For evaluation of the economic analysis listed assumption in table 13, 14 and 15 for CAPEX estimation, OPEX estimation and economic assumption. Therefore, with the adoption of the ion transport membrane technology plant costs would be 31% less compared to the ASU plant which would decrease the cost of DME and power production by tremendously and improve the overall efficiency of the plant by 2-4% [72]. expenditure. For evaluation of the economic analysis listed assumption in table 13, 14 and 15 for CAPEX estimation, OPEX estimation and economic assumption. DCF is based on the concept of the time value of money, all the future cash flows are estimated and discounted by a discounted factor (i) (Table 15), obtaining their present value [69]. The sum of the all discounted cash flows, both positive (revenues) and negative (operation cost, Table 14), gives the net present values (NPV). 𝑁𝑃𝑉= −𝑇𝐴𝑆𝐶+ ∑ (𝑛𝑒𝑡 𝑐𝑎𝑠ℎ 𝑓𝑙𝑜𝑤𝑠)𝑛 (1+𝑖)𝑛 𝑡 𝑛=1 (39) 𝑁𝑃𝑉= −𝑇𝐴𝑆𝐶+ ∑ (𝑛𝑒𝑡 𝑐𝑎𝑠ℎ 𝑓𝑙𝑜𝑤𝑠)𝑛 (1+𝑖)𝑛 𝑡 𝑛=1 (39) (39) A project is acceptable only if the NPV is at least positive. In this particular case, TASC is used to evaluate the total project cost instead of TOC, in order to asses both escalation and interest during construction (Table 13) [59,60]. A sensitivity analysis was performed to evaluate the effect of the selling price of power and DME on the economic performance of the plant. A sensitivity analysis was performed to evaluate the effect of the selling price of power and DME on the economic performance of the plant. The results are shown in Figure 19.a-b. Figure 19: (a) Economic performance varying DME and electricity price. (b) Payback period (PBP) varying DME and electricity price Figure 19: (a) Economic performance varying DME and electricity price. (b) Payback period (PBP) varying DME and electricity price A positive NVP corresponds to a high selling cost of the products. The breakeven point was obtained for an electricity and DME price of at least 190 $/MWh and 72 $/MWh respectively (Figure 19.a), which are higher than the current market price [70,71]. Moreover, a payback period of 20 years was obtained with a selling price of DME and electricity equal to 72 $/MWh and 220 $/MWh (Figure 19.b). However, a more lenient carbon credits and a further development of technologies such as Oxyfuel combustion, air separation and chemical looping will make the proposed polygeneration plant more competitive. By considering oxygen transport reactors system which uses ion transport membranes such as perovskites for oxygen seperation at high temperatures above 700oC producing high purity oxygen relatively a very lower price compare to ASUs and thereby increasing the efficiency and equipment cost. At present with technology it is possible to proudce 2000 tpd which is sufficient for a oxyfuel plant of 110 MW capacity. 7 Exergo-economic analysis Since to obtain a positive NVP a high selling cost of the products are required, an exergo-economic analysis was performed to understand which components are the main contributors to the high costs. Exergo-economic rests on the concept that exergy is the only rational basis for assigning monetary costs to the interactions which a system experiences with the environment and to the sources of thermodynamic inefficiencies within it [73]. The exergo-economic analysis method was first introduced in 1990 by Tsatsaronis and Lin ([74]) and named SPECO (Specified Exergy Costing) in 1995 by Lazzaretto and Andreatta [75]. For analyzing energy plant, this method provides an easy and straightforward scheme and it helps in time-saving by employing a compact matrix formulation [76]. So, the SPECO method was adopted in this work. The starting point of this methodology is the exergy analysis of the plant with all the exergy streams and irreversibilities produced during the process ( Exergy analysis). Secondly, the productive structure has to be the defined i.e. which are the fuel (F), product (P) and loss (L) for each subsystem. Fuel is defined to be equal as i) all the exergy values to be considered at the inlet plus ii) all the exergy decreases between inlet and outlet (i.e. the exergy removals from the respective material streams) minus iii) all the exergy increases (between inlet and outlet) that are not in accord with the purpose of the component. The product is defined to be equal to the sum of i) all the exergy values to be considered at the outlet (including the exergy of energy streams generated in the component) plus ii) all the exergy increases between inlet and outlet that are in accord with the purpose of the component [77]. While flows that leave the unit and the plant, are not subsequently used and do not require a special treatment are denominated as losses [78]. Based on the results of the exergy balance of the plant, an exergy cost E* and unit exergy cost k=E/E is assigned to each stream. The exergy cost of a physical flow is the exergy spent to produce it, irreversibilities comprised. Therefore, is completely connected to the production procedure efficiency, the highest the efficiency is the lowest are the irrevesibilities produced, and the lowest the exergy cost of the stream is. In order to evaluate the exergy cost of each of the ‘m’ flows of a certain plant, it will be necessary to write ‘m’ independent equations. If the plant is sequential, that is all the subsystems are characterized by a single output, since the exergy cost is conservative, it is possible to write as many equations of exergy cost balance as the number of subsystems. Vice versa, if the subsystems are characterized by multiple outlets (bifurcations), additional equations must be written for each unit equations, equal in number to the outlet streams ‘m’ minus the fuel F and losses streams L (x=m-F- L). Valero et al. Exergy analysis). [79] have formulated a rational procedure of exergy cost estimation based on four propositions:  P1 rule: in the absence of an external assessment, the cost of the inlet flow to the plant is equal to the exergy of the stream:  P2 rule: in absence of external assessment, the exergy cost of losses is null:  P2 rule: in absence of external assessment, the exergy cost of losses is null: Ei=0  P3 rule: if an output flow of a device is a part of the fuel, its exergy unit cost is the same of the input flow from which it comes. e: if an output flow of a device is a part of the fuel, its exergy unit cost is the same of input flow from which it comes.  P4 rule: if a unit has as a product a multiple outlet flows, since the formation process is the same, an equal exergy cost is assigned to all the streams. The exergy cost assessment can be formulated in a compacted matrix form as: * [ ]× c e A E Y  (40) * × 0 0 e e x E A                         (41) * 1 [ ] c e E A Y    (42) (40) * × 0 0 e e x E A                         (41) (42) Where:  Ac is the cost matrix (m × m);  Ac is the cost matrix (m × m);  A (n × m) the incident matrix with as many rows, ‘m’, as the number of exchanged flows between the components and the environment and as many columns, ‘n’, as the number of components. It relates to the i-th component with the j-th flow. If the i-j element of the matrix is null, it means there is no correlation between the two elements, if it is equal to –1, the j-th stream is an outlet for the i-th component and, vice versa, if it is equal to 1, the stream is an inlet stream.  αe (e × m) is the external and losses assessment matrix. Exergy analysis). Composed of ‘m’ columns as the number of components and ‘e’ rows as the number of the streams coming from the extern plus the plant losses, Alphae is the matrix representation of the equations coming from the P1 and P2 rules.  αe (e × m) is the external and losses assessment matrix. Composed of ‘m’ columns as the number of components and ‘e’ rows as the number of the streams coming from the extern plus the plant losses, Alphae is the matrix representation of the equations coming from the P1 and P2 rules.  αx (x × m) is the bifurcation matrix. Composed of ‘m’ columns as the number of components and ‘x’ rows as the number of bifurcations, it is the matrix representation of the equations coming from the P4a, P4b rules.  Ye* (m × 1) is the vector of external assessment, composed of ‘n’ elements as a result of proposition P1, e elements with the values of the exergies (ωe) corresponding to the resources of the plant (P1 rule), and m-n-e null elements corresponding to the losses and product bifurcations ( P3 –P4 rule).  E* is the vector containing the exergy cost of all the streams.  E* is the vector containing the exergy cost of all the streams. After the calculation of the exergetic cost, it is possible to evaluate the monetary cost (C) of the fuel and products of the plant. The formation of the economic cost of the internal flows and final products is related to both the thermodynamic efficiency of the process and to the equipment and maintenance cost of the plant. Therefore, the exergo-economic cost of a flow, (Cprod), can be defined as the combination of two contributions: the first comes from the monetary cost of the exergy entering the plant needed to produce this flow (Cfuel), that is its exergy cost, and the second covers the rest of the cost concurred in the production process (Z) (capital cost of equipment, maintenance operating costs, etc.). For a generic component, the cost balance equation can be expressed as: (43) fuel prod C + Z = C The cost assessing procedure is based on the same principles of the exergy cost estimation and can be carried on with same listed rules, replacing the exergy with the monetary cost. Exergy analysis). Considering the whole plant, the cost accounting equations can be described in a matrix form: [ ] c e A C Z   (44) 0 e e x A Z C C                          (45) 1 [ ] c e C A Z    (46) (44) (45) (46) Where Ce is the cost of the external assessment. Where Ce is the cost of the external assessment. After obtaining the cost C and relative specific cost (c=C/E), the exergo economic variables, such as the exergo economic factor (fk) (equation 47) and relative cost difference (rk) (equation 48), can be calculated. The exergo economic variable permits to evaluate the contribution of the investment cost on the product streams, the highest it is the bigger is the component investment cost contribution. While the relative cost difference allows locating the component with the highest difference between product and fuel. While the relative cost difference allows locating the component with the highest difference between product and fuel. k k k D Z f = Z C  (47) P,k F,k k F,k c - c r = c (48) k k k D Z f = Z C  (47) P,k F,k k F,k c - c r = c (48) k k k D Z f = Z C  P,k F,k k F,k c - c r = c (47) (48) P,k F,k F,k c - c c where cF,k and cP,k represent the specific cost of the fuel and product of the the k-th component, while cD is the cost of the destroyed exergy in the k-th component (equation 49): where cF,k and cP,k represent the specific cost of the fuel and product of the the k-th component, while cD is the cost of the destroyed exergy in the k-th component (equation 49): (49) D,k D,k dest,k c = c I In the current study, the cost rate (Z) was calculated using the annual capital cost ACC [80]: ACC=TOC CRF  (50) In the current study, the cost rate (Z) was calculated using the annual capital cost ACC [80]: ACC=TOC CRF  (50) (50) The annual capital cost is a combination of the total overnight cost and the recovery factor (CRF). Exergy analysis). CRF is defined as a ratio of constant annuity to the present value at a time (t) with a specified discount rate i (Error! No s'ha trobat l'origen de la referència.Table 15): ( (1 ) ) / ((1 ) 1) t t CRF i i i      (51) ( (1 ) ) / ((1 ) 1) t t CRF i i i      (51) (51) The cost rate Z comprises the total overnight cost plus the operational costs shown in Table 17. It was given by the overall of this costs divided the annual operational time τ, based on the capacity factor CF (Eq. 52) (Table 15). Z = (ACC + Fixed costs)/ Variable cost [ $/ ] M s  (52) Z = (ACC + Fixed costs)/ Variable cost [ $/ ] M s  (52) (52) s water cost was attributed to the four condensers proportionally to the heat rejected. The process water cost was attributed to the four condensers proportionally to the heat rejected. The process water cost was attributed to the four condensers proportionally to the heat rejected. The analysis was performed considering 64 streams (49 physical streams and 15 energy streams) and 23 components. Hence, 41 auxiliary equations were formulated ( Table 16). The process water cost was attributed to the four condensers proportionally to the heat rejected. The analysis was performed considering 64 streams (49 physical streams and 15 energy streams) and 23 components. Hence, 41 auxiliary equations were formulated ( Table 16). In table 17 are reported in detail the main results of the exergo-economic analysis. The exergo- economic factor (f) ranged from 1 to 0.06. The highest exergo-economic factor was observed for the ASU since the power absorbed is self-produced inside the plant. The component with lowest (f) resulted in the COMB+RED. When evaluating an equipment by an exergo-economic study, a general rule for reducing the final cost of the streams was followed. The subsystems that have the priority for an intervention are the ones with the highest (Z+CD). Among these, the equipment with the highest relative difference cost rk must be selected. The exergo-economic factor identifies the main causes of cost increase: a high value of this parameter (f) indicates a high influence of the cost of investment, a low value indicates a high incidence of the thermodynamic efficiency. Exergy analysis). In the first case a possible solution could be the use of cheaper components (generally characterized by lower efficiency), in the second case a higher efficiency component could be used with a consequent increase of the investment cost. The component with the highest Z+CD factor is the COMB+RED. The exergo- economic factor resulted in 0.06, so the exergy destroyed is the main contributor to the product cost. As reported in the As reported in the Exergy analysis section, in this element are localized the 46.3% of the overall irreversibilities, so an increase of the efficiency is suggested. For example, a different arrangement between the combustor and the reduction reactor from the annulus, might be a solution for proper heat integration. Same consideration for the OXI reactor that has the second highest (Z + CD) factor and a factor (f) equal to 0.09. The HRSG1 has the fourth highest Z+CD, the relative cost difference of 1.77, so the product cost of this unit is bigger than the fuel cost of about 170%. The exergo-economic factor is equal to 0.6 so the capital cost is the main contributor. The compressors and turbines exergo-economic factor ranged from 0.37 to 0.8. Finally, the overall plant presents a relative cost of 1.5 and an exergo- economic factor of 0.37, so a general efficiency improvement is required. Table 16: productive structure of the plant and auxiliary equations. Exergy analysis). COMPONENT FUEL PRODUCTS WASTE AUX.EQS ASU 0+WASU 1+1b - 𝑃1: 𝐸1 = 𝐸1 ∗ 𝑃1: 𝐸𝐴𝑆𝑈= 𝐸𝐴𝑆𝑈 ∗ 𝑃3: 𝑘1𝑏 ∗= 𝑘1 ∗ COMP-2 29+WCOMP-2 30 - 𝑃1: 𝐸𝑊𝐶𝑂𝑀𝑃1 = 𝐸𝑊𝐶𝑂𝑀𝑃1 ∗ COMP-3 36+WCOMP-3 37 - 𝑃1: 𝐸𝑊𝐶𝑂𝑀𝑃2 = 𝐸𝑊𝐶𝑂𝑀𝑃2 ∗ GT 31-32 WGT - 𝑃3: 𝑘31 ∗= 𝑘32 ∗ COND-2 33 35+36+38+43 42 𝑃2: 𝑘42 ∗= 0 𝑃4: 𝑘35 ∗= 𝑘36 ∗ 𝑃4: 𝑘36 ∗= 𝑘38 ∗ 𝑃4: 𝑘38 ∗= 𝑘43 ∗ NGPHX1 28b-29 3-2 - 𝑃1: 𝐸2 = 𝐸2 ∗ 𝑃3: 𝑘28 ∗= 𝑘29 ∗ TURBOEXP 3-4 WTURBEXP - 𝑃3: 𝑘3 ∗= 𝑘4 ∗ NGPHX2 28-28b 5-4 - 𝑃3: 𝑘28 ∗= 𝑘28𝑏 ∗ RED+COMB 9+5+30+1+37+49 7+28+31 - 𝑃4: 𝑘7 ∗= 𝑘28 ∗ 𝑃4: 𝑘28 ∗= 𝑘31 ∗ OXY 7+46+40 10+9 - 𝑃4: 𝑘10 ∗= 𝑘9 ∗ LIQPUMP1 43+WPUMP-1 44 - 𝑃1: 𝐸𝑊𝑃𝑈𝑀𝑃1 = 𝐸𝑊𝑃𝑈𝑀𝑃1 ∗ H2OPHX 10a-10b 46-45 - 𝑃3: 𝑘10𝑎 ∗ = 𝑘10𝑏 ∗ CO2COMP 38+WCOMP-4 39 - 𝑃1: 𝐸𝑊𝐶𝑂𝑀𝑃4 = 𝐸𝑊𝐶𝑂𝑀𝑃4 ∗ CO2HX 10b-11 40-39 - 𝑃3: 𝑘10𝑏 ∗ = 𝑘11 ∗ COND-1 11 13 12 𝑃2: 𝑘12 ∗= 0 COMP-1 13+WCOMP-1 14 - 𝑃1: 𝐸𝑊𝐶𝑂𝑀𝑃1 = 𝐸𝑊𝐶𝑂𝑀𝑃1 ∗ DME Reactor 14+44 15+45 - 𝑃4: 𝑘15 ∗= 𝑘45 ∗ VLS 15 17+49 - 𝑃4: 𝑘17 ∗= 𝑘49 ∗ Distillation Unit ( only columns) 16+Qcond,CO2-COL Qcond,DME-COL +Qreb,CO2-COL + Qreb,DME-COL +Qreb,MeOH-COL 26+23+20 27 𝑃1: 𝐸𝑄𝐶1 = 𝐸𝑄𝐶1 ∗ 𝑃1: 𝐸𝑄𝐶2 = 𝐸𝑄𝐶2 ∗ 𝑃1: 𝐸𝑄𝑅𝐵1 = 𝐸𝑄𝑅𝐵1 ∗ 𝑃1: 𝐸𝑄𝑅𝐵2 = 𝐸𝑄𝑅𝐵2 ∗ 𝑃1: 𝐸𝑄𝑅𝐵3 = 𝐸𝑄𝑅𝐵3 ∗ 𝑃2: 𝑘27 ∗= 0 𝑃4: 𝑘20 ∗= 𝑘23 ∗ 𝑃4: 𝑘23 ∗= 𝑘26 ∗ ST 5-A - 6-A WST - 𝑃3: 𝑘5−𝐴 ∗ = 𝑘6_𝐴 ∗ HRSG1 32-33 5-A – 1-A - 𝑃1: 𝐸1−𝐴= 𝐸1−𝐴 ∗ 𝑃3: 𝑘32 ∗= 𝑘33 ∗ ST2 5-B – 6-B WST2 - 𝑃3: 𝑘5−𝐵 ∗ = 𝑘6−𝐵 ∗ HRSG2 10-10a 5-B - 1-B - 𝑃1: 𝐸1−𝐵= 𝐸1−𝐵 ∗ 𝑃3: 𝑘10 ∗= 𝑘10𝑎 ∗ LOOP FOR CERIA - 𝑘9𝑎 ∗= 𝑘9𝑏 ∗ - 𝑘28𝑏 ∗ = 𝑘28𝑐 ∗ Table 17: main results of exergo-economic analysis. Table 17: main results of exergo-economic analysis. COMPONENT cf [$/MWh] cpr [$/MWh] rk Cd [$/s] Z [$/s] Z+Cd fk ASU 0.00 163.55 0.00 0.00 0.49 0.49 1.00 COMP-2 78.16 79.37 0.02 0.05 0.06 0.11 0.57 COMP-3 35.32 49.22 0.39 0.06 0.21 0.27 0.77 GT 43.96 56.29 0.28 0.07 0.32 0.39 0.81 Table 17: main results of exergo-economic analysis. Exergy analysis). COND-2 43.96 56.23 0.28 0.06 0.12 0.18 0.66 NGPHX1 80.77 123.66 0.53 0.01 0.01 0.02 0.50 TURBEXP 32.66 50.61 0.55 0.08 0.02 0.10 0.16 NGPHX2 80.77 565.56 6.00 0.04 0.04 0.07 0.52 RED+COMB 60.19 80.77 0.34 1.71 0.11 1.82 0.06 OXY 80.77 102.00 0.26 0.71 0.11 0.82 0.13 H2OPHX 102.00 396.19 2.88 0.09 0.01 0.10 0.06 COMP-4 59.35 62.20 0.05 0.00 0.01 0.01 0.91 CO2PHX 102.00 149.22 0.46 0.01 0.01 0.02 0.52 COND-1 102.00 102.00 0.00 0.13 0.01 0.14 0.10 COMP-1 86.69 88.91 0.03 0.02 0.03 0.05 0.65 DME Reactor 88.86 97.49 0.10 0.11 0.06 0.18 0.37 Vapour-Liquid Separator 98.70 98.70 0.00 0.02 0.01 0.02 0.38 Distillation Unit (only colums) 97.44 114.38 0.17 0.15 0.17 0.32 0.52 ST 69.38 93.23 0.34 0.11 0.19 0.30 0.63 HRSG1 43.96 121.79 1.77 0.17 0.25 0.42 0.60 ST2 196.28 236.39 0.20 0.02 0.01 0.03 0.37 HRSG2 102.00 205.12 1.01 0.10 0.03 0.13 0.22 9. Environmental performance 9.1 Water footprint An important parameter for an industrial and/or power plant is its water use. A generally accepted indicator of water use is the water footprint [81], it measures the volume of fresh water used to produce a product over the full supply chain [82]. In this specific case for water footprint is intended the water exploited during the plant operation, i.e. for cooling and other process. The following water withdrawal calculation is based on the model described in [83]. The water use can be expressed as a function of the heat rate HR (kJ/kWh) and three other adjustable parameters: A, B and D: (53) The heat rate (HR) is defined as the amount of energy required to produce one kW of electricity: The heat rate (HR) is defined as the amount of energy required to produce one kW of electricity: HR = Heat Input of Fuel Net Power Output (54) Not all the input heat is converted into electricity, but it is converted in waste heat that has to be dissipated to the environment. The greatest part is rejected through a cooling system, which usually uses water as a transfer medium. In general, water use in a polygeneration plant can be really complicated. However, a good estimation of it doesn’t require a detailed analysis since, according to [84], the cooling process accounts for 73-99% of the water consumption. So that, it is possible to have a good water need assessment by knowing only the heat rate and the type of cooling system used in the analysed plant. The energy going into the plant can leave it only in two ways, as a process heat losses or as a product stream, so the heat sent to the cooling system is given by HR-B. Where B (kJ/kWh) takes into account all the heat losses during the process, i.e steam turbine heat losses, generator heat losses, radiation losses, combustor losses etc. In general, depending on the technology, they ranged from 3% to 5% of the total energy input [83]. The parameter A (L/kJ) is the constant that takes into consideration the water need for the cooling system per unit of energy that has to be rejected. These parameters depend on many factors such as the type of cooling system (cooling tower, one-trough, dry cooling), the design of the cooling system, temperature and humidity of the air and water. 9. Environmental performance 9.1 Water footprint Finally, the parameter D (L/kWh), the parameter D takes into account only the make-up water for the two steam cycle. The values of the described parameter are shown in Table 18: Not all the input heat is converted into electricity, but it is converted in waste heat that has to be dissipated to the environment. The greatest part is rejected through a cooling system, which usually uses water as a transfer medium. In general, water use in a polygeneration plant can be really complicated. However, a good estimation of it doesn’t require a detailed analysis since, according to [84], the cooling process accounts for 73-99% of the water consumption. So that, it is possible to have a good water need assessment by knowing only the heat rate and the type of cooling system used in the analysed plant. The energy going into the plant can leave it only in two ways, as a process heat losses or as a product stream, so the heat sent to the cooling system is given by HR-B. Where B (kJ/kWh) takes into account all the heat losses during the process, i.e steam turbine heat losses, generator heat losses, radiation losses, combustor losses etc. In general, depending on the technology, they ranged from 3% to 5% of the total energy input [83]. The parameter A (L/kJ) is the constant that takes into consideration the water need for the cooling system per unit of energy that has to be rejected. These parameters depend on many factors such as the type of cooling system (cooling tower, one-trough, dry cooling), the design of the cooling system, temperature and humidity of the air and water. Finally, the parameter D (L/kWh), the parameter D takes into account only the make-up water for the two steam cycle. The values of the described parameter are shown in Table 18: Table 18: main assumption made in water footprint calculation and results. Table 18: main assumption made in water footprint calculation and results. 9. Environmental performance 9.1 Water footprint Heat Rate (HR) 11523,45 kJ/kWhe Heat losses 5% B 4176,2 kJ/kWhe A [44] 0,001 L/kJ D [44] 0,02 L/kWhe,gross Wcooling 2834374,4 m3/year Wprocess 24893,7 m3/year Heat Rate (HR) 11523,45 kJ/kWhe Heat losses 5% B 4176,2 kJ/kWhe A [44] 0,001 L/kJ D [44] 0,02 L/kWhe,gross Wcooling 2834374,4 m3/year Wprocess 24893,7 m3/year Heat Rate (HR) 11523,45 kJ/kWhe Heat losses 5% B 4176,2 kJ/kWhe A [44] 0,001 L/kJ D [44] 0,02 L/kWhe,gross Wcooling 2834374,4 m3/year Wprocess 24893,7 m3/year Figure 20 illustrates the water consumption of the proposed plant compared with other technologies. It results in the plant with the highest water consumption, the main reason is that the considered fuel power plants are without CCS. Moreover, the calculated water footprint is based only on the electrical power production, since it produces also a liquid fuel, the water footprint can be related also to the MWh produced of DME. In this case the m3/MWh is equal to 2.33, comparable to a nuclear power plant. Based on the calculation it is found that 73.3 kg/kgDME of water are consumed for cooling application. It has been observed that for DME polygeneration plants with CSP the water consumption for cooling is 103 kg/kgDME, 26.8 kg/kgDME with PV solar and 1561 kg/kgDME with biomass driven cycle [175]. Hence, the water consumption is lower than the renewable technology driven polygeneration DME plant, except PV solar. power production, since it produces also a liquid fuel, the water footprint can be related also to the MWh produced of DME. In this case the m3/MWh is equal to 2.33, comparable to a nuclear power plant. Based on the calculation it is found that 73.3 kg/kgDME of water are consumed for cooling application. It has been observed that for DME polygeneration plants with CSP the water consumption for cooling is 103 kg/kgDME, 26.8 kg/kgDME with PV solar and 1561 kg/kgDME with biomass driven cycle [175]. Hence, the water consumption is lower than the renewable technology driven polygeneration DME plant, except PV solar. Figure 20: water footprint comparison between different plant technology [81]. 0 0,5 1 1,5 2 2,5 3 3,5 4 [m3/MWhe] Wind Solar Natural Gas Geothermal Coal and Lignite Oil Nuclear OxyF-CL-NGPL 0 Figure 20: water footprint comparison between different plant technology [81]. Conclusion A novel natural gas feed polygeneration plant was proposed. The plant consisted in the integration of a chemical looping unit with an oxyfuel combustion unit for the production of power and DME. The results demonstrated the advantages of using a chemical looping process in a polygeneration plant to reduce the efficiency penalty coming from the carbon capture. The plant was able to produce 103 MWe and 2.14 kg/s with an energetic and exergetic efficiency of 50% and 44%, respectively. Through an exergy analysis, the main contributors of exergy destruction were identified; the combustor and reduction system resulted to contribute for 46% of the total generated irreversibilities (221 MW). An economic study was performed to assess the total investment of the plat that resulted in 534 M$. The most expensive equipment resulted in the air separation unit that accounts for 23% of the total investment. A discounted cash flow analysis was performed to assess the economic profitability of the proposed polygeneration plant. A minimum selling price of 190 $/MWh and 72 $/MWh for electricity and DME respectively, was found in order to have a positive NVP. 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Pinkerton, Mark Woods, James Simpson, Marc J. 5864. doi:10.1021/acs.iecr.6b00815. Turner, Elsy Varghese, Cost and Performance Baseline for Fossil Energy Plants Volume 1: Bituminous Coal and Natural Gas to Electricity, Doe/Netl- 2010/1397. 1 (2010). doi:DOE/NETL-2010/1397. [85] R. Dindorf, Estimating potential energy savings in compressed air systems, Procedia Eng. 39 (2012) 204–211. doi:10.1016/j.proeng.2012.07.026. [85] R. Dindorf, Estimating potential energy savings in compressed air systems, Procedia Eng. 39 (2012) 204–211. doi:10.1016/j.proeng.2012.07.026. Supplimentary File Techno-economic, energy and exergy analysis of polygeneration scheme for power and DME production with the integration of chemical looping CO2 and H2O dissociation Figure S1: Detailed plant of the Air Separation Unit. Figure S1: Detailed plant of the Air Separation Unit. Table S1: Thermodynamic properties and composition of ASU streams. Table S1: Thermodynamic properties and composition of ASU streams. 1-C 2-C 3-C 4-C 5-C 6-C 7-C 8-C 9-C 10-C 11-C 12-C T [°C] 25 30 20 20 30 30 30 20 -170 - 173.4 - 189.6 - 176.6 P [bar] 1.01325 5 6.3 6.3 30 30 30 6.3 6.3 5.1 1.2 5 Molar flow [kmol/hr] 11645.1 1164 5.1 1164 5.2 2329. 0 2329. 0 1863. 2 465.8 9316. 1 9316. 1 2279. 2 2279. 2 7036. 8 Mole frac. [%] N2 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.79 0.49 0.49 0.884 O2 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.21 0.50 0.50 0.11 13-C 14-C 15-C 16-C 17-C 18-C 19-C 20-C 21-C 22-C 23-C T [°C] -176.8 - 146.1 - 191.7 - 146.1 - 191.2 - 181.5 - 180.5 - 194.2 - 181.8 -20 0 P [bar] 5 30 1.2 30 1.2 1.2 26 1.2 1.2 1.2 26 Molar flow [kmol/hr] 7036.8 1863. 2 1863. 2 465.8 465.8 2445. 4 2445. 4 9199. 6 9199. 6 9199. 6 2445. 4 Mole frac. [%] N2 0.884 0.79 0.79 0.79 0.79 4.10E -04 4.10E -04 0.99 0.99 0.99 4.10E -04 O2 0.116 0.21 0.21 0.21 0.21 0.99 0.99 1.09E -04 1.09E -04 1.09E -04 0.99 0 1 2 3 4 5 6 7ASUSYNG COMPCO2 COMPGAS TURBINEEXHAUST CONDENSERCO2-H2O SEPNG PHX1TURBOEXPANDERNG PHX2RED REACTOR+COMBUSTOROXY REACTORLIQPUMP1H2O HEATERCO2COMPCO2 HEATERLIQ-SEPARATORDME COMPRESSORDME-REACTORDME COOLING SYSTEMCLEAN-UPSTHRSGST2HRSG2 [$/s] Z+Cd f ri 0 1 2 3 4 5 6 7ASUSYNG COMPCO2 COMPGAS TURBINEEXHAUST CONDENSERCO2-H2O SEPNG PHX1TURBOEXPANDERNG PHX2RED REACTOR+COMBUSTOROXY REACTORLIQPUMP1H2O HEATERCO2COMPCO2 HEATERLIQ-SEPARATORDME COMPRESSORDME-REACTORDME COOLING SYSTEMCLEAN-UPSTHRSGST2HRSG2 [$/s] Z+Cd f ri
https://openalex.org/W2567099642	https://europepmc.org/articles/pmc5285278?pdf=render	English	null	The role of assessment in supporting the movement toward patient-centred care	Perspectives on medical education	2,016	cc-by	1,503	Enable change The Institute of Medicine describes patient-centredness as ‘Providing care that is respectful of and responsive to in- dividual patient preferences, needs, and values, and ensur- ing that patient values guide all clinical decisions [1].’ The World Health Organization expands on this concept by de- scribing people-centred care as being responsive to commu- nities as well as patients [2]. Regardless of the deﬁnition, there is evidence that these forms of delivery improve both patient outcomes and the efﬁciency of care [3]. It is widely agreed that assessment has a signiﬁcant educa- tional effect [5]. It signals what is valued and it motivates trainees to prepare in a fashion that has educational beneﬁt. Given these effects, it can enable the transition to patient- centred care by indicating the needed competencies and en- suring that they will be taught and modelled throughout the educational process. Just as importantly, if these competen- cies are not assessed, it will undermine change even if the competencies are given priority in the curriculum. Unfortunately, the important study by Wilcox and col- leagues in this edition of the journal ﬁnds that student as- sessments of the patient-centredness of the learning envi- ronment decrease as they progress from the ﬁrst to sec- ond half of medical school [4]. The authors correctly con- clude that this highlights the need to explicitly teach these skills throughout medical school with particular attention to the behaviours modelled by faculty during clinical training. These reforms will certainly be an important step forward in underscoring the importance of patient-centred care and enabling its implementation in practice. Interprofessional learning and collaborative practice is an example of a competence necessary for patient-centred care. It has been shown to generate positive health outcomes and increase patient safety while reducing complications and length of stay [6]. If this competence was included in the curriculum, students would be given the opportunity to acquire it. If it was assessed as well, it would signal the value of interprofessional learning and collaboration, increase students’ motivation to develop skills in the area, and enable, though not create, change in clinical practice. Although imperfect in a variety of ways, methods to assess this competency are already available and include written tests of roles and attitudes, simulation of teams, and peer and patient surveys. DOI 10.1007/s40037-016-0318-9 Perspect Med Educ (2017) 6:5–6 COMMENTARY The role of assessment in supporting the movement toward patient-centred care John J. Norcini1 Published online: 16 December 2016 © The Author(s) 2016. This article is available at SpringerLink with Open Access. Published online: 16 December 2016 Enable change In addition to these curricular recommendations, it would also be useful to focus on the role of assessment in the trans- formation of the curriculum to one that supports patient- centred care. Assessment plays a central role in both edu- cation and regulation across the entire continuum of training and practice. It enables change by signalling which compe- tencies are important; it enhances and creates competence when it is used for learning; and it ensures competence when it is used as a measure of learning. 1 Foundation for Advancement and International Medical Education and Research, Philadelphia, USA John J. Norcini jnorcini@faimer.org Enhance and create learning Formative assessment (for learning) can have powerful ef- fects in education and appropriately done it should focus on the needs of the student. It guides and creates learn- ing while increasing the efﬁciency of the educational pro- cess. Reviews of the literature have highlighted the fact that feedback has a signiﬁcant effect on learning and, although John J. Norcini jnorcini@faimer.org J. J. Norcini 6 form they recommend also depends on changing the assess- ments done during and after medical school. Assessment plays a pivotal role in creating change across the continuum of training and practice. It signals what is valued. When used for formative purposes, it supports and creates learn- ing for students and when used for summative purposes it ensures patients that their doctors have the required com- petencies. If assessment is not changed at the same time as the curriculum is changed, reform is doomed to failure. it is not often acknowledged, assessment is the predicate to feedback [7]. Therefore, formative assessment aimed at aspects of patient-centred care should be included as part of any curricular reform. Further, this will not require ad- ditional resources since the current overemphasis on sum- mative assessment in many medical schools can be reduced substantially to provide the means for such a change. Even when feedback is not provided, assessment by it- self has a positive effect on learning. In an ingenious series of studies, Roediger and colleagues demonstrated that the active retrieval of information or performances required by assessment generates learning [8]. This constitutes another argument for increasing the number of formative assess- ments aimed at patient-centred care. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http:// creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. References Summative assessment (of learning) is also a powerful, if overused, force in education and appropriately done it fo- cuses on the needs of various stakeholders (such as pa- tients, the health care system, and educational institutions) rather than students. Inclusion of the knowledge and skills required for patient-centred care in the summative exami- nations given during training will ensure that they become an integral part of the curriculum, that their value is com- municated, and that students achieve competence. 1. Institute of Medicine. Committee on quality of health care in Amer- ica. Crossing the quality chasm: a new health system for the 21st century. Washington, D.C: National Academy Press; 2001. 2. World Health Organization. Retrieved 25 October 2016. http:// www.wpro.who.int/health_services/people_at_the_centre_of_care/ deﬁnition/en/ 3. Doyle C, Lennox L, Bell D. A systematic review of evidence on the links between patient experience and clinical safety and effective- ness. BMJ Open. 2013;3(1):e001570. 4. Wilcox MV, Orlando MS, Rand CS, et al. Medical students’ percep- tions of the patient-centredness of the learning environment. Per- spect Med Educ. 2017. doi:10.1007/s40037-016-0317-x In addition to their use in medical school, national high stakes assessment programmes are central to ensuring com- petence and there is a growing literature indicating that per- formance on such exams is associated with the outcomes of care. For example, studies have demonstrated a decrease in relative risk for mortality among the patients of those doctors who performed well on national assessment pro- grammes [9, 10]. Moreover, inclusion of this content in national examinations will ensure that all medical schools in the country include content relevant to patient-centred care in their curricula. Most importantly, this will reassure patients that their physicians are proﬁcient in this important competence. In addition to their use in medical school, national high stakes assessment programmes are central to ensuring com- petence and there is a growing literature indicating that per- formance on such exams is associated with the outcomes of care. For example, studies have demonstrated a decrease in relative risk for mortality among the patients of those doctors who performed well on national assessment pro- grammes [9, 10]. Moreover, inclusion of this content in national examinations will ensure that all medical schools in the country include content relevant to patient-centred care in their curricula. Most importantly, this will reassure patients that their physicians are proﬁcient in this important competence. 5. Cilliers FJ, Schuwirth LW, Adendorff HJ, et al. John J. Norcini is President and Chief Executive Ofﬁcer of the Foun- dation for Advancement of International Medical Education and Re- search References A model of the pre- assessment learning effects of summative assessment in medical ed- ucation. Adv Health Sci Educ. 2010;15:695–715. 6. Institute of Medicine. Interprofessional education for collabo- ration: Learning how to improve health from interprofessional models across the continuum of education to practice. Washington, D.C: National Academy Press; 2013. 7. Hattie J, Timperley H. The power of feedback. Rev Educ Res. 2007;77(1):81–112. 8. Roediger HL, Karpicke JD. Test-enhanced learning taking memory tests improves long-term retention. Psychol Sci. 2006;17(3):249–55. 9. Norcini JJ, Boulet JR, Dauphinee WD, Opalek A, Krantz ID, An- derson ST. Evaluating the quality of care provided by graduates of international medical schools. Health Aff. 2010;29(8):1461–8. 10. Norcini JJ, Boulet JR, Opalek A, Dauphinee WD. The relation- ship between licensing examination performance and the outcomes of care by international medical school graduates. Acad Med. 2014;89(8):1157–62. In summary, the study by Wilcox et al. in this edition of the journal ﬁnds that the patient-centredness of the learning environment decreases as students move through their un- dergraduate educational experiences [4]. They underscore the need to explicitly teach these skills with particular atten- tion to the behaviours modelled by faculty during clinical training. In this commentary, I have suggested that the re- John J. Norcini is President and Chief Executive Ofﬁcer of the Foun- dation for Advancement of International Medical Education and Re- search
https://openalex.org/W4308820326	https://proceeding.unram.ac.id/index.php/iccs/article/download/23/82	English	null	Persuasive Communication of SintuwuGo Green Hydroponic Community in Community Empowerment Efforts in Palu City	Proceedings of International Conference on Communication Science	2,022	cc-by	2,572	Proceeding 2nd International Conference on Communication Science (ICCS 2022) PERSUASIVE COMMUNICATION OF SINTUWUGO GREEN HYDROPONIC COMMUNITY IN COMMUNITY EMPOWERMENT EFFORTS IN PALU CITY Andi Akifah1, Citra Antasari2, Ahmad fauzan3 1Department of Communication, University of Tadulako (ilkomakifah@gmail.com) 2Department of Communication, University of Tadulako (citraantasari@gmail.com) 3Department of Communication, University of Tadulako (fauzanp3100@gmail.com) ABSTRACT. Hydroponic plants are one of the alternative planting methods that are currently quite popular. For people who are constrained by land that is not so large and the soil is not fertile, the hydroponic method can be one of the options for profitable farming business, which can improve the welfare of the community. As a new system, the promotion requires a lot of effort to persuade the society adopt the method. This study will look at the Persuasive communication used to reach and attract public participation in Hydroponic method. The research uses qualitative descriptive research, which focus on the Sintuwu Gogreencommunity as a research subject. The result shows that to persuade people participating in hydroponic planting, the government use some experts which not only expert in hydroponic but also as a public speakers. Social media is also better used as an alternative medium for persuasive communication because of its unlimited nature and can be accessed anywhere and anytime. The Sintuvu Go Green Community uses any kind of social media such as Facebook and Instagram. This two media has been actively promote their products and their activity. This way will attract not only hydroponic product buyers, but also new farmers. KEYWORDS: hydroponic; Community; persuasive Communication 1 INTRODUCTION Indonesia is rich in natural resources with a very high population. However, this potential is not comparable to the welfare of the average Indonesian people. This condition is almost evenly distributed in various regions of Indonesia, including Central Sulawesi. Moreover, the COVID-19 pandemic, which has lasted for two years, has had an economic impact on almost the entire community. Various efforts have been made by the government to improve the welfare of the community, one of which is by disbursing Direct Cash Assistance for Micro, Small and Medium Enterprises (BLT UMKM). However, it is not an easy thing to invite people to seek to improve their welfare only by disbursing cash. As in previous years, these financial aids often just 'evaporate' and do not last long due to the community's lack of skills in managing businesses. Therefore, efforts are needed in seeking empowerment, so that the community has resilience in global competition(Damsar, 2016). Community empowerment is a process to facilitate and encourage communities to be able to position themselves proportionally and become the main actors in utilizing their strategic environment to achieve sustainability in the long term. Community empowerment has a close relationship with sustainable development. That is the main prerequisite that will lead the community towards a dynamic economic, social and ecological sustainability Palu City, which is known as a five-dimensional city because of its complete natural landscape, including valleys, oceans, rivers, mountains and bays (https://traveling.bisnis.com/read/20160623/224/560506/palu-kota-5-dimensi-di-timur-indonesia), become a separate capital in carrying out empowerment efforts, coupled with advances in information and communication technology which have been proven to be able to assist in business improvement, especially in the field of business and product promotion. g y y (https://traveling.bisnis.com/read/20160623/224/560506/palu-kota-5-dimensi-di-timur-indonesia), become a separate capital in carrying out empowerment efforts, coupled with advances in information and communication technology which have been proven to be able to assist in business improvement, especially in the field of business and product promotion. Various communities have emerged and have become pioneers in helping the government seek to improve people's welfare. These groups are scattered in the community with various uniqueness of 498 each. One community that has played a large role in community empowerment efforts is the Hydroponic Community, which is a group of plant lovers. This group is spread over several regions and even consists of several groups. 1 INTRODUCTION Of the several hydroponic communities, the Sintuvu Go Green Community is one that is actively undertaking community empowerment efforts, which consists of about 50 people. This community has been designated as a Pilot Project for the Implementation of Urban Farming by the Bank Indonesia Representative Office for the Province of Central Sulawesi. Community groups in this program have received assistance in the form of a hydroponic circuit(channelsulawesi.id, 2021). p g y p ( Therefore, this research is aimed to answer the following questions: g q 1.How the Sintuvu Go Green Community persuade people to involve in the community y p p p 2.How is the form of community empowerment carried out by the community? In general, the urgency of this research is to try to provide alternative activities as an effort to empower the community, including persuasive strategies carried out in these efforts. The results of this research can be a recommendation for policy makers and the community in terms of improving people's welfare. 2.1 Persuasive Communication Persuasion comes from the Latin, namely persuasion, which is another word from persuader which means to persuade, invite or seduce. Persuasion can be done by someone rationally and emotionally. In terms of rationality, a person's cognitive components such as ideas and concepts can be affected. Meanwhile, in terms of emotion, what needs to be touched is the affective aspect, which is related to one's emotional life(Megawati, 2018). According to Perloff (1993) cited in Novianti et al. (2020)Persuasive communication involves a symbolic process in which communicators try to convince others to change their attitudes or behaviours regarding an issue through the transmission of messages in an atmosphere of free choice. Persuasive communication requires a lot of interaction in the dialogue, where the perceived trustworthiness and credibility of the communicator can influence the success of influence or compliance techniques(Teven, J. J., & McCroskey, 1999).Mood and age differences can also affect persuasion due to differences in value or topic relevance, successful communicators modify their approach to suit the audience. The determining factor is the persuasiveness of the Communication context(Warren et al., 2017) In persuasive communication, the focus of attention is on efforts to change and strengthen the attitudes or beliefs of the audience or on efforts to invite them to act in a certain way. In other words, persuasion is an attempt to change attitudes through the use of messages and focuses on the characteristics of communicators and listeners(Devito, 2011).The targets of persuasive communication are very diverse, this can be seen from demographic characteristics such as age, gender, social status, economic status, marital status, educational status, and others. 2.2 Hydroponic Community According to Aref et al. (2010) cited in (Gunawijaya & Pratiwi, 2018; Sekarrini & Siswanto, 2020)a community is defined as a group people livingor working within the same environmental area with some shared cultures or commoninterests.Similarly, Wheat (2021)defined community bygeographic location, particularly in a rural setting. In urban and suburban areas, a community may be defined by location, but also maybe defined by specific characteristics. More details, A community is characterized by the presence of some collective dimensions such as emotional connectedness and solidarity, trust, and civic values(Di Napoli et al., 2021). 499 Although, the community is considered a small organization but is able to provide a big influence for its members(Ayuningtyas & Abdullah, 2018). Referring to Manski (1993), one of the effects of community involvement is the correlation of the influence of community behavior on individual behavior(Armananti & Asteria, 2019). Based on the definition of community mentioned above, the hydroponic community is a group of people who have a special interest in one method of planting in agriculture, namely hydroponics. In addition, the Hydroponic Community is an effort by a group of people to foster economic independence for the community, so that the community is able to increase the annualincome. For some people farming and gardening are synonymous with rural communities, and are more in demand by parents. The interest of the younger generation is decreasing in the agricultural sector(Maghfiroh et al., 2019). As a result, the agricultural sector is dominated by the older generation who are generally resistant to change. In addition, in the youth's view, farming is a traditional job that is less prestigious and the results are not immediately available but are also relatively inadequate(Chaidir & Kamelia, 2018). This is where the Hydroponics community takes a role in changing the perception of some of these people. 3 RESEARCH METHOD This research uses qualitative descriptive research, which focus on the Sintuwu Gogreencommunity as a research subject. A qualitative research is an iterative process in which improved understanding to the scientific community is achieved by making new significant distinctions resulting from getting closer to the phenomenon studied(Aspers & Corte, 2019). The descriptive method analyzes the data collected in the form of words, pictures, and not numbers. The data collection taken from interviews, scripts, field notes, photos, video tapes, personal documents, notes or memos, and other official documents. 4 DISCUSSION To convince the society, the local government involved some experts to provide training to the community, also provide some hydroponic tools and greenhouses to the society. Mas’ad, a housewife living in the area around the greenhouses states that before being introduced to hydroponic, she was only a housewife without any income, but now she can help her husband to fulfil the family needs. Social media is also better used as an alternative medium for persuasive communication because of its unlimited nature and can be accessed anywhere and anytime. The Sintuvu Go Green Community uses any kind of social media such as Facebook and Instagram. This two media has been actively promote their products and their activity. This waywill attract not only hydroponic product buyers, but also new farmers. 5 CONCLUSION In conclusion, In order to persuade people participating in hydroponic planting, the government use some experts which not only expert in hydroponic but also as a public speakers. Social media is also better used as an alternative medium for persuasive communication because of its unlimited nature and can be accessed anywhere and anytime. The Sintuvu Go Green Community uses any kind of social media such as Facebook and Instagram. This two media has been actively promote their products and their activity. This way will attract not only hydroponic product buyers, but also new farmers. 4 DISCUSSION Hydroponic plants are one of the alternative planting methods that are currently quite popular. For people who are constrained by land that is not so large and the soil is not fertile, the hydroponic method can be one of the options for profitable farming business(Christianingrum, 2019; Foster et al., 2021). The process of seedling nursery and its care does not take a long time, so it is economically very profitable for capital turnover. The results can also be a solution to the food security – related problem and are very helpful in improving people's nutrition with quality food products because they do not use pesticides and other chemicals. Of all the benefits, however only few people adopt this method due to the lack of understanding and low communication approach. Persuasive communication is one best approach to promote this practice. Sintuwu Go Green Community is one of Hydroponic communities in Palu, South Sulawesi which actively conduct hydroponic planting system. The community that established since 2019 has become the pioneer of Urban Farming in Central Sulawesi. According to Nanda Andriana the founder of the community, this planting system has been proven to help increase the family income. Hydroponic crops are used for public consumption and sold to increase household income. Moreover, this hydroponic program is one way to increase productivity and community cooperation. 500 Figure 1: Selling hydroponic vegetables At the time of introducing the hydroponic method, many people were hesitant to join because of a lack of knowledge about hydroponic farming. The most effective way of influencing people to participate in a hydroponic planting program is to ensure that the method will change their standard of living. To convince the society, the local government involved some experts to provide training to the community, also provide some hydroponic tools and greenhouses to the society. Mas’ad, a housewife living in the area around the greenhouses states that before being introduced to hydroponic, she was only a housewife without any income, but now she can help her husband to fulfil the family needs. Figure 1: Selling hydroponic vegetables Figure 1: Selling hydroponic vegetables At the time of introducing the hydroponic method, many people were hesitant to join because of a lack of knowledge about hydroponic farming. The most effective way of influencing people to participate in a hydroponic planting program is to ensure that the method will change their standard of living. ACKNOWLEDGEMENTS We would like to take this opportunity to thank to DIPA Universitas Tadulako as the Sponsorship of this research and also to the editors and the reviewers for the valuable comments and review for this article. 501 REFERENCES Armananti, S. H., & Asteria, D. (2019). Partisipasi Anggota dan Pemanfaatan Instagram dalam Interaksi Komunitas Brand Ria Miranda. Jurnal Komunikasi, 11(2), 155. https://doi.org/10.24912/jk.v11i2.5266 p g j Aspers, P., & Corte, U. (2019). What is Qualitative in Qualitative Research. Qualitative Sociology, 42(2), 139–160. https://doi.org/10.1007/s11133-019-9413-7 Ayuningtyas, F., & Abdullah, A. Z. (2018). Kognisi Sosial Melalui Situs Jejaring Youtube Pada Komunitas Online (Studi Kasus pada Komunitas Online LinkPictureID). Jurnal Komunikasi, 9(2), 137. https://doi.org/10.24912/jk.v9i2.1076 Chaidir, L., & Kamelia, L. (2018). Pelatihan Bangga Menjadi Petani Hidroponik Pada Komunitas Pemuda Di Desa Cigugur Girang Kabupaten Bandung Barat. 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https://openalex.org/W2933375185	https://www.scielo.br/j/bjps/a/5SHZ5Yy9BFfr7KsHHFkQMWv/?lang=en&format=pdf	English	null	Determination of Gemifloxacin in human plasma by high performance liquid chromatography using Ultra Violet detector and its application to a bioequivalence study	Brazilian Journal of Pharmaceutical Sciences	2,018	cc-by	3,131	Article Brazilian Journal of Pharmaceutical Sciences http://dx.doi.org/10.1590/s2175-97902018000417239 Determination of Gemifloxacin in human plasma by high performance liquid chromatography using Ultra Violet detector and its application to a bioequivalence study Syed Husain Hashemi Mousavi1 1Department of Chemistry, Faculty of Engineering, South Tehran Branch, Islamic Azad University, Tehran, Iran A liquid chromatography method was developed and validated for the determination of gemifloxacin in human plasma using chloramphenicol as internal standard to achieve lower quantification limit. Acetonitrile was used to precipitated and extracted analyte and internal standard from plasma by Protein Precipitation. Analysis was performed isocratically on C18 column using 25% acetonitrile and 75% 0.02 M phosphate buffer as mobile phase. The method was demonstrated to be linear from 0.003 µg/mL to 5 µg/mL with the lower limit of quantitation of 0.003 µg/mL. The method was successfully applied for the bioequivalence study of gemifloxacin after a single oral administration of 320 mg gemifloxacin mesylate tablets to 12 healthy volunteers. Keywords: Gemifloxacin/determination. Bioequivalence. Plasma. HPLC. FIGURE 1 - Chemical structure of gemifloxacin and chloramphenicol. Correspondence: S. H. H. Mousavi. Department of Chemistry, Faculty of Engineering, South Tehran Branch, Islamic Azad University, Tehran, Iran, P. O. Box 11365-4435. Phone number: 0989038400292, 098 21 33030505. E-mail: hashemimosavi@yahoo.com INTRODUCTION Gemifloxacin mesylate is a synthetic broad- spectrum antibacterial agent for oral administration. Gemifloxacin, a compound related to the fluoroquinolone class of antibiotics, is available as the mesylate salt in the sesqui hydrate form. Chemically, gemifloxacin is (R,S)-7[(4Z)-3-(aminomethyl)-4-(methoxyimino)-1- pyrrolidinyl]-1-cyclopropyl-6-fluoro-1,4-dihydro-4- oxo1,8-naphthyridine-3-carboxylic acid (Figure 1). It is used in the treatment of acute bacterial exacerbation of chronic bronchitis and mild-to-moderate pneumonia. Gemifloxacin has been shown to be active against most strains of microorganisms. It is particularly active against Gram-positive organisms including penicillin, macrolide, and quinolone resistant Streptococcus pneumoniae (Calvo, Gimenez, 2002; Oh et al., 1996; Johnson et al., 1999; Berry et al., 2000; Hardy et al., 1999). Gemifloxacin acts by inhibiting DNA synthesis through the inhibition of both deoxyribonucleic acid (DNA) gyrase and topoisomerase IV (TOPO IV), which are essential for bacterial growth. FIGURE 1 - Chemical structure of gemifloxacin and chloramphenicol. Literature survey revealed that analytical methods have been reported for the estimation of gemifloxacin, they include high performance liquid chromatography tandem mass spectrometry (Doyle et al., 2000; Ramji et al., 2001), microchip electrophoresis (Seung et al., 2004), chiral high performance liquid chromatography (Hee et al., 2009) and chiral countercurrent chromatography (Eun, Yoo-Mo, Doo, 2004; Myung et al., 2002). Simple and sensitive ion pairing spectrophotometric methods have been described for the assay of gemifloxacin mesylate by Marothu and Dannana (2008). Barbosa and co-workers (Barbosa et al., 1997) studied dissociation constants of series of compounds including diuretics and quinolones in several acetonitrile: water mixtures, high performance liquid chromatography by UV detector (Sultana et al., 2011), with fluorescence detection (Allen et al., 2000b; Al-Hadiya et al., 2010), high performance thin-layer Page 1 / 6 Page 1 / 6 Page 1 / 6 Braz. J. Pharm. Sci. 2018;54(4):e17239 S. H. H. Mousavi time schedule: before drug administration and at 0.33, 0.67, 1, 1.33, 1.67, 2, 2.33, 2.67, 3, 3.5, 4, 6, 8, 10, 12 and 24 hours after drug administration (18 samples per subject per profile period). time schedule: before drug administration and at 0.33, 0.67, 1, 1.33, 1.67, 2, 2.33, 2.67, 3, 3.5, 4, 6, 8, 10, 12 and 24 hours after drug administration (18 samples per subject per profile period). chromatography (HPTLC) with fluorescence detection (El-Koussi et al., 2014). The aim of the present study was to establish an efficient, reliable, accurate, sensitive and reproducible method for the application in bioequivalence study of gemifloxacin tablets 320 mg. Chromatographic conditions The Knuer EA4300 HPLC pump and Knuer E4310 detector were used for bioanalytical assay of standards and human samples. Mobile phase consisted of phosphate buffer (0.02 M) and acetonitrile (75:25) and the pH of mixture adjust to 3.0 with phosphoric acid and pumped at the flow rate of 1 mL/min through the 250 mm C18 column. The wavelength of detector for monitoring of peaks was 275 nm. Representative chromatograms of blank plasma, plasma of one volunteer and standard chromatogram are shown in Figure 2. MATERIAL AND METHODS A liquid chromatography method was developed and validated for the determination of gemifloxacin in human plasma as per FDA and ICH guidelines. Extraction procedure as described below. Study design and blood samples This was a laboratory-blind, single-dose, randomized, 2-way cross-over study with a wash-out period of 7 days between the clinic (blood sampling) days to compare the pharmacokinetics of gemifloxacin (test product) and Factive® (reference product). Calibration curve d d Standard samples were prepared as mentioned before (Bioanalytical Methods). The method has good linearity (r>0.999) over the concentration rang 0.003 – 5.000 µg/ mL. The equation of line was y=0.53199x+0.02165 and the limit of quantification was 0.003 µg/mL. Inter-day and intra-day precision and accuracy and stability of this method shows that this is the validated method for determination of gemifloxacin in human plasma. Body temperature, heart rate and blood pressure were recorded before drug administration on clinic days. Heart rate and blood pressure recordings were repeated at approximately 4 and 6 hours after drug administration. After insertion of an indwelling venous cannula, pre-dose blood samples had been drawn. Six Subjects received Gemifloxacin Tablets (test product, 320 mg) and the other six subjects received reference Factive® Tablets (320 mg) with 240 mL drinking water. The only food allowed was standardized meals served 6 hours and a standardized breakfast 3 hours after drug administration. No restrictions on the intake of food and fluid applied after the subjects had been discharged from the clinic. Gemifloxacin was assayed in plasma samples collected from 12 subjects, and data from 12 subjects were evaluated. Venous blood samples will be collected into heparinized, glass tubes, labeled as per-treatment phase, according to the following Subjects Twelve healthy male and female volunteers with mean weight of 65.6±11 kg, mean height of 164.6±7.8 cm and mean age of 32.2±8.4 years were enrolled into the study. All volunteers had normal paraclinical parameters. 100 µL chloramphenicol 30 mg/L is added to 1500 µL of plasma as an internal standard and mix. 1500 µL acetonitrile is added to plasma and is vortexed for 10 second then 100 µL sulfuric acid 2M is added. After mixing, sodium chloride is added and centrifuge at 4000 rpm for 10 minutes. The organic phase is dried and then reconstitute with 100 µL of mobile phase. 50 µL of this sample was injected into the HPLC. Exclusion criteria were as follows: Consumption of tobacco in any form in study day, Addiction to alcohol or history of any drug abuse, History of kidney or liver dysfunction, History of jaundice in the past 6 months, History of drug allergy to the test drug or any chemically similar to the drug under investigation, Administration / intake of any prescription or OTC medication for 2 weeks before the study, Patient suffering from any chronic illness such as arthritis and asthma, Subject suffering from any psychiatric (acute or chronic) illness, Participation in any bioavailability / bioequivalence study in the past 12 weeks, Intake of barbiturates or any enzyme – inducing drug in the past 3 months and HIV positive volunteers. Precision and accuracy Accuracy is measured as % bias and precision is measured as coefficient of variation (%CV). For the assignment of a valid calibration range bias is taken as measure of accuracy and coefficient of variation (%CV) is taken as measure of precision. Intra-day accuracy and precision for a valid range must be within 15% but within 20% at the lower limit of quantification. Summarize inter-day and intra-day accuracy and precision during assay validation are in Table I to II. Braz. J. Pharm. Sci. 2018;54(4):e17239 Page 2 / 6 ination of Gemifloxacin in human plasma by high performance liquid chromatography using Ultra Violet detector ity The stability of gemifloxacin in plasma was tigated in samples obtained from a spiked plasma at i h l l l d Analysis of long-term storage stability, three cycle freeze-thaw stability, bench top stability, stock solution stability shows gemifloxacin was stable in plasma for at l h h h d 20 °C (T bl III) RE 2 - Representative chromatograms of a) blank plasma b) standard chromatogram (0.7 µg mL-1) and c) plasma of one teer (3 hours). Determination of Gemifloxacin in human plasma by high performance liquid chromatography using Ultra Violet detector FIGURE 2 - Representative chromatograms of a) blank plasma b) standard chromatogram (0.7 µg mL-1) and c) plasma of one volunteer (3 hours). Analysis of long-term storage stability, three cycle freeze-thaw stability, bench top stability, stock solution stability shows gemifloxacin was stable in plasma for at least three months when stored at −20 °C (Table III). RESULTS Plasma sample from twelve healthy volunteers at 0, 0.33, 0.67, 1, 1.33, 1.67, 2, 2.33, 2.67, 3, 3.5, 4, 6, 8, 10, 12 and 24 hours were collected and were analyzed as mentioned before in section “Bioanalytical Methods”. The mean pharmacokinetic parameters such as maximum concentration (Cmax), time to reach Cmax (Tmax), area under curve from time zero to 24 hours (AUC0-24) and area under curve from time zero to infinite (AUC0-∞) are presented in the Table IV. The mean plasma volunteers curve is shown in Figure 3. i Stability y The stability of gemifloxacin in plasma was investigated in samples obtained from a spiked plasma at two concentrations. Three plasma samples were analysed three months after storage at −20 °C. Page 3 / 6 Braz. J. Pharm. Sci. 2018;54(4):e17239 S. H. H. Mousavi TABLE I - Inter-day precision 0.0030 µg/mL 1.0 µg/mL 5.0 µg/mL 1 0.0025 1.1 5.0 2 0.0032 1.1 5.0 3 0.0028 1.1 5.1 4 0.0034 0.9 5.1 5 0.0036 1.1 5.1 Average 0.0031 1.06 5.06 RSD 14.4 8.4 1.1 TABLE II - Intra-day precision and accuracy 0.0030 µg/mL 1.0 µg/mL 5.0 µg/mL 1 0.0029 1.1 5.0 2 0.0034 1.1 5.1 3 0.0025 0.9 5.0 4 0.0024 1.0 4.9 5 0.0032 1.1 5.1 Average 0.0029 1.04 5.02 RSD 15.0 8.6 1.7 Accuracy 96.7 104 100.4 RESULTS Plasma sample from twelve healthy volunteers at 0, 0.33, 0.67, 1, 1.33, 1.67, 2, 2.33, 2.67, 3, 3.5, 4, 6, 8, 10, 12 and 24 hours were collected and were analyzed as TABLE I - Inter-day precision FIGURE 3 - Mean plasma volunteers curve. FIGURE 3 - Mean plasma volunteers curve. FIGURE 3 - Mean plasma volunteers curve. TABLE II - Intra-day precision and accuracy mean ratio of the pharmacokinetic variables Cmax, Tmax, AUC0-10, AUC0-Inf, fall within the conventional bioequivalence range of 80% to 125%. TABLE II - Intra-day precision and accuracy 0.0030 µg/mL 1.0 µg/mL 5.0 µg/mL 1 0.0029 1.1 5.0 2 0.0034 1.1 5.1 3 0.0025 0.9 5.0 4 0.0024 1.0 4.9 5 0.0032 1.1 5.1 Average 0.0029 1.04 5.02 RSD 15.0 8.6 1.7 Accuracy 96.7 104 100.4 RESULTS The results of this study indicate that the test product with assay of 105.8% for test product of Gemifloxacin is bioequivalent to the reference product (Factive®) with respect to both the rate and extent of absorption of Gemifloxacin. Under Medication Guide of Factive® tablets which has been approved by the U.S. Food and Drug Administration it was reported that following repeat oral doses of 320 mg of Factive® tablets to healthy subjects (FACTIVE® Tablets-FDA), and in another study “The effect of food on the bioavailability of oral gemifloxacin in healthy volunteers” (Allen et al., 2000a, the pharmacokinetics parameters were compare with this study in Table V. Comparison of this results with our study shows its similar to other studies. CONCLUSION It is thus concluded that the proposed method is simple, cost effective, accurate, safe and precise. A specific LC method, with a single step Protein Precipitation procedure, has been developed and validated (as per FDA and ICH forguidelines) for g The 90% confidence intervals for the “test/reference” TABLE III – Gemifloxacin stability Braz. J. Pharm. Sci. 2018;54(4):e17239 TABLE III – Gemifloxacin stability Moreover, it indicated that the two pharmaceutical products showed similar bioavailability profiles and therefore are considered bioequivalent with regard to the extent and rate of absorption and, interchangeable as well, for clinical and therapeutic purposes. l Barbosa J, Marqués I, Fonrodona G, Barrón D, Bergés R. Factor analysis applied to correlation between acidity constants of series of diuretics, quinolones and buffers, with solvatochromic parameters in water-acetonitrile mixtures. Anal Chim Acta. 1997;347(3):385-393. Berry V, Page R, Satterfield J, Singley C, Straub R, Woodnutt G. Comparative in vivo activity of gemifloxacin in a rat model of respiratory tract infection. J Antimicrob Chemother. 2000;45(Suppl 1):79-85. The proposed method to analyze gemifloxacin in plasma by HPLC with UV detection happens to be first of its kind described so far in the literature. This new method with low LOQ (0.003 µg/mL) will be helps for carrying out pharmacokinetic study of gemifloxacin in laboratories that lack sophisticated analytical instrument of LC-MS/MS. Calvo A, Gimenez MJ. Ex vivo serum activity (killing rates) after Gemifloxacin 320 mg versus Trovafloxacin 200 mg single doses against ciprofloxacin-susceptible and -resistant streptococcus pneumoniae. Int J Antimicrob Agents. 2002;20:144-6. TABLE III – Gemifloxacin stability Braz. J. Pharm. Sci. 2018;54(4):e17239 Page 4 / 6l long-term storage stability (three months) freeze-thaw stability (three cycle) bench top stability stock solution stability Spiked concentration (µg/mL) 0.0030 5.0 0.0030 5.0 0.0030 5.0 0.0030 5.0 Average 0.0027 4.9 0.0028 5.0 0.0031 5.0 0.0030 4.9 Accuracy 90.0 98 93.3 100 103.3 100 100.0 98 %CV 0.00152 0.05773 0.00115 0.11547 0.00058 0.05773 0.00058 0.05774 Determination of Gemifloxacin in human plasma by high performance liquid chromatography using Ultra Violet detector TABLE IV - Pharmacokinetic parameters of the gemifloxacin 320 mg test and reference Mean Confidence interval P value Test Reference T-test ANOVA AUC0→t 588.98±155.60 543.59±152.79 103.6%-121.34% 0.478 0.478 AUC0→∞ 701.28±178.09 667.90±189.27 100.5%-118.48% 0.661 0.661 Cmax (µg/mL) 1.95±0.50 1.82±0.57 103.3%-116.99% 0.579 0.579 Tmax (min) 71.67±24.80 78.33±31.29 88.67%-109.65% 0.569 0.569 Kel 0.00220±0.00081 0.00214±0.00082 88.47%-102.29% 0.872 0.872 T ½ (min) 381.823±224.907 396.169±229.044 102.0%-115.05% 0.884 0.884 TABLE IV - Pharmacokinetic parameters of the gemifloxacin 320 mg test and reference TABLE V- Comparison of pharmacokinetic parameters with other studies TABLE V- Comparison of pharmacokinetic parameters with other studies This study Allen et al. study Factive® study AUC0→24 588.98±155.60 595.8±184.2 AUC0→∞ 701.28±178.09 454.2±139.8 Cmax (µg/mL) 1.95±0.50 1.21±0.33 1.61±0.51 Tmax (min) 71.67±24.80 90 (60-240) 30-120 the determination of gemifloxacin in human plasma supporting a pharmacokinetic and bioequivalence study comparison of other study (FACTIVE® Tablets-FDA). The statistical analysis demonstrated that none of the parameters accepted for drug bioavailability (AUC0-t and Cmax) were not significantly different between the treatments for the single dose data. Moreover, it indicated that the two pharmaceutical products showed similar bioavailability profiles and therefore are considered bioequivalent with regard to the extent and rate of absorption and, interchangeable as well, for clinical and therapeutic purposes. l Allen A, Bygate E, Oliver S, Johnson M, Ward C, Cheon A, Choo YS, Kim IC. Pharmacokinetics and tolerability of gemifloxacin (SB-265805) after administration of single oral doses to healthy volunteers. Antimicrob Agents Chemother. 2000b;44(6):1604-1608. Allen A, Bygate E, Oliver S, Johnson M, Ward C, Cheon A, Choo YS, Kim IC. Pharmacokinetics and tolerability of gemifloxacin (SB-265805) after administration of single oral doses to healthy volunteers. Antimicrob Agents Chemother. 2000b;44(6):1604-1608. the determination of gemifloxacin in human plasma supporting a pharmacokinetic and bioequivalence study comparison of other study (FACTIVE® Tablets-FDA). The statistical analysis demonstrated that none of the parameters accepted for drug bioavailability (AUC0-t and Cmax) were not significantly different between the treatments for the single dose data. REFERENCES Doyle E, Fowles SE, Mcdonnell DF, Mccarthy R, White SA. Rapid determination of gemifloxacin in human plasma by high performance liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2000;746(2):191-8. Doyle E, Fowles SE, Mcdonnell DF, Mccarthy R, White SA. Rapid determination of gemifloxacin in human plasma by high performance liquid chromatography-tandem mass spectrometry. J Chromatogr B. 2000;746(2):191-8. Al-Hadiya BMH, Khady AA, Mostafa GAE. Validated liquid chromatographic-fluorescence method for the quantitation of gemifloxacin in human plasma. Talanta. 2010;83(1):110-116. Allen A, Bygate E, Clark D, Lewis A, Pay V. The effect of food on the bioavailability of oral gemifloxacin in healthy volunteers. Antimicrob Agents Chemother. 2000a;16(1):45-50. El-Koussi WM, Atia NN, Mahmoud AM, El-Shabouri SR. HPTLC method for direct determination of gemifloxacin mesylate in human plasma. J Chromatogr B. 2014;967:98-101. Braz. J. Pharm. Sci. 2018;54(4):e17239 Page 5 / 6 Page 5 / 6 This is an open-access article distributed under the terms of the Creative Commons Attribution License. S. H. H. Mousavi Myung HH, Sang CH, Yoon JC, Jong SJ, Wonjae L. Liquid chromatographic resolution of gemifloxacin mesylate on a chiral stationary phase derived from crown ether. Biomed Chromatogr. 2002;16(5):356-360. Eun SK, Yoo-Mo K, Doo SC. Chiral counter-current chromatography of gemifloxacin guided by capillary electrophoresis using (+)-(18-crown-6)-tetracarboxylic acid as a chiral selector. J Chromatogr A. 2004;1045(1-2):119-124. FACTIVE® Tablets FDA. 2008. 34p. Available from: https://www.accessdata.fda.gov/drugsatfda_docs/ label/2008/021158s013lbl.pdf. Oh JI, Paek KS, Ahn MJ, Kim MY, Hong CY, Kim IC, Kwak JH. In vitro and in vivo evaluations of LB20304, a new fluoronaphthyridone. Antimicrob Agents Chemother. 1996;40(6):1564-8. Hardy D, Amsterdam D, Mandell L, Rotstein C. Comparative in vitro activity of gemifloxacin, moxifloxacin, trovafloxacin, sparfloxacin, grepafloxacin, ofloxacin, ciprofloxacin and other antimicrobial agents against bloodstream isolates of Gram- positive cocci. J Antimicrob Chemother. 1999;44(3):802-5. Hardy D, Amsterdam D, Mandell L, Rotstein C. Comparative in vitro activity of gemifloxacin, moxifloxacin, trovafloxacin, sparfloxacin, grepafloxacin, ofloxacin, ciprofloxacin and other antimicrobial agents against bloodstream isolates of Gram- positive cocci. J Antimicrob Chemother. 1999;44(3):802-5. Ramji JV, Austin NE, Boyle GW, Chalker MH, Duncan G, Fairless AJ, et al. The disposition of gemifloxacin a new fluoroquinolone antibiotic, in rats and dogs. Drug Metab Dispos. 2001;29(4 Pt 1):435-442. Seung IC, Jiyeon S, Min-Su K, Yong-Kweon K, Doo SC. On- line sample cleanup and chiral separation of gemifloxacin in a urinary solution using chiral crown ether as a chiral selector in microchip electrophoresis. J. Chromatogr A. 2004;1055(1- 2):241-245. Hee JC, Hwan SC, Sang CH, Myung HH. Braz. J. Pharm. Sci. 2018;54(4):e17239 REFERENCES HPLC of fluoroquinolone antibacterials using chiral stationary phase based on enantiomeric (3,3′-diphenyl-1,1′-binaphthyl)-20- crown-6. J Sep Sci. 2009;32(4):536-541. Johnson DM, Jones RN, Erwin ME. The Quality Control Study Group. Anti-streptococcal activity of SB-265805 (LB20304), a novel fluoronaphthyridone, compared with five other compounds, including quality control guidelines. Diagn Microbiol Infect Dis. 1999;33(2):87-91. Johnson DM, Jones RN, Erwin ME. The Quality Control Study Group. Anti-streptococcal activity of SB-265805 (LB20304), a novel fluoronaphthyridone, compared with five other compounds, including quality control guidelines. Diagn Microbiol Infect Dis. 1999;33(2):87-91. Sultana N, Arayne MS, Shamim S, Akhtar M, Gul S. Validated method for the determination of gemifloxacin in bulk, pharmaceutical formulations and human serum by RP-HPLC: in vitro applications. J Brazil Chem Soc. 2011;22(5):987-992. Received for publication on 25th April 2017 Accepted for publication on 06th April 2018 Marothu VK, Dannana GS. Spectrophotometric determination of gemifloxacin mesylate in pharmaceutical formulations through ion-pair complex formation. E-J. Chem. 2008;5(3):515-520. Braz. J. Pharm. Sci. 2018;54(4):e17239 Page 6 / 6
https://openalex.org/W2103184520	http://www.scielo.br/pdf/csp/v29n7/03.pdf	English	null	O uso de escalas de silhuetas na avaliação da satisfação corporal de adolescentes: revisão sistemática da literatura	null	2,013	cc-by	9,839	1 Faculdade de Ciências Médicas da Santa Casa de São Paulo, São Paulo, Brasil. 2 Centro de Vigilância Epidemiológica, Secretaria Estadual de Saúde de São Paulo, São Paulo, Brasil. 3 Divisão de Vacinas, Sanofi Pasteur, São Paulo, Brasil. Correspondence J. C. Moraes Faculdade de Ciências Médicas da Santa Casa de São Paulo. Rua Frederico von Martius 88, São Paulo, SP 01548-010, Brasil. jcassiom@uol.com.br Should acellular pertussis vaccine be recommended to healthcare professionals? A vacina pertússis acelular deve ser recomendada a proﬁ ssionais de saúde? ¿La vacuna contra la tos ferina acelular se debe recomendar a los profesionales de la salud? José Cassio de Moraes 1 Telma Carvalhanas 2 Lucia Ferro Bricks 3 1277 REVISÃO REVIEW 1277 REVISÃO REVIEW 1277 REVISÃO REVIEW Should acellular pertussis vaccine be recommended to healthcare professionals? Should acellular pertussis vaccine be recommended to healthcare professionals? Pertussis Vaccine; Health Personnel; Occupational Risks José Cassio de Moraes 1 Telma Carvalhanas 2 Lucia Ferro Bricks 3 Pertussis clinical manifestations and diagnosis Pertussis, also known as “whooping cough”, is an infectious disease with a high transmission rate (90%) caused by the bacterium B. pertussis. Hu- mans are the sole hosts of B. pertussis, and trans- mission occurs through contact with respiratory droplets from an infected individual. The incuba- tion period of pertussis ranges from seven to 10 days, and the signs and symptoms of the disease vary according to age, vaccination condition, and time lapse since the last dose of the vaccine. In Brazil, a specific law (NR32) recommends that HCPs receive vaccines against diphtheria, tetanus, measles, mumps, rubella, hepatitis B and influenza and states: “available efficient vaccines against other biological agents to which workers are, or may become, exposed must be provided free of charge by the employer” 2. However, HCP vac- cination policies vary considerably between dif- ferent countries 3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19 and have changed over the two last decades due to new information on the epidemiologi- cal profile of pertussis and licensing of new safe and effective vaccines for use in adolescents and adults, such as the triple acellular (Tdap) vac- cine that, besides protecting against diphtheria and tetanus, also protects against pertussis, and the quadruple vaccine against diphtheria, teta- nus and pertussis combined with the inactivated polio vaccine (Tdap-IPV), already licensed in a number of countries 7,8,9,10,11,12,20,21,22,23,24,25,26,27, 28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49, 50,51,52,53,54,55,56,57. Pertussis typically occurs in the following three phases: 1) Catarrhal (one to two weeks): characterized by a runny nose, intermittent cough and low-grade fever. Symptoms can be not distinguished from other respiratory infections and the infected per- son is most contagious in this phase; 2) The paroxysmal phase (four to six weeks): characterized by paroxysms of intense coughing often followed by vomiting and a loud “whoop”. These symptoms are more frequently seen in in- fants, the most vulnerable group with the high- est rates of complications, such as pneumonia, weight loss, seizures, encephalopathy and even- tually death; 1) Catarrhal (one to two weeks): characterized by a runny nose, intermittent cough and low-grade fever. Symptoms can be not distinguished from other respiratory infections and the infected per- son is most contagious in this phase; Acellular pertussis vaccines especially formu- lated for adolescents may contribute to a sub- stantial reduction in the incidence of the disease 7,57,58,59,60,61,62. Introduction World Health Organization (WHO), Centers for Disease Control and Prevention (CDC), Pan American Health Organization (PAHO) and the Brazilian Ministry of Health websites based on the following key words: vaccine, pertussis, Bor- detella pertussis, health professionals, and vac- cination coverage. World Health Organization (WHO), Centers for Disease Control and Prevention (CDC), Pan American Health Organization (PAHO) and the Brazilian Ministry of Health websites based on the following key words: vaccine, pertussis, Bor- detella pertussis, health professionals, and vac- cination coverage. The terms health care professionals (HCPs) and health care workers (HCWs) are applied to a range of professional categories that includes physicians, nurses, assistants, physiotherapists, dentists, radiologists and technicians and oth- er professional caregivers. Vaccination of these professionals is important to protect their health and reduce the risk of disease transmission to patients. In addition, vaccination reduces the economic impact of diseases associated with morbidity, work absences and loss of productiv- ity due to presenteeism 1. Abstract O objetivo deste artigo é descrever as recentes mudanças na epidemiologia da pertússis e as políticas de vacinação voltadas à prevenção da coqueluche para profissionais de saúde. Os auto- res fizeram um levantamento dos artigos publi- cados no PubMed, SciELO e páginas da Internet da Organização Mundial da Saúde, Organiza- ção Pan-Americana da Saúde, Centers for Disea- se Control and Prevention (Estados Unidos) e do Ministério da Saúde usando as palavras-chave: pertussis, vacinas e profissionais de saúde. A va- cinação de profissionais de saúde contra coque- luche é recomendada pela OMS, OPAS, CDC, e autoridades de saúde de nove países europeus, da Austrália, Hong Kong, Cingapura, Costa Rica, Argentina e Uruguai, e em alguns países é com- pulsória. No Brasil, identificamos apenas um ar- tigo abordando a vacinação de profissionais de saúde contra coqueluche, mas considerando a reemergencia da doença com grande número de hospitalizações e mortes em 2011, consideramos necessário rediscutir as políticas públicas envol- vendo a vacinação dos profissionais de saúde, particularmente daqueles que têm contato fre- quente com lactentes jovens. The aim of this study was to describe recent changes in the epidemiology of pertussis and existing policies regarding recommended and mandatory occupational vaccinations for healthcare professionals (HCPs). The authors carried out an extensive review of references on the PubMed and SciELO databases and the offi- cial sites of the World Health Organization, Pan American Health Organization, Centers for Dis- ease Control and Prevention, and Brazilian Min- istry of Health, using the keywords pertussis, vac- cines and healthcare professionals. Vaccination against pertussis is recommended for HCPs in the United States, Canada, nine European countries, Australia, Hong Kong, Singapore, Costa Rica, Argentina and Uruguay, and in some countries it is compulsory. In Brazil, only one publication discussing the risk of pertussis among HCPs was found. Considering the reemergence of pertussis and the great number of associated hospitaliza- tions and deaths registered in 2011, it is neces- sary to review public policies regarding HCP per- tussis vaccination, particularly among workers in frequent contact with young babies. Correspondence J. C. Moraes Faculdade de Ciências Médicas da Santa Casa de São Paulo. Rua Frederico von Martius 88, São Paulo, SP 01548-010, Brasil. jcassiom@uol.com.br Pertussis Vaccine; Health Personnel; Occupational Risks Vacina Contra Coqueluche; Pessoal de Saúde; Riscos Ocupacionais Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Moraes JC et al. 1278 Pertussis clinical manifestations and diagnosis The Bordetella pertussis and diph- theria toxoid antigen content of these vaccines is lower than whole cell vaccines and the acellular vaccines recommended for children and they are as safe as the double-adult vaccine (dT) 7,9,23,24, 29,38,57,58,59,60,61,62. It is currently recommended in several countries that adolescents and adults receive at least one booster of the dT vaccine in combination with the acellular pertussis (Tdap) vaccine 1,7,57,58,59,60,61,62. Increasing numbers of developed nations prefer IPV, and Tdap-IPV or Tdap + IPV vaccines are specially indicated to people who are in contact with immunocompro- mised people or children immunized with the oral polio vaccine (OPV) 1,14. 3) Convalescence (two to six weeks): the most common symptom is a chronic cough. Pertussis diagnosis is hindered due to the fact that signs and symptoms can not be differenti- ated from other respiratory diseases. Culture is considered the gold standard for pertussis diag- nosis. However, its sensitivity is low, and many countries included PCR and serology to improve pertussis surveillance. Even in regions where these lab tests were introduced the true burden of pertussis is still underestimated 3,4,6,7,19,22,29. Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Vaccine recommendations for HCPs circulation of B. pertussis in the community ac- counts for natural “boost”. However, vaccination leads to a reduction in bacterial circulation and a subsequent loss of protection after natural infec- tion, resulting in an increase in the incidence of the disease among teenagers and young adults, who become an significant source of infection of children 29,30,31,32,33,34,35,36,37,38,39,40. HCP vaccination recommendations vary accord- ing to country, vaccine availability, and to the risk estimates of the different preventable diseases immunization, the latter of which depends on epidemiological information 1,2,3,4,5,6,7,8,9,10,11,12, 13,14,15,16,17,18,19. Most countries recommend the use of the influenza and hepatitis B vaccines; however, after the licensing of acellular vaccines for adolescents and adults, many countries have introduced these vaccines for HCPs. In the Unit- ed States 1,7,8,9,10,11,12, Canada 13, various Europe- an countries 14,15, Australia 16,17 and some Latin America countries 18,19, acellular pertussis vac- cines combined with the tetanus and diphtheria toxoids (Tdap) and IPV standalone or with Tdap (Tdap-IPV) are already recommended for all or some HCPs 1,14 (Tables 2 and 3). Protection against the most serious forms of the disease offered by whole cell vaccines and acellular vaccines is greater in the early years after vaccination. Although the risk of hospi- talization and death due to pertussis is lower in adults, the disease causes substantial morbid- ity in these groups and is one of the most com- mon causes of continuous cough 22,24, 29,32,33,38,42. Separate vaccination of adolescents and adults became possible after the development and ap- proval of specific vaccines for each group. The pertussis vaccines for adults licensed for use in Brazil are listed in Table 1. Only acellular vaccines with reduced quantity of diphtheria toxoid can be administered to adults, and they can be pro- duced individually or in combination with IPV 7,8,9,10,11,12,13,14,29. The inclusion of the acellular pertussis vac- cine is due to an increase in the incidence of per- tussis in certain European countries 28,29,30,31,32,33, 34,35,36,37,38,39,40, the USA 1,78,9,10,11,12,21,24,25,26,27, Canada 13,22,23 and Australia 16,17,41,42,43. In these regions pertussis currently affects all age groups, and incidence rates are particularly high in ado- lescents and young adults 36,38,43. During the last decade, the use of these vac- cines for adolescents and adults, particularly those in contact with young babies, has been in- troduced in several countries 3,6,7,18,19,25,26,27,29,30, 31,32,38,43,46,49,52,57. Pertussis vaccines Pertussis vaccines have been in use since the 1940s, but are not recommended after seven years of age due to their reactogenicity. It is esti- mated that protection after natural infection and/ or pertussis vaccination lasts between 5 and 10 years. In locations with low vaccination coverage, In this paper we review the current HCP vac- cination recommendations, focusing on the per- tussis acellular vaccines, through a data search of the PubMed (January/2000 to March/2012), Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 PERTUSSIS VACCINE FOR HEALTHCARE PROFESSIONALS 1279 FIM: ﬁ mbria; PRN: pertactina; PHA: phytohaemagglutinin. Vaccine recommendations for HCPs The reemergence of pertussis can be associ- ated with a number of factors, including: incom- plete protection provided by vaccines; waning of immunity after 5 to 20 years; introduction of new diagnostic tests, such as PCR and serologic tests; high perception of the disease during out- breaks and high notification rates of suspicious cases; circulation of new strains not covered by Table 1 Table 1 Combined pertussis vaccines for adults. Sanofi Pasteur GSK Presentation Tdap-IPV Tdap Tdap Tdap-IPV Indicated age > 3 years and adults > 4 years and adults > 4 years and adults > 4 years and adults Pertussis toxoid (µg) 2.5 2.5 8.0 8.0 PHA (µg) 5.0 5.0 8.0 8.0 PRN (µg) 3.0 3.0 2.5 2.5 FIM 2 + 3 (µg) 3.0 5.0 - - Diphtheria toxoid ≥ 2 IU (2 Lf) 2 Lf ≥ 2 IU (2.5 Lf) ≥ 2 IU (2.5 Lf) Tetanus toxoid ≥ 20 IU (5 Lf) 5 Lf ≥ 20 IU (5 Lf) ≥ 20 IU (5 Lf) Polio (D-Ag-U) 1 40 - - 40 2 8 - - 8 3 32 - - 32 FIM: ﬁ mbria; PRN: pertactina; PHA: phytohaemagglutinin. Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Moraes JC et al. 1280 Moraes JC et al. Table 2 Table 2 Acellular pertussis vaccines recommended for health care professionals (HCPs) by country and year. Acellular pertussis vaccines recommended for health care professionals (HCPs) by country and year. Acellular pertussis vaccines recommended for health care professionals (HCPs) by country and year. Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Vaccine recommendations for HCPs Country/Region Reference Year Recommendation United States 7 2005 Adults aged between 19 and 64 years and those with contact with babies < 12 months (parents, grandparents aged < 65 years and HPCs), HCPs working in hospitals or ambulatory care settings and have direct patient contact Canada 13 2006 All HCPs in contact with infants ≤ 18 months World (World Health Organization) 6 2011 HCPs working with infants or immunocompromised people, especially in maternity and pediatric units Germany 14 2011 All HCPs Austria 14 2011 Pediatricians and HCPs working in neonatal centers Belgium 14 2011 All HCPs France 14 2011 HCPs in direct patient contact Finland 14 2011 Pediatricians and HCPs in neonatal centers Netherlands 14 2011 All HCPs Luxembourg 14 2011 All HCPs Norway 14 2011 Pediatricians and HCPs in neonatal centers United Kingdom 14 2011 All HCPs Uruguay 18 2012 HCPs, especially those in direct contact with infants < 6 months Americas (Pan American Health Organization) 19 2012 Recommended for HCPs to prevent transmission to infants < 6 months and immunocompromised people Table 3 Countries with recommendations for inactivated polio vaccine (IPV) use among health care professionals (HCPs) 14. Country Recommendation Austria Recommended for specific HCP groups or health care settings Cyprus All HCPs Finland All HCPs France Mandatory for all HCPs Germany Recommended for specific HCP groups or health care settings Ireland Recommended for specific HCP groups or health care settings Lithuania All HCPs Luxembourg All HCPs Malta All HCPs Switzerland All HCPs United Kingdom All HCPs vaccination was limited to these vaccines in the USA, Canada and Europe. The comparison of current trends between countries is limited be- cause different countries use different vaccines, employ different vaccination schedules, have variable coverage rates and use different pertus- sis case research and reporting methods. vaccines; and other unidentified factors. In devel- oped countries, acellular pertussis vaccines were introduced after 1999, and nowadays the majori- ty of pertussis cases are diagnosed in adolescents and adults that received acellular vaccines. There is no doubt that the duration of protection after acellular vaccines is limited. Furthermore, the low duration of protection after whole cell vac- cines and natural infection was observed when The recent reemergence of pertussis in Latin American countries that still use whole cell vac- Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 PERTUSSIS VACCINE FOR HEALTHCARE PROFESSIONALS 1281 worked in a laboratory specialized in pertussis diagnosis 96. Pertussis in HCPs: risks to patients and health workers The risk of contracting pertussis through contact with patients has been very well docu- mented 15,16,17,21,31,61,62,65,66,67,68,69,70,71,72,74, 104,105. In 2003, in the United States, a single baby with pertussis transmitted the disease to 17 HCPs that in turn transmitted it to 307 people 66. HCPs may contract pertussis and transmit the disease to their patients and hospital colleagues, thus causing disease outbreaks 1,8,10,11,15,16,45,48,63, 64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85, 86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105. Residents and those working in emergency rooms are at risk of acquiring pertussis 24; however, oth- er HCPs, such as nurses, physiotherapists and people who are in direct contact with patients are at greater risk of becoming infected 63,64,65,66,67,68, 69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91,92,93,94,95,96,97,98,99,100,101,102,103,104,105. Adults generally develop a milder form of the disease than children, however morbidity and the chanc- es of complications are high 1,7,21,22,23,24,29,30. In adults the disease generally lasts for four weeks, with 25% of patients developing complica- tions 24. It should be noted that even asymptom- atic or mild cases may transmit B. pertussis to oth- ers, including family members 51,66,69,72,73,83,96. During an outbreak in Cincinnati, 2% of the employees (87 out of 3,764) of a pediatric hospi- tal developed pertussis, leading to the following morbidities: 97% (84/87) of the infected employ- ees had a cough for over two weeks; 74% (64/87) had a paroxysmal cough; and 31% (27/87) suf- fered from post-cough vomits 80. Ten patients and five employees of a hospital in Atlanta developed pertussis during a period of only two months. A total of 630 of the 1,330 HCPs were treated with erythromycin prophylaxis and a half dose of the child acellular vaccine (DTaP) in an attempt to control the epidemic, since the specific adult formulation had still not been li- censed at the time 21. In 2003, the investigation of a case of pertus- sis in a premature newborn hospitalized in an in- tensive care unit revealed that the index case was a 36 year-old nurse who had worked with a par- oxysmal cough for three weeks, and was only re- moved from work after developing pneumotho- rax. During the investigation, it was discovered that four out of a total of 72 exposed health pro- fessionals also developed the disease 65. The majority of pertussis outbreaks as- sociated with HCPs have been reported in pe- diatric hospitals 8,63,68,69,87,83,96, particularly in neonatal care units and intensive care services 65,66,67,69,72,88,94,98,103 with hospitalized infants in units that treat immunocompromised or insti- tutionalized patients 64,95,104. Vaccine recommendations for HCPs cines has led to a great number of deaths. How- ever, this reemergence cannot be explained by the introduction of acellular vaccines or new di- agnostic tests, because access to these tests is not universal. Rather, it is probable that Latin Ameri- can countries are experiencing a similar trend to that registered in developed countries over the last decade, where an age shift of pertussis was observed towards adolescents and adults due to high vaccination coverage rates among children. The availability of new diagnostic resources for pertussis, such as PCR and serology, has helped to identify pertussis as one of the main causes of prolonged cough illness in adults 22 and has shown that the disease may exist in mild or as- ymptomatic forms 33,51. The large majority of pertussis cases are reported in young babies, but the disease may affect people of any age group, including the elderly 28,39,34,35,37,41,45,47,50,51,56. Although pertussis hospital outbreaks involve only a small number of individuals, they may be very costly due to the recommended investigation and control measures that involve the temporary removal of the professionals from the working environment, the use of prophylactic antibiotics and laboratory tests of infected cases 65,66,67,68, 69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90, 91,92,93,94,95,96,97,98,99,100,101,102,103,104,105, as well as problems associated with noncompliance with recommended prophylaxis 76,92,93,94,95,97,98,99. Most of the information on pertussis in HCPs comes from the United States, Canada, Europe and Australia, where laboratory resources for the confirmation of cases in adults, such as PCR and specific serology, are more easily available 16,17,57, 58,59,60,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,81,82,83,84, 85,86,87,88,89,90,91,92,93,94,95,97,98,99,100,101,102,103,104. Seroepidemiologic investigations of the disease conducted in different countries show that the annual incidence of pertussis in ado- lescents and young adults varies between 1 and 6% 34,35,37,40,44 and that infection rates are greater among HCPs than in other professionals 24. In the United States, pertussis outbreaks in HCPs were reported more than three decades ago 105 and reports of pertussis outbreaks involving HCPs have become increasingly common since the 1990s, especially during pertussis epidem- ics which occur every three to four years even in vaccinated populations 10,11,66,67,67,68,69,70,71,72,73, 74,75,76,77,78,79,80,81. Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Cost implications of Pertussis outbreaks in hospitals Although pertussis outbreaks that occurred in hospital settings affected relatively few patients (between two and 17) and HCPs (between five and 13), the cost implications were substantial due to the need to investigate the presence of the disease in a large number of people, absenteeism and measures against pertussis 66,68,69,70,80,92. In a study of a case involving four health pro- fessionals diagnosed with pertussis conducted in Atlanta, the average cough duration before the index case was identified varied between 11 and 25 days 65. During an outbreak in Cincinnati, 79 employ- ees had to stay off work for at least five days and 622 people took antibiotics for 14 days 80. Another study concluded that precaution measures (isolation and chemoprophylaxis) were inadequate in 17 out of 28 confirmed pertus- sis cases, resulting in the exposure of 355 HCPs, each of which exposed an average of nine other HCPs to the disease (variation 1 to 86). Adequate precautionary measures were adopted in a little more than one third of cases (11 out of a total of 28 cases) during another outbreak. A total of 355 HCPs were exposed to 17 patients with pertussis without any protection, and it was considered that exposure to pertussis would be “probably in- evitable” in more than 80% of the contacts 69. It is estimated that each pertussis case costs between US$164 and US$357, while the cost of controlling hospital outbreaks varies between US$19,500 and US$195,000, depending on the number of professionals exposed to the disease 66,68,69,70,92. The estimated cost of managing the exposure of 355 HCPs in a tertiary care center for children in Cincinnati during a period of 20 months was US$69,770, while vaccination of the profession- als involved cost approximately US$60,000 69. In France, pertussis is not a disease that re- quires compulsory notification and approxi- mately half of the notified pertussis cases are from hospitals where an HCP is affected 86. Several pertussis intra-hospital transmission cases have also been reported in the last decade. In 1997, a 55 year-old nurse worked for five weeks with a cough until she was eventually diagnosed with pertussis. The investigation showed that nine out of 61 health professionals (15%) who had been in contact with the index case had serological indi- cations of recent pertussis infection 82. Pertussis in HCPs: risks to patients and health workers Recently, a pertus- sis outbreak was also detected among HCPs who Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Moraes JC et al. 1282 In 2004, an investigation of a pertussis out- break that affected six children born between 4 and 16 of June in Texas, identified 29 children under four months of age who met the clinical criteria for pertussis, of which 11 had had con- tact with a nurse who was later confirmed as having pertussis. This professional worked with cough and dyspnea symptoms while in contact with 113 babies, of which 11 (9.7%) developed the disease 72. The investigation of a pertussis outbreak be- tween November 2000 and March 2011 after con- firmation of three cases among hospital health professionals revealed that 91 people were ex- posed to infected patients, including 77 HCPs, 12 patients and two patients’ relatives, generating four additional pertussis cases 83. Another outbreak in a maternity center in- volving five HCPs led to 10 probable cases among a group of 101 HCPs who were exposed to the disease out of a total of 201 professionals that worked in the center and agreed to participate in the investigation. Only 60% of participants took the prescribed antibiotic, 85% of the suspects used masks, and only 46% used the mask cor- rectly 85. One of the reasons for noncompliance with prophylactic measures is the high rate of side effects associated with the use of macrolides (33%) 84. However, the main reason for noncom- pliance is a lack of awareness among HCPs of the risks the disease poses 71,81. Since the symptoms of pertussis in adoles- cents and adults generally consists of an exten- sive cough without fever, it is not always easy to recognize the disease, and the majority of the HCPs maintain their usual activities during the maximum transmissibility phase, thus exposing patients and fellow workers to pertussis 16,17,63,64, 65,66,67,68,69,70,71,72,73,74,75,76,77,85,90,91,92,93,94,95,96,97, 98,99,100,101,102,103,104,105. The growth culture is con- sidered the gold standard for pertussis diagnosis; however, test sensitivity is poor since B. pertus- sis exhibits a slow growth. In locations where the PCR test is not available, the disease is seldom recognized in adults and, therefore, outbreak control measures are seldom taken 68. In addi- tion, recommended hospital outbreak control measures are not always effectively implemented 65,69,72,73,74,75,76,81. Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Vaccine acceptance and the difficulties in implementing other prophylactic measures to prevent the spread of pertussis pertussis, and only 22% knew that the patients’ close contacts should also be tested 75. In a study conducted in France, physicians were more aware of occupational vaccinations than other HCPs and pediatricians were the best informed regarding the need to protected against flu and pertussis 87. In Germany 91 and Austra- lia 97 misconceptions about pertussis were more prevalent among nursing health professionals. It should be pointed out that these professionals have greater direct contact with high risk chil- dren, and have been more frequently identified as primary cases in pertussis outbreaks reported in neonatal units 65,66,67,68,69,72,94,98. Despite recommendations, many HCPs do not get vaccinated 77,87,88,89,90,91,92,93 because they wrongly believe that infection or vaccination during childhood provides permanent pro- tection 87,88,89,90,91,92,93. Studies also show that the following main factors are associated with low vaccine coverage in HCPs: low perception of risk; lack of specific knowledge of vaccines; unawareness of vaccination recommendations and legislation and lack of orientation by the health authorities (Table 4). Lack of access to vaccines in the workplace due to cost factors also hinder compliance with vaccination recom- mendations. This is the case not only with new vaccines, such as Tdap and Tdap-IPV, but also for those vaccines traditionally recommended for HCPs 8,16,17,88,89. In a study that involved almost 2,000 HCPs in Sweden in 2007 71, only 13% of interviewees planned to take the acellular pertussis vac- cine. The main factor associated with vaccine refusal was the belief that vaccination against pertussis during childhood provides permanent protection. The difficulties involved in achieving compli- ance with pertussis vaccination of HCPs in some countries have led to discussions regarding com- pulsory vaccination 10,11,16,17. In Germany, 68.4% of 1,215 HCPs supported mandatory vaccination for HCPs, however, acceptations of mandatory vaccination was lower among nurses, the group with lower immunization coverage rates 91. Low coverage and incomplete vaccination regimens put the exposed individual at risk and generate expenses for employees and for the em- ployers in the form of laboratory tests, treatment and/or immunoprophylaxis. Despite this, pro- phylactic measures to avoid infections are not always adopted in a timely manner 63,64,65,66,67,68, 69,70,71,72,73,74,75,76. Even in some regions in Australia, where the pertussis vaccination is compulsory for health professionals, more than one third of profession- als believed that they remained protected after vaccination during childhood 97. Cost implications of Pertussis outbreaks in hospitals The cost of a pertussis outbreak involving HCPs in France was €46,661, of which 42% was due to loss of productivity 83. An estimate based on the direct and indirect costs (temporary work absence) associated with outbreak control suggested that HCP vaccina- tion could decrease costs by 238% and reduce the chances of infection by 46% 66. PERTUSSIS VACCINE FOR HEALTHCARE PROFESSIONALS 1283 Vaccine acceptance and the difficulties in implementing other prophylactic measures to prevent the spread of pertussis Lack of knowledge of the recommendation regarding the use of chemoprophylaxis after exposure to pertussis is a relevant issue even in countries where vaccines for HCPs are recom- mended 17,76,80,81,82,92. An investigation into the knowledge and at- titudes with regard to pertussis vaccination per- formed in the USA with 63 HCPs showed that only 19% were aware that pertussis is a manda- tory notifiable disease and half (52%) were aware of pertussis vaccination recommendations for HCPs in force in the USA since 2006. Almost half (48%) were not aware of the need to prescribe prophylactic antibiotics for people exposed to CDC: Centers for Disease Control and Prevention. Pertussis in Brazil In Brazil, acellular pertussis vaccines for adoles- cents and adults (Tdap and Tdap-IPV) are only available in private clinics and, although recom- mended by the Brazilian Society of Immuniza- tion and by Medical Associations, they have not yet been included in the NR32 for HCPs 2,3,4,5. Table 4 Factors associated with acceptance and refusal of acellular pertussis vaccines by health care professionals (HCPs) 17,75,81,87,88,89,90,91. Acceptance Refusal 1. Received a recommendation to take vaccination from a physician 1. Lack of a personal recommendation for vaccination 2. Awareness of CDC recommendation for pertussis vaccination for HCPs 2. Misconception that vaccine received previously protects for life 3. Encouraged to take vaccination by coworker 3. Low perception of the personal risks of contracting pertussis 4. Belief that HCPs may spread pertussis to patient and family CDC: Centers for Disease Control and Prevention. actors associated with acceptance and refusal of acellular pertussis vaccines by health care professionals (HCPs) 17,75,81,87,88,89,90,91. Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Moraes JC et al. 1284 Pertussis incidence rates in Brazil are very low compared with many countries and most cases are reported in infants under six months of age 3,4,54,55,56. Pertussis diagnosis is generally carried out using clinical criteria and with or without the culture method 3,4,54,55,56. Although culture is considered the gold standard for pertussis di- agnosis, it rarely gives positive results when the classic criterion for pertussis (a cough lasting for more than two weeks) is adopted. Pertussis is sel- dom suspected in adults and it is generally not possible to confirm diagnosis through growth culture 54,55,56. Lack of data does not mean that the disease does not occur, and recent studies in Latin American countries 45,46,47,48,49,50,51,52, 53,54,55,56, including Brazil 45,56, have shown that young adults (19 to 39 years of age) are a source of infection to children 56. With regard to trans- mission among HCPs, only one publication con- cerning a teaching hospital in the State of São Paulo was found 106. The number of pertussis cases and associated deaths has risen in Brazil. In 2011, there were 2,257 cases and 55 deaths 3. In the State of São Paulo, 913 cases and 23 deaths were recorded, corresponding to about 40% of pertussis cases and deaths registered in Brazil 4. It is important to emphasize that RT-PCR pertussis diagnosis has only been introduced in the State of São Paulo 3,4. rently no minimum interval between dT and Tdap or Tdap-IPV 9,114,120 and data confirms that Tdap can be used safely in the elderly, albeit with a reduction in antibody levels 115,116. Table 4 The Advisory Committee on Immunization Practices (ACIP) recommends that adults aged ≥ 65 years (including HCPs) who have or who anticipate having close contact with an infant < 12 months of age and who have not previously received dTpa should receive a single dose of Tdap to protect against pertussis, regardless of the interval since the last tetanus or diphtheria- toxoid containing vaccine 9. European studies of Tdap-IPV also showed that this vaccine can be safely used after dT 107,108. When necessary (e.g., travel or epidemics), Tdap or Tdap-IPV vaccines may be given to preg- nant women, preferentially after the first trimes- ter of pregnancy 9,12,13,18,19,114. It is estimated that protection lasts for 5 to 10 years 110,111,112,113,117,118,119; however, until now, only a few countries recommend redosing of these vaccines 31. Recent studies confirmed that a dTpa vaccine can be given 5 to 10 years after the previous dose in adults immunized with the same product 117,118,119. Several studies have provided evidence that Tdap and Tdap-IPV may be given simultaneously with other vaccines whether they contain a live agent or not (influenza, triple viral, hepatitis B and HPV) 7,9,109,114. Cad. Saúde Pública, Rio de Janeiro, 29(7):1277-1290, jul, 2013 Conclusions The Tdap and Tdap-IPV vaccines recommended for HCPs are safe and their reactogenicity profile is similar to that of the dT and dT-IPV vaccines 7, 8,9,16,29,100,101,102,103,104,107,108,109,110,111,112,113,114, 115,116,117,118,119,120. Fewer local reactions were re- ported among Tdap or Tdap-IPV recipients com- pared with those who received Tdap or Tdap-IPV. The majority of adverse events were local reac- tions, similar to those observed in children who received four doses of DTaP 7,62,107,108,109,113. Pertussis has reemerged in a number of countries and is considered a risk to HCPs, their patients and family members. HCPs play an important role in suspecting, reporting and treating pertussis. Pertussis out- breaks in health care facilities have been detected in different countries. Measures to investigate, treat and control pertussis clusters are not very effective and generate a huge waste of resources that could be much better employed in disease prevention. There is little information on the efficacy of Tdap and Tdap-IPV vaccines for HCPs. How- ever, immune response is excellent and, based on efficacy data in adolescents and adults, it is estimated that vaccine effectiveness is almost 80% 7,23. Seroconversion occurs after only one week in more then 40% of individuals and high seroconversion rates (> 90%) against diphtheria, tetanus, pertussis and poliovirus have been re- ported after two to four weeks 107,108. HCP pertussis immunization benefits both the community and the individual, since it re- duces the risk of contraction and transmission of the disease. Little information on the risk and acceptance of pertussis vaccines by HCPs is available in Bra- zil, and it is crucial to disseminate information on the risks of this disease and the benefits of vaccination, and improve pertussis surveillance with introduction of new diagnostic tests (RT- PCR and serology). Tdap and Tdap-IPV vaccines were recom- mended only for adolescents and young adults (< 64 years of age) with an interval of at least two years after the use of dT 7; however, there is cur- PERTUSSIS VACCINE FOR HEALTHCARE PROFESSIONALS 1285 PERTUSSIS VACCINE FOR HEALTHCARE PROFESSIONALS 1285 Considering the close association between vaccine acceptance and recommendations that laypeople receive from HCPs, the latter should receive continuing education on this subject to enable them to provide information on the ben- efits offered by the vaccines as recommended by the Medical Associations. Considering the difficulties in diagnosing the disease and delays in the implementation of measures to control the dissemination of B. Vacuna contra las Tos Ferina; Personal de Salud; Riesgos Laborales Contributors El propósito de este artículo es describir los recientes cambios en la epidemiología y políticas de vacunación para la prevención de la tos ferina en los profesionales de la salud. Los autores realizaron un estudio de los ar- tículos publicados en PubMed, sitios como SciELO, de la OMS, OPS, CDC y Ministerio de Salud de Brasil con las siguientes palabras clave: vacunas contra la tos ferina y profesionales de la salud. La vacunación de los traba- jadores de la salud contra la tos ferina es recomendada por la OMS, la OPS, CDC y por las autoridades sanita- rias de 9 países europeos, de Australia, Hong Kong, Sin- gapur, Costa Rica, Argentina y Uruguay, y en algunos países es obligatoria. En Brasil, se ha identificado un solo artículo sobre la vacunación de los trabajadores de la salud contra la tos ferina, sin embargo, frente al re- surgimiento de la enfermedad con un gran número de hospitalizaciones y muertes en 2011, consideramos que es necesario revisar la política pública de vacunación de los profesionales de la salud, especialmente si tienen contacto con niños pequeños. J. C. Moraes and L. F. Bricks contributed to project de- sign and the drafting and final approval of this article. T. Carvalhanas contributed to project design and the critical review and final approval of this article. Acknowledgments The authors are grateful to the Department of Social Medicine of the Faculty of Medical Sciences of the Santa Casa de São Paulo. Conclusions pertussis in hospitals, this study recommends greater attention on the part of health authori- ties with regard to the control and management of pertussis. Cad. 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https://openalex.org/W4297808742	https://uajnas.adenuniv.com/index.php/uajnas/article/download/38/147	Arabic	null	Remote system performance analysis of the virtual applications and virtual desktops by using Parallels 2X RAS technique	Mağallaẗ ğāmi'aẗ 'adan li-l-'ulūm al-ṭabīyyaẗ wa-al-taṭbīqiyyaẗ	2,019	cc-by-sa	5,079	Abstract The fast spread of computer networks and broadband Internet access and also the development of different operating systems make possible the use of different virtualization techniques. Virtualization techniques provide efficient IT solutions in corporate and educational sectors. We present an original structural framework for which the effect of virtualization technology are measured, based on a real campus wide deployment. The Virtual Desktop Infrastructure (VDI) provides academic members access to virtual applications on and off-campus for easy convenient access to academic resources in a server in data centre. In this paper, we present, at the beginning, a comparative performance analysis of Remote applications and Desktop Virtualization based on parallels 2X RAS versus Microsoft RDS. We introduce system architecture for the two tested scenario and test environment with Experiment Setup and implementations. Also, this paper provides analysis and implementation on the perceived and categorical perspectives on the usefulness, effectiveness and values of this technology in an academic environment. The main conclusions of the paper Indicated that students and academic staff have generally improved a remote accessibility to their course documents and academic materials virtually, using their own devices of different platforms which enabled them to perform course work more effectively resulting in improved system reliability, availability and scalability. Key words: Virtualization technology, Parallels 2X RAS, Microsoft RDS, Publishing Remote Apps and VDI, performance monitoring. emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee Remote system performance analysis of the virtual applications and virtual desktops by using Parallels 2X RAS technique Subhi Abdul-Rahim Bahudaila and Amal Abdul-Hafedh Abdu Saeed Department of Information Technology, Faculty of Engineering, Aden-University (Email: drsubhibahudaila@gmail.com, risebluesky6@gmail.com) DOI: https://doi.org/10.47372/uajnas.2019.n2.a12 Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee • Applying the virtualization system, using parallels 2X RAS technique in the laboratories of our Engineering Faculty, by deploying all applications and desktops to all devices in labs with zero setup, and no conflict between different applications or with legacy OS (section 3 & Table3). Related Work Virtualization technology supplements in cloud computing is considered as a set of software tools which divides a server into virtual resources reducing power and expanding computing resource allocation and data storage [11]. Chawdry and Lance [3] discussed computer virtualization techniques used at Californ niversity, highlighting the potential paybacks virtualization can offer to academic institutions. Chawdry and Lance [3] discussed computer virtualization techniques used at California University, highlighting the potential paybacks virtualization can offer to academic institutions. Castiglione, et al. [2] explained how visualization can be used to increase the course learning outcomes by providing exercises to the students accessing, using PCs. G i l [5] d ib d diff h i h l i h b d b ild i bl astiglione, et al. [2] explained how visualization can be used to increase the course learnin utcomes by providing exercises to the students accessing, using PCs. Garcia et al. [5] described different hosting technologies that can be used to build up a suitable virtualization infrastructure in an academic environment. Mohamed K. Watfa, Vincent A. Udoh, and Said M. Al Abdulsalam [9] discussed the fundamental benefits derived from the adoption of this technology in the academic environments. Younes and Soraya Nazarian Library – University of Haifa,[12] which uses Parallels Remote Application Server to efficiently publish 50-odd local applications and provide remote access to its staff, faculty and students at a cost-effective price. Introduction The release and spread of virtualization platforms make possible the development of cost- effective information systems that can provide additional dynamic resource management and simplified system administration [4]. This is due to the capabilities provided by virtualization such as maximizing of applications versatility, high-speed with large-capacity resource consolidation, ease aggregating, dynamics for load balancing enhancement and fault tolerance, ease of management, increased availability from pools of compute resources at less cost and with less complexity, and rapid services deployment to new servers included in a virtual infrastructure environment [8]. Virtualization and cloud computing technologies are often utilized in the teaching-learning process where academic professionals can access the needed academic resources using virtual classrooms. Universities have utilized the benefits of virtual technologies in a number of university level subjects [1]. The development of the IT infrastructure is one of the key factors in determining the attractiveness of the university. One of the implementation solutions is virtualization technology that has a significant influence on the study and research process [6]. Also, in business with today's mobile workforce, employees no longer have the option to be disconnected during the workday — they need access to their data anytime, anywhere, such as using the cloud’s Desktop as a Service (DaaS) that hosts virtualized desktop infrastructure (VDI) [7]. So, by publishing a VDI and applications, organizations can guarantee this enhanced connectivity, ensuring employees the same computing experience wherever they are in the world and increase employee productivity [10]. Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 411 emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee ote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed In the Engineering Faculty of Aden University, we were facing challenges when going to labs for applying some functions and assignments. All programming languages and installed applications in all pc’s and if one of the devices stops working or goes down, the student who is working on his pc will stop his work until the pc is being repaired. Other challenges, if there are any problems in any pc whether locally or remotely, the administrator has to go to the location of pc with the problem to be managed and fixed. Sometimes there is a conflict between different applications, or even with legacy O.S a risk of deleting the applications or editing O.S files or registry will also rise. This research will help us to solve the most important problems facing us in educational sector, especially in training laboratories. The main contributions of this paper are the following: The main contributions of this paper are the following: • Improving the educational methods in laboratories and expanding a connections method and data transmission between different offices accessing the needed academic resources virtually on and off-campus using different platforms. (section 3) • Increasing revenues and decreasing Total Cost of Ownership (TCO), by facilitating centrally administrative tasks. (section 3 & 5.2-Table3) • Improving the security aspects with decreasing the expense of more money, as provided in our research for configuring traditional pc's to be converted into Thin Clients (Desktop Replacement) with more data security. (section 5.2-Table3) • Improving the security aspects with decreasing the expense of more money, as provided in our research for configuring traditional pc's to be converted into Thin Clients (Desktop Replacement) with more data security. (section 5.2-Table3) • Monitoring server performance with the parallels 2X RAS Reporting Engine and how to prevent a data centre performance bottlenecks and overloads (section 6) • Monitoring server performance with the parallels 2X RAS Reporting Engine and how to prevent a data centre performance bottlenecks and overloads (section 6) • Applying the virtualization system, using parallels 2X RAS technique in the laboratories of our Engineering Faculty, by deploying all applications and desktops to all devices in labs with zero setup, and no conflict between different applications or with legacy OS (section 3 & Table3). Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee 2. Publishing App-s Parallels Remote Application Server delivers Windows applications to any device, allowing employees to instantly work on local devices without the necessity of installing the applications. Parallels RAS gives employees seamless access to applications published from RDSH, VDI or Remote PC host. Deliver applications and server-based desktops whenever and wherever you need them, on any device of your choice: mobile phone, desktop, tablet, Raspberry PI, and more. Published applications are also accessible from a web browser using the Parallels HTML5 Client, which enables you to quickly access applications and virtual desktops instantly. Parallels 2X RAS deliver applications, desktops, and data to any user, regardless of the operating system or device. Parallels Client is available for Windows, Linux, Mac®, iOS, Android, Thin Client, Chromebook™, Raspberry Pi and any HTML5 web browser. On-the-go access allows employees to be as productive as if they were working from the office, as shown in Figure 1. Figure 1: A Published Resources on a Client Side Figure 1: A Published Resources on a Client Side Parallels Techniques of publishing App-s and Desktop q p g pp p Parallels 2X RAS is a cost-effective application delivery and VDI solution, which allows your employees to access and use applications, desktops and data from any device. Easy to deploy, configure, and maintain. Parallels RAS provides organizations with a seamless application delivery and VDI experience while reducing TCO and improving security [13]. p g p g 1. Publishing Virtual Desktop Infrastructure (VDI) 1. Publishing Virtual Desktop Infrastructure (VDI) Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 412 Parallels Remote Application Server (RAS) makes VDI quick, easy, and affordable. It enables organizations to deliver fully functioning virtual windows desktops and applications to employees. Parallels 2X RAS allows users to be productive anywhere by providing a windows desktop–like experience on any device. Parallels 2X RAS simplifies the VDI deployment. Despite the benefits, the initial upfront costs of implementing VDI and application publishing are substantial. 412 Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 1.2. Microsoft RDS Microsoft RDS scenario is also presented in Figure 2. In the left part is a Windows service created to make possible multi user connections to a Windows server station. Connected clients use RDP to transfer commands to the server and to receive the graphical user interface of the remote desktop. Connected users have access granted to all the software applications installed on the server station. In this case, the received remote desktop is a Windows Server 2008 R2 desktop. 1.1. Remote APPs and Desktop using Parallels 2X RAS Test environment is presented in Figure 2. Remote applications and desktops virtualization, using parallel 2X RAS, can be seen in the right part of this Figure. Needed components are highlighted with blue color. This scenario uses virtualization in order to host the remote applications and desktops that will be deployed to remote and local users, using parallels 2X RAS console. In the client side, the users can access virtual resources in an easy way via 2X RDP client software installed in client side. Connected users have access granted to published applications and desktops installed on the server station. Published resources, client management, centralized management of end user's desktops configurations and monitoring system performance all that are through remote application server console. -The server PC for testing is LENOVO corei7, 8GBRAM, 1 T.B SATA H.D.D. -Connection media via wireless connection. - The server (host) is running Microsoft windows 2008R2 operating system with terminal server role and RDS and Desktop session Host installed and configured. -The server PC for testing is LENOVO corei7, 8GBRAM, 1 T.B SATA H.D.D. -The server PC for testing is LENOVO corei7, 8GBRAM, 1 T.B SATA H.D.D. -Connection media via wireless connection. 1. Experiment Setup and Implementation 1. Experiment Setup and Implementation The test environment is achieved for the comparison between Microsoft RDS and Parallels 2X RAS based on publishing or deploying a virtualized remote applications and desktops scenario, as shown in Figure 2. We have windows server 2008R2 operating system with remote desktop services rule and Remote Desktop Session Host feature installed and configured on server station. On the left part of Figure 2, there are Microsoft RDS components marked with green color and, on the right part of Figure 2, there is parallels 2X RAS program installed for remotely published APPS and desktops for different clients, managing, monitoring and controlling applications, users and devices locally or remotely marked with blue color. Several types of clients connect and access to server for using published applications and desktops via Parallels 2X RAS such as personal computers, laptops, thin clients and smart phones of different platforms, including IOS, Windows, Android, Raspberry Pi, Linux and MAC.… The connections are realized, using remote desktop protocol (RDP). In addition, we can use any HTML5 browser to access published resources without installing the RDP client. In order to test the performance of both Parallels 2X RAS and Microsoft RDS implementations, the built-in performance monitor tool was used. Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 413 emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee Figure 2: Remote Applications and Desktop virtualization test environment. Figure 2: Remote Applications and Desktop virtualization test environment. Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee All those counters are taken together each time with diff. no. of users accessing and running virtualized remote apps and desktops for getting the actual comparative performance benchmarks to be able for determining the best virtualization solution for virtualized remote apps and desktops that can be used in universities, laboratories, hospitals, banks and other sectors. We started our tests scenarios with 1 user access and run remote virtualized apps in a server, using Microsoft RDS, then the same no. of users (1 user) access the same remote virtualized apps in a server using parallels 2X RAS and recording the results, repeating the same test scenario with increasing consecutively no. of users (2,3,4,5). 2. Performance evaluation tests Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 414 In order to test the performance of both virtualization solution technique, Parallels 2X RAS and Microsoft RDS, the built-in Performance Monitor tool was used. The first challenge was finding the right performance counters that accurately monitor the use of system resources. Three test suites were performed related to memory usage, CPU load and storage drive queue. These parameters are critical when we evaluate system performance. The Committed bytes counter of the Memory object shows the amount of used memory. This can be given also as a percentage of the total amount of memory if we use the %Committed bytes in use counter. Also, at the same time, we investigated other counters such as Avg.Disk Queue Length and the percentage of processor time beside the committed bytes and the percentage of Committed Bytes in Use counters. 414 1. Comparative Performance Results fter applying our test scenarios we have got the following results for both virtualization olutions separately, as shown in the Tables [1-2] and reflected to graph: Table 1. Microsoft RDS reading test values Number Of Users CPU [%] Memory Disk Storage Committed Bytes Committed Bytes In Use [%] 1 5.946 1,780,687,823 21,235 0.016 2 5.656 1,841,438,720 21.959 0.015 3 7.476 1,773,791,134 21.153 0.236 4 12.284 1,941,497,368 23.153 0.13 5 82.024 1,708,559,019 20.375 0.123 Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 41 5 82.024 1,708,559,019 20.375 0.123 Table 2. Parallels 2X RAS reading test values Number Of Users CPU [%] Memory Disk Storage Committed Bytes Committed Bytes In Use [%] 1 3.462 1,559,024,591 18.592 0.014 2 5.047 1,630,458,831 19.443 0.033 3 5.786 1,701,149,745 20.286 0.028 4 6.714 1,713,807,945 20.437 0.032 5 12.602 2,108,680,680 20.146 0.035 Figure 3: CPU Load Test for Virtualized apps and desktops Table 2. Parallels 2X RAS reading test values Number Of Users CPU [%] Memory Disk Storage Committed Bytes Committed Bytes In Use [%] 1 3.462 1,559,024,591 18.592 0.014 2 5.047 1,630,458,831 19.443 0.033 3 5.786 1,701,149,745 20.286 0.028 4 6.714 1,713,807,945 20.437 0.032 5 12.602 2,108,680,680 20.146 0.035 Table 2. Parallels 2X RAS reading test values Number Of Users CPU [%] Memory Disk Storage Committed Bytes Committed Bytes In Use [%] 1 3.462 1,559,024,591 18.592 0.014 2 5.047 1,630,458,831 19.443 0.033 3 5.786 1,701,149,745 20.286 0.028 4 6.714 1,713,807,945 20.437 0.032 5 12.602 2,108,680,680 20.146 0.035 Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 41 Figure 3: CPU Load Test for Virtualized apps and desktops Figure 3: CPU Load Test for Virtualized apps and desktops Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 415 Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed Figure 3 shows our performed test with increasing number of users accessing virtualized remote apps and desktops. At the beginning, we start our test with a user in both scenarios to see the benchmarks of tested performance counters. Then, as the number of users increased, the results were compared and evaluated. Users accessing the virtualized Remote Apps and Desktops using different platforms of different mobile devices and desktop devices. Five test suites were performed related to memory usage, CPU load and storage drive queue. %Total Run Time counter of both virtualized solutions Parallels 2X RAS and Microsoft RDS can be used for CPU test. This counter records the total consumption of CPU resource. Figure 3 shows CPU tests for deploying Apps and desktops of both virtualized system to users. We conclude that full CPU resources are shared to each user accessing virtualized Apps and, when server CPU usage reach its limit, all users perform slower. CPU tests are repeated for both scenarios with increasing in no. of users accessing virtualized Apps and desktops and the results was that both virtualized system solutions are the same when the number of users started from 1 up to 4 users accessing the same virtualized apps in the same interval in each scenario. we found that the %processor time tests seems to be the same with so little difference between the two solutions, but with increasing no. of users we see that CPU usage percentage, in case of Microsoft RDS is a little higher than in Parallels 2X RAS. This is because, at the starting point, CPU usage is near to 0 in both scenarios. Figure 4: Memory utilization Test for Virtualized apps and desktops Figure 4: Memory utilization Test for Virtualized apps and desktops Figure 4 shows Memory management test that was performed in order to determine the way of memory allocation for users accessing and running virtualized remote apps and desktops in a server for both scenarios and monitoring the committed bytes and percentage committed bytes in use counters. The benchmarks in Figure 4 clearly shows that the reading values are close to each other in both virtualized solutions, with little increasing in %committed bytes in use counter for Microsoft RemoteAPP RDS. Univ. Aden J. Nat. and Appl. Sc. Vol. Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed Figure 5 presents storage tests in the two scenarios. Measurements were taken during different no. of users (incremental no.) accessing virtualized remote apps via different platforms of devices. Average disk queue length is one of the most important parameter marked with blue color in the graph. In Microsoft RDS, solution disk queue is higher than that in Parallels 2X RAS, as no. of users increased. Parallels 2X RAS measurements show a low average disk queue length because, once the application is started, it is multiplied in memory for the connected client stations. Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed 23 No.2– October 2019 416 Figure 5: Storage disk queue Test for Virtualized apps and desktops Figure 5: Storage disk queue Test for Virtualized apps and desktops Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 416 Figure 5: Storage disk queue Test for Virtualized apps and desktops Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 416 2. Comparative Results of RAS Features 2. Comparative Results of RAS Features In addition to the above, we compare between various virtualization solutions like (parallels 2X RAS, Microsoft RDS). We obtained other results in the following categories of RAS features as described in Table 3. Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 417 Table 3. Parallels RAS Features VS Microsoft RDS # Feature Parallels 2X RAS Microsoft RDS i. Installation and Configuration 1 Easy to install and manage.  2 Offers a centralized management system  3 Effectively monitor and manage the entire network.  ii. 2X Desktop Replacement 4 extend the lifespan of hardware and delay migration to the latest Oss  5 Disabling the most common local configuration options, while guaranteeing the same level of service and security afforded by thin clients.  iii. Application Publishing 6 Publishing app-s from all servers in a farm  Only publish applications from an installed Remote App Server iv. Cross-Platform Support 7 Support a wide range of devices such as Android, iOS, Linux, Java, Chrome OS and HTML5.  Only support Windows clients and Mac v. Printing and Scanning Redirection 8 using a local printer or scanner without installing drivers on the server  9 Supports printing and scanning  Only supports printing vi. Hypervisor Support 10 Supports all major hypervisors including Hyper- V, Citrix and VMware.  Only supports Hyper-V and MS Virtual Server. vii. Security & Authentication 11 Filtering is done based on the MAC address, IP address, gateway and the client.  viii. High Availability Load Balancing (HALB) 12 Supports built-in load balancer.  Needs to Install Microsoft Network Load Balancing (NLB) services. 13 Provide default auto-configured of HALB. Not only does it check the available servers, it also checks the available gateways for a high availability network.  Installing and deploying NLB requires expertise knowledge as well. ix. Reporting Services of Monitor Server Performance 14 Provides an extensive reporting tool that generates 14 types of reports categorized into five groups.  Table 3. Parallels RAS Features VS Microsoft RDS 417 Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee Parallels 2X RAS Performance Monitoring The Performance Monitor is a new feature introduced with Parallels RAS. It provides the administrator with more in-depth information on how to improve the Parallels RAS infrastructure. By inspecting these reports, the organization can effortlessly enhance the performance of Parallels RAS and Windows Server components. The Performance Monitor enables real-time access to historical and real-time stats on Parallels RAS infrastructure. These extensive reports provide IT administrators with insight into a wide range of key metrics, such as session information and CPU usage. Analysing resource usage, diagnosed bottlenecks, map scalability needs help eliminate potential Parallels RAS misconfigurations, as well as keeping an eye on the general performance of the Parallels RAS environment. This save up to 30% of your hardware infrastructure by knowing how to reallocate your hardware resources [14]. Parallels offers 2X Remote Access Server monitoring via its built-in reporting features. An extensive reporting tool that generates 14 types of reports, categorized into five groups, as shown in Figure 7 & 8, interact with Parallels RAS: • User Reports provide insights into how users interaction. • Group Reports provide insights into how each group interaction. • Device Reports provide insights into how devices interaction. p p g • Server Reports provide insights into server health, CPU and RAM usage • Application Reports provide insights into published applications. • Application Reports provide insights into published applications. Figure 6: Server Health by Server pp p p g p pp Figure 6: Server Health by Server Figure 6: Server Health by Server Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 418 Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed Figure 7: Devices Used to access published Apps and VDI Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed Figure 7: Devices Used to access published Apps and VDI Figure 7: Devices Used to access published Apps and VDI Conclusion In this paper, we have investigated the performance of remote apps and desktop virtualization using two alternative virtualization solutions: parallels 2X RAS and Microsoft RDS. We have implemented the test environment, using an entry level server machine for both scenarios and performed memory, CPU and storage management measurements. In case of CPU and Memory management tests, both systems have almost the same results. CPU usage, in case of Microsoft RDS, is a little bit higher as the number of users increased. Storage management measurement shows a clear advantage for parallels 2X RAS scenario. Average disk queue values are higher than in the order of magnitude when number of users increased to access virtualized apps and desktops, using Microsoft RDS. The main conclusion of the first part of this paper is that the parallels 2X RAS scenario outperforms the Microsoft, based on deploying and accessing virtualized Remote Applications and Desktops both CPU and storage management. Notably, Parallels RAS allows businesses of all sizes to implement high end virtualization solution. We conclude that virtualization is an effective solution in academia. Parallels 2X RAS integrates all virtualization technologies into a single platform. With parallels 2X RAS, businesses can securely and seamlessly deliver highly scalable virtual desktops and applications with greater flexibility to monitor and manage VDI/RDP networks. Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 References 1. BouSaba, C., Burton, L., Fatehi, F. (2010). Using virtualization technology to improve education. In: 2nd International Conference on Education and New Learning Technologies, pp. 201–206. 1. BouSaba, C., Burton, L., Fatehi, F. (2010). Using virtualization technology to improve education. In: 2nd International Conference on Education and New Learning Technologies, pp. 201–206. 2. Castiglione, A., Cattaneo, G., Luigi, C., Ezio, C., CarloFulvio M. (2010). Virtual Lab: a concrete experience in building multi-purpose virtualized labs for Computer Science Education. Dipartimento di Informatica - Universit`adegli Studi di Salerno, Italy, pp. 1–15. 2. Castiglione, A., Cattaneo, G., Luigi, C., Ezio, C., CarloFulvio M. (2010). Virtual Lab: a concrete experience in building multi-purpose virtualized labs for Computer Science Education. Dipartimento di Informatica - Universit`adegli Studi di Salerno, Italy, pp. 1–15. 4. DARABONT, O., KISS, K.J., DOMOKOS, J. (2015). Performance Analysis of Remote Desktop Virtualization based on Hyper-V versus Remote Desk top Services. Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 419 Remote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saeed MACRo 2015 5th Int. Conference on Recent Achievements in Mechatronics, Automation, Computer Science and Robotics. 5. Garcia, C.R., Quesada-Arencibia, A., Candela, S., Carrasco, E., Gonzلlez, A. (2012). Teaching information systems technologies: a new approach based on virtualization and hosting technologies. Int. J. Online Eng. 8(4), 32–41. 6. Miseviciene, R., Plestys, R., Zakarevicius, R., Ambraziene, D., Pazereckas, N. (2013). Virtual Desktop Infrastructure Technology Based Study & Research. Int. Journal of Education and Research Vol. 1No. 4. 7. Murugesan, S., Bojanova, I. (2016). Cloud Computing: An Overview, pp 3-14 In: Encylopedia of Cloud Computing. Wiley-IEEE press 725 pp. 8. Stallings, W. (2018). Virtual Machines, pp. 627-656. In: Operating Systems: Internals and Design Principles, 9th E. © Pearson Education Limited 798 pp. 9. Watfa, M. K., Udoh, V. A., and Al Abdulsalam, S. M. (2015). An Educational Virtualization Infrastructure. © ICST Institute for Computer Sciences, Social Informatics and Telecommunications Engineering 2016Y. Zhang et al. (Eds.): Cloud Comp, LNICST 167, pp. 12–21. 10. 4Ways Desktop Virtualization Improves Employee Efficiency (2017). URL: https://insights.samsung.com/2017/10/24/4-ways-desktop-virtualization-improves-employee- efficiency/. 11. G5 Networks–Technology Partner (2013). URL: http://www.g5networks.net/virtualization.html. 12. The Younes and Soraya Nazarian Library – University of Haifa (2015). URL: https://www.parallels.com/blogs/university-of-haifa/. 12. The Younes and Soraya Nazarian Library – University of Haifa (2015). URL: https://www.parallels.com/blogs/university-of-haifa/. 13. Parallels Techniques of publishing App-s and Desktop Remotely. URL: https://www.parallels.com/products/ras/remote-application-server/ 14. Parallels RAS v16.1 \| Performance Monitor URL: https://www.parallels.com/blogs/ras/performance-monitor 14. Univ. Aden J. Nat. and Appl. Sc. Vol. 23 No.2– October 2019 References Parallels RAS v16.1 \| Performance Monitor URL: https://www.parallels.com/blogs/ras/performance-monitor 420 emote system performance analysis of the virtual ….Subhi A. Bahudaila,Amal A. Abdu Saee حتليل أداء النظا م عن بعد للتطبيقات االفرتاضية واسطح املكتب االفرتاضي باستخدام تقنية Parallels 2X RAS صبحي عبدالرحيم باهذيلة و أمل عبدالحافظ عبده سعيد قسم تكنولوجيا المعلومات، كلية الهندسة– جامعة عدن drsubhibahudaila@gmail.com ، risebluesky6@gmail.com DOI: https://doi.org/10.47372/uajnas.2019.n2.a12 حتليل أداء النظا م عن بعد للتطبيقات االفرتاضية واسطح املكتب االفرتاضي باستخدام تقنية Parallels 2X RAS صبحي عبدالرحيم باهذيلة و أمل عبدالحافظ عبده سعيد قسم تكنولوجيا المعلومات، كلية الهندسة– جامعة عدن drsubhibahudaila@gmail.com ، risebluesky6@gmail.com DOI: https://doi.org/10.47372/uajnas.2019.n2.a12 امللخص امللخص يتيح االنتشار السريع لشبكات الكمبيوتر والوصول إلى اإلنترنت ، وكذلك تطوير أنظمة تشغيل مختلفة ، إلى استخدام تقن يات األنظمة االفتراضية المختلفة. األنظمة االفتراضية توفر الحلول التقنية الفعالة في قطاعي الشركات والتعليم. قدمنا بنية هيكلية يتم من خاللها قياس تأثيرات تطبيق األنظمة االفتراضية عن طريق نشر التطبيقات و سطح ال مكتب للحرم الجامعي. توفر البنية التحتية االفتراضية لسطح المكتب لألعضاء األكاديميين و الطالب الجامعيين الوصول إلى التطبيقات االفتراضية من داخل وخارج الحرم الجامعي من أجل سهولة الوصول إلى الموارد األكاديمية الموجودة في الخادم الرئيسي في مركز البيانات. في هذه الورقة نقدم في البداية تحليل أداء النظام من خالل المقارنة بي ن تقنيات نشر التطبيقات االفتراضية عن بعد وسطح المكتب االفتراضي استنادًا إلى تقنية Parallels 2X RAS مقابل Microsoft RDS. . قدمنا سيناريو اختباري ألداء النظام مع إعداد التجربة والتطبيقات عن طريق اختبار عدد من المعامالت أثناء استخدام التطبيقات وسطح المكتب ا لا فتراضي عن بعد من قبل عدد من المستخدمين . كما توفر هذه الورقة البحثية أيضًا التحليل والتنفيذ .على وجهات النظر المدركة والقاطعة حول فائدة وفعالية وقيمة هذه التقنية االفتراضية في بيئة أكاديمية االستنتاجات الرئيسية للورقة البحثية تشير إلى أن الطالب والموظفين األكاديميين قد قاموا بشكل عام بتحسين إمكانية الوصول عن بعد إلى الدورات الدراسية والمواد األكاديمية باستخدام األجهزة الخاصة بهم ذات األنظمة المختلفة التي مكنتهم من أداء أعمالهم بشكل أكثر فعالية و.وثوقية ال كلمات المفتاحية: تقنية األنظمة االفتراضية ، Parallels 2X RAS ، Microsoft RDS ، نشر التطبيقات و سطح المكتب عن بعد، مراقبة األداء. ال كلمات المفتاحية: تقنية األنظمة االفتراضية ، Parallels 2X RAS ، Microsoft RDS ، نشر التطبيقات و سطح المكتب عن بعد، مراقبة األداء. 421
https://openalex.org/W2150763831	http://old.scielo.br/pdf/pcp/v33n3/v33n3a07.pdf	Portuguese	null	Abuso sexual infanto-juvenil: a atuação do programa sentinela na cidade de Blumenau/SC	Psicologia	2,013	cc-by	10,006	596 Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC Children’s sexual abuse: the performance of the sentinela program in Blumenau / SC Abuso sexual infanto-juvenil: la actuación del programa sentinela en la ciudad de Blumenau/SC Artigo Glauco Anderson Espindola Universidade do Estado de Santa Catarina Vanderléia Batista Universidade Regional de Blumenau 596 596 Glauco Anderson Espindola & Vanderléia Batista Glauco Anderson Espindola & Vanderléia Batista Resumo: A violência sexual contra crianças e adolescentes é um problema de saúde pública que exige uma intervenção adequada. O presente estudo teve como objetivo identificar a atuação do Programa Sentinela, da cidade de Blumenau/SC, diante da violência sexual infanto-juvenil e mapear seus fatores de assistência e de vulnerabilidade. A metodologia empregada foi a análise documental das intervenções realizadas pelo programa. O estudo foi composto por 30 prontuários relativos a todas as crianças e adolescentes em situação de violência sexual atendidos pelo Sentinela que foram desligados do programa no segundo semestre de 2009. Os resultados apontam o programa como uma ferramenta contra a violência sexual infanto-juvenil, mas, da mesma forma, demonstram que ele necessita de melhorias. Entre os fatores de assistência, observou- se a credibilidade do Sentinela, o atendimento aos cuidadores das crianças e adolescentes, a variedade de intervenções e a articulação entre aspectos psicológicos, sociais e legais. Verificou-se, por outro lado, a falta de comunicação entre as instituições para articular as medidas de proteção necessárias, o não planejamento das intervenções, a não realização de intervenção com o autor da violência e a ausência de informações em alguns prontuários como fatores de vulnerabilidade. Abstract: The sexual violence against children and adolescents is a public health problem that demands an adequate intervention. The present study aims to identify the performance of the Sentinela Program of Blumenau/SC due to sexual violence against children and adolescents. The study presents the mapping of service and the vulnerability factors in the program’s resource network. The methodology used in the research was the documentary analysis of the interventions that were performed by the program. The study was composed by 30 medical records for all children and adolescents in situations of sexual violence treated at the Sentinela that have been disconnected from the program in the second semester of 2009. The results revealed that the Sentinela Program is a tool against sexual violence against children and adolescents but at the same time they showed that the program needs improvement. Among the assistance factors observed were the credibility of the Sentinela, the treatment with children and adolescents’ parents, the variety of interventions and the relationship among psychological, social and legal aspects. 596 Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC Children’s sexual abuse: the performance of the sentinela program in Blumenau / SC Abuso sexual infanto-juvenil: la actuación del programa sentinela en la ciudad de Blumenau/SC PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 597 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 598 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista A violência sexual é definida pela Organização Mundial da Saúde (World Health Organization, 2003) como qualquer atividade sexual, tentativa de obtenção do ato sexual, ações de tráfico de mulheres para prostituição ou comentários sexuais indesejáveis realizados por qualquer pessoa conhecida ou desconhecida da vítima pelo uso de coerção, de ameaças ou de força física. Tal conceito denota a não limitação da violência sexual ao ato sexual em si, e inclui desde a prática de carícias, a manipulação de genitália, mama ou ânus, a exploração sexual, o voyeurismo, a pornografia, o exibicionismo, até o ato sexual, com ou sem penetração. Entretanto, esses números representam uma dimensão parcial do fenômeno, uma vez que muitos casos de abuso sexual infanto- juvenil não são denunciados, devido tanto a sentimentos de vergonha e medo da criança ou do adolescente quanto ao sigilo intrafamiliar, como, por exemplo, nos casos em que mães encobrem a violência dos maridos, à relutância de alguns profissionais em reconhecer e relatar o abuso e à insistência de tribunais por regras estritas de evidência (Furniss, 1993). Segundo Furniss (1993), os fatores que influenciam o impacto da violência sexual são a idade de início do abuso, a duração do abuso, o grau de violência ou da ameaça de violência, a diferença de idade entre a pessoa que cometeu o abuso e a criança que sofreu o abuso, o vínculo entre o autor da violência e a criança e a ausência de características parentais protetoras. O impacto da violência sexual é complexo e variado, e envolve consequências físicas, psicobiológicas, psicológicas e sociais. Segundo Habigzang, Koller, Azevedo e Machado (2006), crianças ou adolescentes em situação de violência sexual podem desenvolver quadros de depressão, transtornos de ansiedade, alimentares, dissociativos, hiperatividade e déficit de atenção, transtorno de personalidade borderline e transtorno do estresse pós-traumático; essas crianças e adolescentes podem igualmente apresentar crenças disfuncionais relacionadas com a situação abusiva. O abuso sexual infanto-juvenil, da mesma forma, não se restringe a uma prática realizada por um adulto em relação a uma criança, mas abrange qualquer prática sexual realizada por uma pessoa em relação a outra com menor nível de desenvolvimento psicossexual. PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista On the other hand the vulnerability factors were the lack of communication among institutions to articulate the necessary protection measures, badly planned interventions, failure to complete the intervention with the perpetrator of the violence and the lack of information in some records. Keywords: Child abuse. Abuse sexual. Sex offenses. Public policies. Resumen: La violencia sexual contra niños y adolescentes es un problema de salud pública que exige una intervención adecuada. El presente estudio tuvo como objetivo identificar la actuación del Programa Sentinela, de la ciudad de Blumenau/SC, ante la violencia sexual infanto- juvenil; y, mapear sus factores de asistencia y de vulnerabilidad. La metodología empleada fue el análisis documental de las intervenciones realizadas por el Programa. El estudio estuvo compuesto de 30 historias clínicas relativas a todos los niños y adolescentes en situación de violencia sexual atendidos por el Sentinela que fueron retirados del programa en el segundo semestre de 2009. Los resultados apuntan el Programa Sentinela como una herramienta contra la violencia sexual infanto- juvenil, pero, de la misma forma, demuestran que el Programa necesita mejorías. Entre los factores de asistencia se observó la credibilidad del Sentinela, la atención a los cuidadores de los niños y adolescentes, la variedad de intervenciones y la articulación entre aspectos psicológicos, sociales y legales. Se verificó, por otro lado, la falta de comunicación entre las instituciones para articular las medidas de protección necesarias; la no planificación de las intervenciones, la no realización de intervención con el autor de la violencia; y, la ausencia de informaciones, como, por ejemplo, sobre factores de vulnerabilidad, en algunas historias clínicas. g Palabras clave: Abuso de niños. Abuso sexual. Delitos sexuales. Políticas Públicas. A violência sexual infanto-juvenil é um fenômeno prevalente desde a antiguidade, que, atualmente, devido aos altos índices de incidência e ao seu impacto negativo no desenvolvimento das crianças e adolescentes, se tornou uma questão de saúde pública. Tal fato refletiu a necessidade de se criar serviços especializados e capacitados que possam prestar atendimento a crianças e adolescentes que sofreram abuso sexual. Nesse sentido, o objetivo da presente pesquisa é identificar os mecanismos de funcionamento do Programa Sentinela, da cidade de Blumenau/SC, suas estratégias e técnicas de enfrentamento da violência sexual, bem como identificar os fatores de assistência e de vulnerabilidade do programa, fornecendo subsídios que possibilitem sua qualificação. Glauco Anderson Espindola & Vanderléia Batista Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 599 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Sendo assim, toda a rede de apoio social da criança ou do adolescente, que inclui, conforme Habigzang, Azevedo, Koller e Machado (2006), os órgãos de proteção à criança e ao adolescente, tais como os Conselhos de Direito, Conselhos Tutelares, Promotoria e Juizado da Infância e Adolescência e demais instituições como escolas, postos de saúde, hospitais e abrigos, entre outras, irá influir no impacto da violência sexual. Como denota Furniss (1993), uma intervenção profissional equivocada pode levar a um dano secundário e à revitimização das crianças que sofreram violência sexual, podendo, de tal modo, ocasionar um dano psicológico adicional à criança. Uma rede de apoio social articulada, portanto, constituída por profissionais competentes e qualificados, que possibilite um ambiente protetor e afetivo à criança e ao adolescente e evite a sua revitimização, é de extrema importância na minimização dos efeitos negativos da violência sexual. Sendo assim, toda a rede de apoio social da criança ou do adolescente, que inclui, conforme Habigzang, Azevedo, Koller e Machado (2006), os órgãos de proteção à criança e ao adolescente, tais como os Conselhos de Direito, Conselhos Tutelares, Promotoria e Juizado da Infância e Adolescência e demais instituições como escolas, postos de saúde, hospitais e abrigos, entre outras, irá influir no impacto da violência sexual. Como denota Furniss (1993), uma intervenção profissional equivocada pode levar a um dano secundário e à revitimização das crianças que sofreram violência sexual, podendo, de tal modo, ocasionar um dano psicológico adicional à criança. Uma rede de apoio social articulada, portanto, constituída por profissionais competentes e qualificados, que possibilite um ambiente protetor e afetivo à criança e ao adolescente e evite a sua revitimização, é de extrema importância na minimização dos efeitos negativos da violência sexual. fatores relevantes (World Health Organization, 2003). fatores relevantes (World Health Organization, 2003). Destaca-se, todavia, que a compreensão do nível de risco de danos no ambiente da criança ou do adolescente e o subsequente planejamento de segurança são os primeiros passos na avaliação de casos de abuso sexual (Saunders, Berliner, & Hanson, 2004). Glauco Anderson Espindola & Vanderléia Batista Assim sendo, Arboleta e Duarte (2005) afirmam que o contato sexual entre um adolescente e uma criança menor pode ser considerado abusivo quando há uma diferença significativa de idade, de desenvolvimento ou de tamanho, que não permita ao menor ter condições de dar consentimento consciente para o ato. No entanto, o abuso sexual afeta o desenvolvimento de crianças e adolescentes de maneiras diferentes. Em alguns casos, a criança ou o adolescente pode apresentar efeitos mínimos ou nenhum efeito aparente, e, em outros, podem ocorrer graves problemas emocionais, sociais e/ou psiquiátricos (Saywitz, Mannarino, Berliner, & Cohen, 2000). Segundo Furniss (1993), os fatores que influenciam o impacto da violência sexual são a idade de início do abuso, a duração do abuso, o grau de violência ou da ameaça de violência, a diferença de idade entre a pessoa que cometeu o abuso e a criança que sofreu o abuso, o vínculo entre o autor da violência e a criança e a ausência de características parentais protetoras. Além desses fatores, Habigzang et. al. ressaltam a influência das características pessoais da criança ou do adolescente, da rede de apoio social e dos recursos financeiros. Em relação à epidemiologia da violência sexual, estudos realizados em diferentes partes do mundo sugerem uma prevalência do abuso sexual infanto-juvenil de 7% a 36% entre as meninas, e de 3% a 29% entre os meninos (World Health Organization, 2003). De acordo com dados da Associação Brasileira de Proteção à Infância e à Adolescência (ABRABIA, 2002), estima-se que, no Brasil, 165 crianças sofram abuso sexual por dia ou 7 a cada hora. Na cidade de Blumenau/ SC, a realidade não é diferente. Conforme a Delegacia de Proteção à Mulher, à Criança e ao Adolescente (DPPMCA), entre janeiro de 2006 e agosto de 2008, foram registradas 104 ocorrências de abuso sexual contra crianças e adolescentes (esses dados se restringem aos abusos realizados por maiores de 18 anos). Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Uma vez que muitas crianças e adolescentes abusados sexualmente continuam a viver com o autor da violência ou em famílias onde a violência doméstica ocorre, determinar um ambiente seguro para a criança ou adolescente em situação de violência sexual é uma prioridade. No mais, deve-se preparar adequadamente a criança ou adolescente antes de ela/elecomparecer ao tribunal para testemunhar (Arboleta et al., 2005). Nas intervenções terapêuticas, o acolhimento é o primeiro passo para um tratamento eficiente. Neste, conforme Pfeiffer e Salvagni (2005), é preciso que se crie um bom vínculo, explicando sempre o que será feito e o porquê de isso ser feito, sem que se prometa o que não se pode cumprir, pois a escuta da história da criança ou adolescente, livre de preconceitos, sem interrupções ou solicitações de detalhamentos desnecessários, vai demonstrar respeito a quem foi desrespeitado no que tem de mais precioso, que é seu corpo, sua imagem e seu amor-próprio. Nesse sentido, o abuso sexual infanto-juvenil exige a intervenção coordenada de uma rede de instituições, e inclui aspectos médicos, legais e psicossociais. Por conseguinte, Furniss afirma que os profissionais envolvidos com as questões do abuso sexual infanto-juvenil precisam integrar, em uma abordagem global, aspectos normativos e de saúde mental. Assim, os terapeutas podem ter de confiar no apoio das agências legais para a terapia, tanto quanto os profissionais da lei podem ter que compreender a dimensão psicológica do abuso sexual. Além disso, os aspectos jurídicos e clínicos devem ser conduzidos de uma forma coordenada, para que a criança ou o adolescente não seja revitimizada pela repetição de questionamentos desnecessários e para que a informação não seja perdida ou distorcida. Realizar uma comunicação ajustada para a idade ou para a compreensão da criança e documentar todas as informações, incluindo os estados emocionais da criança ou do adolescente e de sua família são também Além disso, é preciso considerar a singularidade do impacto da violência sexual, tornando-se necessário, segundo Saywitz et. al. (2000) realizar tratamentos em diferentes modalidades (individual, familiar, grupo, farmacológico) e em diferentes níveis de cuidados, para diferentes crianças ou para a mesma criança em diferentes tempos. Assim, um princípio básico de toda a prática clínica é a realização de uma avaliação inicial da criança ou do adolescente em situação de violência sexual. Glauco Anderson Espindola & Vanderléia Batista De tal modo, o plano de tratamento deve ser desenvolvido com base nos resultados da Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 600 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista e pesquisas que apontam os efeitos positivos no tratamento do abuso sexual das modalidades individual, grupal e familiar. Todavia, é de notável importância observar que a modalidade grupal tem apresentado melhores resultados na redução da ansiedade, de sintomas depressivos e de sentimentos de culpabilização e diferenciação (Mcgain & Mckinzey, 1995; Seixas, 1999; Habigzang, 2006), uma vez que prioriza um espaço onde as crianças e os adolescentes podem dividir suas experiências e sentimentos com alguém que passou pela mesma situação que eles. Conforme Habigzang, o grupo tem a função de oferecer apoio e alívio emocional para a criança ou adolescente, fazendo com que eles não se sintam sozinhos. Além disso, através do relato de sentimentos referentes ao abuso, da discussão das crenças de culpa pela experiência abusiva e do desenvolvimento de habilidades preventivas a outras situações abusivas, propicia-se a modificação do autoconceito das crianças e dos adolescentes em situação de abuso sexual de autodesprezo para autovalorização. avaliação, verificando-se as necessidades de cada criança ou adolescente bem como de suas famílias. Da mesma forma, a avaliação é um componente integral do processo contínuo do tratamento. É preciso realizar avaliações periódicas da criança ou do adolescente, para acompanhar o tratamento e identificar os problemas emergentes (Saunders, Berliner, & Hanson, 2004). Na escolha do tipo de atendimento, a literatura aponta os atendimentos psicológicos psicossociais e psicoeducativos, sendo a escolha dependente da avaliação inicial da criança ou do adolescente. Vale destacar que as crianças e adolescentes que aparentemente não apresentam nenhum sintoma podem beneficiar-se do tratamento psicoeducativo para a prevenção de futuras vitimizações e para verificação, durante um período de tempo, de consequências que poderiam estar ainda latentes (Saywitz et. al. 2000). Com relação ao tempo de intervenção clínica, segundo Habigzang (2006), este pode variar de acordo com o referencial teórico que fundamenta a intervenção e os fatores relacionados com a história de abuso e as consequências desse abuso para a criança ou adolescente. Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC Glauco Anderson Espindola & Vanderléia Batista Ressalta-se, no entanto, que o fator de maior relevância na intervenção do abuso sexual infanto-juvenil não é o tempo de duração do tratamento, mas o tempo de duração de seus efeitos, pois um tratamento com maior duração dos efeitos não apenas produz melhora dos sintomas psicológicos mas também tem melhores resultados na suspensão ou até mesmo na reversão dos efeitos psicobiológicos do abuso sexual infanto-juvenil (Cohen, Knudsen, & Mannarino, 2005). Outra consideração especial em casos de abuso sexual é a postura dos cuidadores relativa à acusação da violência. Geralmente, os pais entram ou trazem os filhos para tratamento devido à recomendação ou à exigência das autoridades. Em alguns casos, eles podem estar em busca de tratamento devido apenas ao mandato judicial, podendo contestar as alegações de abuso ou minimizar o impacto negativo sobre a criança ou adolescente. Desse modo, a avaliação da percepção parental e do estágio de prontidão para a mudança é um ingrediente central para desenvolver um plano de tratamento significativo (Saunders, Berliner & Hanson, 2004). Saywitz et. al. (2000) também acrescentam que a melhora da consequência da violência sexual depende não apenas da eficácia do tratamento e das condições da criança ou do adolescente mas também da atuação dos adultos de quem a criança depende. No que se refere à modalidade terapêutica, Habigzang (2006) afirma que diversas modalidades podem ser utilizadas como recursos para a intervenção, havendo estudos Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 601 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista É de extrema importância que o abusador receba alguma forma de tratamento. Furniss compara a pessoa que abusa sexualmente com um alcoolista, sendo o álcool comparado à criança ou ao adolescente. Dessa forma, ele destaca que os autores de violência sexual necessitam de uma prolongada terapia, em que a pré-condição é a admissão inicial do ato abusivo. No mais, o autor afirma que precisamos acreditar que as pessoas que cometem abuso sexual queiram parar de abusar, mas ainda não devemos acreditar que elas não irão recair no abuso futuramente, pois, assim como em outras formas de adição, essas pessoas correm o risco de recaída se não evitarem situações de alto risco. Glauco Anderson Espindola & Vanderléia Batista Outrossim, o autor da violência não deve ser visto apenas como um abusador, mas como um ser humano que cometeu um erro e que precisa de ajuda; portanto, a terapia com o autor da violência, embora seja de extrema complexidade, é necessária, até mesmo como uma forma de compreensão da violência sexual e de prevenção da ocorrência de novos abusos. Como podemos notar, embora seja relevante realizar novas pesquisas sobre formas de intervenção com crianças e adolescentes em situação de violência sexual, os estudos sobre o tema já conseguiram avançar em alguns aspectos. Sendo assim, ressalta-se que, devido a sua complexidade, qualquer intervenção relativa à violência sexual infanto-juvenil deve dar-se através de uma rede articulada de instituições que possibilitem uma ação integrada. Além disso, é primordial que toda criança ou adolescente seja tratado com total respeito, pois não se pode esquecer que estamos diante de indivíduos que tiveram seu corpo violado e, na maioria das vezes, sua inocência roubada. PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Método Atualmente, denomina-se Serviço de Proteção e Atendimento Especializado a Famílias e Indivíduos (Paefi), e destina-se ao atendimento de indivíduos e famílias em situações de violação de direitos, como violência (física, psicológica e negligência, abuso e/ou exploração sexual), tráfico de pessoas, situação de rua, mendicância, abandono, vivência de trabalho infantil, discriminação em decorrência da orientação sexual ou raça/etnia e outras formas de violação de direitos decorrentes de discriminações ou de submissões (Conselho Nacional de Assistência Social, 2009). Para tanto, efetuou-se a leitura dos prontuários, incluindo os atendimentos realizados em cada situação. A análise dos dados foi realizada com base em uma abordagem qualitativa de análise de conteúdo, conforme proposto por Bardin (1977). De acordo com Engers (1994), a análise de conteúdo se caracteriza por um conjunto de instrumentos e técnicas utilizados na análise e na interpretação de dados de uma pesquisa, que são aplicados, principalmente, em pesquisas de documentos escritos, discursos e semelhantes, com o objetivo de se fazer uma leitura crítica e aprofundada, que proporcione a descrição e a interpretação desse material. No entanto, em Blumenau, o programa que foi implantado como Centro de Referência, no mês de setembro de 2005, continua com a denominação Sentinela, e atende cerca de 285 crianças e adolescentes por mês, através de uma equipe de 10 profissionais das áreas de serviço social, Psicologia e Pedagogia (Prefeitura Municipal de Blumenau, 2009). A população total envolvida na pesquisa foi composta por 30 crianças e adolescentes que sofreram violência sexual e que foram atendidos pelo Programa Sentinela. Essa população foi estudada a partir da análise de seus prontuários de atendimento. A amostra foi intencional, e os requisitos de inclusão foram: ser prontuário de criança ou adolescente (faixa etária de zero a 18 anos), conter notificação de violência sexual e a criança ou o adolescente atendido ter sido desligado do programa no segundo semestre de 2009. Dessa forma, a primeira fase do processo de análise incluiu a leitura dos prontuários selecionados na íntegra, de modo a destacar e a selecionar os aspectos relevantes presentes nesses documentos para se atingir os objetivos da pesquisa. Na etapa de exploração do material e de tratamento dos resultados, houve a categorização dos dados em unidades temáticas, conforme protocolo de investigação. Uma vez identificados os temas principais, foi realizada a interpretação dos resultados dessa análise, buscando-se apreender o modo de atuação do Programa Sentinela frente à violência sexual infanto-juvenil. Método Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 602 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Em 2006, com a implantação do Sistema Único de Assistência Social (SUAS), o Sentinela passou a ser compreendido como serviço de média complexidade do Centro de Referência Especial de Assistência Social (CREAS), que obedece às Normas Operacionais Básicas da Política Pública de Assistência Social, e tornou-se um serviço de ação continuada (Conselho Federal de Psicologia, 2009). Atualmente, denomina-se Serviço de Proteção e Atendimento Especializado a Famílias e Indivíduos (Paefi), e destina-se ao atendimento de indivíduos e famílias em situações de violação de direitos, como violência (física, psicológica e negligência, abuso e/ou exploração sexual), tráfico de pessoas, situação de rua, mendicância, abandono, vivência de trabalho infantil, discriminação em decorrência da orientação sexual ou raça/etnia e outras formas de violação de direitos decorrentes de discriminações ou de submissões (Conselho Nacional de Assistência Social, 2009). o qual permitiu a coleta das informações referentes à atuação do Programa Sentinela na cidade de Blumenau/SC. Essas informações compreendiam os seguintes dados: pessoa ou instituição que realizou a denúncia, instituição ou órgão de primeiro contato, intervalo entre denúncia e primeiro atendimento, procedimentos realizados com a criança ou adolescente, procedimentos realizados com o autor da violência, outras intervenções realizadas pelo programa, encaminhamentos feitos pelo Sentinela, profissionais que atuaram na situação, tempo de acompanhamento da família, motivo de desligamento, planejamento das intervenções e ocorrência de avaliação inicial da criança ou adolescente. Assim, a coleta de dados se efetivou pelo preenchimento do protocolo de investigação com as informações contidas nos prontuários. Em 2006, com a implantação do Sistema Único de Assistência Social (SUAS), o Sentinela passou a ser compreendido como serviço de média complexidade do Centro de Referência Especial de Assistência Social (CREAS), que obedece às Normas Operacionais Básicas da Política Pública de Assistência Social, e tornou-se um serviço de ação continuada (Conselho Federal de Psicologia, 2009). Método A presente pesquisa foi realizada no Programa Sentinela, da cidade de Blumenau/SC, e caracteriza-se como um estudo exploratório de caráter descritivo que, segundo Gil, tem “(...) como objetivo primordial a descrição das características de determinada população ou fenômeno (...)” (2002, p. 42). Dadas, por um lado, a especificidade da investigação e, por outro, as questões éticas que envolvem a violência sexual infanto-juvenil, a opção metodológica empregada na investigação foi a análise documental, tendo sido utilizada a abordagem quantitativa-qualitativa, que permitiu a interpretação e a mensuração dos dados. Para finalizar, é importante destacar a existência de poucos estudos controlados que avaliam os resultados de tratamentos com crianças e adolescentes em situação de violência sexual (Saywitz et. al., 2000), fato que se agrava na literatura brasileira (Ferreira & Gonçalves, 2002). Além disso, a maior parte das pesquisas sobre estratégias de atendimento aos casos de abuso sexual infantil focaliza poucos aspectos do atendimento, abordando, sobretudo, o processo de diagnóstico, cuja figura central é o médico, o que reflete a ausência de uma visão integral do atendimento (Alzuguir, Assis, & Souza, 2002). Conforme Pereda, Polo, Grau, Navales e Martínez (2007), tal fato é consequência da falta, na maioria das ocasiões, de um dano físico visível, e da inexistência de um conjunto de sintomas psicológicos que permitam a detecção e o diagnóstico da violência sexual. Por outro lado, acrescenta-se a isso o pacto de silêncio que cerca esses fatos e o escândalo social que envolve as pessoas em questão. O Sentinela é um programa federal de proteção social especial de média complexidade, e abrange 1104 dos 5565 Municípios brasileiros. O programa foi instituído pelo Governo brasileiro em 2001, com o objetivo de prestar atendimento a crianças e adolescentes vítimas de violência física, abuso/violência sexual, violência psicológica e negligência. A ênfase do programa está no abuso e na exploração sexual (Comitê Nacional de Enfrentamento à Violência Sexual contra Crianças e Adolescentes, 2006). PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC Características das situações de violência sexual A análise dos dados coletados demonstrou que, na maioria dos casos, a violência sexual foi denunciada por algum membro da família da criança ou adolescente (Gráfico 1), sendo a mãe responsável pela denúncia em 50% dos casos, e os pais (mãe e pai juntos), em 14%. Método Para a coleta de dados, foi elaborado um protocolo de investigação pré-estabelecido, Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 603 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Por fim, os dados foram analisados quantita- tivamente com auxílio do Microsoft Excel e distribuídos em gráficos e tabelas com frequências e porcentagens, para melhor compreensão dos resultados. O principal órgão do qual procederam os encaminhamentos para o Programa Sentinela foi o Conselho Tutelar, responsável por 73% dos encaminhamentos para o programa, sendo que 40% deles procederam do Conselho Tutelar do Centro, e 33%, do Conselho Tutelar do Garcia. Os encaminhamentos, igualmente, procederam da DPMCA (14%), da polícia civil (7%), do Disque 100 (3%) e do Aviso por Maus-Tratos contra Criança ou Adolescente (APOMT) (3%). Vale ressaltar que, por envolver dados pessoais e privativos de crianças e adolescentes, a coleta de dados foi condicionada à obtenção da autorização da coordenadora do Projeto Sentinela e à assinatura do Termo de Responsabilidade pelos pesquisadores, garantindo-se os cuidados éticos necessários para a elaboração da pesquisa. Em relação ao autor da violência, verifica-se que, em 36% dos casos, a idade do autor da violência era desconhecida ou não constava nos prontuários. Dos casos nos quais a idade do abusador foi identificada, 28% das situações foram provocadas por adultos (entre 25 a 59 anos), 25% por adolescentes (entre 12 a 17 anos), 5%, por crianças (entre 10 a 11 anos), 3%, por jovens (entre 18 a 24 anos) e 3%, por idosos (igual ou superior a 60 anos). PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Aspectos de assistência e vulnerabilidade do Programa Sentinela Nesta seção, serão apresentados os dados relacionados ao funcionamento do Programa Sentinela, identificando os fatores de assistência e de vulnerabilidade do programa. Nesse sentido, os aspectos de assistência do Sentinela são as ações que auxiliam, de maneira eficaz, a criança ou o adolescente em situação de violência sexual, bem como suas famílias, a superar a violência sofrida. Por outro lado, os aspectos de vulnerabilidade referem-se aos fatores de riscos do programa, que dificultam a superação da violência sexual e/ou podem ocasionar um dano secundário para a criança ou para o adolescente e suas famílias. O Gráfico 2 apresenta o intervalo de tempo entre denúncia da violência sexual e realização do atendimento pelo programa. 604 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Como podemos constatar, na maior parte dos casos, a criança ou o adolescente demorou entre 0 a 15 dias ou entre 15 a 30 dias para receber o primeiro atendimento após a revelação do abuso. Gráfico 2. Intervalo de tempo entre denúncia e primeiro atendimento Número de Casos Dias 12 10 8 6 4 2 0 11 6 2 3 1 1 1 1 2 0 0 0 a 15 75 a 90 15 a 30 90 a 105 30 a 45 120 a 135 105 a 120 45 a 60 135 a 150 60 a 75 150 a 165 Não consta 2 Gráfico 2. Intervalo de tempo entre denúncia e primeiro atendimento Em 100% dos casos pesquisados, foi primeiramente realizado acolhimento com a criança ou adolescente e suas famílias, que tinha como objetivo principal o estabelecimento de vínculo, a orientação sobre o programa e as metodologias utilizadas e o esclarecimento sobre o motivo de encaminhamento. Observou-se que os acolhimentos se davam em um ambiente protetor, onde a criança ou adolescente poderiam expressar-se livremente e tirar suas dúvidas em relação ao programa. Nesse primeiro momento, não eram realizadas perguntas pertinentes à situação abusiva, sendo que a criança ou o adolescente relatava o abuso apenas se fosse de sua vontade. No que se refere à avaliação inicial, em nenhum dos prontuários consta se ela ocorreu ou não, porém, em 90% dos prontuários, há relatos das consequências que a violência sexual teve para a criança ou adolescente. Tal fator pode indicar a ocorrência de uma avaliação inicial. Aspectos de assistência e vulnerabilidade do Programa Sentinela Os tipos de atendimento que decorreram da realização do acolhimento foram o atendimento psicossocial, o psicológico e o psicoeducativo (Gráfico 3). Observa-se que com maior frequência as crianças ou adolescentes receberam mais de um tipo de atendimento, havendo prevalência da realização de atendimento psicossocial e psicológico com a mesma criança ou adolescente, que foi realizado em 33% dos casos estudados. Gráfico 3. Tipos de atendimentos realizados pelo Programa Sentinela da cidade de Blumenau 3% 3% 10% 17% 17% 17% 33% Psicossocial e Psicológico Apenas acolhimento Somente psicossocial Psicosocial, psicológico e psicoeducativo Somente psicológico Psicossocial e psicoeducativo Psicológico e psicoeducativo Gráfico 3. Tipos de atendimentos realizados pelo Programa Sentinela da cidade de Blumenau Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 605 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Glauco Anderson Espindola & Vanderléia Batista Entre as modalidades de tratamento, estão a terapia familiar, a individual e com os pais. A terapia familiar foi a modalidade de maior incidência (47%), seguida pela utilização da terapia familiar, individual e com os pais no mesmo caso (30%) e terapia individual e familiar no mesmo caso (14%). Além dessas, observa-se a ocorrência da terapia individual e com os pais, da terapia familiar e com os pais e a realização apenas da modalidade individual, ambas com frequência de 3%. Outras medidas promovidas pelo Sentinela foram: contato telefônico com a família da criança ou do adolescente, visita domiciliar, visita à escola da criança ou do adolescente ou contato com essa instituição de ensino, orientações judiciais, contato com a DPMCA, contato e reunião com o Centro de Atenção Psicossocial Infanto-Juvenil (CAPSI), contato com o Centro de Referência de Assistência Social (CRAS) e com o Conselho Tutelar. Essas medidas foram realizadas em 24 dos 30 casos, e estão distribuídas conforme a Tabela 1. O contato telefônico com a família da criança ou do adolescente teve o objetivo de agendar o atendimento ou de informar sobre a sua ausência nos atendimentos pré-agendados. Nos casos em que não foi possível contato pelo telefone, foi realizada a visita domiciliar, que foi, também, utilizada com o propósito de verificar o meio no qual a criança está inserida, levantando-se questões socioeconômicas e de proteção da criança ou do adolescente. Tabela 1. Outras intervenções realizadas pelo Programa Sentinela da cidade de Blumenau/SC a 1. Outras intervenções realizadas pelo Programa Sentinela da cidade de Blumenau/SC ç p g / Intervenção Freq. Absoluta Freq. Relativa % Contato telefônico com a família da criança ou adolescente Contato telefônico com a DPMCA Contato telefônico e reunião com a CAPSI Contato telefônico com o CRAS Contato telefônico com o Conselho Tutelar Contato telefônico com a escola da criança ou adolescente Orientações judiciais Visita domiciliar Visita a escola da criança ou adolescente TOTAL 12 1 1 1 1 4 4 17 6 47 25 2 2 2 2 9 9 36 13 100 Glauco Anderson Espindola & Vanderléia Batista Glauco Anderson Espindola & Vanderléia Batista As intervenções foram realizadas, geralmente, por uma equipe interdisciplinar que envolvia psicólogo e assistente social em 83% dos casos. Contudo, em alguns casos, verifica-se apenas a atuação do psicólogo (10%) ou do assistente social (7%). Outro fator relevante é que, em 87% dos casos, não houve nenhuma intervenção com o autor da violência. Em dois casos, a pessoa que cometeu a violência sexual teve como medida punitiva a prisão, e apenas dois dos autores da violência sexual receberam alguma forma de tratamento terapêutico, em que se realizou o aconselhamento em uma das situações e o atendimento psicossocial familiar em outra. Os motivos pelos quais as crianças e os adolescentes foram desligados do Programa Sentinela estão distribuídos no Gráfico 4. Em sete dos casos, foi relatado mais de um motivo, e o mais frequente foi que a criança ou adolescente se encontrava com seus direitos garantidos, aparecendo em 23 dos casos analisados, porém, em nenhum dos casos foram descritos os critérios utilizados para tal afirmação. Gráfico 4. Motivos de desligamento do Programa Sentinela da cidade de Blumenau/SC Não apresenta comprometimento decorrentes do fato ocorrido Não adesão ao tratamento Mudança de cidade Encaminhamento para outra Instituição Confirmação de não ocorrência do abuso Criança ou adolescente superou/elaborou a situação de violência Criança ou adolescente encontra com seus direitos garantidos 1 1 3 5 4 3 23 Gráfico 4. Motivos de desligamento do Programa Sentinela da cidade de Blumenau/SC Não apresenta comprometimento decorrentes do fato ocorrido Não adesão ao tratamento Mudança de cidade Encaminhamento para outra Instituição Confirmação de não ocorrência do abuso Criança ou adolescente superou/elaborou a situação de violência Criança ou adolescente encontra com seus direitos garantidos 1 1 3 5 4 3 23 Gráfico 4. Motivos de desligamento do Programa Sentinela da cidade de Blumenau/SC Em relação ao tempo de permanência no Programa Sentinela, o Gráfico 5 demonstra que o tempo durante o qual as crianças e adolescentes receberam tratamento no programa variou entre 1 a 660 dias. Os tempos prevalentes foram de 330 a 440 dias (em 8 casos), de 110 a 220 (em 7 casos) e de 1 a 110 dias (em 6 casos). Gráfico 5. Intervenção Para maior efetividade do tratamento, o Programa Sentinela realizou encaminhamentos para outras instituições em 16 dos 30 casos pesquisados. A instituição que recebeu maior número de encaminhamentos foi o Programa Saúde da Família (PSF), com (32%) dos encaminhamentos, que atendeu as questões médicas relacionadas com a criança ou o adolescente e suas famílias. As famílias, igualmente, foram encaminhadas para a Secretaria de Assistência Social da Criança e do Adolescente (SEMASCRI), em 17% dos encaminhamentos, e para o CAPSI, com 11%, atendendo as questões sociais e de saúde mental, respectivamente. Os outros encaminhamentos foram para atendimento pedagógico, avaliação neurológica, defesa civil, Programa Pró-Trabalhador, Programa Egresso, Diretoria de Articulação, Reinserção Profissional e Captação de Recursos, DPPMCA e Pronto Atendimento Médico, ambos com 5% de frequência. Ressalta-se, todavia, que em nenhum dos prontuários consta o relato do planejamento das intervenções realizadas com a criança ou o adolescente e com suas famílias no que tange ao tipo e à modalidade de atendimento que seriam utilizados, bem como aos encaminhamentos e às medidas necessários para a superação da violência, o que sugere o não planejamento das intervenções por parte das equipes do Programa Sentinela. Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 606 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Discussão O notificante é o elemento que torna pública a violência, e pode ser considerado um ator importante na rede de proteção à criança. O presente trabalho constatou que, em 50% dos casos, a mãe foi o notificante, e os pais (mãe e pai juntos) o foram em 14% dos casos. Dados semelhantes foram encontrados por Habigzang et al (2005). Em sua pesquisa sobre violência sexual, os autores também constataram que a violência sexual foi denunciada com maior frequência pela mãe da criança ou do adolescente (37,6% dos casos) e por outros parentes (15,1%). Nesse sentido, o elevado percentual de notificação por pessoas com algum vínculo familiar com a criança ou adolescente sugere quão difícil é para a sociedade apresentar denúncias de situações de suspeita ou de confirmação de violência sexual. Percebe-se, assim, que a violência sexual ainda é vista como um tabu e um escândalo social, pertencente ao meio familiar no qual ela ocorre e de responsabilidade desse mesmo meio. Após a revelação do abuso sexual, é importante que a criança ou adolescente envolvido na situação e sua família recebam alguma forma de intervenção o mais rápido possível, já que, conforme Furniss (1993), a revelação do abuso sexual contra a criança conduz a uma crise imediata nas famílias, visto que a simples nomeação do abuso estabelece o abuso como um fato para a criança e sua família. Nesse contexto, durante a análise dos dados, observou- se que 37% dos casos notificados levaram até quinze dias para receber atendimento. Já na literatura de apoio não foram detectados estudos que mencionassem o intervalo de tempo entre denúncia e atendimento. No entanto, visto o fator de risco que o abuso sexual representa, apesar de a maior parte dos casos pesquisados ter recebido atendimento em tempo hábil, esse número ainda não é expressivo. Dessa forma, é relevante que o Sentinela continue a trabalhar para aumentar o número de casos em que há um pequeno intervalo de tempo entre revelação do abuso e primeiro atendimento. Em relação ao órgão ou instituição de procedência dos encaminhamentos ao Programa Sentinela, observou-se que o programa recebeu denúncias de abuso sexual advindas do Conselho Tutelar, da polícia civil, do APOMT, do Disque 100 e da DPMCA, o que demonstra a credibilidade que vem sendo conquistada por esse programa. Entretanto, a maioria dos encaminhamentos procedeu do Conselho Tutelar. Glauco Anderson Espindola & Vanderléia Batista Tempo de permanência no Programa Sentinela da cidade de Blumenau/SC Número de Casos 10 8 6 4 2 0 6 7 5 3 1 1 a 110 110 a 220 220 a 330 330 a 440 440 a 550 550 a 660 8 Gráfico 5. Tempo de permanência no Programa Sentinela da cidade de Blumenau/SC Em todos os casos, houve acompanhamento do Conselho Tutelar, que foi realizado através de trocas de relatórios. Assim, o desfecho de cada caso deu-se através do encaminhamento ao Conselho Tutelar de um relatório referente à situação atual da criança ou adolescente, procedimentos realizados pelo Sentinela e motivo de desligamento. Percebe-se, enfim, a ausência de informações em alguns prontuários. Tal fator comprometeu a análise de algumas variáveis, especialmente no que se refere às informações sobre a realização ou não da Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 607 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista avaliação inicial da criança ou adolescente, o planejamento das intervenções e as técnicas e os critérios utilizados pelo profissional para avaliar o desligamento da criança ou adolescente do programa. Observou-se, ainda, a falta de uniformidade nos dados, visto que em alguns casos os atendimentos foram relatados apenas de maneira sucinta. Conselho Tutelar foi o meio mais utilizado para a realização das denúncias. Igualmente, Baptista, Brito, Costa e França (2008) descrevem que 68% dos casos de abuso sexual infanto-juvenil foram notificados pelo Conselho Tutelar em estudo realizado em Campina Grande/PB. Tal fator, além de representar a obrigatoriedade, instituída pelo Estatuto da Criança e do Adolescente (ECA), da notificação de casos de violência sexual ao Conselho Tutelar, é reflexo do modo de funcionamento do Programa Sentinela, pois, de acordo com as diretrizes de funcionamento do programa, este “deve receber os casos encaminhados pelo Conselho Tutelar do Município para análise e estudo da situação” (Ministério do Desenvolvimento Social, 2006, p. 5). Esse dado também denota a importância que o Conselho Tutelar ocupa na rede e na comunidade na qual está inserido. Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Discussão Outros estudos, do mesmo modo, destacam esse órgão como o principal órgão de encaminhamento ou de primeiro contato. Ferriani, Garbin e Ribeiro (2004), em pesquisa realizada em Ribeirão Preto/SP, relatam que o A literatura aponta o acolhimento como parte primordial do atendimento, sendo ele importante para estabelecer um vínculo com a criança e o adolescente, e deve ser realizado Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 608 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista livre de preconceitos e sem interrupções ou solicitações de detalhamento desnecessário (Pfeiffer & Salvagni, 2005; Habigzang, 2006). Desse modo, a realização de acolhimento em todos os casos pesquisados, sendo ele realizado em um ambiente protetor, onde a criança ou adolescente poderiam expressar-se livremente, representa um dos fatores de assistência do Programa Sentinela. Destaque-se, todavia, que a literatura ressalta a modalidade grupal como excelente modalidade na redução das consequências advindas da violência sexual (Mcgain & Mckinzey 1995; Seixas, 1999; Habigzang, 2006). Entretanto, essa modalidade não é realizada pelo Sentinela. Assim, sugere-se, como medida que possa contribuir para a qualificação do programa, a introdução da modalidade grupal nos atendimentos realizados. Com base nos resultados do presente estudo, as crianças e adolescentes em situação de violência sexual atendidas pelo Programa Sentinela receberam com maior frequência mais de uma forma de tratamento, destacando- se o atendimento psicossocial e psicológico. Lueneberg, Machado, , Régis e Nunes (2005), igualmente, verificaram que o tipo predominante de atendimento prestado a crianças e adolescentes em situação de violência sexual é o psicossocial (26,80%). Por outro lado, a realização de tratamento com a família da criança ou adolescente em situação de violência sexual representa um fator de assistência do programa, pois o tratamento familiar é apontado como essencial para desenvolver um plano de tratamento significativo (Furniss, 1993; Saywitz et al., 2000; King et al., 2000, Saunders, Berliner, & Hanson., 2004). Além dessas, o Sentinela promove outras medidas de intervenção, como contato telefônico e visita domiciliar em caso da ausência da criança ou adolescente nos atendimentos pré-agendados. Tais medidas representam uma não negligência por parte do programa com as famílias em atendimento. PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Discussão Observa- se, no presente estudo, que essa articulação é feita através de encaminhamentos para outras instituições, porém, com exceção do Conselho Tutelar, o programa realizou contato com as instituições que compõem o Sistema de Garantia de Direitos em apenas 3 dos 19 encaminhamentos realizados. Percebe-se, assim, a falta de uma ação articulada, o que representa um fator de vulnerabilidade do programa. as diretrizes do programa, “(...) esgotadas todas as possibilidades de intervenção, sem mudança dos padrões violadores de direitos, a autoridade competente deverá ser informada por meio de relatório circunstanciado, para que sejam tomadas as medidas pertinentes” (Ministério do Desenvolvimento Social, 2006, p. 20), o que efetivamente ocorre no contexto estudado. Em relação ao tempo de permanência no Programa Sentinela, o estudo demonstrou que a maioria das crianças e adolescentes receberam tratamento no programa durante 1 ano e 3 meses. Na literatura utilizada, não localizamos dados sobre o tempo médio de permanência na instituição. No processo de análise, percebeu-se, por fim, que algumas fichas apresentavam ausência de informações, o que representa um fator de vulnerabilidade do programa, uma vez que, conforme a Organização Mundial de Saúde (2003) é de extrema importância documentar todas as informações. No mais, Lueneberg, Machado, Régis, e Nunes (2005) afirmam que um fator importante para a prevenção desse tipo de violência é o preenchimento correto e completo dos dados que compõem a ficha de atendimento, pois, se ela estiver incompleta, poderá dificultar a assistência e impedirá o diagnóstico precoce dos fatores de risco para possível prevenção da violência sexual. Como sugestão, fica a descrição dos critérios de desligamento e da avaliação inicial em cada protocolo, justificando-os de maneira detalhada. Além desse, outro fator de vulnerabilidade do Sentinela é a não realização, em 87% dos casos, de intervenção com o autor da violência, fato esse de extrema gravidade, já que, em 30% dos casos estudados, o autor da violência tinha entre 10 e 17 anos. Furniss (1993) ressalta a necessidade dessa intervenção, como forma de se compreender e de prevenir a violência sexual. Outro fato ao qual devemos dar suma importância é a impunidade com que a violência sexual é tratada no Brasil, pois em apenas dois casos a pessoa que cometeu a violência sexual teve como medida punitiva a prisão. Discussão A visita domiciliar, também utilizada com o propósito de verificar o meio no qual a criança está inserida, é outro aspecto importante, uma vez que a coexistência de diferentes formas de violência em caso de abuso sexual tem sido frequentemente apontada pelos pesquisadores da área (Cohen & Mannarino, 2000; Habigzang & Caminha, 2004; Kellog & Menard, 2003). Além dessas, o Sentinela promove outras medidas de intervenção, como contato telefônico e visita domiciliar em caso da ausência da criança ou adolescente nos atendimentos pré-agendados. Além disso, na escolha ao tipo de atendimento, a literatura destaca os atendimentos psicológicos, psicossociais e psicoeducativos (Saywitz et. al., 2000). De acordo com a análise dos resultados, esses tipos de atendimento são prestados pelo Programa Sentinela, o que indica um fator de assistência do programa. Contudo, nos casos pesquisados, não se observa um planejamento relativo ao tipo de atendimento que será utilizado. De acordo com Saywitz et. al., a escolha do tipo de atendimento depende da avaliação inicial da criança ou do adolescente; o não planejamento da escolha do tratamento é, portanto, um fator de vulnerabilidade do Sentinela. Entre as outras formas de intervenção, destaca- se, do mesmo modo, a realização de orientações judiciais feitas pelos profissionais do programa às famílias em atendimento. Observa-se, desse modo, que os profissionais do Sentinela, em seus atendimentos, unem os domínios legais, de saúde e de saúde mental, fator de extrema relevância para que se evite o processo de revitimização (Furniss, 1993; Amazarray & Koller, 1998). Como previsto nas diretrizes de funcionamento do Sentinela, os atendimentos são feitos na modalidade familiar, individual e com os pais. Habigzang (2006) e Saywitz et al. (2000) ressaltam, como já apontamos, que é necessário realizar tratamentos em diferentes modalidades para diferentes crianças ou para a mesma criança em diferentes tempos, o que indica um fator de assistência do programa. Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC 609 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Conforme as diretrizes de funcionamento do programa, este deve manter estreita articulação com os demais serviços de proteção social básica e especial, com as demais políticas públicas e instituições que compõem o Sistema de Garantia de Direitos (Ministério do Desenvolvimento Social, 2006). Discussão Nas diretrizes de funcionamento do Sentinela, constata-se que o programa deve contar com equipe de profissionais que trabalhem de maneira interdisciplinar, o que pudemos verificar a partir do estudo realizado, pois grande parte dos atendimentos foram realizados por psicólogos e assistentes sociais. PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista Os resultados revelam que o Sentinela, de maneira satisfatória, consegue cumprir seu objetivo de prestar atendimento às crianças e adolescentes em situação de violência sexual, bem como aos seus familiares. Para tanto, o programa atua em uma rede integrada com o Conselho Tutelar, prestando atendimento aos casos notificados no Conselho. Ao chegar ao Sentinela, primeiramente é realizado um acolhimento com a criança ou adolescente em situação de violência sexual e sua família, estabelecendo-se vínculos e esclarecendo- se a metodologia de trabalho. Em seguida, são proporcionados atendimentos a essas crianças e adolescentes, dentre os quais estão os atendimentos psicológicos, psicossociais e psicoeducativos, realizados na modalidade individual ou familiar. Quando necessário, para maior efetividade do tratamento, o Sentinela realiza encaminhamentos para outras instituições de garantia de direitos da criança e do adolescente, que são desligados do programa quando se encontram com seus direitos garantidos, permanecendo em média 1 ano e 3 meses no programa. Ao ser realizado o desligamento das crianças e adolescentes, o Conselho Tutelar é informado através de um relatório. a insistência para que a família compareça aos atendimentos, a verificação do meio no qual a criança ou adolescente está inserido (visita domiciliar), a articulação entre domínios legais, de saúde e de saúde mental, o desligamento do programa somente após a garantia dos direitos da criança ou do adolescente e as trocas de informações (laudos e relatórios) com o Conselho Tutelar. Por outro lado, os fatores de vulnerabilidade do programa foram: não planejamento das intervenções, não realização de intervenção com o autor da violência, a falta de uma ação articulada entre o programa, os serviços de proteção social básica e especial, as demais políticas públicas e instituições que compõem o Sistema de Garantia de Direitos da criança e do adolescente e a ausência de informação em alguns prontuários, principalmente na avaliação inicial e na descrição dos critérios sociais e psicológicos de desligamento. Sugere-se, por fim, como medida para qualificação do Sentinela, a introdução da modalidade grupal nos atendimentos, uma vez que a grupoterapia tem apresentado resultados relevantes no tratamento de crianças e adolescentes em situação de violência sexual (Mcgain & Mckinzey, 1995; Seixas, 1999; Cohen & Mannarino, 2005; Habigzang, 2006). A análise dos documentos ainda possibilitou o mapeamento de fatores de assistência e de vulnerabilidade do Programa Sentinela no atendimento de criança ou adolescente em situação de violência sexual. 610 610 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Glauco Anderson Espindola & Vanderléia Batista PSICOLOGIA: CIÊNCIA E PROFISSÃO, 2013, 33 (3), 596-611 Conclusões As informações sobre os casos de violência sexual contra crianças e adolescentes coletadas a partir da análise dos prontuários disponíveis no Programa Sentinela, da cidade de Blumenau/SC, permitiram compreender a atuação do Sentinela frente ao abuso sexual e identificar os fatores de assistência e de vulnerabilidade envolvidos no seu contexto. Do mesmo modo, possibilitaram observar a impunidade com que a questão da violência sexual é tratada no Brasil e o tabu que cerca essa violência. Durante a análise dos dados, verificou-se, como fator de assistência, o fato de que, de maneira geral, os casos de atendimento somente foram desligados do programa com a constatação de que a criança ou adolescente se encontrava com seus direitos garantidos. Outro fator de assistência é o acompanhamento do Conselho Tutelar, realizado através de trocas de relatórios entre as duas instituições. Conforme Abuso Sexual Infanto-Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC Glauco Anderson Espindola & Vanderléia Batista Nesse contexto, entre os fatores de assistência identificados, destacam-se: a credibilidade do programa, a realização de acolhimento em um ambiente de total respeito à criança ou adolescente, a variedade de tipos e de modalidades de atendimento utilizados, o atendimento com os cuidadores da criança ou do adolescente, Diante do exposto, verificamos que o presente estudo conseguiu atingir seus objetivos, identificando a atuação do Programa Sentinela e mapeando os fatores de assistência e de vulnerabilidade do programa. Assim sendo, observou-se que o Sentinela constitui uma ferramenta contra a violência sexual, entretanto, possui alguns pontos que necessitam ser otimizados. Recebido 12/12/2011, 1ª Reformulação 04/04/2013, Aprovado 11/04/2013. Glauco Anderson Espindola Especialista em Metodologias de Atendimento a Criança e ao Adolescente em Situação de Risco pela Universidade do Estado de Santa Catarina, Blumenau– SC – Brasil. E-mail: glaucopsi@gmail.com Vanderléia Batista Psicóloga formada pela Fundação Universidade Regional de Blumenau, Blumenau – SC – Brasil. E-mail: letista19@hotmail.com Endereço para envio de correspondência: Rua Harry Wruck, nº 61, Fortaleza. CEP: 89056-210. Blumenau, SC. Abuso Sexual Infanto Juvenil: A Atuação do Programa Sentinela na Cidade de Blumenau/SC Recebido 12/12/2011, 1ª Reformulação 04/04/2013, Aprovado 11/04/2013. Glauco Anderson Espindola Especialista em Metodologias de Atendimento a Criança e ao Adolescente em Situação de Risco pela Universidade do Estado de Santa Catarina, Blumenau– SC – Brasil. E-mail: glaucopsi@gmail.com Vanderléia Batista Psicóloga formada pela Fundação Universidade Regional de Blumenau, Blumenau – SC – Brasil. 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https://openalex.org/W2110157263	https://pure.rug.nl/ws/files/17475555/PLoS_ONE_2015_10_e0121374.pdf	English	null	Adapting to a Warmer Ocean—Seasonal Shift of Baleen Whale Movements over Three Decades	PloS one	2,015	cc-by	9,554	Adapting to a warmer ocean Ramp, Christian; Delarue, Julien; Palsbøll, Per J.; Sears, Richard; Hammond, Philip S. DOI: 10.1371/journal.pone.0121374 DOI: 10.1371/journal.pone.0121374 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ramp, C., Delarue, J., Palsbøll, P. J., Sears, R., & Hammond, P. S. (2015). Adapting to a warmer ocean: Seasonal shift of baleen whale movements over three decades. PLoS ONE, 10(3), Article e0121374. https://doi.org/10.1371/journal.pone.0121374 Copyright Other than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons). University of Groningen Adapting to a warmer ocean Ramp, Christian; Delarue, Julien; Palsbøll, Per J.; Sears, Richard; Hammond, Philip S. Published in: PLoS ONE DOI: 10.1371/journal.pone.0121374 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ramp, C., Delarue, J., Palsbøll, P. J., Sears, R., & Hammond, P. S. (2015). Adapting to a warmer ocean: Seasonal shift of baleen whale movements over three decades. PLoS ONE, 10(3), Article e0121374. https://doi.org/10.1371/journal.pone.0121374 University of Groningen Adapting to a warmer ocean Ramp, Christian; Delarue, Julien; Palsbøll, Per J.; Sears, Richard; Hammond, Philip S. Published in: PLoS ONE DOI: 10.1371/journal.pone.0121374 IMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite from it. Please check the document version below. Document Version Publisher's PDF, also known as Version of record Publication date: 2015 Link to publication in University of Groningen/UMCG research database Citation for published version (APA): Ramp, C., Delarue, J., Palsbøll, P. J., Sears, R., & Hammond, P. S. (2015). Adapting to a warmer ocean: Seasonal shift of baleen whale movements over three decades. PLoS ONE, 10(3), Article e0121374. https://doi.org/10.1371/journal.pone.0121374 University of Groningen Adapting to a Warmer Ocean—Seasonal Shift of Baleen Whale Movements over Three Decades Christian Ramp1,2, Julien Delarue1, Per J. Palsbøll3, Richard Sears1, Philip S. Hammond2 Christian Ramp1,2, Julien Delarue1, Per J. Palsbøll3, Richard Sears1, Philip S. Hammond2 1 Mingan Island Cetacean Study, St. Lambert, Quebec, Canada, 2 Sea Mammal Research Unit, Scottish Oceans Institute, University of St. Andrews, St. Andrews, United Kingdom, 3 Marine Evolution and Conservation, Centre for Ecological and Evolutionary Studies, University of Groningen, Groningen, The Netherlands 1 Mingan Island Cetacean Study, St. Lambert, Quebec, Canada, 2 Sea Mammal Research Unit, Scottish Oceans Institute, University of St. Andrews, St. Andrews, United Kingdom, 3 Marine Evolution and Conservation, Centre for Ecological and Evolutionary Studies, University of Groningen, Groningen, The Netherlands * car@rorqual.com * car@rorqual.com OPEN ACCESS Citation: Ramp C, Delarue J, Palsbøll PJ, Sears R, Hammond PS (2015) Adapting to a Warmer Ocean— Seasonal Shift of Baleen Whale Movements over Three Decades. PLoS ONE 10(3): e0121374. doi:10.1371/journal.pone.0121374 Academic Editor: Elliott Lee Hazen, UC Santa Cruz Department of Ecology and Evolutionary Biology, UNITED STATES Academic Editor: Elliott Lee Hazen, UC Santa Cruz Department of Ecology and Evolutionary Biology, UNITED STATES Received: June 2, 2014 Accepted: February 11, 2015 Published: March 18, 2015 Copyright: © 2015 Ramp et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the Supporting Information Files. Data Availability Statement: All relevant data are within the Supporting Information Files. Funding: The authors have no support or funding to report. Funding: The authors have no support or funding to report. Abstract Global warming poses particular challenges to migratory species, which face changes to the multiple environments occupied during migration. For many species, the timing of migra- tion between summer and winter grounds and also within-season movements are crucial to maximise exploitation of temporarily abundant prey resources in feeding areas, themselves adapting to the warming planet. We investigated the temporal variation in the occurrence of fin (Balaenoptera physalus) and humpback whales (Megaptera novaeangliae) in a North At- lantic summer feeding ground, the Gulf of St. Lawrence (Canada), from 1984 to 2010 using a long-term study of individually identifiable animals. These two sympatric species both shifted their date of arrival at a previously undocumented rate of more than 1day per year earlier over the study period thus maintaining the approximate 2-week difference in arrival of the two species and enabling the maintenance of temporal niche separation. However, the departure date of both species also shifted earlier but at different rates resulting in in- creasing temporal overlap over the study period indicating that this separation may be start- ing to erode. Our analysis revealed that the trend in arrival was strongly related to earlier ice break-up and rising sea surface temperature, likely triggering earlier primary production. The observed changes in phenology in response to ocean warming are a remarkable exam- ple of phenotypic plasticity and may partly explain how baleen whales were able to survive a number of changes in climate over the last several million years. However, it is question- able whether the observed rate of change in timing can be maintained. Substantial modifica- tion to the distribution or annual life cycle of these species might be required to keep up with the ongoing warming of the oceans. Copyright strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of th or copyright holder(s), unless the work is under an open content license (like Creative Commons). The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license. More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne- amendment. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. Take-down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to and investigate your claim. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons the number of authors shown on this cover page is limited to 10 maximum. Download date: 24-10-2024 RESEARCH ARTICLE Introduction Migration occurs in all major branches of the animal kingdom, takes place in the air, at sea and on land, and exists at extreme temporal and spatial scales[1]. It can take many forms ranging Competing Interests: The authors have declared that no competing interests exist. 1 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Seasonal Shift of Whale Movement due to Warmer Ocean from diurnal vertical migration of plankton to the migration of diadromous fish species to and from their spawning sites [2,3]. The main driving force is typically the use of resources such as food, mates or shelter varying over time and space [4].We focus here on the “classic” annually reoccurring seasonal large-scale migration between summer and winter areas [1,5]. Such mi- gratory species may be subject to climate-induced changes in resources at different seasonal and life cycle stages to which they must adapt, or adjust their migratory behaviour [6]. The tim- ing of these changes may differ in direction, severity and speed [7], potentially resulting in a temporal mismatch between resources and migratory cycles [8]. Seasonally migrating species have been shown to change their home ranges in both summer and wintering areas [9,10] or alter their timing in response to changes in their environment [11]. Numerous migratory species utilize high-latitude summer regions to benefit from the temporarily available high productivity and some of them reproduce during that period [6,12,13]. The timing of arrival at such summer foraging areas often corresponds with the oc- currence of one or several prey species on which the adults or their offspring depend [8]. In- creasing spring temperatures have shifted the phenology of plants and insects so that species at higher trophic levels have modified their timing patterns accordingly [14]. However, some avian species have failed to change their arrival timing sufficiently to maintain synchrony with their prey species [15], and similar trophic mismatches have been observed in the marine eco- system [16]. While most migratory species show a unidirectional reaction to warmer tempera- tures in spring, the effect on autumn departure varies. Short-distance migrants tend to depart later in autumn whereas long-distance migrants depart earlier [11,17]. Generally, long-distant migrants seem to adapt less well to climate change than short-distant migrants potentially lead- ing to reduced fitness and population declines. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Introduction Animals cannot predict environmental condi- tions thousands of kilometers away and potential wrong timing of movements causing a mismatch with prey occurrence might reduce feeding success, and thus reproductive success and ultimately survival [17,18]. Most baleen whales undertake seasonal migrations ranging from a few hundreds to thou- sands of kilometers [19–21] alternating between low latitude winter breeding grounds and high latitude summer feeding grounds [22,23]. Baleen whales have existed for several million years [24] and have thus survived several glacial and interglacial periods, including the Dans- gaard-Oeschger cycles, when temperature over Greenland increased 8–15°C in the span of a few decades on multiple occasions during the last 80,000 years [25,26]. These species have life- spans of at least 40 to well over 100 years [27,28] and individual whales thus experience more environmental variation during their life-time than individuals of most other species. The large body size of baleen whales helps buffer individuals against short-term variation in environmen- tal conditions and reduces the relative costs of locomotion [29,30], facilitating long-range sea- sonal migration and extensive movements across the summer range [31,32]. With such advantages, how are baleen whales adapting to global warming? Migratory spe- cies have been described as a ‘paradox’ because their mobility allows them to react even to rapid changes but they also depend on suitable habitat in multiple locations [6]. Predictions for the response of marine mammals range from a more pole-ward distribution and the earlier ar- rival in feeding areas to follow changing prey distribution [33,34] to a longer residency time of some migratory species in higher latitudes in response to enhanced productivity [33]. Several studies suggest that entire ecosystems or communities will move pole-ward following rising SST [35,36]. Populations of fin (Balaenoptera physalus) and humpback whales (Megaptera novaean- gliae), the target species of this study, spend part of the summer in the Gulf of St. Lawrence in the North Atlantic Ocean. These two species are sympatric and feed on a wide, mostly overlap- ping, variety of zooplankton and schooling fish [37]. They exhibit some niche separation in the 2 / 15 Seasonal Shift of Whale Movement due to Warmer Ocean Gulf of St. Lawrence; fin whales arrive earlier and feed, on average, at a lower trophic level and wider niche than humpback whales, which arrive later and feed on higher trophic prey that are more available later in the season [38,39]. Introduction Northwest Atlantic humpback whales breed in the West Indies during winter [40] after traveling between 2,000 and 8,000 kilometers from their summer feeding grounds, which include the Gulf of Maine, eastern Canada, and western Greenland [41,42]. The fin whales found in the summer in the Gulf of St. Lawrence are believed to overwinter off Nova Scotia just outside the pack ice [43], but their winter distribution is poorly known [44]. Indeed, most of their entire range and migration routes are unknown and they are thought to be more pelagic because they are not observed as close to the shore as are humpback whales and do not seem to aggregate in distinct breeding grounds. The best documented effects of recent changes in climate are the warming of the oceans and the reduction in sea ice [45]. The Gulf of St. Lawrence has a subarctic climate with seasonal ice cover in winter and spring, representing the southernmost extent of sea ice in the Northern Hemisphere [46]. Over the last 30 years, sea surface temperature (SST) in the Gulf of St. Lawrence has increased [47] while temporal ice coverage has decreased [48]. We investigat- ed whether, and in which direction, fin and humpback whales have changed their temporal oc- currence in this area in response to the warming climate. We used the timing of the first and last sighting of photographically identified individuals as proxies for the arrival and departure dates of these two species in the main feeding area in the Gulf of St. Lawrence over a period of 27 years. Many individuals were present in the area throughout the study period. Therefore, we not only use these individuals as samples to explore the potential response to climate change of the two populations, but also investigate the behavioural change or adaption of individuals. We also investigate whether changes in the timing of occurrence are linked to changing en- vironmental conditions in the area and, using this case study as a proxy for a feeding ground in general, explore possible future responses and implications for these species, faced with pre- dicted continuing ocean warming. Our analysis reveals that the earlier arrival of fin and hump- back whales in the Gulf of St. Introduction Lawrence by approximately one day per year over 27 years was strongly related to an earlier ice break-up and rising sea surface temperature, indicating a re- markable phenotypic plasticity to a changing environment. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Data The Gulf of St. Lawrence is a semi-enclosed sea that connects the Great Lakes to the North At- lantic and is a summer feeding destination for several species of rorqual whales [49]. The data were collected in the Jacques-Cartier Passage and adjacent waters (roughly 49.5°N to 50.3°N and 63°W to 66°W (Fig. 1)) between 1984 and 2010. A field season lasted between the begin- ning of June and mid/end of October with an average of 65 survey days and ~500 hours of observation. We used standard photo-identification techniques to identify individual humpback and fin whales from their natural markings [50,51]. The primary aim of the study was the estimation of population parameters using mark-recapture techniques and thus we maximised the effort to photo-identify all animals present. The sighting effort remained relatively stable over the study period. The starting date of the seasonal fieldwork remained relatively constant (S1 Table), while the end date depended on the animals; surveying stopped when no animals had been sighted in the study area for approximately two weeks. The study was conducted under annual permits from Department of Fisheries and Oceans, Canada. Between 1984–2010, we identified 450 fin whales, which were sighted on a total of 1,404 oc- casions, and 270 individual humpback whales sighted 1,075 times in total. Not all juvenile 3 / 15 PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Seasonal Shift of Whale Movement due to Warmer Ocean Fig 1. The Study area. The Gulf of St. Lawrence and eastern Canadian waters with 500m bathymetric line representing the shelf edge. The research area is marked as JCP (Jacques Cartier Passage). The other boxes show the extent of the areas for which SST data were used. doi:10.1371/journal.pone.0121374.g001 Fig 1 The Study area The G lf of St La rence and eastern Canadian aters ith 500m bath metric lin Fig 1. The Study area. The Gulf of St. Lawrence and eastern Canadian waters with 500m bathymetric line representing the shelf edge. The research area is marked as JCP (Jacques Cartier Passage). The other boxes show the extent of the areas for which SST data were used. doi:10.1371/journal.pone.0121374.g001 Fig 1. The Study area. The Gulf of St. Lawrence and eastern Canadian waters with 500m bathymetric line representing the shelf edge. The research area is marked as JCP (Jacques Cartier Passage). The other boxes show the extent of the areas for which SST data were used. Data doi:10.1371/journal.pone.0121374.g001 humpback whales migrate to the breeding grounds [52] and might remain closer to the feeding grounds during winter. Therefore, we restricted our analysis to mature animals under the as- sumption that mature animals consistently migrate to the breeding grounds each year. Accord- ingly, we included only sightings of animals when they were known for at least 5 years, the age at which humpback whales are presumed to be sexually mature [53]. This reduced data set con- tained 96 individual humpback whales sighted 677 times in total. For fin whales, it is unknown whether or not all animals migrate but we assumed they do and used all sighting data in our analysis. However, calves (lactating young of the year) of both species were omitted because their occurrence depends on their mothers. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Analysis We used the first and last date in each year (1984–2010) that an individual whale was sighted and identified as proxies for its arrival and departure date, respectively, and the mean first and last sighting dates as estimates of the population mean arrival and departure date, respectively. Individual whales were not necessarily detected when present and true arrival and departure dates of each individual whale in each year were unknown. Mean date of first sighting is thus a potentially biased (late) estimate of population arrival date, mean last sighting date is a poten- tially biased (early) estimate of population departure date and residency times are thus poten- tially underestimated. This bias might be reduced by limiting analysis to data for animals seen multiple times, for example by removing animals seen only once in a year and thus with identi- cal arrival and departure dates. We conducted analyses with datasets reduced in this way and PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 4 / 15 Seasonal Shift of Whale Movement due to Warmer Ocean obtained similar results to the simple regressions (S2 Table). We therefore used all data to max- imise sample size and to avoid the risk of introducing unknown bias by setting a threshold for the amount of data available for individuals. Mean first and last sighting dates were each regressed on year to estimate the annual rates of change. These dates for the same individual in different years could potentially be viewed as re- peated measures, thus violating assumptions of independence when estimating trends over time. To test for bias, we randomly selected one observation per individual from the total data- set, calculated the annual mean first sighting day for that reduced dataset, regressed mean first sighting date against year, and repeated this procedure 1,000 times. We then compared the mean slope and 95% confidence interval (CI) of the reduced resampled data set with the regres- sion estimated using the full dataset. The difference in slope between the full and the reduced resampled data sets was negligible and the slopes of the linear models using all data fell within the 95% confidence interval of the replicated data sets (S1 and S2 Figs.). Thus we based our analyses on the full data set. We repeated this bootstrap procedure for the last sighting (depar- ture) date (S3 and S4 Figs.), which gave similar results. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Analysis PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 5 / 15 Seasonal Shift of Whale Movement due to Warmer Ocean Data Center and GHRSST (http://pathfinder.nodc.noaa.gov). The PFV5.2 data are an updated version of the Pathfinder Version 5.0 and 5.1 collection described in [54]. The North Atlantic Oscillation (NAO) monthly anomalies were downloaded directed from NOAA:ftp://ftp.cpc. ncep.noaa.gov/wd52dg/data/indices/tele_index.nh. GSL6 is a region along the Quebec North Shore, covering our study area [55]. SST was only available from 1985 to 2009 and therefore we used the same time frame for all other variables. Data Center and GHRSST (http://pathfinder.nodc.noaa.gov). The PFV5.2 data are an updated version of the Pathfinder Version 5.0 and 5.1 collection described in [54]. The North Atlantic Data Center and GHRSST (http://pathfinder.nodc.noaa.gov). The PFV5.2 data are an updated version of the Pathfinder Version 5.0 and 5.1 collection described in [54]. The North Atlantic Oscillation (NAO) monthly anomalies were downloaded directed from NOAA:ftp://ftp.cpc. ncep.noaa.gov/wd52dg/data/indices/tele_index.nh. GSL6 is a region along the Quebec North Shore, covering our study area [55]. SST was only available from 1985 to 2009 and therefore we used the same time frame for all other variables. We also wanted to test the hypothesis that time of arrival is also influenced by the condi- tions of the area the animals are leaving. The winter and spring distribution of fin whales is not well known, but some reports suggest that they spend the winter just outside the Gulf of St. Lawrence [43] and large whales are reported on Grand Banks in spring [56]. Therefore, we also included as candidate covariates SST data from several regions outside the Gulf (Fig. 1 and Table 1). There are time lags of several weeks between the ice break up, rising temperatures, start and peak of primary production and the occurrence of prey species. Therefore we investi- gated monthly means of SST of 1 to 9 months prior to the earliest arrival (October to June). SST data were not available for 1984 and 2010. Thus, we restricted this analysis to 1985 to 2009, for which data were available for all covariates. We used simple least squares regression and examined the variance explained in models for each covariate alone. Monthly mean SST is not independent within regions and seasons, nor like- ly to be between regions, so we included only a single SST variable in each model. Analysis Similarly, ice breakup, duration and coverage are also highly correlated and we used only a single ice-related covariate in each model. Although ice breakup, coverage and duration are also correlated with sea surface temperature and all of these are correlated with climate variables such as the NAO index, we decided to test two variables at a time and to choose which of the SST and ice-related variables to include based on significance tests and variance explained by the models. Model selection was based on Akaike’s Information Criterion [57]. All statistical analysis was undertaken in R [58]. Analysis As an alternative way of addressing this issue, we also fitted mixed effects models, which gave very similar results to the simple regres- sions (S2 Table). We subsequently developed a set of linear regression models with annual mean first sighting date as the response variable to investigate the explanatory power of a range of covariates (Table 1) [54,55]. The covariates included several measures of research effort to test that the trend was not influenced by effort (S1 Table). The environmental covariates included the large- scale North Atlantic Oscillation (NAO) index, sea surface temperatures (SST), and measures of ice coverage in the Gulf of St. Lawrence. Timing of arrival of whales in the St. Lawrence is as- sumed to depend on the availability of prey. Prey data were unavailable for our research area and chlorophyll concentration was available only for the later years of the study so we used the ice and SST covariates as a proxy for the onset of annual primary productivity [46]. Sea ice data were obtained from Environment Canada Ice Services (http://ice-glaces.ec.gc. ca/). We obtained mean and maximum monthly Sea Surface Temperatures (SST) using Sea ice data were obtained from Environment Canada Ice Services (http://ice-glaces.ec.gc. ca/). We obtained mean and maximum monthly Sea Surface Temperatures (SST) using AVHRR Pathfinder Version 5.2 (PFV5.2) data, obtained from the US National Oceanographic Table 1. Covariates used in the linear regression modeling. Covariate Data period Comment Effort: First survey day of the year 1985–2009 Number of survey days in June 1985–2009 Number of survey days in July 1985–2009 Number of survey days in June+July 1985–2009 Sea ice data First week ice free 1985–2009 < 1% ice coverage No. of weeks with ice 1985–2009 > 1% ice coverage Week number with maximum ice coverage 1985–2009 Climate data NAO monthly anomalies 1985–2009 9 monthly means prior arrival Mean and Maximum monthly SST 1985–2009 SST in GSL6 [55] 1985–2009 9 monthly means prior arrival SST in Cabot Strait (CB) (Fig. 1) 1985–2009 9 monthly means prior arrival SST in Grand Banks (GB) (Fig. 1) 1985–2009 9 monthly means prior arrival SST in Scotian Shelf (Fig. 1) 1985–2009 9 monthly means prior arrival doi:10.1371/journal.pone.0121374.t001 Table 1. Covariates used in the linear regression modeling. Table 1. Covariates used in the linear regression modeling. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Results The mean first sighting date of fin whales shifted significantly earlier over the study period (slope = -1.062, SE = 0.093, p<0.001), from day 229 (17 August) in 1984 to day 201 (20 July) in 2010 (Fig. 2), thus one month earlier over 27 years. The mean first sighting date of mature humpback whales (Fig. 2) also shifted significantly earlier (slope = -1.201, SE = 0.135, p <0.001); from day 245 (2 September) in 1987, to day 217 (5 August) in 2010 (Fig. 2). Plots of the standardized residuals of the models showed no departure from normality nor evidence for temporal correlation (S5–S8 Figs.). The mean last sighting date of fin whales also moved earlier in time (slope = -0.435, SE = 0.09, p<0.001) from day 233 (21 August) to day 222 (10 August) but the change was not as great as for first sighting date (Fig. 2). Thus fin whales extended the period between first and last sighting date by an average of 16 days across the study period. The mean day of the last sighting for mature humpback whales also changed over the study period (slope = -1.196, SE = 0.136, p<0.001) from day 269 (26 September) to day 242 (30 August), resulting in a con- stant average period between first and last sighting during the 27-year study period. In the regression models for fin whales (Table 2) many covariates were significant but they accounted for only a small fraction of the variance in date of first sighting. No effort variable was significant. We tested multiple combinations of significant covariates but only two im- proved the model fit. The best-supported model (adjusted r2 = 0.51) included the first ice-free week in the Gulf of St Lawrence (slope = 0.32, SE = 0.11, p = 0.013, Fig. 3) and sea surface tem- perature (SST) in Cabot Strait in January (slope = -8.5, SE = 2.6, p = 0.003). NAO was PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 6 / 15 Seasonal Shift of Whale Movement due to Warmer Ocean Fig 2. Mean arrival and departure date of fin (Bp) and humpback whales (Mn). The difference between trends in arrival and departure represent average measured residency time. Fig 2. Mean arrival and departure date of fin (Bp) and humpback whales (Mn). The difference between trends in arrival and departure represent average Fig 2. doi:10.1371/journal.pone.0121374.t002 Selection of models for ﬁn whale arrival (1985–2009) ordered by AICc. r2 values given only for models in which the covariates was signiﬁcant. ns stands for non-signiﬁcant covariates Results Mean arrival and departure date of fin (Bp) and humpback whales (Mn). The difference between trends in arrival and departure represent average measured residency time doi:10.1371/journal.pone.0121374.g002 significant as a single variable but not in combination with any other covariate. For mature humpback whales, many of the covariates were also significant in the models and explained some variation in the first sighting date (Table 3). No effort variable was significant. The best- supported model (adjusted r2 = 0.47) included a single covariate, the SST at the research site in January (slope = -33.66, SE = 7.45, p = 0.001). No combination of additional covariates im- proved the overall model fit. Table 2. Model results Fin whales. model intercept slope r2 AICc ΔAICc AICc weight ice free + SST.CB.JAN 175.9 0.328 / -8.52 0.513 187.9 0 0.792 SST.CB.JAN 215.8 -10.97 0.377 192.3 4.39 0.088 SST.GS6L.OCT 259.4 -6.705 0.345 193.6 5.66 0.047 ice free + SST.GSL6.OCT 214.3 0.248 / -4.38 0.372 194.3 6.35 0.033 ice free 159.7 0.464 0.31 194.9 6.96 0.024 No weeks ice 167.3 2.502 0.22 197.7 9.77 0.006 SST.GSL6.JAN 192.7 -19.79 0.202 198.5 10.54 0.004 NAO March 213.1 6.246 0.173 199.4 11.5 0.003 Week max ice 250.4 -0.577 0.166 199.6 11.71 0.002 Days effort July 236 -1.106 ns 203.5 15.53 0 Total effort J/J 225.9 -0.358 ns 204.3 16.38 0 First day effort 176 0.245 ns 204.4 16.46 0 Days effort July 218.7 -0.271 ns 205 17.04 0 Selection of models for ﬁn whale arrival (1985–2009) ordered by AICc. r2 values given only for models in which the covariates was signiﬁcant. ns stands for non-signiﬁcant covariates Table 2. Model results Fin whales. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 7 / 15 Seasonal Shift of Whale Movement due to Warmer Ocean Fig 3. Mean annual arrival date of fin whales (Bp) and the first week the Gulf of St. Lawrence was ice-free. d i 10 1371/j l 0121374 003 Fig 3. Mean annual arrival date of fin whales (Bp) and the first week the Gulf of St. Lawrence was ice-free. d i 10 1371/j l 0121374 003 Fig 3. Mean annual arrival date of fin whales (Bp) and the first week the Gulf of St. Lawrence was ice-free. doi:10.1371/journal.pone.0121374.g003 doi:10.1371/journal.pone.0121374.g003 Selection of models for mature humpback whale arrival ordered by AICc. r2 values given only for models in which the covariate was signiﬁcant. ns stands for non-signiﬁcant covariates. Only the covariates of the ﬁrst ﬁve models were signiﬁcant. No combination with two covariates yielded signiﬁcant results. d i 10 1371/j l 0121374 t003 Selection of models for mature humpback whale arrival ordered by AICc. r2 values given only for models in which the covariate was signiﬁcant. ns stands for non-signiﬁcant covariates. Only the covariates of the ﬁrst ﬁve models were signiﬁcant. No combination with two covariates yielded signiﬁcant results. doi:10.1371/journal.pone.0121374.t003 Seasonal Shift of Whale Movement due to Warmer Ocean other species [11,17]. Humpback whale departure shifted at the same pace as arrival, thus keep- ing the residency time almost constant. The trend towards an earlier departure date of fin whales was less pronounced than for the arrival date, increasing the residency time, but that increase is subject to small sample bias in the first two years. Thus, there is only weak evidence that fin whales increased their residency time in the study area, and none for humpback whales. The Jacques Cartier Passage is an important summer feeding area for both species, with many individuals returning every year, as indicated by high recapture rates [59,60], but it rep- resents only a fraction of the potential summer range for both populations. Individuals of both species thus likely spent only a part of the summer in our study area and the residence times calculated here do not represent their entire feeding season. Fin whales are thought to spend the winter outside the Gulf of St. Lawrence and move in with the retreating ice [43] and then follow the plankton bloom further north [61], but their winter distribution is unknown. For humpback whales, we know when they depart from the breeding grounds [62] and it is clear that the arrival date calculated here does not represent the arrival direct from the wintering grounds. They arrive in the feeding latitudes already in May and June and stay until October or even later [62]. However, whether we consider the classic migration from the winter grounds or the within season movements between different summer feeding areas, both species showed the same behavioural adaptation and advanced their temporal occurrence in the Jacques Car- tier Passage by one month. The observed change in phenology of these populations is based on the observation of many re-sighted whales over the study period, showing remarkable adaptation of the individual ani- mals to changing conditions and highlighting the phenotypic plasticity of these species. We ob- served many of the same individuals at the beginning and the end of the study period, thus there is no evidence that animals left the study area and moved pole-ward, as some studies have predicted [33]. However, our results are limited by the size of the study area and animals could have probed further north before or after their occurrence in the study area. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Discussion Inferring from the results for mean first sighting date, fin and humpback whales arrived earlier in the study area over the 27 years of the study, in line with general predictions [34]. However, the rate of change of >1day earlier per year is, to our knowledge, undocumented. Both species also left the study area earlier, as inferred from mean last sighting date, as observed in many Table 3. Model Results Humpback whales. model intercept slope r2 AICc ΔAICc AICc weight SST.GSL6.JAN 192.6 -33.67 0.47 178.3 0 0.76 SST.GB.JAN 253.5 -6.50 0.27 180.9 2.62 0.205 SST.CB.JAN 231.7 -9.94 0.2 185.7 7.46 0.018 NAO.OCT 234.4 6.51 0.15 187.8 9.54 0.006 SST.GSL.DEC 238 -10.01 0.09 189.1 10.86 0.003 First day effort 336.1 -0.65 ns 190.6 12.37 0.002 SST.CB.DEC 243.2 -4.50 ns 191.2 12.92 0.001 No weeks ice 197.4 1.74 ns 191.4 13.18 0.001 First week ice free 200.9 0.25 ns 191.5 13.29 0.001 Days effort June 223.1 0.78 ns 192.2 13.99 0.001 Week max ice 216.8 -0.20 ns 192.8 14.57 0.001 Total effort J/J 243.3 0.05 ns 193.3 15.01 0 Days effort July 230.4 0.04 ns 193.6 15.33 0 Table 3. Model Results Humpback whales. Table 3. Model Results Humpback whales. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 8 / 15 Seasonal Shift of Whale Movement due to Warmer Ocean Humpback whales arrive in the Gulf of St Lawrence about 2 weeks later than fin whales. Analysis of the isotopic niche in this area between 1992 and 2010 revealed that humpback whales fed at a higher trophic level compared to fin whales [38] implying that the difference in timing of arrival may reflect a difference in prey preference. A possible explanation for the ob- served temporal separation between fin and humpback whales is niche partitioning to reduce competition. Our results show that the temporal separation between these two species has so far largely been maintained despite the shift towards earlier arrival times. However, our results also show that this temporal separation may have started to erode due to the weak evidence for longer residency times of fin whales. Short-distance migrants seem to adapt better to climate change than long-distant migrants [17,18]. Some Northern Hemisphere bird species have shortened their migration distance by establishing wintering grounds further north [10,63], while other bird species have ceased to migrate altogether [64]. For baleen whales, gray whale (Eschrichtius robustus) calls have been recorded in the winter off Alaska, indicating that some individuals may have ceased to migrate annually [65]. While bird species breed and feed at high latitudes during the summer, most ba- leen whales feed only during the summer. When sea ice covers the Gulf of St. Lawrence, fin whales are assumed to winter only a few hundred kilometers away off the coast of Nova Scotia [43]. As the winter sea ice coverage decreases in the Gulf of St. Lawrence, so may the need for fin whales to migrate. A subpopulation of fin whales in the Mediterranean Sea that is distinct from conspecifics in the North Atlantic does not migrate long distances but feeds during the winter and may reproduce year-round [66–68]. If the patterns described here for fin whales in the Gulf of St. Lawrence continue, and noting that they show flexibility regarding where they give birth [23], it is tempting to speculate that continuing warming could lead to a discrete year-round population of fin whales in the Gulf of St Lawrence if parts of it become ice-free in winter. There is, to our knowledge, no indication that humpback whales have shifted their timing of arrival at or departure from their breeding grounds in the West Indies. PLOS ONE \| DOI:10.1371/journal.pone.0121374 March 18, 2015 Fin whale arrival in the Gulf of St. Lawrence followed the shift in the date of the ice break up, as first suggested many years ago [43]. The influence of SST inside Cabot Strait (Fig. 1) is likely related to the spatial and temporal ice coverage and could serve as a signal to the whales that it is time to move back into the Gulf of St. Lawrence. There was a time lag of 13–15 weeks between when this area became totally ice-free and the arrival of the fin whales in the Jacques Cartier Passage. This period is similar to the time lag estimated in the Azores, where fin and humpback whales appear to arrive 15 weeks after the onset of the spring bloom to feed on eu- phausiid species when en route to high latitude summer feeding grounds [61]. For humpback whales, we assume that the influence of SST in January in the Gulf of St. Lawrence on arrival date must constitute a proxy for larger scale environmental variation because at this time humpback whales are in the West Indies on the breeding grounds approximately 4,000 kilome- ters south of the Gulf of St. Lawrence. However, the only large-scale climate variable included in our models, the North Atlantic Oscillation index, did not correlate well with the observed change in arrival date. It is unknown where humpback whales are located between their arrival in higher latitudes from the breeding grounds and their arrival in the Jacques Cartier Passage but environmental changes may have triggered an earlier departure from this unknown loca- tion and thus earlier arrival in the Gulf of St. Lawrence. Fin and humpback whales are generalist feeders and the arrival of the whales in the Gulf of St Lawrence is related to the temporal and spatial distribution of the arrival of their prey. The start of the spring phytoplankton bloom depends on temperature and light conditions [16] and earlier ice break-up coupled with higher SST leads to a progressively earlier bloom followed by the earlier growth of populations of primary and then secondary consumers. Thus, the earlier arrival of fin and humpback whales enables timely feeding on these prey species. 9 / 15 The results presented here indicate, rather, an earlier inshore movement within northern latitudes. Humpback whale feeding aggregations throughout the North Atlantic show a wide variation in migration dis- tances [41]. Those populations feeding at temperate latitudes may simply extend their annual migration further north as their prey retreat northbound in response to elevated SST [69], as several studies have predicted, thus increasing interspecific competition with northern species [70,71]. The continuing rise in ocean temperatures could eventually cause problems for long distance migrating humpback whales to time their arrival in the feeding grounds with the oc- currence of their main prey. Extreme changes, such as shown in this study, and pressure to adapt further to accommodate ongoing rising SST are likely to affect population dynamics as shown for other marine migrants [72,73] and future studies should test for effects of climate change on population dynamics and health. Fin and humpback whales in the Gulf of St. Lawrence have shifted their phenology at a pre- viously undocumented pace over the last 27 years. Both species have adapted their seasonal movement to the shift in productivity in one of their prime feeding grounds in the North At- lantic. Whether this pattern can continue as ocean temperatures increase is an open question and the implications for these two species in the region are uncertain but could be profound. The phenotypic plasticity of these long-lived marine predators shown in our analysis is notable and may explain how they have coped with past fluctuations in climate. However, it remains questionable for how much longer they can adapt to further rapid environmental change. Sub- stantial modification to their distribution or annual life cycle might be required to keep up with the continuing warming of the oceans, and the implications might be more severe for the humpback whale with more distant breeding grounds. 10 / 15 Seasonal Shift of Whale Movement due to Warmer Ocean Supporting Information S1 Fig. Arrival date of individual fin whales. All first sightings with the linear trend (red line, slope = -1.062, SE = 0.093) laying within the upper and lower 95% confidence intervals (dashed black lines) of the resampled data. Annual trend of resampled data as black line (bootstrapped data slope = -1.097, SE = 0.006). (PDF) S1 Fig. Arrival date of individual fin whales. All first sightings with the linear trend (red line, slope = -1.062, SE = 0.093) laying within the upper and lower 95% confidence intervals (dashed black lines) of the resampled data. Annual trend of resampled data as black line (bootstrapped data slope = -1.097, SE = 0.006). (PDF) S2 Fig. Arrival date of individual (mature) humpback whales. All first sightings with the lin- ear trend (red line, slope = -1.201, SE = 0.135) laying within the upper and lower 95% confi- dence intervals (dashed line) of the resampled data. Annual trend of resampled data as black line (bootstrapped data slope = -1.041, SE = 0.016). (PDF) S3 Fig. Departure date of individual fin whales. All first sightings with the linear trend (red line) laying within the upper and lower 95% confidence intervals (dashed black lines) of the resampled data. (PDF) S4 Fig. Departure date of individual humpback whales. All first sightings with the linear trend (red line) laying within the upper and lower 95% confidence intervals (dashed line) of the resampled data. (PDF) S5 Fig. Standardized residuals over standardized predicted values of the linear model (arri- val~year) for the fin whale data set. (PDF) S6 Fig. Histogram of standardized residuals of the linear model (arrival~year) with theoret- ical normal distribution of the fin whale data set. (PDF) S7 Fig. Standardized residuals over standardized predicted values of the linear model (arri- val~year) for the mature humpback whale data set. (PDF) S8 Fig. Histogram of standardized residuals of the linear model (arrival~year) with theoret- ical normal distribution for the humpback whale data set. (PDF) S1 File. Raw data. Arrival and Departure Dates for all Individuals. (PDF) S1 Table. Dataset. Mean arrival and departure date for fin and (mature) humpback whales and the effort covariates according to Table 1 (No of survey days in June, July, June and July, and the first survey day of the year) (DOCX) S2 Table. Model Results. References 1. Dingle H, Drake VA. What Is Migration? BioScience. 2007; 57: 113–121. 1. Dingle H, Drake VA. What Is Migration? BioScience. 2007; 57: 113–121. 2. Meyers GS. 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https://openalex.org/W3110417735	https://link.springer.com/content/pdf/10.1007/s12268-020-1484-1.pdf	German	null	Terrestrische Cyanobakterien als Quelle für antimikrobielle Wirkstoffe	BIOspektrum	2,020	cc-by	2,575	Besonderheit terrestrischer Cyanobakterien MARCO WITTHOHN1, ANNA SCHWARZ1, DORINA STRIETH2, SELINA LENZ2, ROLAND ULBER2, KAI MUFFLER1 1 FACHBEREICH 1 – LIFE SCIENCES AND ENGINEERING, TH BINGEN 2 LEHRGEBIET BIOVERFAHRENSTECHNIK, TU KAISERSLAUTERN MARCO WITTHOHN1, ANNA SCHWARZ1, DORINA STRIETH2, SELINA LENZ2, ROLAND ULBER2, KAI MUFFLER1 1 FACHBEREICH 1 – LIFE SCIENCES AND ENGINEERING, TH BINGEN 2 LEHRGEBIET BIOVERFAHRENSTECHNIK, TU KAISERSLAUTERN Bisher wurden vor allem aquatische Cyano- bakterien als reiche Quelle für bioaktive Naturstoffe beschrieben. Doch auch terrestri- sche Cyanobakterien produzieren eine Viel- zahl medizinisch und biotechnologisch inte- ressanter Substanzen. Innerhalb der Ordnun- gen Chroococcales, Oscillatoriales, Pseudo- anabaenales und Nostocales kommen neben aquatischen auch terrestrisch lebende Cyanobakterien vor [4]. Es ist bereits länger bekannt, das s Arten der Ordnungen Chroo- coccales, Oscillatoriales und Nostocales die meisten bioaktiven Sekundärmetabolite pro- duzieren [3]. Darunter befinden sich vor allem Tox ine, antivirale, antimykotische und antibakterielle Substanzen, aber auch Stoffe mit nachgewiesener Wirkung gegen Krebs- zellen, höhere Pﬂ anzen sowie Algen. Neben einem breiten Spektrum an potenziell inter- essanten bioaktiven Sekundärmetaboliten haben terrestrische Cyanobakterien noch einige biotechnologische Vorteile gegenüber den aquatischen Arten zu bieten. Trotz ihrer natürlichen Lebensweise an Land können die meisten terrestrischen Cyanobakterien sub- mers in (Photo-) Bioreaktoren kultiviert wer- den. Dabei weisen sie eine hohe Toleranz gegenüber schwankenden Kultivierungsbe- dingungen auf, wie Temperatur, Lichteintrag und Begasung [4]. Selbst eine komplette Aus- trocknung überste hen diese Organismen unbeschadet, was ganz neue Möglichkeiten im Bereich der Bioreaktortechnik ermöglicht [5]. Terrestrische Cyanobakterien sind also zum ei nen eine vielversprechende Quelle noch unbekannter bioaktiver Substanzen und besitzen zum anderen einzigartige phy- siologische Eigenschaften, die sie höchst interessant für biotechnologische Prozesse machen. Dieses Potenzial soll im Forschungs- cluster iProcess weiter ausgeschöpft werden. Cyanobacteria developed an enormous reservoir of bioactive secondary metabolites in order to prevail against competitive microorganisms and harsh environmental impacts. Many cyanobacterial substances with vast economical, medical and biotechnological potential have been described in the past. However, most of the examined bacteria are aquatic strains. We want to take a closer look on their terrestrial rela- tives which also possess a rich secondary metabolome that is still to explore. DOI: 10.1007/s12268-020-1484-1 © Die Autoren 2020 DOI: 10.1007/s12268-020-1484-1 © Die Autoren 2020 DOI: 10.1007/s12268-020-1484-1 © Die Autoren 2020 unterschiedliche Synthesewege für Energie- speicher entwickelt, was in einer großen Fülle unterschiedlicher Speicherstoffe resul- tiert. Darunter beﬁ nden sich vor allem lang- kettige ungesättigte Fettsäuren, wie z. B. Gamma-Linolensäure, Arachidonsäure, oder Docosahexaensäure, die aufgrund ihrer gesundheitsfördernden Wirkung ein gefrag- ter Nahrungsmittelzusatz sind [3]. 794 BIOTECHNOLOGIE 794 BIOTECHNOLOGIE Besonderheit terrestrischer Cyanobakterien Des Wei- teren produz ieren Cyanobakterien eine Viel- zahl biotechnologisch relevanter Substanzen, wie z. B. Lipide, Polysaccharide, Aminosäu- ren, Vitamine, Sterole und Enzyme. Unter den von Cyanobakterien produzierten Natur- stoffen beﬁ nden sich häuﬁ g Toxine, wie von den alljährlich auftretenden „Cyanobakteri- en-Blüten“ in heimischen Badeseen bekannt ist. Das von Anabaena produzierte Anatoxin A sowie von Microcystis gebildete Microcys- tine gehören zu den stärksten natürlich vor- kommenden Giften und können auch bei Menschen schwere gesundheitliche Schäden zur Folge haben. Weitaus nützlicher sind die vielzähligen cyanobakteriellen Sekundärme- tabolite, bei denen unter anderem antimikro- bielle oder antitumorale Wirkungen nachge- wiesen werden konnten. Interessant ist dabei vor allem die Diversität der unterschiedlichen Substanzklassen. Diese reichen über Peptide, Polyketide, Alkaloide und Isoprenoide, bis hin zu antiviralen Polysacchariden. ó Cyanobakterien gehören zu den ältesten Organismen unseres Planeten, fossilen Fun- den zufolge entstanden sie vor etwa 3,5 Mil- liarden Jahren [1] . Als oxygen phototrophe Organismen haben sie einen großen Beitrag zur Entstehung der sauerstoffreichen Erdat- mosphäre geleistet. Somit können Cyanobak- terien als Motor für diesen folgenschweren und richtungsweisenden Evolutionsprozess angesehen werden. Hochwertige Syntheseprodukte Cyanobakterien sind sehr anpassungsfähige und robuste Bakterien, die in fast allen Lebensräumen vorkommen. Isolate wurden in Salz- und Süßwasser, Wüsten, Gletschern und heißen Quellen gefunden. Als Folge dieser Anpassungen produzieren Cyanobak- terien eine Vielzahl hochwertiger Substan- zen mit enormem kommerziellen Potenzial. Cyanobakterien sind im Gegensatz zu Pﬂ an- zen dazu in der Lage, Licht der Wellenlänge von 490–620 Nanometer photosynthetisch zu nutzen. Dafür haben sich spezielle Chro- mophore entwickelt, die Phycobiline. Auch verschiedene Carotinoide tragen zur Erwei- terung des nutzbaren Lichtspektrums in Cyanobakterien bei. Beide Stoffklassen wer- den seit einiger Zeit erfolgreich in der Lebensmittelindustrie eingesetzt [2]. Gleich- zeit ig haben sich bei Cyanobakterien viele iProcess: intelligente Prozess- entwicklung – von der Modellierung bis zum Produkt iProcess: intelligente Prozess- entwicklung – von der Modellierung bis zum Produkt Das Land Rheinland-Pfalz intensiviert seit 2018 über das Instrument der Forschungs- BIOspektrum \| 07.20 \| 26. Jahrgang BIOspektrum \| 07.20 \| 26. Jahrgang 795 ein relativ breites Wirkspektrum abgedeckt werden. ˚ Abb. 1: Erreichte Maximal-OD595nm der Indikatororganismen Candida auris, Escherichia coli und Micrococcus luteus nach Zugabe von Kulturüberständen der terrestrischen Cyanobakterien Nostoc ﬂ agelliforme, Synechococcus elongatus, Nostoc punctiforme und Scytonema stuposum (1:1). Alle Cyanobakterien wurden als Flüssigkultur phototroph (30 μmol m–2 s–1) bei 27 °C und 120 rpm in BG-11-Medium kultiviert. Proben wurden nach 2, 5 und 7 Tagen entnommen. Dar gestellt sind die Daten der Proben mit höchster Inhibitionswirkung. Rot: Negativkontrolle mit BG-11-Me- dium. Fehlerbalken zeigen Standardabweichung, n = 4. Die Kulturüberstände von S. elongatus, N. punctiforme und S. stuposum zeigen die deutlichsten Hemmwirkungen. Das Wachs- tum der Hefe C. auris wurde durch ins Me dium sekretierte Substanzen von S. elon- gatus um fast 50 Prozent inhibiert, jenes der Bakterien besonders durch Kulturüberstände von N. punctiforme und S. stuposum. Dabei hemmten von N. punctiforme gebildete Stoffe vor allem das Gram-positive Bakterium M. luteus. Kulturüberstände von S. stuposum wirkten sich auf das Wachstum beider Bak- terien negativ aus. Die antiSMASH-Analyse konnte einige putativ von den ausgewählten terrestrischen Cyanobakterien produzierte Substanzen auf- zeigen, welche die in Abbildung 1 gezeigten Hemmwirkungen auslösen könnten (Tab. 1). In den Cyanobakterien-Genomen wurden potenzielle Biosynthesecluster für Substan- zen gefunden, die eine antibiotische Wirkung aufweisen könnten. Dabei ist es ebenfalls möglich, dass als cytotoxisch eingestufte Stoffe bisher noch nicht auf ihre Wirksam- keit gegen Mikroorganismen getestet wur- den und demnach nicht als Antibiotika beschrieben sind. Für viele von antiSMASH gefundene Cluster konnte kein putatives Genprodukt gefunden werden, demnach kön- nen die Metabolome der untersuchten Cyano- bakterien noch eine Vielzahl unentdeckter Substanzen unterschiedlicher Stoffklassen und Bioaktivität enthalten Die antiSMASH-Analyse konnte einige putativ von den ausgewählten terrestrischen Cyanobakterien produzierte Substanzen auf- zeigen, welche die in Abbildung 1 gezeigten Hemmwirkungen auslösen könnten (Tab. 1). ˚ Abb. 1: Erreichte Maximal-OD595nm der Indikatororganismen Candida auris, Escherichia coli und Micrococcus luteus nach Zugabe von Kulturüberständen der terrestrischen Cyanobakterien Nostoc ﬂ agelliforme, Synechococcus elongatus, Nostoc punctiforme und Scytonema stuposum (1:1). Alle Cyanobakterien wurden als Flüssigkultur phototroph (30 μmol m–2 s–1) bei 27 °C und 120 rpm in BG-11-Medium kultiviert. Proben wurden nach 2, 5 und 7 Tagen entnommen. Dar gestellt sind die Daten der Proben mit höchster Inhibitionswirkung. Rot: Negativkontrolle mit BG-11-Me- dium. Fehlerbalken zeigen Standardabweichung, n = 4. Screening ausgewählter g p g den durch die Ähnlichkeit einzelner Gene innerhalb der cyanobakteriellen Genom-Sequenz zu Genen bereits bekannter Biosynthesecluster in anderen Organismen ermittelt. NRPS: nicht-ribosomale Peptid- synthetase; PKS: Polyketidsynthase. Organismus Biosynthesecluster Putatives Genprodukt Übereinstimmung [%] Stoffklasse Bioaktivität N. ﬂ agelliforme NRPS/PKS Nostophycin (27) Cycl. Peptid cytotoxisch [7] PKS Minutissamid A/C/D (46) Cycl. Depsipeptid antiproliferativ, cytotoxisch [8] N. punctiforme NRPS-like/PKS Merocyclophane C/D (33) Polyketid cytotoxisch [9] NRPS/PKS/Bacteriocin Nostophycin (27) Cycl. Peptid cytotoxisch [7] PKS Minutissamid A/C/D (46) Cycl. Depsipeptid antiproliferativ, cytotoxisch [8] NRPS/PKS Cyanopeptolin (75) Peptid cytotoxisch [10] BIOspektrum \| 07.20 \| 26. Jahrgang iProcess: intelligente Prozess- entwicklung – von der Modellierung bis zum Produkt In den Cyanobakterien-Genomen wurden potenzielle Biosynthesecluster für Substan- zen gefunden, die eine antibiotische Wirkung aufweisen könnten. Dabei ist es ebenfalls möglich, dass als cytotoxisch eingestufte Stoffe bisher noch nicht auf ihre Wirksam- keit gegen Mikroorganismen getestet wur- den und demnach nicht als Antibiotika beschrieben sind. Für viele von antiSMASH gefundene Cluster konnte kein putatives Genprodukt gefunden werden, demnach kön- nen die Metabolome der untersuchten Cyano- bakterien noch eine Vielzahl unentdeckter Substanzen unterschiedlicher Stoffklassen und Bioaktivität enthalten. biotische Wirkung getestet und vorhandene Genomsequenzen mittels der Biosynthese- cluster-Analyse-Software antiSMASH auf Gencluster mit eventuell bioaktiven Genpro- dukten hin überprüft [6]. Die Produktion antimikrobieller Sekundärmetabolite wurde mittels Wac hstumstests in Mikrotiterplatten untersucht, in denen die Indikatororganis- men Escherichia coli, Micrococcus luteus und Candida auris mit dem jeweiligen Cyanobak- terien-Kulturüberstand versetzt wurden (Abb. 1). Durch die Wahl eines Gram-negati- ven und eines Gram-positiven Bakteriums sowie einer Hefe als Testorganismen kann kollegs die wissenschaftliche Zusammen- arbeit von Hochschulen und Universitäten, wobei innerhalb von Verbundprojekten die Durchführung kooperativer Promotionen der wissenschaftlichen Mitarbeiter/innen der beteiligten Hochschulen ermöglicht wird. Zu den ersten geförderten Verbünden zählt das interdisziplinäre Forschungskolleg „iPro- cess“ (https://www.mv.uni-kl.de/iprocess), in dem sich Arbeitsgruppen der HS Trier, der TH Bingen und der TU Kaiserslautern ver- netzt haben, um die verfahrenstechnischen Grundlagen zu erarbeiten, damit Pilze und Cyanobakterien als Produktionsorganismen für pharmazeutisch wirkende Substanzen genutzt werden können. Die gezeigten Ergebnisse deuten bereits an, dass terrestrische Cyanobakterien großes Potenzial für eine ganze Bandbreite wert- voller Sekundärmetabolite bergen, die nicht zuletzt im Kampf gegen Viren und multi- Tab. 1: Ergebnisse der antiSMASH-Analyse, beispielhaft für zwei der genutzten Cyanobakterien- Stämme. Aufgeführt ist eine Auswahl bioaktiver Stoffe mit putativ antibiotischer Wirkung. Diese wur- den durch die Ähnlichkeit einzelner Gene innerhalb der cyanobakteriellen Genom-Sequenz zu Genen bereits bekannter Biosynthesecluster in anderen Organismen ermittelt. NRPS: nicht-ribosomale Peptid- synthetase; PKS: Polyketidsynthase. Organismus Biosynthesecluster Putatives Genprodukt Übereinstimmung [%] Stoffklasse Bioaktivität N. ﬂ agelliforme NRPS/PKS Nostophycin (27) Cycl. Peptid cytotoxisch [7] PKS Minutissamid A/C/D (46) Cycl. Depsipeptid antiproliferativ, cytotoxisch [8] N. punctiforme NRPS-like/PKS Merocyclophane C/D (33) Polyketid cytotoxisch [9] NRPS/PKS/Bacteriocin Nostophycin (27) Cycl. Peptid cytotoxisch [7] PKS Minutissamid A/C/D (46) Cycl. Depsipeptid antiproliferativ, cytotoxisch [8] NRPS/PKS Cyanopeptolin (75) Peptid cytotoxisch [10] Tab. 1: Ergebnisse der antiSMASH-Analyse, beispielhaft für zwei der genutzten Cyanobakterien- Stämme. Aufgeführt ist eine Auswahl bioaktiver Stoffe mit putativ antibiotischer Wirkung. Diese wur- den durch die Ähnlichkeit einzelner Gene innerhalb der cyanobakteriellen Genom-Sequenz zu Genen bereits bekannter Biosynthesecluster in anderen Organismen ermittelt. iProcess: intelligente Prozess- entwicklung – von der Modellierung bis zum Produkt NRPS: nicht-ribosomale Peptid- synthetase; PKS: Polyketidsynthase. terrestrischer Cyanobakterien nach geeigneten Substanzen Ein Teilbereich von iProcess beschäftigt sich mit der Produktion antimikrobieller Wirk- stoffe mittels terrestrischer Cyanobakterien. Dabei soll der gesamte Prozess entwickelt werden – von der Modellierung über den Upstream-Bereich und die Entwicklung geeigneter Reaktoren bis hin zum Down- stream-Prozess. Für die Entwicklung eines Produktionspro- zesses muss zunächst eine geeignete Ziel- substanz gefunden werden. Dafür wurden cyanobakterielle Kulturüberstände auf anti- BIOspektrum \| 07.20 \| 26. Jahrgang BIOTECHNOLOGIE 796 Roland Ulber, Dorina Strieth, Selina Lenz (links) Kai Mufﬂ er, Anna Schwarz, Marco Witthohn (rechts) resistente Keime den entscheidenden Beitrag leisten könnten. [5] Mufﬂ er K, Lakatos M, Schlegel C et al. (2014) Application of bioﬁ lm bioreactors in white biotechnology BT. In: Mufﬂ er K, Ulber R (Hrsg.) Productive bioﬁ lms. Springer International Publishing, Basel, 123–161 [5] Mufﬂ er K, Lakatos M, Schlegel C et al. (2014) Application of bioﬁ lm bioreactors in white biotechnology BT. In: Mufﬂ er K, Ulber R (Hrsg.) Productive bioﬁ lms. Springer International Publishing, Basel, 123–161 [6] Medema MH, Blin K, Cimermancic P et al. (2011) AntiSMASH: rapid identiﬁ cation, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res 39:339–346 [7] Fujii K, Sivonen K, Kashiwagi T et al. (1999) Nostophycin, a novel cyclic peptide from the toxic Cyanobacterium Nostoc. J Org Chem 64:5777–5782 Danksagung Dieses Projekt ist Teil des Forschungs- clusters „iProcess“ und wird ﬁ nanziell vom Land Rheinland-Pfalz unterstützt. Wir dan- ken Prof. Dr. Burkhard Büdel (Abt. Pﬂ anzen- ökologie und Systematik, TU Kaiserslautern) für die freundliche Bereitstellung der Cyano- bakterien. Wir danken ebenfalls der DFG für die ﬁ nanzielle Unterstützung der Projekte MU 2985/3-1 und UL 170/16-1. [8] Kang HS, Krunic A, Shen Q et al. (2011) Minutissamides A-D, antiproliferative cyclic decapeptides from the cultured cyanobacterium Anabaena minutissima. J Nat Prod 74:1597– 1605 [9] May D, Luo S, Krunic A et al. (2015) Isolation and struc- ture elucidation of merocyclophane C from the cultured cyan- bacterium Nostoc sp. (UIC 10110). Planta Med 81:PT12 [10] Faltermann S, Zucchi S, Kohler E et al. (2014) Molecular effects of the cyanobacterial toxin cyanopeptolin (CP1020) occurring in algal blooms: Global transcriptome analysis in zebraﬁ sh embryos. Aquat Toxicol 149:33–39 Open Access: Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenzveröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungs gemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/licenses/by/4.0/deed.de. [1] Farrokh P, Sheikhpour M, Bavandi R (2019) Cyano- bacteria as an eco-friendly resource for biofuel production : A critical review. Biotechnol Prog 35:1–16 [2] Borowitzka MA (1995) Microalgae as sources of pharma- ceuticals and other biologically active compounds. J Appl Phycol 7:3–15 [3] Burja AM, Banaigs B, Abou-Mansour E et al. (2007) Marine cyanobacteria – a proliﬁ c source of natural products. Tetrahedron Lett 57:9347–9377 [4] Lakatos M, Strieth D (2008) Terrestrial microalgae: novel concepts for biotechnology and applications. In: Cánovas FM, Lüttge U, Matyssek R (Hrsg.) Progress in botany. Springer International Publishing, Basel, 269–312 Literatur Literatur [1] Farrokh P, Sheikhpour M, Bavandi R (2019) Cyano- bacteria as an eco-friendly resource for biofuel production : A critical review. Biotechnol Prog 35:1–16 [2] Borowitzka MA (1995) Microalgae as sources of pharma- ceuticals and other biologically active compounds. J Appl Phycol 7:3–15 [3] Burja AM, Banaigs B, Abou-Mansour E et al. (2007) Marine cyanobacteria – a proliﬁ c source of natural products. Tetrahedron Lett 57:9347–9377 [4] Lakatos M, Strieth D (2008) Terrestrial microalgae: novel concepts for biotechnology and applications. In: Cánovas FM, Lüttge U, Matyssek R (Hrsg.) Progress in botany. Springer International Publishing, Basel, 269–312 [1] Farrokh P, Sheikhpour M, Bavandi R (2019) Cyano- bacteria as an eco-friendly resource for biofuel production : A critical review. Biotechnol Prog 35:1–16 [2] Borowitzka MA (1995) Microalgae as sources of pharma- ceuticals and other biologically active compounds. J Appl Phycol 7:3–15 [1] Farrokh P, Sheikhpour M, Bavandi R (2019) Cyano- bacteria as an eco-friendly resource for biofuel production : A critical review. Biotechnol Prog 35:1–16 Funding note: Open Access funding enabled and organized by Projekt DEAL. Roland Ulber, Dorina Strieth, Selina Lenz (links) Kai Mufﬂ er, Anna Schwarz, Marco Witthohn (rechts) Open Access: Dieser Artikel wird unter der Creative Commons Namensnennung 4.0 International Lizenzveröffentlicht, welche die Nutzung, Vervielfältigung, Bearbeitung, Verbreitung und Wiedergabe in jeglichem Medium und Format erlaubt, sofern Sie den/die ursprünglichen Autor(en) und die Quelle ordnungs gemäß nennen, einen Link zur Creative Commons Lizenz beifügen und angeben, ob Änderungen vorgenommen wurden. Die in diesem Artikel enthaltenen Bilder und sonstiges Drittmaterial unterliegen ebenfalls der genannten Creative Commons Lizenz, sofern sich aus der Abbildungslegende nichts anderes ergibt. Sofern das betreffende Material nicht unter der genannten Creative Commons Lizenz steht und die betreffende Handlung nicht nach gesetzlichen Vorschriften erlaubt ist, ist für die oben aufgeführten Weiterverwendungen des Materials die Einwilligung des jeweiligen Rechteinhabers einzuholen. Weitere Details zur Lizenz entnehmen Sie bitte der Lizenzinformation auf http://creativecommons.org/licenses/by/4.0/deed.de. Korrespondenzadresse: Prof. Dr.-Ing. Kai Mufﬂ er Fachbereich 1 – Life Sciences & Engineering Technische Hochschule Bingen D-55411 Bingen am Rhein k.mufﬂ er@th-bingen.de [4] Lakatos M, Strieth D (2008) Terrestrial microalgae: novel concepts for biotechnology and applications. In: Cánovas FM, Lüttge U, Matyssek R (Hrsg.) Progress in botany. Springer International Publishing, Basel, 269–312
https://openalex.org/W1748893942	https://napier-repository.worktribe.com/file/172800/1/The%20future%20internet%3A%20a%20world%20of%20secret%20shares.	English	null	The Future Internet: A World of Secret Shares	Future internet	2,015	cc-by	9,814	m J. Buchanan , David Lanc, Elochukwu Ukwandu, Lu Fan, Gordon Russell and William J. Buchanan , David Lanc, Elochukwu Ukwandu, Lu Fan, Gordon Russell and Owen Lo Centre for Distributed Systems and Security, Edinburgh Napier University, Edinburgh EH10 5DT, UK; E-Mails: david_lanc@payfont.com (D.L.); e.ukwandu@napier.ac.uk (E.U.); l.fan@napier.ac.uk (L.F.); g.russell@napier.ac.uk (G.R.); o.lo@napier.ac.uk (O.L.) * Author to whom correspondence should be addressed; E-Mail: w.buchanan@napier.ac.uk; Tel.: +44-131-455-2759. * Author to whom correspondence should be addressed; E-Mail: w.buchanan@napier.ac.uk; Tel.: +44-131-455-2759. Academic Editors: Steven Furnell and Nathan Clarke Received: 27 April 2015 / Accepted: 12 October 2015 / Published: 24 November 2015 Abstract: The Public Key Infrastructure (PKI) is crumbling, partially due to the lack of a strong understanding of how encryption actually works, but also due to weaknesses in its implementation. This paper outlines an Internet storage technique using secret sharing methods which could be used to overcome the problems inherent with PKI, while supporting new types of architectures incorporating such things as automated failover and break-glass data recovery. The paper outlines a novel architecture: SECRET, which supports a robust cloud-based infrastructure with in-built privacy and failover. In order to understand the performance overhead of SECRET, the paper outlines a range of experiments that investigate the overhead of this and other secret share methods. Keywords: secret shares; PKI; cloud computing future internet ISSN 1999-5903 www.mdpi.com/journal/futureinternet OPEN ACCESS future internet ISSN 1999-5903 www.mdpi.com/journal/futureinternet OPEN ACCESS Future Internet 2015, 7, 445-464; doi:10.3390/fi7040445 Future Internet 2015, 7, 445-464; doi:10.3390/fi7040445 Future Internet 2015, 7 Future Internet 2015, 7 Future Internet 2015, 7 446 encryption keys or the implementation of multi-factor authentication techniques. Additionally, the public cloud still suffers from doubts around large-scale outage risks and other security concerns. One of the major risks in scaling existing systems into the Cloud is the current reliance on PKI (Public Key Infrastructure) to secure information, where there are risks around the possible existence of back-doors, front-doors, and a general lack of true understanding of encryption methods. Many encryption methods use a key pair, such as with Rivest Shamir and Adleman (RSA) algorithm, to protect the symmetric key which is then used to encrypt data into the Cloud. While most common methods are considered free of major vulnerabilities, provided the key size is sufficiently large to make it safe from brute force attacks, it is the loss of the private key which can cause major data loss concerns. Many public cloud-based systems are at risk of major data loss through private key loss (and thus data loss), especially through the growth of APTs (Advanced Persistent Threats), certificate cracking, and insider threats. The Future Internet thus requires in-built data protection, either with a strong protective shell, such as that provided by sticky policies, or with sharing methods which break data into secure fragments, and which have a strong enforcement policy to rebuild the data shares without relying on traditional encryption. 1. Introduction Cloud Computing has seen one of the most radical shifts within Information Technology, and the shift is most apparent both in the move from migrating private networks to virtualized networks, as well as the move from private cloud systems onto public ones. Unfortunately these moves have not really changed the methods of providing security and robustness, and instead often rely on the addition of 2. Founding Principles around Secure Data Storage Protecting data in Cloud-based storage systems is a key challenge [1], and existing architectures often fail to properly control access rights to such data. The insider threat, especially from the likes of a System Administrator, and from unforeseen implementation issues, causes Cloud-based systems to be flawed from many viewpoints. There are thus some key driving forces to ensure that data is protected in future Cloud-based infrastructures, including:  Keyless encryption method. While the methods employed in modern encryption are often theoretically watertight, it is the actual implementation in real-life systems which are flawed. Most systems still rely on digital certificates holding a key-pair, and these certificates are all too easily stolen or compromised through brute force attacks. The Superfish compromise highlights a weakness introduced by the software developers, where a digital certificate with both the public and private key was distributed with the software, and which also had a default password based on the name of the company who developed the software [2].  Self-destruct. Some techniques support the concept of a self-destructing data system. With this, all the stored information and their copies, as well as decryption keys, will become unusable after a user-specified time period and without any user involvement. SeDas [3] causes sensitive information, such as account numbers, passwords and notes to irreversibly self-destruct, without any action on the user’s part. This technique is applicable in a multiple server-based system, admittedly incurring an operational overhead.  Break-glass data recovery. Many systems have strict access control policies for data, but in some circumstances it is important that access to data can be granted in emergency situations. This is typical in life-threatening scenarios, such as in health and social care. The loss of an encryption key can also cause major problems in data recovery, which would hinder a break-glass requirement. Future Internet 2015, 7 447  In-built failover protection. For instance, data which needs redundancy can be replicated and encoded across multiple cloud-based storage systems using an any k-from-n sharing policy. So for example, with a storage system striped across three Cloud storage platforms, a policy of 2-from-3 can support a single Cloud storage systems failure, while still allowing the original data to be recovered. 2. Founding Principles around Secure Data Storage So for example, with a storage system striped across three Cloud storage platforms, This paper looks at a range of techniques for tackling these issues using a secret sharing technique, where the information to be made secret is encoded and distributed across multiple data shares, where each share of the information is stored on a different cloud provider. 3.1. Basic Data Striping and RAID Storage Systems RAID stands for Redundant Array of Independent Disks. It is a well-known method of combining several hard disk drives into one logical unit, so as to address the fault-tolerance and performance limitations of an individual hard disk drive. The key notions behind a RAID storage system are Data Striping and various levels of Redundancy. The striping process takes large data blocks and splits them into blocks of a defined size, such as 4 KB, which are then spread across each of the disks in the array. Thi i th f f t t di k (i t i i ) id RAID stands for Redundant Array of Independent Disks. It is a well-known method of combining several hard disk drives into one logical unit, so as to address the fault-tolerance and performance limitations of an individual hard disk drive. The key notions behind a RAID storage system are Data Striping and various levels of Redundancy. The striping process takes large data blocks and splits them into blocks of a defined size, such as 4 KB, which are then spread across each of the disks in the array. This can increase the performance of a storage system as a n-disk array (i.e., n-way striping) provides n times the read and write speed improvement of a single disk (although in practice the actual performance achieved tends to be less than n times due to control overheads). However, the trade-off is reliability, as the failure of any individual disk would result in the failure of the entire array. Formally, assume that each independent hard disk drive has an identical rate of failure r, then the overall failure rate of an array of n disks is: 1 − (1 − r)n Basic data striping over an array of n-disks without redundancy is referred to as RAID0, which is only suitable to situations where the highest I/O performance is desired, whilst the resulting increased probability of data loss can be tolerated. In other situations, reliability of data may outweigh system performance, and thus instead of striping the data over n disks, the same data is duplicated (or mirrored) to n disks. This strategy is referred to as RAID1, which is suitable to ensure the reliability of critical data. 3.1. Basic Data Striping and RAID Storage Systems In comparison to RAID0, the overall failure rate of a RAID1 array of n disks becomes: However, when the same set of data is duplicated over n disks, it would result in a storage overhead of n − 1 disks. X = A XOR B then the following will be true: A = X XOR B B = X XOR A Using this property, we can say that the following expressions are also true (and this can be repeated for an infinite amount of terms): A XOR B XOR C XOR D = X X XOR B XOR C XOR D = A A XOR X XOR C XOR D = B A XOR B XOR X XOR D = C A XOR B XOR C XOR X = D A XOR B XOR C XOR D = X X XOR B XOR C XOR D = A A XOR X XOR C XOR D = B A XOR B XOR X XOR D = C A XOR B XOR C XOR X = D A XOR B XOR C XOR D = X X XOR B XOR C XOR D = A A XOR X XOR C XOR D = B A XOR B XOR X XOR D = C A XOR B XOR C XOR X = D A XOR B XOR C XOR D = X X XOR B XOR C XOR D = A A XOR X XOR C XOR D = B A XOR B XOR X XOR D = C A XOR B XOR C XOR X = D Put simply, if we calculate a XOR of a number sequence, we can substitute X for any number in this sequence, recalculate the XOR, and recover the original number. This is the principle for building a RAID5 array, of which an example is shown in Figure 1. Each of the four drives in Figure 1 are divided into four blocks, each belonging to a stripe on the same level across each drive. Each drive and each stripe have a parity block (stored in disk 4) which is the XOR of the other three blocks. So, say Disk 2 failed, then the XOR of A1 with A2 and A4 would get the remaining A3 for the A stripe; B1 with B2 and B3 to get the B4 parity block; etc. Generally speaking, by introducing a single parity bit, RAID5 is able to survive the failure of any single hard disk drive in any array which contains a minimum of three drives. Future Internet 2015, 7 448 Another RAID level, namely RAID5, attempts to mitigate the high costs of RAID01/10. RAID5 utilizes striping and parity techniques to achieve simultaneously a similar I/O performance to RAID0 with a significantly lower failure rate of: 1 − (1 − r)n − nr(1 − r)n−1 A parity bit (or check bit) is a bit added to the end of a string of binary code that indicates whether the number of bits in the string with the value 1 is even or odd. In computing and telecommunications, a parity bit is calculated via an XOR sum of all the previous bits, yielding 0 for even parity and 1 for odd parity. Let A and B being two binary sequences. If: However, when the same set of data is duplicated over n disks, it would result in a storage overhead of n − 1 disks. RAID0 and RAID1 can be combined into a RAID01 or a RAID10 configuration. The former means a mirrored configuration of multiple striped sets (i.e., mirror of stripes); and the latter means a stripe across a number of mirrored sets (i.e., stripe of mirrors). Both strategies are able to provide very good performance and reliability, whereas both are expensive solutions as considerable amount of hard disk storage is committed to maintaining redundancy information. Future Internet 2015, 7 Future Internet 2015, 7 Future Internet 2015, 7 3.2. Reed-Solomon Error Correcting Code Reed-Solomon’s error correcting code [4] was developed by Irving Reed and Gustave at MIT Lincoln Laboratory in 1960. By adding t check symbols to the data, the RS code can detect any combination of up to t erroneous symbols, and correct up to t/2 symbols or t known erasures. RS code is often used to correct burst errors on storage media, such as for CD-ROMs. For example, RAID6 extends RAID5 by using two RS parity blocks to recover from the failure of any two hard disk drives in an array of a minimum of four drives. RS code is suitable for applying to secret sharing, because the t-out-of-n property of a secret sharing scheme is equivalent to the loss of (n − t) shares, with the positions of the missing shares known in advance. In other words, (n − t) check symbols can be introduced at the point when the shares were created, e.g. use two check symbols to ensure that any three out of the five shares can be combined together to reconstruct the original data. A secret sharing scheme implemented using the RS code algorithm can be highly efficient, but unfortunately not very secure. A secure secret sharing scheme should distribute shares so that anyone with fewer than t shares has no extra information about the secret than someone with zero shares. For example consider the secret sharing scheme in which the secret phrase “password” is divided into the following shares (plus RS code shares): “pa------”, “--ss----”, “----wo--”, “------rd” A person with 0 of these shares may only perhaps know that the password consists of eight letters. For this they would have to guess the password from 208 billion combinations (268). With one share, however, they would only have 308 million combinations (as to guess only the six letters—266), and so on as more shares became available. RS code is not a secure secret sharing scheme, because it is possible for a player with fewer than t secret-shares to partially identify some of the original data. X = A XOR B The RAID5 data striping mechanism can be applied to secret sharing of data in the Cloud, where each cloud storage provider represents a disk storing one share of the secret data, but the limitation of this lies in that it always requires n − 1 shares to reconstruct the original data, rather than a more desirable and flexible arbitrary k-out-of-n shares. A1 B1 C1 D1 A2 B2 C2 D2 A3 B3 C3 D3 A4 B4 C4 D4 Disk 1 Disk 2 Disk 3 Disk 4 Figure 1. A 4-disk array in RAID5 format. A1 B1 C1 D1 A2 B2 C2 D2 A3 B3 C3 D3 A4 B4 C4 D4 Disk 1 Disk 2 Disk 3 Disk 4 Figure 1. A 4-disk array in RAID5 format. Figure 1. A 4-disk array in RAID5 format. 449 4. Basic Data Striping Schemes in Cloud-Based Systems The major concerns of moving towards Cloud-based storage have been the security and availability of data when accessed. Several researchers have opinioned that a multi-cloud storage platform might improve security and resilience [5–7]. Based on this, to ensure effective data splitting and security of stored data, a secret sharing algorithm such as Shamir secret sharing has been proposed for multi-cloud storage [8–13]. Previous methods split the data into pieces and then reconstruct when needed, while replicating data at different storage areas using encryption to safeguard them. This type of methodology can be costly (in terms of network bandwidth and storage facilities), and not quite safe in scenarios involving poor key management. Dodis [6] states that a basic secret sharing scheme which lacks redundancy is given by two algorithms: Sharing (Share); and Recovery (Rec). Share takes a message M and split it into n pieces. Since M is secret, Share must introduce randomness (that is, Share is probabilistic); Rec is a deterministic algorithm which recreates the message from some or all of the shares: 450 Future Internet 2015, 7 where → depicts randomness. where → depicts randomness. where → depicts randomness. Secret sharing schemes are important in cryptography and are a key building block for many secure protocols, such as for: Threshold Cryptography; Access Control; Attribute-Based encryption; Byzantine agreement; generalised oblivious transfer; and general protocol for Multi-Party computation [14]. For general algorithms, the following thresholds are useful:  tp—the privacy threshold. This describes the maximum number of servers that cannot determine the secret, even when they combine their shares together.  tf—the fault-tolerance threshold. With this, supposing that every server is honest, this is the minimum number of servers you need to recover the secret (if the other servers are absent).  tr—the robustness threshold. This is arguably the most important, as this is the minimum number of correct shares you need to recover the secret if all other servers are compromised.  ts—the soundness threshold. This determines the minimum number of correct shares such that it is not possible to recover the wrong secret.  ti—the information rate threshold. This describes the amount of information that the server needs to keep on a secret and determines the efficiency and idealness of a secret sharing scheme. 4.1. Sharing Methods There are several relevant methods which could be used for secret sharing and these are outlined in the following sections. The most relevant methods are then evaluated at the end of this paper. There are several relevant methods which could be used for secret sharing and these are outlined in the following sections. The most relevant methods are then evaluated at the end of this paper. 4.1.1. Perfect Secret Sharing Scheme (PSS) • The Recovery Algorithm: (Sʹ1, Sʹ2, …. Sʹn, pub) = Mʹ. The correctness property of the algorithm defines that, for any message M, Rec(Share(M)) = M h d i d (Sʹ1, Sʹ2, …. Sʹn, pub) = Mʹ. The correctness property of the algorithm defines that, for any message M, Rec(Share(M)) = M h d i t d (Sʹ1, Sʹ2, …. Sʹn, pub) = Mʹ. The correctness property of the algorithm defines that, for any message M, Rec(Share(M)) = M • The Sharing Algorithm: Share(M) → (S1, S2, …. Sn, pub). The secrets S1, S2, …. Sn are distributed securely among servers 1 through to n, and pub is a public share. Share(M) → (S1, S2, …. Sn, pub). The secrets S1, S2, …. Sn are distributed securely among servers 1 through to n, and pub is a public share. • The Recovery Algorithm: 4.1.1. Perfect Secret Sharing Scheme (PSS) 4.1.1. Perfect Secret Sharing Scheme (PSS) Shamir (1979) provides a good example of a perfect secret sharing [15]. It requires two conditions to be perfect: if, and only if, t − 1 shares provide absolutely no information regarding the hidden secret, and when the ratio of the length of the secret to the length of each of the shares (known as the information rate) is equivalent to 1. Shamir’s PSS relies on the idea that on the principle that you can define a straight line with two points, three points for a quadratic equation, and so on, to give t points to define a polynomial of degree t − 1. Hence, a method for t-out-of-n secret sharing can thus use a polynomial with a t − 1 degree using a secret for the first coefficient, and then random values for the remaining coefficients. Next, find n points on the curve and give one to each of the players. As a result, when at least t out of the n players reveal their points, there is sufficient information to fit a (t − 1)th degree polynomial to them, in which the first coefficient is the secret. 451 Future Internet 2015, 7 To illustrate this construction technique, consider a concrete example. Assume that a secret value is split into five parts, three of which are needed to reconstruct the original data (i.e., n = 5, t = 3). The first step is to create a second degree polynomial: y = a0 × x0 + a1 × x1 + a2 × x2 The second step is to assign the secret value to the first coefficient (i.e., a0) and choose random values for the remaining coefficients (i.e., a1 and a2). Suppose that the secret value is: 42, a1 = 1 and a2 = 2, then the polynomial becomes: The second step is to assign the secret value to the first coefficient (i.e., a0) and choose random values for the remaining coefficients (i.e., a1 and a2). Suppose that the secret value is: 42, a1 = 1 and a2 = 2, then the polynomial becomes: y = 42 + x + 2 × x2 y = 42 + x + 2 × x2 The third step is to calculate any five (x, y) pairs, for instance: The third step is to calculate any five (x, y) pairs, for instance: x ______y 1 42 + 1 + 2 × 1 45 2 42 + 2 + 2 × 4 52 3 42 + 3 + 2 × 9 63 4 42 + 4 + 2 × 16 78 5 42 + 5 + 2 × 25 97 Finally, distribute one pair to each player, e.g., Player1 receives (1, 45), Player2 receives (2, 52), and so on. No player can thus tell anything about the original secret, unless at least three players exchange their information and yield equations like: Finally, distribute one pair to each player, e.g., Player1 receives (1, 45), Player2 receives (2, 52), and so on. No player can thus tell anything about the original secret, unless at least three players exchange their information and yield equations like: Finally, distribute one pair to each player, e.g., Player1 receives (1, 45), Player2 receives (2, 52), and so on. Future Internet 2015, 7 For example, consider the example of p = 61: x p y 1 61 (42 + 1 + 2 × 1) % 61 45 2 61 (42 + 2 + 2 × 4) % 61 52 3 61 (42 + 3 + 2 × 9) % 61 2 4 61 (42 + 4 + 2 × 16) % 61 17 5 61 (42 + 5 + 2 × 25) % 61 36 Shamir’s PSS is a promising approach to secret sharing for cloud storage, and provides many advantages, including: Shamir’s PSS is a promising approach to secret sharing for cloud storage, and provides many advantages, including:  Secure—Anyone with fewer than t shares has no extra information about the secret than someone with zero shares.  Extensible—When n is fixed, new shares can be dynamically added or deleted without affecting the existing shares.  Dynamic—With this it is possible to modify the polynomial and construct new shares without changing the secret.  Flexible—In organisations where hierarchy is important, it is possible to supply each participant different number of shares according to their importance. However, the PSS scheme also has the following drawbacks: However, the PSS scheme also has the following drawbacks:  Computational overheads—The preparation of the polynomial, the generation of shares, and the reconstruction of the secret may consume significant computational resources.  Storage and transmission overheads—Given t − 1 shares, no information whatsoever can be determined about the secret. Hence, the final share must contain as much information as the secret itself, which means effectively each share of the secret must be at least as large as the secret itself. The storage and transmission of the shares requires an amount of storage space and network bandwidth equivalent to the size of the secret times the total number of shares. 4.1.2. Share Distribution Future Internet 2015, 7 No player can thus tell anything about the original secret, unless at least three players exchange their information and yield equations like: 45 = a0 + a1 × 1 + a2 × 12 52 = a0 + a1 × 2 + a2 × 22 63 = a0 + a1 × 3 + a2 × 32 45 = a0 + a1 × 1 + a2 × 12 52 = a0 + a1 × 2 + a2 × 22 63 = a0 + a1 × 3 + a2 × 32 45 = a0 + a1 × 1 + a2 × 12 52 = a0 + a1 × 2 + a2 × 22 63 = a0 + a1 × 3 + a2 × 32 Hence, the three players together can work out that the secret value a0 is 42. This method works fine, but from security point of view a player can still obtain more information about the secret with every pair on the polynomial that they find. For example, player Eve finds two pairs (2, 52) and (4, 78). Although these are not enough to reveal the secret value, Eve could combine them together and get: 76 = a0 + a1 × 4 + a2 × 42 52 = a0 + a1 × 2 + a2 × 22 Therefore, Eve can work out that: a0 = 26 + 8 × a2 So, Eve starts to replace a2 with 0, 1, 2, 3… to find all possible values of a0. This problem can be fixed by using finite field arithmetic in a field of size r where: r > ai, r > N, r = pk, p ∈ P where P is the set of primes and k is a positive integer. r > ai, r > N, r = pk, p ∈ P where P is the set of primes and k is a positive integer. Then, calculate the pairs as: y = f(x) mod(p) y = f(x) mod(p) 452 4.1.3. Information Dispersal Algorithm (IDA) 4.1.3. Information Dispersal Algorithm (IDA) 4.1.3. Information Dispersal Algorithm (IDA) Rabin (1989) suggested splitting a secret S into n pieces such that a person can obtain the secret only if k < n of these pieces are available, where k is the threshold. Here, each secret Si, i ≤ n, is of size \|S\|/k, where \|S\| is the size of the secret [16]. The total sizes of all the secrets are: (n/k) × \|S\| Thus, with Rabin’s Information Dispersal Algorithm, the storage complexity of a secret sharing system can be significantly reduced in comparison to Shamir (1979) perfect secret sharing (PSS) scheme. But, the security flaw in this method is that, if the data exhibits some pattern frequently, and that the attacker gets hold of m < k slices, there are great possibilities for the attacker gaining the secret S. Both the split and combine algorithms operate by performing matrix multiplication over the input. In the case of the split operation, the transform matrix has n rows (n = number of shares) and k columns (k = quorum), while, in the case of combine operation, the transform matrix has k rows and k columns. Either operation is described simply as the matrix multiplication: Transform matrix × Input matrix = Output matrix Transform matrix × Input matrix = Output matrix In the case of the split operation, the Transform Matrix has n rows and t columns, while in the case of combine operation, the Transform Matrix has t rows and t columns. The Input Matrix always has t rows, while the Output Matrix will have n rows for the split operation, or t rows for the combine operation. The Transform Matrix must have the property that any subset of t rows represent linearly independent basis vectors. If this is not the case then the transform cannot be reversed. To generate a Transform Matrix, a key must be defined as a list of distinct elements, i.e.,: x1, x2, …, xn, y1, y2, …, yt Then, the Transform Matrix is created as: Then, the Transform Matrix is created as: 4.1.2. Share Distribution An honest Dealer (D) selects n distinct, non-zero elements of ℤp (the finite field of size p), denoted by xi where i ∈ [1,n] and it is required that p ≥ n + 1 because in a field of ℤp, there can be at most p − 1 participants. D spreads the value xi to Pi and these xi values are public. The Dealer selects the secret k ∈ ℤp, secretly, and independently, at random, selects t − 1 elements of ℤp denoted by a1, a2, … at−1. For i ∈ [1,n], dealer computes yi = a(xi), where:      1 1 ) (mod ) ( t j j j p x a k x a (1) (1) For i ∈ [1,n], dealer gives the Share S = yi to Pi. For i ∈ [1,n], dealer gives the Share S = yi to Pi. To recover the information, a group of participants A, which are members of the access structure Γ defined as (ܣᇱ⊂A: ܣᇱ can reconstruct the secret ݇ሻ are required to meet to recover the secret. A ∈ Γ and can reconstruct the secret k = a(0) by substituting x = 0 into LaGrangian interpolation formula: Future Internet 2015, 7 453 p x x x y k t j j n t n i i i i j n n j mod 1 , 1                 (2) (2) 4.1.5. Rabin’s and Ben-Or’s Publicly Verifiable Secret Sharing (PVSS) Some applications of a secret sharing scheme, such as e-auction, e-voting and multiparty computation, require public verifiability. Namely, it must be publicly verifiable without revealing the secret or any of its shares that all the n shares are consistently generated from a unique share-generating polynomial such that any t of them can reconstruct the same secret. This capability is crucial to overcome the problem of dishonest share dealers. Tal Rabin and Michael Ben-Or [19] devised a publicly verifiable secret sharing system that supports dishonest detection for the dealer, or for one-third of the players, even if those players are coordinated by an adaptive attacker who can change strategies in real-time depending on what information has been dishonest detection for the dealer, or for one-third of the players, even if those players are coordinated by an adaptive attacker who can change strategies in real-time depending on what information has been revealed. The PVSS is essentially a combination of secret sharing and publicly verifiable encryption and works as follows: (1) The dealer creates shares S1, S2, ..., Sn for each participant P1, P2, ..., Pn respect (2) The dealer encrypts the shares for each participant using their public keys and publishes the ciphertexts Ei(Si); (3) The dealer also publishes a string ProofD to show that each Ei encrypts Si and the reconstruction protocol will result in the same secret S; Anybody knowing the public keys for the encryption methods Ei, can verify the shares (5) If one or more verifications failed, the dealer fails and the protocol is aborted; (6) Each participant Pi decrypts their shares Si using Ei(Si); The participant releases Si plus a string ProofPi to show that the released share is corre Dishonest participants who failed to decrypt Si or to generate ProofPi will be excluded The secret S can be securely reconstructed from the shares of any qualified set of partic Kun Peng [20] carried out a critical survey of five existing PVSS algorithms which claimed strong security and high efficiency. Surprisingly, in practice none of the algorithms can achieve efficiency as high as O(tn) exponentiations, which is widely believed to be feasible. Then, the Transform Matrix is created as:                              1 1 2 1 2 2 2 1 2 1 2 1 1 1 1 ... 1 1 . ... . . 1 ... 1 1 1 ... 1 1 y x y x y x y x y x y x y x y x y x n n t t 4.1.4. Krawczyk’s Computational Secret Sharing (CSS) 4.1.4. Krawczyk’s Computational Secret Sharing (CSS) Hugo Krawczyk [17] proposed the Computational Secret Sharing (CSS) technique (a.k.a. secret sharing made short), which combines Rabin’s IDA with Shamir’s PSS. Data is first encrypted with a randomly generated key, using a symmetric encryption algorithm. Next this data is split into n fragments using Rabin’s IDA with a threshold t configured. In this case, the scheme is t times more efficient than 454 Future Internet 2015, 7 Shamir’s PSS. The final step is to use Shamir’s PSS to produce shares of the randomly generated symmetric key (which is typically of the order of 64 to 256 bits) and then give one share and one fragment to each shareholder. A related approach, known as AONT-RS [18], applies an All-Or-Nothing transform (AONT) to the data as a pre-processing step to the IDA. AONT guarantees that any number of shares less than the threshold is insufficient to decrypt the data. 5. Secret Sharing in a Multi-Cloud Environment Application Platform: Determines the access structure; encodes secret; sends same to main multi‐cloud proxy server for distribution to multi‐ cloud service providers as well keeps the secret. Data storage E with self destruct ability S2 CSP2 CSP4 Data storage A with self destruct ability Data storage D with self destruct ability Data storage C with self destruct ability Data storage B with self destruct ability Figure 2. Proposed architecture of a Secret Sharing Scheme in a Multi-cloud environment. 5. Secret Sharing in a Multi-Cloud Environment The secret sharing methods show potential in being used in Cloud-based infrastructures, as they inherently preserve the data without the requirement for private keys. Figure 2 outlines an example of a proposed architecture known as SECRET which supports a secret sharing scheme in a multi-cloud environment. It has four main elements: 455 Future Internet 2015, 7  Application platform. Its function is to: determines the access structure; encodes secrets; sends secrets to the main multi-cloud proxy server for distribution to multi-cloud service providers, as well as keeping the secret shares when recovered.  Main multi-cloud proxy server with router. This splits and distributes encoded shares to multi-cloud based on pre-determined access structure and manages the fail-over protection of shares.  Main multi-cloud proxy server with router. This splits and distributes encoded shares to multi-cloud based on pre-determined access structure and manages the fail-over protection of shares.  Metadata server. This includes the functionality of: User management; Server management; Session management; and File metadata management [3].  Multi-cloud proxy server. This Gathers shares and reconstruct secret as well manages break-glass data recovery.  Sub-routers. This creates a path between the Cloud Service Provider (CSP) (considered here as front-end) with other Cloud Service providers (considered here as the Back-ends) thereby creating a quick and alternative recovery path for all the shares. For example: R4 connects with R3 + R2 + R1, so R4 is a path for CSP1 + R3 + R2 + R1 + S1, and so on. Application Platform Multi‐cloud Proxy Server Metadata server Main Multi‐Cloud Proxy Server with Router S5 CSP5 S4 CSP4 S3 CSP3 S2 CSP2 R3 R4 R1 R2 Data storage E with self destruct ability Data storage D with self destruct ability Data storage C with self destruct ability Data storage B with self destruct ability Data storage A with self destruct ability R1 – Sub‐router 1 R2 – Sub‐router 2 R3 – Sub‐router 3 R4 – Sub‐router 4 R5 – Sub‐router 5 S1‐S5 – Shares 1‐5 CSP1‐5 Cloud Service Providers 1‐5 Application Platform: Determines the access structure; encodes secret; sends same to main multi‐cloud proxy server for distribution to multi‐ cloud service providers as well keeps the secret. S1 CSP1 Figure 2. Proposed architecture of a Secret Sharing Scheme in a Multi-cloud environment. 5.2. How It Works At the application platform, the data owner determines n and t values (see Section 6) and using both calls up the application to be used, selects algorithm of choice based on our Evaluation in Section 6 after a successful sign in to the system and access level determined. It will be nice to base the choice of algorithm on performance as it relates to security, overhead cost and data size. Figure 2 shows a 3-out-of-5 access structure, while Section 6 use 4-out-of-10 and 2-out-of-5. The encoded data is sent to the local main multi-cloud proxy server with router for onward dissemination to the CSPs. The proxy splits the encoded data according to a secret-sharing scheme determined access structure, and distributes each share over the Internet to different CSPs. The retrieval process is similar to the storage process as the metadata server helps to keep track of the siblings of the shares. The proxy retrieves enough corresponding shares from the cloud service providers. This retrieval involves authentication to the cloud providers. The retrieved shares are sent back to the application platform software, which decodes them and verifies their authenticity before reconstructing the data. The design incorporates unique feature in a multi-cloud environment as it uses secret sharing scheme to implement near keyless encryption using SeDaS. This is done by breaking the secret into chunks called (k-out-of-n) threshold in such a manner that less than k shares cannot recover the secret, thus using it for data distribution in object storage system. This is also used to implement self-destruct with equal divided shares. The incorporation of a Self-Destructive system solves the problem of cloud user’s privacy as there is no way the user’s data can be accessed, copied, cached or used without the data owner’s consent within a pre-determined time-frame as all data and their copies become destructed or unreadable after a user-specified time, without any user intervention [3]. The self-destructive system defines two modules: a self-destruct method object; and survival time parameter for each secret key part. In this case, a secret sharing algorithm is used to implement share distribution in object storage system so as to ensure safe destruct with equally divided shares. Based on active storage framework, object-based storage interface will be used to store and manage the equal divided shares. Future Internet 2015, 7 Future Internet 2015, 7 among users due to wider coverage areas and increased in data confidentiality, availability and integrity. In many other similar works like [21,22], the use in exchange of Electronic Health Records were discussed extensively and the prove that such deployment can provide an increased data availability, confidentiality and integrity of the documents stored in the cloud, and so on were provided. SECRET architecture tends to extend these features by adding the possibility of a break-glass data recovery system, near keyless encryption with improved data privacy and security leading to the elimination of threats in poor key management by incorporating Self-Destructive Data System (SeDaS) [3] on the data to be distributed among the cloud service providers. This therefore enhances the security and privacy of data at every stage (in motion, at rest or in use) within and outside the cloud environment. 5.1. The SECRET Architecture SECRET is one of the efforts geared towards accelerating the adoption of multiple cloud storage among enterprises due to its multiple benefits especially as it concerns its pay-as-you-use concept, reduction of capital expenditures in personnel hiring for data management; purchase of huge infrastructure for data storage through data outsourcing; the simplification of wider information sharing 456 Future Internet 2015, 7 There is also an incorporation of a Break-Glass data recovery system, implemented using one of the Proxy Servers. Just as in Secret Sharing, a trusted dealer can decide to give more shares to a more trusted and reliable participant, which will enable the secret to be reconstructed by fewer participants. SECRET leverages on this concept to implement Break-Glass data recovery by establishing an access to Multi-Cloud proxy Server II, which entails an access to CSPs 1, 3 and 5 and this in turn ensures a quick recovery of shares in order to reconstruct the secret as it is a quick link to all other CSPs and moreover, following the access structure, such access ensures the possibility of reconstructing the secret in an emergency situation. This is an important feature as there could be a period of cloud outage as stated in [23], and in such situation, data recovery could be done from 3-out-of-5 Cloud service providers being used for data storage. That is to say, if 2-out-of-the-5 cloud service providers fail, data recovery is still possible in such an extreme condition. Following the above analysis, SECRET therefore shows that the architecture enhances privacy, confidentiality, integrity, availability and security of data at every state (rest, in motion or in use). There is also a reduced overhead and time lags associated with use of Shamir’s algorithm by supporting an integration of Rabin’s IDA with perhaps Krawczyk’s CSS. The proposition of near keyless encryption thereby avoiding the problem associated with key management is also an added advantage. Therefore, the main advantages of the proposed scheme includes: ast and efficient data/key distribution to multi-cloud service providers.  Fast and efficient data/key distribution to multi-cloud service providers.  Keyless encryption and by extension data security and reduced key management  Keyless encryption and by extension data security and reduced key management hassles.  Provide data owner’s privacy by implementing SeDas, as it meets all the privacy-preserving goals [3].  SeDas supports security erasing files and random storage in drives (Cloud, HDD or SSD) respectively [3].  Backup operational mode in which the function of 5 CSPs can be assumed by 3 CSPs when 2-out-of-the-5 CSPs become unavailable either through failure or scheduled down time.  Break-glass data recovery.  Break-glass data recovery. 5.2. How It Works The use and implementation of a threshold system in cloud services are a deliberate act towards implementing failover protection in the model. In normal circumstances all the service providers are used in share storage as well as secret reconstruction, but in an extreme and desperate situation, 2-out-of-5 can be made redundant. That is to say if 2-out-of-5 CSPs fails, data/secret storage and data reconstruction are still possible. 457 6. Evaluation Secret sharing schemes have been used successfully in data splitting and reconstruction, thereby providing data security in a keyless manner. This section outlines an experiment involving three of the main contenders for Secret Sharing schemes in Cloud-based systems: Adi Shamir’s Perfect Secret Sharing Scheme (PSS), Hugo Krawczyk’s Secret Sharing made short or Computational Secret Sharing scheme (CSS) and Rabin’s Information Dispersal Algorithm (IDA), see Tables 1–8. The experiments were conducted in collaboration with a commercial partner, Payfont Limited, that has further validated the results below in a highly scaled, commercial technical infrastructure. The evaluator in this case is the performance overhead at increasing thresholds and data sizes show varied behaviours that depict their strengths and weaknesses at different application scenarios. Figures 3–6 outline the results. e Internet 2015, 7 Figure 3 Share creation and recreation against increasing data sizes (n = 5; t =2) 458 Future Internet 2015, 7 e Internet 2015, 7 Figure 3. Share creation and recreation against increasing data sizes (n = 5; t =2). Figure 4. Share creation and recreation against increasing data sizes (n = 10; t = 4). (a) (b) Figure 5 Graphs of Time taken against Data sizes in share creation (a) and recreation (b re Internet 2015, 7 Figure 3. Share creation and recreation against increasing data sizes (n = 5; t =2). Figure 4. Share creation and recreation against increasing data sizes (n = 10; t = 4) Figure 4 Share creation and recreation against increasing data si es ( 10; t 4) Figure 4. Share creation and recreation against increasing data sizes (n = 10; t = 4). (a) (b) Figure 5. Graphs of Time taken against Data sizes in share creation (a) and recreation (b) when n = 10, t = 6. Figure 4. Share creation and recreation against increasing data sizes (n = 10; t = 4). Figure 4. Share creation and recreation against increasing data sizes (n = 10; t = 4). Figure 4. Share creation and recreation against increasing data sizes (n = 10; t = 4). (a) (b) Figure 5. Graphs of Time taken against Data sizes in share creation (a) and recreation (b) when n = 10, t = 6. (b) Figure 5. Graphs of Time taken against Data sizes in share creation (a) and recreation (b) when n = 10, t = 6. (b) ture Internet 2015, 7 (a) (b) Figure 6. Future Internet 2015, 7 Future Internet 2015, 7 number of shares to create while the variable t relates to the number of shares required for recreation of the original arbitrary data (using each SSS algorithm). Time taken for share creation and recreation unit is presented in milliseconds. From Figures 3–6 and Tables 1–8, it can be clearly demonstrated that IDA is the fastest algorithm regardless of data size. CSS comes second in terms of time taken for share creation and recreation while PSS comes last. Some significant observations in the results presented is that PSS demonstrates greater issues in regards to scalability as the data size increases in comparison with the other two algorithms and as it gets beyond 131,072 KB on machine (b) shares could no longer be created nor recreated as the machine throws up out of memory exceptions. Additionally, as we increase the parameters from n = 5; t = 2 to n = 10; t = 4 and from n = 5; t = 3; n = 10; t = 6 it can be demonstrated that only share creation will produce significant increase in performance time. Although IDA has demonstrated the fastest time in results presented in this paper it would be naive to simply use this algorithm from these results alone. Depending on the context and application, there may be a need to strike a fine balance between ensuring strong security and acceptable level of performance. Thus, ultimately, the decision on which SSS algorithm to use will be most dependent on the use-case scenario at hand. Table 1. Time taken (ms) of share creation (n = 5; t =2). Algorithm 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) IDA 14.42 31.90 65.83 126.52 274.43 CSS 21.27 43.29 97.25 174.97 380.53 PSS 172.21 304.94 622.16 1065.41 2228.30 Table 2. Time taken (ms) of share recreation (n = 5; t =2). Algorithm 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) IDA 26.35 63.39 116.69 223.60 445.53 CSS 35.01 76.88 121.42 251.43 536.36 PSS 45.69 104.73 171.81 428.29 867.35 Table 3. Time taken (ms) of share creation (n = 10; t = 4). Algorithm 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) IDA 19.97 58.90 185.20 333.22 518.00 CSS 26.80 70.81 192.48 367.04 871.62 PSS 298.25 567.11 1169.94 3013.11 6091.91 Table 4. Time taken (ms) of share recreation (n = 10; t = 4). 6. Evaluation Graph of Time taken against Data sizes in share creation (a) and recreation (b) when n = 5, t = 3. 459 Future Internet 2015, 7 (b) (b) Figure 6. Graph of Time taken against Data sizes in share creation (a) and recreation (b) when n = 5, t = 3. Two different experiments were performed on two different machines. The aim of the experiments were to demonstrate the implication data size variations have on the performance of each secret sharing scheme (SSS) algorithm in terms of share creation and share recreation. Data sizes from 1024 KB to 16,384 KB and 2 KB to 262, 144 KB were evaluated on machines (a) and (b) respectively. The data generated are arbitrary due to the fact that the evaluations are not catered for in relation to one specific area where SSS algorithms may be applied in. The test machines are (a) a D-Series 3 specification Microsoft Azure virtual machine which consists of 4 vCores, 14 GB of RAM and a 200 GB SSD; and (b) an HP EliteDesk 800 G1 USDT, X64-based PC running on Intel® Core ™i5-4570S CPU @ 2.90 GHz, 2901 MHz, 4 cores, 4 GB RAM and a 460 GB SATA drive. Four primary sets of results were presented which use the parameters of (n = 5, t = 2) and (n = 10, t = 4) on machine (a) and (n = 5, t = 3) and (n = 10, t = 6) on machine (b). The variable n relates to the 460 Future Internet 2015, 7 Algorithm 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) IDA 21.04 45.11 104.75 163.78 362.32 CSS 26.83 59.10 122.94 210.51 470.08 PSS 56.79 139.10 222.47 455.58 932.91 Table 1. Time taken (ms) of share creation (n = 5; t =2). Algorithm 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) IDA 14.42 31.90 65.83 126.52 274.43 CSS 21.27 43.29 97.25 174.97 380.53 PSS 172.21 304.94 622.16 1065.41 2228.30 Table 1. Time taken (ms) of share creation (n = 5; t =2). Table 2. Time taken (ms) of share recreation (n = 5; t =2). Table 3. Time taken (ms) of share creation (n = 10; t = 4). Table 4. Time taken (ms) of share recreation (n = 10; t = 4). 461 44 B) 75 90 4 5. Time take in (ms) for share creation (n = 10, t = 6). 128 (KB) 256 (KB) 512 (KB) 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) 32768 (KB) 65536 (KB) 131072 (KB) 2621 (KB 44.94 56.65 63.39 77.71 90.95 125.22 203.46 350.05 637.28 1337.67 2492.91 5005. 30.82 31.14 36.97 44.93 59.78 87.37 156.65 268.90 505.48 1109.95 2042.17 4030. 70.71 106.58 175.70 306.61 564.58 1067.90 2078.61 4129.68 8163.94 15999.61 36832.71 - . Time take in (ms) for share recreation (n = 10, t = 6). 128 (KB) 256 (KB) 512 (KB) 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) 32768 (KB) 65536 (KB) 131072 (KB) 2621 (KB 29.21 33.62 41.61 46.66 77.40 104.98 162.85 305.54 639.02 1104.15 2103.79 4050. 33.58 32.53 34.03 48.91 62.42 96.28 135.73 263.71 535.24 832.55 1595.89 4722. 32.96 48.69 52.16 88.83 149.98 272.39 492.71 908.36 1779.57 3341.85 6930.38 - 7. Time take in (ms) for share creation (n = 5, t = 3). 128 (KB) 256 (KB) 512 (KB) 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) 32768 (KB) 65536 (KB) 131072 (KB) 2621 (KB 42.57 55.58 61.69 68.02 79.97 115.65 175.01 277.30 501.91 1040.84 1943.00 3765. 24.93 27.22 32.88 45.00 49.39 67.36 119.23 204.49 377.09 792.22 1468.66 3034. 52.80 68.67 106.28 166.79 277.36 529.73 971.72 1902.44 3792.03 7311.64 14605.74 63156 8. Time take in (ms) for share recreation (n = 5, t = 3). Future Internet 2015, 7 Future Internet 2015, 7 Future Internet 2015, 7 462 Author Contributions The paper builds on patented dynamic fragmentation for non-linear authentication, authored by David Lanc. David Lanc initially collaborated with Bill Buchanan to investigate the application of dynamic fragmentation to data at rest, eliminating the need for reliance on keys. Initial research and prototyping was performed by Lu Fan and Owen Lo. This was then extended as part of Elochukwu Ukwandu’s PhD research, with contributions from his supervisory team William J. Buchanan and Gordon Russell. David Lanc and Payfont Limited have further contributed to this work through commercial research focusing on high-scalability, high-resilience, architecture agnostic applications. 7. Conclusions The modern cloud-based systems that are commonly produced are often scaled from private platforms into public cloud infrastructures, with the addition of some degree of encryption. As the data is more accessible, many systems now suffer from a loss of the symmetric key which had been used to encrypt the data. Too often a single key is used to encrypt large-scale data storage elements, where a single breach could potentially release vast amounts of sensitive information. The usage of secret share, though, may provide new and better methods of protecting data, and in providing robustness. An important element is the break-glass property, as this allows access to information within a critical incident, while similar studies conducted in [21,22] lend support to the fact that data storage using our multi-provider cloud architecture coupled with secret sharing scheme for data dissemination to these cloud storage devices has great potential in providing increased confidentiality, integrity and availability of data stored in the cloud. In all SECRET tries to lend a voice that using this scheme, a more robust Disaster Recovery system would have been implemented as a failover protection system different from the already existing Redundancy and Backup strategies [24], which we may refer to as Threshold Disaster Recovery System. Moreso, as the founding principles of SECRET, the incorporation of SeDaS for security and privacy tends to deviate from the use of encryption alone for data security and privacy implementation in cloud-based systems. All the same, this paper has shown that IDA and CSS provide the best performance in creating shares, and are ideal companions with the SECRET technique. Conflicts of Interest The authors declare no conflicts of interest. Future Internet 2015, 7 128 (KB) 256 (KB) 512 (KB) 1024 (KB) 2048 (KB) 4096 (KB) 8192 (KB) 16384 (KB) 32768 (KB) 65536 (KB) 131072 (KB) 2621 (KB 29.64 33.98 37.52 47.30 69.14 109.34 181.29 300.20 707.48 1106.48 2215.46 4826. 29.58 34.79 37.39 44.34 64.87 98.49 152.63 269.23 561.52 885.50 1711.26 3466. 29.25 35.07 40.46 60.76 89.60 145.85 265.34 498.64 906.07 1935.26 3692.24 113498 2 3 3 63 2 4 3 11 5.24 79.57 768 KB) 1.91 7.09 92.03 768 KB) 7.48 1.52 6.07 . Time take in (ms) for s 128 (KB) 256 (KB) 512 (KB) 102 (KB 44.94 56.65 63.39 77.7 30.82 31.14 36.97 44.9 70.71 106.58 175.70 306. Time take in (ms) for sh 128 (KB) 256 (KB) 512 (KB) 102 (KB 29.21 33.62 41.61 46.6 33.58 32.53 34.03 48.9 32.96 48.69 52.16 88.8 7. Time take in (ms) for 128 (KB) 256 (KB) 512 (KB) 1024 (KB 42.57 55.58 61.69 68.0 24.93 27.22 32.88 45.0 52.80 68.67 106.28 166.7 . 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https://openalex.org/W2994150797	https://europepmc.org/articles/pmc6888937?pdf=render	English	null	Identifying gene-specific subgroups: an alternative to biclustering	BMC bioinformatics	2,019	cc-by	10,207	Branders et al. BMC Bioinformatics (2019) 20:625 https://doi.org/10.1186/s12859-019-3289-0 Branders et al. BMC Bioinformatics (2019) 20:625 https://doi.org/10.1186/s12859-019-3289-0 Open Access © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Abstract Background: Transcriptome analysis aims at gaining insight into cellular processes through discovering gene expression patterns across various experimental conditions. Biclustering is a standard approach to discover genes subsets with similar expression across subgroups of samples to be identified. The result is a set of biclusters, each forming a specific submatrix of rows (e.g. genes) and columns (e.g. samples). Relevant biclusters can, however, be missed when, due to the presence of a few outliers, they lack the assumed homogeneity of expression values among a few gene/sample combinations. The Max-Sum SubMatrix problem addresses this issue by looking at highly expressed subsets of genes and of samples, without enforcing such homogeneity. Results: We present here the K-CPGC algorithm to identify K relevant submatrices. Our main contribution is to show that this approach outperforms biclustering algorithms to identify several gene subsets representative of specific subgroups of samples. Experiments are conducted on 35 gene expression datasets from human tissues and yeast samples. We report comparative results with those obtained by several biclustering algorithms, including CCA, xMOTIFs, ISA, QUBIC, Plaid and Spectral. Gene enrichment analysis demonstrates the benefits of the proposed approach to identify more statistically significant gene subsets. The most significant Gene Ontology terms identified with K-CPGC are shown consistent with the controlled conditions of each dataset. This analysis supports the biological relevance of the identified gene subsets. An additional contribution is the statistical validation protocol proposed here to assess the relative performances of biclustering algorithms and of the proposed method. It relies on a Friedman test and the Hochberg’s sequential procedure to report critical differences of ranks among all algorithms. Conclusions: We propose here the K-CPGC method, a computationally efficient algorithm to identify K max-sum submatrices in a large gene expression matrix. Comparisons show that it identifies more significantly enriched subsets of genes and specific subgroups of samples which are easily interpretable by biologists. Experiments also show its ability to identify more reliable GO terms. These results illustrate the benefits of the proposed approach in terms of interpretability and of biological enrichment quality. Open implementation of this algorithm is available as an R package. Keywords: Gene expression analysis, Biclustering, Gene enrichment analysis, Identification of signifi Identifying gene-specific subgroups: an alternative to biclustering Vincent Branders* , Pierre Schaus and Pierre Dupont Correspondence: vincent.branders@uclouvain.be Université catholique de Louvain - ICTEAM/INGI - Machine Learning Group, Place Sainte Barbe 2, 1348 Louvain-la-Neuve, Belgium Background of a number of genes. Biclustering, also known as co- clustering, is one of the most common approaches for such a task as it identifies specific subsets of rows and of columns which jointly form homogeneous entries [1, 2]. of a number of genes. Biclustering, also known as co- clustering, is one of the most common approaches for such a task as it identifies specific subsets of rows and of columns which jointly form homogeneous entries [1, 2]. A substantial number of biclustering methods and applications have been described since the application of biclustering, introduced in [3], to gene expression data analysis [4]. Several biclustering algorithms reviews have been published emphasizing on various characteristics of the biclustering algorithms, applications, or results. Gene expression data is typically represented as a large matrix of gene expression levels across various samples. The study of such data is a valuable tool to improve the understanding of the underlying biological processes. A frequent objective of gene expression analysis is to group genes according to their expression under certain con- ditions or to group conditions based on the expression A substantial number of biclustering methods and applications have been described since the application of biclustering, introduced in [3], to gene expression data analysis [4]. Several biclustering algorithms reviews have been published emphasizing on various characteristics of the biclustering algorithms, applications, or results. Correspondence: vincent.branders@uclouvain.be Université catholique de Louvain - ICTEAM/INGI - Machine Learning Group, Place Sainte Barbe 2, 1348 Louvain-la-Neuve, Belgium Page 2 of 13 Page 2 of 13 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics For example, Madeira et al. study in [1] a collection of sixteen biclustering methods and categorize them accord- ing to the structures and patterns of biclusters they can find, the methods used to perform the search and the approach used to evaluate the solution. In [5], the survey mentioned above is updated and extended to forty-seven biclustering algorithms. Each method is further catego- rized based on the use, or not, of evaluation metrics within the search. solution of existing biclustering algorithms or of our own method. From a biological viewpoint, there might be several biclusters to be identified from the same original data matrix. Indeed, a single gene may participate in multi- ple pathways which may or may not be co-active under several conditions [1]. Specific genes may also be rep- resentative of expression patterns among some samples, while other genes would be more informative for other subsets of samples. In other words, one typically looks at several biclusters which might partially overlap in terms of genes or of samples they contain. Padilha et al. conduct, in [6], a comparative study on seventeen algorithms on a large collection of synthetic and real datasets. They conclude that algorithms only achieved satisfactory results in a specific context, and that best results are obtained by selecting an algorithm depending on the specific task at hand. A similar conclu- sion is presented in [7], based on results from a compar- ative study on twelve algorithms on a suite of synthetic datasets and eight real datasets. The contributions of this work are: 1 the introduction of the max-sum submatrix optimization problem as an alternative to biclustering, 2 a greedy extension to the CPGC method proposed in [8] to produce several, possibly overlapping, biclusters of maximal sums, A systematic summary of basic and advanced applica- tions of biclustering for biological and biomedical data is presented in [2]. Guidance on the appropriate algorithms and tools to effectively analyze specific data type and to generate valuable biological knowledge is provided. The max-Sum submatrix problem (MSSM) Given a matrix M ∈Rm×n consisting of m rows and n columns, let R = {1, . . . , m} and C = {1, . . . , n} be index sets for rows and for columns respectively, find the max- sum submatrix (I∗; J∗) , with I∗⊆R and J∗⊆C, such that: (I∗; J∗) = arg max I⊆R,J⊆C f (I, J) = arg max I⊆R,J⊆C i∈I,j∈J Mi,j. (1) (1) Problem definition The max-sum submatrix problem consists in finding a rectangular submatrix, not necessarily made of contigu- ous rows or columns, of a large matrix with maximal sum of the selected entries. As an alternative, the max-sum submatrix problem seeks for subsets of rows and of columns with globally high values. In biological terms, one looks for a subset of biomarkers which are, after appropriate normalization, relatively highly expressed among a subset of samples. One could also look for patterns of low expression simply by considering the opposite values of a normalized ver- sion of the original matrix. By default, we will look for high expression patterns. Both subsets of selected genes and of selected samples are a priori unknown and must be identified. They form a rectangular, and not neces- sarily contiguous, submatrix of the original data matrix exactly like biclusters do. Yet, the mathematical criterion used to find such submatrix differs and is less influ- enced by the presence of some outliers. In the sequel, we use the terms submatrix and bicluster interchange- ably and, depending on the context, they refer to the 3 a rigorous statistical validation protocol to assess the performances of 6 well-known biclustering methods compared between them and with our proposal, 4 practical experiments on 17 gene expression cDNA microarray datasets from Saccharomyces cerevisiae samples under various controlled conditions, Biclustering is typically applied on a dataset in the form of a matrix M where the entry Mi,j represents the value of a specific row i (e.g. a gene) obtained for a specific col- umn j (e.g. a sample). A bicluster is a submatrix of M defined by a subset of selected rows and a subset of selected columns. The selected rows or selected columns need not be contiguous in the original matrix M. 5 a gene enrichment analysis showing that our proposed method outperforms biclustering algorithms to find biologically relevant biclusters, 6 a freely available R package implementing the proposed approach. Biclustering algorithms tend to produce biclusters shar- ing similar expression values, for example by minimizing the variance across the selected genes and selected sam- ples. However, some relevant biclusters may be missed when, due to the presence of a few outliers, they lack the assumed homogeneity of expression values among a few gene/sample combinations. Interpretation The data matrix typically represents gene expression val- ues in a continuous range, for instance on a logarithmic scale and properly normalized: negative values, respec- tively positive values, represent expression values below, respectively above, a threshold θ. For example, θ may cor- respond to the median expression level over the whole Page 3 of 13 Page 3 of 13 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics (2019) 20:625 samples that should arguably be selected as in Fig. 1b. Our experimental results reported in the “Results” section illustrate the benefits of the proposed approach to extract biologically relevant gene subsets. data matrix, or a row-specific value representing the aver- age expression level of a gene across all samples. After such normalization, positive values are considered as the interesting ones. By default, they correspond to the high levels of expression one is interested to find in the data matrix. One could also look for low levels of expression by replacing such a normalized matrix M by its opposite −M. In any case, the objective function f (I, J) is the sum of the entries of a submatrix (I; J). The maximization of this objective rewards, respectively penalizes, matrix entries with positive, respectively negative, values. Finding a submatrix of maximal sum, as formalized in problem (1), is an NP-hard combinatorial optimization problem. This can be shown from a reduction to the max- imum edge weight biclique problem [9], by considering the rows and columns of the original matrix as the two sets of nodes of a bipartite graph. This problem is dif- ficult to solve, especially in gene expression analysis, as one is typically interested in solving it for large instances made of thousands of genes (rows) and possibly hun- dreds of samples (columns). Polynomial algorithms have been proposed in the restricted case of finding a subma- trix made of contiguous rows and contiguous columns of the original matrix [10–12]. Such a restriction is how- ever not justified in the general context of transcriptomics since it would require to know in advance unique and spe- cific orders in which the genes and the samples can be clustered. Figure 1a depicts a toy example of such a normalized data matrix. Positive values (in red) are considered to have high expression levels and negative values (in blue) corre- spond to low expression levels. Results (2) (2) Table 1 gives a global overview of the ability of various algorithms to find significantly enriched biclusters among 35 gene expression datasets from human tissues and Sac- charomyces cerevisae. The eight algorithms and 35 gene expression datasets considered in this work are detailed in the “Methods” section. The K-CPGC algorithm clearly outperforms the other approaches in this global overview: it is the best in terms of the number of enriched biclusters found. Some algorithms are only able to produce a lim- ited number of distinct biclusters, even less enriched ones. This is due to the specifics of each algorithm. For instance, several random initializations used by ISA do not guaran- tee to find distinct solutions. Plaid only returns biclus- ters that offers a better fit to their underlying statistical model than those obtained through random permutations of the original matrix. As for K-CPGC, a slight increase of the threshold θ would lead to producing more biclusters while constraining further the objective of finding high expression patterns. The reported setting looks sound anyway as the prescribed number of 10 biclusters for each dataset is very close to being found with this approach. The results presented in Table 2 lead to reject the null hypothesis of no difference between the ν = 8 algorithms over N = 35 datasets with an associated p-value equal to 1.33 × 10−11. It should be highlighted that some algo- rithms present performances discrepancies regarding the data collections. Namely, ISA, QUBIC and Spectral present higher enrichment performances on the Human tissues collection than on the Yeast collection. Each of the other approaches provides comparable performances on both collections of datasets. We proceed with a post hoc test, the Hochberg’s sequential procedure [16], to determine whether K-CPGC significantly outperforms the other algorithms. Figure 2 reports a diagram of critical differences between the ranks of the various algorithms. The horizontal lines in bold represent the differences between ranks that are required for statistical significance. Such intervals increase as more approaches are included in the comparison following the Hochberg’s correction for multiple testing. In conclusion, K-CPGC has a significantly better rank compared to all other approaches. A non-parametric Friedman test [14] is routinely used in the machine learning literature to assess the relative performance of various classification algorithms across several datasets [15]. We adopt here the same method- ology to compare biclustering algorithms and our own K-CPGC method. Results For each dataset, the algorithms under study are ranked according to the number of enriched biclusters they return. Table 2 reports the number of enriched biclusters identified per dataset by each algo- rithm and its associated rank. The last row reports the average rank RA of algorithm A over all datasets. The Friedman statistic has a χ2 F distribution with ν −1 degrees of freedom where N is the number of datasets and ν the number of algorithms being tested: The analysis so far has been focusing on the number of biclusters for which the subset of genes they con- tain is associated with at least one significant GO term. Since K-CPGC and CCA are the best methods accord- ing to this analysis, one looks now at all significant GO terms identified by both algorithms on all 35 datasets. Figure 3 reports the difference metric between p-values of these GO terms according to equation (4). It shows that K-CPGC outperforms CCA in this regard since it exhibits a positive difference in 638 out of 1054 cases. In other words, K-CPGC identifies gene subsets which are gener- ally estimated more significant as they correspond more often to lower p-values. To complement the analysis, we evaluate the p-values differences on a per dataset basis. Comparing p-values of terms found by K-CPGC and CCA would have lit- tle sense whenever the enriched terms differ completely between both approaches, however. As a consequence, we first report in Fig. 4a the number of terms found by the two approaches, as well as the number of terms that are common to both approaches. Figure 4b reports the num- ber of the terms common to K-CPGC and CCA for which an algorithm presents a smaller p-value as compared to the other algorithm. It shows that when K-CPGC and CCA return several GO terms in common, the significance of such terms is typically better with our K-CPGC approach (see Fig. 4b). Interpretation BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics Page 4 of 13 χ2 F = 12N ν(ν + 1) ν A=1 R2 A −ν(ν + 1)2 4 (2) Interpretation Figure 1b represents the optimal solution to the maximal sum objective. It defines a specific rectangular submatrix, or bicluster, of genes and samples, maximizing the sum of its entries. It can include a few outliers in terms of high expression levels. For exam- ple, the -4.1 entry (row 4, column 4) is included in the optimal solution because such a low value is compensated along its row and its column by other positive values, hence all selected rows and selected columns contribute positively to the objective function. In contrast, as one looks for a rectangular submatrix, a positive entry may be excluded from the optimal solution if it is penalized by the presence of negative values along its row and its column. This is the case, for example, for the entry 4.0 in row 3 and column 3 of this toy example. Our previous work [8] presents several algorithms to address problem (1) without any restriction. They include a mixed integer linear programming (MILP) and two constraint programming (CP) approaches. The first CP method combined with large neighborhood search is an improved version of the work proposed in [13]. Gene expression analysis results were already reported in this earlier work but the proposed approach looks for fully disjoint biclusters. We argue that some overlap may exist between various expression patterns extracted from the same data matrix. The second method, denoted CPGC, includes a global constraint and proved, in our previous work, to be the most efficient one to solve problem (1). We study here its applicability to discover several, and possibly overlapping, biclusters from gene expression data and we show its benefits compared to existing biclustering algorithms. Figure 1c and d represents the results obtained with two different biclustering algorithms, namely CCA and ISA (further described in the “Methods” section), starting from the same toy example (Fig. 1a). Both their solu- tions strongly differ from the one represented in Fig. 1b. In particular, the CCA solution includes many negative entries as they imply a lower variance along selected rows and selected columns. In contrast, the ISA solution only includes positive entries but is missing several genes and Fig. 1 Toy example. a Illustration of a full, normalized, matrix, b the associated submatrix of maximal sum, c bicluster returned by CCA and d bicluster return by ISA uster returned by CCA and d Branders et al. Results When no such overlap exists between the enriched terms reported by both approaches, this is essen- tially due to the fact that CCA hardly returns any enriched Table 1 Total number of identified and enriched biclusters Algorithm Biclusters Enriched biclusters CCA 349 108 ISA 163 90 K-CPGC 342 177 Plaid 102 57 QUBIC 269 107 Spectral 147 44 xMOTIFs 309 60 CPGC 35 35 Results reported for each algorithm on the 35 gene expression datasets from human tissues and Saccharomyces cerevisae. The defined target is K = 10 biclusters for each dataset, for a maximum of 350 biclusters overall. A bicluster is considered significantly enriched if the subset of genes it contains is associated to at least one GO term with an FDR corrected p-value below 5% Page 5 of 13 Branders et al. BMC Bioinformatics (2019) 20:625 Page 5 of 13 Branders et al. Results BMC Bioinformatics Table 2 Number of enriched biclusters found by each algorithm on each dataset dataset CCA ISA K-CPGC Plaid QUBIC Spectral xMOTIFs CPGC 1 1 (5.0) 7 (1.0) 6 (2.0) 1 (5.0) 0 (7.5) 0 (7.5) 2 (3.0) 1 (5.0) 2 0 (7.5) 8 (1.0) 6 (2.0) 1 (5.0) 1 (5.0) 0 (7.5) 2 (3.0) 1 (5.0) 3 1 (6.0) 8 (1.0) 7 (2.0) 1 (6.0) 5 (3.0) 0 (8.0) 3 (4.0) 1 (6.0) 4 1 (5.5) 2 (2.5) 1 (5.5) 2 (2.5) 0 (8.0) 5 (1.0) 1 (5.5) 1 (5.5) 5 2 (4.0) 6 (1.5) 2 (4.0) 1 (6.5) 6 (1.5) 0 (8.0) 2 (4.0) 1 (6.5) 6 1 (5.5) 7 (1.0) 5 (2.0) 2 (4.0) 0 (7.5) 0 (7.5) 3 (3.0) 1 (5.5) 7 2 (5.0) 3 (4.0) 8 (1.0) 1 (7.0) 7 (2.5) 7 (2.5) 1 (7.0) 1 (7.0) 8 3 (5.0) 8 (1.5) 8 (1.5) 1 (7.5) 6 (3.0) 5 (4.0) 2 (6.0) 1 (7.5) 9 1 (7.0) 2 (4.5) 7 (1.0) 1 (7.0) 6 (2.5) 6 (2.5) 2 (4.5) 1 (7.0) 10 1 (5.0) 1 (5.0) 5 (1.0) 1 (5.0) 0 (8.0) 2 (2.0) 1 (5.0) 1 (5.0) 11 0 (7.5) 1 (4.5) 4 (1.5) 0 (7.5) 4 (1.5) 1 (4.5) 1 (4.5) 1 (4.5) 12 2 (3.5) 8 (1.0) 7 (2.0) 0 (7.5) 2 (3.5) 0 (7.5) 1 (5.5) 1 (5.5) 13 0 (7.0) 3 (1.5) 3 (1.5) 0 (7.0) 2 (3.5) 0 (7.0) 2 (3.5) 1 (5.0) 14 2 (4.5) 3 (2.5) 1 (6.5) 0 (8.0) 2 (4.5) 10 (1.0) 3 (2.5) 1 (6.5) 15 3 (2.0) 3 (2.0) 2 (4.5) 0 (8.0) 3 (2.0) 2 (4.5) 1 (6.5) 1 (6.5) 16 1 (5.5) 8 (1.5) 8 (1.5) 0 (7.5) 4 (3.0) 0 (7.5) 2 (4.0) 1 (5.5) 17 0 (7.0) 3 (2.0) 1 (4.5) 0 (7.0) 3 (2.0) 0 (7.0) 3 (2.0) 1 (4.5) 18 2 (2.0) 1 (4.0) 3 (1.0) 0 (7.0) 1 (4.0) 0 (7.0) 0 (7.0) 1 (4.0) 19 8 (2.5) 0 (7.0) 9 (1.0) 6 (4.0) 8 (2.5) 0 (7.0) 0 (7.0) 1 (5.0) 20 6 (2.0) 3 (4.5) 10 (1.0) 3 (4.5) 4 (3.0) 0 (8.0) 2 (6.0) 1 (7.0) 21 2 (4.0) 1 (6.5) 8 (1.0) 4 (2.0) 2 (4.0) 0 (8.0) 2 (4.0) 1 (6.5) 22 6 (2.0) 1 (6.5) 8 (1.0) 0 (8.0) 3 (4.5) 5 (3.0) 3 (4.5) 1 (6.5) 23 2 (2.5) 0 (7.0) 4 (1.0) 0 (7.0) 2 (2.5) 0 (7.0) 1 (4.5) 1 (4.5) 24 4 (1.5) 0 (6.5) 4 (1.5) 0 (6.5) 0 (6.5) 0 (6.5) 1 (3.5) 1 (3.5) 25 5 (1.5) 0 (7.0) 5 (1.5) 3 (3.0) 0 (7.0) 0 (7.0) 1 (4.5) 1 (4.5) 26 4 (1.5) 0 (7.5) 4 (1.5) 2 (4.5) 2 (4.5) 0 (7.5) 3 (3.0) 1 (6.0) 27 4 (1.0) 1 (6.5) 3 (2.0) 2 (4.0) 2 (4.0) 0 (8.0) 2 (4.0) 1 (6.5) 28 5 (1.0) 0 (7.0) 4 (2.0) 2 (3.5) 2 (3.5) 0 (7.0) 0 (7.0) 1 (5.0) 29 3 (3.5) 1 (6.0) 6 (1.0) 3 (3.5) 4 (2.0) 0 (8.0) 1 (6.0) 1 (6.0) 30 5 (1.0) 0 (7.5) 2 (2.5) 1 (5.0) 1 (5.0) 0 (7.5) 2 (2.5) 1 (5.0) 31 4 (2.5) 1 (5.5) 5 (1.0) 4 (2.5) 1 (5.5) 0 (8.0) 1 (5.5) 1 (5.5) 32 8 (1.5) 0 (7.5) 5 (4.5) 7 (3.0) 8 (1.5) 0 (7.5) 5 (4.5) 1 (6.0) 33 6 (3.0) 0 (8.0) 7 (2.0) 3 (4.0) 9 (1.0) 1 (6.0) 1 (6.0) 1 (6.0) 34 6 (2.0) 0 (7.5) 4 (3.0) 3 (4.5) 7 (1.0) 0 (7.5) 3 (4.5) 1 (6.0) 35 7 (1.0) 0 (6.5) 5 (2.0) 2 (3.0) 0 (6.5) 0 (6.5) 0 (6.5) 1 (4.0) avg. Results rank 3.7 4.5 2.1 5.4 3.9 6.2 4.7 5.6 Numbers in parentheses are the associated ranks. In case of ties, average ranks are assigned. The last row corresponds to the algorithm ranks averaged over the 35 datasets. Best performances are highlighted in bold. It is observed that all enriched biclusters have different GO enrichment. Note that CPGC is the original algorithm identifying a single submatrix of maximal sum per dataset Numbers in parentheses are the associated ranks. In case of ties, average ranks are assigned. The last row corresponds to the algorithm ranks averaged over the 35 datasets. Best performances are highlighted in bold. It is observed that all enriched biclusters have different GO enrichment. Note that CPGC is the original algorithm identifying a single submatrix of maximal sum per dataset terms (see Fig. 4a). Such a per dataset analysis further supports the benefits of our proposed method. identifies in each dataset are consistent with the con- trolled conditions under which these experiments were conducted. The full analysis is detailed in the supple- mentary materials associated with this manuscript [see Additional file 1]. It shows that the GO terms identified We further analyze the actual gene subsets identified by K-CPGC from Saccharomyces cerevisae samples to check whether the 20 most significantly enriched GO terms it Branders et al. BMC Bioinformatics (2019) 20:625 Page 6 of 13 Page 6 of 13 (2019) 20:625 Branders et al. BMC Bioinformatics Fig. 2 Critical difference of ranks. Comparison between the average rank of each algorithm over N = 35 datasets, with a 5% level of significance and Hochberg’s correction for multiple testing While this work focuses on the biological relevance of identified submatrices, it must be stressed that K-CPGC usually finds the best solutions in less than a minute1. On average, K-CPGC requires 14.7 s, the median being equal to 1.7 s. The longest run is performed within 195.7 s on dataset 18 (Yeoh-v1). Discussion The experiments and results reported in this work show that the K-CPGC method outperforms six well-known biclustering algorithms to identify biologically relevant gene subsets among subgroups of samples. The various algorithms are compared essentially based on their ability to return gene subsets which are associated with signifi- cantly enriched GO terms. One could consider that such a performance assessment validates only part of the results as it focuses on the genes (rows) and does not validate a posteriori the identified subgroups of samples (columns). This is actually a common limitation of the assessment of biclustering methods from gene expression data [6]. For the Saccharomyces cerevisae experiments reported here, there is no gold standard in terms of subgroups of sam- ples to be identified. Yet, these subgroups of samples are, at least indirectly, validated because their compo- nents directly influence the subsets of genes which are returned. This is particularly clear for the CPGC approach as one looks for a rectangular submatrix of maximal sum and the returned genes are directly constrained by the selected samples in such a submatrix. This is also true for biclustering algorithms since, for instance, they look Fig. 2 Critical difference of ranks. Comparison between the average rank of each algorithm over N = 35 datasets, with a 5% level of significance and Hochberg’s correction for multiple testing in the first four datasets, representative of cell cycles, are associated with some form of biogenesis, including ribosome, RNA, peptide and macromolecules synthe- sis. The GO terms identified in the next 12 datasets are indeed associated with various forms of response to stress-induced environments, including many representa- tives of the response to the stimulus, oxidation-reduction processes, and cellular responses to stress. Some GO terms also refer to generic responses to stress which are less specific to the controlled condition. For example, in the complete DTT dataset, many GO terms relate to alter- ation in the general patterns of protein biosyntheses as reported by Miller et al. [17]. The last experiment related to yeast sporulation includes GO terms referring to cell cycle, sporulation, and reproductive processes. Fig. 3 Comparison of K-CPGC and CCA p-values for enriched GO terms. This figure presents the (logarithmic) ratio of corrected p-values associated to each GO term identified by both K-CPGC and CCA on all the 35 datasets. Positive values (638 GO terms) are in favor of K-CPGC Fig. Discussion 3 Comparison of K-CPGC and CCA p-values for enriched GO terms. This figure presents the (logarithmic) ratio of corrected p-values associated to each GO term identified by both K-CPGC and CCA on all the 35 datasets. Positive values (638 GO terms) are in favor of K-CPGC Branders et al. BMC Bioinformatics (2019) 20:625 Page 7 of 13 Branders et al. BMC Bioinformatics Fig. 4 Comparison of terms enriched by K-CPGC and CCA for each of the 35 datasets. a Number of terms enriched by K-CPGC and CCA for each of the 35 datasets. The horizontal line reports the number of terms that are enriched by both approaches. b Number of times that the adjusted p-value of a term found by an algorithm is smaller than the adjusted p-value of the term found by the other algorithm. Only terms enriched by both algorithms are considered Fig. 4 Comparison of terms enriched by K-CPGC and CCA for each of the 35 datasets. a Number of terms enriched by K-CPGC and CCA for each of the 35 datasets. The horizontal line reports the number of terms that are enriched by both approaches. b Number of times that the adjusted p-value of a term found by an algorithm is smaller than the adjusted p-value of the term found by the other algorithm. Only terms enriched by both algorithms are considered Fig. 4 Comparison of terms enriched by K-CPGC and CCA for each of the 35 datasets. a Number of terms enriched by K-CPGC and CCA for each of the 35 datasets. The horizontal line reports the number of terms that are enriched by both approaches. b Number of times that the adjusted p-value of a term found by an algorithm is smaller than the adjusted p-value of the term found by the other algorithm. Only terms enriched by both algorithms are considered be interested to play with this parameter as it influences indirectly the sizes of the biclusters found. In the limit, if θ is set below the minimal expression value, all entries of the normalized matrix will be positive and the solution to the maximal sum problem is trivially identified as the full matrix. Similarly, if θ is set above the maximal expres- sion value, all entries of the normalized matrix become negative and the optimal solution is the empty matrix. An intermediate θ value between these extreme cases is typ- ically chosen. Discussion For a fixed data matrix increasing θ tends to produce smaller biclusters. The actual bicluster sizes found is difficult to predict exactly, as it also depends on the actual distribution of expression value in the matrix, but the analyst may easily play with θ to find biclusters of interest. for homogeneous expression patterns both across rows and columns. Notwithstanding, in a medical context, for example, the actual samples are typically associated with specific patients. In such a case, direct validation of the identified subgroups of samples could be performed by comparing these subgroups with actual clinical annota- tions. Interpreting the evaluation of unsupervised method on their ability to recover an expected structure is diffi- cult, however. As an illustrative example, Padilha et al. [6] evaluated the ability of several biclustering algorithms to recover the predefined sample classes. They showed that the best methods are biased towards methods that force every row and every column to be biclustered. The proposed K-CPGC method could be considered as including two control parameters: the number K of biclus- ters one looks for and the threshold θ defining the level of expression above which interesting patterns are searched. In the present work, we fix θ to the 75th percentile of expression values and we argue that this is a reasonable choice to find high expression patterns. Yet, the user may In the present study, for a fair comparison between all algorithms, we fix the maximal number of biclusters to be found to K = 10. In practice, however, this is not a criti- cal choice since the analyst can start with K = 1 and use the proposed gene enrichment analysis to check whether Branders et al. BMC Bioinformatics (2019) 20:625 Page 8 of 13 Page 8 of 13 Page 8 of 13 Branders et al. BMC Bioinformatics the successive biclusters returned by increasing K are still significantly enriched [see Additional file 2]. This could even be automated in a script and the actual value of K automatically determined in this way. biclustering algorithms, it does not look for homoge- neous gene expression values. The specific search order it follows could also be easily adapted to discover small relevant submatrices rather than large biclusters, hence focusing on rare but relevant expression patterns. Conclusions We propose a novel algorithm, K-CPGC, to find K non- redundant and possibly overlapping submatrices of max- imal sum from a large gene expression matrix. The returned solutions have the same bi-dimensional struc- ture as biclusters produced by existing biclustering algo- rithms. Yet, the mathematical objective is different and more explicitly optimized with the proposed methodol- ogy. Indeed, the role of a matrix entry Mi,j in a submatrix is clear: its contribution to the decision of including gene i and sample j in the submatrix is Mi,j. It follows that the contribution of each gene in the definition of a gene sub- set, respectively each sample, can be easily computed as the sum of matrix entries for each of the selected samples, respectively genes. Discussion As already mentioned in the “Interpretation” section, the K-CPGC approach can also be used to find low expres- sion patterns instead of high-level ones simply by consid- ering the opposite of the normalized data matrix. These are two obvious possibilities but it is straightforward to generalize this approach. For instance, if one would be interested in finding patterns of average expression values (neither over-expressed nor under-expressed), one can easily transform the original matrix to a new one, e.g. according to a Gaussian or RBF kernel, in which a higher value would represent an original entry closer to the aver- age expression value. This average (or median) value can be computed overall, row-wise or column-wise. Countless variants are easy to define and illustrate the flexibility of this approach. The K-CPGC method and the biclustering algorithms it is compared to are unsupervised methods since they do not require any particular annotation of the ana- lyzed samples. A different and interesting setting arises when the samples, or at least a fraction of them, are labeled according to various conditions or clinical vari- ables. In such a context, a new objective would be to identify subsets of genes that are maximally relevant to discriminate between subsets of samples from different conditions. Methods This section briefly presents six biclustering algorithms frequently cited in the literature and for which software implementations are publicly available [6, 7, 19–22]. Next, we present our own constraint programming approach, CPGC, to identify a submatrix of maximal sum and its extension to extract K submatrices. Our evaluation proto- col, including the data collection and experimental setup, is also detailed. Implicit search space Iterative Signature Algorithm (ISA) starts from a ran- domly selected bicluster and greedily adds or removes columns and rows till reaching some prescribed minimal average value TC (TR) across the selected columns (rows) [24]. Several biclusters can be found by restarting from another randomly selected bicluster. Any feasible solution to the problem is defined by a com- plete assignment of the selected rows and columns. Such a complete assignment defines a specific rectangular sub- matrix (I ⊆R; J ⊆C) of the original matrix. The full search space includes 2\|R\| −1 × 2\|C\| −1 non- empty feasible solutions. Yet, for any full assignment of the columns (no unbound Cj variable), the optimal assign- ment of the rows can be directly computed. Indeed, for any of the two dimensions being fixed, optimization along the other dimension is straightforward since it amounts to select only the subset of entries whose contribution is positive. For a fixed subset of columns J ⊆C, the optimal subset of rows I∗ J ⊆R that maximizes the objective value is identified as I∗ J = {i ∈R \| j∈J Mi,j ≥0}. QUalitative BIClustering (QUBIC) discretizes the orig- inal matrix and builds a graph where each node corre- sponds to a gene, and each edge weight is the number of samples for which two genes have the same nonzero discretized value. It then searches for biclusters corre- sponding to heavy subgraphs [19]. Plaid fits a generative statistical model with K compo- nents from which each entry Mij of the original matrix is assumed to have been generated [25]. K Mij = B + K k=1(μk + αik + βjk)ρikκjk + εij where B is a background level, μk is a specific bicluster effect, αik and βjk are row and column effects, ρ and κ are cluster mem- berships respectively along the rows and the columns, ε is a random noise. The Plaid algorithm fits such an addi- tive model by minimizing a mean square error between the modeled and observed data [26]. This algorithm may actually return less than K biclusters because a specific bicluster is returned only if it offers a better fit (= a lower residue) than biclusters found from random permutations of the original matrix. Efficient search and filtering The CPGC algorithm includes several refinements to speed up the search for an optimal solution. It uses a branch and bound strategy to avoid the exploration of proven suboptimal solutions. A CP algorithm usu- ally updates a best so far lower bound to the objective whenever it reaches a leaf of its search tree, that is when every decision variable is bound. For the MSSM problem, any partial assignment of the decision variables can be extended as a complete solution for which the unbound variables are set to 0. It is thus possible to update the best lower bound so far at each node of the search tree, which can improve the filtering of suboptimal solutions. Spectral relies on singular value decomposition to clus- ter genes and samples simultaneously after a specific normalization of rows and columns [27]. It looks for distinctive checkerboard patterns which form biclusters including contiguous rows and contiguous columns. The net result is a set of biclusters of low variance such that each gene and each sample exactly belong to a single bicluster. Implicit search space j In the gene expression analysis context, with order(s) of magnitude more rows (genes) than columns (samples), the actual search space is explored only over the column assignments. For each of the O(2\|C\|) column assignments, the optimal subset of rows can then be computed in linear time. Biclustering algorithms Cheng and Church’s Algorithm (CCA) is based on itera- tively adding or removing rows and columns to a current bicluster in order to minimize the variance within it [4]. The variance in a bicluster (I; J) is evaluated as a mean squared residue MSR = 1 \|I\|\|J\| i∈I,j∈J(Mij −MiJ −MIj + MIJ)2, where Mi,J is the average of the ith row in the bicluster, MI,j the average of the jth column, and MI,J the average of all elements in the bicluster. A parameter δ defines a threshold of maximum MSR for a bicluster to be accepted. The identification of multiple biclusters is achieved iteratively by replacing all entries of the previ- ously identified bicluster(s) by random values within the range of the original data matrix. Through enrichment analysis performed on 35 gene expression datasets from human tissues and Saccha- romyces cerevisae samples, we show that K-CPGC out- performs biclustering algorithms when looking for bio- logically relevant gene subsets. Not only is our approach efficient, but it also identifies more enriched biclusters than other biclustering methods. The K-CPGC approach provides stronger results (lower p-values of gene subsets or GO terms) than these alternative algorithms. These results illustrate the benefits of the proposed approach in terms of biological enrichments and biological relevance. Conserved Gene Expression Motifs (xMOTIFs) finds biclusters with simultaneously conserved genes in sub- sets of samples in a discretized data matrix [23]. Each discretized entry corresponds to a continuous range of expression values from the original matrix. Genes are considered conserved across a subset of samples if the discretized expression values are identical. This approach greedily searches for a largest xMOTIF start- ing from various random seeds. When such an xMO- TIF is found, the corresponding samples are removed from the original matrix and the whole process is iter- ated. This approach is thus constrained to return biclus- ters without overlap between the respective samples they contain. The K-CPGC is, however, not limited to gene expression analysis. For example, Liu and Wang [18] use a drug activ- ity dataset consisting of a matrix of 10,000 compounds with 30 features for each compound. The K-CPGC algo- rithm could be used to identify subsets of compounds presenting highly valued entries in subsets of features. This method has the potential to find relevant gene subsets across various -omics technologies since, unlike Page 9 of 13 Page 9 of 13 Branders et al. Biclustering algorithms BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics The CPGC method The CPGC method has been introduced in [8] to solve the Max-Sum Submatrix Problem (1) through constraint programming (CP). A solution to the MSSM problem is represented by two vectors of boolean decision variables R = (R1, . . . , Rm) for the rows and C = (C1, . . . , Cn) for the columns, with Ri ∈{0, 1} and Cj ∈{0, 1}. When a decision variable is equal to 1, its corresponding row or column is selected in the solution. When it is equal to 0, its corresponding row or column is not part of the selected submatrix. The algorithm searches through the space of possible variable assignments in the form of a tree as depicted in Fig. 5. Initially, at the root, all deci- sions variables are unbound and the algorithm explores such a tree in a depth-first fashion. Any configuration with no unbound variable defines a specific submatrix and is called a feasible solution. The goal is to find an opti- mal solution, i.e. a solution of maximal sum, among the feasible solutions. The complexity of this approach is defined by the num- ber of nodes explored and the complexity of the methods executed at each node. The CPGC approach explores O 2\|C\| nodes, or possible assignments of column vari- ables. The time complexity of the methods performed at each node of the search tree is in O(\|R\| × \|C\|). The global time complexity of CPGC is therefore in O 2\|C\| × \|R\| × \|C\| . The space complexity of the nodes is in O(\|R\| + \|C\|). The number of nodes to maintain effectively is in O(\|C\|), by virtue of the depth-first search exploration strategy. The global space complexity of CPGC is therefore in O (\|C\| × (\|R\| + \|C\|)). These bounds on the space and time complexities do not take into account the substantial reduction of the search space induced by the filtering procedures. In experiments with instance matrices of 10,000 rows by 1000 columns, the best solutions are found within short periods of time, Branders et al. BMC Bioinformatics (2019) 20:625 (2019) 20:625 Page 10 of 13 Branders et al. BMC Bioinformatics Fig. 5 Search tree. This figure illustrates the search tree defined on the set of possible submatrices. Identification of K biclusters The CPGC algorithm looks for a single submatrix of max- imal sum from an original data matrix while there might be several biclusters to be identified. In gene expres- sion analysis, the same gene may indeed participate in multiple pathways. Hence one would like to identify K biclusters with possible overlaps between them. The con- trol parameter K must be chosen by the data analyst (e.g. K = 10) but, as illustrated in the “Results” section, a biological interpretation of the biclusters found may help in this regard. Formally, any row and any column of the original data matrix may belong to zero, one up to K biclusters. Hence, each decision variable can now take 2K values. The extension of the MSSM problem to identify K solutions would thus lead to a search space con- taining O 2K\|R\| × 2K\|C\| feasible solutions. A completed assignment of the column variables does not help as in the “Implicit search space” section. Indeed, each decision for row i in submatrix k depends on the decisions on row i for the K −1 other submatrices. Consequently, one can no longer hope to find optimal solutions in a reasonable time from real gene expression datasets. Instead, we pro- pose to follow a greedy strategy as commonly adopted in several biclustering algorithms [1, 4, 5]. The time complexity of the method is computed as K times the complexity of the CPGC subroutine. The greedy procedure does not alter the space complexity. Identifying K submatrices with a large total sum is performed within a reasonable time (in the order of a minute), which is unsur- prising given the performances of the CPGC subroutine. An implementation of this greedy algorithm, called K-CPGC, is freely available as an R package from https:// github.com/vbranders/mssm. 1All reports of time in the present work are computed from experiments on a MacBook Pro (OS version 10.10.5) laptop (Intel i7-2720 CPU 2.20-3.30GHz, 1GB RAM per run) with a single thread. The CPGC method A question mark refers to an unbound variable that can be equal to 0 or 1 usually less than 10 s1. Moreover, providing more time (up to 1000 s) never improves the objective value. entries in this solution are replaced in the original matrix by zeros. A zero value is indeed neutral with respect to the maximal sum objective. In other words, any particular entry that has already been selected can again be selected but without any benefit nor loss in the objective value. Such a strategy allows for a possible overlap between sev- eral biclusters, neither forcing such overlap nor discarding it a priori. This process can be iterated till producing K biclusters. These results suggest that CPGC is scalable to tackle reasonably large problems from biological to biomedical domains. The interested reader is invited to consult [8] for further technical details about this approach. An evaluation study on human tissues and on Saccharomyces cerevisiae In this study, we look for biologically relevant biclus- ters computed from thirty-five publicly available gene expression microarray datasets. The first 18 datasets were obtained from human tissues using single-channel Affymetrix chips (Affy), proposed and preprocessed by de Souto et al. [28]. Similarly to the latter work, expres- sion values are transformed prior to further analyzes: M∗ i,j ←log2(Mi,j/mi) where mi is the median of row i and M∗ i,j is the value in row i and column j after trans- formation. The subsequent 17 datasets, proposed and preprocessed by [29], were obtained from Saccharomyces cerevisiae samples under various controlled conditions using double-channel cDNA (cDNA) technology. These expression values are left unaltered. Table 3 summarizes this collection by reporting the number of genes and samples measurements in each dataset. A first submatrix is found by solving the optimization problem (1) with CPGC. Next, the values of the selected Page 11 of 13 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. An evaluation study on human tissues and on Saccharomyces cerevisiae BMC Bioinformatics Table 3 Data collection summary Name Chip Genes Samples Organism Tissue/Condition 1 armstrong-v1 Affy 1081 72 Human Blood 2 armstrong-v2 Affy 2194 72 Human Blood 3 bhattacharjee Affy 1543 203 Human Lung 4 chowdary Affy 182 104 Human Breast, Colon 5 dyrskjot Affy 1203 40 Human Bladder 6 gordon Affy 1626 181 Human Lung 7 laiho Affy 2202 37 Human Colon 8 nutt-v1 Affy 1377 50 Human Brain 9 nutt-v2 Affy 1070 28 Human Brain 10 nutt-v3 Affy 1152 22 Human Brain 11 pomeroy-v1 Affy 857 34 Human Brain 12 pomeroy-v2 Affy 1379 42 Human Brain 13 ramaswamy Affy 1363 190 Human Multi-tissue 14 shipp Affy 798 77 Human Blood 15 singh Affy 339 102 Human Prostate 16 su Affy 1571 174 Human Multi-tissue 17 west Affy 1198 49 Human Breast 18 yeoh-v1 Affy 2526 248 Human Bone marrow 19 alpha factor cDNA 1099 18 Yeast Cell cycle synchronisation 20 cdc 15 cDNA 1086 24 Yeast Cell cycle synchronisation 21 cdc 28 cDNA 1044 17 Yeast Cell cycle synchronisation 22 elutriation cDNA 935 14 Yeast Cell cycle synchronisation 23 1mM menadione cDNA 1050 9 Yeast Environmental modification 24 1M sorbitol cDNA 1030 7 Yeast Environmental modification 25 15mM diamide cDNA 1038 8 Yeast Environmental modification 26 25mM DTT cDNA 991 8 Yeast Environmental modification 27 constant 32nM H2O2 cDNA 976 10 Yeast Environmental modification 28 diauxic shift cDNA 1016 7 Yeast Environmental modification 29 complete DTT cDNA 962 7 Yeast Environmental modification 30 heat shock 1 cDNA 988 8 Yeast Environmental modification 31 heat shock 2 cDNA 999 7 Yeast Environmental modification 32 nitrogen depletion cDNA 1011 10 Yeast Environmental modification 33 YPD 1 cDNA 1011 12 Yeast Environmental modification 34 YPD 2 cDNA 1022 10 Yeast Environmental modification 35 Yeast sporulation cDNA 1006 7 Yeast Sporulation Table 3 Data collection summary To compare all approaches on a fair basis, we look for (up to) K = 10 biclusters for each controlled experi- ment. As detailed below, some algorithms do not produce so many solutions while others, including K-CPGC, could be tuned to produce more solutions. Ten biclusters from each data matrix are also considered as reasonable for the subsequent biological interpretation of the results. Funding h g The UCLouvain has fully supported the design of the study, the analysis and interpretation of data as well as the writing of the manuscript. Results are reported on free and publicly available datasets. For each GO term, or functional class C, we calculate the p-value of the current submatrix enrichment as the probability of selecting at random at least c genes of this functional class C in the submatrix, where c is the actual number of genes from this class present in the current submatrix [19]. The smaller the p-values of the terms asso- ciated with a submatrix, the more likely the selected genes come from the same biological process. Availability of data and materials The K-CPGC approach is freely available as an R package: Evaluation In order to evaluate the biological relevance of the biclus- ters returned by the various algorithms in this study, a gene enrichment analysis is performed from the selected genes in each bicluster. Specifically, we perform an enrich- ment step for the selected genes through the Gene Ontol- ogy (GO; considering the Biological Process Ontology) [32] using the clusterProfiler R package [22]. Additional file 1: Gene subsets identified by K-CPGC. Additional_file_1.pdf provides tables of the 20 most enriched GO terms identified by K-CPGC on Saccharomyces cerevisiae samples from 17 different conditions. One table is provided per condition. Additional file 2: Evolution of the number of gO terms identified. Additional_file_2.pdf illustrates the evolution of the number of enriched biclusters and enriched GO terms as K increases. For each of the 35 datasets, each of the 8 algorithms pro- duces up to 10 biclusters. For each bicluster, the enrich- ment step provides a list of GO terms and FDR corrected p-values [33]. This p-value refers to the probability of selecting at random n genes out of the N genes from the original expression matrix, with c out of n being associated to the same functional class C. Let s be the true propor- tion of the N genes associated to the functional class C, the p-value associated to a GO term, or functional class C, is computed as: Supplementary information pp y Supplementary information accompanies this paper at https://doi.org/10.1186/s12859-019-3289-0. pp y Supplementary information accompanies this paper at y Supplementary information accompanies thi https://doi.org/10.1186/s12859-019-3289-0. Supplementary information accomp Experimental setup Instead, when comparing two algorithms A1 and A2, for any GO term considered significantly enriched (FDR corrected p-value < 5%) by both algorithms, one computes a performance difference as: diff(A1, A2) = −log pA1 pA2 (4) (4) The larger diff(A1, A2), the smaller the corrected p-value of pA1 compared to pA2 with a positive difference when- ever A1 outperforms A2. Experimental setup Our objective is to assess to which extent biclustering algorithms and our own K-CPGC approach are able to find biclusters representative of the controlled conditions in our evaluation study. To do so, we analyze the gene sub- sets found by each approach and we check which of them are significantly enriched. Page 12 of 13 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics (2019) 20:625 Branders et al. BMC Bioinformatics All algorithms used in this work are available through R packages: biclust [30], isa2 [31] and https://github. com/vbranders/mssm for K-CPGC. By default, the con- trol parameters of each biclustering algorithm are those recommended by their original authors. For example, as proposed by the authors of CCA, the original data matri- ces are initially multiplied by 100 to match the range of data values their control parameters are assuming. The discretization step of xMOTIFs is performed with 10 equally spaced intervals from minimum to maximum. The K-CPGC threshold θ (see Interpretation within Prob- lem definition) is set to the 75th percentile of expression values, specifically to each dataset. We consider such a threshold as representative of the objective of capturing high expression patterns. Given the performances of the CPGC approach on larger datasets, the K-CPGC method waits for convergence of the CPGC method. In other words, each call to the CPGC method is interrupted when- ever the solution is proved optimal, or the best solution has not been improved for 10 s. We additionally compare the performances of the CPGC subroutine to the other approaches. According to the methodology proposed in [6, 7, 19, 20], a specific bicluster is considered enriched if there is at least one GO term with a FDR corrected p-value below 5%. An algorithm is considered better if it produces more enriched biclusters. A refined analysis has also been proposed in [6, 34] through pairwise comparison of the smallest p-value among the GO terms found from the selected genes returned by each algorithm. Such a comparison could be criticized as it is limited to a single p-value for each algorithm, not necessarily computed for comparable GO terms. Authors’ contributions VB implemented and performed the experiments. VB and PD designed the study and analyzed the obtained results. 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https://openalex.org/W4206539984	https://link.springer.com/content/pdf/10.1007/JHEP12(2021)051.pdf	English	null	Flavored leptogenesis and neutrino mass with A4 symmetry	The Journal of high energy physics/The journal of high energy physics	2,021	cc-by	13,883	Published for SISSA by Springer Received: June 22, 2021 Revised: November 15, 2021 Accepted: November 21, 2021 Published: December 9, 2021 Received: June 22, 2021 Revised: November 15, 2021 Accepted: November 21, 2021 Published: December 9, 2021 Open Access, c⃝The Authors. Article funded by SCOAP3. Published for SISSA by Springer 4 Leptogenesis 4.1 Generation of mass splitting and CP asymmetry 4.1 Generation of mass splitting and CP 4.1.1 Lifting the mass degeneracy 4.1.2 Estimating CP asymmetry 4.2 Solution of Boltzmann equation 5 Conclusion 5 Conclusion A A4 multiplication rules Flavored leptogenesis and neutrino mass with A4 symmetry https://doi.org/10.1007/JHEP12(2021)051 Contents 1 Introduction 1 2 Structure of the model 3 3 Neutrino phenomenology 8 3.1 Constraining the parameter space 8 3.2 Implications for light neutrino masses and low energy phase 10 3.3 Neutrinoless double beta decay 11 3.4 Lepton ﬂavor violation 12 4 Leptogenesis 12 4.1 Generation of mass splitting and CP asymmetry 13 4.1.1 Lifting the mass degeneracy 14 4.1.2 Estimating CP asymmetry 15 4.2 Solution of Boltzmann equation 17 5 Conclusion 20 A A4 multiplication rules 21 Contents 1 Introduction 1 2 Structure of the model 3 3 Neutrino phenomenology 8 3.1 Constraining the parameter space 8 3.2 Implications for light neutrino masses and low energy phase 10 3.3 Neutrinoless double beta decay 11 3.4 Lepton ﬂavor violation 12 4 Leptogenesis 12 4.1 Generation of mass splitting and CP asymmetry 13 4.1.1 Lifting the mass degeneracy 14 4.1.2 Estimating CP asymmetry 15 4.2 Solution of Boltzmann equation 17 5 Conclusion 20 A A4 multiplication rules 21 Contents 1 Introduction 1 2 Structure of the model 3 3 Neutrino phenomenology 8 3.1 Constraining the parameter space 8 3.2 Implications for light neutrino masses and low energy phase 10 3.3 Neutrinoless double beta decay 11 3.4 Lepton ﬂavor violation 12 4 Leptogenesis 12 4.1 Generation of mass splitting and CP asymmetry 13 4.1.1 Lifting the mass degeneracy 14 4.1.2 Estimating CP asymmetry 15 4.2 Solution of Boltzmann equation 17 5 Conclusion 20 A A4 multiplication rules 21 1 Introduction 2 Structure of the model 3 Neutrino phenomenology 3.1 Constraining the parameter space 3.2 Implications for light neutrino masses and low energy phase JHEP12(2021)051 3.2 Implications for light neutrino masses and low energy phase 3.3 Neutrinoless double beta decay 3.3 Neutrinoless double beta decay 3.3 Neutrinoless double beta decay 3.4 Lepton ﬂavor violation Flavored leptogenesis and neutrino mass with A4 symmetry JHEP12(2021)051 Arghyajit Datta,a Biswajit Karmakarb,c and Arunansu Sila aDepartment of Physics, Indian Institute of Technology, Guwahati, Assam-781039, India bDepartment of Physics, Indian Institute of Technology, Hyderabad, 502285 Telangana, India cInstitute of Physics, University of Silesia, 40-007 Katowice, Poland E-mail: datta176121017@iitg.ac.in, biswajit.karmakar@us asil@iitg.ac.in Arghyajit Datta,a Biswajit Karmakarb,c and Arunansu Sila aDepartment of Physics, Indian Institute of Technology, Guwahati, Assam-781039, India bDepartment of Physics, Indian Institute of Technology, Hyderabad, 502285 Telangana, India cInstitute of Physics, University of Silesia, 40-007 Katowice, Poland E-mail: datta176121017@iitg.ac.in, biswajit.karmakar@us.edu asil@iitg.ac.in Arghyajit Datta,a Biswajit Karmakarb,c and Arunansu Sila aDepartment of Physics, Indian Institute of Technology, Guwahati, Assam-781039, India bDepartment of Physics, Indian Institute of Technology, Hyderabad, 502285 Telangana, India cInstitute of Physics, University of Silesia, 40-007 Katowice, Poland E-mail: datta176121017@iitg.ac.in, biswajit.karmakar@us.e asil@iitg.ac.in Abstract: We propose a minimal A4 ﬂavor symmetric model, assisted by Z2×Z3 symme- try, which can naturally takes care of the appropriate lepton mixing and neutrino masses via Type-I seesaw. It turns out that the framework, originated due to a speciﬁc ﬂavor structure, favors the normal hierarchy of light neutrinos and simultaneously narrows down the range of Dirac CP violating phase. It predicts an interesting correlation between the atmospheric mixing angle and the Dirac CP phase too. While the ﬂavor structure indicates an exact degeneracy of the right-handed neutrino masses, renormalization group running of the same from a high scale is shown to make it quasi-degenerate and a successful ﬂa- vor leptogenesis takes place within the allowed parameter space obtained from neutrino phenomenology. Keywords: Beyond Standard Model, Neutrino Physics ArXiv ePrint: 2106.06773 Open Access, c⃝The Authors. Article funded by SCOAP3. 1 Introduction Over the last couple of decades, we have witnessed remarkable success in neutrino experi- ments [1–9] indicating that neutrinos are indeed massive. Furthermore, mixing parameters have been measured with great precision. In fact, two of the three mixing angles namely solar (θ12) and atmospheric (θ23) ones are found to be large while the other one, reactor (θ13) mixing angle, is relatively small. Such a ﬁnding clearly shows the distinctive feature associated to the lepton sector in contrast to the quark one where all the three mixing angles are measured to be small. To have a deeper understanding of it, one needs to in- vestigate the origin of the neutrino mass by looking at the neutrino mass matrix as well as the charged lepton sector from a symmetry perspective. To address the tiny neutrino mass issue, various seesaw mechanisms [10–17] have been proposed by extending the Standard Model (SM) with heavy fermions/scalars. Among these, the type-I seesaw mechanism provides perhaps the simplest explanation of tiny neutrino mass where the SM is extended by three singlet right-handed neutrinos (RHN) [10– 12]. Involvement of ﬂavor symmetries within this simple setup is oﬀcourse an interesting possibility in order to explain the typical mixing pattern in the lepton sector. Non-abelian discrete groups (like S3, A4, S4, A5, ∆(27) etc.) in this regard have been extensively used (see reviews [18–25] and references therein). – 1 – Among the various discrete groups, A4 turns out to be the most economical one.1 It is a group of even permutations of four objects having three inequivalent one-dimensional representations (1, 1′ and 1′′) as well as one three-dimensional representation (3). In- terestingly, the three generations (or ﬂavors) of right-handed charged lepton singlets can naturally ﬁt into these three inequivalent one-dimensional representations of A4 while the three SM lepton doublets can be accommodated into the triplet representation of A4 [28– 30]. Works along this direction [28, 29, 31, 32] showed that type-I seesaw model with A4 ﬂavor symmetry in general leads to a typical tri-bimaximal (TBM) lepton mixing (sin2 θ12 = 1/3, sin2 θ23 = 1/2 and θ13 = 0) pattern [33, 34] in presence of SM singlet (though charged under A4) ﬂavon ﬁelds. Though such TBM pattern received a great deal of attention due to its close proximity with experimental observation prior to 2012, it fails to accommodate the recent observation of small, but non-zero θ13 [8, 35, 36]. 1A4 group was initially proposed as an underlying family symmetry for quark sector by [26, 27]. 1 Introduction Subsequently, modiﬁcations over models based on A4 (and other discrete groups) are suggested to accom- modate non-zero θ13 either by considering additional ﬂavon ﬁelds or including corrections to vaccuum alignments of the ﬂavons [37, 38] or considering contributions to additional mixing from the charged lepton sector [39]. JHEP12(2021)051 In this work, we particularly focus on a framework where a non-trivial contribution to lepton mixing is originated from charged lepton sector. We do not consider any ad- ditional ﬂavon ﬁeld apart from those ones incorporated in the original Altarelli-Feruglio (AF) model [28]. While the RHN mass matrix turns out to be diagonal as a result of the ﬂavor symmetry imposed, the structure of the charged lepton mass matrix becomes such that it can be diagonalized by a complex ‘magic’ matrix [29]. Interestingly, an antisym- metric contribution to the Dirac neutrino mass matrix, originated from the product of two A4 triplets, plays a crucial role in generating non-zero θ13 [40–43] in our model which was overlooked in an earlier attempt [44]. In doing the analysis, we ﬁnd the atmospheric mixing angle θ23 ≤45◦i.e. to lie in lower octant (LO). We also note that only normal hierarchy (NH) of light neutrino masses are allowed in this model. This turns out to be another salient feature of our construction. These predictions can be tested in ongoing and future neutrino experiments as ambiguities are still present in determining octant for θ23 as well as hierarchies of light neutrino masses. Additionally, we also discuss the aspects of leptogenesis [45–49] from the CP-violating decays of RHNs in this A4 based type-I seesaw scenario in line with observations [50–57]. In doing so, since the involvement of the neutrino Yukawa matrix in the charged-lepton mass diagonal basis is necessary, the speciﬁc ﬂavor symmetric construction of it is expected to play an important role. In fact, due to this symmetry, exactly degenerate heavy RHNs result at tree level, thereby indicating the breaking of the perturbative ﬁeld theory involved in CP asymmetry generation [58]. Following [44], we are able to show that running of the parameters involved in the neutrino sector from the ﬂavor symmetry breaking scale to the RHN mass scale actually eliminates such exact degeneracies and as a result, leptogenesis can indeed be possible. 1 Introduction The present study of matter-antimatter asymmetry generation via leptogenesis taking into account the eﬀect of running however diﬀers from that of [44] by – 2 – Fields ℓ eR µR τR NR H ϕ Φ Ψ SM (2, 1/2) (1, 1) (1, 1) (1, 1) (1, 0) (2, −1/2) (1, 0) (1, 0) (1, 0) A4 3 1 1′ 1′′ 3 1 1 3 3 Z2 1 1 1 1 −1 1 −1 1 −1 Z3 ω 1 1 1 1 1 ω ω ω Table 1. Representations of the ﬁelds under SU(2)L × U(1)Y × A4 × Z2 × Z3 symmetry Table 1. Representations of the ﬁelds under SU(2)L × U(1)Y × A4 × Z2 × Z3 symmetry two aspects. Firstly, we use less number of ﬂavon ﬁelds and secondly, we present a detailed analysis of ﬂavored leptogenesis by solving the relevant Boltzmann equations. JHEP12(2021)051 The rest of the paper is organized as follows. In section 2 we present detail structure of the model including the analysis of the mixing matrices involved. Section 3 deals with phenomenology of neutrino mixing. Constrains and predictions on neutrino parameters (including neutrinoless double beta decay) involved are presented here. In section 4 we perform a detailed study on leptogenesis solving ﬂavored Boltzmann equations. Finally in section 5 we summarize the results and make ﬁnal conclusion. 2 Structure of the model To realize the canonical type-I seesaw mechanism, we ﬁrst consider an extension of the SM by including three singlet RHN ﬁelds (NR). Additionally, three ﬂavon ﬁelds namely Φ, Ψ, ϕ and a discrete symmetry A4 × Z2 × Z3 are also incorporated to probe the typical ﬂavor structure involved in the lepton sector. Note that same ﬁelds content was also present in the original AF [18] construction. Here NR and the ﬂavon ﬁelds Φ, Ψ transform as triplet, whereas ϕ transforms as a singlet under A4. A judicious choice of additional Z2 × Z3 symmetry assists the leptonic mass matrices to take speciﬁc forms and hence forbid several unwanted contributions. In table 1, we present transformation properties of all the relevant SM ﬁelds, NR and ﬂavons involved in the analysis. The relevant eﬀective Lagrangian involving charged leptons and neutrinos can be writ- ten as L ⊃yℓ 1 Λ ¯ℓΦ 1 H eR + yℓ 2 Λ ¯ℓΦ 1′′ H µR + yℓ 3 Λ ¯ℓΦ 1′ H τR (2.1) + yν 1 Λ h (¯ℓNR)s Ψ i 1 ˜H + yν 2 Λ h (¯ℓNR)a Ψ i 1 ˜H + yν 3 Λ ¯ℓNR 1 ϕ ˜H + 1 2M Nc RNR + h.c., (2.1) where yℓ,ν i=1,2,3 are the respective coupling constants, M is the mass parameter of RHNs and Λ is the cut-oﬀscale of the theory. In the ﬁrst line of eq. (2.1), terms in the ﬁrst parentheses represent products of two A4 triplets forming a one-dimensional representation which further contract with 1, 1′ and 1′′ of A4, corresponding to eR, µR and τR respectively, to make a true singlet under A4. On the other hand, in the second line of eq. (2.1), the subscripts s, a correspond to symmetric and anti-symmetric parts of triplet products in the S diagonal basis of A4. The essential multiplication rules of the A4 group elements are elaborated in appendix A. – 3 – From table 1 it is evident that the tree level contribution to charged lepton Yukawa in- teraction, ¯ℓHαR (with α = e, µ, τ), gets forbidden. Instead, such interactions are eﬀectively generated once the ﬂavon Φ gets a vacuum expectation value (vev) via the dimension-5 op- erators (present in ﬁrst line of eq. (2.1)). 2Such vev alignments of the ﬂavons is widely used and can be realised in a natural way by minimisi the scalar potential following the approach of [18, 40, 44, 59–62]. 2 Structure of the model Similarly in the neutrino sector, the renormalizable Dirac Yukawa coupling is forbidden as the lepton doublet ℓis charged under Z3 whereas both NR and H transform trivially under it. However such eﬀective Yukawa coupling is generated from dimension-5 operators involving ﬂavons Ψ and ϕ, after they obtain vevs. Presence of Z2 symmetry is important in identifying Φ from Ψ (both being A4 triplet) so that they contribute to the charged lepton and Dirac neutrino Yukawa couplings diﬀerently. Th ﬂ ﬁld b k th ﬂ t A Z Z h th i l 2 JHEP12(2021)051 JHEP12(2021)051 ⟨ϕ⟩= vϕ , ⟨Φ⟩= vΦ (1, 1, 1) , ⟨Ψ⟩= vΨ (0, 1, 0) , (2.2) (2.2) as a result of which the part of the Lagrangian contributing to the charged lepton sector can be written as Ll = yℓ 1vΦ Λ (¯ℓe+¯ℓµ+¯ℓτ)H eR+yℓ 2vΦ Λ (¯ℓe+ω¯ℓµ+ω2¯ℓτ)H µR+yℓ 3vΦ Λ (¯ℓe+ω2¯ℓµ+ω¯ℓτ)H τR. (2. Using the above Lagrangian one obtains the charged lepton mass matrix after the elec- troweak symmetry breaking as Using the above Lagrangian one obtains the charged lepton mass matrix after the elec- troweak symmetry breaking as Y ℓ= v      fℓ 1 fℓ 2 fℓ 3 fℓ 1 ω fℓ 2 ω2 fℓ 3 fℓ 1 ω2 fℓ 2 ω fℓ 3     ; fℓ i = vΦ Λ yℓ i with i = 1, 2, 3, (2.4) (2.4) where v= 174 GeV stands for the vev of the SM Higgs. where v= 174 GeV stands for the vev of the SM Higgs. In a similar way, the Lagrangian for neutrino sector after breaking of the ﬂavor sym- metries can be written as Lν = yν 3 Λ vϕ ¯ℓe N1R + ¯ℓµ N2R + ¯ℓτ N3R ˜H + (yν 1 −yν 2) vΨ Λ ¯ℓe N3R ˜H + (yν 1 + yν 2) vΨ Λ ¯ℓτ N1R ˜H + M Nc1RN1R + Nc2RN2R + Nc3RN3R + h.c.. with fν i = vΨ Λ yν i , i = 1, 2, 3. with fν i = vΨ Λ yν i , i = 1, 2, 3. 2 Structure of the model (2.5) (2.5) This yields the corresponding Dirac and Majorana mass matrices as Y ν =      fν 3 0 fν 1 −fν 2 0 fν 3 0 fν 1 + fν 2 0 fν 3     , (2.6) MR =      M 0 0 0 M 0 0 0 M     , (2.7) (2.6) (2.7) with fν i = vΨ Λ yν i , i = 1, 2, 3. 2Such vev alignments of the ﬂavons is widely used and can be realised in a natural way by minimising the scalar potential following the approach of [18, 40, 44, 59–62]. 2Such vev alignments of the ﬂavons is widely used and can be realised in a natural way by minimising the scalar potential following the approach of [18, 40, 44, 59–62]. – 4 – Let us now discuss the diagonalization of the charged lepton and neutrino mass matrices so as to obtain the lepton mixing matrix. First we note that the charged lepton mass matrix given in eq. (2.4) can be diagonalized by a bi-unitary transformation Y ℓ= V dℓI3×3 with dℓ= √ 3v diag (fℓ 1, fℓ 2, fℓ 3), (2.8) (2.8) where I3×3 is a 3 × 3 identity matrix and where I3×3 is a 3 × 3 identity matrix and V = 1 √ 3      1 1 1 1 ω ω2 1 ω2 ω     , (2.9) (2.9) JHEP12(2021)051 where ω (= e2iπ/3) is the cube root of unity. From eq. (2.7), it is evident that the right- handed Majorana neutrino mass matrix MR is diagonal having degenerate mass eigenvalues (M) to start with. where ω (= e2iπ/3) is the cube root of unity. From eq. (2.7), it is evident that the right- handed Majorana neutrino mass matrix MR is diagonal having degenerate mass eigenvalues (M) to start with. On the other hand, fν 1 and fν 2 appearing in eq. (2.6) are the symmetric and antisym- metric contributions to the Dirac neutrino Yukawa respectively, originated as products of two A4 triplets ℓand NR which further contract with Φ (see the product rules eq. (A.5) and (A.6). This antisymmetric part plays an instrumental role3 in realizing correct neutrino oscillation data. 2 Structure of the model Here it is worth mentioning that in the vanishing limit of fν 2 →0, (keeping the structure of the charged lepton and Majorana mass matrix intact) one can reproduce the TBM mixing as discussed in [44]. The eﬀective light neutrino mass4 matrix can be obtained within the type-I seesaw framework as mν = −mDM−1 R mT D, (2.10) (2.10) where the structure of MR is given in eq. (2.7). Now, from eq. (2.6) and (2.8), in the basis where the charged leptons are diagonal, the Dirac neutrino mass matrix can be written as, where the structure of MR is given in eq. (2.7). Now, from eq. (2.6) and (2.8), in the basis where the charged leptons are diagonal, the Dirac neutrino mass matrix can be written as, mD = vV †Y ν = vYν. (2.11) (2.11) Therefore, substituting eq. (2.11) in the type-I seesaw formula given by eq. (2.10) one obtains the light neutrino mass matrix as mν = −v2V †Y νM−1 R Y νT V ∗, (2.12) = −1 M V †(v2Y νY νT )V ∗. (2.13) (2.12) (2.13) 3Earlier the role of such antisymmetric contributions was analyzed in the context of Dirac neutrinos [40– 3Earlier the role of such antisymmetric contributions was analyzed in the context of Dirac neutrinos [40– 43]. 4Wi h h i i d i bl 1 i i i l h ill b ib i h ﬀ i li h y y [ 43]. 4With the symmetries mentioned in table 1 in principle there will be a contribution to the eﬀective light ] 4With the symmetries mentioned in table 1 in principle, there will be a contribution to the eﬀective light neutrino mass via a dim-6 operator given by yeﬀ Λ2 (ℓHℓHΦ). However, in the limit vΦ > M, this additional contribution can be neglected compared to the dominant type-I contribution considered here. – 5 – Clearly, to get the mass eigenvalues of light neutrinos we need to diagonalize Y νY νT where Clearly, to get the mass eigenvalues of light neutrinos we need to diagonalize Y νY νT where  2 2  Y νY νT =      (fν 1 −fν 2 )2 + fν2 3 0 2fν 1 fν 3 0 fν2 3 0 2fν 1 fν 3 0 (fν 1 + fν 2 )2 + fν2 3     . 2 Structure of the model (2.14) (2.14) Though Y ν is in general a complex matrix, Y νY νT being a complex symmetric matrix can be diagonalized by an orthogonal transformation (in the (1, 3) plane) through the relation UT 13(Y νY νT )U13 = d2 D = diag(λ1, λ2, λ3), (2.15) (2.15) JHEP12(2021)051 where the rotation matrix U13 (parametrised by angle θ and phase ψ) is given by U13 =      cos θ 0 e−iψ sin θ 0 1 0 −eiψ sin θ 0 cos θ     . (2.16) (2.16) The complex eigenvalues are given by The complex eigenvalues are given by λ1 = fν2 1 + fν2 2 + fν2 3 −2 q fν2 1 (fν2 2 + fν2 3 ), (2.17) λ2 = fν2 3 , (2.18) λ3 = fν2 1 + fν2 2 + fν2 3 + 2 q fν2 1 (fν2 2 + fν2 3 ). (2.19) λ1 = fν2 1 + fν2 2 + fν2 3 −2 q fν2 1 (fν2 2 + fν2 3 ), (2.17) λ2 = fν2 3 , (2.18) λ3 = fν2 1 + fν2 2 + fν2 3 + 2 q fν2 1 (fν2 2 + fν2 3 ). (2.19) Now substituting eq. (2.15) in eq. (2.13), we get mν = −V †U13 v2d2 D M ! UT 13V ∗, (2.20) = −V †U13 (dν) UT 13V ∗, (2.21) (2.20) (2.21) where dν = v2d2 D/M is a diagonal matrix having diagonal elements v2λi/M (i = 1, 2, 3), representative of three complex light neutrino mass eigenvalues. In order to extract the real and positive light neutrino mass eigenvalues, we choose the following representations of the parameters fν 1,2,3(= \|fν 1,2,3\|eiφ1,2,3 and φ1,2,3 are the three phases associated) as fν 1 fν 3 = \| fν 1 \| \| fν 3 \|ei(φ1−φ3) = χ1eiγ1, (2.22) fν 2 fν 3 = \| fν 2 \| \| fν 3 \|ei(φ2−φ3) = χ2eiγ2, (2.23) (2.22) (2.23) where χ1 = \|fν 1 /fν 3 \|, χ2 = \|fν 2 /fν 3 \| and (φ1 −φ3) = γ1, (φ2 −φ3) = γ2 are the redeﬁned parameters used for the rest of our analysis. Now we are in a position to deﬁne the rotation angle θ and phase ψ of U13 matrix (see eq. 2 Structure of the model (2.16)) as: Now we are in a position to deﬁne the rotation angle θ and phase ψ of U13 matrix (see eq. (2.16)) as: tan 2θ = 2χ1 2χ1χ2 cos γ2 cos ψ − χ2 1 sin γ1 + χ2 2 sin(2γ2 −γ1) −sin γ1 sin ψ, (2.24) −2χ1χ2 sin γ2 tan 2θ = 2χ1 2χ1χ2 cos γ2 cos ψ − χ2 1 sin γ1 + χ2 2 sin(2γ2 −γ1) −sin γ1 sin ψ, (2.24) tan ψ = −2χ1χ2 sin γ2 cos γ1 + χ2 2 cos(2γ2 −γ1) + χ2 1 cos γ1 . (2.25) (2.24) (2.25) – 6 – – 6 – Similarly, the real and positive light neutrino masses can also be expressed in terms of χ1,2 and γ1,2 after we extract the phases from the complex eigenvalues. To proceed, note that eq. (2.21) can be rewritten as Similarly, the real and positive light neutrino masses can also be expressed in terms of χ1,2 and γ1,2 after we extract the phases from the complex eigenvalues. To proceed, note that eq. (2.21) can be rewritten as mν ≡Udiag(m1, m2, m3)UT , (2.26) (2.26) with with with U = V †U13ei π 2 Up. (2.27) th U = V †U13ei π 2 Up. 2 Structure of the model (2.27) (2.27) Here Up stands for a diagonal phase matrix given by Up = diag(1, eiβ21/2, eiβ31/2), and the real positive light neutrino masses are given by: Here Up stands for a diagonal phase matrix given by Up = diag(1, eiβ21/2, eiβ31/2), and the real positive light neutrino masses are given by: JHEP12(2021)051 m1 = v2 M \| fν2 3 \| q o2r + o2 i , (2.28) m2 = v2 M \| fν2 3 \|, (2.29) m3 = v2 M \| fν2 3 \| q n2r + n2 i , (2.30) m1 = v2 M \| fν2 3 \| q o2r + o2 i , (2.28) m2 = v2 M \| fν2 3 \|, (2.29) m3 = v2 M \| fν2 3 \| q n2r + n2 i , (2.30) (2.28) (2.29) (2.30) where or, oi, nr and ni can be written in terms of the associated parameters (χ1, χ2, γ1 and γ2) in our model as or = χ2 1 cos 2γ1 + χ2 2 cos 2γ2 + 1 −2Aχ1 cos γ1 + 2Bχ1 sin γ1, (2.31) oi = χ2 1 sin 2γ1 + χ2 2 sin 2γ2 −2Aχ sin γ1 −2Bχ1 cos γ1, (2.32) nr = χ2 1 cos 2γ1 + χ2 2 cos 2γ2 + 1 + 2Aχ1 cos γ1 −2Bχ1 sin γ1, (2.33) ni = χ2 1 sin 2γ1 + χ2 2 sin 2γ2 + 2Aχ sin γ1 + 2Bχ1 cos γ1., (2.34) A = r 1 + χ2 2 cos 2γ2 + q 1 + χ4 2 + 2χ2 2 cos 2γ2 √ 2 , B = χ2 2 sin 2γ2 2A . (2.35) A = r 1 + χ2 2 cos 2γ2 + q 1 + χ4 2 + 2χ2 2 cos 2γ2 √ 2 , B = χ2 2 sin 2γ2 2A . (2.35) The phases β21(31) involved in Up are given by, β21 = −tan−1 oi or , β31 = tan−1 ni nr −tan−1 oi or . (2.36) (2.36) Therefore using eqs. (2.9), (2.16) and (2.27), the ﬁnal form of the mixing matrix U which diagonalises the eﬀective light neutrino mass matrix (in the charged lepton diagonal basis) can now be written as Therefore using eqs. 2 Structure of the model (2.37) and (2.38) we get the correlation between the neutrino mixing angles (and Dirac CP phase) appearing in UPMNS and the model parameters as [40] \| s13 \|2 = 1 + sin 2θ cos ψ 3 , tan δ = sin θ sin ψ cos θ + sin θ cos ψ, (2.39) s2 12 = 1 3(1−\| s13 \|2), tan 2θ23 cos δ = 1 −2 \| s13 \|2 \| s13 \| p 2 −3 \| s13 \|2 . (2.40) \| s13 \|2 = 1 + sin 2θ cos ψ 3 , s2 12 = 1 3(1−\| s13 \|2), tan δ = sin θ sin ψ cos θ + sin θ cos ψ, (2.39) tan 2θ23 cos δ = 1 −2 \| s13 \|2 \| s13 \| p 2 −3 \| s13 \|2 . (2.40) (2.39) Additionally, the two Majorana phases α21 and α31 are identiﬁed as α21 = β21, and α31 = β31 (ignoring the irrelevant common phase). These correlations given in eq. (2.39)– (2.40) are the keys to the subsequent analysis of neutrino phenomenology. 5The Majorana phases are insensitive to neutrino oscillation experiments. However, they may play an important role in neutrinoless double beta decay [65]. 2 Structure of the model (2.9), (2.16) and (2.27), the ﬁnal form of the mixing matrix U which diagonalises the eﬀective light neutrino mass matrix (in the charged lepton diagonal basis) can now be written as U =      cos θ−eiψ sin θ √ 3 1 √ 3 cos θ+e−iψ sin θ √ 3 cos θ−ωeiψ sin θ √ 3 ω2 √ 3 ω cos θ+e−iψ sin θ √ 3 cos θ−ω2eiψ sin θ √ 3 ω √ 3 ω2 cos θ+e−iψ sin θ √ 3     eiπ/2Up. (2.37) (2.37) U is therefore the lepton mixing matrix, called the Pontecorvo-Maki-Nakagawa-Sakata (UPMNS) matrix, the standard form of which is given by [63], UPMNS =      c12c13 s12c13 e−iδs13 −s12s23 −eiδc12s13s23 c12c23 −eiδs12s13s23 c13s23 s12s23 −eiδc12s13s23 −c12c23 −eiδs12s13s23 c13c23     Um, (2.38) (2.38) – 7 – – 7 – parameters best ﬁt value 3σ range sin2 θ12 0.304 0.269 →0.343 sin2 θ23 0.573 0.415 →0.616 sin2 θ13 0.02219 0.02032 →0.02410 δCP /◦ 197 120 →369 ∆m2 21 10−5 eV 2 7.42 6.82 →8.04 ∆m2 31 10−3 eV 2 +2.517 +2.435 →+2.598 Table 2. neutrino oscillation data obtained from NuFIT [64] for NH scenario of light neutrino mass. Table 2. neutrino oscillation data obtained from NuFIT [64] for NH scenario of light neutrin mass. Table 2. neutrino oscillation data obtained from NuFIT [64] for NH scenario of light neutrino mass. JHEP12(2021)051 where cij = cos θij and sij = sin θij, and δ is the CP violating Dirac phase. Also, Um = diag(1, eiα21/2, eiα31/2) is a phase matrix which contains two Majorana phases α21 and α31. Comparing above two matrices given in eq. (2.37) and (2.38) we get the correlation between the neutrino mixing angles (and Dirac CP phase) appearing in UPMNS and the model parameters as [40] where cij = cos θij and sij = sin θij, and δ is the CP violating Dirac phase. Also, Um = diag(1, eiα21/2, eiα31/2) is a phase matrix which contains two Majorana phases α21 and α31. Comparing above two matrices given in eq. 3.1 Constraining the parameter space As seen from eqs. (2.39) and (2.40) in conjugation with eqs. (2.24) and (2.25), all the mixing angles (θ13, θ12, θ23) and the Dirac CP phase (δ) involved in the lepton mixing matrix UPMNS are ﬁnally determined by the model parameters χ1, χ2, γ1 and γ2. Hence, using the 3σ allowed ranges of the three mixing angles (θ13, θ12, θ23) from neutrino oscillation data5 presented in table 2, we can restrict parameter space for χ1,2 and γ1,2. This parameter space of the current set-up can be further constrained using the 3σ allowed ranges of the mass-squared diﬀerences (see table 2). For that purpose, we introduce a dimensionless quantity r, deﬁned as the ratio of solar to atmospheric mass squared diﬀerence for normal hierarchy, i.e., r = ∆m2 21 ∆m2 31 with ∆m2 21 = m2 2 −m2 1 and ∆m2 31 = m2 3 −m2 1 . Using the three light neutrino mass eigenvalues given in eq. (2.28)–(2.30), we are able to rewrite it as r = ∆m2 21 ∆m2 31 = 1 −o2 r −o2 i n2r + n2 i −o2r −o2 i . (3.1) (3.1) 5The Majorana phases are insensitive to neutrino oscillation experiments. However, they may play an important role in neutrinoless double beta decay [65]. 5The Majorana phases are insensitive to neutrino oscillation experiments. However, they may play an important role in neutrinoless double beta decay [65]. – 8 – Figure 1. Allowed parameter spaces of χ1-χ2 (left panel) and γ1-γ2 (right panel) using 3σ ranges of neutrino oscillation parameters [64]. The light blue dots in both the panels correspond to 3σ allowed values for the mixing angles while the darker patches in each panel further satisfy constraints coming from the mass-squared diﬀerences, their ratio and sum of absolute masses. ⋆, ▲, ■, ♦marks of the right panel are indicative of four benchmark points (BP) used in section 4. JHEP12(2021)051 Figure 1. Allowed parameter spaces of χ1-χ2 (left panel) and γ1-γ2 (right panel) using 3σ ranges of neutrino oscillation parameters [64]. The light blue dots in both the panels correspond to 3σ allowed values for the mixing angles while the darker patches in each panel further satisfy constraints coming from the mass-squared diﬀerences, their ratio and sum of absolute masses. ⋆, ▲, ■, ♦marks of the right panel are indicative of four benchmark points (BP) used in section 4. Substituting or,i, nr,i from eq. 3.1 Constraining the parameter space (2.31)–(2.34) into eq. (3.1), we note that r now becomes function of χ1, χ2, γ1 and γ2. Apart from the satisfaction of r value obtained from the ratio of the best ﬁt values of mass-squared diﬀerences, we must satisfy both the individual mass-squared diﬀerences, ∆m2 21 and ∆m2 31, independently within their 3σ allowed ranges using eqs. (2.28)–(2.30). There also exists a cosmological upper bound on sum of the light neutrinos masses as P i mi ≤0.11 eV [66, 67] which will also constrain the parameter space. Note that in order to evaluate P i mi, we need to get an estimate of the pre-factor \|fν2 3 \|v2/M (see eqs. (2.28)–(2.30)) which can be obtained by using the relation \|fν2 3 \|v2/M = q ∆m2 21/(1 −o2r −o2 i ), (3.2) (3.2) with the known value of ∆m2 21 from current global analysis [64]. Equipped with all these, we provide a range of the allowed parameter space of our model in ﬁgure 1. In the left panel, we ﬁrst indicate the correlation between two of the parameters χ1 −χ2 while the same for γ1 −γ2 is shown in the right panel, indicated by the light blue points. The corresponding values of the parameters (light blue points) satisfy the 3σ allowed ranges of the lepton mixing angles, θ13, θ12, θ23. In obtaining these points, we varied parameters within a large range. For example, χ1,2 are varied from 0 to 2 while γ1,2 are considered within their full range: 0-360◦. Once we also incorporate the constraints following from the mass-squared diﬀerences as well as the one on the sum of the light neutrino masses, the entire allowed parameter space is reduced to a smaller region indicated by the dark blue patch on the left panel (in χ1 −χ2 plane) and four cornered patches (red, magenta, brown and purple) on the right panel (in γ1 −γ2 plane). From ﬁgure 1, we ﬁnd 0.584 ≲χ1 ≲1.462 whereas the ratio of the magnitudes of the antisymmetric contribution to the diagonal one (in view of eq. (2.6)) falls in a range: 0.470 ≳χ2 ≳0.145. Turning into the right panel, we ﬁnd that γ1 and γ2 both are pushed to- ward four cornered regions represented by red, magenta, brown and purple patches respec- tively. Here we ﬁnd that for 0◦≤γ1 ≤69◦the allowed regions for γ2 are (57◦−152◦) and – 9 – (a) (b) Figure 2. 3.1 Constraining the parameter space Correlations within δ −θ23 (left panel) and P i mi −m1 (right panel) are presented while allowed ranges for χ1, χ2, γ1 and γ2 are used from ﬁgure 1. (b) (a) (b) (a) JHEP12(2021)051 Figure 2. Correlations within δ −θ23 (left panel) and P i mi −m1 (right panel) are presented while allowed ranges for χ1, χ2, γ1 and γ2 are used from ﬁgure 1. (200◦−282◦). Whereas for 291◦≤γ1 ≤360◦, the allowed regions for γ2 are limited within (78◦−161◦) and (206◦−287◦). Here we also note that, in the right panel of ﬁgure 1, ⋆, ▲, ■ and ♦represent four unique benchmark points in the parameter space {χ1, χ2, γ1, γ2} given by BP1 = (1.37, 0.399, 21.53◦, 135.59◦), BP2 = (0.978, 0.235, 301.81◦, 119.1◦), BP3 = (1.417, 0.372, 341.6◦, 260.83◦) and BP4 = (0.707, 0.209, 68.62◦, 231.15◦). It is important to note that so far the analysis presented here is applicable only for normal hierarchy of light neutrino mass. In the present setup, due to the special ﬂavor structure of the model an inverted hierarchy of light neutrino mass spectrum however can not be accommodated. This is an interesting prediction that will undergo tests in several ongoing and near-future experiments. (200◦−282◦). Whereas for 291◦≤γ1 ≤360◦, the allowed regions for γ2 are limited within (78◦−161◦) and (206◦−287◦). Here we also note that, in the right panel of ﬁgure 1, ⋆, ▲, ■ and ♦represent four unique benchmark points in the parameter space {χ1, χ2, γ1, γ2} given by BP1 = (1.37, 0.399, 21.53◦, 135.59◦), BP2 = (0.978, 0.235, 301.81◦, 119.1◦), BP3 = (1.417, 0.372, 341.6◦, 260.83◦) and BP4 = (0.707, 0.209, 68.62◦, 231.15◦). It is important to note that so far the analysis presented here is applicable only for normal hierarchy of light neutrino mass. In the present setup, due to the special ﬂavor structure of the model an inverted hierarchy of light neutrino mass spectrum however can not be accommodated. This is an interesting prediction that will undergo tests in several ongoing and near-future experiments. 3.2 Implications for light neutrino masses and low energy phase From the previous part of the analysis, we have an understanding on the allowed regions for the χ1, χ2, γ1 and γ2 which satisfy all the constrains in the form of mass square diﬀerences, mixing angles and sum of the light neutrino masses. Hence, we are now in a position to study the implications of this allowed parameter space toward the predictions involving sum of the light neutrino masses, and phases. We already have correlation between the Dirac CP phase δ and the atmospheric mixing angle θ23 as seen from eqs. (2.39) and (2.40), both of which are functions of χ1,2, γ1,2 as evident from eqs. (2.24), (2.25). In ﬁgure 2a, we have plotted this correlation in δ −θ23 plane where only the allowed set of points for χ1, χ2, γ1 and γ2 are employed (as in ﬁgure 1). The model seems to predict δ to be in the range 33◦(213◦) ≲δ ≲80◦(260◦) and 100◦(280◦) ≲δ ≲147◦(327◦) which correspond to the atmospheric mixing angle θ23 in the lower octant. Similarly, in ﬁgure 2a, we use eqs. (2.28), (2.29), (2.30) along with eq. (3.2) to indicate the predictions related to the sum of the light neutrino masses against m1, the lightest neutrino mass, indicated by the blue patch. The region between the black dotted lines represents 3σ allowed range for P i mi and the blue patch within it represents the predicted region in our framework. The red shaded region with P i mi ≤0.11 eV is disallowed by cosmological observation mentioned earlier. This plot shows that the lightest neutrino mass is O(10−3) eV whereas the sum of the – 10 – Figure 3. Correlation between mββ and lightest neutrino mass m1 (for NH) with allowed ranges for χ1, χ2, γ1 and γ2 obtained from ﬁgure 1. Here the light blue shaded region represents the combined upper limit of GERDA and KamLAND-Zen experiments whereas the brown and blue dashed lines stand for future sensitivities of the LEGEND and nEXO experiments respectively. JHEP12(2021)051 Figure 3. Correlation between mββ and lightest neutrino mass m1 (for NH) with allowed ranges for χ1, χ2, γ1 and γ2 obtained from ﬁgure 1. Here the light blue shaded region represents the combined upper limit of GERDA and KamLAND-Zen experiments whereas the brown and blue dashed lines stand for future sensitivities of the LEGEND and nEXO experiments respectively. light neutrino masses is around O(0.06) eV. 3.2 Implications for light neutrino masses and low energy phase On top of this, the present set up excludes the possibility of having maximum CP violation (δ = 90◦/270◦) and at the same time favors θ23 to be below maximal mixing, i.e. θ23 < 45◦. These are the salient features of our proposal. 3.4 Lepton ﬂavor violation Due to the existence of active-sterile neutrino mixing, the possibility of rare lepton ﬂavor violating processes should arise in our framework. Out of all the processes, contribution to µ →eγ is the most important one as it is signiﬁcantly constrained. In the weak basis, i.e. where charged and RHN mass matrix is diagonal, the branching ratio of the same process can be written as [68, 69]: JHEP12(2021)051 B(µ →eγ) = 3α 8π X i ReiR† iµF M2 i M2 W 2 , (3.4) (3.4) where α = e2/4π is the ﬁne structure constant, MW stands for W ± mass, R = mDM−1 R is the mixing matrix representing active-sterile mixing, Mi is the mass of RHN mass eigenstates Ni and F(x) = x(1−6x+3x2+2x3−6x2 ln x) 2(1−x)4 , with x = Mi/MW . The current upper bound on the branching ratio of the µ →eγ is found to be BR(µ →eγ) ≲4.2 × 10−13 (at 90% C.L.) [63]. In our analysis, with the allowed ranges for χ1, χ2, γ1 and γ2 (obtained from ﬁgure 1) and Mi in the TeV scale, the contribution towards the branching ratio for µ →eγ turns out to be insigniﬁcant (O(10−35)) compared to the experimental limit. 3.3 Neutrinoless double beta decay It is pertinent to also shed light on the eﬀective neutrino mass parameter, mββ, involved in the half life of neutrinoless double beta decay in our set-up, which is given by [63] mββ = \|m1c2 12c2 13 + m2s2 12c2 13eiα21 + m3s2 13ei(α31−2δ)\|. (3.3) (3.3) Note that for the normal hierarchy of light neutrino masses, one can write m2 = q (m2 1 + ∆m2 21), and m3 = q (m2 1 + ∆m2 31). Recall also that we have already elaborated on our ﬁnding for lightest neutrino masses m1 (see ﬁgure 2b), and δ (see ﬁgure 2a) in the last subsections corresponding to the allowed parameter space of {χ1, χ2, γ1, γ2} from ﬁgure 1. Using the same, we could also estimate the respective allowed ranges of Majorana phases α21 and α31 via eq. (2.36) (as α21 = β21 and α31 = β31) and in turn we can evaluate mββ as function of m1 (substituting m2 and m3 in eq. (3.3)). With the allowed ranges for χ1, χ2, γ1 and γ2 satisfying all the neutrino data inclusive of the cosmological mass bounds (i.e. corresponding to the dark blue patch of left panel, and four cornered patches of right panel of ﬁgure 1), we therefore plot mββ as a function of lightest neutrino masses m1 for normal hierarchy as presented in ﬁgure 3 by the red patch. The background light red patch indicates the allowed region in general when mixing angles, mass squared diﬀerences along with δ are allowed to vary within their 3σ range. Hence from this mββ vs m1 plot (red patch), we notice that for m1 within the range (0.001-0.027) eV (allowed in our set-up as per ﬁgure 2b), the eﬀective mass parameter is predicted to be: 0.002 ≲mββ ≲0.021 eV. – 11 – This prediction lies well within the limits on mββ by combined analysis of GERDA and KamLAND-Zen experiments denoted by the light blue shade. The horizontal brown and blue dashed lines stand for future sensitivity by the LEGEND and nEXO experiments. 4 Leptogenesis The presence of RHNs in the seesaw realization of light neutrino mass provides an oppor- tunity to study leptogenesis from the CP-violating out-of-equilibrium decay of RHNs into lepton and Higgs doublets in the early universe [45, 49, 70]. The lepton asymmetry created is expected to be converted to a baryon asymmetry via the sphaleron process [71, 72]. In the previous part of our analysis, we have found that the phenomenology of the neutrino sector is mainly dictated by four parameters i.e χ1, χ2, γ1, and γ2 which in turn deter- mine most of the observables in the neutrino sector. However we also notice the presence of the prefactor \|fν2 3 \|v2/M associated to the light neutrino mass eigenvalues as given in eq. (2.28)–(2.30). Using eq. (3.2), though this prefactor can be evaluated, we can’t have speciﬁc estimate for the degenerate mass of the RHNs (M) as fν 3 remains undetermined. To have a more concrete picture, we provide a plot for \|fν2 3 \|v2/M against one of the param- eters, χ1, in ﬁgure 4 obtained using the correlation with other parameters ﬁxed by neutrino oscillation and cosmological data. Hence barring the ambiguity in determining fν 3 apart from a conservative limit \|fν 3 \| < O(1), M is seen to be anywhere from a very large value (say 1014−15 GeV) to a low one (say TeV). Furthermore, the RHNs are exactly degenerate in our framework. Hence unless we break this exact degeneracy, no CP asymmetry can be generated [58] . Below we proceed to discuss leptogenesis mechanism in the present framework keeping in mind that we need to remove the exact degeneracy of RHN masses and study of ﬂavored leptogenesis becomes essential (as M can be below 1012 GeV). – 12 – Figure 4. Correlation between \|f ν 3 \|2v2 M and χ1 for NH. JHEP12(2021)051 Figure 4. Correlation between \|f ν 3 \|2v2 M and χ1 for NH. 4.1.1 Lifting the mass degeneracy The exact mass degeneracy of heavy Majorana neutrinos is the result of the ﬂavor symmetry imposed in our construction. To remove this degeneracy, here we adopt the renormalization group eﬀects into consideration [76, 77]. Considering the discrete A4 × Z3 × Z2 symmetry breaking scale close to the GUT scale ∼Λ (the cut-oﬀscale introduced in eq. (2.1)), we determine the running of the RHN mass matrix MR and Dirac neutrino Yukawa matrix Yν from GUT scale to seesaw scale M (assuming M < Λ). Using renormalisation group equations, the evolution of the RHN mass matrix M (= diag(M1, M2, M3)) and Dirac neutrino Yukawa matrix Yν (in charged lepton Y ℓdiagonal basis) at one-loop can be written as [76–78] JHEP12(2021)051 dMi dt = 2Mi Hii , (4.5) dYν dt = {T −3 4g2 1 −9 4g2 2}I3 −3 2 Y ℓY ℓ† −YνYν† Yν + YνR , (4.6) (4.5) (4.6) with T = 3Tr(YuY † u ) + 3Tr(YdY † d ) + Tr(Y ℓY ℓ†) + Tr(YνYν†), (4.7) (4.7) where Yu,d are the up-quark and down-quark Yukawa matrices respectively, g1,2 are the gauge couplings and I3 is the identity matrix of order 3 × 3. Here the matrix R is anti- hermitian deﬁned by [77] R11 = R22 = R33 = 0, Rji = −R∗ ij (i ̸= j), Rij = 2 + δij δij Re (Hij) + i δij 2 + δij Im (Hij) , (4.8) (4.8) δij = Mj Mi −1 is the degeneracy parameter for the RHN masses and t = 1 16π2 ln Λ M . Now as the RHNs are exactly degenerate at scale Λ, the right hand side (ﬁrst term) of eq. (4.8) becomes singular unless we impose Re(Hij) = 0. Note that, in our construction, H12 and H23 are already zero due to the ﬂavor symmetry imposed. 4.1 Generation of mass splitting and CP asymmetry The CP asymmetry parameter generated as a result of the interference between the tree and one loop level decay amplitudes of RHN Ni decaying into a lepton doublet with speciﬁc ﬂavor lα and Higgs (H) is deﬁned by: ϵα i = Γ(Ni →ℓαH) −Γ(Ni →ℓα ¯H) Γ(Ni →ℓαH) + Γ(Ni →ℓα ¯H). (4.1) (4.1) Considering the exact mass degeneracy is lifted by some mechanism (will be discussed soon), the general expression for such asymmetry can be written as [73, 74]: Considering the exact mass degeneracy is lifted by some mechanism (will be discussed soon), the general expression for such asymmetry can be written as [73, 74]: ϵα i = 1 8πHii X j̸=i Im[Hij(Yν†)iα(Yν)αj] " f(xij) + √xij(1 −xij) (1 −xij)2 + H2 jj 64π2 # + 1 8πHii X j̸=i Im[Hji(Yν†)iα(Yν)αj] " (1 −xij) (1 −xij)2 + H2 jj 64π2 # , (4.2) (4.2) where Yν (≡V †Y ν in our case, see eq. (2.11)) is the neutrino Yukawa matrix in charge lepton diagonal basis, H and the loop factor f(xij) are given by H = Yν†Yν = Y ν†Y ν; (4.3) f(xij) = √xij 1 −(1 + xij) ln 1 + xij xij , (4.4) (4.3) (4.4) with xij = M2 j M2 i where Mi are the masses of the RHNs after the degeneracy is removed. This is applicable for both hierarchical as well as quasi-degenerate mass spectrum of RHNs [73]. For the hierarchical RHNs, one neglects H2 jj 64π2 compared to (1 −xij)2 while the entire ex- pression of eq. (4.2) can be used for quasi-degenerate case inclusive of resonance situation for which (1 −xij)2 ≃ H2 jj 64π2 [74, 75]. Below we discuss the mass splittings induced by the running of the heavy RHNs. with xij = M2 j M2 i where Mi are the masses of the RHNs after the degeneracy is removed. This is applicable for both hierarchical as well as quasi-degenerate mass spectrum of RHNs [73]. For the hierarchical RHNs, one neglects H2 jj 64π2 compared to (1 −xij)2 while the entire ex- pression of eq. (4.2) can be used for quasi-degenerate case inclusive of resonance situation for which (1 −xij)2 ≃ H2 jj 64π2 [74, 75]. Below we discuss the mass splittings induced by the running of the heavy RHNs. – 13 – 4.1.1 Lifting the mass degeneracy Hence the above condi- tion should be exercised only to realize Re(H13) = 0 in our case which can be materialized if we choose to use ˜Yν, obtained by performing an orthogonal rotation (by a matrix O say) on Dirac Yukawa matrix Yν as, ˜Yν = YνO , with O =      cos Θ 0 sin Θ 0 1 0 −sin Θ 0 cos Θ     , (4.9) (4.9) having the rotation angle Θ determined by the relation tan 2Θ = 2Re (H13) H33 −H11 = −cos γ1 χ2 cos (γ1 −γ2). (4.10) (4.10) In obtaining the rightmost expression above, we employ eqs. (2.6), (2.22), (2.23) in eq. (4.3). This ﬂexibility in using ˜Yν prevails due the following reason. Note that, if we rotate the Yν in this manner, the neutrino Yukawa Lagrangian gets modiﬁed to: In obtaining the rightmost expression above, we employ eqs. (2.6), (2.22), (2.23) in eq. (4.3). This ﬂexibility in using ˜Yν prevails due the following reason. Note that, if we rotate the Yν in this manner, the neutrino Yukawa Lagrangian gets modiﬁed to: ¯ℓLYν ˜HNR = ¯ℓL ˜YνOT ˜HNR. (4.11) ¯ℓLYν ˜HNR = ¯ℓL ˜YνOT ˜HNR. (4.11) – 14 – – 14 – Figure 5. Variation of mass splitting δM ij with respect to scale M for the benchmark points BP1, BP2, BP3 and BP4 respectively. Figure 5. Variation of mass splitting δM ij with respect to scale M for the benchmark points BP1, BP2, BP3 and BP4 respectively. We can now redeﬁne NR by: ˜NR = OT NR , i.e. if we rotate RHN ﬁelds by OT , RH mass term will not change as NC R MRNR = ˜NC R MR ˜NR due to the orthogonal property O matrix. We can now redeﬁne NR by: ˜NR = OT NR , i.e. if we rotate RHN ﬁelds by OT , RHN mass term will not change as NC R MRNR = ˜NC R MR ˜NR due to the orthogonal property of O matrix. We can now redeﬁne NR by: ˜NR = OT NR , i.e. if we rotate RHN ﬁelds by OT , RHN mass term will not change as NC R MRNR = ˜NC R MR ˜NR due to the orthogonal property of O matrix. JHEP12(2021)051 The eqs. 4.1.1 Lifting the mass degeneracy (4.5) and (4.6) can now be rewritten in terms of ˜H = OT HO and ˜Yν by using the above relations. The form of ˜H can be obtained by ˜H = ˜Yν† ˜Yν = OT HO =      H11 −∆ 0 i Im (H13) 0 H22 0 −i Im (H13) 0 H33 + ∆     , (4.12) (4.12) where ∆≡tan Θ Re (H13). As seen from the eq. (4.5) (with right hand side written in terms of ˜H now), we ﬁnd that a mass splitting generated at a scale (M) as δM ij = 2( ˜Hii −˜Hjj)t , (4.13) (4.13) thanks to the eﬀect of running. Using eq. (4.6), we also get a oﬀ-diagonal contribution (HR ij, i ̸= j) to ˜H [77], ˜HM ij = ˜Hij + HR ij; HR ij ≃3y2 τ ˜Yν∗ 3i ˜Yν 3j t; (i ̸= j) (4.14) (4.14) while ˜HM ii = ˜Hii. As mentioned earlier, the seesaw scale M remains undetermined even after applying neutrino mass and mixing constraints, we have shown in ﬁgure 5 how such splitting δM 12 varies with the degenerate RHN mass M due to running corresponding to benchmark points: BP1, BP2, BP3 and BP4 allowed by the neutrino data. We ﬁnd that below M ≃1012 GeV, δM ij become smaller than O(10−4) implying that the masses of the three RHNs fall in the quasi-degenerate category [73]. Such a small splitting, although crucial for generation of CP asymmetry, won’t alter our ﬁndings of the neutrino section. Note that the estimated splitting does not correspond to the requirement of resonant leptogenesis. We are now in a position to evaluate the CP asymmetry generated at scale M, as discussed below. 4.1.2 Estimating CP asymmetry Starting with exact degeneracy of RHN masses, we have shown that the running of involved parameters from a typical high scale to the scale of the heavy neutrino masses leads to a quasi-degenerate spectrum of RHNs. Hence we can now estimate the CP asymmetry created at a scale M by using eq. (4.2) while replacing H by ˜HM and δij by δM ij in view – 15 – Figure 6. Variation of individual components of CP asymmetry with respect to model parameter χ1 for three diﬀerent scales M = 1013 GeV (top most plot), M = 1011 GeV (plots from second row) and M = 108 GeV (plots from third row). JHEP12(2021)051 Figure 6. Variation of individual components of CP asymmetry with respect to model parameter χ1 for three diﬀerent scales M = 1013 GeV (top most plot), M = 1011 GeV (plots from second row) and M = 108 GeV (plots from third row). of our discussion above. Furthermore, it can be shown that maximum contribution to CP asymmetry comes from self energy diagram [48, 79, 80]. Therefore, the asymmetry expression of eq. (4.2) gets modiﬁed to of our discussion above. Furthermore, it can be shown that maximum contribution to CP asymmetry comes from self energy diagram [48, 79, 80]. Therefore, the asymmetry expression of eq. (4.2) gets modiﬁed to ϵα i ≃− 1 16π ˜HM ii X j̸=i δM ij (δM ij )2 + ˜ HM jj 16π 2 n Im[ ˜HM ij ˜Yν∗ αi ˜Yν αj] + Im[ ˜HM ji ˜Yν∗ αi ˜Yν αj] o . (4.15) (4.15) Now, using eqs. (4.12) to (4.14) and employing them in eq. (4.15), we estimate for the cp asymmetry parameter for the heavy RHNs decaying into various ﬂavors which will be useful to evaluate the ﬁnal lepton asymmetry taking the ﬂavor eﬀects into account. Since all the entities of eq. (4.15) are function of set of parameters {χ1, χ2, γ1, γ2} and M, we can make use of the allowed parameter space from neutrino phenomenology (refer to ﬁgure 1) and ﬁnally calculate the CP asymmetries produced from all three RHN decays (i = 1, 2, 3) to diﬀerent ﬂavors of lepton doublets and Higgs. 4.1.2 Estimating CP asymmetry For representation purpose, in ﬁgure 6, we depict the variation of individual ﬂavor components of CP asymmetry with respect to χ1 at three diﬀerent RHN mass scales: M = 1013 (top panel), 1011 (middle panel), 108 (bottom panel) GeV respectively. Since the ﬂavor – 16 – eﬀects are known not to be important beyond T ∼M ≃1012 GeV, we estimate asymmetries produced by individual RHNs only for top panel. It is found that maximum asymmetry falls in the ballpark of \|ϵi=1,3\|max ∼6 × 10−7 whereas (\|ϵ2\|)max remains subdominant. At T = 1011 GeV (and above 108 GeV), tau Yukawa comes to equilibrium, so eﬀectively the scenario with M = 1011 GeV becomes a two ﬂavor scenario (τ and another orthogonal direction, say a) and the corresponding CP asymmetries are marked by: {ϵτ i , ϵa=µ+e i }. At this scale, \|ϵτ,a i=2,3\|max ∼2 × 10−6 (middle panel of ﬁgure 6) and \|ϵτ,a 1 \|max becomes relatively small. We also estimate CP asymmetry at M = 108 GeV(bottom panel of ﬁgure 6). At this temperature (or scale), all Yukawa couplings are in equilibrium and hence contributions to CP asymmetries from all the three ﬂavors, {ϵe i, ϵµ i , ϵτ i }, become important. We ﬁnd \|ϵτ i=2,3\|max ∼3 × 10−6 and \|ϵτ 1\|max < \|ϵτ i=2,3\|max. An analogous pattern is observed for ϵµ i . CP asymmetry along electron ﬂavor is shown in the third plot of the bottom panel of ﬁgure 6 and is found to be \|(ϵe i=2,3)max\| ∼1.5 × 10−6 , \|(ϵe 1)max\| ∼5 × 10−7. With these various ﬂavor dependent CP asymmetries, we can now proceed for evaluation of baryon asymmetry by solving the Boltzmann equations as illustrated below. JHEP12(2021)051 4.2 Solution of Boltzmann equation (γDi +2γNis +4γNi t ) + X j̸=i YNi Y eq Ni YNj Y eq Nj −1 ! (γ(1) NiNj +γ(2) NiNj)   , (4.16)  sHz dY∆α dz = −    X i YNi Y eq Ni −1 ! ϵα i γDi − X β "X i 1 2 Cℓ αβ −CH β γα Di (4.17) + Cℓ αβ YNi Y eq Ni − CH β 2 ! γNis + 2Cℓ αβ − CH β 2 1+ YNi Y eq Ni !! γNi t ! + X γ   Cℓ αβ +Cℓ γβ −2CH β γ(1)αγ N +γ(2)αγ N X i,j Cℓ αβ −Cℓ γβ γ(1)αγ NiNj    Y∆β Y eq   , JHEP12(2021)051 where z = Mi/T and α = e, µ, τ. In the above, Y∆α(Ni) = n∆α(Ni)/s denotes the density of ∆α = B 3 −Lα (relevant heavy neutrino) with respect to the entropy s, Y eq’s are the respective number densities while in thermal equilibrium. Here, total decay rate density of Ni is given by γDi = X α [γ(Ni →ℓα + H) + γ(Ni →¯ℓα + ¯H)] = neq Ni K1(z) K2(z)Γi, (4.18) (4.18) where Γi is the total decay rate of Ni at tree level and written as Γi = X α [Γ(Ni →ℓα + H) + Γ(Ni →¯ℓα + ¯H)], (4.19) (4.19) and γNis, γNi t (both are Higgs mediated scattering process with change in lepton number ∆L = 1), γ(1) NiNj, γ(2) NiNj (both are neutrino pair annihilation process) are the reaction rate densities for the scattering processes: [Ni+ℓ↔Q+ ¯U]s, [Ni+ ¯Q ↔¯ℓ+ ¯U]t+[Ni+U ↔¯ℓ+ ¯Q]t, [Ni + Nj ↔ℓ+ ¯ℓ] and [Ni + Nj ↔H + ¯H] respectively [47, 87]. Here in eq. (4.18), K1(z) and K2(z) are the modiﬁed Bessel functions. With all the ingredients at hand, we ﬁrst substitute the evaluated CP asymmetry (from eq. (4.15)) in eq. (4.17) and proceed for solving the coupled Boltzmann equations in order to ﬁnd out the ﬁnal lepton asymmetry as well as ﬁnal baryon asymmetry. In doing so, we divide the temperature range into three zones so as to take care of the ﬂavor eﬀects as discussed before while taking into account the ∆L = 1 processes (and ignoring ∆L = 2 processes). 4.2 Solution of Boltzmann equation It is worth mentioning that while estimating the ﬁnal lepton asymmetry, one needs to take care of decays and inverse decays of heavy RHNs as well as various scattering processes. As stated earlier, we consider the contributions of all three RHNs having Mi ≲1012 GeV. Hence ﬂavor eﬀects have to be considered [81] as with the mass equivalent temperature regime T ∼ 1012 GeV, decay rate of τ (Γτ ∼5×10−3y2 τT) [82, 83] becomes comparable to the Hubble ex- pansion rate. Below this temperature, the relation becomes Γτ > H indicative of the start of equilibrium era for τ Yukawa interactions and τ lepton doublet becomes distinguishable. In a similar way, for the temperature regime 108 GeV ≲T ≲1011 GeV, muon Yukawa inter- action comes to equilibrium (and both µ and τ ﬂavors of lepton doublets are distinguishable henceforth) and ﬁnally below T ≲108 GeV, e Yukawa interaction are in equilibrium. In our analysis, therefore, we include these ﬂavor eﬀects into consideration while con- structing the Boltzmann equations. We work in a most general setup for leptogenesis, where all three RHNs are contributing to the asymmetry due to their quasi degenerate spectrum of masses. As standard, the produced lepton doublets from the RHN decay needs to be appropriately projected to ﬂavor states in the three above mentioned temperature regimes diﬀerently where the related respective lepton asymmetries are characterized by the Cℓ matrices (CH stands for that of Higgs) [84, 85]. For example, when only the τ Yukawa interaction is in equilibrium (1011 GeV ≲T ≲1013 GeV), eﬀectively the scenario becomes a two ﬂavor case (as the ﬂavor space is spanned by ℓτ and another orthogonal direction) and so Cl is a matrix of order 2×2. For a further smaller temperature, the situation comprises of three eﬀective ﬂavors and so Cℓis of 3×3. Below we write down the relevant Boltzmann equations to study the time evolution of the lepton-number asymmetries (for a system of – 17 – three RHNs) as [84–86] three RHNs) as [84–86] sHz dYNi dz = −    YNi Y eq Ni −1 ! (γDi +2γNis +4γNi t ) + X j̸=i YNi Y eq Ni YNj Y eq Nj −1 ! (γ(1) NiNj +γ(2) NiNj)   , (4 1 sHz dYNi dz = −    YNi Y eq Ni −1 ! 4.2 Solution of Boltzmann equation We have considered diﬀerent benchmark values for RHN degenerate mass M (splittings are automatically taken cared by running in terms of other parameters): M = 109, 106, 105 GeV so that the eﬀects of ﬂavor can be visible. These benchmark values of M are so chosen that they can produce requisite amount of baryon asymmetry corresponding to a speciﬁc choice of parameters: {χ1, χ2, γ1, γ2}. In ﬁgure 7, we present our ﬁndings in terms of estimate of the evolution of the B −L asymmetry (denoted by red dotted line) as well as B asymmetry (denoted by Magenta solid line) for speciﬁc choices of the parameters {χ1, χ2, γ1, γ2} which correctly produce – 18 – (a) (b) (c) (d) Figure 7. Variation of YB, YB−L, Y∆e, Y∆µ, Y∆τ (denoted by solid magenta, dotted red, dashed blue, dashed pink and dashed green lines respectively) presented as function of z = M/T. Here we have considered one benchmark point from each of the four patches of γ2 vs γ1 plot for the light neutrino parameters of the model (from ﬁgure 1). (a) JHEP12(2021)051 (b) (b) (d) (a) (c) (c) (d) Figure 7. Variation of YB, YB−L, Y∆e, Y∆µ, Y∆τ (denoted by solid magenta, dotted red, dashed blue, dashed pink and dashed green lines respectively) presented as function of z = M/T. Here we have considered one benchmark point from each of the four patches of γ2 vs γ1 plot for the light neutrino parameters of the model (from ﬁgure 1). Figure 8. Variation of ﬁnal YB with respect to M (neglecting ∆L = 2 processes) for four bench- mark point BP1, BP2, BP3, BP4, from each of the four patches of γ2 vs γ1 plot for the light neutrino parameters of the model (from ﬁgure 1). Here the horizontal patch (light greenish-blue) indicates the observed value of baryon asymmetry [67]. Figure 8. Variation of ﬁnal YB with respect to M (neglecting ∆L = 2 processes) for four bench- mark point BP1, BP2, BP3, BP4, from each of the four patches of γ2 vs γ1 plot for the light neutrino parameters of the model (from ﬁgure 1). Here the horizontal patch (light greenish-blue) indicates the observed value of baryon asymmetry [67]. – 19 – neutrino data as discussed in section 3. 4.2 Solution of Boltzmann equation Figure 7a, 7b, 7c, 7d represent the benchmark points BP1, BP2, BP3, and BP4 respectively from the allowed cornered patches of γ1 and γ2 plot of ﬁgure 1. Asymmetries of individual ﬂavors are also drawn in these ﬁgures. While solving the Boltzmann equations, we have assumed that initially the abundance of all the RHNs was very less and they were out of equilibrium. Then due to annihilation of bath particles it gets produced and comes to equilibrium. Around M T ∼1, the produc- tion rate and decay rate of the RHN become almost equal and afterward the decay rate dominates over the production rate and hence it’s abundance starts to fall. The correct baryon asymmetry can be produced with M ≲106 GeV for BP1, M ≲105 GeV for BP2 and BP4, M ≲109 GeV for BP3 region respectively. For these individual sets of parameters, we have checked the variation of ﬁnal baryon asymmetry, YB, with respect to mass of M as shown in ﬁgure 8. From this ﬁgure 8, we also see that ﬁnal YB is increasing with the decreasing of M. There seems to be two discontinuities for each such plot. For example, with blue-dotted line, these are observed at or around M = 1011 GeV and at M = 106 GeV. These are indicative of the eras where diﬀerent ﬂavors of lepton doublets enter in (or exit from) equilibrium and the Boltzmann equations get modiﬁed. JHEP12(2021)051 5 Conclusion In this analysis, we present an economical, predictive ﬂavor symmetric setup based on A4 × Z3 × Z2 discrete group to explain neutrino masses, mixing via type-I seesaw mecha- nism while matter-antimatter asymmetry is also addressed via leptogenesis. In the original AF model, TBM mixing scheme was realized introducing three ﬂavon ﬁelds. With similar ﬁelds content, here we show that correct neutrino mixing and mass-squared diﬀerences are originated from non-trivial structure of the neutrino Dirac Yukawa coupling and diago- nal RHN mass matrix, thanks to the contribution from the charged lepton sector too. In particular, the antisymmetric contribution in the Dirac Yukawa coupling plays an instru- mental role in generating the non-zero θ13. Using the current experimental observation on neutrino oscillation and other cosmological limits, we ﬁnd the allowed parameter space for parameters χ1, χ2, γ1, γ2 which in turn not only restricts some of the observables as- sociated to neutrinos like Dirac CP phase, neutrino-less double beta decay, lepton ﬂavor violating decays, estimation of Majorana phases etc. but also are helpful in determining the matter-antimatter asymmetry of the universe. More speciﬁcally, we ﬁnd that this model is highly predictive in nature. Only normal mass hierarchies are found to be allowed in the current setup. Interestingly the atmospheric mixing angle θ23 lies in the lower octant while the leptonic Dirac CP phase falls within the range 33◦(213◦) ≲δ ≲80◦(260◦) and 100◦(280◦) ≲δ ≲147◦(327◦). Apart from these predictions for absolute neutrino mass and eﬀective mass parameter appearing the neutrino-less double beta decay have also been made. The model also predicts an interesting correlation between the atmospheric mixing angle θ23 and the Dirac CP phase which is a feature of the speciﬁc ﬂavor symmetry consid- ered here. At high scale, owing to the symmetry of the model, the heavy RHNs are found to be exactly degenerate apparently forbidding the generation of baryon asymmetry via leptogenesis. However, this is accomplished here elegantly by considering the renormaliza- – 20 – tion group eﬀects into the picture. A tiny mass splitting produced as a result of running from a high scale (GUT scale) to the scale of RHN mass opens the room for leptogenesis. We have incorporated the ﬂavor eﬀects in leptogenesis as our working regime of RHN mass falls near or below 109 GeV. Acknowledgments JHEP12(2021)051 The work by BK is supported by the Polish National Science Centre (NCN) under the Grant Agreement 2020/37/B/ST2/02371, funds granted under the Research Excellence Initiative of the University of Silesia in Katowice and DST, Govt. of India (SR/MF/PS- 01/2016-IITH/G). BK also acknowledges the support provided by the Institute of High Energy Physics and the University of Chinese Academy of Sciences, Beijing, China, where part of the work has been completed. AD would like to thank Rishav Roshan and Dibyendu Nanda for fruitful discussions. 5 Conclusion Finally, we ﬁgure out that the parameter space allowed by the neutrino data in fact is good enough to generate suﬃcient amount of baryon asymmetry of the universe with RHN mass as low as 105 GeV. 6Here S is a 3 × 3 diagonal generator of A4. A A4 multiplication rules It has four irreducible representations: three one-dimensional and one three dimensional which are denoted by 1, 1′, 1′′ and 3 respectively. The multiplication rules of the irreducible representations are given by [18] 1 ⊗1 = 1, 1′ ⊗1′ = 1′′, 1′ ⊗1′′ = 1, 1′′ ⊗1′′ = 1′, 3 ⊗3 = 1 + 1′ + 1′′ + 3a + 3s (A.1) (A.1) where a and s in the subscript corresponds to anti-symmetric and symmetric parts respec- tively. Now, if we have two triplets as A = (a1, a2, a3)T and B = (b1, b2, b3)T respectively, their direct product can be decomposed into the direct sum mentioned above. The product rule for this two triplets in the S diagonal basis6 can be written as where a and s in the subscript corresponds to anti-symmetric and symmetric parts respec- tively. Now, if we have two triplets as A = (a1, a2, a3)T and B = (b1, b2, b3)T respectively, their direct product can be decomposed into the direct sum mentioned above. The product rule for this two triplets in the S diagonal basis6 can be written as (A × B)1 ∽a1b1 + a2b2 + a3b3, (A.2) (A × B)1′ ∽a1b1 + ω2a2b2 + ωa3b3, (A.3) (A × B)1′′ ∽a1b1 + ωa2b2 + ω2a3b3, (A.4) (A × B)3s ∽(a2b3 + a3b2, a3b1 + a1b3, a1b2 + a2b1), (A.5) (A × B)3a ∽(a2b3 −a3b2, a3b1 −a1b3, a1b2 −a2b1), (A.6) here ω (= e2iπ/3) is the cube root of unity. here ω (= e2iπ/3) is the cube root of unity. here ω (= e2iπ/3) is the cube root of unity. Open Access. 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https://openalex.org/W4282542798	https://www.researchprotocols.org/2022/7/e37934/PDF	English	null	Burnout and Associated Psychological Problems Among Teachers and the Impact of the Wellness4Teachers Supportive Text Messaging Program: Protocol for a Cross-sectional and Program Evaluation Study	JMIR research protocols	2,022	cc-by	9,871	Abstract Background: Stress, burnout, anxiety, and depression continue to be a problem among teachers worldwide. It is not presently known what the prevalence and correlates for these psychological problems are among teachers in Alberta and Nova Scotia. It is also not known if a supportive text message program (Wellness4Teachers) would be effective in reducing stress, burnout, anxiety, or depression symptoms among teachers. Objective: The goal of this study is to evaluate the prevalence and correlates of stress, burnout, symptoms of anxiety, depression, and low resilience among elementary and high school teachers in Alberta and Nova Scotia, Canada. It also aims to determine if daily supportive text messages can help reduce the prevalence of these psychological problems in teachers. Methods: This is a cross-sessional mixed methods study with data to be collected from subscribers of Wellness4Teachers using a web-based survey at baseline (onset of text messaging), 6 weeks, the program’s midpoint (3 months), and end point (6 months). Teachers can subscribe to the Wellness4Teachers program by texting the keyword “TeachWell” to the program phone number. Outcome measures will be assessed using standardized rating scales and key informant interviews. Data will be analyzed with descriptive and inferential statistics using SPSS and thematic analysis using NVivo. Results: The results of this study are expected 24 months after program launch. It is expected that the prevalence of stress, burnout, anxiety, depression, and low resilience among teachers in Alberta and Nova Scotia would be comparable to those reported in other jurisdictions. It is also expected that factors such as gender, number of years teaching, grade of teaching, and school type (elementary vs high school) will have an association with burnout and other psychological disorders among teachers. Furthermore, it is expected that Wellness4Teachers will reduce the prevalence and severity of psychological problems in teachers, and subscriber satisfaction will be high. Conclusions: The Wellness4Teachers project will provide key information regarding prevalence and correlates of common mental health conditions in teachers in Alberta and Nova Scotia, as well as the impact of daily supportive text messages on these mental health parameters. Information from this study will be useful for informing policy and decision-making concerning psychological interventions for schoolteachers. (JMIR Res Protoc 2022;11(7):e37934) doi: 10.2196/37934 Protocol Burnout and Associated Psychological Problems Among Teachers and the Impact of the Wellness4Teachers Supportive Text Messaging Program: Protocol for a Cross-sectional and Program Evaluation Study Belinda Agyapong1, MEd; Yifeng Wei1, PhD; Raquel da Luz Dias2,3, PhD; Vincent Israel Opoku Agyapong2, MD, PhD 1Department of Psychiatry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada 2Department of Psychiatry, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada 3Nova Scotia Health Authority, Halifax, NS, Canada Agyapong et al JMIR RESEARCH PROTOCOLS JMIR RESEARCH PROTOCOLS Agyapong et al https://www.researchprotocols.org/2022/7/e37934 JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 1 (page number not for citation purposes) Protocol Burnout and Associated Psychological Problems Among Teachers and the Impact of the Wellness4Teachers Supportive Text Messaging Program: Protocol for a Cross-sectional and Program Evaluation Study Belinda Agyapong1, MEd; Yifeng Wei1, PhD; Raquel da Luz Dias2,3, PhD; Vincent Israel Opoku Agyapong2, MD, PhD 1Department of Psychiatry, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, A 2Department of Psychiatry, Faculty of Medicine, Dalhousie University, Halifax, NS, Canada 3Nova Scotia Health Authority, Halifax, NS, Canada Corresponding Author: Vincent Israel Opoku Agyapong, MD, PhD Department of Psychiatry Faculty of Medicine Dalhousie University 5909 Veterans' Memorial Lane 8th Floor Abbie J Lane Memorial Building QEII Health Sciences Centre Halifax, NS, B3H 4R2 Canada Phone: 1 7807144315 Email: vincent.agyapong@nshealth.ca Corresponding Author: Vincent Israel Opoku Agyapong, MD, PhD Department of Psychiatry Faculty of Medicine Dalhousie University 5909 Veterans' Memorial Lane 8th Floor Abbie J Lane Memorial Building QEII Health Sciences Centre Halifax, NS, B3H 4R2 Canada Phone: 1 7807144315 Email: vincent.agyapong@nshealth.ca Background A recent study by Li et al [10] reported that 53.2 % of teachers identified work as a source of long-term stress, leading to burnout. The results of a transversal study conducted in Tunisia reported that most teachers (66.4%) acknowledged being stressed at work, and burnout syndrome was found in 21% of those teachers [3]. A systematic review indicated that burnout was an important predictor of both physical and psychological consequences, including insomnia, depressive symptoms, and mortality below the age of 45 years; hospitalization for mental disorders; and psychological ill-health symptoms [11]. Another study reported that physical illnesses are more common among individuals with burnout compared with those without (64% vs 54%; P<.001) with increased prevalence of diseases associated with severity of burnout [12]. A study finding also showed that high burnout was associated with a high level of emotional exhaustion, low personal accomplishment, and depersonalization, while low burnout was linked to high personal accomplishment [7]. The professional outcomes of burnout include job dissatisfaction and absenteeism [11]. Absence due to sickness was more prevalent among employees with burnout compared with those without burnout [13]. The level of satisfaction is a significant factor that impacts the mental and physical health of teachers as well as other workers. Those with low job satisfaction are more susceptible to experiencing burnout, high anxiety levels, depression, and low self-esteem [7,14]. A study also showed that participants who reported high social anxiety levels reported high burnout levels as well [15]. A survey conducted in Canada and the United States confirmed that workplace improvements could prevent adverse sequelae, improve health outcomes, and reduce health care expenditures [16]. Data on the prevalence of burnout and other psychological problems among teachers in Canada In a longitudinal study, individual teachers who experienced an increase in the states of burnout had an increase in depression in comparison to those with decrease in burnout, which corresponded to less depression [7,24]. This was also observed in a study where 86% of the teachers who identified as burned-out met the criteria for a provisional diagnosis of depression [25]. The results of a study in Quebec, Canada found that the proportion of teachers who reported a high level of psychological distress was twice as high (40%) than that reported for a Quebec-wide sample (20%) [26], an indication that teachers in Canada are also prone to stress. Background Educators and the public alike see stress and burnout as a distinct problem of the teaching profession [1,2]. Burnout is defined as a state of emotional, mental, and physical exhaustion resulting from prolonged and lengthy stress at work [3,4], as well as a response to chronic emotional and interpersonal stressors. Burnout is defined by the 3 dimensions of emotional exhaustion, cynicism, and inefficacy [5]. Emotional exhaustion represents emotional depletion and loss of energy; depersonalization or cynicism is the interpersonal dimension of burnout and refers to a negative, callous, or excessively detached response to other people. Reduced accomplishment describes the self-evaluation dimension of burnout and refers to feelings of incompetence and a lack of achievement and productivity at work [5,6]. In a cross-sectional study, 33.3% of teachers reported high burnout while 27.6% were at risk for having moderate burnout [7]. Similarly, 34.9% of teachers indicated they might be threatened by burnout syndrome [8]. The relationship between burnout and health exhibits complicated pattern in the sense that poor health contributes to burnout, and burnout contributes to poor health [17]. Burnout is a risk factor for poor physical and mental well-being, and it may adversely affect health [11,18]. There is increasing evidence that burnout as a stress response represents a risk factor not only for depression but also for cardiovascular and other somatic diseases [19]. Burnout is occupational-specific dysphoria, which is different from depression—a general mental illness [5]. Burnout is regarded as a stress-related state, and the rates of clinical depression increase with the severity of burnout [19]. Burnout is also job related and dependent on the situation or condition, while depression is more general and context‐free [6]. Cross-sectional studies showed that there is a relationship between burnout and depression with greater risk of major depressive disorder (MDD) when burnout is severe [12]. Teachers with MDD also had higher levels of perceived stress, anxiety, disorder, and lower quality of life [20]; moreover, poor workplace environment was a factor associated with both increased anxiety and depressive symptoms [21]. The published results of a study reported a high prevalence of depression (49.1%) among teachers [22] as well as a relatively high prevalence of anxiety 68.0% [23]. Literature shows that teachers experience considerable stress in the workplace resulting in higher burnout rates, which poses a health risk [7,9]. (JMIR Res Protoc 2022;11(7):e37934) doi: 10.2196/37934 https://www.researchprotocols.org/2022/7/e37934 https://www.researchprotocols.org/2022/7/e37934 XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Agyapong et al KEYWORDS burnout; stress; Wellness4Teachers; anxiety; depression; e-mental health; teachers; support; text message; mental health; SMS; high school; elementary school; prevalence; psychological intervention; school burnout; stress; Wellness4Teachers; anxiety; depression; e-mental health; teachers; support; text message; mental health; SMS; high school; elementary school; prevalence; psychological intervention; school are limited in the literature. It has been suggested that this is because North American jurisdictions have been hesitant to recognize burnout as a clinical diagnosis, partly due to concerns about increasing requests for disability coverage [6]. https://www.researchprotocols.org/2022/7/e37934 JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 2 (page number not for citation purposes) Background The ability to be able to cope under stress and pressure is an important factor to reduce burnout. Self-efficacy is viewed as a significant personal resource associated with coping with stress, and teacher’s self-efficacy was positively correlated with general self-efficacy but negatively with job burnout [27]. According to psychologists, resilience is the process of adapting well in the face of adversity, trauma, tragedy, threats, or significant sources of stress including workplace stressors [28]. Low resilience is predicted to reduce the individual’s ability to cope with stress and therefore will lead to increased levels of burnout. Increased levels of job support, however, were a protective factor against emotional exhaustion [29]. A study’s results also indicated that the prevalence of resilience was exclusively XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Agyapong et al predicted by factors including the participant’s level of trauma exposure, social support, and recent and past life stressors [30]. is currently unknown whether supportive text messages will be helpful in reducing stress, anxiety, and depression while improving resilience among elementary and high school teachers. Given the generally high psychological burden among teachers and the evidence of effectiveness of supportive text message interventions in the literature, we propose to use Wellness4Teachers, a supportive text message program to help reduce stress, burnout, anxiety, and depression and to improve resilience among teachers. Elementary and high school teachers must constantly deal with students’ discipline issues while at the same time ensuring the timely delivery of the curriculum. Teachers must also deal with their own personal and family day-to-day stressors while helping to support students under their care. This leads to heightened levels of occupational stress and amplified risk of mental disorders [1]. The stress of the job sometimes takes a toll on the teachers, giving rise to burnout, increased anxiety and depression, and sometimes reduced resilience [5,17,31]. Meditation is effective in improving resilience, psychological distress, fatigue, and burnout. Thus, teachers may benefit from in-school wellness programs that incorporate mindfulness and meditation [31]. Additionally, the quest for professional fulfillment is more challenging in schools with high discipline issues and poor classroom settings. Furthermore, in the last 2 years, the global pandemic has led to changes in the school system, including intermittent web-based learning, closure of schools, and cancellation of provincial exams, thus creating major uncertainties for both students and teachers. Wellness4Teachers Program Wellness4Teachers is a self-subscription daily supportive text message intervention program designed to address stress, burnout, anxiety, and depression, and to build resilience and improve professional satisfaction in teachers. The Wellness4Teachers program is powered by the ResilienceNHope web-based application [40] and provided by the Global Psychological eHealth Foundation [41]. ResilienceNHope is an evidence-informed e–mental health program that incorporates cognitive behavioral therapy based on daily supportive messages (mobile text or email), weekly mental health literacy information, web-based mental health self-assessments, and other mental health resources to help address part of the mental health literacy and the psychological treatment gap for individuals and communities globally. The Wellness4Teachers application will deliver one-way (noninteractive) psychological intervention messages to mobile phones. Subscribers will be made aware of the noninteractive nature of the supportive messaging program through the welcome and introductory message they receive upon subscribing to the program. They will also be offered the phone number of the mental health crisis service for their province or region to call if they are in crisis. The daily supportive messages delivered through the Wellness4Teachers program were crafted by psychiatrists, mental health therapists, and psychologists based on the cognitive behavioral therapy principles. The messages were further reviewed by the lead author, who is an education specialist, before they were built into the Wellness4Teachers program. Different messages will be received daily from a bank of messages. Examples of the text messages are as follows: p g y The daily supportive messages delivered through the Wellness4Teachers program were crafted by psychiatrists, mental health therapists, and psychologists based on the cognitive behavioral therapy principles. The messages were further reviewed by the lead author, who is an education specialist, before they were built into the Wellness4Teachers program. Different messages will be received daily from a bank of messages. Examples of the text messages are as follows: Deep breathing is a skill. You may need to practice it often and for more than 5 minutes to feel calmer. When you are feeling stressed or tense, shrug your shoulders up to your ears, hold for 5 seconds, release a n d r e p e a t 2 m o r e t i m e s . https://www.youtube.com/watch?v=cOOd-wlMMRg. As a teacher, it is very important for your well-being to spend time talking, laughing, and sharing with your colleagues. Background It is currently unknown if the additional stressors experienced from the pandemic impacts the levels of stress, anxiety, depression, burnout, and resilience in teachers in elementary and high schools. Given the aforementioned, teachers may benefit from in-school intervention and wellness programs to alleviate their stress and burnout. Meditation techniques such as mindfulness have been suggested to help teachers cope with stress and burnout. Mindfulness is the practice of bringing awareness to the here and now using a variety of methods [32], and it has been suggested as beneficial in coping with job-related stress and burnout in the teaching profession [33,34]. Mindfulness can also improve the interpersonal faculties of teachers’ sense of efficacy and perceived stress [33], as well as reducing depression [34]. However, the nature of these programs requires focusing and concentration, and this can be challenging to pursue in the busyness of the school environment and especially when teachers are stressed or burnt-out. Thus, the teachers may not be in the position to complete these programs. As mentioned, these programs can be time-consuming and may require teachers to consciously set time apart to participate. In addition, mindfulness may not be easily accessible and scalable in all schools. Furthermore, mindfulness may not be suitable or convenient for some teachers. An innovative way to offer intervention to teachers is through mobile text technology. https://www.researchprotocols.org/2022/7/e37934 Wellness4Teachers Program Subscribers of Text4Hope (launched in Alberta during the COVID-19 pandemic [44]), who had been enrolled for 6 weeks (intervention group) had a significantly lower prevalence of moderate-to-high stress (78.8% vs 88.0%); moderate-to-high anxiety symptoms (31.4% vs 46.5%); and moderate-to-high depression symptoms (36.8% vs 52.1%), suicidal ideation (16.9% vs 26.6%), and disturbed sleep (76.9% vs 85.1%) compared to new subscribers, respectively, during the same time period (control group) [45]. Furthermore, there were statistically significant reductions in both the prevalence and mean scores on standardized measures for stress, anxiety, and depression at 6 weeks and 3 months for subscribers to the Text4Hope program [36,37]. receive supportive text messages. Teachers will receive supportive text messages at no cost to themselves or their institutions and will not receive any reimbursement or incentives for participating. Wellness4Teachers supportive text messages will be delivered to subscriber cell phones at 12 PM Mountain Standard Time in Alberta and 9 AM Atlantic Standard Time in Nova Scotia each day, and subscribers will receive the daily messages for 6 months. Based on a 10% dropout rate recorded for the Text4Hope program [36,37], the dropout rate expected for the Wellness4Teachers program is less than 15% [42]. receive supportive text messages. Teachers will receive supportive text messages at no cost to themselves or their institutions and will not receive any reimbursement or incentives for participating. Wellness4Teachers supportive text messages will be delivered to subscriber cell phones at 12 PM Mountain Standard Time in Alberta and 9 AM Atlantic Standard Time in Nova Scotia each day, and subscribers will receive the daily messages for 6 months. Based on a 10% dropout rate recorded for the Text4Hope program [36,37], the dropout rate expected for the Wellness4Teachers program is less than 15% [42]. The effectiveness of the ResilienceNHope suite of programs have been evaluated and established through several randomized controlled clinical trials and evaluations of population level programs. In a randomized controlled trial (RCT) in Fort McMurray, Alberta, Canada, involving 73 patients diagnosed with MDD, the intervention group (n=35, 48%) received twice-daily supportive text messages for 3 months (intervention group) as part of their outpatient treatment, while the control group (n=38, 52%) received a single thank-you message (control group) every fortnight (20.8, SD 11.7 vs 24.9, SD 11.5, respectively; F1,60=4.83; P=.03, ηp 2=0.07). The mean difference in the Beck Depression Inventory (BDI) score change was significant with an effect size (Cohen d) of 0.67 [43]. Wellness4Teachers Program If you feel overwhelmed, take time to do something kind for yourself such as a cup of tea, or nature walk. Deep breathing is a skill. You may need to practice it often and for more than 5 minutes to feel calmer. When you are feeling stressed or tense, shrug your shoulders up to your ears, hold for 5 seconds, release a n d r e p e a t 2 m o r e t i m e s . https://www.youtube.com/watch?v=cOOd-wlMMRg. Mobile text technology is a unique and innovative way that offers a convenient, low-cost, and easily accessible way of delivering psychological interventions to the general public with mental health problems [35]. Supportive text messages can be used to supplement mental health therapy and hence indirectly reduce waitlist and the number of days required to attend group-based or face-to-face therapy programs that span months and require a lot of human resources for their delivery. Text messages have been effectively used to support the mental health of the general Albertan population and was effective in reducing stress, anxiety, and depression [36,37]. Supportive text messaging has also been used to reduce depression and increase abstinence duration in alcohol use disorder [38,39]. It As a teacher, it is very important for your well-being to spend time talking, laughing, and sharing with your colleagues. If you feel overwhelmed, take time to do something kind for yourself such as a cup of tea, or nature walk. Visualize yourself coping with the current challenging student behavior or workload. See yourself confidently facing these challenges. You can do it! No matter the challenges. Teachers in both Alberta and Nova Scotia can subscribe to the Wellness4Teachers program by texting “TeachWell” to a designated phone number to be automatically registered to JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 3 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Agyapong et al adjusting for baseline scores, there was a statistically significant difference in the 3-month BDI-II scores between the intervention and control groups (8.5, SD 8.0 vs 16.7, SD 10.3, respectively; F1,49=9.54; P=.003, ηp 2=0.17. The mean difference in change BDI-II scores was –7.9 (95% CI –13.06 to –2.76; Cohen d=0.85) [39]. Ethics Approval The study has approval from the University of Alberta Ethics Review Board (Pro00117558) and is currently seeking approval from the Dalhousie University Human Research Ethics Review Board. Consent to participate will be implied when participants complete and submit the web-based survey responses. The web-based questionnaires will be designed to collect demographic, professional, and clinical variables including stress, burnout, anxiety, depression, and resilience. Follow-up surveys will also include subscriber experience and satisfaction questions. Teachers in Alberta and Nova Scotia will be invited to subscribe to the Wellness4Teachers program through an advertisement organized in collaboration with Alberta Teachers Association, the Alberta School Boards Association, the Nova Scotia School Boards Association, and the Nova Scotia Teachers Study Design This study will use a mixed-methods quantitative and qualitative cross-sectional survey design. Quantitative data will be collected using web-based–administered questionnaires through the University of Alberta REDCap platform [46], a secure web application for building and managing web-based surveys and databases. Qualitative data will be collected through key informant interviews. Wellness4Teachers Program In an earlier RCT in Dublin, Ireland, 54 patients with MDD and comorbid alcohol use disorder were also randomized to receive either twice-daily supportive text messages (intervention group) or a thank-you text message (control group) for 3 months. After Main objectives • To determine the prevalence and correlates of burnout, moderate to high stress, likely generalized anxiety disorder, likely major depressive disorder, and low resilience among elementary and high school teachers in Alberta and Nova Scotia • To determine if the daily supportive text messaging program, Wellness4Teachers, can reduce the prevalence and severity of stress, burnout, anxiety, and depression, and to improve resilience among elementary and high school teachers in Alberta and Nova Scotia • To assess Wellness4Teachers program subscriber experience and satisfaction with the daily supportive text messaging program • To determine the prevalence and correlates of burnout, moderate to high stress, likely generalized anxiety disorder, likely major depressive disorder, and low resilience among elementary and high school teachers in Alberta and Nova Scotia • To determine if the daily supportive text messaging program, Wellness4Teachers, can reduce the prevalence and severity of stress, burnout, anxiety, and depression, and to improve resilience among elementary and high school teachers in Alberta and Nova Scotia • To assess Wellness4Teachers program subscriber experience and satisfaction with the daily supportive text messaging Union. The web-based surveys will be distributed to subscribers upon enrollment, at 6 weeks, 3 months, and 6 months. Key informant interview questionnaire will be developed to assess and explore the factors that contribute to stress, burnout, anxiety, and depression among teachers, the impact of the Wellness4Teachers program on the levels of these mental health variables in subscribers, and subscriber satisfaction with the daily supportive text messaging program. Union. The web-based surveys will be distributed to subscribers upon enrollment, at 6 weeks, 3 months, and 6 months. Key informant interview questionnaire will be developed to assess and explore the factors that contribute to stress, burnout, anxiety, and depression among teachers, the impact of the Wellness4Teachers program on the levels of these mental health variables in subscribers, and subscriber satisfaction with the daily supportive text messaging program. https://www.researchprotocols.org/2022/7/e37934 Study Aims One goal of this study is to evaluate the prevalence and correlates of burnout, probable mental health disorders, and low resilience among elementary and high school teachers in Alberta and Nova Scotia, Canada. Another goal of this study is to determine if daily supportive text messages can help reduce the prevalence of burnout, stress, symptoms of anxiety, and depression, and improve resilience among elementary and high school teachers (Textbox 1). Textbox 1. Study objectives. e tbo . Study object ves. Main objectives • To determine the prevalence and correlates of burnout, moderate to high stress, likely generalized anxiety disorder, likely major depressive disorder, and low resilience among elementary and high school teachers in Alberta and Nova Scotia • To determine if the daily supportive text messaging program, Wellness4Teachers, can reduce the prevalence and severity of stress, burnout, anxiety, and depression, and to improve resilience among elementary and high school teachers in Alberta and Nova Scotia • To assess Wellness4Teachers program subscriber experience and satisfaction with the daily supportive text messaging program Hypothesis We hypothesize that the prevalence of stress, burnout, symptoms of anxiety and depression, as well as low resilience among teachers in Alberta and Nova Scotia would be comparable to those reported in other jurisdictions [8,66]. Outcomes and Measures Clinical outcomes will be assessed using validated screening scales for self-reported symptoms, including Perceived Stress Scale (PSS-10; a score of ≥10 indicates a likely moderate or high stress) [55], the 7-item GAD scale (GAD-7; a score of ≥10 indicates likely generalized anxiety disorder) [56], the Patient Health Questionnaire-9 (PHQ-9; a score ≥10 indicates likely MDD) [57], and the Brief Resilience Scale (BRS; mean scores ranging from 1.00 to 2.99 indicate low resilience, from 3.00 to 4.30 suggest normal resilience, and from 4.31 to 5.00 suggest high resilience) [58,59].Validated scales have been chosen to better understand self-reported symptoms and potential symptom severity and to screen for the likely presence of psychopathology. These scales are not intended as diagnostic tools but instead are to identify risk factors and early symptoms of potential mental disorders such as depression and anxiety disorders. In addition, we will assess burnout using the Maslach Burnout Inventory (MBI) [60]. The MBI-Educators Survey will be used in this case, which is a version of the original MBI for use with educators, including teachers, administrators, other staff members, and volunteers working in any educational setting [61]. The primary outcome measures will be prevalence of moderate-to-high stress, burnout, likely GAD, likely MDD, and low resilience at baseline in subscribers of Wellness4Teachers. Other primary outcome measures will be changes in prevalence of moderate-to-high stress, burnout, likely GAD, likely MDD, and low resilience as well as changes in mean scores on the PSS-10, MBI, GAD-7, PHQ-9, and the BRS from baseline to 6 weeks, 3 months, and 6 months. Secondary outcome measures will include sociodemographic, clinical, and professional correlates of moderate-to-high stress, burnout, likely MDD, likely GAD, and low resilience among subscribers of Wellness4Teachers at baseline. An exploratory outcome will be a measurement of the adoption of the Wellness4Teachers program by teachers in Alberta and Nova Scotia. This will be assessed by measuring the proportion of the target population (teachers in Alberta and Nova Scotia) who subscribe to the daily supportive text messages. We will analyze qualitative data (obtained by audio recording and transcribing responses from key informant interviews) using thematic analysis with NVivo (version 9; QSR International). The results will be reported as themes and subthemes supported by verbatim quotes. Statistical Analysis Quantitative data from the surveys will be analyzed using SPSS (version 25, IBM Corp) [65]. Descriptive statistics will be provided for demographic, clinical, and burnout-related variables based on the province of residence. Cross-tabular analyses using the chi-square test will explore the differences between elementary and high school teachers with respect to demographic, clinical, and professional variables. Descriptive characteristics will be presented as numbers and percentages, and a 2-tailed P≤.05 will be used to determine statistical significance for all analyses. We will use the chi-square test and logistic regression analysis to identify demographic, clinical, and work-related correlates of anxiety, depression, stress, low resilience, and burnout for elementary and high school teachers separately. Furthermore, we will assess the impact of the Wellness4Teachers program in reducing moderate-to-high stress, burnout, likely GAD, likely MDD, and improving resilience among Wellness4Teachers subscribers by assessing the mean changes in these parameters from baseline to 6 weeks, 3 months, and 6 months using the 2-tailed paired t test. To assess the effects of the intervention against a control group, we will choose a defined period of 3 months during the study period (eg, from the beginning of November 2022 to the end of January 2023) and compare the prevalence and mean scores on standardized scales for stress, burnout, anxiety, and depression at 6 weeks for subscribers who have received the daily supportive text messages for 6 weeks (intervention group) to the baseline prevalence and mean scores on the same scales for new subscribers during the period (control group). Surveys with more than 50% missing responses will be omitted from data analysis. For the included survey responses, there will be no imputation for missing data, and the analysis and results will be based on completed survey data. Study Setting The study will occur in Alberta and Nova Scotia. Alberta is a province in Western Canada, with an estimated population of 4,067,175 in 2016 [47]. Elementary and high schools in Alberta are run by 61 school boards [48]. In 2013, Alberta’s school JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 4 (page number not for citation purposes) XSL•FO RenderX XSL•FO RenderX JMIR RESEARCH PROTOCOLS Agyapong et al jurisdictions employed approximately 35,000 full-time equivalent teachers. Alberta has more than 150 private school authorities, which operate about 180 schools and serve more than 38,000 students [49]. Nova Scotia is a province in Eastern Canada, with a population of 1 million residents in 2021, according to Statistics Canada [50]. Elementary and high schools in Nova Scotia are located in 8 school districts with 7 English-language school boards and 1 French–first language school board. Public schools in Nova Scotia are managed by the provincial department of education, called Education and Early Childhood Development. There were 372 public schools in Nova Scotia in the 2017-18 school year [51,52]. Nova Scotia also has more than 20 private or independent schools, many of which are in Halifax, the provincial capital [53]. There are more than 10,000 public school teachers in Nova Scotia, who are represented by the Nova Scotia Teachers Union [54]. that the sample size needed for our prevalence estimates with a 95% CI and 3% margin of error for moderate-to-high stress, burnout, likely GAD, likely MDD, and low resilience among teachers in Alberta and Nova Scotia is 1043. Based on the response rates achieved for the Text4Hope and Text4Mood programs in Alberta [35,63,64], we expect a maximum of 20% survey completion rate for the Wellness4Teachers program. Thus, to achieve the 1043 completed surveys at baseline, we expect to enroll 5515 teachers on the Wellness4Teachers program within 12 months. JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 5 (page number not for citation purposes) https://www.researchprotocols.org/2022/7/e37934 Sample Size Estimation We also hypothesize that factors such as gender, sex, age, marital status, number of years teaching, grade of teaching, and school type (elementary vs high school) will have an association With a teacher population of approximately 45,000 in Alberta and Nova Scotia, using a web-based script [62], we estimate JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 5 (page number not for citation purposes) https://www.researchprotocols.org/2022/7/e37934 XSL•FO RenderX JMIR RESEARCH PROTOCOLS Agyapong et al with burnout and other psychological disorders in teachers [67-72]. with burnout and other psychological disorders in teachers [67-72]. school policy and decision makers regarding psychological interventions for teachers, especially during the ongoing COVID-19 pandemic or similar stressful situations. We hope that the outcome of this study will promote the integration of supportive text messaging into many organizations’occupational health programs to provide readily available psychological support to individuals who need it, while at the same time improving their overall resilience and promoting professional fulfilment. Finally, we hypothesize that the Wellness4Teachers program will reduce the prevalence and severity of stress, anxiety, depression, burnout, and low resilience symptoms among teachers by at least 20% [43]. This specific hypothesis is based on related research findings. In the 2 RCTs conducted in Ireland and Canada, there was a greater than 20% reduction in depression symptom scores in the intervention group compared to the control group [73,74]. Furthermore, for the Text4Hope program in Alberta, there was a greater than 20% reduction in anxiety symptom scores in subscribers at 6 weeks and 3 months [36,37]. Conclusion The outcome of this study will establish the prevalence and correlates of the common risk factors for psychological disorders under study in teachers in Alberta and Nova Scotia. The study will have a significant impact on the management of stress, burnout, anxiety, depression, and low resilience among teachers. If the findings are positive, the Wellness4Teachers program can be promoted as a tool to support the mental health of teachers in Canada and internationally. The study outcome will also compliment policy decision-making for health care resource allocation in support of the education sector. The outcomes of this study will be evaluated with standardized and empirically validated questionnaires. The findings will contribute to knowledge on eHealth approaches in the education sector and will provide key information about the prevalence rates of stress, burnout, anxiety, depression, and low resilience as well as their correlates in teachers. The findings will also provide evidence of effectiveness for the use of daily supportive text messaging programs to address stress, burnout, anxiety, depression, and low resilience among teachers. Information from this study will thus be critical and useful for informing Expected Findings Stress, burnout, anxiety, and depression can have a significant impact on the health, lifestyle, psychological safety, and well-being of teachers, leading to low levels of resilience and reduced professional fulfilment. The psychological impact is likely to be more significant for those with prior mental health conditions or those who have been exposed to previous traumas. Mental health support for groups such as teachers require innovative techniques that can provide support for more teachers. This protocol outlines the use of mobile health technology as a convenient, cost-effective, and accessible means for implementing a psychological intervention for teachers who may be experiencing stress, burnout, anxiety, and depression, and improve their overall resilience. Limitations of the Study First, the self-report scales used to assess mental health variables such as likely MDD, although standardized, are not meant to be diagnostic. Second, it is possible that participants’ demographics in the study may not reflect the demographics of the teachers’population in Alberta or Nova Scotia, and therefore the study findings may not be generalizable to all teachers in the 2 provinces. Furthermore, web-based surveys with survey links delivered through text messages usually achieve a response rate of less than 20% [42,63,64,75-78], and therefore it is possible that we may not achieve our desired sample size. Lastly, the supportive text messages will be delivered for 6 months, and the outcome measures will be evaluated at 6 weeks, 3 months, and the end point of 6 months. It is unclear what the effects of the intervention would be if it were prolonged. It is also unclear if the benefits of the intervention would wane with the cessation of the daily supportive text messages. These limitations notwithstanding, this study is the first to examine the prevalence and correlates of stress, burnout, anxiety, and depression among teachers in the provinces of Alberta and Nova Scotia in Canada, aided by a text message program. This study is also the first globally to assess if daily supportive text messages delivered through the Wellness4Teachers program can reduce the prevalence and severity of stress, burnout, anxiety, and depression, and improve resilience among elementary and high school teachers. The findings would therefore be of interest to policy makers, especially those working in the education sector. Results The Wellness4Teachers program is expected to be launched in September 2022 when the new academic year is scheduled to begin. Enrollment will last for approximately 1 year, and data collection will continue for another 6 months. Study results will be disseminated with stakeholders in the education sector in Alberta and Nova Scotia and globally through workshops, conference presentations, and peer-reviewed publications. JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 6 (page number not for citation purposes) Acknowledgments This study is being funded by the Douglas Harding Trust Fund, the Alberta Mental Health Foundation, and the Global Psychological eHealth Foundation. This study is being funded by the Douglas Harding Trust Fund, the Alberta Mental Health Foundation, and the Global Psychological eHealth Foundation. References 1. Hillert A, Koch S, Lehr D. 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JMIR Ment Health 2020 Sep 25;7(9):e22408 [FREE Full text] [doi: 10.2196/22408] [Medline: 32915764] Please cite as: Agyapong B, Wei Y, da Luz Dias R, Agyapong VIO Burnout and Associated Psychological Problems Among Teachers and the Impact of the Wellness4Teachers Supportive Text Messaging Program: Protocol for a Cross-sectional and Program Evaluation Study JMIR Res Protoc 2022;11(7):e37934 URL: https://www.researchprotocols.org/2022/7/e37934 doi: 10.2196/37934 PMID: 76. Lawal MA, Shalaby R, Chima C, Vuong W, Hrabok M, Gusnowski A, et al. COVID-19 Pandemic: Stress, Anxiety, and Depression Levels Highest amongst Indigenous Peoples in Alberta. Behav Sci (Basel) 2021 Aug 24;11(9):115 [FREE Full text] [doi: 10.3390/bs11090115] [Medline: 34562953] Abbreviations by T Leung; submitted 12.03.22; peer-reviewed by K Adapa, J Beames; comments to author 10.05.22; revised version receive 22; accepted 13.06.22; published 14.07.22 ©Belinda Agyapong, Yifeng Wei, Raquel da Luz Dias, Vincent Israel Opoku Agyapong. Originally published in JMIR Research Protocols (https://www.researchprotocols.org), 14.07.2022. This is an open-access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work, first published in JMIR Research Protocols, is properly cited. The complete bibliographic information, a link to the original publication on https://www.researchprotocols.org, as well as this copyright and license information must be included. https://www.researchprotocols.org/2022/7/e37934 https://www.researchprotocols.org/2022/7/e37934 JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 10 (page number not for citation purposes) JMIR Res Protoc 2022 \| vol. 11 \| iss. 7 \| e37934 \| p. 10 (page number not for citation purposes) XSL•FO RenderX
https://openalex.org/W3006526054	https://hal.science/hal-03353449/document	English	null	Inhibition of Carbohydrate Hydrolysing Enzymes, Antioxidant Activity and Polyphenolic Content of Beilschmiedia Species Extracts	IOP conference series. Materials science and engineering	2,020	cc-by	6,121	PAPER • OPEN ACCESS PAPER • OPEN ACCESS IOP Conference Series: Materials Science and Engineering IOP Conference Series: Materials Science and Engineering Inhibition of Carbohydrate Hydrolysing Enzymes, Antioxidant Activity and Polyphenolic Content of Beilschmiedia Species Extracts To cite this article: Y N Tay et al 2020 IOP Conf. Ser.: Mater. Sci. Eng. 716 012007 View the article online for updates and enhancements. This content was downloaded from IP address 157.136.47.200 on 24/09/2021 at 08:35 This content was downloaded from IP address 157.136.47.200 on 24/09/2021 at 08:35 This content was downloaded from IP address 157.136.47.200 on 24/09/2021 at 08:35 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 Content from this work may be used under the terms of the Creative Commons Attribution 3.0 licence. Any further distribution of this work must maintain attribution to the author(s) and the title of the work, journal citation and DOI. nder licence by IOP Publishing Ltd 1 1 Bioprocess Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800, Minden, Gelugor, Penang, Malaysia 1 Bioprocess Technology Division, School of Industrial Technology, Universiti Sains Malaysia, 11800, Minden, Gelugor, Penang, Malaysia 2 School of Chemical Sciences, Universiti Sains Malaysia, 11800, Minden, Gelugor, Penang, Malaysia 3Centre for Natural Product Research and Drug Discovery (CENAR), Department of Chemistry, Faculty of Science, University of Malaya, 50603 Kuala Lumpur, Malaysia 4 Institut de Chimie des Substances Naturelles, CNRS-ICSN UPR2301, Univ. Paris-Sud 11, av. de la Terrasse, 91198 Gif-sur-Yvette, France Email: mhafizi88@usm.my Email: mhafizi88@usm.my Abstract. The goal of the present study was to provide in vitro evidence for potential inhibition of carbohydrate hydrolysing enzymes and antioxidant activities of methanol and ethyl acetate extracts from barks of two different Beilschmiedia - -glucosidase inhibitory activities, mode of enzyme inhibition, total polyphenolic content (TPC) and antioxidant capabilities. Methanolic bark extract of Beilschmiedia insignis --glucosidase with IC50 values of 3.233 µg/mL and 12.357 µg/mL, respectively. Further analysis of inhibition mode revealed that the extract demonstrated a mixed inhibition against both enzymes. In comparison to other extracts, methanolic bark extract of Beilschmiedia insignis demonstrated the highest TPC content of 420.393 mg GAE/g extract, lowest IC50 value of 12.103 µg/mL for DPPH radical scavenging ability and highest FRAP value of 1904.247 µM Fe (II)/mg extracts, indicating the antioxidant potential of the extract. A significant strong correlation coefficient was observed between TPC with FRAP (r = 0.994, p < 0.01) and TPC with DPPH (r = -0.860, p < 0.01), signifying that antioxidant activity and reducing capability were contributed by the polyphenolic compounds present in the crude extract. Collectively, methanolic bark extract of Beilschmiedia insignis possessed significant carbohydrate hydrolyzing enzyme inhibitory effects and antioxidant activity, suggesting its possible alternative application for diabetes and postprandial hyperglycemia treatment. Inhibition of Carbohydrate Hydrolysing Enzymes, Antioxidant Activity and Polyphenolic Content of Beilschmiedia Species Extracts Y N Tay1, M H A Bakar1, M N Azmi2, N A Saad2, K Awang3, M Litaudon4, and M A Kassim1 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 leading cause of death in women [1]. DM is characterized by high glucose level in the blood (hyperglycemia) associated by disturbances in carbohydrates, proteins and fats metabolism. Such defects in these metabolic pathways may lead to complete or partial insufficiency of insulin actionand/ or insulin resistance with macro-microvascular complications [2]. Previous studies reported that prolonged hyperglycemia is the key to these DM complications as it causes an increase in oxidative stress due to excess generation of free radicals through protein glycation, glucose oxidation and lipid peroxidation [3]. Therefore, the therapeutic approach for DM should be based on targeting glucose metabolism and oxidative stress caused by hyperglycemia-related conditions [3]. There are numerous conventional drugs available for DM, which varies in mechanism of treatment [4]. One of the pharmacological approaches is by using carbohydrate enzyme inhibitor drugs such as acarbose, voglibose and miglitol [5]. These drugs inhibit both -amylase and -glucosidase, which are enzymes responsible for the breakdown of carbohydrates. Such inhibition can be achieved by delaying the carbohydrate digestion, which decrease the rate of glucose absorption, thus maintaining blood glucose level as close to normal as possible. However, these current antidiabetic drugs suffer from a number of undesirable side effects, leading researchers to seek traditional medicinal plants as alternatives for diabetic treatment [4]. Beilschmiedia is a genus of approximately 250 species that are trees or shrubs in the family Lauraceae [6]. One of the previous phytochemical investigations of plants of the genus Beilschmiedia reported the presence of polyphenols which are the secondary metabolites of plants [7,8]. Polyphenols are organic chemicals characterized by the presence of large multiple phenol structural units. They are among the naturally occurring antidiabetic agents because polyphenols exhibit various important biochemical properties including carbohydrate hydrolysing enzyme inhibition and their capabilities to act as antioxidants [9]. In the present study, the methanol and ethyl acetate extracts from barks of Beilschmiedia lumutensis and Beilschmiedia insignis were evaluated on carbohydrate hydrolysing enzyme inhibition potential, polyphenolic content and antioxidant activities. 2.1. Plant material and preparation crude extracts 2.1. Plant material and preparation crude extracts The bark of Beilschmiedia lumutensis and Beilschmiedia insignis were collected at Machang, Kelantan on 2006 and at Kuala Lipis, Pahang on 2007, respectively. This plant was identified by T. Leong Eng, a botanist in University of Malaya. The voucher specimens (KL-5269 and KL-5368, respectively) have been deposited at the Herbarium of the Department of Chemistry, Faculty of Science, University of Malaya, Kuala Lumpur, Malaysia. The air-dried barks of Beilschmiedia lumutensis and Beilschmiedia insignis (each 200 g) were sliced, ground and extracted with EtOAc (3 × 0.5 L) followed by MeOH (3 × 0.5 L) at room temperature using maceration method. The solvents were filtered and removed using rotary evaporator to gave the respective extracts. Collectively, four crude extracts were produced for biological evaluation in this study, BLM: Beilschmiedia lumutensis-methanol; BLEA: Beilschmiedia lumutensis-ethyl acetate; BIM: Beilschmiedia insignis-methanol; BIEA: Beilschmiedia insignis-ethyl acetate. The extracts were carried out for carbohydrate hydrolysing enzyme inhibition potential, polyphenolic content and antioxidant activities. 2. Materials and method 2.1. Plant material and preparation crude extracts 1. Introduction In 2012, it was estimated that 1.5 million deaths worldwide were caused by diabetes mellitus (DM), causing it to be ranked number eight among the leading cause of death among both sexes and the fifth In 2012, it was estimated that 1.5 million deaths worldwide were caused by diabetes mellitus (DM), causing it to be ranked number eight among the leading cause of death among both sexes and the fifth The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Publishing 2.2. -amylase inhibition assay The -amylase inhibition assay was determined according to Kazeem et al., [10] with minor ! different concentrations (0.004-0.333 "!# ! "! - $% phosphate buffer (pH 6.9). The mixture was pre-incubated for 10 minutes at 25°C in a water bath. Then, ! &' $% () +# incubated for 10 minutes at 25°C. The reaction was terminated by adding 500 ! of DNS reagent, 2 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 e Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Publ The Third Bioprocessing and Biomanufacturing Symposium 2019 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 P Conf. Series: Materials Science and Engineering 716 (2020) 012007 doi:10.1088/1757-899X/716/1/01 $! distilled water and the absorbance was measured at ,$ using the Hitachi U-1900 spectrophotometer. Acarbose was used as a positive control. The percentage of enzyme inhibition was calculated by using the following equation: $! distilled water and the absorbance was measured at ,$ using the Hitachi U-1900 spectrophotometer. Acarbose was used as a positive control. The percentage of enzyme inhibition was calculated by using the following equation: Inhibition (%) = x 100% (1) (1) IC50 which is defined as the concentration of extracts required to achieve 50% of enzyme inhibition was calculated using GraphPad Prism. 2.3. -glucosidase inhibition assay g y The -glucosidase inhibition assay was performed accordingly to a previous method with slight modifications [11]. Yeast -glucosidase (1U/mL) and pNPG (3 mM) were prepared in 0.02 M phosphate buffer. An amount of 100 ! of -glucosidase enzyme (1U/mL) was added with 10 ! of plant extracts with different concentrations ranging from 0.004 mg/mL to 0.333 mg/mL and incubated for 10 minutes -./0 !123 37 °C for 20 minutes. The reaction was terminated by adding 2 mL 0.1 M sodium carbonate. The absorbance reading of the yellow p-nitrophenol was measured at 405 nm using the Hitachi U-1900 spectrophotometer. Acarbose was used as the positive control. Phosphate buffer was used to replace the enzyme for the blank and for negative control, plant extracts were replaced by phosphate buffer. The percentage of enzyme inhibition for -glucosidase was calculated using equation (1) and IC50 was calculated using GraphPad Prism. 2.4. -amylase inhibition The extract with the lowest IC50 4 -amylase inhibition assay was evaluated for mode of inhibition [10]. Firstly, 250 ! of the extract was mixed with 250 ! of -amylase enzyme. For control !)+ pre-incubated for 10 minutes at 25 °C in a ! concentrations ranging from 0.30 to 5.0 mg/mL was added to both set of the reaction mixtures to initiate the reaction. The mixture was further incubated for 10 minutes at 25 °C and 500 ! of DNS reagent was added to terminate the reaction. The amount of reducing sugars released was determined spectrophotometrically at 540 nm using a maltose standard curve generated using the maltose assay by DNS method. The mode of inhibition on -amylase was determined by analyzing the Lineweaver-Burk plot using Michaelis-Menten kinetics. The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 2.6. Determination of total polyphenolic content (TPC) 2.6. Determination of total polyphenolic content (TPC) The amount of total polyphenolic content (TPC) was determined by using the Folin–Ciocalteu reagent with slight modifications [12]. Each of the methanol and ethyl acetate Beilschmiedia extracts was dissolved in DMSO to a final concentration of 0.2 mg/mL. Then, 0.5 mL of the plant extract was dissolved in 2.5 mL of the Folin- Ciocalteu reagent (1/10 dilution) and the mixture was topped up to 10 mL with distilled water. The mixture was left for 5 minutes at room temperature, followed by the addition of 2 mL of 7.5% Na2CO3 solution. The final mixture was vortexed and incubated for 2 hours in a dark room at room temperature. Absorbance of the samples were measured at 765 nm using the Hitachi U-1900 spectrophotometer. A gallic acid standard curve was prepared ranging from gallic acid concentrations of 0.2-1.0 mg/mL and TPC was expressed in terms of mg of gallic acid equivalents (GAE) per gram of extract (mg GAE/g extract). The equation below was used for thecalculation: TPC (mg GAE/g extract) = C X (2) (2) where C represents the concentration determined from the gallic acid standard curve (mg/mL), V represents the volume of extracts used in the assay (mL) and M represents the mass of extracts used in the assay (g). 2.7. DPPH radical scavenging assay DPPH is a dark- purple coloured crystalline powder composed of stable free-radical molecules. This assay is used to determine the antioxidant ability of the extract being tested by observing the change in colour from purple to yellow by the reduction process caused by antioxidants present in the extract. The DPPH radical scavenging assay was performed accordingly with minor modifications [5]. An amount of 1.5 mL of 0.1 mM DPPH prepared in methanol was added to 0.5 mL of the sample extracts of different concentrations (0.004 to 0.333 mg/mL) in a test tube. The mixture was vortexed for 30 seconds and left to stand in the dark at room temperature for 30 minutes. Then, the absorbance was measured at 517nm using the Hitachi U-1900 spectrophotometer. The positive control used was ascorbic acid. The negative control was prepared by replacing the extracts with methanol and the blank was prepared by replacing DPPH with methanol. The IC50 values were determined using GraphPad Prism and total DPPH radical scavenging ability (%) was calculated using the equation: DPPH radical scavenging ability (%) = x 100% (3) DPPH radical scavenging ability (%) = x 100% (3) (3) 2.9. Statistical analysis -glucosidase inhibition The extract with the lowest IC50 value from -glucosidase inhibition assay was evaluated for mode of 5&67 & ! & !-glucosidase enzyme. The mixture was pre-incubated for 10 minutes at 37 °C in a water bath while pH 6.9 phosphate !1234$% phosphate buffer (pH 6.9) was added at increasing concentrations ranging from 0.63 to 2.0 mg/mL to both set of the reaction mixtures to initiate the reaction. The mixture was further incubated for 10 minutes at 37 °C and $ ! of sodium carbonate was addedto stop the reaction. The amount of reducing sugars released was determined spectrophotometrically at 405 nm by using a p-nitrophenol standard 4 -glucosidase was determined by analyzing the Lineweaver-Burk plot using Michaelis-Menten kinetics. 3 2.8. Ferric ion reducing antioxidant power (FRAP) assay 2.8. Ferric ion reducing antioxidant power (FRAP) assay The FRAP assay was performed with slight modifications [13]. In brief, an amount of 3 mL of FRAP 8& ! (---"!#4 was placed in a water bath at 37°C for 30 minutes in a dark condition and the absorbance was measured at 593 nm using the Hitachi U-1900 spectrophotometer. Ascorbic acid was used as the positive control. A ferrous sulphate standard curve ranging from 5-100 µM was constructed. The absorbance reading obtained was substituted into the standard curve to obtain the concentration of ferrous sulphate and the 9:;24( %9(77#" extracts). 3 Results and discussion The barks of Beilschmiedia lumutensis and Beilschmiedia insignis were extracted using two different solvents which were methanol and ethyl acetate. The resulting IC50 values of each extracts were calculated and presented in Table 1, where a lower IC50 value indicates higher enzyme inhibition activity. The bark methanolic extract of Beilschmiedia insignis was observed to exhibit as promising -amylase and -glucosidase inhibitor. For -amylase inhibition, BIM extract had the lowest IC50 value of 3.233 ± 0.512 µg/mL, which explains the better potency of BIM extract in -amylase, thus delaying the breakdown and absorption of carbohydrates and lowering the glucose peaks encountered in postprandial glucose [14]. As for -glucosidase inhibition, BIM extract demonstrated lowest IC50 value of 12.357 ± 1.785 µg/mL compared to other extracts. Plant species are known to contain a broad supply of bioactive compounds or secondary metabolites that are devoted to the beneficial medicinal effects [15]. These bioactive compounds differ in solubility when extracted using different extraction solvents such as methanol and ethyl acetate. Therefore, different active compounds extracted differently by exhibiting different yields when different extraction solvents were used [16]. In the current study, the bioactive compound in the crude extract responsible for -amylase and -glucosidase inhibition was more likely to be extracted by methanol. This indicates that the inhibitory activity was influenced by the choice of solvent used during extraction and the solubility of the bioactive compounds. Table 1. IC50 values for -amylase and -glucosidase inhibitory activity of two Beilschmiedia species (Beilschmiedia lumutensis and Beilschmiedia insignis) extracted using two different solvents (methanol and ethyl acetate). Extract IC50 v -amylase (μg/mL) IC50 -glucosidase (μg/mL) BLM 7.398 ± 1.091 58.377 ± 6.675 BLEA 7.905 ± 1.002 21.913 ± 3.933* BIM 3.233 ± 0.512 12.357 ± 1.785 * BIEA 19.203 ± 1.892 46.700 ± 4.963* Acarbose 13.784 ± 3.703 4148.333 ± 650.177 Each value represent the mean IC50 value (N=3, mean ± SEM). * Values are significantly different (p < 0.05) as compared to acarbose. * Values are significantly different (p < 0.05) as compared to acarbose. BIM extract with the lowest IC50 4< 4 - -glucosidase activities. The results in the form of Lineweaver-Burk plot were shown in Fig. 1 and 2. It was shown that both the Vmax and Km were influenced by the presence of BIM extract which indicated a mixed inhibition. 2.9. Statistical analysis y All experiments were performed in triplicates and data was presented in terms of mean ± standard error of mean (SEM). The IC50 values were calculated using GraphPad Prism. The differences between the data obtained and positive control were assessed using t-test and a p value of less than 0.05 is considered 4 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 as significantly different. Pearson correlation was used to correlate TPC and biological activities of extracts accordingly. as significantly different. Pearson correlation was used to correlate TPC and biological activities of extracts accordingly. . Series: Materials Science and Engineering 716 (2020) 012007 doi:10.1088/1757-899X/716/1/012007 In mixed inhibition, inhibitors tend to attach to the allosteric site of the enzyme instead of the active site. Therefore, it can be concluded that the active compounds in BIM extract interact with the enzyme by binding to the allosteric site of the free enzyme, causing conformational changes to the active site of the enzyme and lowers the enzyme affinity for the substrate, which explains the increase in Km value. The decrease in Vmax value observed indicated such inihibition that can be resulted in a retarded breakdown of disaccharides into absorbable monosaccharides, which lowers hyperglycemia peaks in type 2 diabetes [17]. Figure 2. %-glucosidase inhibition by BIM extract using Lineweaver-Burk plot. Figure 1. % -amylase inhibition by BIM extract using Lineweaver-Burk plot. Figure 2. %-glucosidase inhibition by BIM extract using Lineweaver-Burk plot. Figure 1. % -amylase inhibition by BIM extract using Lineweaver-Burk plot. Figure 2. %-glucosidase inhibition by BIM extract using Lineweaver-Burk plot. Figure 2. %-glucosidase inhibition by BIM extract using Lineweaver-Burk plot. Figure 2. %-glucosidase inhibition by BIM extract using Lineweaver-Burk plot. Figure 1. % -amylase inhibition by BIM extract using Lineweaver-Burk plot. In mixed inhibition, inhibitors tend to attach to the allosteric site of the enzyme instead of the active site. Therefore, it can be concluded that the active compounds in BIM extract interact with the enzyme by binding to the allosteric site of the free enzyme, causing conformational changes to the active site of the enzyme and lowers the enzyme affinity for the substrate, which explains the increase in Km value. The decrease in Vmax value observed indicated such inihibition that can be resulted in a retarded breakdown of disaccharides into absorbable monosaccharides, which lowers hyperglycemia peaks in type 2 diabetes [17]. In the present study, the TPC of three Beilschmiedia species extracted using two different solvents were determined and shown in Table 2. All four extracts contained polyphenolic compounds. The TPC of all four extracts ranged from 60.439 to 420.393 mg GAE/g extract. The results gathered followed the order: BIM > BLM > BIEA > BLEA. All methanolic extracts had a higher TPC value than their respective ethyl acetate extracts. This result indicated that methanol is a preferred extraction solvent when compared to ethyl acetate in the extraction of polyphenolic compounds due to its higher polarity index. 3 Results and discussion The active compounds that contributed to -amylase and - glucosidase inhibition present in BIM extract is believed to bind preferably to the free enzyme instead of the enzyme-substrate complex. 5 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Publishing The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 p g g y p IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 g doi:10.1088/1757-899X/716/1/012007 . Series: Materials Science and Engineering 716 (2020) 012007 doi:10.1088/1757-899X/716/1/012007 This confirmed that choice of solvent, the polarity index and the solubility of TPC in the solvent affects the recovery of polyphenolic contents [18]. The antioxidant activity of polyphenolic compounds result from their redox properties, enabling them to neutralize free radicals, act as reducing agents, hydrogen donors, quenching singlet and triplet oxygen and metal chelators [12]. Polyphenolic compounds were also reported to contribute towards enzyme inhibition due to their hydroxyl group that is able to bind with proteins, which explains the ability of most Beilschmiedia extracts in the current research in inhibiting -amylase and -glucosidase [19]. Table 2. Total polyphenolic content of three Beilschmiedia species extracted using two extraction solvents, methanol and ethyl acetate. Extract Total phenolic content (mg GAE/g extract) BLM (bark) 344.202 ± 9.275 BLEA (bark) 60.439 ± 4.684 BIM (bark) 420.393 ± 8.010 BIEA (bark) 82.836 ± 7.928 Table 2. Total polyphenolic content of three Beilschmiedia species extracted using two extraction solvents, methanol and ethyl acetate. 6 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Publishing IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 doi:10.1088/1757-899X/716/1/012007 The resulting IC50 values for DPPH radical scavenging ability of each extracts were presented in Table 3 in comparison to ascorbic acid (AA). All extracts were weaker than ascorbic acid in scavenging the DPPH radical and had IC50 values that were significantly higher (p < 0.05) than ascorbic acid. BIM extract with the lowest IC50 value of 12.103 ± 0.312 µg/mL demonstrated the strongest DPPH radical scavenging ability whereas BLEA extract with the highest IC50 value of 245.867 ± 35.220 µg/mL demonstrated the weakest DPPH radical scavenging ability. The results of the current study proved that TPC of the extracts were obtained in a more polar solvent with increased level of antioxidants. This finding is in line with previous findings where the antioxidant property of the plant extract was correlated to the levels of polyphenolic content and that antioxidant activity of plants were dependent and influenced by the choice of extracting solvents [20]. This finding is of significant as plant extracts with DPPH scavenging ability are capable to donate their hydrogen atom or electrons, acting as primary antioxidants, thus neutralizing the free radicals in hyperglycemia-induced oxidative stress that leads to complications in type 2 diabetes [21] Table 3. IC50 values for DPPH radical scavenging ability of three Beilschmiedia species. . Series: Materials Science and Engineering 716 (2020) 012007 doi:10.1088/1757-899X/716/1/012007 Extracts IC50 values for DPPH (µg/mL) BLM 13.170 ± 0.935* BLEA 245.867 ± 35.220* BIM 12.103 ± 0.312* BIEA 137.950 ± 15.650* AA 4.930 ± 0.316 * Values are significantly different (p < 0.05) as compared to AA. Table 3. IC50 values for DPPH radical scavenging ability of three Beilschmiedia species. The reducing potential of all Beilschmiedia extracts were evaluated for their ability to reduce the straw colour TPTZ-Fe (III) complex into blue colour, TPTZ-Fe (II). The results revealed that all of the Beilschmiedia extracts possessed reducing power, indicated by their respective FRAP values ranging from 499.212 ± 18.884 to 1904.247 ± 24.588 µM Fe (II)/mg extracts (Fig. 3). The results obtained were in the order: AA > BIM > BLM > BIEA > BLEA, which was almost similar to current findings inTPC and DPPH assay. Based on Figure 3, it was observed that both methanolic extracts exhibited higher FRAP values than their respective ethyl acetate extracts, which reconfirmed the trend in our previous findings in TPC and DPPH assay. Highest FRAP value was exhibited by BIM extract. This result was in parallel with a previous study [22], which discovered that the extraction of active compounds was solvent dependent and polar solvent (methanol) is an efficient solvent for extraction of antioxidants and polyphenolic compounds. Therefore, it can be deduced that the high reducing ability of BIM extract was mostly contributed by the polyphenolic compounds present [23]. 7 7 The Third Bioprocessing and Biomanufacturing Symposium 2019 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 doi:10.1088/1757-899X/716/1/012007 6 0 0 0 4 0 0 0 2 0 0 0 0 B L M B L E A B I M B I E A A A E x t r a c t s Figure 3. FRAP value of two Beilschmiedia species extracted using two extraction solvents, methanol and ethyl acetate. * Values are significantly different (p < 0.05) as compared to AA. * * * * F R A P v a l u e ( μ M F e ( I I ) / m g e x t r a c t s ) Figure 3. FRAP value of two Beilschmiedia species extracted using two extraction solvents, methanol and ethyl acetate. . Series: Materials Science and Engineering 716 (2020) 012007 doi:10.1088/1757-899X/716/1/012007 * Values are significantly different (p < 0.05) as compared to AA. Pearson correlation analysis was conducted to analyze the correlations between TPC and some biological activities. Based on Table 4, the highest correlation coefficient, r was found between TPC and FRAP activity and the lowest correlation coefficient was found between TPC and -glucosidase inhibitory activity. TPC had significant strong correlations with both DPPH and FRAP antioxidant assays with respective correlation coefficient of -0.860 and 0.994. Similar results were reported from a previos report [24], where a strong correlation between TPC and both DPPH and FRAP antioxidant assays were observed with correlation coefficients of 0.921 and 0.868 respectively. This indicated that polyphenolic content found in Beilschmiedia extracts used in the current research strongly contributed to the radical scavenging capability and reducing strength seen in both antioxidant assays, which is in line with a previous analysis [25]. TPC and enzyme inhibitory activities were also correlated. it was observed that a higher correlation coefficient (-0.589, p > 0.01) was seen between TPC and -amylase inhibitory activity compared to TPC and -glucosidase inhibitory activity ( -0.191, p > 0.01). There is a -amylase inhibition than to -glucosidase inhibition. Therefore, -glucosidase inhibition activity demonstrated by Beilschmiedia extracts in the current study may be contributed by other phytochemicals or secondary metabolites but not in the form of polyphenolic compounds. Pearson correlation analysis was conducted to analyze the correlations between TPC and some biological activities. Based on Table 4, the highest correlation coefficient, r was found between TPC and FRAP activity and the lowest correlation coefficient was found between TPC and -glucosidase inhibitory activity. TPC had significant strong correlations with both DPPH and FRAP antioxidant assays with respective correlation coefficient of -0.860 and 0.994. Similar results were reported from a previos report [24], where a strong correlation between TPC and both DPPH and FRAP antioxidant assays were observed with correlation coefficients of 0.921 and 0.868 respectively. This indicated that polyphenolic content found in Beilschmiedia extracts used in the current research strongly contributed to the radical scavenging capability and reducing strength seen in both antioxidant assays, which is in line with a previous analysis [25]. TPC and enzyme inhibitory activities were also correlated. Conclusion It can be concluded that BIM extract serves as a promising candidate for further evaluation as diabetic --glucosidase by mixed inhibition. Along with promising enzyme inhibition potential, BIM extract also possessed antioxidant activity. BIM extract demonstrated the highest TPC content, the lowest IC50 in DPPH radical scavenging assay and highest FRAP value out of all tested extracts. This revealed the strong correlation between TPC and both DPPH and FRAP antioxidant assays. Taken together, all these findings supported the potential of methanolic bark extract of Beilschmiedia insignis to be part of the potential medicinal plants that may serve as one of the alternative treatments for hyperglycemia-related conditions. . Series: Materials Science and Engineering 716 (2020) 012007 doi:10.1088/1757-899X/716/1/012007 it was observed that a higher correlation coefficient (-0.589, p > 0.01) was seen between TPC and -amylase inhibitory activity compared to TPC and -glucosidase inhibitory activity ( -0.191, p > 0.01). There is a -amylase inhibition than to -glucosidase inhibition. Therefore, -glucosidase inhibition activity demonstrated by Beilschmiedia extracts in the current study may be contributed by other phytochemicals or secondary metabolites but not in the form of polyphenolic compounds. Table 4. Pearson correlation coefficient, r between total polyphenolic content (TPC) and the biological activities of Beilschmiedia species. Table 4. Pearson correlation coefficient, r between total polyphenolic content (TPC) and the biological activities of Beilschmiedia species. Biological Activities Pearson correlation coefficient, r TPC DPPH scavenging ability -0.860 FRAP ability 0.994 -amylase inhibitory activity -0.589 -glucosidase inhibitory activity -0.191 ** significant correlation at p < 0.01. 8 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 The Third Bioprocessing and Biomanufacturing Symposium 2019 The Third Bioprocessing and Biomanufacturing Symposium 2019 IOP Conf. Series: Materials Science and Engineering 716 (2020) 012007 IOP Publishing doi:10.1088/1757-899X/716/1/012007 IOP Conf. 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J. 12 5 [22]Barchan A, Bakkali M, Arakrak A, Pagán R and Laglaoui A 2014 The effects of solvents polarity on the phenolic contents and antioxidant activity of three Mentha species extracts Int J Curr Microbiol App Sci 3 399–412 [23]Sadeghi Z, Valizadeh J, Shermeh O A and Akaberi M 2015 Antioxidant activity and total phenolic content of Boerhavia elegans (choisy) grown in Baluchestan, Iran Avicenna J. phytomedicine 5 1 [24] Hyun T K, Kim H-C, Ko Y-J and Kim J-@&*;-glucosidase inhibitory and anti- inflammatory effects of aerial parts extract from Korean crowberry (Empetrum nigrum var. japonicum) Saudi J. Biol. Sci. 23 181–8 [25] Liu H, Qiu N, Ding H and Yao R 2008 Polyphenols contents and antioxidant capacity of 68 Chinese herbals suitable for medical or food uses Food Res. Int. 41 363–70 Acknowledgement This study was supported by the TWAS-COMSTECH Joint Research Grant-18-420 RG/PHA/AS_C, Universiti Sains Malaysia Short Term Grants (Reference no: 304/PTEKIND/6313329 and 304/PKIMIA/6313330) and RUI USM 1001/PKIMIA/8012310. This research was conducted within the frame of the auspices of CNRS-UM; Associated International Laboratory (LIA) under International French Malaysian Natural Product Laboratory (IFM-NatProLab). We thank Nor, D.M., Syamsir, R., and Teo, L.E. (UM) for the collection and identification of plant material. 10 10
https://openalex.org/W2052868483	https://zenodo.org/record/2240608/files/article.pdf	English	null	SOME NEW SPECIES OF THE GENUS LONCHÆA (DIPTERA, LONCHÆIDÆ)	The Canadian entomologist/Canadian Entomologist	1,920	public-domain	1,010	Lonchaea nigrocaerulea, sp. n. iWale.-Blue-black, almost metallic in colour. Antennz and palpi black; arista pale a t base; upper orbits and ocellar triangle glossy. Legs black. Wings, calyptrz, and halteres as in major. Frons about 1.5 as long as wide, sparsely hairy; upper orbits highly glossy smooth; third antennal segment about four times as long as wide, extending below mouth margin; arista as in major; palpi dilated, slightly protruded. Praescutellar acrostichals very long; scutellum as in major; pteropleura bare; no bristly hairs adjacent to stigmatal bristle. Abdomen with longer hairs than in major, especially a t apex of fourth tergite; fourth tergite about twice as long as third. Venation as in major, but the antepenultimate section of fourth vein is only two-thirds as long as penultimate, whereas in major it is three- fourths as long. Hind femur with a few distinct anteroventral bristles. Length 4 mm. ~~pe.-darbados (E. F. Becher). Type in British Museum. ~~pe.-darbados (E. F. Becher). Type in British Museum. ~~pe.-darbados (E. F. Becher). Type in British Museum. Lonchaea major, sp. n. ~Vale.~Shining black, with a slight bluish tinge. A n t e n n ~ and palpi black; arista yellowish at base. Legs pitchy black, the hind tarsi hardly paler than their tibiz. Wings hyaline, veins brownish yellow, whitish a t bases. Calyptrz white, fringes concolorous except a t junction of upper and lower, where there are some long, black, setulose hairs. Halteres black. Eyes bare; frons about twice as long as wide, parallel-sided, surface with short, moderately dense hairs; upper orbits slightly rugose above, bare; frontal lunule hairy; longest hairs on arista over twice as long as its basal diameter; third antennal segment about four times as long as its width, extending to mouth margin. Scutellum with setulose hairs on margins and between apical bristles. the disc bare; pteropleura bare; no hairs adjacent to the stigmatal bristle. Abdomen broad, with rather dense, short, setulose hairs on dorsum; fourth tergite about 1.5 as long as third. Hind femur without distinct anteroventral bristles. Auxiliary vein almost fused with first a t its apex. Length 6.5 mm. TJJ@~.-Amazon, South America. Type in British Museum. 246 246 THE CANADI-4N ENTObIOLOGIST Lonchaea striatifrons, sp. n. Male.-Glossy steel-blue, the abdomen paler than the thorax. Frons shining black, ocellar spot and upper orbits glossy blue; third antennal segment reddish a t base below. Legs black, tarsi with the exception of the apical two or three segments reddish testaceous. Wings clear, yellow a t bases. C a l y p t r ~ white, fringes concolorous. Halteres black. Frons less than twice as long as wide a t anterior margin, not distinctly November. 1920 247 THE CASADIAX EKTOMOLOGIST narrowed anteriorly, the surface of interfrontalia finely striate on upper half laterally; orbits narrow, distinctly separated from ocellar triangle posteriorly their surfaces rnicroscopically diagonally striate anteriorly; hairs on frons short and sparse; third antennal segment three times as long as wide; cheek narrow, without dense hairs or strong bristles. Thorax as in poliin Say. Basal abdominal sternite with some hairs on each side. Legs and wings as in polita. Fringes of calyptrz longer than usual, about six of the hairs at apex of the fold setulose and longer than the calyptrz. narrowed anteriorly, the surface of interfrontalia finely striate on upper half laterally; orbits narrow, distinctly separated from ocellar triangle posteriorly their surfaces rnicroscopically diagonally striate anteriorly; hairs on frons short and sparse; third antennal segment three times as long as wide; cheek narrow, without dense hairs or strong bristles. Thorax as in poliin Say. Basal abdominal sternite with some hairs on each side. Legs and wings as in polita. Fringes of calyptrz longer than usual, about six of the hairs at apex of the fold setulose and longer than the calyptrz. Female.-Frons at least one-third of the head-width and less than 1.5 a long as wide, not so distinctly striated as in male. Ovipositor as in politz Say Length 4-4.5 mm. Type.-Male, allotype, and one male paratypc, Santa Clara, Cal. (Baker) ; one male, San Diego, Cal. (Harkins collection); one male, Palo Alto, Cal., August 3. Type in collection of University of California. Lonchaea bakeri, sp. n. Female.-Glossy black, without distinct bluish tinge, the frons greenish blue anteriorly. Face and cheeks with whitish pruinosity; from shining upper, orbits and ocellar triangle glossy; antennz brownish black. Legs black, tarsi yellow, apical two segments darkened. Wings clear, veins yellow. Calyptrz and their fringes white. Frons a t vertex one-third of the head-width, not narrowed anteriorly, its length about 1.5 of its width, surface microscopically striate on upper half granulose anteriorly, with sparse hairs: upper orbits almost imperceptibly striate; third antennal segment not twice as long as wide; face not carinate, cheek with 3 or 4 strong, but not very long bristles on lower margin anteriorly. A strong bristle on upper margin of mesopleura a little behind spiracle and considerably cephalad of the vertical series at anterior margin of haired area; scutellum without hairs between apical bristles. Ovipositor not broad, the apical hairs very short. Hind femora without antero-ventral bristles; antero- dorsal setulz on hind tibiz stronger than usual. Veins 3 and 4 very slightly convergent apically. Fringes of calyptrz normal. Length 3 mm., exclusive of ovipositor. Type.-Chinangega, Nicaragua (F. C. Baker). Type.-Chinangega, Nicaragua (F. C. Baker). Named in honour of the collector. Named in honour of the collector. This species is closely allied to albiceps Malloch, having the same armature of the mesopleura, and the same type of frons but without the transverse de- pression, and the calyptrz white, and venation different. Type in collection of University of California. AN ADDITION TO BIBLIOGRAPHY ON AGRILUS (Coleop.). Through an oversight on our part, a recent paper of Burke's was omitted from the Agrilzu bibliography, (Can. Ent., Sept., 1920, p. 204), and is given below. Burke, Jour. Econ. Ent., Vol. 10, No. 3, pp. 325-332, 1917. Contains notes on food plants and habits of Agrilus angelicus Horn, A. niaeiventris Horn, A. granztlatus Say, A. anxius Gory, A. acutipennis Mann., A. politus Say. C. A. FROST and H. B. WEISS.
https://openalex.org/W4238053870	https://osf.io/z3tsd/download	English	null	Perceptual Boundaries Cause Mnemonic Trade-Offs Between Local Boundary Processing and Across-Trial Associative Binding	null	2,017	cc-by	21,276	unning head: PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY unning head: PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Perceptual boundaries cause mnemonic trade-oﬀs between local boundary processing and across-trial associative binding Andrew C. Heusser1, Youssef Ezzyat1, Ilana Shiﬀ1, and Lila Davachi1,2 1Department of Psychology, New York University 2Center for Neural Science, New York University Aﬃliation Author Note Corresponding Author: Lila Davachi Email: lila.davachi@nyu.edu Phone Number: (212) 992-9612 Address: New York University Department of Psychology 6 Washington Pl. Room 871A New York, NY 10003 This research was supported by NIMH RO1 MH074692 to Lila Davachi. Perceptual boundaries cause mnemonic trade-oﬀs between local boundary processing and across-trial associative binding Andrew C. Heusser1, Youssef Ezzyat1, Ilana Shiﬀ1, and Lila Davachi1,2 1Department of Psychology, New York University 2Center for Neural Science, New York University Aﬃliation Andrew C. Heusser1, Youssef Ezzyat1, Ilana Shiﬀ1, and Lila Davachi1,2 Author Note Author Note Corresponding Author: Lila Davachi Email: lila.davachi@nyu.edu Phone Number: (212) 992-9612 This research was supported by NIMH RO1 MH074692 to Lila Davachi. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 2 2 Abstract PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 3 Perceptual boundaries cause mnemonic trade-oﬀs between local boundary processing and across-trial associative binding Perceptual boundaries cause mnemonic trade-oﬀs between local boundary processing and across-trial associative binding Abstract Episodic memories are not veridical records of our lives, but rather are better described as organized summaries of experience. Theories and empirical research suggest that shifts in perceptual, temporal and semantic information lead to a chunking of our continuous experiences into segments, or ‘events’. However, the consequences of these contextual shifts on memory formation and organization remains unclear. In a series of three behavioral studies, we introduced context shifts (or ‘event boundaries’) between trains of stimuli and then examined the inﬂuence of the boundaries on several measures of associative memory. In Experiment 1, we found that perceptual event boundaries strengthened associative binding of item-context pairings present at event boundaries. In Experiment 2, we observed reduced temporal order memory for items encoded in distinct events relative to items encoded within the same event, and a trade-oﬀbetween the speed of processing at boundaries, and temporal order memory for items that ﬂanked those boundaries. Finally, in Experiment 3 we found that event organization imprinted structure on the order in which items were freely recalled. These results provide insight into how boundary- and event-related organizational processes during encoding shape subsequent representations of events in episodic memory. Keywords: episodic memory, event boundaries, temporal order memory, associative memory PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Introduction Although our experiences unfold in a forward and continuous manner, our memories for those experiences are structured and organized around speciﬁc events. For example, imagine spending a night out in New York City. You might go out to dinner with friends, stop at a bar for a cocktail, and then proceed to an evening concert. Prior research suggests that the experiential details within a speciﬁc event (i.e. dinner, bar, or concert) are more tightly linked in memory than details experienced in distinct events (Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2011). One possible explanation for this ﬁnding is that associative processes that serve to link adjacent representations of an event are enhanced when contextual aspects of an experience are shared across time. Thus, the contents of a memory may be structured and grouped by the context in which the experience occurred. However, it is also known that contextual novelty is beneﬁcial for some forms of encoding and, thus, memory may also beneﬁt from distinctiveness in contextual representations (Ranganath & Rainer, 2003; Restorﬀ, 1933). Across a series of studies, we aimed to understand how contextual stability and distinctiveness both beneﬁt memory. Speciﬁcally, the work is focused on elucidating the diﬀerent forms of associative memory that might beneﬁt from shared and distinctive context. A large body of prior work supports the idea that organizational processes engaged at encoding modulate the structure of our memories for those experiences (Atkinson & Shiﬀrin, 1971; Farrell, 2012; Lee & Estes, 1981; Lehman & Malmberg, 2013; Miller, 1956; Murdock, 1983; Raaijmakers & Shiﬀrin, 1981). One computational account for how this occurs suggests that the contents of our experiences are maintained in a limited-capacity buﬀer during encoding (Lehman & Malmberg, 2009, 2013). Item and context representations that co-occupy the buﬀer become associatively bound. When those maintained representations are no longer of use to the participants, a ‘compartmentalization’ operation is hypothesized to clear the contents of the buﬀ ‘compartmentalization’ operation is hypothesized to clear the contents of the buﬀer. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 4 Thus, episodic ‘events’ may become bound in long-term memory as a downstream consequence of encoding-related organizational processes that compartmentalize information through a selective integration mechanism. Another conceptually related framework called Event Segmentation Theory (EST) hypothesizes that ‘segmentation’ processes parse ongoing experience into events and serve to guide eﬃcient allocation of cognitive processing resources in the moment (Reynolds, Zacks, & Braver, 2007; Zacks, Speer, Swallow, Braver, & Reynolds, 2007). This model proposes that incoming perceptual information and prior experience are actively integrated in working memory to generate predictions about what is likely to occur in the near future. At event boundaries, when future input may be unpredictable or surprising, attention is drawn to novel perceptual features in the environment and the prior event model is abandoned. Prior behavioral and neuroimaging results support the notion that people can segment ongoing experience into events and do so in a similar fashion (Radvansky, 2012; Speer & Zacks, 2005; Swallow, Zacks, & Abrams, 2009; Zacks et al., 2001, 2007). Furthermore, there is evidence that information contained at event boundaries is better remembered than information contained within an event (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004). While somewhat diﬀerent in their implementation, both of these models make the unique and interesting prediction that shifts in context may lead to enhanced memory for boundary information at the expense of ongoing integration processes. Put another way, while event boundaries may lead to a memory enhancement for information encountered at the contextual shift, they may in fact have a detrimental eﬀect on associative, or sequential, memory for pairs of items that ﬂank that boundary due to an interruption of ongoing maintenance/integration processes. Prior research has typically focused on the positive eﬀects of boundaries on item memory (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004). However, this series of experiments was designed to test the idea that contextual shifts may be good for ‘in-the-moment’ (within-trial) associative memory encoding at the expense of across-trial sequence memory. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 6 encoded lists of object stimuli that were grouped into smaller mini-lists (i.e. events) by a shared background color. This simple perceptual manipulation of the background color allowed us to ask whether low-level perceptual shifts in context have consequences for later memory. In Experiment 1, we tested whether items at perceptual event boundaries are more strongly bound to their context (i.e. the color background), relative to trials where there was no contextual shift. In Experiment 2, we tested whether event structuring led to reduced binding between items encountered in distinct events vs. within events, and also whether the speed of processing at boundaries is related to the cost in across-event associative memory for pairs of items. Finally, in Experiment 3 we utilize a more naturalistic retrieval task (a free recall paradigm) to ask whether and how recall behavior is organized according to the event structure of the experiment. encoded lists of object stimuli that were grouped into smaller mini-lists (i.e. events) by a shared background color. This simple perceptual manipulation of the background color allowed us to ask whether low-level perceptual shifts in context have consequences for later memory. In Experiment 1, we tested whether items at perceptual event boundaries are more strongly bound to their context (i.e. the color background), relative to trials where there was no contextual shift. In Experiment 2, we tested whether event cturing led to reduced binding between items encountered in distinct events vs. within events, and also whether the speed of processing at boundaries is related to the cost in across-event associative memory for pairs of items. Finally, in Experiment 3 we utilize a more naturalistic retrieval task (a free recall paradigm) to ask whether and how recall behavior is organized according to the event structure of the experiment. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 5 First, we predicted that items encountered at event boundaries would be more tightly bound to their context, as attention may be shifted away from the maintenance of previous ‘within-event’ information to the changing contextual features in the environment. While previous studies suggest that that item memory at boundaries may be boosted (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004), here we explore speciﬁcally whether these items are more tightly bound to their associated contexts at event boundaries. We addressed this question in our ﬁrst experiment. Second, we predicted that integration processes across contiguous items that share the same context (i.e. an ‘event’) will be enhanced relative to pairs of items that were encountered in neighboring events. Put another way, while event boundaries may lead to a memory enhancement for information encountered at the contextual shift, they may in fact have a detrimental eﬀect on relational memory for pairs of items that ﬂank that boundary due to an interruption of ongoing item-item integration processes. Recent work using narrative cued recall (Ezzyat & Davachi, 2011), recency discrimination for sequences of visual images (Dubrow & Davachi, 2013; DuBrow & Davachi, 2016), and temporal proximity judgments (Ezzyat & Davachi, 2014) is consistent with this prediction, showing that several measures of associative memory are reduced for pairs stimuli that ﬂank an event boundary (compared to items within the same event). Finally, we test whether these two memory eﬀects are in fact related. Speciﬁcally, we test whether there is a trade-oﬀbetween item-context processing at event boundaries and across-event integration processes, such that a stronger boundary eﬀect may result in worse across-event integration. We hypothesize that, at boundaries, attentional resources are redirected from the integration/maintenance of previous within-event representations to changing features in the environment. In other words, the subjective strength of the event boundary should inﬂuence associative memory for pairs of items studied across that boundary, such that a ‘larger’ boundary should result in worse across event associative memory. The present set of experiments was designed to test these predictions by assessing multiple forms of associative memory. In each of these experiments, participants PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 7 object-color associative memory. We predicted that the memory enhancement would be speciﬁc to the boundary object-color pair. A pattern of this nature would suggest a transient memory eﬀect, perhaps driven by a boundary-driven allocation of attentional resources (Kurby & Zacks, 2008). Experiment 1: Perceptual boundaries facilitate object-color associative memory. In Experiment 1, we sought to understand how perceptual boundaries inﬂuence associative memory for the local information presented at boundaries. Participants encoded lists of objects that were embedded in a colored frame (Figure 1). On each trial, they were instructed to imagine the displayed object in the color of the background frame and to make a pleasant/unpleasant judgment on the object-color combination. Importantly, the color of the frame did not change for six consecutive trials before switching to a new background color. We operationalized an “event” as consecutive trials where the color of the frame stayed the same. After each encoding list, participants performed an object-color associative memory test. “Boundary” trials were deﬁned as trials on which the presented color frame was diﬀerent from the previous trial; “non-boundary” trials were all other trials (i.e. the presented color matched the color from the preceding trial). Boundary objects were therefore objects that were encoded concurrently with a color frame switch, while non-boundary objects were all other objects. The goal of Experiment 1 was to assess whether perceptual event boundaries increased PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Methods Participants. Participants were 26 individuals (ages 18-35) recruited from New York University and the greater New York Metropolitan Area. All participants gave informed written consent in accordance with the University Committee on Activities Involving Human Subjects (UCAIHS) and participated in exchange for monetary compensation. For all three experiments, the sample size was chosen based on a power analysis (power=.8, alpha=.05) of a separate study on boundary-related memory eﬀects from (Dubrow & Davachi, 2013). Materials. For all experiments, we used a stimulus set consisting of 576 gray-scale pictures of objects from various online databases. A subset of these stimuli was used for Experiment 1 (432 objects). Each one was resized to a ﬁxed size of 350x350 pixels. To generate colors for the frames, 24 unique colors were selected from color continuum ranging from [0,0,0] to [255, 255, 255] RGB values. Lists of colors for each block were generated, such that no two colors that occurred consecutively at encoding could be perceptually similar. For example, if the previous event color was red, the next color could not be orange, but it could be blue or green. Furthermore, the same color could not appear in a list more than once and colors were recycled after every four lists. Stimulus order varied for each subject and stimulus-color pairings were randomized across subject. Design and Procedure. Before the experiment began, there was a brief practice version of the experiment to ensure that participants understood the task. For each of 12 study lists, participants intentionally encoded lists of 36 trial-unique gray-scale objects that were embedded on a colored frame. Participants were instructed to imagine the object in the color of the frame and decide if the object-color pair was pleasing. The PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 8 participants were instructed to respond as soon as they made a pleasantness decision by pressing one of two buttons on the keyboard (‘j’ or ‘k’). The color of the frame was identical for six consecutive objects before switching to a new color for the next six objects. An ‘event’ was deﬁned as six consecutive objects with the same colored frame. There were six events per study list. On boundary trials, the frame color updated at trial onset (i.e. concurrently with the object). All other trials (event positions 2-6) were called non-boundary trials. Objects were presented for a ﬁxed time of 2.5 seconds (s) with a ﬁxed 2 s inter-trial interval (ITI) followed by a .5 s ﬁxation cross before the onset of the next trial. The colored frame remained on the screen continuously during the presentation of each event (including the 2 s ITI and a .5 s ﬁxation cross) and only changed concurrently with boundary objects. It is important to note that the objects remained on the screen for a ﬁxed period of time (2.5 seconds). Thus, encoding response times in the rest of this manuscript refer to the amount of time it took for the participant to complete the pleasantness decision, not the duration that the stimulus was on the screen. participants were instructed to respond as soon as they made a pleasantness decision by pressing one of two buttons on the keyboard (‘j’ or ‘k’). The color of the frame was identical for six consecutive objects before switching to a new color for the next six objects. An ‘event’ was deﬁned as six consecutive objects with the same colored frame. There were six events per study list. On boundary trials, the frame color updated at trial onset (i.e. concurrently with the object). All other trials (event positions 2-6) were called non-boundary trials. Objects were presented for a ﬁxed time of 2.5 seconds (s) with a ﬁxed 2 s inter-trial interval (ITI) followed by a .5 s ﬁxation cross before the onset of the next trial. The colored frame remained on the screen continuously during the presentation of each event (including the 2 s ITI and a .5 s ﬁxation cross) and only changed concurrently with boundary objects. It is important to note that the objects remained on the screen for a ﬁxed period of time (2.5 seconds). PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 9 during the test (HC left color, LC left color, HC, right color, LC right color). Test trials were self-paced and advanced as soon as a response was given, with a ﬁxed .5 s ITI between test trials. Half of the items in each encoding list were tested: We alternated between testing of even and odd trials on each list. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Thus, encoding response times in the rest of this manuscript refer to the amount of time it took for the participant to complete the pleasantness decision, not the duration that the stimulus was on the screen. Following each encoding list, we tested object-color associative memory. To minimize recency memory eﬀects, the test was structured such that objects presented in the ﬁrst half of the list (1:18) were tested ﬁrst and objects presented in the second half of the list (19:36) were tested second. However, within each half, the test trials were randomized. For each test trial, participants were shown a previously studied object with a grey border presented above two colors that were positioned on the left and right side of the computer screen (Figure 1). One of these colors (target) was originally paired with the object while the other color (lure) was always one of the colored frames that had immediately preceded or followed the target color at encoding. The lure was counterbalanced such that it was equally likely to precede or follow the target color. Targets and lures were also equally likely to appear in the left or right positions on screen. In one step, participants were asked to indicate which of the two colors had been paired with the object at encoding and also to indicate their conﬁdence in their decision (high/low conﬁdence, HC/LC). Thus, there were a total of 4 possible responses PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 10 correct boundary trials were speeded relative to non-boundary trials, suggesting that information studied at boundaries is more accessible than non-boundary information. Encoding Response times. RTs during encoding varied as a function of event position [F(5,115) = 60.7, p<.001, η2=.72]. A planned contrast conﬁrmed that RTs to boundary trials (event position 1) were signiﬁcantly slower than those on non-boundary trials (positions 2-6; t(25)=9.52, p<.001, Cohen’s d=2.86). Follow-up pairwise t-tests also show that boundary RTs are signiﬁcantly slower than all other tested non-boundary positions [1 vs. 2: t(25)=9.5, p<.001, Cohen’s d=.97; 1 vs. 3: t(25)=9.21, p<.001, Cohen’s d=1.03; 1 vs. 4: t(25)=9.44, p<.001, Cohen’s d=1.14, 1 vs. 5: t(25)=8.98, p<.001, Cohen’s d=1.14; 1 vs. 6: t(25)=8.29, p<.001, Cohen’s d=1.07]. correct boundary trials were speeded relative to non-boundary trials, suggesting that information studied at boundaries is more accessible than non-boundary information. Encoding Response times. RTs during encoding varied as a function of event position [F(5,115) = 60.7, p<.001, η2=.72]. A planned contrast conﬁrmed that RTs to boundary trials (event position 1) were signiﬁcantly slower than those on non-boundary trials (positions 2-6; t(25)=9.52, p<.001, Cohen’s d=2.86). Follow-up pairwise t-tests also show that boundary RTs are signiﬁcantly slower than all other tested non-boundary positions [1 vs. 2: t(25)=9.5, p<.001, Cohen’s d=.97; 1 vs. 3: t(25)=9.21, p<.001, Cohen’s d=1.03; 1 vs. 4: t(25)=9.44, p<.001, Cohen’s d=1.14, 1 vs. 5: t(25)=8.98, p<.001, Cohen’s d=1.14; 1 vs. 6: t(25)=8.29, p<.001, Cohen’s d=1.07]. Results Eﬀect of perceptual boundaries on color memory performance.. We found that memory for the object-color association varied as a function of event position [Figure 2; F(5,125)=4.27, p=.001, η2=.15]. The position by conﬁdence interaction was not signiﬁcant [F(5,125)=1.28, p>.1], so we collapsed across high and low conﬁdence trials. A planned contrast revealed that color memory was signiﬁcantly better for the boundary trials compared to non-boundary trials [t(25)=4.68, p<.001, Cohen’s d=.41] and follow up pairwise t-tests show that memory for the boundary condition was signiﬁcantly better than each non-boundary condition [1 vs. 2: t(25)=2.87, p=.008, Cohen’s d=.33; 1 vs. 3: t(25)=3.44, p<.002, Cohen’s d=.42; 1 vs. 4: t(25)=4.29, p<.001, Cohen’s d=.42; 1 vs. 5: t(25)=3.69, p=.001, Cohen’s d=.40; 1 vs. 6: t(25)=3.5, p=.002, Cohen’s d=.36]. Thus, object-color associative memory at boundaries was signiﬁcantly enhanced relative to trials that are not studied at perceptual boundaries. Next, we asked whether perceptual boundaries also impacted RTs during the successful retrieval of the object-color associations. We found that RTs to correctly remembered trials varied as a function of event position [F(5,125) = 4.18, p=.001, η2=.143]. The position by conﬁdence interaction was not signiﬁcant, so we collapsed across conﬁdence [F(5,125)=1.23, p>.1]. A planned contrast revealed that retrieval of items from the boundary condition was signiﬁcantly faster than the non-boundary conditions [t(25)=2.95, p=.007, Cohen’s d=.22]. Subsequent pairwise t-tests revealed that position 1 was signiﬁcantly faster than positions 3, 4 and 6, and a trend for an eﬀect for position 2 and 5 [1 vs. 2: t(25)=1.88, p=.07, Cohen’s d=.18; 1 vs. 3: t(25)=2.15, p=.04, Cohen’s d=.15; 1 vs. 4: t(25)=3.62, p=.001, Cohen’s d=.22; 1 vs 5: t(25)=1.84, p=.08, Cohen’s d=.20; 1 vs. 6: t(25)=3.56, p=.002, Cohen’s d=.35]. Thus, during retrieval, RTs to [t(25)=2.95, p=.007, Cohen’s d=.22]. Subsequent pairwise t-tests revealed that position 1 was signiﬁcantly faster than positions 3, 4 and 6, and a trend for an eﬀect for position 2 and 5 [1 vs. 2: t(25)=1.88, p=.07, Cohen’s d=.18; 1 vs. 3: t(25)=2.15, p=.04, Cohen’s d=.15; 1 vs. 4: t(25)=3.62, p=.001, Cohen’s d=.22; 1 vs 5: t(25)=1.84, p=.08, Cohen’s d=.20; 1 vs. 6: t(25)=3.56, p=.002, Cohen’s d=.35]. Thus, during retrieval, RTs to PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Discussion Experiment 1 revealed that associative memory was enhanced for trials that appeared at perceptual event boundaries. Prior studies that have reported better overall memory for information studied at event boundaries (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004), used clips from studied movies as retrieval cues and these cues taken from event boundaries contained more diagnostic information about the clips, which could ultimately account for the memory beneﬁt. By contrast, in the present study, the only diﬀerence between the boundary and non-boundary conditions was a change in the color of the background frame in the boundary condition. Therefore, during retrieval, the amount of available perceptual information on each test trial was the same and so, any diﬀerential eﬀects we see in boundary memory must be related to processes that occurred during encoding. Thus, the current ﬁnding is consistent with these prior results but importantly extend them to a situation where the retrieval content is matched, which implicates that processes that occur at the time of the boundary itself are responsible for enhancing memory for boundary information. In one relevant study, Swallow et al. (2009) found that memory for objects occurring at event boundaries was better than memory for non-boundary objects when object recognition memory was probed very shortly after ( 5 seconds) an event boundary, but Therefore, during retrieval, the amount of available perceptual information on each test trial was the same and so, any diﬀerential eﬀects we see in boundary memory must be related to processes that occurred during encoding. Thus, the current ﬁnding is consistent with these prior results but importantly extend them to a situation where the retrieval content is matched, which implicates that processes that occur at the time of the boundary itself are responsible for enhancing memory for boundary information. In one relevant study, Swallow et al. (2009) found that memory for objects occurring at event boundaries was better than memory for non-boundary objects when object recognition memory was probed very shortly after ( 5 seconds) an event boundary, but PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 11 11 not when memory was probed within the same event. The authors interpreted this eﬀect to suggest that retrieving across event boundaries relies on the access of long-term item representations (as opposed to accessing working memory representations). They reasoned that since EST predicts better encoding of boundary information into long-term memory, the recognition memory diﬀerence for boundary and non-boundary objects should be maximal when the test occurs after an event boundary. The results of the current experiment are consistent with the results of this prior study. However, our study is novel in a few critical ways: First, we test memory after a substantially longer delay ( 3-5 minutes as compared to 5 seconds). This conﬁrms the claim that long-term boundary memory is enhanced, rather than a diﬀerence in working memory accessibility between boundary and non-boundary information. Second, we test associative memory between an object and its accompanying color background rather than item memory. Thus, our ﬁndings extend previous work to suggest that at event boundaries, items are more strongly bound to their context (i.e. the color background). Finally, while the previous study used naturalistic movies as their stimuli, we opted for a simpler stimulus set consisting of objects and color backgrounds. While there are undoubtedly beneﬁts to using naturalistic stimuli, our choice of simple object and color associations allowed us to carefully control for the quantity and quality of information available at event boundaries. Thus, the current experiment supports and extends previous work on boundary-related memory enhancements. not when memory was probed within the same event. The authors interpreted this eﬀect to suggest that retrieving across event boundaries relies on the access of long-term item representations (as opposed to accessing working memory representations). They reasoned that since EST predicts better encoding of boundary information into long-term memory, the recognition memory diﬀerence for boundary and non-boundary objects should be maximal when the test occurs after an event boundary. The results of the current experiment are consistent with the results of this prior study. However, our study is novel in a few critical ways: First, we test memory after a substantially longer delay ( 3-5 minutes as compared to 5 seconds). This conﬁrms the claim that long-term boundary memory is enhanced, rather than a diﬀerence in working memory accessibility between boundary and non-boundary information. Second, we test associative memory between an object and its accompanying color background rather than item memory. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 12 items within the same list (as opposed to lists with no boundaries). Another notable diﬀerence between the studies is that Polyn utilized a free recall task to probe memory, items within the same list (as opposed to lists with no boundaries). Another notable diﬀerence between the studies is that Polyn utilized a free recall task to probe memory, whereas, in the current study, we used a forced-choice associative recognition test. If free recall of the boundary item ‘reinstates’ its associated temporal (Howard & Kahana, 2002; Polyn, Norman, & Kahana, 2009a) or source (Frost, 1971; Hintzman, Block, & Inskeep, 1972; Murdock & Walker, 1969; Nilsson, 1974; Polyn et al., 2009b) context, the context reinstatement could act as a cue to facilitate retrieval of neighboring items, and result in enhanced memory for items that neighbored the boundary item. Thus, the enhancement of items following the boundary item in the free recall study could be driven by organizational processes during retrieval, rather than boundary-driven segmentation during encoding. In contrast, retrieval processes are unlikely to interact with the boundary enhancements reported in the current study because we probed associative recognition memory and the amount of retrieval content was matched between conditions. However, importantly, both studies employ a shift in processing during study to evoke a boundary and see that information encountered at a boundary are more likely to be remembered. diﬀerence between the studies is that Polyn utilized a free recall task to probe memory, whereas, in the current study, we used a forced-choice associative recognition test. If free recall of the boundary item ‘reinstates’ its associated temporal (Howard & Kahana, 2002; Polyn, Norman, & Kahana, 2009a) or source (Frost, 1971; Hintzman, Block, & Inskeep, 1972; Murdock & Walker, 1969; Nilsson, 1974; Polyn et al., 2009b) context, the context reinstatement could act as a cue to facilitate retrieval of neighboring items, and result in enhanced memory for items that neighbored the boundary item. Thus, the enhancement of items following the boundary item in the free recall study could be driven by organizational processes during retrieval, rather than boundary-driven segmentation during encoding. In contrast, retrieval processes are unlikely to interact with the boundary enhancements reported in the current study because we probed associative recognition memory and the amount of retrieval content was matched between conditions. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Thus, our ﬁndings extend previous work to suggest that at event boundaries, items are more strongly bound to their context (i.e. the color background). Finally, while the previous study used naturalistic movies as their stimuli, we opted for a simpler stimulus set consisting of objects and color backgrounds. While there are undoubtedly beneﬁts to using naturalistic stimuli, our choice of simple object and color associations allowed us to carefully control for the quantity and quality of information available at event boundaries. Thus, the current experiment supports and extends previous work on boundary-related memory enhancements. Another related study showed that switching encoding tasks midway through a short list of words resulted in a signiﬁcant increase in the free recall of words that followed the task switch (speciﬁcally, n and n+1) relative to recall of items (in the same serial position) of a control list with only one task (Polyn, Norman, & Kahana, 2009b). Although interpreted as evidence for the notion that task context serves as a retrieval cue, the task switch may have additionally acted as an event boundary thus, facilitating the encoding of words that followed it. While the eﬀect reported in (Polyn et al., 2009b) compared lists containing a task switch to lists with no task switch, the boundary-related memory enhancement reported here is relative to other non-boundary PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 13 the memory beneﬁt could be a result of novelty driven attentional priority. In summary, these results add to a growing body of literature suggesting that contextual novelty, or event boundaries, promote memory encoding. One ﬁnal, but important, point is that in this experiment and the two that follow, the color background was a task-relevant feature of the experiment. Participants made pleasantness judgments on the object-color pairing. When the context is task relevant, we see a boost in item-context binding at event boundaries. Whether or not task relevance of the context is a necessary part of the experimental design should be addressed in future research. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY However, importantly, both studies employ a shift in processing during study to evoke a boundary and see that information encountered at a boundary are more likely to be remembered. This enhancement in memory observed at event boundaries is also reminiscent of the Von Restorﬀeﬀect, the empirical ﬁnding that items with features that are novel within the local context of an experiment are better remembered (Ranganath & Rainer, 2003; Restorﬀ, 1933). However, this study is unique in that we don’t test item memory, but rather we measure associative memory between the encoded item and the contextual feature that changed (a colored background in this case). Furthermore, it emphasizes the transient nature of novelty-driven associative memory encoding: we observed an associative memory enhancement speciﬁcally at the boundaries where associative memory for the remaining non-boundary positions is reduced and not signiﬁcantly diﬀerent from one another. One explanation for the boundary-related memory enhancements we observed here is that the switch in context drives attention toward the novel feature in the environment (the colored background) and since attentional priority is high for this trial, binding between the object and the color is boosted. Thus, PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 14 modiﬁed the design of Experiment 1 to include a temporal order memory test (while keeping the test of object-color background memory). The encoding task and parameters in Experiment 2 were identical to encoding during Experiment 1, except that we encouraged subjects to associate items across time to increase temporal order memory performance. After each encoding list, we tested temporal order memory for object pairs studied within the same event (the ‘within-event’ condition) and compared that to temporal order memory for objects studied in two adjacent events (the ‘across-event’ condition), keeping the actual lag between tested items the same. We also tested object-color memory after each list. We predicted that perceptual boundaries would 1) increase object-color associative memory for boundary trials (replicating Experiment 1), and 2) result in reduced temporal order memory for objects studied in adjacent events. Furthermore, we hypothesized that the magnitude of the boundary eﬀect (i.e. the time spent processing the boundary item) should be directly related to the decrement in across-event temporal order memory. Experiment 2: Perceptual boundaries facilitate object-color binding, but reduce across-event temporal order memory. Experiment 1 provided evidence that associative binding is enhanced for local representations encountered at event boundaries compared to those encountered in the midst of an event (i.e. non-boundary items). The enhancement in binding representations present at event boundaries is consistent with the notion that attention to boundary representations is enhanced. If this is the case, then we reasoned that another form of memory, namely temporal order memory, may be disrupted. If perceptual boundaries caused a shift in attention to the novel color information, then the associative binding between pairs of items ﬂanking that boundary would be disrupted. Indeed, prior experiments using temporal shifts in narrative as well as category and task switches at boundaries has shown this to be the case (Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2011, 2014). Thus, we aimed to extend that work and see if boundaries as deﬁned in this paradigm (i.e. the color shifts) are associated with reduced temporal order memory. Thus, in Experiment 2, we assessed the eﬀect of perceptual boundaries on temporal order memory, as well as object-color associative memory. Furthermore, this experimental design allowed us to ask whether temporal order memory for pairs items encountered across an event boundary is related to the enhanced processing of the boundary information itself. We boundary is related to the enhanced processing of the boundary information itself. We PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 15 color condition. Objects that were studied in the middle of the list (speciﬁcally, in the 4th position of the event) made up the “non-boundary” color condition. To assess temporal order memory, participants made order judgments on pairs of items: objects studied in the second and sixth positions of each event were paired together and made up the “within-event” order condition. Objects that were studied in the ﬁfth and third positions of two adjacent events were paired together and made up the “across-event” order condition. Note that a given item was never tested more than once. An item was either tested for color memory or temporal order memory (but never both). There were a total of 80 test trials for each of the four conditions. The only procedural diﬀerence during encoding (compared to Experiment 1) was that participants were encouraged to adopt an associative memory strategy to promote later temporal order memory. Speciﬁcally, we told participants to imagine the objects interacting with each other over time. Critically, participants were instructed to associate objects irrespective of the presence of diﬀerent color frames. We added this The only procedural diﬀerence during encoding (compared to Experiment 1) was that participants were encouraged to adopt an associative memory strategy to promote later temporal order memory. Speciﬁcally, we told participants to imagine the objects interacting with each other over time. Critically, participants were instructed to associate objects irrespective of the presence of diﬀerent color frames. We added this additional instruction since we reasoned that after the ﬁrst temporal order memory test, participants may adopt this kind of strategy to be successful on temporal order memory judgments in subsequent lists. There was a short practice session to assure that subjects understood the task instructions. After each study list, color memory was tested ﬁrst, with the same design as Experiment 1. Following completion of the color memory test, participants were tested on their memory for the temporal order of pairs of objects. For each test trial, two previously studied objects appeared side by side on the screen (Figure 1). Participants were asked to indicate which of the two objects appeared earlier in the list. The tested pairs of objects were chosen such that there were always three intervening objects between them at encoding. Critically, this was the case for both within-event and across-event trial-pairs. Methods Participants. Participants were 31 individuals (ages 18-35) recruited from New York University and the greater New York Metropolitan Area. All participants gave informed written consent in accordance with the University Committee on Activities Involving Human Subjects (UCAIHS) and participated in exchange for monetary compensation. One participant was excluded for failure to press any buttons during encoding. The remaining 30 participants were used for all analyses. Materials. Materials were the same as Experiment 1. The entire stimulus set was used for Experiment 2 (576 objects). Design and Procedure. The design of the encoding was very similar to Experiment 1, except that we had 16 study/test blocks (compared to 12 in Experiment 1). Design and Procedure. The design of the encoding was very similar to Experiment 1, except that we had 16 study/test blocks (compared to 12 in Experiment 1). Following each encoding list, we tested object-color memory followed by temporal order memory. To assess object-color memory, we tested two items from each event. Objects that appeared concurrently with a change in the color frame made up the “boundary” Following each encoding list, we tested object-color memory followed by temporal order memory. To assess object-color memory, we tested two items from each event. Objects that appeared concurrently with a change in the color frame made up the “boundary” PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 16 conﬁdence in their decision (high/low). Test trials were self-paced and advanced as soon a response was given, with a ﬁxed .5 s ITI included between test trials. conﬁdence in their decision (high/low). Test trials were self-paced and advanced as soon a response was given, with a ﬁxed .5 s ITI included between test trials. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Thus, the actual number of intervening items between the two conditions was constant (3 intervening items) but the across-event test-pairs were studied with a boundary between them while within-event pairs were studied with the same color frame (i.e. in the same event; Figure 1). Participants were again asked to indicate their PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Results Eﬀect of perceptual boundaries on object-color and temporal order memory. Accuracy for object-color memory and temporal order memory was calculated in two ways: First, we looked at the overall proportion of correct responses as a function of test condition. Secondly, if the data varied by conﬁdence, then we separated correct trials by conﬁdence (HC/LC) and calculated memory accuracy for each condition. Finally, we assessed whether there was an interaction between boundary/non-boundary status and memory test type (color/order). We found that object-color memory accuracy for boundary trials was signiﬁcantly better than non-boundary trials [t(29)=6.84, p<.001, Cohen’s d=.61; see Figure 3a]. There was no condition by conﬁdence interaction, so we did not separate the data by conﬁdence [F(1,29)=.73, p>.1]. These analyses suggest that the event boundary signiﬁcantly increased memory for the object-color association. This result replicates the boundary-related memory enhancement reported in Experiment 1. Eﬀect of perceptual boundaries on object-color and temporal order memory. Accuracy for object-color memory and temporal order memory was calculated in two ways: First, we looked at the overall proportion of correct responses as a function of test condition. Secondly, if the data varied by conﬁdence, then we separated correct trials by conﬁdence (HC/LC) and calculated memory accuracy for each condition. By contrast, temporal order memory was signiﬁcantly better for the within-event condition relative to the across-event condition [t(29)=4.82, p<.001, Cohen’s d=1.02]. The condition by conﬁdence interaction was not signiﬁcant [F(1,29)=2.71, p>.1]. Taken together with the data above, this suggests that the boundaries led to a reduction in associative memory across events while simultaneously increasing associative binding of representations at the boundary itself. To directly test for this interaction, we performed a two-way repeated measures ANOVA and found a signiﬁcant test type (color/order) by condition (Boundary/Across-event and Non-boundary/Within-event) interaction [Figure 3a; F(1,29)=45.34, p<.001, η2=.61]. This data shows that perceptual boundaries improved object-color memory at the boundary itself, but disrupted temporal order memory for object pairs that spanned the boundary. Together, these ﬁndings suggest a boundary-related trade-oﬀ, where memory for boundary By contrast, temporal order memory was signiﬁcantly better for the within-event condition relative to the across-event condition [t(29)=4.82, p<.001, Cohen’s d=1.02]. The condition by conﬁdence interaction was not signiﬁcant [F(1,29)=2.71, p>.1]. Taken together with the data above, this suggests that the boundaries led to a reduction in associative memory across events while simultaneously increasing associative binding of representations at the boundary itself. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 17 information is enhanced, perhaps at the cost of across-event associative binding. Across-event temporal order memory negatively related to boundary processing. As a further means for asking whether there is a trade-oﬀbetween boundary processing and these diﬀerent forms of memory, our next analysis assessed whether response times to boundaries at encoding were related to later color and order memory. We reasoned that increased encoding response times to boundary items might reﬂect greater attention to the item and color on boundary trials and may, in turn, be related to the temporal order reductions. Thus, we hypothesized that longer boundary response times may result in worse order memory for pairs of objects that spanned the boundary. Crucially, however, we expected no such relationship between non-boundary response times and temporal order memory for within-event trial pairs. Across-event temporal order memory negatively related to boundary processing. As a further means for asking whether there is a trade-oﬀbetween boundary processing and these diﬀerent forms of memory, our next analysis assessed whether response times to boundaries at encoding were related to later color and order memory. We reasoned that increased encoding response times to boundary items might reﬂect greater attention to the item and color on boundary trials and may, in turn, be related to the temporal order reductions. Thus, we hypothesized that longer boundary response times may result in worse order memory for pairs of objects that spanned the boundary. Crucially, however, we expected no such relationship between non-boundary response times and temporal order memory for within-event trial pairs. To test this hypothesis, we divided boundary encoding response times into terciles and assessed temporal order memory separately for each bin. This analysis allowed us to visualize the data as well as reduce some of the variance of response times driven by noise. To test whether boundary (but not non-boundary) response times are related to temporal order memory, we conducted a two-way repeated measures ANOVA. There was a signiﬁcant condition (boundary/non-boundary) by response time tercile (slowest, middle, fastest) interaction [F(2,58)=3.52, p=.036, η2 = .11]. This interaction was driven by the fact that order memory performance was the lowest in the slowest tercile of response times and was the best in the fastest tercile of boundary response times [Figure 3b; fastest vs. slowest: t(29)=2.8; p=.009, Cohen’s d=.60]. Results To directly test for this interaction, we PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Crucially, no eﬀect of response time on order memory for the analogous intervening trial in the non-boundary condition was evident [fastest vs. slowest: t(29)=-1.01; p=.32]. That is, the response time to the item in the 4th event position was not related to within-event order memory (between the items in the 2nd and 6th event position). Furthermore, there was a signiﬁcant linear trend for across-boundary temporal order memory as a function of boundary response tercile [F(2,58)=7.86, p=.007, η2=.12] that was not present for the analogous within-event condition [F(2,58)=.91, p=.34], suggesting that the linear trend was speciﬁc to the across-boundary condition. These data support the PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 18 notion that event boundaries disrupt temporal order memory and provide evidence that response time variability on boundary trials is related to the outcome of temporal order memory for the trials that span the boundary. In contrast, response times to an intervening item within events do not appear to be related to temporal order memory. We also performed a logistic regression using boundary response times (i.e. task responses to position 1 items) and non-boundary response times (task responses to position 4 items) to predict within-event/across-boundary temporal order accuracy (position 5 and 3 items). Neither regression was signiﬁcant [boundary: coeﬃcient=-.04, z(29)=-.51, p=.61; non-boundary: coeﬃcient=.13, z(29)=1.61, p=.11], but the interaction was [coeﬃcient=.45, z(29)=4.9, p<.001]. We then ran a separate control analysis to test whether this relationship between boundary response time and temporal order memory was speciﬁc to the boundary item. It’s possible that rather than temporal order memory being speciﬁcally related to processing at the boundary, memory could vary as a function of the overall speed of processing across the sequence of items. In other words, order memory could vary as a function of the mean encoding response time across the tested items (i.e. all trials that intervened the later tested items), rather than to the boundary item speciﬁcally. To address this, we averaged encoding response times across sequences of items that crossed a boundary, where the ﬂanking items of the sequence were boundary temporal order test pairs. Importantly, we excluded the boundary response itself, but included the other intervening items. Then, we again sorted the data into terciles, and computed temporal order memory separately for each response time bin. If the relationship between the speed of boundary response time and temporal order memory were speciﬁc to the processing of the boundary item, then the average response time across the sequence would not predict temporal order memory. However, if temporal order memory co-varied with the mean response time across a sequence, than we would expect a pattern similar to the previous analysis, where longer response times predicted worse order memory performance. A one-way ANOVA revealed that mean response time for sequences of trials that crossed an event boundary was not predictive of PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 19 temporal order memory [F(1,85)=1.59, p>.1], providing further evidence that boundary processing is speciﬁcally related to across-trial associative encoding. Discussion The reduction in temporal order memory observed is consistent with previous work showing event boundary-related reductions in cued recall for narrative stimuli (Ezzyat & Davachi, 2011), as well as an inﬂuence of boundaries on temporal memory for visual images (Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2014). Interestingly, our temporal order memory eﬀects emerged using a simple color manipulation, demonstrating that boundary-related temporal memory disruptions can result from simple changes in a perceptual feature of an event. Moreover, we found that slower boundary encoding response times were associated with worse temporal order memory for objects spanning those boundaries. In other words, the larger the magnitude of the “boundary eﬀect” (as indexed by response time), the worse the temporal order memory for pairs of objects that spanned the boundary. The current data supports previous work by replicating the boundary-related reduction of across-event temporal order memory and importantly extends it to suggest a trade-oﬀ between boundary processing itself and across-event order memory. In other words, this data highlights the idea that orienting to something salient in the environment comes at the cost of the ongoing maintenance of items over time. Our ﬁnding of relatively better temporal order memory for within-event items as compared to across-event items is also consistent with buﬀer models of episodic memory (Atkinson & Shiﬀrin, 1968; Lehman & Malmberg, 2009, 2013; Raaijmakers & Shiﬀrin, 1981). Lehman and Malmberg (2013) buﬀer model hypothesizes that during encoding, a buﬀer process exists that can maintain or drop information depending upon the goals of the subject or the demands of the task. Items and contexts that are maintained in the buﬀer become associatively linked, while information experienced in diﬀerent buﬀers does not become as strongly associated. In our task, we hypothesize that items encountered in the same color are maintained in the same buﬀer whereas a switch in color (i.e. perceptual event The results of the Experiment 2 provide evidence that while associative memory was enhanced for stimuli presented at perceptual boundaries, these boundaries resulted in a decrease in temporal order memory for items studied in adjacent events (Figure 3a). PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 20 PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Together with the accuracy data described above, these results argue for a trade-oﬀbetween boundary processing and across-event mnemonic binding. Encoding response times. Replicating the results from Experiment 1, we see that response times during encoding varied as a function of within-event position [Figure 3c; F(5,155)=54.89, p<.001, η2=.66], and a planned contrast revealed that task responses on boundary items (position 1) were signiﬁcantly slower than non-boundary items [position 2-6; t(29)=8.60, p<.001, Cohen’s d=.61]. Retrieval response times. Finally, we compared response times for correct color and order memory retrieval as a function of condition. The goal of this analysis was to see whether the relative increase in memory performance we observed in the previous color and order accuracy analyses (i.e. Figure 3a) was also accompanied by a speeding of the retrieval response. To test whether the pattern of response times across conditions was dependent on conﬁdence, we conducted a repeated measures two-way ANOVA and assessed the condition by conﬁdence interaction. The interaction was not signiﬁcant for order [F(1,28)=.06, p>.1] or color [F(1,28)=.07, p>.1], so we collapsed across conﬁdence. We found that response times for correct color memory retrieval trials were signiﬁcantly faster for boundary trials relative to non-boundary trials [t(29)=6.07, p<.001, Cohen’s d=.52; Figure 3d]. On the order memory test, better performance on the within-event trials was accompanied by faster response times relative to the across-event trials [t(29)=3.85, p<.001, Cohen’s d=.40]. Additionally, a two-way repeated measures ANOVA revealed a signiﬁcant memory test by condition interaction [F(1,29)=28.62, p< .001, η2=.50]. In sum, mnemonic representations in conditions with better memory (boundary object color and within-event temporal order) were also accessed more quickly at retrieval. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Discussion Discussion The results of the Experiment 2 provide evidence that while associative memory was enhanced for stimuli presented at perceptual boundaries, these boundaries resulted in a decrease in temporal order memory for items studied in adjacent events (Figure 3a). The reduction in temporal order memory observed is consistent with previous work showing event boundary-related reductions in cued recall for narrative stimuli (Ezzyat & Davachi, 2011), as well as an inﬂuence of boundaries on temporal memory for visual images (Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2014). Interestingly, our temporal order memory eﬀects emerged using a simple color manipulation, demonstrating that boundary-related temporal memory disruptions can result from simple changes in a perceptual feature of an event. Moreover, we found that slower boundary encoding response times were associated with worse temporal order memory for objects spanning those boundaries. In other words, the larger the magnitude of the “boundary eﬀect” (as indexed by response time), the worse the temporal order memory for pairs of objects that spanned the boundary. The current data supports previous work by replicating the boundary-related reduction of across-event temporal order memory and importantly extends it to suggest a trade-oﬀ between boundary processing itself and across-event order memory. In other words, this data highlights the idea that orienting to something salient in the environment comes at the cost of the ongoing maintenance of items over time. Our ﬁnding of relatively better temporal order memory for within-event items as compared to across-event items is also consistent with buﬀer models of episodic memory (Atkinson & Shiﬀrin, 1968; Lehman & Malmberg, 2009, 2013; Raaijmakers & Shiﬀrin, 1981). Lehman and Malmberg (2013) buﬀer model hypothesizes that during encoding, a buﬀer process exists that can maintain or drop information depending upon the goals of the subject or the demands of the task. Items and contexts that are maintained in the buﬀer become associatively linked, while information experienced in diﬀerent buﬀers does not become as strongly associated. In our task, we hypothesize that items encountered in the same color are maintained in the same buﬀer whereas a switch in color (i.e. perceptual event Discussion The results of the Experiment 2 provide evidence that while associative memory was enhanced for stimuli presented at perceptual boundaries, these boundaries resulted in a decrease in temporal order memory for items studied in adjacent events (Figure 3a). Discussion The reduction in temporal order memory observed is consistent with previous work showing event boundary-related reductions in cued recall for narrative stimuli (Ezzyat & Davachi, 2011), as well as an inﬂuence of boundaries on temporal memory for visual images (Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2014). Interestingly, our temporal order memory eﬀects emerged using a simple color manipulation, demonstrating that boundary-related temporal memory disruptions can result from simple changes in a perceptual feature of an event. Moreover, we found that slower boundary encoding response times were associated with worse temporal order memory for objects spanning those boundaries. In other words, the larger the magnitude of the “boundary eﬀect” (as indexed by response time), the worse the temporal order memory for pairs of objects that spanned the boundary. The current data supports previous work by replicating the boundary-related reduction of across-event temporal order memory and importantly extends it to suggest a trade-oﬀ between boundary processing itself and across-event order memory. In other words, this data highlights the idea that orienting to something salient in the environment comes at the cost of the ongoing maintenance of items over time. Our ﬁnding of relatively better temporal order memory for within-event items as compared to across-event items is also consistent with buﬀer models of episodic memory (Atkinson & Shiﬀrin, 1968; Lehman & Malmberg, 2009, 2013; Raaijmakers & Shiﬀrin, 1981). Lehman and Malmberg (2013) buﬀer model hypothesizes that during encoding, a buﬀer process exists that can maintain or drop information depending upon the goals of the subject or the demands of the task. Items and contexts that are maintained in the buﬀer become associatively linked, while information experienced in diﬀerent buﬀers does not become as strongly associated. In our task, we hypothesize that items encountered in the same color are maintained in the same buﬀer whereas a switch in color (i.e. perceptual event PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 21 boundary) may serve as a cue for the brain to ﬂush the contents of the current buﬀer, thus creating an associative disconnect between items studied in diﬀerent contexts. Thus, one possible explanation for our observed diﬀerence between within and across event temporal order memory is that items encountered within the same event occupy the same buﬀer and thus are more strongly bound to each other than items encountered in diﬀerent events (i.e. distinct buﬀers). Buﬀer models of episodic memory have been quite successful at accounting for patterns in episodic memory, particularly in studies with an intentional forgetting component (Lehman & Malmberg, 2013). Typically in these studies, at the end of an encoding list, subjects are instructed on whether or not to forget the previously studied list. The model predicts that the cue to forget causes a ﬂushing of the previously studied items from the buﬀer (a buﬀer operation the authors call “compartmentalization”). One major diﬀerence between our study and these prior studies is that we do not explicitly instruct participants to forget between events. In fact, we encourage participants to bind together items irrespective of the background color. Thus, a possible interpretation of our ﬁndings is that perceptual event boundaries serve as a bottom-up cue that triggers the brain to remove the contents of the current buﬀer. Thus, compartmentalization may occur spontaneously if there is suﬃcient perceptual change in the environment. Future work could be conducted to characterize whether the nature of the task (i.e. top-down vs. bottom-up compartmentalization) has diﬀerential consequences for the organization of events in episodic memory. Finally, the memory “trade-oﬀs” reported here can be contrasted with a body of literature investigating block-level or instruction-level trade-oﬀs in item vs. associative in formation in memory (Einstein & Reed, 1980; Gronlund & Ratcliﬀ, 1989; Hockley & Cristi, 1996; Hunt & Einstein, 1981; Sharps & Tindall, 1992). In this literature, the overarching theme is that when item encoding is prioritized over associative or relational encoding, item memory beneﬁts and associative memory suﬀers. In contrast, when associative memory is prioritized, there is a beneﬁt for associative memory but interestingly, item memory remains intact. In the current work, we show that at event boundaries, when attention is presumably redirected from item-item associative boundary) may serve as a cue for the brain to ﬂush the contents of the current buﬀer, thus creating an associative disconnect between items studied in diﬀerent contexts. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Thus, one possible explanation for our observed diﬀerence between within and across event temporal order memory is that items encountered within the same event occupy the same buﬀer and thus are more strongly bound to each other than items encountered in diﬀerent events (i.e. distinct buﬀers). Buﬀer models of episodic memory have been quite successful at accounting for patterns in episodic memory, particularly in studies with an intentional forgetting component (Lehman & Malmberg, 2013). Typically in these studies, at the end of an encoding list, subjects are instructed on whether or not to forget the previously studied list. The model predicts that the cue to forget causes a ﬂushing of the previously studied items from the buﬀer (a buﬀer operation the authors call “compartmentalization”). One major diﬀerence between our study and these prior studies is that we do not explicitly instruct participants to forget between events. In fact, we encourage participants to bind together items irrespective of the background color. Thus, a possible interpretation of our ﬁndings is that perceptual event boundaries serve as a bottom-up cue that triggers the brain to remove the contents of the current buﬀer. Thus, compartmentalization may occur spontaneously if there is suﬃcient perceptual change in the environment. Future work could be conducted to characterize whether the nature of the task (i.e. top-down vs. bottom-up compartmentalization) has diﬀerential consequences for the organization of events in episodic memory. Finally, the memory “trade-oﬀs” reported here can be contrasted with a body of literature investigating block-level or instruction-level trade-oﬀs in item vs. associative in formation in memory (Einstein & Reed, 1980; Gronlund & Ratcliﬀ, 1989; Hockley & Cristi, 1996; Hunt & Einstein, 1981; Sharps & Tindall, 1992). In this literature, the overarching theme is that when item encoding is prioritized over associative or relational encoding, item memory beneﬁts and associative memory suﬀers. In contrast, when associative memory is prioritized, there is a beneﬁt for associative memory but boundary) may serve as a cue for the brain to ﬂush the contents of the current buﬀer, thus creating an associative disconnect between items studied in diﬀerent contexts. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 22 processing to more local item-context processing, across-event temporal order memory suﬀers, while boundary item-context memory is enhanced. To coalesce these ﬁndings, it appears that regardless of whether the manipulation is top-down and extended in time (such as an instructional manipulation) or bottom-up and dynamic (such as a perceptual event boundary in the current design), attention to “in-the-moment” representations trades oﬀwith more temporally extended relational encoding (i.e. focusing on item-item relationships). Thus, in contrast to previous work, the results reported in the current study highlight the temporally dynamic nature of item and relational tradeoﬀs during episodic memory encoding. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 23 with an accompanying verbal label, and after a short distractor task were instructed to verbally recall as many items as possible. Critically, like the previous experiments, the studied objects were embedded on a colored frame that periodically changed in color. Experiment 3: Perceptual boundaries impose structure on verbal free recall So far, we’ve provided evidence that perceptual boundaries enhance item-context associative memory, while simultaneously reducing across-event temporal order memory, and that the magnitude of the boundary eﬀect is predictive of the cost in temporal order memory. One intuitive explanation for this pattern of results is that when encountering a perceptual boundary, there is a shift in attention to the processing of the novel color information, which trades oﬀwith the integration of the previous item representation (i.e. the pre-boundary trial) and the current (boundary) trial. We reasoned that if within-event item representations are more strongly linked than items that span a boundary, then one might expect within-event items may be represented more similarly in memory. If asked to recall the items, this could lead to a greater likelihood of sequentially recalling within-event items relative to across event items. Thus, in Experiment 3, we tested the hypothesis that the likelihood of making a local forward transition (e.g. n+1, n+2, n+3) from boundary items to other within-event items would be greater than the likelihood of a local forward transition from pre-boundary items, where forward transitions would be to items in a new event. To test this hypothesis, we employed a modiﬁed version of the encoding paradigm used in Experiments 1 and 2 that was optimized to test memory using verbal free recall (see Design for details). In this experiment, participants encoded lists of visual objects embedded in a colored frame PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Methods Participants. Participants were 24 individuals (ages 18-35) recruited from New York University and the greater New York Metropolitan Area. All participants gave informed written consent in accordance with the University Committee on Activities Involving Human Subjects (ACAIHS) and participated in exchange for monetary compensation. One person was excluded for not completing the experiment, leaving 23 participants for all analyses. Materials. For this experiment, we used a subset of the objects (totaling 384 grey-scale objects) used in Experiments 1 and 2. We also included a written label displayed below each presented object to encourage participants to use the same label during verbal recall. Each list was designed to have minimal conceptual overlap, to minimize confusion during free recall scoring as well as semantic clustering at retrieval. Design. In this experiment, participants studied lists of 24 objects (along with a written label) embedded in a colored frame that changed to a new color after every 4 trials. Like the previous experiments, there were 6 ‘events’ and 5 ‘event boundaries’ per list. We chose to present 24 items per list rather than 36 (as in Experiment 1 and 2) due to pilot data suggesting poor free recall performance when longer study lists were used. Additionally, we reduced event length from 6 to 4 items to ensure adequate power for the free recall analyses (i.e. to have suﬃcient boundary trials). Like Experiments 1 and 2, trials that were studied concurrently with a color change are considered “boundary” trials and objects studied in the other event positions (2-4) are called “non-boundary” trials. rocedure. For each list (12 total), participants encoded 24 object-color pairs. Procedure. For each list (12 total), participants encoded 24 object-color pairs. Participants were instructed to imagine the object in the color of the frame and make a pleasantness judgment on the object/color combination. After each study list, participants completed a distractor task in which they were asked to indicate whether PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 24 arithmetic problems were correct or incorrect (e.g. “5+4+2=11?”). The numbers 1-6 were used for the arithmetic problems, and the probability of the answer being correct was 50%. Each problem was presented for 3 seconds for a total of 10 problems during each distractor period (30 seconds). They were also given immediate feedback to encourage engagement. Following the distractor task, participants were presented with a screen prompting them to say “Next Block” and then verbally recall as many words as they could. They were given a minimum of 90 seconds and told to use more time if they felt they could recall more words. Audio was recorded throughout the entire session. Unclear responses were excluded from the analysis. Synonyms to words that were actually studied were not considered correct responses. For example, if the correct response was “panther” and the subject reported “leopard”, this would not be considered a correct response. However, partial answers were counted as correct. For example, if the correct response was “toy train” and the subject responded “train”, it would be marked as correct. The total number of excluded trials (combining across synonyms, unclear responses and prior list intrusions) was small (3.7%). Analysis of the free recall audio ﬁles was performed by one author and one research assistant using Penn TotalRecall (http://memory.psych.upenn.edu/TotalRecall). Using this program, the onset and serial recall order of each retrieved object was recorded. Crucially, scorers were blind to the encoding condition to which each object belonged. After each free recall period, participants were given an object-color memory task following the same protocol as Experiments 1 and 2. The object-color memory test was self-paced. For this experiment, we tested color memory for all 24 studied items in the list. Across the entire experiment, there were a total of 36 color memory test trials for each condition (event positions: 1-4). Results Overall Free Recall Performance. Participants recalled an average of 31.30% (SD=12.6%) of the items presented for each list (or 7.51 out of 24 items per list). As PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 25 expected, free recall varied as a function of serial position of the list [F(23,506)=6.82, p<.001, η2=.237, Figure 4a], where items at the beginning and the end of the list were more likely to be recalled than items in the middle of the list (Murdock, 1962). We also computed free recall performance as a function of event position within an event, but did not observe an eﬀect [F(3,66)=.96, p>.1], meaning that all event positions 1-4 were equally likely to be recalled. We discuss the implication of this ﬁnding in more detail in the general discussion. Then, we computed the mean lag-conditional response probability (lag-CRP) curve (Figure 4b; see Kahana (1996)) which showed that, given free recall of an item, Then, we computed the mean lag-conditional response probability (lag-CRP) curve (Figure 4b; see Kahana (1996)) which showed that, given free recall of an item, participants were most likely to make free recall transitions to items in neighboring list positions, consistent with previous reports (Howard & Kahana, 2002; Kahana, 1996). participants were most likely to make free recall transitions to items in neighboring list positions, consistent with previous reports (Howard & Kahana, 2002; Kahana, 1996). PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 26 items recalled. The results of this analysis are plotted in Figure 5a. Consistent with our hypothesis, relatively more local forward transitions were made from boundary items than pre-boundary items [t(22)=1.88, p= .07, Cohen’s d = .71]. These data is consistent with the idea that perceptual event boundaries inﬂuence the structure of free recall behavior. Next, we tested the idea that local backward transitions from pre-boundary items might be relatively more likely than local backwards transitions from boundary items because for pre-boundary items, local backwards transitions were to other within-event items. Next, we tested the idea that local backward transitions from pre-boundary items might be relatively more likely than local backwards transitions from boundary items because for pre-boundary items, local backwards transitions were to other within-event items. We performed the same analysis as described above, but now for only local backwards transitions as a function of within-event position (Figure 5b). Interestingly, we did not observe the predicted eﬀect that local backward transitions would be relatively more likely for pre-boundary items as compared to boundary items [t(22)=.10, p>.9]. To summarize, while local forward transitions were more likely from boundary relative to pre-boundary items, there was no diﬀerence in the likelihood of backward transitions. We performed the same analysis as described above, but now for only local backwards transitions as a function of within-event position (Figure 5b). Interestingly, we did not observe the predicted eﬀect that local backward transitions would be relatively more likely for pre-boundary items as compared to boundary items [t(22)=.10, p>.9]. To summarize, while local forward transitions were more likely from boundary relative to pre-boundary items, there was no diﬀerence in the likelihood of backward transitions. Distal Transition Probabilities in Free Recall. The intriguing result that there were no diﬀerences in backward transition probabilities led us to the follow question: If participants are not transitioning from pre-boundary items to other within-event items (i.e. local backward transitions), to which items are they are more likely to transition? We reasoned that if local forward transitions from pre-boundary items are relatively unlikely (as compared to boundary items) and there were no diﬀerences in local backward transitions, then transitions from pre-boundary items must be to more distal items in the list. To quantify this, we computed the average transition distance as a function of position (Figure 5c). Local Transition Probabilities in Free Recall. To measure the inﬂuence of Local Transition Probabilities in Free Recall. To measure the inﬂuence of perceptual event boundaries on free recall, our next analysis focused on local transition probabilities. That is, given the recall of item n, what is the likelihood of the next item recalled being locally forward (i.e. n+1, n+2, or n+3) or locally backward (i.e. n-1, n-2, or n-3)?. First, we focused on local forward transitions up to a lag of three because for boundary items, local forward transitions would be to other within-event items whereas for pre-boundary items (i.e. position 4 items), local forward transitions would be to items in the next event. We predicted that there would be a greater likelihood of local forward transitions from boundary items as compared to pre-boundary items due to the fact that local forward transitions from boundary items would be to other within-event items. To test this prediction, for each within-event position, we computed the likelihood of a local forward transition (i.e. the sum of the number of transitions from n to n+1, n+2, and n+3) divided by the number of transitions to all other items. This resulted in a measure representing the proportion of the time participants made a local forward transition relative to all other transitions to items elsewhere in the list. Critically there were no diﬀerences in the total number of items recalled for each Critically, there were no diﬀerences in the total number of items recalled for each within-event position (see Free Recall Performance Section) and thus, the above analysis is only sensitive to the order of free recall, rather than the total number of PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY That is, given the recall of item n, what is the average lag of the next recalled item? For this analysis, our hypothesis was that transitions from pre-boundary items would be to more distal items relative to all other within-event positions. Put another way, when one recalls the last item in an event, we predicted that they would be relatively more likely to transition to a distal item. To test this hypothesis, we computed a contrast of the average transition distance from pre-boundary items relative to the other within-event positions. The analysis revealed PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 27 that the average transition distance from pre-boundary items was signiﬁcantly more distal than the average transition distance from other within-event positions [t(22)=2.63, p<.05, Cohen’s d=.48]. Furthermore, the direct comparison of average transition distance from pre-boundary and boundary items revealed that pre-boundary transitions are signiﬁcantly more distal than boundary transitions [t(22)=2.55, p<.05, Cohen’s d=.39]. Thus, compared to other within-event positions, free recall transitions from pre-boundary trials are to relatively more distal items providing further support for the idea that perceptual event boundaries inﬂuence the structure of free recall behavior. Transitions from Pre-boundary Items. Our last free recall analysis was designed to test the idea that transitions from pre-boundary items would be disproportionately more likely to boundary items relative to items in other within-event positions. We predicted that given the recall of the last item in an event (and thus terminating the recall of a particular episode), one might initiate a memory search process in an attempt to recall additional items. If boundary items somehow ‘stand out’ in memory (Radvansky, 2012; Zacks et al., 2007), then the likelihood of transitioning to a boundary item given the recall of a pre-boundary item may be relatively higher than transitioning to other within-event positions. In this way, boundary items may serve as a ‘gateway’ into an episodic event. To test this prediction, we computed the conditional likelihood of transitioning to each within-event position given the recall of a pre-boundary item (Figure 5d). A contrast of pre-boundary transitions to boundary items relative to items in other positions suggests that following the recall of a pre-boundary item, there is a trending eﬀect for boundary items to be recalled next [t(22)=1.89, p<.10, Cohen’s d=.79]. One potential issue with the analysis described above is that the eﬀect could be driven solely by local forward transitions (i.e. a temporal contiguity eﬀect). To rule out this explanation, we performed the analysis again, now removing all local transitions (n +/- 1, 2 or 3). When analyzing only transitions from pre-boundary items to other distal items (greater than 3 positions away), the trend remained [t(22)=1.73, p<.10, Cohen’s d=.60]. Thus consistent with our prediction (albeit a trend), following the recall of the last item in an event, distal boundary items are more likely to be recalled next (as PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 28 compared to other within event positions). Together with the free recall analyses described above, these data support the idea that perceptual boundaries introduce structure into free recall behavior. Color Memory Performance. Analysis of variance revealed a trend for color memory to vary as a function of within-event position [F(3,66)=2.57, p=.06, η2=.11]. A planned contrast demonstrated object-color memory was better for boundary trials than non-boundary trials [t(22)=2.67, p=.01, Cohen’s d=.80]. Post-hoc pairwise t-tests Color Memory Performance. Analysis of variance revealed a trend for color memory to vary as a function of within-event position [F(3,66)=2.57, p=.06, η2=.11]. A planned contrast demonstrated object-color memory was better for boundary trials than non-boundary trials [t(22)=2.67, p=.01, Cohen’s d=.80]. Post-hoc pairwise t-tests revealed that memory was signiﬁcantly better for boundary trials than all non-boundary trials [1 vs. 2: t(23)=2.1, p<.05, Cohen’s d=.24; 1 vs. 3: t(23)=2.84, p<.05, Cohen’s d=.31; 1 vs. 4: t(23)=2.1, p<.05, Cohen’s d=.22]. The interaction between conﬁdence and condition was not signiﬁcant. Note that in this experiment, we replicate Experiments 1 and 2 by showing that boundary object-color memory accuracy was better than non-boundary memory. While the eﬀect was modest in this version of the paradigm, together with the color memory enhancements observed in both Experiments 1 and 2, these results provide consistent evidence for boundary-related memory enhancements. Response times for correct color memory retrieval trials also varied as a function of within-event position [F(3,66)=10.29, p<.001, η2=.319]. A planned contrast between boundary and non-boundary color retrieval RTs revealed that boundary RTs were signiﬁcantly faster than non-boundary RTs [t(22)=-4.24, p<.001, Cohen’s d=1.26]. Post-hoc tests showed that color retrieval RTs were signiﬁcantly faster for boundary trials compared to all non-boundary trials [1 vs. 2: t(23)=4.71, p<.001, Cohen’s d=.48; 1 vs. 3: t(23)=2.47, p<.05, Cohen’s d=.29; 1 vs. 4: t(23)=4.08, p<.001, Cohen’s d=.5]. There was no signiﬁcant interaction between condition and conﬁdence, so these analyses were performed on data collapsed across condition. Encoding Response Times. There was a signiﬁcant eﬀect of event position on encoding response times [F(3,66)=28.90, p<.001, η2=.57]. A planned contrast reveals that boundary response times were signiﬁcantly slower than non-boundary [t(22)=6.72, p<.001, Cohen’s d=2.04]. Pairwise t-tests revealed that boundary encoding response PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 29 times were signiﬁcantly slower than all non-boundary trials [1 vs. 2: t(23)=7.08, p<.001, Cohen’s d=.55; 1 vs. 3: t(23)=5.21, p<.001, Cohen’s d=.54; 1 vs. 4: t(23)=7.11, p<.001, Cohen’s d=.55] and there were no other diﬀerences. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY This ﬁnding replicates Experiments 1 and 2 and extends it to include shorter events (four as compared with six items in Experiments 1 and 2). Math Accuracy. Accuracy on the math task was high (mean=.89, SD=.08) and all participants performed statistically above chance. Discussion In the current experiment, we aimed to examine whether and how free recall organization was inﬂuenced by perceptual event boundaries. Overall, we found that transition likelihoods varied as a function of within-event position (Figure 5). First, our results suggest that local forward transitions were signiﬁcantly more likely from boundary items as compared to preboundary items whereas there were no diﬀerences between conditions for local backward transitions (Figure 5a, b). Second, we found that transitions from preboundary items were on average to more distal items than transitions from other within-event positions. Lastly, transitions from pre-boundary items were most likely to be to boundary items as compared to other within-event positions. These ﬁndings argue that perceptual event boundaries inﬂuence the structure of free recall behavior. Our ﬁnding that local forward transitions (n+1, n+2, n+3) were more likely from boundary items than from pre-boundary items is consistent with the idea that items encountered in the same event are more strongly associated to each other than items in distinct events. For boundary items, forward local transitions were all to other within event items, whereas for pre-boundary items local forward transitions were all to items in the neighboring event. This result is in line with temporal context models of memory (Howard & Kahana, 2002; Manning, Polyn, Baltuch, Litt, & Kahana, 2011; Polyn, Natu, Cohen, & Norman, 2005; Polyn et al., 2009a), that propose that during the study of a list of stimuli, items are bound to a slowly-changing representation of context. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 30 Although the particular features (e.g. semantic, source, temporal) that are included in the context representation vary according to the speciﬁc model, all retrieved context models propose that when an item is recalled from memory, the context representation is reinstated and used to guide the retrieval process. Because the context Although the particular features (e.g. semantic, source, temporal) that are included in the context representation vary according to the speciﬁc model, all retrieved context models propose that when an item is recalled from memory, the context representation is reinstated and used to guide the retrieval process. Because the context representations of neighboring items are most similar, this leads to free recall transitions between neighboring items, and the overall lag-CRP pattern. Our data are in line with such a retrieval interpretation to the extent that the perceptual details associated with each item are also reinstated during recall. Thus, reinstatement of the color associated with an object may make it more likely that participants will recall other objects encoded with the same color. Although the particular features (e.g. semantic, source, temporal) that are included in the context representation vary according to the speciﬁc model, all retrieved context models propose that when an item is recalled from memory, the context representation is reinstated and used to guide the retrieval process. Because the context representations of neighboring items are most similar, this leads to free recall transitions between neighboring items, and the overall lag-CRP pattern. Our data are in line with such a retrieval interpretation to the extent that the perceptual details associated with each item are also reinstated during recall. Thus, reinstatement of the color associated with an object may make it more likely that participants will recall other objects encoded with the same color. Although the particular features (e.g. semantic, source, temporal) that are included in the context representation vary according to the speciﬁc model, all retrieved context models propose that when an item is recalled from memory, the context representation is reinstated and used to guide the retrieval process. Because the context representations of neighboring items are most similar, this leads to free recall transitions between neighboring items, and the overall lag-CRP pattern. Our data are in line with such a retrieval interpretation to the extent that the perceptual details associated with each item are also reinstated during recall. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Thus, reinstatement of the color associated with an object may make it more likely that participants will recall other objects encoded with the same color. In a previous study, Polyn et al. (2009b) found that when freely recalling a short list of 12 words that included one task switch at the halfway point in the list, participants tended to cluster their recall responses by items that shared an encoding task. That is, more free recall transitions were made between words that were encoded using the same task as compared to words encoded under two diﬀerent tasks. Our results are consistent with that result and extend it to suggest that the change in a perceptual feature during experience is suﬃcient to impose structure in the free recall of events. While Polyn and colleagues focused their analyses on the eﬀect of task boundaries on the likelihood of within- vs. across-event transitions (irrespective of the list position of the item), we speciﬁcally asked whether items that ﬂanked a perceptual boundary would show a within-event bias. This analytic approach allowed us to determine precisely how boundaries modulate the likelihood of within-event recall transitions. Interestingly, while we found that local forward transitions are signiﬁcantly more likely from boundary items relative to pre-boundary items, we found no diﬀerence between the two conditions in local backwards transitions. If shared context results in stronger associative binding between items in the same event, one might expect local backwards transitions to be relatively greater for pre-boundary items compared to other within-event positions, since local backwards transitions would be to other within-event items. While we note that the aforementioned result is a null ﬁnding (and therefore not PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 31 31 directly interpretable), we can oﬀer a few speculations as to why we might not expect to see an eﬀect. Previous studies suggest that free recall is reliably bias in the forward direction (Howard & Kahana, 2002; Kahana, 1996) and backwards transitions are relatively more rare. We see this pattern in our data as well (Figure 4b, 5a, b). Thus, it’s possible that perceptual boundaries exert a substantially stronger eﬀect on forward transition probabilities. An alternative explanation is that our experimental design was not sensitive enough to detect the eﬀect of boundaries on backward transitions (e.g., lists too long, events too short or boundaries not strong enough). Nonetheless, the fact that that we do observe a greater likelihood of local forward transitions from boundary items as compared to pre-boundary items is evidence that perceptual boundaries do indeed shape free recall behavior. A second notable observation from this experiment was that the average transition distance from pre-boundary items was signiﬁcantly further than from boundary items. On other words, given the successful recall of a pre-boundary item, participants were more likely to next recall a more distal item in the list. One possible explanation for this ﬁnding is that perceptual event boundaries result in a weak associative link between a pre-boundary item and its neighboring boundary item (Dubrow & Davachi, 2013; Ezzyat & Davachi, 2011). Because of this weak associative link, after the successful recall of a pre-boundary item, a memory search process might be initiated and as a consequence, more distal item may be subsequently recalled. This ﬁnding is consistent with the idea that event boundaries disrupt associative binding between items and suggest that transitions following the recall of the last item in an event are on average more distal than transitions fro other within-event positions. A second notable observation from this experiment was that the average transition distance from pre-boundary items was signiﬁcantly further than from boundary items. On other words, given the successful recall of a pre-boundary item, participants were more likely to next recall a more distal item in the list. One possible explanation for this ﬁnding is that perceptual event boundaries result in a weak associative link between a pre-boundary item and its neighboring boundary item (Dubrow & Davachi, 2013; Ezzyat & Davachi, 2011). PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 32 tends to be a boundary item. An intriguing interpretation of this ﬁnding is that event boundaries may serve as a ‘gateway’ into an episodic event. In other words, after successfully recalling the end of a previous event and during a mnemonic search for more items, boundary items might stand out as entry points into other mnemonic episodes. Consistent with this interpretation, we found that on average transitions from pre-boundary items were more distal than from other positions and out of those distal transitions, transitions to boundary items were most probable. While the ‘gateway’ idea is certainly attractive, future studies will be necessary to determine if this is the most likely explanation for this pattern of results. Finally, event segmentation theory suggests that items encountered at event boundaries may be more memorable than items encountered elsewhere in an event, possibly due to increased attention at boundaries (Radvansky, 2012; Zacks et al., 2007). All three experiments are consistent with this idea by showing that object-color memory is better at event boundaries than at other within-event positions. Interestingly, however, in Experiment 3 we did not observe an overall increase in free recall of boundary items, as one might have predicted from EST. While we hesitate to over interpret this null ﬁnding, we can oﬀer some speculation to why we observed this eﬀect. Rather than boundaries generally boosting memory for boundary information, boundaries may selectively increase associative binding between a boundary item and its context. A mechanism of this nature would predict greater object-color associative memory, but not necessarily better encoding of boundary items alone. Alternatively, perceptual boundaries may generally enhance encoding, but our study may not have been sensitive enough to detect it. Future work will be necessary to disentangle whether event boundaries diﬀerentially inﬂuence item memory vs. item-context associative binding. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Because of this weak associative link, after the successful recall of a pre-boundary item, a memory search process might be initiated and as a consequence, more distal item may be subsequently recalled. This ﬁnding is consistent with the idea that event boundaries disrupt associative binding between items and suggest that transitions following the recall of the last item in an event are on average more distal than transitions fro other within-event positions. Another interesting ﬁnding from this experiment was that following the successful recall of a pre-boundary item, the next item recalled is most likely to be a boundary item. Given that pre-boundary items are neighbored by boundary items and that participants generally tend to transition forward in free recall, this may not seem entirely surprising. However, this eﬀect remains signiﬁcant after removing local items from the analysis, suggesting that when one transitions from a pre-boundary item to a more distal item, it Another interesting ﬁnding from this experiment was that following the successful recall of a pre-boundary item, the next item recalled is most likely to be a boundary item. Another interesting ﬁnding from this experiment was that following the successful recall of a pre-boundary item, the next item recalled is most likely to be a boundary item. Given that pre-boundary items are neighbored by boundary items and that participants PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY General Discussion The studies presented here demonstrate that perceptual boundaries inﬂuence the organization of events stored in long-term memory. In Experiment 1, we found that perceptual boundaries enhanced associative binding between an object and a color PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Finally, in Experiment 3, we found that participants exhibited recall behavior that was structured by perceptual event boundaries. Taken together, this work provides compelling evidence that perceptual boundaries have a lasting inﬂuence on the structure of our memories. background. In Experiment 2, we show that while boundary-related associative memory was enhanced, temporal order memory for pairs of items that span a perceptual boundary was disrupted relative to order memory for within-event item pairs. background. In Experiment 2, we show that while boundary-related associative memory was enhanced, temporal order memory for pairs of items that span a perceptual boundary was disrupted relative to order memory for within-event item pairs. Furthermore, the magnitude of the boundary eﬀect (i.e. the response time to the boundary) predicted the cost in temporal order memory suggesting a trade-oﬀbetween boundary processing and across-event temporal order memory. Finally, in Experiment 3, we found that participants exhibited recall behavior that was structured by perceptual event boundaries. Taken together, this work provides compelling evidence that perceptual boundaries have a lasting inﬂuence on the structure of our memories. These results add to a growing body of literature characterizing the inﬂuence of event segmentation on long-term memory (Baldassano et al., 2017; Boltz, 1992; Chen et al., 2016; Davachi & DuBrow, 2015; Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2011, 2014; Heusser, Poeppel, Ezzyat, & Davachi, 2016; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004; Zacks, Speer, Vettel, & Jacoby, 2006). Previous studies using naturalistic stimuli have provided data consistent with the idea that information experienced at event boundaries is better encoded in memory (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004). However, there is some concern that these eﬀects could be explained by diﬀerences in the amount of diagnostic background. In Experiment 2, we show that while boundary related associative memory was enhanced, temporal order memory for pairs of items that span a perceptual boundary was disrupted relative to order memory for within-event item pairs. Furthermore, the magnitude of the boundary eﬀect (i.e. the response time to the boundary) predicted the cost in temporal order memory suggesting a trade-oﬀbetween boundary processing and across-event temporal order memory. Finally, in Experiment 3, we found that participants exhibited recall behavior that was structured by perceptual event boundaries. Taken together, this work provides compelling evidence that perceptual boundaries have a lasting inﬂuence on the structure of our memories. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY These results add to a growing body of literature characterizing the inﬂuence of event segmentation on long-term memory (Baldassano et al., 2017; Boltz, 1992; Chen et al., 2016; Davachi & DuBrow, 2015; Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2011, 2014; Heusser, Poeppel, Ezzyat, & Davachi, 2016; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004; Zacks, Speer, Vettel, & Jacoby, 2006). Previous studies using naturalistic stimuli have provided data consistent with the idea that information experienced at event boundaries is better encoded in memory (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004). However, there is some concern that these eﬀects could be explained by diﬀerences in the amount of diagnostic information between conditions; that is, if boundary test items provide a better ‘summary’ of a particular event than non-boundary test items, one might expect higher boundary memory that is not driven by event segmentation processes, per se. By contrast, in the current study, we carefully matched the boundary and non-boundary conditions during retrieval, such that the only diﬀerence between the two was their position within an event during encoding. Thus, the best explanation for the boundary-related memory enhancement in our study is an inﬂuence during encoding of the perceptual boundary on associative binding (between the object and color). This eﬀect, which we see across three experiments is consistent with previous research demonstrating boundary-related memory enhancements, and conﬁrms that segmentation processes during encoding lead to better memory for information encoded Furthermore, the magnitude of the boundary eﬀect (i.e. the response time to the boundary) predicted the cost in temporal order memory suggesting a trade-oﬀbetween boundary processing and across-event temporal order memory. Finally, in Experiment 3, we found that participants exhibited recall behavior that was structured by perceptual event boundaries. Taken together, this work provides compelling evidence that perceptual boundaries have a lasting inﬂuence on the structure of our memories. that perceptual boundaries have a lasting inﬂuence on the structure of our memories. These results add to a growing body of literature characterizing the inﬂuence of event segmentation on long-term memory (Baldassano et al., 2017; Boltz, 1992; Chen et al., 2016; Davachi & DuBrow, 2015; Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2011, 2014; Heusser, Poeppel, Ezzyat, & Davachi, 2016; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004; Zacks, Speer, Vettel, & Jacoby, 2006). PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 33 background. In Experiment 2, we show that while boundary-related associative memory was enhanced, temporal order memory for pairs of items that span a perceptual boundary was disrupted relative to order memory for within-event item pairs. Furthermore, the magnitude of the boundary eﬀect (i.e. the response time to the boundary) predicted the cost in temporal order memory suggesting a trade-oﬀbetween boundary processing and across-event temporal order memory. Finally, in Experiment 3, we found that participants exhibited recall behavior that was structured by perceptual event boundaries. Taken together, this work provides compelling evidence that perceptual boundaries have a lasting inﬂuence on the structure of our memories. These results add to a growing body of literature characterizing the inﬂuence of event segmentation on long-term memory (Baldassano et al., 2017; Boltz, 1992; Chen et al., 2016; Davachi & DuBrow, 2015; Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2011, 2014; Heusser, Poeppel, Ezzyat, & Davachi, 2016; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004; Zacks, Speer, Vettel, & Jacoby, 2006). Previous studies using naturalistic stimuli have provided data consistent with the idea that information experienced at event boundaries is better encoded in memory (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004). However, there is some concern that these eﬀects could be explained by diﬀerences in the amount of diagnostic information between conditions; that is, if boundary test items provide a better ‘summary’ of a particular event than non-boundary test items, one might expect higher boundary memory that is not driven by event segmentation processes, per se. By contrast, in the current study, we carefully matched the boundary and non-boundary conditions during retrieval, such that the only diﬀerence between the two was their position within an event during encoding. Thus, the best explanation for the boundary-related memory enhancement in our study is an inﬂuence during encoding of the perceptual boundary on associative binding (between the object and color) This background. In Experiment 2, we show that while boundary-related associative memory was enhanced, temporal order memory for pairs of items that span a perceptual boundary was disrupted relative to order memory for within-event item pairs. Furthermore, the magnitude of the boundary eﬀect (i.e. the response time to the boundary) predicted the cost in temporal order memory suggesting a trade-oﬀbetween boundary processing and across-event temporal order memory. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 34 at event boundaries. at event boundaries. These results are also consistent with studies of contextual novelty that ﬁnd that items that are in someway deviant from the local surroundings show a boost in memory (Cimbalo, 1978; Fabiani & Donchin, 1995; Lin, Pype, Murray, & Boynton, 2010; Swallow & Jiang, 2011, 2013; Wallace, 1965). Typically, these studies have observed diﬀerences in item memory based on deviance status. The present study is novel in that we ﬁnd enhanced item-context associative binding for contextually novel trials (i.e. object-color pairs encoded at perceptual event boundaries). While this distinction may seem subtle, it does not follow that better item encoding should necessarily entail better item-context associative memory. For instance, compared to neutral stimuli, These results are also consistent with studies of contextual novelty that ﬁnd that items that are in someway deviant from the local surroundings show a boost in memory (Cimbalo, 1978; Fabiani & Donchin, 1995; Lin, Pype, Murray, & Boynton, 2010; Swallow & Jiang, 2011, 2013; Wallace, 1965). Typically, these studies have observed diﬀerences in item memory based on deviance status. The present study is novel in that we ﬁnd enhanced item-context associative binding for contextually novel trials (i.e. object-color pairs encoded at perceptual event boundaries). While this distinction may seem subtle, it does not follow that better item encoding should necessarily entail better item-context associative memory. For instance, compared to neutral stimuli, emotionally arousing stimuli show better item memory and worse item-context associative memory (Bisby & Burgess, 2014; Madan, Caplan, Lau, & Fujiwara, 2012). Furthermore, it is now well established that diﬀerent neural structures support the encoding of item information vs. the binding of an item to its context (Davachi, 2006; Davachi, Mitchell, & Wagner, 2003; Ranganath et al., 2004). Therefore, it is conceivable that item memory and item-context associative memory are dissociable memory representations. Future work should test the relationship between item memory and item-context associative memory at perceptual boundaries. In Experiment 2, we found that temporal order memory was relatively worse for across-event trial pairs relative to within-event trial pairs. This is consistent with previous results that show that cued recall (Ezzyat & Davachi, 2011) and temporal order memory decisions (Dubrow & Davachi, 2013) are more accurate for test items that were from the same ‘event’ compared to those from adjacent ‘events’ – even though the actual temporal lag was the same in both conditions. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Previous studies using naturalistic stimuli have provided data consistent with the idea that information experienced at event boundaries is better encoded in memory (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004). However, there is some concern that these eﬀects could be explained by diﬀerences in the amount of diagnostic information between conditions; that is, if boundary test items provide a better ‘summary’ of a particular event than non-boundary test items, one might expect higher boundary memory that is not driven by event segmentation processes, per se. By These results add to a growing body of literature characterizing the inﬂuence of event segmentation on long-term memory (Baldassano et al., 2017; Boltz, 1992; Chen et al., 2016; Davachi & DuBrow, 2015; Dubrow & Davachi, 2013; DuBrow & Davachi, 2016; Ezzyat & Davachi, 2011, 2014; Heusser, Poeppel, Ezzyat, & Davachi, 2016; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004; Zacks, Speer, Vettel, & Jacoby, 2006). Previous studies using naturalistic stimuli have provided data consistent with the idea that information experienced at event boundaries is better encoded in memory (Boltz, 1992; Newtson & Engquist, 1976; Schwan & Garsoﬀky, 2004). However, there is some concern that these eﬀects could be explained by diﬀerences in the amount of diagnostic information between conditions; that is, if boundary test items provide a better ‘summary’ of a particular event than non-boundary test items, one might expect higher boundary memory that is not driven by event segmentation processes, per se. By contrast, in the current study, we carefully matched the boundary and non-boundary conditions during retrieval, such that the only diﬀerence between the two was their position within an event during encoding. Thus, the best explanation for the boundary-related memory enhancement in our study is an inﬂuence during encoding of the perceptual boundary on associative binding (between the object and color). This eﬀect, which we see across three experiments is consistent with previous research demonstrating boundary-related memory enhancements, and conﬁrms that segmentation processes during encoding lead to better memory for information encoded PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY The current study complements these previous results that used more complex boundaries (e.g. task/stimulus class switches, narrative temporal boundaries) by demonstrating that simple perceptual boundaries are suﬃcient to induce event segmentation processes that result in better within-event versus across-event associative memory. It’s worthwhile to note that some theories of recency memory might predict the opposite pattern of results In Experiment 2, we found that temporal order memory was relatively worse for across-event trial pairs relative to within-event trial pairs. This is consistent with previous results that show that cued recall (Ezzyat & Davachi, 2011) and temporal order memory decisions (Dubrow & Davachi, 2013) are more accurate for test items that were from the same ‘event’ compared to those from adjacent ‘events’ – even though the actual temporal lag was the same in both conditions. The current study complements these previous results that used more complex boundaries (e.g. task/stimulus class switches, narrative temporal boundaries) by demonstrating that task/stimulus class switches, narrative temporal boundaries) by demonstrating that simple perceptual boundaries are suﬃcient to induce event segmentation processes that result in better within-event versus across-event associative memory. It’s worthwhile to note that some theories of recency memory might predict the opposite pattern of results PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 35 (see Friedman (1993) for review). For instance, if participants were using an item-strength or contextual overlap retrieval strategy to recover recency information, a change in context between items could be beneﬁcial for performance. On the other hand, if participants are performing recency discrimination by recovering associative information among a set of items, one might predict that within-event temporal order memory would be greater than across-event temporal order memory since shared contextual features can help to bind items across time. The latter prediction is consistent with our results, suggesting that rather than a distance-based retrieval strategy, participants may be relying on retrieving the associative links between a series of items. Further evidence for this type of retrieval mechanism comes from a study that found that during a recency discrimination test, the speed of retrieval for items that intervened the recency test pair during encoding is facilitated relative to items that did not intervene the test pair (DuBrow & Davachi, 2014). Furthermore, in a related fMRI experiment, the category of items that intervened the recency test probes during encoding was “reactivated” during successful recency discrimination (DuBrow & Davachi, 2014). Together, these ﬁndings suggest that in at least some cases (i.e. when the lag between the tested items is short), recency discrimination can be successfully performed by recovering the items that intervened the test pair. (see Friedman (1993) for review). For instance, if participants were using an item-strength or contextual overlap retrieval strategy to recover recency information, a change in context between items could be beneﬁcial for performance. On the other hand, if participants are performing recency discrimination by recovering associative information among a set of items, one might predict that within-event temporal order memory would be greater than across-event temporal order memory since shared contextual features can help to bind items across time. The latter prediction is consistent with our results, suggesting that rather than a distance-based retrieval strategy, participants may be relying on retrieving the associative links between a series of items. Further evidence for this type of retrieval mechanism comes from a study that found that during a recency discrimination test, the speed of retrieval for items that intervened the recency test pair during encoding is facilitated relative to items that did not intervene the test pair (DuBrow & Davachi, 2014). PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY One interpretation of this result is that when a participant recalls an item that occurred just prior to an event boundary, the associative link to the next item was severed by the perceptual boundary, thus prompting a memory search to recall other items in the list. A mechanism of this nature could lead to selectively more distal transitions for pre-boundary items relative to other within-event positions. Lastly, we found that following the successful recall of a pre-boundary item, there was a trend for boundary items to be recalled next. This was evident for local transitions as well as distal transitions. One explanation for this pattern of results is that following the recall of the end of an event, participants may initiate a memory search process in an eﬀort to recover additional list items. If boundary items ‘stand out’ in memory due to better overall encoding or better item-context binding, then the likelihood of a boundary transition would be greater than the likelihood of transitioning to other list positions. This last preliminary data point is consistent with the idea that event boundaries may act as ‘gateways’ into an episodic events. That is, during free recall, boundaries may stand out as entry points into an episodic memory and facilitate the retrieval of additional within-event information. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY Furthermore, in a related fMRI experiment, the category of items that intervened the recency test probes during encoding was “reactivated” during successful recency discrimination (DuBrow & Davachi, 2014). Together, these ﬁndings suggest that in at least some cases (i.e. when the lag between the tested items is short), recency discrimination can be successfully performed by recovering the items that intervened the test pair. Finally, the results from our free recall data (Experiment 3) underscore the inﬂuence of perceptual event boundaries on memory organization. Namely, we found that perceptual boundaries modulated free recall transition probabilities: local forward transitions from boundary items were more likely as compared to local forward transitions from pre-boundary items (Figure 5). This is likely due to the fact that for boundary items, local forward transitions were to other within-event items where as local forward transitions from pre-boundary items were to items in a diﬀerent event. This result is consistent with previous studies that ﬁnd evidence for recall organization according to the source of the stimuli (Frost, 1971; Hintzman et al., 1972; Murdock & Walker, 1969; Nilsson, 1974; Polyn et al., 2009b). Interestingly, however, we did not ﬁnd that local backwards transitions we more likely for pre-boundary items, as one might Finally, the results from our free recall data (Experiment 3) underscore the inﬂuence of perceptual event boundaries on memory organization. Namely, we found that perceptual boundaries modulated free recall transition probabilities: local forward transitions from boundary items were more likely as compared to local forward transitions from pre-boundary items (Figure 5). This is likely due to the fact that for boundary items, local forward transitions were to other within-event items where as local forward transitions from pre-boundary items were to items in a diﬀerent event. This result is consistent with previous studies that ﬁnd evidence for recall organization This result is consistent with previous studies that ﬁnd evidence for recall organization according to the source of the stimuli (Frost, 1971; Hintzman et al., 1972; Murdock & Walker, 1969; Nilsson, 1974; Polyn et al., 2009b). Interestingly, however, we did not ﬁnd that local backwards transitions we more likely for pre-boundary items, as one might PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 36 expect if recall organization is strongly inﬂuenced by source information. However, since this was a null ﬁnding, its interpretation is not entirely straight-forward as many factors can inﬂuence the lack of a ﬁnding. Nonetheless, the fact that we see a larger proportion of local forward transitions from boundary items compared to pre-boundary items is evidence that perceptual event boundaries structure memory recall behavior. We also found that transitions from pre-boundary items were more likely to be to distal items (as compared to transitions from boundary items). One interpretation of this result is that when a participant recalls an item that occurred just prior to an event boundary, the associative link to the next item was severed by the perceptual boundary, thus prompting a memory search to recall other items in the list. A mechanism of this nature could lead to selectively more distal transitions for pre-boundary items relative to other within-event positions. Lastly, we found that following the successful recall of a pre-boundary item, there was a trend for boundary items to be recalled next. This was evident for local transitions as well as distal transitions. One explanation for this pattern of results is that following the recall of the end of an event, participants may initiate a memory search process in an eﬀort to recover additional list items. If boundary items ‘stand out’ in memory due to better overall encoding or better item-context binding, then the likelihood of a boundary transition would be greater than the likelihood of transitioning to other list positions. This last preliminary data point is consistent with the idea that event boundaries may act as ‘gateways’ into an episodic events. That is, during free recall, boundaries may stand out as entry points into an episodic memory and facilitate the retrieval of additional within-event information. expect if recall organization is strongly inﬂuenced by source information. However, since this was a null ﬁnding, its interpretation is not entirely straight-forward as many factors can inﬂuence the lack of a ﬁnding. Nonetheless, the fact that we see a larger proportion of local forward transitions from boundary items compared to pre-boundary items is evidence that perceptual event boundaries structure memory recall behavior. We also found that transitions from pre-boundary items were more likely to be to distal items (as compared to transitions from boundary items). Conclusions Together, Experiments 1 and 2 suggest that while perceptual boundaries may enhance some forms of memory (i.e. object-color associative memory), this comes at the cost of reduced across-event associative memory (temporal order memory). In other words, shifting one’s attention to a novel stimulus in the environment trades oﬀwith the ongoing maintenance and integration of representations into an event model, and this PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 38 PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 37 causes a disruption in the associative binding of items encountered across time. 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Schematic of the Task. Participants made pleasant/unpleasant judgments on object-color pairs. Critically, the color switched every 6 trials. Object-color associative memory test. (Bottom, left) After encoding, participants performed a two alternative forced choice object-color memory test. The object-color test was in both Experiment 1 and 2. Temporal order memory test. (Bottom, right) After the color test, temporal order memory was tested using a two alternative forced choice task. Participants indicated which of two studied objects appeared ﬁrst in the list. The temporal order test was only in Experiment 2. Figure 1. Schematic of the Task. Participants made pleasant/unpleasant judgments on object-color pairs. Critically, the color switched every 6 trials. Object-color associative memory test. (Bottom, left) After encoding, participants performed a two alternative forced choice object-color memory test. The object-color test was in both Experiment 1 and 2. Temporal order memory test. (Bottom, right) After the color test, temporal order memory was tested using a two alternative forced choice task. Participants indicated which of two studied objects appeared ﬁrst in the list. The temporal order test was only in Experiment 2. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 45 1 2 3 4 5 6 position 0.5 0.6 0.7 0.8 0.9 1.0 mean(accuracy) * A 1 2 3 4 5 6 position 1.0 1.2 1.4 1.6 1.8 2.0 2.2 mean(response time) * B Figure 2. Experiment 1 Results. Object-color associative memory accuracy (A) and response times are shown as a function of within-event position (B). Error bars represent 95% conﬁdence intervals. p < .05, p < .005, *p < .001. 2B Figure 2. Experiment 1 Results. Object-color associative memory accuracy (A) and response times are shown as a function of within-event position (B). Error bars represent 95% conﬁdence intervals. p < .05, p < .005, p < .001. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 46 object-color temporal order test 0.5 0.6 0.7 0.8 0.9 1.0 mean(accuracy) * * A boundary nonboundary boundary nonboundary condition 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 mean(accuracy) * n.s. * B fastest middle slowest 1 2 3 4 5 6 position 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 mean(response time) * C object-color temporal order test 1 2 3 4 5 6 mean(response time) * * * D boundary nonboundary Figure 3. Experiment 2 Results. (A) Memory accuracy is shown as a function of test and condition. (B) Temporal order memory accuracy split into terciles by encoding response time of intervening boundary (for across-event) or non-boundary (for within-event) trial. (C) Task response times during encoding as a function of event position. (D) Retrieval response times for correct trials as a function of condition and memory test. All error bars represent 95% conﬁdence intervals. p<.05, p<.005, p<.001. boundary nonboundary condition 0.50 0.55 0.60 0.65 0.70 0.75 0.80 0.85 0.90 mean(accuracy) * n.s. * B fastest middle slowest D object-color temporal order test 0.5 0.6 0.7 0.8 0.9 1.0 mean(accuracy) * * * A boundary nonboundary C 0 B condition object-color temporal order test 1 2 3 4 5 6 mean(response time) * * * D boundary nonboundary 6 D C 1 2 3 4 5 6 position 1.0 1.2 1.4 1.6 1.8 2.0 2.2 2.4 mean(response time) *** C Figure 3. Experiment 2 Results. (A) Memory accuracy is shown as a function of test and condition. (B) Temporal order memory accuracy split into terciles by encoding response time of intervening boundary (for across-event) or non-boundary (for within-event) trial. (C) Task response times during encoding as a function of event position. (D) Retrieval response times for correct trials as a function of condition and memory test. All error bars represent 95% conﬁdence intervals. p<.05, p<.005, p<.001. PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 47 A B Figure 4. Experiment 3: Serial Position Curve and Lag-CRP (A) Memory accuracy broken down by list position. (B) Given the recall of an item in position n, this graph plots the probability of recalling a neighboring items next in the recall sequence (+/- 5). B A B Figure 4. Experiment 3: Serial Position Curve and Lag-CRP (A) Memory accuracy broken down by list position. (B) Given the recall of an item in position n, this graph plots the probability of recalling a neighboring items next in the recall sequence (+/- 5). Figure 4. Experiment 3: Serial Position Curve and Lag-CRP (A) Memory accuracy broken down by list position. (B) Given the recall of an item in position n, this graph plots the probability of recalling a neighboring items next in the recall sequence (+/- 5). PERCEPTUAL BOUNDARIES AND ASSOCIATIVE MEMORY 48 1 2 3 4 position 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 mean(transition probability) ~ A 1 2 3 4 position 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 mean(transition probability) n.s. B 1 2 3 4 position 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 mean(transition distance) C 1 2 3 4 position 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 mean(transition probability) ~ D Figure 5. Experiment 3 Results. (A) Local forward (sum of n+1, n+2, n+3) transition likelihood as a function of within-event position. (B) Local backward (sum of n-1, n-2, n-3) transition likelihood as a function of within-event position. (C) Average transition distance as a function of within-event position. (D) Likelihood of transition from pre-boundary items as a function of within-event position. Error bars represent 95% conﬁdence intervals. ~p<.10, p<.05, p<.005. 1 2 3 4 position 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 mean(transition probability) ~ A C 1 2 3 4 position 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 mean(transition probability) n.s. B D B D Figure 5. Experiment 3 Results. (A) Local forward (sum of n+1, n+2, n+3) transition likelihood as a function of within-event position. (B) Local backward (sum of n-1, n-2, n-3) transition likelihood as a function of within-event position. (C) Average transition distance as a function of within-event position. (D) Likelihood of transition from pre-boundary items as a function of within-event position. Error bars represent 95% conﬁdence intervals. ~p<.10, p<.05, **p<.005.
https://openalex.org/W4220967738	https://www.researchsquare.com/article/rs-893209/latest.pdf	English	null	A multi-environment framework to evaluate the adaptation of wheat (Triticum aestivum) to heat stress	Theoretical and applied genetics	2,022	cc-by	24,978	Authors: 3 Assessing adaptation to abiotic stresses such as high temperature conditions across multiple 12 environments presents opportunities for breeders to target selection for broad adaptation and 13 specific adaptation. 14 Assessing adaptation to abiotic stresses such as high temperature conditions across multiple 12 environments presents opportunities for breeders to target selection for broad adaptation and 13 specific adaptation. 14 Assessing adaptation to abiotic stresses such as high temperature conditions across multiple 12 environments presents opportunities for breeders to target selection for broad adaptation and 13 specific adaptation. 14 Research Article Keywords: Heat-stress, QTL, multi-environment, adaptation Posted Date: September 21st, 2021 DOI: https://doi.org/10.21203/rs.3.rs-893209/v1 License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Keywords: Heat-stress, QTL, multi-environment, adaptation License:   This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Version of Record: A version of this preprint was published at Theoretical and Applied Genetics on January 20th, 2022. See the published version at https://doi.org/10.1007/s00122-021-04024-5. A multi-environment framework to evaluate the adaptation of wheat 1 (Triticum aestivum) to heat stress 2 Authors: 3 Paul Telfer1,2, James Edwards1,2, Julian Taylor2, Jason A. Able2, Haydn Kuchel1,2 4 1 Australian Grain Technologies, 20 Leitch Road, Roseworthy, SA 5371, Australia 5 2 School of Agriculture, Food and Wine, The University of Adelaide, Waite Campus, PMB 1 Glen 6 Osmond, Adelaide, SA 5064, Australia 7 Corresponding author. E-mail address: paul.telfer@agtbreeding.com.au 8 Keywords: 9 Heat-stress, QTL, multi-environment, adaptation 10 Key Message: 11 Assessing adaptation to abiotic stresses such as high temperature conditions across multiple 12 environments presents opportunities for breeders to target selection for broad adaptation and 13 specific adaptation. 14 Supplementary Material 15 Supplementary Table 1. Consensus map and individual linkage maps for all populations affixed with 16 RefSeq physical positions of QTL identified. 17 Supplementary Table 2. All QTL identified by trait measured, QTL type (responsive or performance; if 18 responsive the climatic co-variate to which responsiveness was found), interval position (cM) 19 (consensus map position), the effect of each QTL, P-Value, LOD, and physical position (RefSeq). 20 Conflict of interest statement 21 Supplementary Material 15 Supplementary Table 1. Consensus map and individual linkage maps for all populations affixed with 16 RefSeq physical positions of QTL identified. 17 Supplementary Table 1. Consensus map and individual linkage maps for all populations affixed with 16 RefSeq physical positions of QTL identified. 17 Supplementary Table 2. All QTL identified by trait measured, QTL type (responsive or performance; if 18 responsive the climatic co-variate to which responsiveness was found), interval position (cM) 19 (consensus map position), the effect of each QTL, P-Value, LOD, and physical position (RefSeq). 20 On behalf of all authors, the corresponding author states that there is no conflict of interest. 22 Data availability 23 The datasets generated during the current study are available from the corresponding author on 24 reasonable request. 25 On behalf of all authors, the corresponding author states that there is no conflict of interest. 22 On behalf of all authors, the corresponding author states that there is no conflict of interest. The datasets generated during the current study are available from the corresponding author on 24 reasonable request. 25 The datasets generated during the current study are available from the corresponding author on 24 reasonable request. 25 26 Abstract 27 Adaptation of wheat to heat stress is an important component of adaptation in variable climates such 28 as the cereal producing areas of Australia. However, in variable climates stress conditions may not be 29 present in every season or is present at different levels, at different times during the season. Such 30 conditions complicate plant breeder’s ability to select for adaptation to abiotic stress. This study 31 presents a framework for the assessment of the genetic basis of adaptation to heat stress conditions 32 with improved relevance to breeder’s selection objectives. The framework was applied here with the 33 evaluation of 1225 doubled haploid lines from five populations across six environments (three 34 environments selected for contrasting temperature stress conditions during anthesis and grain fill 35 periods, over two consecutive seasons), using regionally best practice planting times to evaluate the 36 role of heat stress conditions in genotype adaptation. Temperature co-variates were determined for 37 each genotype, in each environment, for the anthesis and grain fill periods. Genome wide QTL analysis 38 identified performance QTL for stable effects across all environments, and QTL that illustrated 39 responsiveness to heat stress conditions across the sampled environments. A total of 199 QTL were 40 identified, including 60 performance QTL, and 139 responsiveness QTL. Of the identified QTL, 99 41 occurred iseparate to the 21 anthesis date QTL identified. Assessing adaptation to heat stress 42 conditions as the combination of performance and responsiveness offers breeders opportunities to 43 select for grain yield stability across a range of environments, as well as genotypes with higher relative 44 yield in stress conditions. 45 Adaptation of wheat to heat stress is an important component of adaptation in variable climates such 28 as the cereal producing areas of Australia. However, in variable climates stress conditions may not be 29 present in every season or is present at different levels, at different times during the season. Such 30 conditions complicate plant breeder’s ability to select for adaptation to abiotic stress. This study 31 presents a framework for the assessment of the genetic basis of adaptation to heat stress conditions 32 with improved relevance to breeder’s selection objectives. Abstract 27 The framework was applied here with the 33 evaluation of 1225 doubled haploid lines from five populations across six environments (three 34 environments selected for contrasting temperature stress conditions during anthesis and grain fill 35 periods, over two consecutive seasons), using regionally best practice planting times to evaluate the 36 role of heat stress conditions in genotype adaptation. Temperature co-variates were determined for 37 each genotype, in each environment, for the anthesis and grain fill periods. Genome wide QTL analysis 38 identified performance QTL for stable effects across all environments, and QTL that illustrated 39 responsiveness to heat stress conditions across the sampled environments. A total of 199 QTL were 40 identified, including 60 performance QTL, and 139 responsiveness QTL. Of the identified QTL, 99 41 occurred iseparate to the 21 anthesis date QTL identified. Assessing adaptation to heat stress 42 conditions as the combination of performance and responsiveness offers breeders opportunities to 43 select for grain yield stability across a range of environments, as well as genotypes with higher relative 44 yield in stress conditions. 45 46 Introduction 47 Many regions throughout the world experience heat, drought, and frost stress that limit crop 48 production. In the Mediterranean-type climates of southern Australia heat stress conditions during 49 the sensitive stage of anthesis and early grain filling are common (Zheng et al. 2012), often co- 50 occurring with other abiotic stresses such as drought and wind (Machado and Paulsen 2001; Shah and 51 Paulsen 2003). In southern Australia heat stress conditions during anthesis and early grain fill are 52 typically short periods, of up to a few days in length, with daily maximum temperatures in excess of 53 35ᵒC accompanied with winds in excess of 40 km h-1 (Alexander et al. 2010; Talukder et al. 2013). The 54 impacts of such stress events can be significant, with Kuchel et al. (2007a) and Bennett et al. (2012b) 55 reporting yield loss of up to 187 kg ha-1 for every one-degree increase in average temperature during 56 anthesis and grain fill in field experiments conducted across southern Australia. This was confirmed 57 by Telfer et al. (2018), who identified a reduction in grain yield of 161 kg ha-1 for each day with a 58 maximum temperature in excess of 30ᵒC during grain fill, and a reduction of 302 kg ha-1 for each day 59 with maximum temperature in excess of 30ᵒC during anthesis. 60 Stress conditions, like those described, impact negatively on a range of developmental stages and 61 physiological processes (Wahid et al. 2007). Plant mechanisms that manage antioxidants, heat shock 62 proteins, maintenance of cell membrane stability, and maintenance of protein stability and function 63 often underpin a plant's ability to cope with stress (Dolferus et al. 2011; Wahid et al. 2007). In the 64 field, heat stress results in reduced pollen viability and seed set when it occurs during anthesis 65 (Dolferus et al. 2011; Saini et al. 1999). Stress during grain filling leads to reduced starch and protein 66 accumulation (Bhullar and Jenner 1985; Zahedi et al. 2004), accelerated plant development, 67 premature leaf senescence, and reduced photosynthetic rate and capacity (Stone and Nicolas 1995; 68 Tewolde et al. 2006), which ultimately reduces grain size (Sharma et al. 2008; Stone and Nicolas 1995; 69 Talukder et al. 2014a; Wardlaw 1994) and grain yield (Talukder et al. 2014a; Tewolde et al. 2006). Introduction 47 70 Stress conditions, like those described, impact negatively on a range of developmental stages and 61 physiological processes (Wahid et al. 2007). Plant mechanisms that manage antioxidants, heat shock 62 proteins, maintenance of cell membrane stability, and maintenance of protein stability and function 63 often underpin a plant's ability to cope with stress (Dolferus et al. 2011; Wahid et al. 2007). In the 64 field, heat stress results in reduced pollen viability and seed set when it occurs during anthesis 65 (Dolferus et al. 2011; Saini et al. 1999). Stress during grain filling leads to reduced starch and protein 66 accumulation (Bhullar and Jenner 1985; Zahedi et al. 2004), accelerated plant development, 67 premature leaf senescence, and reduced photosynthetic rate and capacity (Stone and Nicolas 1995; 68 Tewolde et al. 2006), which ultimately reduces grain size (Sharma et al. 2008; Stone and Nicolas 1995; 69 Talukder et al. 2014a; Wardlaw 1994) and grain yield (Talukder et al. 2014a; Tewolde et al. 2006). 70 Tolerance to heat stress in wheat has been previously reported. This includes the Australian wheat 71 varieties; Halberd (Hays et al. 2007) and Gladius (Fleury et al. 2010; Talukder et al. 2014a), as well as 72 breeding and research line, RAC875 (Bennett et al. 2012a; Izanloo et al. 2008). This was also confirmed 73 by Telfer et al. (2018) who showed that Halberd expressed tolerance to heat stress during flowering, 74 and Gladius and RAC875 that expressed tolerance to heat stress experienced during grain filling. 75 Additionally, Gladius and RAC875 have been reported as drought-tolerant (Bennett et al. 2012a; Fleury 76 et al. 2010; Izanloo et al. 2008; Shirdelmoghanloo et al. 2016a). 77 Tolerance to heat stress in wheat has been previously reported. This includes the Australian wheat 71 varieties; Halberd (Hays et al. 2007) and Gladius (Fleury et al. 2010; Talukder et al. 2014a), as well as 72 breeding and research line, RAC875 (Bennett et al. 2012a; Izanloo et al. 2008). This was also confirmed 73 by Telfer et al. (2018) who showed that Halberd expressed tolerance to heat stress during flowering, 74 and Gladius and RAC875 that expressed tolerance to heat stress experienced during grain filling. 75 Additionally, Gladius and RAC875 have been reported as drought-tolerant (Bennett et al. 2012a; Fleury 76 et al. 2010; Izanloo et al. 2008; Shirdelmoghanloo et al. 2016a). Introduction 47 77 Various methods have been used to evaluate performance under heat stress conditions, using 78 controlled environments or in-field conditions, including Telfer et al. (2018) who compared a 79 controlled environment assay and a field assay for the evaluation of adaption to heat stress conditions. 80 Various methods have been used to evaluate performance under heat stress conditions, using 78 controlled environments or in-field conditions, including Telfer et al. (2018) who compared a 79 controlled environment assay and a field assay for the evaluation of adaption to heat stress conditions. 80 Controlled environment conditions offer many advantages; including ensuring a consistent and 81 repeatable stress environment and being able to manage a range of confounding factors that may be 82 present in field conditions. The oftentimes confounding effects of maturity can be managed using 83 controlled environments, in contrast to field experiments where material that develops at a different 84 rate may be exposed to different temperature stress conditions making comparisons challenging. 85 Despite the advantages of controlled environments, validation is required under representative field 86 conditions. To ensure a high incidence of heat stress conditions, delayed sowing has been a commonly 87 used methodology to ensure that sensitive developmental stages coincide with heat stress conditions 88 typically experienced later in the season (Bennett et al. 2012b; Esten Mason et al. 2013; Pinto et al. 89 2010; Reynolds et al. 2007; Sadras et al. 2015). Unfortunately in this system plants are exposed to 90 growing conditions that are not representative of agronomic practices employed by grain producers, 91 such as longer photoperiod and altered plant available water (Sadras et al. 2015). With phenological 92 development a primary driver of adaptation, and development rate interacting strongly with sowing 93 date, assessments of heat stress adaptation using sowing date variation are consequently 94 confounded, precluding clear conclusions being drawn from such studies. 95 Various methods have been used to evaluate performance under heat stress conditions, using 78 controlled environments or in-field conditions, including Telfer et al. (2018) who compared a 79 controlled environment assay and a field assay for the evaluation of adaption to heat stress conditions. 80 Controlled environment conditions offer many advantages; including ensuring a consistent and 81 repeatable stress environment and being able to manage a range of confounding factors that may be 82 present in field conditions. Introduction 47 (2018) previously discussed an alternative approach whereby the heat stress tolerance of 103 wheat germplasm is evaluated across a multi-environment (contrasting for heat stress) study. Such a 104 system enables the material to be grown in representative growing environments using regional best 105 practice agronomy and using the natural variation in temperature across the environments to evaluate 106 genotype response to heat stress. This provides the opportunity to evaluate adaptation to heat stress 107 conditions in large-scale breeding trials across a range of environments, subsequently enabling the 108 identification and validation of genetic loci using genetic mapping populations. Kuchel et al. (2007a) 109 also reported similar results, having evaluated doubled haploid bi-parental material across multiple 110 representative environments. In that study, variable expression of QTL for grain yield, and associated 111 traits, were attributable to environmental factors such as temperature and rainfall conditions varying 112 across the environments sampled. Similarly, Tura et al. (2020) was able to identify QTL-by- 113 environment interactions for grain yield and related traits. 114 Telfer et al. (2018) previously discussed an alternative approach whereby the heat stress tolerance of 103 wheat germplasm is evaluated across a multi-environment (contrasting for heat stress) study. Such a 104 system enables the material to be grown in representative growing environments using regional best 105 practice agronomy and using the natural variation in temperature across the environments to evaluate 106 genotype response to heat stress. This provides the opportunity to evaluate adaptation to heat stress 107 conditions in large-scale breeding trials across a range of environments, subsequently enabling the 108 identification and validation of genetic loci using genetic mapping populations. Kuchel et al. (2007a) 109 also reported similar results, having evaluated doubled haploid bi-parental material across multiple 110 representative environments. In that study, variable expression of QTL for grain yield, and associated 111 traits, were attributable to environmental factors such as temperature and rainfall conditions varying 112 across the environments sampled. Similarly, Tura et al. (2020) was able to identify QTL-by- 113 environment interactions for grain yield and related traits. 114 A number of studies have identified QTL for adaptation to heat stress conditions that potentially 115 confer improved grain yield (Bennett et al. 2012b; Bhusal et al. 2017; Esten Mason et al. 2013; Liu et 116 al. 2019; Paliwal et al. 2012; Pinto et al. 2016; Pinto et al. 2010; Tadesse et al. 2019; Vijayalakshmi et 117 al. Introduction 47 The oftentimes confounding effects of maturity can be managed using 83 controlled environments, in contrast to field experiments where material that develops at a different 84 rate may be exposed to different temperature stress conditions making comparisons challenging. 85 Despite the advantages of controlled environments, validation is required under representative field 86 conditions. To ensure a high incidence of heat stress conditions, delayed sowing has been a commonly 87 used methodology to ensure that sensitive developmental stages coincide with heat stress conditions 88 typically experienced later in the season (Bennett et al. 2012b; Esten Mason et al. 2013; Pinto et al. 89 2010; Reynolds et al. 2007; Sadras et al. 2015). Unfortunately in this system plants are exposed to 90 growing conditions that are not representative of agronomic practices employed by grain producers, 91 such as longer photoperiod and altered plant available water (Sadras et al. 2015). With phenological 92 development a primary driver of adaptation, and development rate interacting strongly with sowing 93 date, assessments of heat stress adaptation using sowing date variation are consequently 94 confounded, precluding clear conclusions being drawn from such studies. 95 Field evaluation of heat stress adaptation has also been carried out using portable heat chambers in 96 the field to induce heat stress treatments (Alexander et al. 2010; Talukder et al. 2013; Thistlethwaite 97 et al. 2020). Such methodology provides the relevance of a field trial, being managed to agronomic 98 best practice, with the added benefit of controlled environment evaluation through consistent heat 99 stress treatments and fewer confounding factors arising from variation in phenology. The physical 100 encumbrance of handling heat chambers in the field, however, largely limits such systems to smaller 101 exploratory experiments with far fewer genotypes. 102 Field evaluation of heat stress adaptation has also been carried out using portable heat chambers in 96 the field to induce heat stress treatments (Alexander et al. 2010; Talukder et al. 2013; Thistlethwaite 97 et al. 2020). Such methodology provides the relevance of a field trial, being managed to agronomic 98 best practice, with the added benefit of controlled environment evaluation through consistent heat 99 stress treatments and fewer confounding factors arising from variation in phenology. The physical 100 encumbrance of handling heat chambers in the field, however, largely limits such systems to smaller 101 exploratory experiments with far fewer genotypes. 102 Telfer et al. Introduction 47 2010), grain size (Ali et al. 2013; Bennett et al. 2012b; Bhusal et al. 2017; Esten Mason et al. 2010; 118 Guan et al. 2018; Liu et al. 2019; Mason et al. 2011; Mohammadi et al. 2008; Paliwal et al. 2012; Pinto 119 et al. 2016; Pinto et al. 2010; Shirdelmoghanloo 2014; Shirdelmoghanloo et al. 2016b; Tadesse et al. 120 2019), grain number (Bhusal et al. 2017; Esten Mason et al. 2010; Guan et al. 2018; Liu et al. 2019; 121 Mason et al. 2011; Pinto et al. 2016; Pinto et al. 2010; Telfer et al. 2021), grain fill rate (Paliwal et al. 122 2012; Pinto et al. 2016; Shirdelmoghanloo 2014), harvest index (Shirdelmoghanloo 2014), senescence 123 rate (Pinto et al. 2016; Shirdelmoghanloo 2014; Vijayalakshmi et al. 2010) and maturation rate (Bhusal 124 et al. 2017; Paliwal et al. 2012; Shirdelmoghanloo 2014). QTL for physiological traits potentially related 125 to heat stress tolerance have also been identified, including leaf chlorophyll content (Ali et al. 2013; 126 Liu et al. 2019; Pinto et al. 2016; Pinto et al. 2010; Shirdelmoghanloo 2014; Talukder et al. 2014b; 127 Vijayalakshmi et al. 2010), canopy temperature (Ali et al. 2013; Bennett et al. 2012b; Liu et al. 2019; 128 Paliwal et al. 2012; Pinto et al. 2016; Pinto et al. 2010), photosystem II efficiency (Fv/Mv) (Sharma et 129 al. 2017; Vijayalakshmi et al. 2010), NDVI (Liu et al. 2019; Pinto et al. 2016; Pinto et al. 2010) and 130 membrane damage (Talukder et al. 2014b). These studies have primarily been undertaken using 131 delayed sowing systems in the field (Bennett et al. 2012b; Bhusal et al. 2017; Esten Mason et al. 2013; 132 Liu et al. 2019; Paliwal et al. 2012; Pinto et al. 2016; Pinto et al. 2010) or in controlled environment 133 assays (Mason et al. 2011; Mohammadi et al. 2008; Shirdelmoghanloo 2014; Talukder et al. 2014b; 134 Telfer et al. 2021; Vijayalakshmi et al. 2010). 135 2019), grain number (Bhusal et al. 2017; Esten Mason et al. 2010; Guan et al. 2018; Liu et al. 2019; 121 Mason et al. 2011; Pinto et al. 2016; Pinto et al. 2010; Telfer et al. 2021), grain fill rate (Paliwal et al. 122 2012; Pinto et al. 2016; Shirdelmoghanloo 2014), harvest index (Shirdelmoghanloo 2014), senescence 123 rate (Pinto et al. 2016; Shirdelmoghanloo 2014; Vijayalakshmi et al. 2010) and maturation rate (Bhusal 124 et al. Introduction 47 2017; Paliwal et al. 2012; Shirdelmoghanloo 2014). QTL for physiological traits potentially related 125 to heat stress tolerance have also been identified, including leaf chlorophyll content (Ali et al. 2013; 126 Liu et al. 2019; Pinto et al. 2016; Pinto et al. 2010; Shirdelmoghanloo 2014; Talukder et al. 2014b; 127 Vijayalakshmi et al. 2010), canopy temperature (Ali et al. 2013; Bennett et al. 2012b; Liu et al. 2019; 128 Paliwal et al. 2012; Pinto et al. 2016; Pinto et al. 2010), photosystem II efficiency (Fv/Mv) (Sharma et 129 al. 2017; Vijayalakshmi et al. 2010), NDVI (Liu et al. 2019; Pinto et al. 2016; Pinto et al. 2010) and 130 membrane damage (Talukder et al. 2014b). These studies have primarily been undertaken using 131 delayed sowing systems in the field (Bennett et al. 2012b; Bhusal et al. 2017; Esten Mason et al. 2013; 132 Liu et al. 2019; Paliwal et al. 2012; Pinto et al. 2016; Pinto et al. 2010) or in controlled environment 133 assays (Mason et al. 2011; Mohammadi et al. 2008; Shirdelmoghanloo 2014; Talukder et al. 2014b; 134 Telfer et al. 2021; Vijayalakshmi et al. 2010). 135 As discussed by Lemerle et al. (2006), tolerance has previously been defined as the difference in trait 136 expression between an unstressed control and a stressed treatment. Usually there is a strong 137 correlation between trait expression in both stressed and unstressed conditions, making this a 138 potentially misleading definition. Lemerle et al. (2006) and Dolferus et al. (2019) further defined 139 tolerance as a positive deviation from the expected response between the stressed and unstressed 140 treatments. As discussed by Telfer et al. (2021), this deviation from the expected response under 141 stress conditions is more accurately interpreted as responsiveness to stress conditions, and should be 142 considered as an independent factor in adaptation in combination with performance value across all 143 conditions. 144 This study builds on the research carried out by Telfer et al. (2021), by applying a framework for the 145 evaluation of the genetic basis of adaptation to heat stress conditions in representative field 146 conditions, and considers adaptation as the combination of performance and responsiveness. Telfer 147 et al. (2021) identified QTL for performance and heat stress responsiveness in controlled environment 148 conditions. This study uses the same populations to explore adaptation to heat stress conditions in 149 representative field conditions. Methods and Materials 154 Germplasm and genotyping 155 Eight doubled haploid populations were used to evaluate adaptation to heat stress conditions in 156 Mediterranean-type environments of southern Australia, and to identify QTL for grain yield and 157 related traits across multiple representative environments. The populations used are described by 158 Telfer et al. (2021) and are summarised in Table 1. In brief, these were developed to encompass 159 historical germplasm pools representative of the Australian breeding germplasm pool, as well as to 160 evaluate potential novel sources of heat stress tolerance identified in a heat stress FIGS set as 161 mentioned by Telfer et al. (2021). All lines evaluated in this study from each population were 162 genotyped using a custom AxiomTM Affymetrix array containing 18,101 SNP markers as described by 163 Norman et al. (2017). Further, the linkage maps used for QTL analysis (Supplementary Table 1) were 164 created through the R statistical environment (R Core Team 2018) using a combination of the R/qtl 165 (Broman and Sen 2009; Broman and Wu 2015) and R/ASMap (Taylor and Butler 2017) package 166 (Norman et al. 2017), resulting in excess of 5000 polymorphic markers for each population (exact 167 numbers shown in Table 1). To prepare the linkage maps for analysis the functionality of the WGAIM 168 R package (Taylor and Verbyla 2011) was used to numerically encode the alleles (AA = 1, BB = -1), 169 impute missing values using the rules of Martínez and Curnow (1994) and generate unique interval 170 markers using Verbyla et al. (2007). 171 Introduction 47 Here, QTL are discussed in relation to their role in adaptation to heat 150 stress conditions as experienced in Mediterranean-type environments of southern Australia, and their 151 relevance to breeding objectives. 152 This study builds on the research carried out by Telfer et al. (2021), by applying a framework for the 145 evaluation of the genetic basis of adaptation to heat stress conditions in representative field 146 conditions, and considers adaptation as the combination of performance and responsiveness. Telfer 147 et al. (2021) identified QTL for performance and heat stress responsiveness in controlled environment 148 conditions. This study uses the same populations to explore adaptation to heat stress conditions in 149 representative field conditions. Here, QTL are discussed in relation to their role in adaptation to heat 150 stress conditions as experienced in Mediterranean-type environments of southern Australia, and their 151 relevance to breeding objectives. 152 153 Experimental design and plot management 172 The methodology described by Telfer et al. (2018) to evaluate adaptation to heat stress conditions 173 under representative field conditions across multiple environments was used as the basis for this 174 study. Genetic material was evaluated at three cereal producing environments in South Australia; 175 Angas Valley, Roseworthy, and Winulta, across two seasons in 2015 and 2016 (the details of each 176 experiment are shown in Table 2). These environments were targeted to achieve a range in heat stress 177 conditions, with Winulta having a maritime climate with relatively mild conditions during anthesis and 178 grain filling compared to the inland site of Angas Valley which typically has warmer conditions. 179 Roseworthy historically is intermediate in anthesis and grain fill conditions as is demonstrated by the 180 temperature conditions during anthesis and grain filling in Table 2. 181 grain filling compared to the inland site of Angas Valley which typically has warmer conditions. 179 Roseworthy historically is intermediate in anthesis and grain fill conditions as is demonstrated by the 180 temperature conditions during anthesis and grain filling in Table 2. 181 Populations were assigned to separate experiments except for the MG, SM, and SG populations, which 182 due to common parentage across the populations, were grouped and referred to as the GSM 183 populations. Fewer lines were evaluated in 2016 compared to 2015. For 2016, entries are a subset of 184 the 2015 entries selected to remove extreme maturity types as identified in the 2015 season. Lines 185 with other extreme adverse phenotypes such as plant height were also removed. No direct selection 186 occurred for grain yield and physical quality grain attributes which were the focus of this study. Field 187 plots within experiments were sown in a grid format, and the experiment details are shown in Table 188 2. The experimental design used the principles of partially replicated design as discussed by (Cullis et 189 al. 2006), with each doubled haploid line in each population present in each environment and each 190 doubled haploid line replicated in one of the three environments of that year. Consequently, doubled 191 haploid lines were replicated on average 1.33 times at each environment, with check varieties 192 (doubled-haploid parents and locally adapted varieties) fully replicated in each environment (Table 2). Experimental design and plot management 172 193 Within each experiment, plots were sown 1.32 m wide by 5 m long, with plots reduced to a length of 194 3.2 m before anthesis using herbicide. Each experiment was sown to achieve 200 seeds m-2. Field 195 experiments were managed using regional best practice agronomy, encompassing sowing and harvest 196 times, crop nutrition, and pest (weed, insect, and fungal) management. 197 Populations were assigned to separate experiments except for the MG, SM, and SG populations, which 182 due to common parentage across the populations, were grouped and referred to as the GSM 183 populations. Fewer lines were evaluated in 2016 compared to 2015. For 2016, entries are a subset of 184 the 2015 entries selected to remove extreme maturity types as identified in the 2015 season. Lines 185 with other extreme adverse phenotypes such as plant height were also removed. No direct selection 186 occurred for grain yield and physical quality grain attributes which were the focus of this study. Field 187 plots within experiments were sown in a grid format, and the experiment details are shown in Table 188 2. The experimental design used the principles of partially replicated design as discussed by (Cullis et 189 al. 2006), with each doubled haploid line in each population present in each environment and each 190 doubled haploid line replicated in one of the three environments of that year. Consequently, doubled 191 haploid lines were replicated on average 1.33 times at each environment, with check varieties 192 (doubled-haploid parents and locally adapted varieties) fully replicated in each environment (Table 2). 193 Within each experiment, plots were sown 1.32 m wide by 5 m long, with plots reduced to a length of 194 3.2 m before anthesis using herbicide. Each experiment was sown to achieve 200 seeds m-2. Field 195 experiments were managed using regional best practice agronomy, encompassing sowing and harvest 196 times, crop nutrition, and pest (weed, insect, and fungal) management. 197 At Roseworthy, in each year of the study, each plot was assessed to determine spike emergence date 199 (50% of spikes fully emerged from the flag leaf sheath) of each plot. Experimental design and plot management 172 Along with temperature data 200 collected at each environment (explained in further detail later), a degree-day model (Sadras and 201 Monzon 2006) was used to estimate the anthesis date of each line in each environment, using daily 202 At Roseworthy, in each year of the study, each plot was assessed to determine spike emergence date 199 (50% of spikes fully emerged from the flag leaf sheath) of each plot. Along with temperature data 200 collected at each environment (explained in further detail later), a degree-day model (Sadras and 201 Monzon 2006) was used to estimate the anthesis date of each line in each environment, using daily 202 mean temperature >0ᵒC. Relative maturity observations were also made at each location in each year 203 of the study to confirm the accuracy of the modelled anthesis date as described by Telfer et al. (2018). 204 All experiments were harvested and weighed after reaching physiological maturity, to determine grain 205 yield (kg ha-1). The grain was further evaluated for screenings percentage assessed by determining the 206 weight of matter that passes through a 2 mm slotted sieve and test weight (TWT, kg hl-1) assessed by 207 weighing a 500 mL sample. 208 Statistical methods - Baseline model 228 For each of the populations, assume a set of 𝑟 progeny varieties were sown in 𝑡 environments and let 229 𝒚= (𝒚1 𝑇… 𝒚𝑡 𝑇)𝑇 be a vector of observed responses such as grain yield, screenings or TWT. The 230 response was then analysed using a baseline multi-environment linear mixed model (ME-LMM) of the 231 form 232 For each of the populations, assume a set of 𝑟 progeny varieties were sown in 𝑡 environments and let 229 𝒚= (𝒚1 𝑇… 𝒚𝑡 𝑇)𝑇 be a vector of observed responses such as grain yield, screenings or TWT. The 230 response was then analysed using a baseline multi-environment linear mixed model (ME-LMM) of the 231 form 232 𝒚= 𝑿𝝉+ 𝒁𝒖+ 𝒁𝑔𝒖𝑔+ 𝒆 (1) 233 (1) where 𝑿𝝉 was a fixed component containing a vector of fixed effects for estimating experiment means 234 as well as an overall mean for the population, parents, and controls specific to each environment. 𝒁𝒖 235 was a random component consisting of effects 𝒖 that were conformably partitioned to account for 236 environment specific variation such as aspects of the experimental designs including blocks or 237 replicates, and also variation due to potential extraneous non-linear trends potentially existing across 238 the Row or Column of the experiment. To further improve the flexibility of the ME-LMM to model 239 local spatial variability, the residuals were partitioned to 𝒆= (𝒆1 𝑇… 𝒆𝑡 𝑇)𝑇 where the residuals within 240 the 𝑗th environment were assumed to be distributed 𝒆𝑗 ~ 𝑁(𝟎, 𝜎𝑗 2𝑹𝑗) with 𝑹𝑗 parameterized as a 241 separable auto-regressive structure of order one in the Row and Column direction. The important 242 focus of this ME-LMM is the random genotype by environment interaction term 𝒁𝑔𝒖𝑔, with effects 243 𝒖𝑔 of length 𝑟× 𝑡 and indicator matrix 𝒁𝑔 that maps the genotypes to the plots within each 244 experiment. These effects were assumed to be distributed 𝒖𝑔 ~ 𝑁(𝟎, 𝚫⊗𝑰𝑟) where 𝚫 is a 𝑡× 𝑡 245 covariance matrix consisting of environment-specific variances on its diagonal to capture genetic 246 variation of the progeny lines within each environment and covariances on its off diagonals to model 247 the genetic relatedness of the progeny between each pair of environments. Climatic co-variates to describe crop stress conditions 209 Temperature data was collected in each environment for the duration of the growing season using a 210 single factory-calibrated temperature logger (TinyTagTM Talk2), logging on half-hourly intervals. The 211 temperature logger was situated immediately adjacent to the block of experiments all situated 212 adjacent to each other in each location, at 1 m in height to replicate approximate crop height at 213 anthesis and grain fill. 214 Temperature data was collected in each environment for the duration of the growing season using a 210 single factory-calibrated temperature logger (TinyTagTM Talk2), logging on half-hourly intervals. The 211 temperature logger was situated immediately adjacent to the block of experiments all situated 212 adjacent to each other in each location, at 1 m in height to replicate approximate crop height at 213 anthesis and grain fill. 214 Temperature observations were used to calculate climatic co-variates to describe the temperature 215 conditions experienced by each line in each environment during the key developmental periods, 216 anthesis (300 °Cd before anthesis to 100 °Cd post-anthesis), and grain filling (100 °Cd to 600 °Cd post 217 anthesis). Climatic co-variates calculated for each line in each environment included average 218 maximum temperature (°C), number of days >30°C, and number of days >35°C, during both anthesis 219 and grain filling (Dreccer et al. 2008; Telfer et al. 2018). Growing season rainfall (mm for May to 220 October rainfall), was also recorded in each environment. In the 2015 season this was taken from the 221 nearest Australian Bureau of Meteorology (BOM) weather station; Angas Valley sourced from Cambrai 222 BOM station (No. 024513), Roseworthy sourced from Roseworthy BOM station (No. 023021) and 223 Winulta sourced from Ardrossan BOM station (No. 022021)). In the 2016 season, this was measured 224 on-site using a factory-calibrated DavisR Vantage Vue Weather Station situated adjacent to the 225 experiments logging on 15-minute intervals from before planting until harvest. The mean climatic co- 226 variate observed across all lines in each experiment is shown in Table 2. 227 Statistical methods - Baseline model 228 Similar 269 to the previous sections, where necessary, the marker based additive and residual genotype by 270 environment effects were both parsimoniously approximated by a Factor Analytic model. 271 In the second run, the focus was on detecting QTL displaying significant responsiveness across the 272 numerical range of the climatic covariate. If 𝒄 is the climatic covariate then the climate covariate QTL 273 model used an extended ME LMM with 𝒄𝒎𝑏 replacing the first term on the right hand side of (2) 274 For a given population, let 𝑴 be an 𝑟× 𝑝 matrix of unique interval markers. For each trait from this 255 population, a QTL analysis was conducted using a whole genome scanning approach with two separate 256 runs. In the first run the focus was to obtain overall performance QTL across environments. Let 𝒎𝑖𝑗 257 be the 𝑗th interval marker in the 𝑖th chromosome then the performance QTL model was an extension 258 of the ME-LMM defined in (1) where the total genetic effects were partitioned as 259 For a given population, let 𝑴 be an 𝑟× 𝑝 matrix of unique interval markers. For each trait from this 255 population, a QTL analysis was conducted using a whole genome scanning approach with two separate 256 runs. In the first run the focus was to obtain overall performance QTL across environments. Let 𝒎𝑖𝑗 257 be the 𝑗th interval marker in the 𝑖th chromosome then the performance QTL model was an extension 258 of the ME-LMM defined in (1) where the total genetic effects were partitioned as 259 𝒖𝑔= 𝒎𝑖𝑗𝑎𝑖𝑗+ 𝒖𝑎+ 𝒖𝑝 (2) 260 𝒖𝑔= 𝒎𝑖𝑗𝑎𝑖𝑗+ 𝒖𝑎+ 𝒖𝑝 (2) 260 𝒖𝑔= 𝒎𝑖𝑗𝑎𝑖𝑗+ 𝒖𝑎+ 𝒖𝑝 (2) (2) In this extended genetic model 𝑎𝑖𝑗 represents the main or overall performance effect of the interval 261 marker 𝒎𝑖𝑗 across the 𝑡 environments. We used the strategy of Rincent et al. (2014) for improving 262 the power of detecting significant marker or marker by climatic interaction effects by including 263 additional genomic and residual genetic terms in (2). Specifically, we include 𝒖𝑎, a vector of random 264 marker based additive genotype by environment effects that are assumed to be distributed 𝒖𝑎 ~ 𝑁(𝟎, 265 𝚫𝑎⊗𝑮−𝑖) where 𝑮−𝑖= 𝑴−𝑖𝑴−𝑖 𝑇 is an 𝑟× 𝑟 genomic relationship matrix with the interval markers 266 from the 𝑖th chromosome removed and 𝚫𝑎 is an 𝑡× 𝑡 additive genetic covariance matrix. Statistical methods - Baseline model 228 When 𝑡 was moderately 248 large the estimation of 𝚫 became problematic and we sought the use of a parsimonious approximation 249 by defining the genotype by environment effects to have a Factor Analytic (FAk) model (Smith et al. 250 2001; Smith et al. 2005) with variance 𝚲𝚲𝑇+ 𝚿≈𝚫 where 𝚲 is 𝑡× 𝑘 matrix of environment loadings 251 and 𝚿 is a diagonal matrix containing environment specific variances. The number of factors 𝑘 used 252 in the FA model depended on the number of environments the population progeny was sown. 253 and 𝚿 is a diagonal matrix containing environment specific variances. The number of factors 𝑘 used 252 in the FA model depended on the number of environments the population progeny was sown. 253 in the FA model depended on the number of environments the population progeny was sown. 253 Statistical methods - QTL analysis 254 For a given population, let 𝑴 be an 𝑟× 𝑝 matrix of unique interval markers. For each trait from this 255 population, a QTL analysis was conducted using a whole genome scanning approach with two separate 256 runs. In the first run the focus was to obtain overall performance QTL across environments. Let 𝒎𝑖𝑗 257 be the 𝑗th interval marker in the 𝑖th chromosome then the performance QTL model was an extension 258 of the ME-LMM defined in (1) where the total genetic effects were partitioned as 259 𝒖𝑔= 𝒎𝑖𝑗𝑎𝑖𝑗+ 𝒖𝑎+ 𝒖𝑝 (2) 260 In this extended genetic model 𝑎𝑖𝑗 represents the main or overall performance effect of the interval 261 marker 𝒎𝑖𝑗 across the 𝑡 environments. We used the strategy of Rincent et al. (2014) for improving 262 the power of detecting significant marker or marker by climatic interaction effects by including 263 additional genomic and residual genetic terms in (2). Specifically, we include 𝒖𝑎, a vector of random 264 marker based additive genotype by environment effects that are assumed to be distributed 𝒖𝑎 ~ 𝑁(𝟎, 265 𝚫𝑎⊗𝑮−𝑖) where 𝑮−𝑖= 𝑴−𝑖𝑴−𝑖 𝑇 is an 𝑟× 𝑟 genomic relationship matrix with the interval markers 266 from the 𝑖th chromosome removed and 𝚫𝑎 is an 𝑡× 𝑡 additive genetic covariance matrix. We also 267 include 𝒖𝑝, a vector of random polygenic residual genotype by environment effects that are assumed 268 to be distributed 𝒖𝑝 ~ 𝑁(𝟎, 𝚫𝑝⊗𝑰𝑟) where 𝚫𝑝 is a 𝑡× 𝑡 residual genetic covariance matrix. Statistical methods - Baseline model 228 For each of the QTL, a linked marker was used to identify the RefSeq physical 292 position (Alaux et al. 2018), with the base pair position at the start of the candidate marker sequence. 293 Analysis to identify QTL for anthesis data was only conducted using the described methodology to 294 identify performance QTL, as this data was only collected in the experiments collected at Roseworthy 295 in each year of the study. 296 Statistical methods - Baseline model 228 We also 267 include 𝒖𝑝, a vector of random polygenic residual genotype by environment effects that are assumed 268 to be distributed 𝒖𝑝 ~ 𝑁(𝟎, 𝚫𝑝⊗𝑰𝑟) where 𝚫𝑝 is a 𝑡× 𝑡 residual genetic covariance matrix. Similar 269 to the previous sections, where necessary, the marker based additive and residual genotype by 270 environment effects were both parsimoniously approximated by a Factor Analytic model. 271 In the second run, the focus was on detecting QTL displaying significant responsiveness across the 272 numerical range of the climatic covariate. If 𝒄 is the climatic covariate then the climate covariate QTL 273 model used an extended ME-LMM with 𝒄𝒎𝑖𝑗𝑏𝑖𝑗 replacing the first term on the right-hand side of (2) 274 In this new term 𝑏𝑖𝑗 represents the interval marker by climatic covariate interaction effect or 275 responsiveness effect of the interval marker across environments. 276 The two QTL models were then used to scan the complete 𝑝 interval markers across the 21 277 chromosomes of the wheat genome and Wald statistics were calculated for each performance and 278 responsiveness effect. Initial effect significance was determined using the thresholding technique of 279 Li and Ji (2005) outlined for QTL ME-LMMs in Bonneau et al. (2013). Interval marker effects above the 280 significance threshold were omitted from further analysis if they were within a window of 30 cM 281 adjacent to an interval marker effect with greater significance. The remaining set of 𝑛𝑠 significant 282 interval markers were then considered linked to putative QTL and additively included in a final QTL 283 model where, for the significant performance QTL for example, was 284 𝒖𝑔= ∑ 𝒎𝑠𝑎𝑠+ 𝒏𝒔 𝒔=𝟏 𝒖𝑎∗+ 𝒖𝑝 (3) 𝒖𝑔= ∑ 𝒎𝑠𝑎𝑠+ 𝒏𝒔 𝒔=𝟏 𝒖𝑎∗+ 𝒖𝑝 (3) 285 (3) where 𝒖𝑎∗ ~ 𝑁(𝟎, 𝚫𝑎⊗𝑮−𝑠) and 𝑮−𝑠= 𝑴−𝑠𝑴−𝑠 𝑇 is the genomic relationship matrix with interval 286 markers excluded if they were within a 30 cM window of the interval markers in the final model. The 287 significant performance and responsiveness QTL were then summarized with their chromosome, 288 genetic distance, LOD score, and additive effect size. To aid in the interpretation of the size of the 289 additive effect for responsiveness, a normalisation was conducted that multiplied each additive effect 290 by the numerical range of the covariate over the environments. The normalised additive effect size 291 was also provided. QTL identified for performance 310 In total 199 QTL were found in this study, of which 60 were found to be important for performance, 311 with a stable effect across all environments sampled. QTL were spread across all chromosomes except 312 for chromosome 3D, as shown in Figure 1 (additional details for each QTL identified are shown in 313 Supplementary Table 2). Of the 60 QTL for trait performance, 21 were identified for anthesis date, 14 314 for grain yield, 18 for TWT, and seven for screenings. Performance QTL were identified in all 315 populations, although not for each trait in each population. Grain yield QTL were not identified in the 316 RG population, TWT QTL were not identified in the SG population, while screenings QTL were not 317 identified in the MG and SM populations. Anthesis date QTL were identified in all populations. 318 QTL identified for responsiveness to climatic co-variates 319 A further 139 QTL (Figure 2 and Supplementary Table 2) were identified that showed a significant 320 interaction with the climatic co-variates measured for each genotype in each environment. These QTL 321 are termed ‘responsive’, indicating that there is variable trait expression in response to the climatic 322 co-variates from each environment. Grain yield accounted for 44 of the QTL for responsiveness 323 identified across all populations and climatic co-variates. For screenings, 43 responsiveness QTL were 324 identified in the MG, SM, RG, and L2G populations, while none were identified in the SG population. 325 A further 52 responsiveness QTL were found for TWT across all populations and all climatic co-variates, 326 except for the MG population where only one responsiveness QTL was found for grain fill days >35 °C. 327 Statistical methods - Computations 297 Baseline ME-LMMs were fitted using the flexible linear mixed modelling software package ASReml-R 298 (Butler et al. 2018) available in the R statistical computing environment and downloadable through 299 VSNi website from https://www.vsni.co.uk/software/asreml. The ASReml-R package contains a suite 300 of functionality for fitting and diagnosing complex linear mixed models and uses the REML algorithm 301 of Patterson and Thompson (1971) to estimate model parameters. Diagnostic assessment of models 302 was conducted using ASReml-R functions as well as functions from the post-processing package 303 ASExtras available for download from https://mmade.org/. QTL analyses were conducted using the 304 GWASReml R software package available from https://github.com/DrJ001/GWASReml. The package 305 provides functionality for conducting one-stage QTL and GWAS analyses through extensions of the 306 baseline ASReml-R model. 307 Baseline ME-LMMs were fitted using the flexible linear mixed modelling software package ASReml-R 298 (Butler et al. 2018) available in the R statistical computing environment and downloadable through 299 VSNi website from https://www.vsni.co.uk/software/asreml. The ASReml-R package contains a suite 300 of functionality for fitting and diagnosing complex linear mixed models and uses the REML algorithm 301 of Patterson and Thompson (1971) to estimate model parameters. Diagnostic assessment of models 302 was conducted using ASReml-R functions as well as functions from the post-processing package 303 ASExtras available for download from https://mmade.org/. QTL analyses were conducted using the 304 GWASReml R software package available from https://github.com/DrJ001/GWASReml. The package 305 provides functionality for conducting one-stage QTL and GWAS analyses through extensions of the 306 baseline ASReml-R model. 307 308 Clusters of QTL 328 This included a genomic region on chromosome 2B where three anthesis 342 date QTL were identified between 34.2 cM and 38.8 cM in the RG, MG, and SG populations, and these 343 clustered with 24 other QTL between 29.8 cM and 43.5 cM. Although not specifically mapped in this 344 study, this region likely aligns with the Ppd-B1 photoperiod gene found on chromosome 2B (Scarth 345 and Law 1983). QTL associated with this cluster were encompassed by each of the populations 346 evaluated, each trait assessed, and each climatic co-variate measured. Similar but smaller clusters 347 were found on chromosome 2D (likely associated with Ppd-D1 (Law et al. 1978), chromosome 5B 348 (likely associated with Vrn-B1 (Iwaki et al. 2002)), chromosome 5D (likely associated with Vrn-D1 (Law 349 et al. 1976)), as well as chromosomes 7A, 7B and 7D likely associated with the FT gene (Bonnin et al. 350 2008). On chromosome 5A three anthesis date QTL were found to be clustered with one TWT QTL 351 (QTwt.agt-RG.5A), which likely aligns to Vrn-A1 (Law et al. 1976). These regions will not be discussed 352 in depth as there are well understood associations with crop performance and phenology genes, as 353 well as a strong confounding influence on adaptation to heat stress effect because anthesis date will 354 influence the level of stress experienced. 355 Of the 21 QTL identified for anthesis date, all except one (QFlt.agt-SM.7B on chromosome 7B) 341 clustered with other QTL. This included a genomic region on chromosome 2B where three anthesis 342 date QTL were identified between 34.2 cM and 38.8 cM in the RG, MG, and SG populations, and these 343 clustered with 24 other QTL between 29.8 cM and 43.5 cM. Although not specifically mapped in this 344 study, this region likely aligns with the Ppd-B1 photoperiod gene found on chromosome 2B (Scarth 345 and Law 1983). QTL associated with this cluster were encompassed by each of the populations 346 evaluated, each trait assessed, and each climatic co-variate measured. Similar but smaller clusters 347 were found on chromosome 2D (likely associated with Ppd-D1 (Law et al. 1978), chromosome 5B 348 (likely associated with Vrn-B1 (Iwaki et al. 2002)), chromosome 5D (likely associated with Vrn-D1 (Law 349 et al. 1976)), as well as chromosomes 7A, 7B and 7D likely associated with the FT gene (Bonnin et al. 350 2008). Clusters of QTL 328 On chromosome 5A three anthesis date QTL were found to be clustered with one TWT QTL 351 (QTwt.agt-RG.5A), which likely aligns to Vrn-A1 (Law et al. 1976). These regions will not be discussed 352 in depth as there are well understood associations with crop performance and phenology genes, as 353 well as a strong confounding influence on adaptation to heat stress effect because anthesis date will 354 influence the level of stress experienced. 355 Clusters of QTL 328 Of the 199 QTL identified, 18 occurred independently of any other QTL identified. The remaining 181 329 QTL occurred in clusters with one or more other QTL (within 10 cM of the interval associated with 330 other QTL), with up to 27 QTL clustering together. QTL clusters included those characterised as 331 performance QTL as well as responsiveness QTL. 332 Of the 199 QTL identified, 18 occurred independently of any other QTL identified. The remaining 181 329 QTL occurred in clusters with one or more other QTL (within 10 cM of the interval associated with 330 other QTL), with up to 27 QTL clustering together. QTL clusters included those characterised as 331 performance QTL as well as responsiveness QTL. 332 There were 99 QTL that occurred in combination with each other, but which were not associated with 333 QTL for anthesis date. These clusters in many cases combined QTL for performance and 334 responsiveness for a range of climatic co-variates, and a range of traits. However, there were other 335 regions identified where performance QTL only clustered with performance QTL and responsiveness 336 QTL only clustered with responsiveness QTL. The QTL found to not be associated with anthesis date 337 will be discussed in more depth as they offer opportunities to understand adaptation to heat stress 338 conditions independently from anthesis date, which although important, are often considered 339 differently by plant breeders as anthesis date is also an important selection target. 340 There were 99 QTL that occurred in combination with each other, but which were not associated with 333 QTL for anthesis date. These clusters in many cases combined QTL for performance and 334 responsiveness for a range of climatic co-variates, and a range of traits. However, there were other 335 regions identified where performance QTL only clustered with performance QTL and responsiveness 336 QTL only clustered with responsiveness QTL. The QTL found to not be associated with anthesis date 337 will be discussed in more depth as they offer opportunities to understand adaptation to heat stress 338 conditions independently from anthesis date, which although important, are often considered 339 differently by plant breeders as anthesis date is also an important selection target. 340 Of the 21 QTL identified for anthesis date, all except one (QFlt.agt-SM.7B on chromosome 7B) 341 clustered with other QTL. Interpreting QTL with performance and responsiveness effects 356 Of the QTL identified, there are examples of performance QTL and responsiveness QTL occurring 357 independently from other QTL, as well as performance QTL and responsiveness QTL occurring at the 358 same position. Adding an additional layer of complexity, these regions also encompassed QTL for 359 various phenotypic traits and response to a range of climatic co-variates. This is demonstrated in 360 Figure 2(A-E), where responsiveness QTL collocating with a performance QTL for TWT on chromosome 361 4A (QTwt.agt-L2G.4A), shows the effect of each responsiveness QTL plotted against the range of each 362 climatic co-variate experienced. Figure 2(A-D) shows the positive effect of the responsiveness QTL on 363 TWT to increasing anthesis average maximum temperature, grain fill average maximum temperature, 364 number of days >30°C, and number of days >35°C, as well as TWT decreasing in response to increasing 365 growing season rainfall (Figure 2E). However, given that these QTL co-located with a performance QTL 366 (QTwt.agt-L2G.4A), they need to be considered simultaneously. Figure 2F shows the result of selecting 367 either the high or low performance allele at this locus and the resulting responsiveness QTL allele that 368 would be selected. In this situation the increased performance selected by targeting the Gladius allele 369 of the QTwt.agt-L2G.4A locus would be associated with a negative response to increasing anthesis 370 average maximum temperature, grain fill average maximum temperature, number of days >30°C, and 371 number of days >35°C, and a positive response in TWT to increasing growing season rainfall. In other 372 words, selecting for the higher performance allele would also lead to greater sensitivity to hot and dry 373 conditions. 374 Of the QTL identified, there are examples of performance QTL and responsiveness QTL occurring 357 independently from other QTL, as well as performance QTL and responsiveness QTL occurring at the 358 same position. Adding an additional layer of complexity, these regions also encompassed QTL for 359 various phenotypic traits and response to a range of climatic co-variates. This is demonstrated in 360 Figure 2(A-E), where responsiveness QTL collocating with a performance QTL for TWT on chromosome 361 4A (QTwt.agt-L2G.4A), shows the effect of each responsiveness QTL plotted against the range of each 362 climatic co-variate experienced. Interpreting QTL with performance and responsiveness effects 356 Figure 2(A-D) shows the positive effect of the responsiveness QTL on 363 TWT to increasing anthesis average maximum temperature, grain fill average maximum temperature, 364 number of days >30°C, and number of days >35°C, as well as TWT decreasing in response to increasing 365 growing season rainfall (Figure 2E). However, given that these QTL co-located with a performance QTL 366 (QTwt.agt-L2G.4A), they need to be considered simultaneously. Figure 2F shows the result of selecting 367 either the high or low performance allele at this locus and the resulting responsiveness QTL allele that 368 would be selected. In this situation the increased performance selected by targeting the Gladius allele 369 of the QTwt.agt-L2G.4A locus would be associated with a negative response to increasing anthesis 370 average maximum temperature, grain fill average maximum temperature, number of days >30°C, and 371 number of days >35°C, and a positive response in TWT to increasing growing season rainfall. In other 372 words, selecting for the higher performance allele would also lead to greater sensitivity to hot and dry 373 conditions. 374 375 Discussion 376 Assessing the contribution of performance and responsiveness QTL to heat stress adaptation 377 In the first approach to genetic analysis of heat stress adaptation used in this study, performance QTL 378 were found that had consistent effects across a range of environments in the cereal growing areas of 379 South Australia. In the second approach, regions were identified that interacted with climatic co- 380 variates in their effects on the yield related traits researched herein. This dual approach to genetic 381 analysis allows the framework proposed by Telfer et al. (2021) to be applied to the results of a multi- 382 environment field QTL study. In this framework the authors propose that attempting to characterise 383 a QTL as tolerant fails to completely describe the nature of the genetic control being exerted by the 384 locus. Telfer et al. (2021) suggested that adaptation to heat stress conditions (and likely other abiotic 385 stresses) is better described as the combination of overall performance and responsiveness to stress 386 conditions. Armed with this understanding, the optimal genetic combination can be selected by 387 breeders for the specific heat stress environment being targeted. Alternatively, selection for QTL with 388 high-performance value and no responsiveness may offer stable adaptation across a broad range of 389 environments. 390 ssessing the contribution of performance and responsiveness QTL to heat stress adaptation In this study several of the performance and responsiveness QTL identified were found to occur co- 391 located or closely located (<10 cM), to anthesis date QTL identified in this study (Figure 1 and 392 Supplementary Table 2). Anthesis date, is a complex trait determined by several mechanisms; 393 photoperiod, vernalisation, and earliness per se, each controlled by several genes, and is a key 394 component of variety adaptation and crop performance (Eagles et al. 2014; Kuchel et al. 2006). 395 Favourable alleles at these loci are selected by breeders to ensure anthesis occurs at favourable times 396 to minimise exposure to stresses such as frost, drought, and heat while maximising grain yield (Flohr 397 et al. 2017). While this is an important topic, here the discussion is restricted to loci (totalling 116 QTL 398 and described in Supplementary Table 2) that can be selected to improve adaptation to heat stress 399 without affecting time to anthesis. 400 In this study several of the performance and responsiveness QTL identified were found to occur co- 391 located or closely located (<10 cM), to anthesis date QTL identified in this study (Figure 1 and 392 Supplementary Table 2). Anthesis date, is a complex trait determined by several mechanisms; 393 photoperiod, vernalisation, and earliness per se, each controlled by several genes, and is a key 394 component of variety adaptation and crop performance (Eagles et al. 2014; Kuchel et al. 2006). 395 Favourable alleles at these loci are selected by breeders to ensure anthesis occurs at favourable times 396 to minimise exposure to stresses such as frost, drought, and heat while maximising grain yield (Flohr 397 et al. 2017). While this is an important topic, here the discussion is restricted to loci (totalling 116 QTL 398 and described in Supplementary Table 2) that can be selected to improve adaptation to heat stress 399 without affecting time to anthesis. 400 It was frequently observed that QTL alleles with a negative performance effect responded positively 401 to increasing temperature and negatively to increasing rainfall (the reverse being true, logically, for 402 the alternate allele). This response type is not rare (Telfer et al. 2018) and represents the classic 403 genotype-by-environment response of heterogeneity of variance (Finlay and Wilkinson 1963; 404 Malosetti et al. 2013). ssessing the contribution of performance and responsiveness QTL to heat stress adaptation This was demonstrated by QTwt.agt-L2G.4A a performance QTL for TWT on 405 chromosome 4A (Figure 2), that collocated with responsiveness QTL for temperature co-variates and 406 growing season rainfall (QTwt.agt-RG.4A-5). When considering the parental source of the allele 407 conferring a positive trait effect for a performance QTL effect, it was the opposite allele that conferred 408 positive responsiveness to increasing temperature conditions (and negative response to increasing 409 rainfall). The negative association at the 4A locus between responsiveness to increasing temperature 410 and increasing rainfall is not surprising as high temperature conditions and low rainfall are often 411 associated with stressed environments (Lobell et al. 2015). In this case, breeders can select for either 412 high overall performance, or adaptation to heat and drought stress. When considering the framework 413 proposed in Figure 2H, this would be classified as a positive response to stressed conditions and low 414 performance value. The importance of this region for TWT has been highlighted previously by Huang 415 et al. (2006), but also for grain yield (Tura et al. 2020; Yu et al. 2018; Zhang et al. 2018) and grain 416 weight traits (Cui et al. 2014; McCartney et al. 2005; Tura et al. 2020; Zhang et al. 2018). Furthermore, 417 studies targeting heat stress adaption for grain yield have also been reported (Pinto et al. 2010; 418 Tadesse et al. 2019). The region is likely to be TaCWI (Jiang et al. 2015), which is located in the 419 centromeric region of chromosome 4A. The commercial varieties and breeding line parents used for 420 the mapping populations have been screened for this gene and Gladius carries the large grain allele 421 while RAC1548 carries the small grain allele. The results of this study align the large grain allele of 422 Gladius with improved TWT but a negative response to stressed conditions. Interestingly, a screenings 423 performance QTL identified in the L2G population attributed the grain size advantage to AUS17840 424 rather than Gladius, something that will require further investigation. 425 It was frequently observed that QTL alleles with a negative performance effect responded positively 401 to increasing temperature and negatively to increasing rainfall (the reverse being true, logically, for 402 the alternate allele). This response type is not rare (Telfer et al. 2018) and represents the classic 403 genotype-by-environment response of heterogeneity of variance (Finlay and Wilkinson 1963; 404 Malosetti et al. 2013). ssessing the contribution of performance and responsiveness QTL to heat stress adaptation Here a performance QTL for grain yield (QYld.agt- 428 SM.5B-1) was identified collocated with a range of responsiveness QTL for temperature co-variates 429 during both anthesis and grain fill, with the Mace parent providing the preferred allele for both 430 performance and responsiveness to increasing temperature. Similarly, Mace also provided the 431 favourable drought (low rainfall) responsive allele at collocated QTL (QScn.agt-SM.5B-2 and QScn.agt- 432 MG.5B-3) associated with screenings. When considering the framework posed in Figure 2H, this would 433 be classified as positive response with high-performance value. This is a rare combination, but one 434 eagerly sought after by breeders as an opportunity to improve both overall performance and relative 435 performance under heat and drought stress. This region was also described by Bennett et al. (2012b) 436 for grain yield under delayed sowing conditions. Additionally, this region has previously been 437 described for grain yield (Yu et al. 2018) and grain weight traits (Cui et al. 2014; Huang et al. 2003; 438 Ramya et al. 2010; Tsilo et al. 2010; Yu et al. 2018; Zhang et al. 2018). 439 An example identified in this study where a performance QTL was positively associated with 426 responsiveness QTL for rainfall and temperature conditions under stressed conditions was found on 427 chromosome 5B in the SM and MG populations. Here a performance QTL for grain yield (QYld.agt- 428 SM.5B-1) was identified collocated with a range of responsiveness QTL for temperature co-variates 429 during both anthesis and grain fill, with the Mace parent providing the preferred allele for both 430 performance and responsiveness to increasing temperature. Similarly, Mace also provided the 431 favourable drought (low rainfall) responsive allele at collocated QTL (QScn.agt-SM.5B-2 and QScn.agt- 432 MG.5B-3) associated with screenings. When considering the framework posed in Figure 2H, this would 433 be classified as positive response with high-performance value. This is a rare combination, but one 434 eagerly sought after by breeders as an opportunity to improve both overall performance and relative 435 performance under heat and drought stress. This region was also described by Bennett et al. (2012b) 436 for grain yield under delayed sowing conditions. Additionally, this region has previously been 437 described for grain yield (Yu et al. 2018) and grain weight traits (Cui et al. 2014; Huang et al. 2003; 438 Ramya et al. 2010; Tsilo et al. 2010; Yu et al. 2018; Zhang et al. 2018). ssessing the contribution of performance and responsiveness QTL to heat stress adaptation This was demonstrated by QTwt.agt-L2G.4A a performance QTL for TWT on 405 chromosome 4A (Figure 2), that collocated with responsiveness QTL for temperature co-variates and 406 growing season rainfall (QTwt.agt-RG.4A-5). When considering the parental source of the allele 407 conferring a positive trait effect for a performance QTL effect, it was the opposite allele that conferred 408 positive responsiveness to increasing temperature conditions (and negative response to increasing 409 rainfall). The negative association at the 4A locus between responsiveness to increasing temperature 410 and increasing rainfall is not surprising as high temperature conditions and low rainfall are often 411 associated with stressed environments (Lobell et al. 2015). In this case, breeders can select for either 412 high overall performance, or adaptation to heat and drought stress. When considering the framework 413 proposed in Figure 2H, this would be classified as a positive response to stressed conditions and low 414 performance value. The importance of this region for TWT has been highlighted previously by Huang 415 et al. (2006), but also for grain yield (Tura et al. 2020; Yu et al. 2018; Zhang et al. 2018) and grain 416 weight traits (Cui et al. 2014; McCartney et al. 2005; Tura et al. 2020; Zhang et al. 2018). Furthermore, 417 studies targeting heat stress adaption for grain yield have also been reported (Pinto et al. 2010; 418 Tadesse et al. 2019). The region is likely to be TaCWI (Jiang et al. 2015), which is located in the 419 centromeric region of chromosome 4A. The commercial varieties and breeding line parents used for 420 the mapping populations have been screened for this gene and Gladius carries the large grain allele 421 while RAC1548 carries the small grain allele. The results of this study align the large grain allele of 422 Gladius with improved TWT but a negative response to stressed conditions. Interestingly, a screenings 423 performance QTL identified in the L2G population attributed the grain size advantage to AUS17840 424 rather than Gladius, something that will require further investigation. 425 An example identified in this study where a performance QTL was positively associated with 426 responsiveness QTL for rainfall and temperature conditions under stressed conditions was found on 427 chromosome 5B in the SM and MG populations. Regions of performance and their role in adaptation 452 Also, on 7A, QYld.agt-SG.7A occurred 470 adjacent to a TWT performance QTL (QTwt.agt-RG.7A) identified in the RG population, with the 471 Gladius parent being the source of the higher performance allele in both populations and for both 472 traits. 473 Ten performance QTL were identified for grain yield, screenings, and TWT independent of any 462 collocated responsiveness QTL (Figure 1 and Supplementary Table 2). Grain yield QTL were identified 463 on chromosome 2A (QYld.agt-SG.2A) and 7A (QYld.agt-L2G.7A and QYld.agt-SG.7A), with the Gladius 464 parent providing the higher performing allele in each instance. QYld.agt-SG.2A on chromosome 2A 465 occurs in region previously described for grain yield and thousand grain weight QTL by Tura et al. 466 (2020) and Tsilo et al. (2010). On 7A, QYld.agt-L2G.7A occurs in a region where QTL for grain yield, 467 kernel weight and TWT QTL have been previously reported (Cabral et al. 2018; Huang et al. 2003; 468 Maphosa et al. 2014; Tura et al. 2020; Yu et al. 2018) including in heat stress studies (Bennett et al. 469 2012b; Guan et al. 2018; Mason et al. 2013; Pinto et al. 2016). Also, on 7A, QYld.agt-SG.7A occurred 470 adjacent to a TWT performance QTL (QTwt.agt-RG.7A) identified in the RG population, with the 471 Gladius parent being the source of the higher performance allele in both populations and for both 472 traits. 473 ssessing the contribution of performance and responsiveness QTL to heat stress adaptation 439 Alternatively, QTL for responsiveness to increasingly hot conditions not associated with rainfall would 440 allow for the selection of genotypes that can cope with increasing terminal temperature stress 441 conditions and retain performance under a range of rainfall environments. An example of this 442 occurred on chromosome 7A, identified in the SM population, where a performance QTL for grain 443 yield (QYld.agt-SM.7A) occurred in a similar location to heat responsive QTL for TWT (QTwt.agt- 444 SM.7A-1, QTwt.agt-SM.7A-2, and QTwt.agt-SM.7A-3). Here the Mace parent provided the allele 445 conferring both positive performance and positive responsive to stress. This region appears to align 446 with a grain yield QTL identified in delayed sowing conditions by Bennett et al. (2012b) and Pinto et 447 al. (2016), grain number and grain weight per spike by (Guan et al. 2018), as well as a leaf chlorophyll 448 content QTL by (Talukder et al. 2014b). Additionally, this region has been described in non-heat stress 449 literature for its role in grain yield (Narjesi et al. 2015; Tura et al. 2020; Yu et al. 2018) and thousand 450 i i h (C i l 2014 G l 2003 H l 2004) 451 Regions of performance and their role in adaptation 452 Regions conferring a performance advantage in the absence of responsiveness to climatic conditions 453 may provide breeders opportunities to select for broad adaptation to a range of stressed and 454 unstressed environments. When considered within the framework described in Figure 2H, this would 455 be classified as having high-performance value and being non-responsive. This would be an advantage 456 as many environments do not experience stressed conditions every season, or to the same level each 457 season. A performance QTL that is independent of responsiveness would allow a lower risk alternative 458 that would allow elite crop performance under both stressed and unstressed conditions as discussed 459 by Telfer et al. (2021) and fits with the broad adaptation model proposed by Finlay and Wilkinson 460 (1963). 461 Regions conferring a performance advantage in the absence of responsiveness to climatic conditions 453 may provide breeders opportunities to select for broad adaptation to a range of stressed and 454 unstressed environments. When considered within the framework described in Figure 2H, this would 455 be classified as having high-performance value and being non-responsive. This would be an advantage 456 as many environments do not experience stressed conditions every season, or to the same level each 457 season. A performance QTL that is independent of responsiveness would allow a lower risk alternative 458 that would allow elite crop performance under both stressed and unstressed conditions as discussed 459 by Telfer et al. (2021) and fits with the broad adaptation model proposed by Finlay and Wilkinson 460 (1963). 461 Ten performance QTL were identified for grain yield, screenings, and TWT independent of any 462 collocated responsiveness QTL (Figure 1 and Supplementary Table 2). Grain yield QTL were identified 463 on chromosome 2A (QYld.agt-SG.2A) and 7A (QYld.agt-L2G.7A and QYld.agt-SG.7A), with the Gladius 464 parent providing the higher performing allele in each instance. QYld.agt-SG.2A on chromosome 2A 465 occurs in region previously described for grain yield and thousand grain weight QTL by Tura et al. 466 (2020) and Tsilo et al. (2010). On 7A, QYld.agt-L2G.7A occurs in a region where QTL for grain yield, 467 kernel weight and TWT QTL have been previously reported (Cabral et al. 2018; Huang et al. 2003; 468 Maphosa et al. 2014; Tura et al. 2020; Yu et al. 2018) including in heat stress studies (Bennett et al. 469 2012b; Guan et al. 2018; Mason et al. 2013; Pinto et al. 2016). Regions of responsiveness and their role in adaptation 474 (2019) who reported QTL for grain yield and thousand grain weight in the same region. 487 In most instances where clusters of responsive QTL were found, there were QTL responsive to 488 temperature and QTL responsive to growing season rainfall collocated. However, one example of 489 collocated responsiveness QTL independent of growing season rainfall was identified for grain yield 490 on chromosome 5B in the SM population (grain fill average maximum temperature - QYld.agt-SM.5B- 491 3 and grain fill number of days >35°C - QYld.agt-SM.5B-4). This is a region not previously described for 492 adaptation to heat stress conditions but has been associated with grain yield (Tura et al. 2020; Yu et 493 al. 2018) and thousand grain weight (Tura et al. 2020). Each of the QTL reported by Tura et al. (2020) 494 were found to have QTL by environment interactions, potentially identifying similar qualities for 495 adaptation that have been determined to be responsive to changing temperature conditions in the 496 current study. 497 In most instances where clusters of responsive QTL were found, there were QTL responsive to 488 temperature and QTL responsive to growing season rainfall collocated. However, one example of 489 collocated responsiveness QTL independent of growing season rainfall was identified for grain yield 490 on chromosome 5B in the SM population (grain fill average maximum temperature - QYld.agt-SM.5B- 491 3 and grain fill number of days >35°C - QYld.agt-SM.5B-4). This is a region not previously described for 492 adaptation to heat stress conditions but has been associated with grain yield (Tura et al. 2020; Yu et 493 al. 2018) and thousand grain weight (Tura et al. 2020). Each of the QTL reported by Tura et al. (2020) 494 were found to have QTL by environment interactions, potentially identifying similar qualities for 495 adaptation that have been determined to be responsive to changing temperature conditions in the 496 current study. 497 Regions of responsiveness and their role in adaptation 474 This is a region not previously described for 492 adaptation to heat stress conditions but has been associated with grain yield (Tura et al. 2020; Yu et 493 al. 2018) and thousand grain weight (Tura et al. 2020). Each of the QTL reported by Tura et al. (2020) 494 were found to have QTL by environment interactions, potentially identifying similar qualities for 495 adaptation that have been determined to be responsive to changing temperature conditions in the 496 current study. 497 Comparing field results to controlled environment evaluation of heat stress adaptation 498 The populations evaluated across multiple environments in this study to investigate adaptation to 499 heat stress conditions were also evaluated previously in controlled environmental conditions by Telfer 500 independent of performance QTL may provide opportunities to breed specific adaptation but would 477 be more limited in their application and situations in which they confer an advantage. Under extreme 478 stress conditions, such genomic regions may offer opportunities for improvements in adaption, 479 especially if combined with other QTL for performance. When considering the framework proposed 480 in Figure 2H, this would be classified as a positive response and mean performance value. 481 To demonstrate, a region identified on chromosome 5A in the SG population where a grain yield QTL 482 for responsiveness to grain fill number of days >30°C (QYld.agt-SG.5A-2) and grain fill number of days 483 >35°C (QYld.agt-SG.5A-1) were found to collocate with the grain yield QTL responsive to growing 484 season rainfall (QYld.agt-SG.5A-3) with the Scout parent providing the stress adapted allele in each 485 instance. This is a similar result to previous work by Tura et al. (2020), Zhang et al. (2018) and Tadesse 486 et al. (2019) who reported QTL for grain yield and thousand grain weight in the same region. 487 To demonstrate, a region identified on chromosome 5A in the SG population where a grain yield QTL 482 for responsiveness to grain fill number of days >30°C (QYld.agt-SG.5A-2) and grain fill number of days 483 >35°C (QYld.agt-SG.5A-1) were found to collocate with the grain yield QTL responsive to growing 484 season rainfall (QYld.agt-SG.5A-3) with the Scout parent providing the stress adapted allele in each 485 instance. This is a similar result to previous work by Tura et al. (2020), Zhang et al. (2018) and Tadesse 486 et al. Regions of responsiveness and their role in adaptation 474 Regions associated with responsiveness were identified separate from performance QTL, accounting 475 for 42 of the QTL identified (Figure 1 and Supplementary Table 2). Regions of responsiveness 476 Regions associated with responsiveness were identified separate from performance QTL, accounting 475 for 42 of the QTL identified (Figure 1 and Supplementary Table 2). Regions of responsiveness 476 independent of performance QTL may provide opportunities to breed specific adaptation but would 477 be more limited in their application and situations in which they confer an advantage. Under extreme 478 stress conditions, such genomic regions may offer opportunities for improvements in adaption, 479 especially if combined with other QTL for performance. When considering the framework proposed 480 in Figure 2H, this would be classified as a positive response and mean performance value. 481 independent of performance QTL may provide opportunities to breed specific adaptation but would 477 be more limited in their application and situations in which they confer an advantage. Under extreme 478 stress conditions, such genomic regions may offer opportunities for improvements in adaption, 479 especially if combined with other QTL for performance. When considering the framework proposed 480 in Figure 2H, this would be classified as a positive response and mean performance value. 481 To demonstrate, a region identified on chromosome 5A in the SG population where a grain yield QTL 482 for responsiveness to grain fill number of days >30°C (QYld.agt-SG.5A-2) and grain fill number of days 483 >35°C (QYld.agt-SG.5A-1) were found to collocate with the grain yield QTL responsive to growing 484 season rainfall (QYld.agt-SG.5A-3) with the Scout parent providing the stress adapted allele in each 485 instance. This is a similar result to previous work by Tura et al. (2020), Zhang et al. (2018) and Tadesse 486 et al. (2019) who reported QTL for grain yield and thousand grain weight in the same region. 487 In most instances where clusters of responsive QTL were found, there were QTL responsive to 488 temperature and QTL responsive to growing season rainfall collocated. However, one example of 489 collocated responsiveness QTL independent of growing season rainfall was identified for grain yield 490 on chromosome 5B in the SM population (grain fill average maximum temperature - QYld.agt-SM.5B- 491 3 and grain fill number of days >35°C - QYld.agt-SM.5B-4). Comparing field results to controlled environment evaluation of heat stress adaptation 498 The populations evaluated across multiple environments in this study to investigate adaptation to 499 heat stress conditions were also evaluated previously in controlled environmental conditions by Telfer 500 et al. (2021). In that study adaptation to heat stress was investigated during grain filling by exposing 501 plants to three consecutive eight hour days with an air temperature of 36ᵒC and a wind speed of 40 502 km h-1. QTL analysis was conducted to identify performance and responsiveness in a similar framework 503 to the current study. When comparing the results of Telfer et al. (2021), 86 QTL from the current study 504 were found to be collocated with 49 QTL identified in controlled environment conditions (excluding 505 anthesis date QTL). 506 plants to three consecutive eight hour days with an air temperature of 36ᵒC and a wind speed of 40 502 km h-1. QTL analysis was conducted to identify performance and responsiveness in a similar framework 503 to the current study. When comparing the results of Telfer et al. (2021), 86 QTL from the current study 504 were found to be collocated with 49 QTL identified in controlled environment conditions (excluding 505 anthesis date QTL). 506 Key regions of crossover between that discussed by Telfer et al. (2021) and the current study include 507 five performance QTL identified by Telfer et al. (2021) between 50.4 and 63.3 cM on chromosome 5B 508 in the SM population, for thousand kernel weight, grain yield per spike, spikelet fertility, grain number 509 per spike, and spikelet number per spike, where Mace was the source of the high performance allele. 510 In the current study, this is a region where numerous QTL were identified in the L2G, SM, MG, and SG 511 populations. A grain yield performance QTL (QYld.agt-SM.5B-1) identified in the SM population, 512 clustered with screenings QTL responsive to numerous climatic co-variates and growing season 513 rainfall. As with Telfer et al. (2021), Mace was the source of adaptation for stressed environments at 514 this locus. 515 Key regions of crossover between that discussed by Telfer et al. (2021) and the current study include 507 five performance QTL identified by Telfer et al. Comparing field results to controlled environment evaluation of heat stress adaptation 498 (2021) between 50.4 and 63.3 cM on chromosome 5B 508 in the SM population, for thousand kernel weight, grain yield per spike, spikelet fertility, grain number 509 per spike, and spikelet number per spike, where Mace was the source of the high performance allele. 510 In the current study, this is a region where numerous QTL were identified in the L2G, SM, MG, and SG 511 populations. A grain yield performance QTL (QYld.agt-SM.5B-1) identified in the SM population, 512 clustered with screenings QTL responsive to numerous climatic co-variates and growing season 513 rainfall. As with Telfer et al. (2021), Mace was the source of adaptation for stressed environments at 514 this locus. 515 In the current study a grain yield performance QTL (QYld.agt-SM.2B-2) and TWT performance QTL 516 (QTwt.agt-SM.2B), clustered with QTL for screenings and TWT responsive to various temperature co- 517 variates and growing season rainfall. These QTL mapped similarly to six yield related QTL identified in 518 the SM and MG populations identified by Telfer et al. (2021). In both studies, Scout was responsible 519 for the stress-adapted allele in the SM population and Gladius in the MG population where it was 520 responsible for increased thousand grain weight. 521 Telfer et al. (2021) identified three performance QTL between 3.5 and 12.9 cM on chromosome 7A in 522 the SG population for grain yield per spike, spikelet fertility, and grain number per spike where the 523 adapted allele was sourced from the Scout parent. This corresponds to QTL identified in the current 524 study between 13.3 and 23.1 cM in the SM population on chromosome 7A; a grain yield performance 525 QTL (QYld.agt-SM.7A) and TWT QTL responsive to grain fill temperature co-variates, with the Mace 526 allele being the allele conferring adaption to stressed conditions. The parents conferring the 527 favourable allele were different for the QTL identified in the two different populations and studies. 528 This makes interpretation of the favourable allele combination difficult and will require additional 529 investigation. This is a region that has previously shown potential for adaptation to heat stress for 530 grain yield in delayed sowing studies (Bennett et al. 2012b; Pinto et al. 2016), as well as chlorophyll 531 content (Talukder et al. 2014a). Comparing field results to controlled environment evaluation of heat stress adaptation 498 532 In the current study, a grain yield performance QTL (QYld.agt-L2G.7A) on chromosome 7A in the L2G 533 population was identified flanking a TWT QTL (QTwt.agt-SG.7A) responsive to grain fill average 534 maximum temperature in the SG population. For both QTL Gladius was the source of the elite or stress 535 adapted allele. These two QTL were identified on either side of two performance QTL identified by 536 Telfer et al. (2021) for spikelet number per spike identified in the SG and L2G populations where Scout 537 and AUS17840 provided the elite allele respectively in controlled environment conditions. This region 538 has been described by Bennett et al. (2012b) and Mason et al. (2013) for improved grain yield under 539 heat stress conditions induced by delayed sowing, as well as thousand grain weight (Bennett et al. 540 2012b; Guan et al. 2018), grain number per spike ((Guan et al. 2018), and stay green (Mason et al. 541 2013). This region was also found to be important for grain yield determination in other adaptation 542 studies (Tura et al. 2020) and thousand grain weight (Huang et al. 2004; Sun et al. 2008; Tsilo et al. 543 2010; Tura et al. 2020). 544 Telfer et al. (2021) identified two performance QTL on chromosome 6A (QSfi.agt-SG.6A.2 and QSfi.agt- 545 L2G.6A) that were flanked by three QTL identified in the current study (QYld.agt-L2G.6A-1, QYld.agt- 546 L2G.6A-2 and QTwt.agt-RG.6A-2). The populations involved within the two studies were not the same 547 but did share Gladius as a common parent. The TWT response to stress is in repulsion to grain yield 548 performance and responsive to temperature stress, while grain yield in the field appears to be linked 549 positively to spikelet fertility in controlled environment conditions. This locus has been reported 550 numerous times (Cui et al. 2014; Kuchel et al. 2007b; Mir et al. 2012; Pinto et al. 2016; Sun et al. 2008; 551 Telfer et al. (2021) identified two performance QTL on chromosome 6A (QSfi.agt-SG.6A.2 and QSfi.agt- 545 L2G.6A) that were flanked by three QTL identified in the current study (QYld.agt-L2G.6A-1, QYld.agt- 546 L2G.6A-2 and QTwt.agt-RG.6A-2). The populations involved within the two studies were not the same 547 but did share Gladius as a common parent. Comparing field results to controlled environment evaluation of heat stress adaptation 498 The TWT response to stress is in repulsion to grain yield 548 performance and responsive to temperature stress, while grain yield in the field appears to be linked 549 positively to spikelet fertility in controlled environment conditions. This locus has been reported 550 numerous times (Cui et al. 2014; Kuchel et al. 2007b; Mir et al. 2012; Pinto et al. 2016; Sun et al. 2008; 551 Tura et al. 2020; Yu et al. 2018; Zhang et al. 2018) and is likely to be TaGW2 (Su et al. 2011) which 552 confers a grain size advantage. Although TaGW2 was not mapped in the DH lines in this study, it was 553 known to be segregating in some crosses, with Scout carrying the allele for large grain and Gladius the 554 small grain allele. This suggests that the positive response in grain yield to heat stress seen in this study 555 is linked to the TaGW2 allele for large grain. 556 Although some consistency between the field and in controlled environment condition studies can be 557 demonstrated, there were more points of inconsistency. Likewise, links were found with other field 558 studies conducted using delayed sowing, but these too were limited. This lack of commonality 559 between the assay types suggests that the value of controlled environment studies is comparatively 560 poor. It highlights the value of field-based genetic dissection of tolerance traits. 561 Heat stress adaptation in the context of breeding objectives 562 As proposed by Telfer et al. (2021) and further developed here, heat stress adaptation should be 563 considered as the combination of overall performance as well as responsiveness to stress. A definition 564 that similarly could be applied to other stress conditions. 565 There are two types of QTL identified in this study; performance QTL which were found to be 566 significant across the breadth of the environments observed, and responsive QTL that varied in 567 expression in response to the abiotic stress encountered across the environments tested. QTL that 568 combines positive performance with positive response to stress would be most desirable to breeders. 569 There are two types of QTL identified in this study; performance QTL which were found to be 566 significant across the breadth of the environments observed, and responsive QTL that varied in 567 expression in response to the abiotic stress encountered across the environments tested. QTL that 568 combines positive performance with positive response to stress would be most desirable to breeders. 569 While this study identified examples of a positive performance allele for grain yield also conferring a 570 positive response to screenings or TWT QTL, these were rare and support the conclusion that breeding 571 for heat stress tolerance is complex. Perhaps more realistically, a breeder seeking heat stress tolerance 572 could target performance QTL that has been shown not to be responsive to heat stress. The results of 573 this study certainly support this as a more achievable target with three QTL for grain yield identified 574 that meet this criterion. Similarly, QTL that only show responsiveness to heat stress, and are not linked 575 to performance, may offer opportunities to breed varieties that are specifically adapted to heat prone 576 There are two types of QTL identified in this study; performance QTL which were found to be 566 significant across the breadth of the environments observed, and responsive QTL that varied in 567 expression in response to the abiotic stress encountered across the environments tested. QTL that 568 combines positive performance with positive response to stress would be most desirable to breeders. 569 While this study identified examples of a positive performance allele for grain yield also conferring a 570 positive response to screenings or TWT QTL, these were rare and support the conclusion that breeding 571 for heat stress tolerance is complex. Heat stress adaptation in the context of breeding objectives 562 Perhaps more realistically, a breeder seeking heat stress tolerance 572 could target performance QTL that has been shown not to be responsive to heat stress. The results of 573 this study certainly support this as a more achievable target with three QTL for grain yield identified 574 that meet this criterion. Similarly, QTL that only show responsiveness to heat stress, and are not linked 575 to performance, may offer opportunities to breed varieties that are specifically adapted to heat prone 576 environments. This study highlights the importance of considering both performance and 577 responsiveness when investigating tolerance to abiotic stress tolerance. Either in isolation may lead 578 to incomplete or even worse, misguided conclusions regarding genetic selection strategy. 579 Conclusions 580 This study demonstrates that adaptation to heat and drought can be assessed as the combination of 581 performance and responsiveness to stress conditions. Furthermore, this two-dimensional framework 582 for understanding and breeding for stress tolerance can be easily extended to other complex abiotic 583 stresses. Loci for performance, with broad adaption across both stressed and unstressed 584 environments in the absence of responsiveness, represent useful targets for breeders, with examples 585 for grain yield identified in this study on chromosomes 2A and 7A. The responsiveness QTL identified 586 offer opportunities to breed for specific adaption, including the grain yield locus found independent 587 of performance on 5A that combines adaptation to heat stress during grain fill and drought stress. 588 Finally, grain yield loci on 2B and 5B provide unique pathways to combine high performance with 589 positive responsiveness to temperature and drought stress. 590 This study demonstrates that adaptation to heat and drought can be assessed as the combination of 581 performance and responsiveness to stress conditions. Furthermore, this two-dimensional framework 582 for understanding and breeding for stress tolerance can be easily extended to other complex abiotic 583 stresses. Loci for performance, with broad adaption across both stressed and unstressed 584 environments in the absence of responsiveness, represent useful targets for breeders, with examples 585 for grain yield identified in this study on chromosomes 2A and 7A. The responsiveness QTL identified 586 offer opportunities to breed for specific adaption, including the grain yield locus found independent 587 of performance on 5A that combines adaptation to heat stress during grain fill and drought stress. 588 Finally, grain yield loci on 2B and 5B provide unique pathways to combine high performance with 589 positive responsiveness to temperature and drought stress. 590 These genetic regions represent opportunities for marker assisted selection by breeders. Additionally, 591 the multi-environment framework used herein could be applied in the development of two- 592 dimensional (performance and responsiveness) genomic prediction calibrations. This would enable 593 the extension of current genomic selection principles, that are being increasingly used by breeding 594 programs, to be extended into selection of adaptation to abiotic stress. 595 These genetic regions represent opportunities for marker assisted selection by breeders. Additionally, 591 the multi-environment framework used herein could be applied in the development of two- 592 dimensional (performance and responsiveness) genomic prediction calibrations. Acknowledgments 597 The authors thank the technical team from the AGT Roseworthy breeding facility, for assistance in 598 planting, management, harvest, and collecting data on post-harvest measurements. This project was 599 jointly funded by the South Australian Grains Industry Trust (SAGIT) and Australian Grain Technologies 600 Pty Ltd. 601 The authors thank the technical team from the AGT Roseworthy breeding facility, for assistance in 598 planting, management, harvest, and collecting data on post-harvest measurements. This project was 599 jointly funded by the South Australian Grains Industry Trust (SAGIT) and Australian Grain Technologies 600 Pty Ltd. 601 Conclusions 580 This would enable 593 the extension of current genomic selection principles, that are being increasingly used by breeding 594 programs, to be extended into selection of adaptation to abiotic stress. 595 596 Author Contributions 602 PT: Manuscript preparation, phenotypic data collection & data analysis. JE: PhD co-supervisor of PT, 603 direction on research & manuscript content. JT: Development of statistical framework, development 604 of analysis code & assistance with preparing the analysis section of the manuscript. JAA: PhD co- 605 supervisor of PT, direction on research & manuscript content. HK: PhD principal supervisor of PT, 606 direction on research & manuscript content. 607 PT: Manuscript preparation, phenotypic data collection & data analysis. JE: PhD co-supervisor of PT, 603 direction on research & manuscript content. JT: Development of statistical framework, development 604 of analysis code & assistance with preparing the analysis section of the manuscript. JAA: PhD co- 605 supervisor of PT, direction on research & manuscript content. HK: PhD principal supervisor of PT, 606 direction on research & manuscript content. 607 609 References 610 Theoretical and Applied Genetics 125:255-271 623 Bennett D, Reynolds M, Mullan D, Izanloo A, Kuchel H, Langridge P, Schnurbusch T (2012b) Detection 624 of two major grain yield QTL in bread wheat (Triticum aestivum L.) under heat, drought and high 625 yield potential environments. Theoretical and Applied Genetics 125:1473-1485 626 Bennett D, Reynolds M, Mullan D, Izanloo A, Kuchel H, Langridge P, Schnurbusch T (2012b) Detection 624 of two major grain yield QTL in bread wheat (Triticum aestivum L.) under heat, drought and high 625 yield potential environments. 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Genome Biology 19:111 615 g p gy Alexander BM, Hayman PT, McDonald GK, Talukder ASMHM, Gill GS (2010) Characterising the risk of 616 heat stress on wheat in South Australia: meteorology, climatology and the design of a field heating 617 chamber. Proceedings of the 15th Australian Agronomy Conference, Lincoln, New Zealand 618 Ali MB, Ibrahim AH, Malla S, Rudd J, Hays DB (2013) Family-based QTL mapping of heat stress 619 tolerance in primitive tetraploid wheat (Triticum turgidum L.). Euphytica 192:189-203 620 Bennett D, Izanloo A, Reynolds M, Kuchel H, Langridge P, Schnurbusch T (2012a) Genetic dissection 621 of grain yield and physical grain quality in bread wheat (Triticum aestivum L.) under water-limited 622 g p gy Alexander BM, Hayman PT, McDonald GK, Talukder ASMHM, Gill GS (2010) Characterising the risk of 616 heat stress on wheat in South Australia: meteorology, climatology and the design of a field heating 617 chamber. Proceedings of the 15th Australian Agronomy Conference, Lincoln, New Zealand 618 Ali MB, Ibrahim AH, Malla S, Rudd J, Hays DB (2013) Family-based QTL mapping of heat stress 619 tolerance in primitive tetraploid wheat (Triticum turgidum L.). Euphytica 192:189-203 620 Bennett D, Izanloo A, Reynolds M, Kuchel H, Langridge P, Schnurbusch T (2012a) Genetic dissection 621 p p ( g ) p y Bennett D, Izanloo A, Reynolds M, Kuchel H, Langridge P, Schnurbusch T (2012a) Genetic dissection 621 of grain yield and physical grain quality in bread wheat (Triticum aestivum L.) under water-limited 622 environments. References 610 Springer New York roman K, Wu H (2015) qtl: tools for analayzing QTL experiments. R package version 1.36-6. Broman K, Wu H (2015) qtl: tools for analayzing QTL experiments. 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Australian 668 Journal of Agricultural Research 14:742-754 669 Fleury D, Jefferies S, Kuchel H, Langridge P (2010) Genetic and genomic tools to improve drought 670 tolerance in wheat. Journal of Experimental Botany 61:3211-3222 671 p y Flohr BM, Hunt JR, Kirkegaard JA, Evans JR (2017) Water and temperature stress define the optimal 672 flowering period for wheat in south-eastern Australia. Field Crops Research 209:108-119 673 Groos C, Robert N, Bervas E, Charmet G (2003) Genetic analysis of grain protein-content, grain yield 674 and thousand-kernel weight in bread wheat. Theoretical and Applied Genetics 106:1032-1040 675 Guan P, Lu L, Jia L, Kabir MR, Zhang J, Lan T, Zhao Y, Xin M, Hu Z, Yao Y, Ni Z, Sun Q, Peng H (2018) 676 Global QTL Analysis Identifies Genomic Regions on Chromosomes 4A and 4B Harboring Stable Loci 677 for Yield-Related Traits Across Different Environments in Wheat (Triticum aestivum L.). Frontiers in 678 Plant Science 9 679 Hays DB, Do JH, Mason RE, Morgan G, Finlayson SA (2007) Heat stress induced ethylene production 680 in developing wheat grains induces kernel abortion and increased maturation in a susceptible 681 cultivar. Plant Science 172:1113-1123 682 Hays DB, Do JH, Mason RE, Morgan G, Finlayson SA (2007) Heat stress induced ethylene production 680 in developing wheat grains induces kernel abortion and increased maturation in a susceptible 681 cultivar. Plant Science 172:1113-1123 682 Huang XQ, Cloutier S, Lycar L, Radovanovic N, Humphreys DG, Noll JS, Somers DJ, Brown PD (2006) 683 Molecular detection of QTLs for agronomic and quality traits in a doubled haploid population 684 derived from two Canadian wheats (Triticum aestivum L.). Theoretical and Applied Genetics 685 113:753-766 686 Huang XQ, Cloutier S, Lycar L, Radovanovic N, Humphreys DG, Noll JS, Somers DJ, Brown PD (2006) 683 Molecular detection of QTLs for agronomic and quality traits in a doubled haploid population 684 derived from two Canadian wheats (Triticum aestivum L.). wheat cross RL4452/‘AC Domain’. PLOS ONE 13:e0190681 Cui F, Zhao C, Ding A, Li J, Wang L, Li X, Bao Y, Li J, Wang H (2014) Construction of an integrative 645 linkage map and QTL mapping of grain yield-related traits using three related wheat RIL populations. 646 Theoretical and Applied Genetics 127:659-675 647 Cullis BR, Smith AB, Coombes NE (2006) On the design of early generation variety trials with 648 correlated data. Journal of Agricultural, Biological, and Environmental Statistics 11:381 649 Dolferus R, Ji X, Richards RA (2011) Abiotic stress and control of grain number in cereals. Plant 650 Science 181:331-341 651 Dolferus R, Thavamanikumar S, Sangma H, Kleven S, Wallace X, Forrest K, Rebetzke G, Hayden M, 652 Borg L, Smith A, Cullis B (2019) Determining the Genetic Architecture of Reproductive Stage Drought 653 Tolerance in Wheat Using a Correlated Trait and Correlated Marker Effect Model. G3: 654 Genes\|Genomes\|Genetics 9:473-489 655 Dreccer MF, Chapman SC, Ogbonnaya FC, Borgognone MG, Trethowan RM (2008) Crop and 656 environmental attributes underpinning genotype by environment interaction in synthetic-derived 657 bread wheat evaluated in Mexico and Australia. Australian Journal of Agricultural Research 59:447- 658 460 659 Eagles HA, Cane K, Trevaskis B, Vallance N, Eastwood RF, Gororo NN, Kuchel H, Martin PJ (2014) 660 Ppd1, Vrn1, ALMT1 and Rht genes and their effects on grain yield in lower rainfall environments in 661 southern Australia. Crop and Pasture Science 65:159-170 662 Esten Mason R, Hays D, Mondal S, Ibrahim AH, Basnet B (2013) QTL for yield, yield components and 663 canopy temperature depression in wheat under late sown field conditions. Euphytica 194:243-259 664 Esten Mason R, Mondal S, Beecher FW, Pacheco A, Jampala B, Ibrahim AMH, Hays DB (2010) QTL 665 associated with heat susceptibility index in wheat (Triticum aestivum L.) under short-term 666 reproductive stage heat stress. Euphytica 174:423-436 667 Finlay K, Wilkinson G (1963) The analysis of adaptation in a plant-breeding programme. Australian 668 Journal of Agricultural Research 14:742-754 669 Fleury D, Jefferies S, Kuchel H, Langridge P (2010) Genetic and genomic tools to improve drought 670 tolerance in wheat. Journal of Experimental Botany 61:3211-3222 671 Flohr BM, Hunt JR, Kirkegaard JA, Evans JR (2017) Water and temperature stress define the optimal 672 flowering period for wheat in south-eastern Australia. Field Crops Research 209:108-119 673 Groos C, Robert N, Bervas E, Charmet G (2003) Genetic analysis of grain protein-content, grain yield 674 and thousand-kernel weight in bread wheat. wheat cross RL4452/‘AC Domain’. PLOS ONE 13:e0190681 Theoretical and Applied Genetics 106:1032-1040 675 Guan P, Lu L, Jia L, Kabir MR, Zhang J, Lan T, Zhao Y, Xin M, Hu Z, Yao Y, Ni Z, Sun Q, Peng H (2018) 676 Global QTL Analysis Identifies Genomic Regions on Chromosomes 4A and 4B Harboring Stable Loci 677 for Yield-Related Traits Across Different Environments in Wheat (Triticum aestivum L.). Frontiers in 678 Plant Science 9 679 Hays DB, Do JH, Mason RE, Morgan G, Finlayson SA (2007) Heat stress induced ethylene production 680 in developing wheat grains induces kernel abortion and increased maturation in a susceptible 681 cultivar. Plant Science 172:1113-1123 682 Huang XQ, Cloutier S, Lycar L, Radovanovic N, Humphreys DG, Noll JS, Somers DJ, Brown PD (2006) 683 Molecular detection of QTLs for agronomic and quality traits in a doubled haploid population 684 derived from two Canadian wheats (Triticum aestivum L.). Theoretical and Applied Genetics 685 113:753-766 686 Huang XQ, Cöster H, Ganal MW, Röder MS (2003) Advanced backcross QTL analysis for the 687 identification of quantitative trait loci alleles from wild relatives of wheat (Triticum aestivum L.). 688 Theoretical and Applied Genetics 106:1379-1389 689 Huang XQ, Kempf H, Ganal MW, Röder MS (2004) Advanced backcross QTL analysis in progenies 690 derived from a cross between a German elite winter wheat variety and a synthetic wheat (Triticum 691 aestivum L.). Theor Appl Genet 109:933-943 692 Iwaki K, Nishida J, Yanagisawa T, Yoshida H, Kato K (2002) Genetic analysis of Vrn-B1 for vernalizatio 693 requirement by using linked dCAPS markers in bread wheat (Triticum aestivum L.). Theoretical and 694 Applied Genetics 104:571-576 695 Izanloo A, Condon AG, Langridge P, Tester M, Schnurbusch T (2008) Different mechanisms of 696 adaptation to cyclic water stress in two South Australian bread wheat cultivars. Journal of 697 Experimental Botany 59:3327-3346 698 Jiang Y, Jiang Q, Hao C, Hou J, Wang L, Zhang H, Zhang S, Chen X, Zhang X (2015) A yield-associated 699 gene TaCWI, in wheat: its function, selection and evolution in global breeding revealed by haplotype 700 analysis. Theoretical and Applied Genetics 128:131-143 701 Kuchel H, Hollamby G, Langridge P, Williams K, Jefferies SP (2006) Identification of genetic loci 702 associated with ear-emergence in bread wheat. Theoretical and Applied Genetics 113:1103-1112 703 Kuchel H, Williams K, Langridge P, Eagles HA, Jefferies SP (2007a) Genetic dissection of grain yield in 704 bread wheat. II. QTL-by-environment interaction. wheat cross RL4452/‘AC Domain’. PLOS ONE 13:e0190681 Theoretical and Applied Genetics 115:1015-1027 705 Kuchel H, Williams KJ, Langridge P, Eagles HA, Jefferies SP (2007b) Genetic dissection of grain yield in 706 bread wheat. I. QTL analysis. Theoretical and Applied Genetics 115:1029-1041 707 Law CN, Sutka J, Worland AJ (1978) A Genetic study of day-length response in wheat. Heredity 708 41:185-191 709 Eagles HA, Cane K, Trevaskis B, Vallance N, Eastwood RF, Gororo NN, Kuchel H, Martin PJ (2014) 660 Ppd1, Vrn1, ALMT1 and Rht genes and their effects on grain yield in lower rainfall environments in 661 southern Australia. Crop and Pasture Science 65:159-170 662 Esten Mason R, Hays D, Mondal S, Ibrahim AH, Basnet B (2013) QTL for yield, yield components and 663 canopy temperature depression in wheat under late sown field conditions. Euphytica 194:243-259 664 Esten Mason R, Mondal S, Beecher FW, Pacheco A, Jampala B, Ibrahim AMH, Hays DB (2010) QTL 665 associated with heat susceptibility index in wheat (Triticum aestivum L.) under short-term 666 reproductive stage heat stress. Euphytica 174:423-436 667 Finlay K, Wilkinson G (1963) The analysis of adaptation in a plant-breeding programme. Australian 668 Journal of Agricultural Research 14:742-754 669 Fleury D, Jefferies S, Kuchel H, Langridge P (2010) Genetic and genomic tools to improve drought 670 tolerance in wheat. Journal of Experimental Botany 61:3211-3222 671 Flohr BM, Hunt JR, Kirkegaard JA, Evans JR (2017) Water and temperature stress define the optimal 672 flowering period for wheat in south-eastern Australia. Field Crops Research 209:108-119 673 Groos C, Robert N, Bervas E, Charmet G (2003) Genetic analysis of grain protein-content, grain yield 674 and thousand-kernel weight in bread wheat. Theoretical and Applied Genetics 106:1032-1040 675 Guan P, Lu L, Jia L, Kabir MR, Zhang J, Lan T, Zhao Y, Xin M, Hu Z, Yao Y, Ni Z, Sun Q, Peng H (2018) 676 Global QTL Analysis Identifies Genomic Regions on Chromosomes 4A and 4B Harboring Stable Loci 677 for Yield-Related Traits Across Different Environments in Wheat (Triticum aestivum L.). Frontiers in 678 Plant Science 9 679 Hays DB, Do JH, Mason RE, Morgan G, Finlayson SA (2007) Heat stress induced ethylene production 680 in developing wheat grains induces kernel abortion and increased maturation in a susceptible 681 cultivar. wheat cross RL4452/‘AC Domain’. PLOS ONE 13:e0190681 Plant Science 172:1113-1123 682 Huang XQ, Cloutier S, Lycar L, Radovanovic N, Humphreys DG, Noll JS, Somers DJ, Brown PD (2006) 683 Molecular detection of QTLs for agronomic and quality traits in a doubled haploid population 684 derived from two Canadian wheats (Triticum aestivum L.). Theoretical and Applied Genetics 685 113:753-766 686 Huang XQ, Cöster H, Ganal MW, Röder MS (2003) Advanced backcross QTL analysis for the 687 identification of quantitative trait loci alleles from wild relatives of wheat (Triticum aestivum L.). 688 Theoretical and Applied Genetics 106:1379-1389 689 Huang XQ, Kempf H, Ganal MW, Röder MS (2004) Advanced backcross QTL analysis in progenies 690 derived from a cross between a German elite winter wheat variety and a synthetic wheat (Triticum 691 aestivum L.). Theor Appl Genet 109:933-943 692 Iwaki K, Nishida J, Yanagisawa T, Yoshida H, Kato K (2002) Genetic analysis of Vrn-B1 for vernalization 693 requirement by using linked dCAPS markers in bread wheat (Triticum aestivum L.). Theoretical and 694 Applied Genetics 104:571-576 695 Izanloo A, Condon AG, Langridge P, Tester M, Schnurbusch T (2008) Different mechanisms of 696 adaptation to cyclic water stress in two South Australian bread wheat cultivars. Journal of 697 Experimental Botany 59:3327-3346 698 Jiang Y, Jiang Q, Hao C, Hou J, Wang L, Zhang H, Zhang S, Chen X, Zhang X (2015) A yield-associated 699 gene TaCWI, in wheat: its function, selection and evolution in global breeding revealed by haplotype 700 analysis. Theoretical and Applied Genetics 128:131-143 701 Kuchel H, Hollamby G, Langridge P, Williams K, Jefferies SP (2006) Identification of genetic loci 702 associated with ear-emergence in bread wheat. Theoretical and Applied Genetics 113:1103-1112 703 Kuchel H, Williams K, Langridge P, Eagles HA, Jefferies SP (2007a) Genetic dissection of grain yield in 704 bread wheat. II. QTL-by-environment interaction. Theoretical and Applied Genetics 115:1015-1027 705 Kuchel H, Williams KJ, Langridge P, Eagles HA, Jefferies SP (2007b) Genetic dissection of grain yield in 706 bread wheat. I. QTL analysis. Theoretical and Applied Genetics 115:1029-1041 707 Law CN, Sutka J, Worland AJ (1978) A Genetic study of day-length response in wheat. Heredity 708 41:185-191 709 sten Mason R, Hays D, Mondal S, Ibrahim AH, Basnet B (2013) QTL for yield, yield components a anopy temperature depression in wheat under late sown field conditions. Euphytica 194:243-25 canopy temperature depression in wheat under late sown field conditions. wheat cross RL4452/‘AC Domain’. PLOS ONE 13:e0190681 Theoretical and Applied Genetics 685 113:753-766 686 Huang XQ, Kempf H, Ganal MW, Röder MS (2004) Advanced backcross QTL analysis in progenies 690 derived from a cross between a German elite winter wheat variety and a synthetic wheat (Triticum 691 aestivum L.). Theor Appl Genet 109:933-943 692 Huang XQ, Kempf H, Ganal MW, Röder MS (2004) Advanced backcross QTL analysis in progenies 690 derived from a cross between a German elite winter wheat variety and a synthetic wheat (Triticum 691 aestivum L.). Theor Appl Genet 109:933-943 692 Iwaki K, Nishida J, Yanagisawa T, Yoshida H, Kato K (2002) Genetic analysis of Vrn-B1 for vernalization 693 requirement by using linked dCAPS markers in bread wheat (Triticum aestivum L.). Theoretical and 694 Applied Genetics 104:571-576 695 Iwaki K, Nishida J, Yanagisawa T, Yoshida H, Kato K (2002) Genetic analysis of Vrn-B1 for vernalization 693 requirement by using linked dCAPS markers in bread wheat (Triticum aestivum L.). Theoretical and 694 Applied Genetics 104:571-576 695 Izanloo A, Condon AG, Langridge P, Tester M, Schnurbusch T (2008) Different mechanisms of 696 adaptation to cyclic water stress in two South Australian bread wheat cultivars. Journal of 697 Experimental Botany 59:3327-3346 698 Izanloo A, Condon AG, Langridge P, Tester M, Schnurbusch T (2008) Different mechanisms of 696 adaptation to cyclic water stress in two South Australian bread wheat cultivars. Journal of 697 Experimental Botany 59:3327-3346 698 Experimental Botany 59:3327-3346 698 Experimental Botany 59:3327-3346 Jiang Y, Jiang Q, Hao C, Hou J, Wang L, Zhang H, Zhang S, Chen X, Zhang X (2015) A yield-associated 699 gene TaCWI, in wheat: its function, selection and evolution in global breeding revealed by haplotype 700 analysis. Theoretical and Applied Genetics 128:131-143 701 Kuchel H, Hollamby G, Langridge P, Williams K, Jefferies SP (2006) Identification of genetic loci 702 associated with ear-emergence in bread wheat. Theoretical and Applied Genetics 113:1103-1112 703 Kuchel H, Williams K, Langridge P, Eagles HA, Jefferies SP (2007a) Genetic dissection of grain yield in 704 bread wheat. II. QTL-by-environment interaction. Theoretical and Applied Genetics 115:1015-1027 705 Kuchel H, Williams KJ, Langridge P, Eagles HA, Jefferies SP (2007b) Genetic dissection of grain yield in 706 bread wheat. I. QTL analysis. Theoretical and Applied Genetics 115:1029-1041 707 Law CN, Sutka J, Worland AJ (1978) A Genetic study of day-length response in wheat. Heredity 708 41:185-191 709 Law CN, Worland AJ, Giorgi B (1976) The genetic control of ear-emergence time by chromosomes 5A 710 and 5D of wheat. Heredity 36:49-58 711 Lemerle D, Smith A, Verbeek B, Koetz E, Lockley P, Martin P (2006) Incremental crop tolerance to 712 weeds: A measure for selecting competitive ability in Australian wheats. Euphytica 149:85-95 713 Li J, Ji L (2005) Adjusting multiple testing in multilocus analyses using the eigenvalues of a correlation 714 matrix. Heredity 95:221-227 715 Liu C, Sukumaran S, Claverie E, Sansaloni C, Dreisigacker S, Reynolds M (2019) Genetic dissection of 716 heat and drought stress QTLs in phenology-controlled synthetic-derived recombinant inbred lines in 717 spring wheat. Molecular Breeding 39:34 718 Lobell DB, Hammer GL, Chenu K, Zheng B, McLean G, Chapman SC (2015) The shifting influence of 719 drought and heat stress for crops in northeast Australia. Global Change Biology 21:4115-4127 720 Machado S, Paulsen G (2001) Combined effects of drought and high temperature on water relations 721 of wheat and sorghum. Plant and Soil 233:179-187 722 Malosetti M, Ribaut J-M, van Eeuwijk FA (2013) The statistical analysis of multi-environment data: 723 modeling genotype-by-environment interaction and its genetic basis. Frontiers in Physiology 4 724 Maphosa L, Langridge P, Taylor H, Parent B, Emebiri LC, Kuchel H, Reynolds MP, Chalmers KJ, Okada 725 A, Edwards J, Mather DE (2014) Genetic control of grain yield and grain physical characteristics in a 726 bread wheat population grown under a range of environmental conditions. Experimental Botany 59:3327-3346 Theoretical and Applied 727 Genetics 127:1607-1624 728 Martínez O, Curnow RN (1994) Missing markers when estimating quantitative trait loci using 729 regression mapping. Heredity 73:198-206 730 Mason ER, Mondal S, Beecher F, Hays D (2011) Genetic loci linking improved heat tolerance in wheat 731 (Triticum aestivum L.) to lower leaf and spike temperatures under controlled conditions. Euphytica 732 180:181-194 733 Mason RE, Hays DB, Mondal S, Ibrahim AMH, Basnet BR (2013) QTL for yield, yield components and 734 canopy temperature depression in wheat under late sown field conditions. Euphytica 194:243-259 735 McCartney CA, Somers DJ, Humphreys DG, Lukow O, Ames N, Noll J, Cloutier S, McCallum BD (2005) 736 Mapping quantitative trait loci controlling agronomic traits in the spring wheat cross RL4452x'AC 737 Domain'. Genome 48:870-883 738 Mir RR, Zaman-Allah M, Sreenivasulu N, Trethowan R, Varshney RK (2012) Integrated genomics, 739 physiology and breeding approaches for improving drought tolerance in crops. Theoretical and 740 Applied Genetics 125:625-645 741 Mohammadi M, Zali AA, Bihamta MR (2008) Mapping QTLs for Heat Tolerance in Wheat Journal of 742 Agricultural Science and Technology 10:261-267 743 Narjesi V, Mardi M, Hervan EM, Azadi A, Naghavi MR, Ebrahimi M, Zali AA (2015) Analysis of 744 Quantitative Trait Loci (QTL) for Grain Yield and Agronomic Traits in Wheat (Triticum aestivum L.) 745 Under Normal and Salt-Stress Conditions. Plant Molecular Biology Reporter 33:2030-2040 746 Norman A, Taylor J, Tanaka E, Telfer P, Edwards J, Martinant J-P, Kuchel H (2017) Increased genomic 747 prediction accuracy in wheat breeding using a large Australian panel. Theoretical and Applied 748 Genetics 130:2543-2555 749 Paliwal R, Röder MS, Kumar U, Srivastava J, Joshi AK (2012) QTL mapping of terminal heat tolerance 750 in hexaploid wheat (T. aestivum L.). Theoretical and Applied Genetics 125:561-575 751 Patterson HD, Thompson R (1971) Recovery of interblock information when block sizes are unequal. 752 Biometrika 58:545-554 753 Pinto RS, Lopes MS, Collins NC, Reynolds MP (2016) Modelling and genetic dissection of staygreen 754 under heat stress. Theoretical and Applied Genetics 129:2055-2074 755 Pinto RS, Reynolds MP, Mathews KL, McIntyre CL, Olivares-Villegas J-J, Chapman SC (2010) Heat and 756 drought adaptive QTL in a wheat population designed to minimize confounding agronomic effects. 757 Theoretical and Applied Genetics 121:1001-1021 758 R Core Team (2018) R: A language and environment for statistical computing. Experimental Botany 59:3327-3346 R Foundation for 759 Statistical Computing, Vienna, Austria 760 Law CN, Worland AJ, Giorgi B (1976) The genetic control of ear-emergence time by chromosomes 5A 710 and 5D of wheat. Heredity 36:49-58 711 Lemerle D, Smith A, Verbeek B, Koetz E, Lockley P, Martin P (2006) Incremental crop tolerance to 712 weeds: A measure for selecting competitive ability in Australian wheats. Euphytica 149:85-95 713 Li J, Ji L (2005) Adjusting multiple testing in multilocus analyses using the eigenvalues of a correlation 714 matrix. Heredity 95:221-227 715 Liu C, Sukumaran S, Claverie E, Sansaloni C, Dreisigacker S, Reynolds M (2019) Genetic dissection of 716 heat and drought stress QTLs in phenology-controlled synthetic-derived recombinant inbred lines in 717 spring wheat. Molecular Breeding 39:34 718 Lobell DB, Hammer GL, Chenu K, Zheng B, McLean G, Chapman SC (2015) The shifting influence of 719 drought and heat stress for crops in northeast Australia. Global Change Biology 21:4115-4127 720 Machado S, Paulsen G (2001) Combined effects of drought and high temperature on water relations 721 of wheat and sorghum. Plant and Soil 233:179-187 722 Malosetti M, Ribaut J-M, van Eeuwijk FA (2013) The statistical analysis of multi-environment data: 723 modeling genotype-by-environment interaction and its genetic basis. Frontiers in Physiology 4 724 Maphosa L, Langridge P, Taylor H, Parent B, Emebiri LC, Kuchel H, Reynolds MP, Chalmers KJ, Okada 725 A, Edwards J, Mather DE (2014) Genetic control of grain yield and grain physical characteristics in a 726 bread wheat population grown under a range of environmental conditions. Theoretical and Applied 727 Genetics 127:1607-1624 728 Martínez O, Curnow RN (1994) Missing markers when estimating quantitative trait loci using 729 regression mapping. Heredity 73:198-206 730 Mason ER, Mondal S, Beecher F, Hays D (2011) Genetic loci linking improved heat tolerance in wheat 731 (Triticum aestivum L.) to lower leaf and spike temperatures under controlled conditions. Euphytica 732 180:181-194 733 Mason RE, Hays DB, Mondal S, Ibrahim AMH, Basnet BR (2013) QTL for yield, yield components and 734 canopy temperature depression in wheat under late sown field conditions. Euphytica 194:243-259 735 McCartney CA, Somers DJ, Humphreys DG, Lukow O, Ames N, Noll J, Cloutier S, McCallum BD (2005) 736 Mapping quantitative trait loci controlling agronomic traits in the spring wheat cross RL4452x'AC 737 Domain'. Experimental Botany 59:3327-3346 Genome 48:870-883 738 Mir RR, Zaman-Allah M, Sreenivasulu N, Trethowan R, Varshney RK (2012) Integrated genomics, 739 physiology and breeding approaches for improving drought tolerance in crops. Theoretical and 740 Applied Genetics 125:625-645 741 Mohammadi M, Zali AA, Bihamta MR (2008) Mapping QTLs for Heat Tolerance in Wheat Journal of 742 Agricultural Science and Technology 10:261-267 743 Narjesi V, Mardi M, Hervan EM, Azadi A, Naghavi MR, Ebrahimi M, Zali AA (2015) Analysis of 744 Quantitative Trait Loci (QTL) for Grain Yield and Agronomic Traits in Wheat (Triticum aestivum L.) 745 Under Normal and Salt-Stress Conditions. Plant Molecular Biology Reporter 33:2030-2040 746 Norman A, Taylor J, Tanaka E, Telfer P, Edwards J, Martinant J-P, Kuchel H (2017) Increased genomic 747 prediction accuracy in wheat breeding using a large Australian panel. Theoretical and Applied 748 Genetics 130:2543-2555 749 Paliwal R, Röder MS, Kumar U, Srivastava J, Joshi AK (2012) QTL mapping of terminal heat tolerance 750 in hexaploid wheat (T. aestivum L.). Theoretical and Applied Genetics 125:561-575 751 Patterson HD, Thompson R (1971) Recovery of interblock information when block sizes are unequal. 752 Biometrika 58:545-554 753 Pinto RS, Lopes MS, Collins NC, Reynolds MP (2016) Modelling and genetic dissection of staygreen 754 under heat stress. 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Euphytica 732 180:181-194 733 Mason RE, Hays DB, Mondal S, Ibrahim AMH, Basnet BR (2013) QTL for yield, yield components and 734 canopy temperature depression in wheat under late sown field conditions. Experimental Botany 59:3327-3346 Euphytica 194:243-259 735 McCartney CA, Somers DJ, Humphreys DG, Lukow O, Ames N, Noll J, Cloutier S, McCallum BD (2005) 736 Mapping quantitative trait loci controlling agronomic traits in the spring wheat cross RL4452x'AC 737 Domain'. Genome 48:870-883 738 Mir RR, Zaman-Allah M, Sreenivasulu N, Trethowan R, Varshney RK (2012) Integrated genomics, 739 physiology and breeding approaches for improving drought tolerance in crops. Theoretical and 740 Applied Genetics 125:625-645 741 Mir RR, Zaman-Allah M, Sreenivasulu N, Trethowan R, Varshney RK (2012) Integrated genomics, 739 physiology and breeding approaches for improving drought tolerance in crops. Theoretical and 740 Applied Genetics 125:625-645 741 Applied Genetics 125:625-645 Crop Science 59:199-211 809 Ramya P, Chaubal A, Kulkarni K, Gupta L, Kadoo N, Dhaliwal HS, Chhuneja P, Lagu M 761 Ramya P, Chaubal A, Kulkarni K, Gupta L, Kadoo N, Dhaliwal HS, Chhuneja P, Lagu M, Gupt V (2010) 761 QTL mapping of 1000-kernel weight, kernel length, and kernel width in bread wheat (Triticum 762 ti L ) J l f A li d G ti 51 421 429 763 QTL mapping of 1000-kernel weight, kernel length, and kernel width in bread wheat 762 aestivum L.). Journal of Applied Genetics 51:421-429 763 Reynolds MP, Pierre CS, Saad ASI, Vargas M, Condon AG (2007) Evaluating Potential Genetic Gains in 764 Wheat Associated with Stress-Adaptive Trait Expression in Elite Genetic Resources under Drought 765 and Heat Stress. 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Genetics 197:375-387 770 Sadras VO, Monzon JP (2006) Modelled wheat phenology captures rising temperature trends: 771 Shortened time to flowering and maturity in Australia and Argentina. Field Crops Research 99:136- 772 146 773 Sadras VO, Vadez V, Purushothaman R, Lake L, Marrou H (2015) Unscrambling confounded effects of 774 sowing date trials to screen for crop adaptation to high temperature. Field Crops Research 177:1-8 775 Saini HS, Westgate ME, Donald LS (1999) Reproductive Development in Grain Crops during Drought. 776 Sadras VO, Vadez V, Purushothaman R, Lake L, Marrou H (2015) Unscrambling confounded effects of 774 sowing date trials to screen for crop adaptation to high temperature. Applied Genetics 125:625-645 Plant Breeding 127:241-248 786 Shirdelmoghanloo H (2014) Genetic and physiological studies of heat toleracne in hexaploid wheat 787 (Triticum aestivum L.). School of Agricultrure, Food and Wine. The University of Adelaide 788 Shirdelmoghanloo H, Cozzolino D, Lohraseb I, Collins NC (2016a) Truncation of grain filling in wheat 789 (Triticum aestivum) triggered by brief heat stress during early grain filling: association with 790 senescence responses and reductions in stem reserves. Functional Plant Biology 43:919-930 791 Shirdelmoghanloo H, Taylor JD, Lohraseb I, Rabie H, Brien C, Timmins A, Martin P, Mather DE, 792 Emebiri L, Collins NC (2016b) A QTL on the short arm of wheat (Triticum aestivum L.) chromosome 793 3B affects the stability of grain weight in plants exposed to a brief heat shock early in grain filling. 794 BMC Plant Biology 16:100 795 Smith A, Cullis B, Thompson R (2001) Analyzing variety by environment data using multiplicative 796 mixed models and adjustments for spatial field trend. Biometrics 57:1138-1147 797 Smith AB, Cullis BR, Thompson R (2005) The analysis of crop cultivar breeding and evaluation trials: 798 an overview of current mixed model approaches. The Journal of Agricultural Science 143:449-462 799 Stone PJ, Nicolas ME (1995) Effect of timing of heat stress during grain filling on two wheat varieties 800 differing in heat tolerance. I. grain growth. Australian Journal of Plant Physiology 22:927-934 801 Su Z, Hao C, Wang L, Dong Y, Zhang X (2011) Identification and development of a functional marker 802 of TaGW2 associated with grain weight in bread wheat (Triticum aestivum L.). Theoretical and 803 Applied Genetics 122:211-223 804 Sun X-Y, Wu K, Zhao Y, Kong F-M, Han G-Z, Jiang H-M, Huang X-J, Li R-J, Wang H-G, Li S-S (2008) QTL 805 analysis of kernel shape and weight using recombinant inbred lines in wheat. Euphytica 165:615 806 Tadesse W, Suleiman S, Tahir I, Sanchez-Garcia M, Jighly A, Hagras A, Thabet S, Baum M (2019) Heat- 807 Tolerant QTLs Associated with Grain Yield and Its Components in Spring Bread Wheat under Heat- 808 Stressed Environments of Sudan and Egypt. Applied Genetics 125:625-645 Ramya P, Chaubal A, Kulkarni K, Gupta L, Kadoo N, Dhaliwal HS, Chhuneja P, Lagu M, Gupt V (2010) 761 QTL mapping of 1000-kernel weight, kernel length, and kernel width in bread wheat (Triticum 762 aestivum L.). Journal of Applied Genetics 51:421-429 763 Ramya P, Chaubal A, Kulkarni K, Gupta L, Kadoo N, Dhaliwal HS, Chhuneja P, Lagu M, Gupt V (2010) 761 QTL mapping of 1000-kernel weight, kernel length, and kernel width in bread wheat (Triticum 762 aestivum L.). Journal of Applied Genetics 51:421-429 763 Reynolds MP, Pierre CS, Saad ASI, Vargas M, Condon AG (2007) Evaluating Potential Genetic Gains in 764 Wheat Associated with Stress-Adaptive Trait Expression in Elite Genetic Resources under Drought 765 and Heat Stress. Crop Science 47:S-172-S-189 766 Rincent R, Moreau L, Monod H, Kuhn E, Melchinger AE, Malvar RA, Moreno-Gonzalez J, Nicolas S, 767 Madur D, Combes V, Dumas F, Altmann T, Brunel D, Ouzunova M, Flament P, Dubreuil P, Charcosset 768 A, Mary-Huard T (2014) Recovering power in association mapping panels with variable levels of 769 linkage disequilibrium. Genetics 197:375-387 770 Sadras VO, Monzon JP (2006) Modelled wheat phenology captures rising temperature trends: 771 Shortened time to flowering and maturity in Australia and Argentina. Field Crops Research 99:136- 772 146 773 Sadras VO, Vadez V, Purushothaman R, Lake L, Marrou H (2015) Unscrambling confounded effects of 774 sowing date trials to screen for crop adaptation to high temperature. Field Crops Research 177:1-8 775 Saini HS, Westgate ME, Donald LS (1999) Reproductive Development in Grain Crops during Drought. 776 Advances in Agronomy. Academic Press, pp 59-96 777 Scarth R, Law CN (1983) The location of the photoperiod gene, Ppd2 and an additional genetic factor 778 for ear-emergence time on chromosome 2B of wheat. Heredity 51:607-619 779 Shah NH, Paulsen GM (2003) Interaction of drought and high temperature on photosynthesis and 780 grain-filling of wheat. Plant and Soil 257:219-226 781 Sharma DK, Torp AM, Rosenqvist E, Ottosen C-O, Andersen SB (2017) QTLs and Potential Candidate 782 Genes for Heat Stress Tolerance Identified from the Mapping Populations Specifically Segregating for 783 Fv/Fm in Wheat. Frontiers in Plant Science 8 784 Sharma RC, Tiwary AK, Ortiz-Ferrara G (2008) Reduction in kernel weight as a potential indirect 785 selection criterion for wheat grain yield under terminal heat stress. Applied Genetics 125:625-645 Field Crops Research 177:1-8 775 Saini HS, Westgate ME, Donald LS (1999) Reproductive Development in Grain Crops during Drought. 776 Advances in Agronomy. Academic Press, pp 59-96 777 vances in Agronomy. Academic Press, pp 59-96 Scarth R, Law CN (1983) The location of the photoperiod gene, Ppd2 and an additional genetic factor 778 for ear-emergence time on chromosome 2B of wheat. Heredity 51:607-619 779 Shah NH, Paulsen GM (2003) Interaction of drought and high temperature on photosynthesis and 780 grain-filling of wheat. Plant and Soil 257:219-226 781 Sharma DK, Torp AM, Rosenqvist E, Ottosen C-O, Andersen SB (2017) QTLs and Potential Candidate 782 Genes for Heat Stress Tolerance Identified from the Mapping Populations Specifically Segregating for 783 Fv/Fm in Wheat. Frontiers in Plant Science 8 784 Sharma DK, Torp AM, Rosenqvist E, Ottosen C-O, Andersen SB (2017) QTLs and Potential Candidate 782 Genes for Heat Stress Tolerance Identified from the Mapping Populations Specifically Segregating for 783 Fv/Fm in Wheat. Frontiers in Plant Science 8 784 Smith A, Cullis B, Thompson R (2001) Analyzing variety by environment data using multiplicative 796 mixed models and adjustments for spatial field trend. Biometrics 57:1138-1147 797 Smith AB, Cullis BR, Thompson R (2005) The analysis of crop cultivar breeding and evaluation trials: 798 an overview of current mixed model approaches. The Journal of Agricultural Science 143:449-462 799 Stone PJ, Nicolas ME (1995) Effect of timing of heat stress during grain filling on two wheat varieties 800 differing in heat tolerance. I. grain growth. Australian Journal of Plant Physiology 22:927-934 801 Su Z, Hao C, Wang L, Dong Y, Zhang X (2011) Identification and development of a functional marker 802 of TaGW2 associated with grain weight in bread wheat (Triticum aestivum L.). Theoretical and 803 Applied Genetics 122:211-223 804 Sun X-Y, Wu K, Zhao Y, Kong F-M, Han G-Z, Jiang H-M, Huang X-J, Li R-J, Wang H-G, Li S-S (2008) QTL 805 analysis of kernel shape and weight using recombinant inbred lines in wheat. Euphytica 165:615 806 Tadesse W, Suleiman S, Tahir I, Sanchez-Garcia M, Jighly A, Hagras A, Thabet S, Baum M (2019) Heat- 807 Tolerant QTLs Associated with Grain Yield and Its Components in Spring Bread Wheat under Heat- 808 Stressed Environments of Sudan and Egypt. Applied Genetics 125:625-645 Crop Science 59:199-211 809 Talukder ASMHM, McDonald GK, Gill GS (2013) Effect of short-term heat stress prior to flowering 810 and at early grain set on the utilization of water-soluble carbohydrate by wheat genotypes. Field 811 Crops Research 147:1-11 812 Talukder ASMHM, McDonald GK, Gill GS (2014a) Effect of short-term heat stress prior to flowering 813 and early grain set on the grain yield of wheat. Field Crops Research 160:54-63 814 Talukder SK, Babar MA, Vijayalakshmi K, Poland J, Prasad PVV, Bowden R, Fritz A (2014b) Mapping 815 QTL for the traits associated with heat tolerance in wheat (Triticum aestivumL.). BMC Genetics 15:97 816 Taylor J, Butler D (2017) R package ASMap: efficient genetic linkage map construction and diagnosis. 817 Journal of Statistical Software 79 818 Taylor J, Verbyla A (2011) R Package wgaim: QTL Analysis in Bi-Parental Populations Using Linear 819 Mixed Models. Journal of Statistical Software 40 820 Telfer P, Edwards J, Bennett D, Ganesalingam D, Able J, Kuchel H (2018) A field and controlled 821 environment evaluation of wheat (Triticum aestivum) adaptation to heat stress. Field Crops 822 Research 229:55-65 823 Telfer P, Edwards J, Norman A, Bennett D, Smith A, Able JA, Kuchel H (2021) Genetic analysis of 824 wheat (Triticum aestivum) adaptation to heat stress. Theoretical and Applied Genetics 134:1387- 825 1407 826 Tewolde H, Fernandez CJ, Erickson CA (2006) Wheat Cultivars Adapted to Post-Heading High 827 Temperature Stress. Journal of Agronomy and Crop Science 192:111-120 828 Thistlethwaite RJ, Tan DKY, Bokshi AI, Ullah S, Trethowan RM (2020) A phenotyping strategy for 829 evaluating the high-temperature tolerance of wheat. Field Crops Research 255:107905 830 Tsilo TJ, Hareland GA, Simsek S, Chao S, Anderson JA (2010) Genome mapping of kernel 831 characteristics in hard red spring wheat breeding lines. Theor Appl Genet 121:717-730 832 Tura H, Edwards J, Gahlaut V, Garcia M, Sznajder B, Baumann U, Shahinnia F, Reynolds M, Langridge 833 P, Balyan HS, Gupta PK, Schnurbusch T, Fleury D (2020) QTL analysis and fine mapping of a QTL for 834 yield-related traits in wheat grown in dry and hot environments. Theoretical and Applied Genetics 835 133:239-257 836 Verbyla AP, Cullis BR, Thompson R (2007) The analysis of QTL by simultaneous use of the full linkage 837 map. Applied Genetics 125:625-645 Theoretical and Applied Genetics 116:95 838 Vijayalakshmi K, Fritz AK, Paulsen GM, Bai G, Pandravada S, Gill BS (2010) Modeling and mapping 839 QTL for senescence-related traits in winter wheat under high temperature. Molecular Breeding 840 26:163-175 841 Wahid A, Gelani S, Ashraf M, Foolad MR (2007) Heat tolerance in plants: An overview. Environmental 842 and Experimental Botany 61:199-223 843 Wardlaw I (1994) The Effect of High Temperature on Kernel Development in Wheat: Variability 844 Related to Pre-Heading and Post-Anthesis Conditions. Functional Plant Biology 21:731-739 845 Yu M, Mao S-L, Hou D-B, Chen G-Y, Pu Z-E, Li W, Lan X-J, Jiang Q-T, Liu Y-X, Deng M, Wei Y-M (2018) 846 Analysis of contributors to grain yield in wheat at the individual quantitative trait locus level. Plant 847 Breeding 137:35-49 848 Zahedi M, McDonald G, Jenner C, F. (2004) Differential responses to high temperatures of starch and 849 nitrogen accumulation in the grain of four cultivars of wheat. Australian Journal of Agricultural 850 Research 55:551-564 851 Zhang J, Gizaw SA, Bossolini E, Hegarty J, Howell T, Carter AH, Akhunov E, Dubcovsky J (2018) 852 Identification and validation of QTL for grain yield and plant water status under contrasting water 853 treatments in fall-sown spring wheats. Theoretical and Applied Genetics 131:1741-1759 854 Zheng B, Chenu K, Fernanda Dreccer M, Chapman SC (2012) Breeding for the future: what are the 855 potential impacts of future frost and heat events on sowing and flowering time requirements for 856 Australian bread wheat (Triticum aestivium) varieties? Global Change Biology 18:2899-2914 857 alukder ASMHM, McDonald GK, Gill GS (2014a) Effect of short-term heat stress prior to flowerin nd early grain set on the grain yield of wheat. Field Crops Research 160:54-63 Telfer P, Edwards J, Bennett D, Ganesalingam D, Able J, Kuchel H (2018) A field and controlled 821 environment evaluation of wheat (Triticum aestivum) adaptation to heat stress. Field Crops 822 Research 229:55-65 823 Telfer P, Edwards J, Norman A, Bennett D, Smith A, Able JA, Kuchel H (2021) Genetic analysis of 824 wheat (Triticum aestivum) adaptation to heat stress. Theoretical and Applied Genetics 134:1387- 825 1407 826 Tewolde H, Fernandez CJ, Erickson CA (2006) Wheat Cultivars Adapted to Post-Heading High 827 Temperature Stress. Journal of Agronomy and Crop Science 192:111-120 828 Thistlethwaite RJ, Tan DKY, Bokshi AI, Ullah S, Trethowan RM (2020) A phenotyping strategy for 829 evaluating the high-temperature tolerance of wheat. Applied Genetics 125:625-645 Field Crops Research 255:107905 830 Tsilo TJ, Hareland GA, Simsek S, Chao S, Anderson JA (2010) Genome mapping of kernel 831 characteristics in hard red spring wheat breeding lines. Theor Appl Genet 121:717-730 832 Tura H, Edwards J, Gahlaut V, Garcia M, Sznajder B, Baumann U, Shahinnia F, Reynolds M, Langridge 833 P, Balyan HS, Gupta PK, Schnurbusch T, Fleury D (2020) QTL analysis and fine mapping of a QTL for 834 yield-related traits in wheat grown in dry and hot environments. Theoretical and Applied Genetics 835 133:239-257 836 Vijayalakshmi K, Fritz AK, Paulsen GM, Bai G, Pandravada S, Gill BS (2010) Modeling and mapping 839 QTL for senescence-related traits in winter wheat under high temperature. Molecular Breeding 840 26:163-175 841 858 Figures and Tables 859 Table 1. The DH populations evaluated in this study, summarising population parents and genetic map 860 details. 861 Population Name Pedigree No. lines in Map No. Polymorphic SNP markers No. Unique positions Genetic length (cM) Mean interval* MG MACE/GLADIUS 176 5047 1429 3009 2.1 SM SCOUT/MACE 226 4950 1360 3030 2.2 SG SCOUT/GLADIUS 369 5143 1761 2998 1.7 RG RAC1548/GLADIUS 132 5133 1183 3055 2.6 L2G AUS17840/GLADIUS 124 5514 1132 3144 2.8 * Mean interval (cM) between unique map positions 862 863 Table 1. The DH populations evaluated in this study, summarising population parents and genetic map 860 d il 861 Table 1. The DH populations evaluated in this study, summarising population parents and genetic map 860 details. 861 Table 1. The DH populations evaluated in this study, summarising population parents and genetic map 860 details. 861 Population Name Pedigree No. lines in Map No. Polymorphic SNP markers No. Unique positions Genetic length (cM) Mean interval* Table 2. The field experiments conducted as a part of the study. Summarised by location, the populations and number of lines included in each experimen 864 experiment dimensions, sowing date, mean anthesis date for each experiment, and mean maximum daily temperature, number of days >30°C, number o 865 days >35°C during anthesis and grain fill. 866 Table 2. The field experiments conducted as a part of the study. Summarised by location, the populations and number of lines included in each experiment, 864 experiment dimensions, sowing date, mean anthesis date for each experiment, and mean maximum daily temperature, number of days >30°C, number of 865 days >35°C during anthesis and grain fill. Table 1. The DH populations evaluated in this study, summarising population parents and genetic map 860 d il 861 866 y g g ment Year Population Location GPS position Plots Columns Rows Reps DH Genotypes Check Genotypes Sowing Date Mean Anthesis Date Cd.AD Mean Grain Yield kgha-1 May - Oct Rainfall Mean Anthesis Average Maximum Temperature (°C) Mean Anthesis Number of Days >30°C Mean Grain Fill Average Maximum Temperature (°C) Mean Grain Fill Number of Days >30°C Grain Fill Number of Days >35°C M151 2015 MG, SM, SG Angas Valley -34.75, 139.27 1296 12 108 1.4 922 7 15 May 2015 1451 2277 102 24.3 5.0 31.9 16.7 7.9 M161 2016 MG, SM, SG Angas Valley -34.70, 139.25 336 24 14 1.37 245 7 1 June 2016 1511 3379 221 20.8 2.0 26.0 8.8 0.9 M152 2015 MG, SM, SG Roseworthy -34.51, 138.68 1296 12 108 1.4 922 7 21 May 2015 1453 2843 190 24.2 4.2 30.6 10.8 6.0 M162 2016 MG, SM, SG Roseworthy -34.50, 138.68 336 24 14 1.37 245 7 15 May 2016 1511 6203 480 20.9 0.0 23.1 1.8 0.0 M153 2015 MG, SM, SG Winulta -34.30, 137.90 1296 12 108 1.4 922 7 12 May 2015 1458 2591 208 22.8 3.0 28.1 11.1 4.5 M163 2016 MG, SM, SG Winulta -34.26, 137.90 336 24 14 1.37 245 7 18 May 2016 1510 6972 381 19.5 0.0 23.2 2.9 0.0 2151 2015 L2G Angas Valley -34.750, 139.27 216 12 18 1.52 142 8 15 May 2015 1379 1644 102 22.5 3.0 31.9 16.8 7.6 2161 2016 L2G Angas Valley -34.70, 139.25 144 24 6 1.67 86 9 1 June 2016 1555 3298 221 21.5 2.6 26.5 9.3 1.4 2152 2015 L2G Roseworthy -34.51, 138.68 216 12 18 1.52 142 8 22 May 2015 1387 2489 190 22.8 3.0 30.8 12.2 7.2 2162 2016 L2G Roseworthy -34.50, 138.68 144 24 6 1.67 86 9 15 May 2016 1556 6226 480 20.8 0.0 23.6 2.5 0.0 2153 2015 L2G Winulta -34.30, 137.90 216 12 18 1.52 142 8 12 May 2015 1382 2447 208 21.5 1.3 28.0 11.5 5.1 2163 2016 L2G Winulta -34.26, 137. Table 1. The DH populations evaluated in this study, summarising population parents and genetic map 860 d il 861 90 144 24 6 1.67 86 9 18 May 2016 1552 6734 381 19.6 0.0 23.5 3.3 0.3 151 2015 RG Angas Valley -34.75, 139.27 240 12 20 1.49 161 8 15 May 2015 1380 2181 102 22.4 2.8 32.2 17.1 7.8 161 2016 RG Angas Valley -34.70, 139.25 192 24 8 1.62 117 8 1 June 2016 1565 3540 221 21.6 2.7 26.6 9.3 1.5 152 2015 RG Roseworthy -34.51, 138.68 240 12 20 1.49 161 8 22 May 2015 1379 2738 190 22.5 2.5 31.0 12.4 7.5 162 2016 RG Roseworthy -34.50, 138.68 192 24 8 1.62 117 8 15 May 2016 1566 7202 480 20.7 0.0 23.8 2.6 0.0 4153 2015 RG Winulta -34.30, 137.90 240 12 20 1.49 161 8 12 May 2015 1381 3043 208 21.4 1.1 27.9 11.7 5.1 4163 2016 RG Winulta -34.26, 137.90 192 24 8 1.62 117 8 18 May 2016 1567 7903 381 19.6 0.0 23.7 3.4 0.4 Note: the 2015 GSM experiments consisted of 154 MG, 203 SM and 360 SG lines. In 2016 there were 42 MG, 44 SM and 92 SG lines. In each year the remaining plots in each experiment were made up of material not used in the analysis for 67 this study. 68 869 Figure 1. QTL identified for performance and responsiveness to the climatic co-variates measured in 870 each environment mapped against their position (cM) on the consensus map. Colours indicate QTL 871 type and to which climatic co-variate the responsiveness QTL interact. 872 Figure 1. QTL identified for performance and responsiveness to the climatic co-variates measured in 870 each environment mapped against their position (cM) on the consensus map. Colours indicate QTL 871 type and to which climatic co-variate the responsiveness QTL interact. 872 type and to which climatic co-variate the responsiveness QTL interact. Table 1. The DH populations evaluated in this study, summarising population parents and genetic map 860 d il 861 Legend Performance Responsive - Growing season rainfall (mm) Responsive - Anthesis average maximum temperature (°C) Responsive - Anthesis number of days >30°C Responsive - Grain fill average maximum temperature (°C) Responsive - Grain fill number of days >30°C Responsive - Grain fill number of days >35°C yp p Q yp p Q 873 873 Legend Performance Responsive - Growing season rainfall (mm) Responsive - Anthesis average maximum temperature (°C Responsive - Anthesis number of days >30°C Responsive - Grain fill average maximum temperature (°C Responsive - Grain fill number of days >30°C Responsive - Grain fill number of days >35°C Legend Performance Responsive - Growing season rainfall (mm) Responsive - Anthesis average maximum temperature Responsive - Anthesis number of days >30°C Responsive - Grain fill average maximum temperature Responsive - Grain fill number of days >30°C Responsive - Grain fill number of days >35°C Legend Performance Responsive - Growing season rainfall (mm) Responsive - Anthesis average maximum temperature (°C) Responsive - Anthesis number of days >30°C Responsive - Grain fill average maximum temperature (°C) Responsive - Grain fill number of days >30°C Responsive - Grain fill number of days >35°C Responsive Anthesis average maximum temp Responsive - Anthesis number of days >30°C Responsive - Grain fill average maximum temp Responsive - Grain fill number of days >30°C Responsive - Grain fill number of days >35°C Figure 2. To demonstrate the relationship between performance and responsiveness QTL, an example of TWT 874 responsiveness QTL for various co-variates found to cluster with the TWT performance QTL QTwt.agt-L2G.4A, 875 on chromosome 4A, are shown. For each responsiveness QTL, the additive effect on TWT for the RAC1548 allele 876 is shown for the range observed for each climatic co-variate for; (A) QTwt.agt-RG.4A-1, (B) QTwt.agt-RG.4A-2, 877 (C) QTwt.agt-RG.4A-3, (D) QTwt.agt-RG.4A-4 and (E) QTwt.agt-RG.4A-5. (F) Illustrates the impact on 878 responsiveness by selecting for either the favourable performance allele (Gladius shown by open dots) or the 879 alternative allele (RAC1548 allele represented by closed dots). (F) can be interpreted within the framework 880 proposed by Telfer et al. (2021) shown in (G). 881 Figure 2. To demonstrate the relationship between performance and responsiveness QTL, an example of TWT 874 responsiveness QTL for various co-variates found to cluster with the TWT performance QTL QTwt.agt-L2G.4A, 875 on chromosome 4A, are shown. Table 1. The DH populations evaluated in this study, summarising population parents and genetic map 860 d il 861 For each responsiveness QTL, the additive effect on TWT for the RAC1548 allele 876 is shown for the range observed for each climatic co-variate for; (A) QTwt.agt-RG.4A-1, (B) QTwt.agt-RG.4A-2, 877 (C) QTwt.agt-RG.4A-3, (D) QTwt.agt-RG.4A-4 and (E) QTwt.agt-RG.4A-5. (F) Illustrates the impact on 878 responsiveness by selecting for either the favourable performance allele (Gladius shown by open dots) or the 879 alternative allele (RAC1548 allele represented by closed dots). (F) can be interpreted within the framework 880 proposed by Telfer et al. (2021) shown in (G). 881 F B D G A C E Legend Growing season rainfall (mm) Grain fill average maximum temperature (°C) Anthesis average maximum temperature (°C) Grain fill number of days >30°C Anthesis number of days >30°C Grain fill number of days >35°C Low performance +’ve response High performance +’ve response High performance -’ve response Low performance -’ve response A F Legend Low performance +’ve response High performance +’ve response High performance -’ve response Low performance -’ve response F B B B C C G G G D D E E SupplementaryMaterial.xlsx Supplementary Files This is a list of supplementary ¦les associated with this preprint. Click to download. SupplementaryMaterial.xlsx
W2102219322.txt	http://www.scielo.br/pdf/rlae/v14n5/v14n5a18.pdf	en		A EDUCAÇÃO DE ENFERMAGEM: BUSCANDO A FORMAÇÃO CRÍTICO-REFLEXIVA E AS COMPETÊNCIAS PROFISSIONAIS	null	2,006	cc-by	4,212	Rev Latino-am Enfermagem 2006 setembro-outubro; 14(5):755-61 www.eerp.usp.br/rlae Nursing education: seeking... Silva KL, Sena RR. 755 NURSING EDUCATION: SEEKING CRITICAL-REFLEXIVE EDUCATION AND PROFESSIONAL COMPETENCIES Kênia Lara Silva1 Roseni Rosângela de Sena2 Silva KL, Sena RR. Nursing education: seeking critical-reflexive education and professional competencies. Rev Latino-am Enfermagem 2006 setembro-outubro; 14(5):755-61. The study describes changes that are noted in students during training and which contribute to define a professional profile. We carried out a descriptive-exploratory study with a qualitative approach, based on dialectics as a theoretical-methodological framework. The data was obtained from documented analysis and through focal groups with teachers, students and service nurses. The results show the student’s position as an active subject in the teaching-learning process, through a movement of transformation of academic structures. A correlation was found between the movement that seeks students’ greater political, active and critical participation as a way of determining and guiding the profile of the generalist nurse and greater social insertion. We conclude that, despite efforts, training guidelines and the definition of the professional profile in the study settings is directed at the demands of the labor market, and that competency-based training is still incipient. DESCRIPTORS: education; nursing professional competence; problem-based learning LA EDUCACIÓN DE ENFERMERÍA: BÚSQUEDA DE LA FORMACIÓN CRÍTICA Y REFLEXIVA Y DE LAS COMPETENCIAS PROFESIONALES El estudio describe los cambios en los estudiantes durante la formación y que apuntan para la definición de un perfil profesional. Estudio descriptivo exploratorio con aproximación cualitativa, que utilizase la dialéctica como referencial teórico-metodológico. Los datos fueran obtenidos con documentación y grupos focales. Los resultados indican la posición del estudiante como sujeto activo en el proceso enseñanza-aprendizaje desde el movimiento de cambio de las estructuras académicas. Se identificó una correlación entre el movimiento de búsqueda de mayor participación política, activa y crítica de los estudiantes como factor que determina y orienta un perfil del enfermero generalista y de mayor inserción social. Se concluye que, no obstante los esfuerzos, la orientación de la formación y la definición del perfil profesional en los escenarios del estudio están dirigidas a las normas del mercado de trabajo, siendo insuficiente la formación baseada en competencias. DESCRIPTORES: educación en enfermería; competencia profesional; aprendizaje basado en problemas A EDUCAÇÃO DE ENFERMAGEM: BUSCANDO A FORMAÇÃO CRÍTICO-REFLEXIVA E AS COMPETÊNCIAS PROFISSIONAIS O estudo descreve resultados de uma pesquisa que retrata mudanças que são percebidas nos estudantes durante a formação e que contribuem para a definição de um perfil profissional. Estudo descritivo-exploratório, com abordagem qualitativa, que se ancora na dialética como referencial teórico-metodológico. Os dados foram obtidos de análise documental e através da realização de grupos focais com docentes, estudantes e enfermeiros de serviço. Os resultados demonstram a posição do estudante como sujeito ativo no processo ensinoaprendizagem, a partir de um movimento de transformação das estruturas acadêmicas. Identificou-se correlação entre o movimento de busca de maior participação política, ativa e crítica dos estudantes como fator que determina e orienta um perfil do enfermeiro generalista e de maior inserção social. Conclui-se que, apesar dos esforços, a orientação da formação e a definição do perfil profissional nos cenários do estudo estão voltadas às exigências do mercado de trabalho, sendo incipiente a formação baseada em áreas de competências. DESCRITORES: educação em enfermagem; competência profissional; aprendizagem baseada em problemas 1 RN. M.Sc. in Nursing, Faculty, Pontifícia Universidade Católica de Minas Gerais, e-mail: kenialara17@yahoo.com.br; 2 RN, PhD in Nursing, Adjunct Professor (Retired), Minas Gerais Federal University Disponible en castellano/Disponível em língua portuguesa SciELO Brasil www.scielo.br/rlae Nursing education: seeking... Silva KL, Sena RR. Rev Latino-am Enfermagem 2006 setembro-outubro; 14(5):755-61 www.eerp.usp.br/rlae INTRODUCTION 756 Health workers’ training, inserted in the context of professional training, must be guided by T eaching-learning experiences are the definition of competency areas (knowledge, skills determined by the political, social, cultural and and attitudes) that allow for multiprofessional action economic context they are inserted in. The modern and interaction. General guidelines for health world requires professionals to have a multivalent professional education in the 21 century state that training, oriented towards the globalizing view of competency development should be oriented towards (1) reality and a permanent attitude of learning to learn st . the search for health care integrality, contributing to The deregulation of national economies, the formation of professionals who join decision- imposed by the international division of work, making, communication, leadership, management commends the adoption of privatization programs in and permanent education aptitudes(4). the public sector, including in health and education. In order to cope with rapidly emerging and These transformations change work relations which, modifying demands in health professional training, in combination with technological innovations, impose there is a need for changes in the teaching-learning new relations in the labor world and, consequently, process, adapting it to contemporary reality and to new requirements in terms of workers’ profile (2) . In the health sector, transformations occur in the complexity and unforeseeability characteristic of health work. work organization, entailing repercussions for Preparing professionals for the labor world technological incorporation, associated with changes requires the development of knowledge, ideas, in the Brazilian population’s epidemiological profile abilities and, also, dispositions, attitudes, interests and and context, behaviors. These should adapt to the possibilities and modifications should be acknowledged which derive demands of work modes and the way they are from the implantation of new technological and care organized(5). demographic pattern. In this models, posing demands in terms of professional profile. In this perspective, significant mobilization efforts have been made to transform professional In Brazil, these modifications occur in the training models, seeking to integrate the university framework of the process to consolidate the Single with segments of civil society and communities, in a Health System - SUS, including efforts to take form partnership that raises the potential of alternatives in ethical, theoretical, organizational and operative for pedagogical and organizational changes and principles related to health, defined as a right of all institutional interactions. This is necessary for the citizens and a duty of the State, which is responsible academy to demonstrate its relevance in the social for assuring social and economic public policies that context and to allow for student training based on guarantee the population’s physical, mental and social concrete, real problems. well-being. Attempts to revert traditional teaching models The theories and principles guiding the SUS demonstrate that different actors must participate in are addressed in Decree 8.080(3), especially: universal the definition of a professional profile: graduates, health service access at all care levels; care integrality; faculty, students, service professionals, health service preservation of people’s autonomy in defense of their managers, members of class organizations and civil physical and moral integrity; equality of health care, society, in a movement that guides training and the without any kind of prejudices or privileges; political- definition of the professional profile, based on administrative decentralization, with one single competency areas. management sphere; Defining competencies is a hard task. It is regionalization and hierarchization of the health in each government important to move from the analysis of practices to a service network; problem-solving capacity of services reasonable inventory of competencies considered at all care levels. essential, In the attempt to consolidate the SUS, efforts profession (6) which constitute the body of the . have been made to construct a model that gives social A competency is defined as the aptitude to responses to health problems and needs, in view of cope with a series of analogue situations, correctly, Brazil’s heterogeneity and political, economic and rapidly, pertinently and creatively mobilizing several cultural diversity. cognitive resources: knowledge; skills; micro- Rev Latino-am Enfermagem 2006 setembro-outubro; 14(5):755-61 www.eerp.usp.br/rlae Nursing education: seeking... Silva KL, Sena RR. competencies; information; values; attitudes; and perception, assessment and reasoning schemes (7) . 757 Marília Medical School, Bahia Federal University and Rio Grande do Norte Federal University. In terms of health professionals, besides this The Project presented a proposal to teaching concept, the need should be added to incorporate a and service institutions and their actors, to give up prospective analysis of professional practices, in the existing paradigm in capitalist society, in which contexts of technological innovations, changes in professionals are educated to attend to needs related health services and in the population’s epidemiological to profit and capital accumulation, and prioritize human profile and demographic pattern. needs, the population’s health needs and the In the context of these transformations, the improvement of sanitary environments, assuming the notion of competency emerges to reorder the commitment to train professionals who are willing to understanding of the work/education relation, work towards quality of life as a whole, without deviating the focus from employments and tasks to a abandoning the valuation of the dialectic construction reference framework centered on human praxis and of the health-disease process(12). increasing the potential of worker emancipation actions(8-9). We decided to adopt a qualitative approach, based on dialectics(13) as a theoretical-methodological Applying the notion of competency implies reference framework, which reveals a belief in the institutionalizing new forms of educating / training movement process that permanently exists in society, workers and managing organizations and the labor as well as in the historical, cultural and social market internally, in view of economic-productive, construction of health professionals’ education social-historical, cultural and political mediations in the processes and in the capacity to transform and determination of the teaching-learning process (8-9) . overcome contradictions through new practices. Thus, we defend that the nursing training In order to capture reality, we used primary model should be anchored in the reference framework data, collected through the realization of a three-hour of critical-reflexive pedagogy (1,10-11) , contributing to the focus group (14) in each scenario. In total, 27 faculty, construction of professional competencies that allow eight students and eight service professionals for actions focused on integral care. participated. Their discourse was recorded, transcribed and then submitted to Discourse Analysis (13,15) . Secondary data were obtained from reports and METHODOLOGY documents made available by the research institutions, which helped us to understand the change This study presents partial results of the process in nursing training at each of the four research “UNI Project as the scenario for new institutions. In the results section, discourse is experiences in the transformation of nursing identified by the letter C and a number (1, 2, 3 or 4), education”* realized by researchers from the Study which randomly indicate the research scenarios. and Research Group on Nursing Teaching and Practice Before starting data collection in the field, the - NUPEPE at Minas Gerais Federal University between research project was approved by the Ethics and 2000 and 2003. In this research, we attempted to Research Committee at Minas Gerais Federal analyze the change process in nursing education University - COEP/UFMG, in accordance with experienced by nursing courses at Brazilian institutions Resolution 196/96 by the National Research Ethics that developed the UNI Project. Commission. Participants’ testimonies were only The UNI Project - A New Initiative in Health Professional Education, a political-pedagogical recorded after they agreed to participate and signed the Free and Informed Consent Term. program that had been encouraging innovations in This study describes the changes manifested health professional formation since the early 1990’s, and/or perceived during the student training process in partnership with health services and the community, and which can be demonstrated as elements was developed in Brazil by the following higher contributing to the definition of a professional profile. education institutions: Londrina State University, We attempted to describe the change movement in * Research project funded by the Minas Gerais State Research Support Foundation - FAPEMIG Nursing education: seeking... Silva KL, Sena RR. Rev Latino-am Enfermagem 2006 setembro-outubro; 14(5):755-61 www.eerp.usp.br/rlae 758 nursing training, indicating shared points in subjects’ for future professionals to attend to a new dynamics thinking and practice in each scenario, which in health work (C3), requiring professionals to be able supported the construction of the empirical category: to act in the progressive health care network, “From critical student to generalist nurse” and how mobilizing knowledge in individual and collective this movement has contributed to the definition of preventive and curative actions, in order to guarantee the professional competency area. health care quality (C4). In scenario 1, participants indicated that changes in teaching occurred to attend to nurses’ RESULTS AND DISCUSSION training needs to work in the Family Health Program. Subjects’ discourse in this scenario displayed constant The focus group participants’ discourse in the concern about inverting the hospital and disease- four scenarios revealed, at these institutions, the centered model, to construct a health model based student’s position as an active subject in the teaching- on social health production. learning process, with room to take positions and The Family Health Program is a strategy to propose changes. This condition has been facilitated reorganize basic care in Brazil, whose actions are by academic structure transformations, by the directed to the needs of families within its territory, construction of a political-pedagogical project based as a geopolitical space, in a continuous, tailor-made on critical and reflexive reference frameworks, the and active way; emphasizing health promotion and adoption of active teaching methodologies, the problem prevention, but without ignoring the curative- expansion and diversification of teaching-learning rehabilitating focus, with a high problem-solving level; scenarios at low direct and indirect economic or social costs and attempts to adopt formative assessment. and articulated with other sectors determining health. Focus group participants indicated that the In scenario 3, participants affirmed that the changes in the teaching model contributed to the change in the care model, through the implantation construction of a political-pedagogical project in which of the Family Health Program, started to require students are considered as active subjects in the pedagogical practices that emphasize integral health construction of their own knowledge (C1), thus care, which influenced the change in the political- becoming critical, inquiring, innovative subjects with pedagogical project, incorporating new concepts and more mature attitudes (C3). new teaching practices to accompany this tendency. Participants affirmed that, during training, Participants in scenario 4 mentioned their attempts have been made to construct the profile of concern about implementing a training process to a reflexive professional, who knows the ethical, contribute to social changes, related to education and political, cultural nurses’ insertion in the labor market. They affirmed determinants of the profession (C4), in line with the that, to accompany this “pattern”, i.e. the Family Health Ministry of Education’s guidelines for nursing Program, the course has been guided by the principle education historical, (4) ideological and . Discourse in the four scenarios contained quotations that disclose the movement seeking of introducing the student into real practice scenarios of current health models, prioritizing health surveillance actions. students’ greater political, active and critical Discourse analysis reveals that training and participation, as a factor that determines and guides professional profile definition in the study scenarios a generalist nursing profile with greater social insertion. are directed at labor market requirements, while This consideration is remarkable for the training based on competency areas is in an initial reorganization of teaching and health care processes, stage. These areas go beyond the transitory and as professional training exerts positive effects, to a momentary requirements of a market and affirm the greater or lesser extent, on health service practice, construction of a set of abilities and attitudes that will in consonance with the established teaching-learning allow process. unforeseeable daily work situations. professionals to act in different and The changes described in discourse about Therefore, the nursing training process should students’ attitude reflect labor market requirements. occur in view of context dynamics and future needs This market stimulated the construction of a profile in health and education, to which professional training Rev Latino-am Enfermagem 2006 setembro-outubro; 14(5):755-61 www.eerp.usp.br/rlae Nursing education: seeking... Silva KL, Sena RR. 759 should be oriented by means of a competency area- noteworthy in the possibility to construct proposals based education, in which market demands are taken that go beyond the understanding that it is necessary into consideration. However, this factor should not be to attend to the labor market and strengthen the the priority element in the definition of the professional modus operandi of schools and pedagogical practices, profile, preparing nursing professionals not only to sustained attend to labor market demands, but mainly to transmission and conditioning. transform the conditions imposed by this market. by the pedagogical conception of Therefore, the teaching-learning process Focus group participants mentioned that must be assumed on the basis of a critical-reflexive professional training is excessively privileging pedagogical conception that guides methodological knowledge of social areas, to the detriment of options directed at changing the centralism of the knowledge about technical areas. They indicate that teaching-learning process in the teacher, in order to trained nurses are professionals who are very critical transfer it, make it accountable and establish a new with respect to social problems, but do not master meaning in the student(11,17). the technical knowledge and instruments needed to develop nursing practice (C3). The change and redefinition of nurses’ professional profile has been established on the basis This finding goes against the premises of the of opportunities students are offered during the training model in force until that time, which privileged course, to act in multiprofessional activities, using the curative and individual action, based on the hospital- interdisciplinary focus in work teams active in new centered biomedical model and strongly characterized teaching-learning scenarios (C4). by a technicist orientation. However, participants Various proposals are being applied in nursing perceived that social and political issues are education, such as curricular integration, teacher-care emphasized to the detriment of technical skills integration, competency-based curriculum, aimed at development of “health clinic”. Therefore, we believe training professionals who are committed to health that the model is still fragmented and does not allow policies and at developing specific competencies and students to develop themselves simultaneously in skills to exert an impact(18). technical-scientific and political-social competency These experiences have been the fruit of areas, mobilizing them to guarantee integrality in their nursing education programs’ dissatisfaction and training and health care process. fragility at all levels. This fragility can be understood This reveals the need to restructure the as a lack of theoretical bases for action, attested by training process, adopting an integral view of the the traditional lack of reflection about why and what health-disease process and of receiving care and, for, by the obsession to innovate for the mere sake of thus, overcoming the fragmentation between biology innovation, in short, by education processes with little / social, curative / preventive, clinical / epidemiologic, adherence to reality, where the dichotomy between subjectivity / sociability in the construction of a theory and practice is still remarkable and where the teaching-learning future conception of the health and disease process professional subordinated to biological sciences and individual care professionals process develop that specific makes competencies, combining soft-hard, like clinical technology, and soft technologies like bonding, welcoming and accountability(16). is perpetually reproduced(18). Focus group participants also manifested that the financial support of the UNI Project contributed to The analysis of documents from the four the exercise of research methodology among students scenarios allows us to affirm that the professional (C3). The documentary analysis also reveals that profile expressed in the political-pedagogical project support was directed at strategic-institutional plans, was designed on the basis of an analysis of the labor constructed by the teaching / service partnership to market, of the population’s demands and health needs design change processes. and future projections about nurses’ insertion into Documentary data from the four scenarios health service production scenarios, oriented towards affirmed that the approximation between teaching and the consolidation of the Single Health System. research is part of mechanisms to try and integrate However, schools continue structuring their curricula basic and professional teaching and articulate theory by contents and objectives, which is characteristic of and practice, contributing to the definition of a traditional teaching models. This difference seems professional profile that integrates knowledge from Nursing education: seeking... Silva KL, Sena RR. Rev Latino-am Enfermagem 2006 setembro-outubro; 14(5):755-61 www.eerp.usp.br/rlae different disciplines and destructures the dichotomy 760 FINAL CONSIDERATIONS between teaching and care practice. In line with this analysis, practice analysis The research results disclosed that adopting and research are training methods that allow for the a critical-reflexive focus in the training process can construction the affect the health and nursing work process, being able development of a meta-competency: knowing how to affirm care delivery by subjects with values, culture to analyze(7). and ideology, committed to solving the population’s of professionalism, through This seems to be a differentiating element in and health services’ concrete health problems. the search for critical-reflexive training, in which Therefore, this movement should not only be guided learning is the fundamental attribute in the relations by technical and instrumental rationality, but basically between the subjects involved in interactions in the by new possibilities for communication, organization teaching-learning process, through students’ active as well as intersubjective and care relations. participation, the problematization of reality and the theory/practice articulation, in a permanent movement of learning to learn. Thus, we need to think about ways to flexibilize the teaching system, constructing processes that help to structure proposals that are more adapted to In this perspective, students can construct requirements in the modern and future world of knowledge based on problems from concrete reality, uncertainties. This more flexible teaching system should articulating knowledge from different areas, based be manifested by the introduction of new contents but, on interdisciplinarity and on the integration of contents mainly, it should give a meaning to students as subjects and actions. This training has the potential to support who need to be capable of thinking creatively, have actions in the complexity and unforeseeability that self-esteem, can face professional changes and are characteristic of daily health work. construct their own network of beliefs and values. The analysis of focus group participants’ This indicates the need to reorient professional discourse reveals that the teaching / work relation nursing training towards solving concrete and real stimulates transformations in teaching which are problems, as an alternative to develop the capacity nourished by the reorganization of the health care to create, recreate and project new things from the model, i n t e g r a l i t y. perspective of creative practice, allowing for subjects’ Participants revealed that the relation between development in their multiple dimensions: social, teaching historical, cultural and holistic. in the and search service for has care been a positive experience for all actors, allowing for the training Thus, we consider that nursing education of professionals who are in line with the reality of should distinguish itself by the search for integral care, health service and with the population’s health constructed during training, on the basis of critical- demands and needs, considering the complexity reflexive references in the definition of professional and transformations of health work. nursing competencies. 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Sena-Chompré RR, Egry EY. A Enfermagem nos Projetos UNI: contribuição para um novo projeto político para a Enfermagem Brasileira. São Paulo (SP): Hucitec; 1998. 13. Minayo MCS. O desafio do conhecimento: pesquisa qualitativa em saúde. 8 ed. São Paulo (SP): Hucitec; 2004. 14. Dall’agnol CM, Trench MH. Grupos focais como estratégia metodológica em pesquisas na enfermagem. Rev Gaúch Enfermagem 1999 janeiro; 20(1):5-25. 15. Bourdieu P, organizador. A miséria do mundo. 2 ed. Petrópolis (RJ): Vozes; 1998. 16. Merhy EE. Saúde: a cartografia do trabalho vivo. São Paulo (SP): Hucitec; 2002. 17. Vilela EM, Mendes IJM. Interdisciplinaridade e saúde: estudo bibliográfico. Rev Latino-am Enfermagem 2003 julhoagosto; 11(4):525-31. 18. Takahashi OC, organizadora. Formação de recursos humanos em enfermagem na América Latina e Caribe. Documento apresentado à Rede Latino-americana de Enfermería - REAL, Londrina (PR): UEL; 2001. Recebido em: 25.11.2004 Aprovado em: 12.7.2006 Nursing education: seeking... 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https://openalex.org/W3177084595	https://www.frontiersin.org/articles/10.3389/fpsyg.2021.657705/pdf	English	null	Parsing Model and a Rational Theory of Memory	Frontiers in psychology	2,021	cc-by	15,415	Parsing Model and a Rational Theory of Memory Jakub Dotla ˇcil 1* and Puck de Haan 2 1 Utrecht Institute of Linguistics, Utrecht University, Utrecht, Netherlands, 2 Artiﬁcial Intelligence, University of Amsterdam, Amsterdam, Netherlands This paper explores how the rational theory of memory summarized in Anderson (1991) can inform the computational psycholinguistic models of human parsing. It is shown that transition-based parsing is particularly suitable to be combined with Anderson’s theory of memory systems. The combination of the rational theory of memory with the transition-based parsers results in a model of sentence processing that is data-driven and can be embedded in the cognitive architecture Adaptive Control of Thought-Rational (ACT-R). The predictions of the parser are tested against qualitative data (garden-path sentences) and a self-paced reading corpus (the Natural Stories corpus). Keywords: computational psycholinguistics, cognitively constrained parsers, memory retrieval, rational theory of memory, modeling reading data Reviewed by: John Hale, University of Georgia, United States Christian Huyck, Middlesex University, United Kingdom Richard Futrell, University of California, Irvine, United States One particularly successful case of the rational theory was its application to the study of human memory, as summarized in Anderson (1991). Assuming that the human memory should strive to provide information that is needed at a particular situation and that it is costly and takes time to retrieve elements from memory, we would expect that the retrieval of an element be related to the probability that it is needed. That is, elements that are most likely to be needed at a particular situation will be prioritized in retrieval. Since retrieval is ordered by need probabilities, it is expected that less needed items require more time to be recalled. Furthermore, if retrieval is abandoned when the cost for retrieval exceeds some threshold, we expect the less needed an item is, the more likely it is that its recall fails. These predictions have been largely conﬁrmed, see Anderson (1991). Specialty section: This article was submitted to Language Sciences, a section of the journal Frontiers in Psychology Received: 23 January 2021 Accepted: 03 May 2021 Published: 23 June 2021 Citation: Dotla ˇcil J and de Haan P (2021) Parsing Model and a Rational Theory Specialty section: This article was submitted to Language Sciences, a section of the journal Frontiers in Psychology The rational theory of memory played an important role in the development of the cognitive architecture Adaptive Control of Thought-Rational, ACT-R (Anderson and Lebiere, 1998; Anderson et al., 2004), which in turn played an important role in psycholinguistic models of parsing (Lewis and Vasishth, 2005; Lewis et al., 2006; Reitter et al., 2011; Engelmann et al., 2013; Vogelzang et al., 2017; Brasoveanu and Dotlaˇcil, 2020). Lewis and Vasishth (2005) and subsequent works showed, in particular, that the rational theory of memory implemented in ACT-R is insightful in analyzing the pattern of recall in forming dependencies during parsing, for example, subject-verb dependency as in (1-a) and antecedent-reﬂexive dependency as in (1-b) (see also Lewis et al., 2006; Van Dyke, 2007; Wagers et al., 2009; Dillon et al., 2013; Kush et al., 2015; Lago et al., 2015; Jäger et al., 2017; Jäger et al., 2020; Nicenboim et al., 2018; Villata et al., 2018; Engelmann et al., 2019; Vasishth et al., 2019; Smith and Vasishth, 2020, among others). Reviewed by: John Hale, Received: 23 January 2021 Accepted: 03 May 2021 Published: 23 June 2021 ORIGINAL RESEARCH published: 23 June 2021 doi: 10.3389/fpsyg.2021.657705 ORIGINAL RESEARCH 1. INTRODUCTION Edited by: Matthew W. Crocker, Saarland University, Germany In the rational theory of cognition, it is argued that cognitive functions are largely shaped by our adaptation to the environment. In this view, it is assumed that various aspects of our behavior can be explained as the result of the optimization to the structure of the environment. The rational theory of cognition has been fruitful in explaining regularities in categorization, learning, communication and reasoning, among others (Anderson, 1990, 1991; Oaksford and Chater, 1994, 2007; Tenenbaum et al., 2011; Franke and Jäger, 2016; Piantadosi et al., 2016). Saarland University, Germany Reviewed by: John Hale, University of Georgia, United States Christian Huyck, Middlesex University, United Kingdom Richard Futrell, University of California, Irvine, United States Correspondence: Jakub Dotla ˇcil Keywords: computational psycholinguistics, cognitively constrained parsers, memory retrieval, rational theory of memory, modeling reading data 1The procedural memory system stores the knowledge exhibited in automatized, sequential behavior. This type of memory plays less important role in our models of parsing. We will brieﬂy come back to it in section 4. (antecedent-reﬂexive) In ACT-R, the history component is called base- level activation, abbreviated as Bi, and the context component is called spreading activation, which we will abbreviate as Si. We can rewrite the formula as follows2: 2ACT-R activation also standardly includes noise parameter. In (5), we ignore the noise parameter ǫ, so that activation is deterministic. (antecedent-reﬂexive) (2) Example of a chunk stored and retrieved in (1-a):   Form students Function SUBJECT Number PLURAL   (2) Example of a chunk stored and retrieved in (1-a):   (2) Example of a chunk stored and retrieved in (1-a): This brings us to the research topic of this paper, namely, studying whether other aspects in which parsing has to rely on memory can also be seen as ﬁtting the research programme of the rational theory of cognition. In particular, during comprehension and production, native speakers have to continuously rely on their past knowledge of parsing rules. For instance, in (1), readers would not be able to comprehend the sentences correctly unless they recall that subjects normally precede verbs in English, verbs are followed by objects, English has prepositions (not post- positions) etc. From the perspective of the rational theory of memory, it is expected that the retrieval of parsing rules, such as these should follow the general considerations highlighted above, i.e., parsing rules should be retrieved in the order of their need probability and the order should monotonically correlate with latencies and accuracies. We will show that it is indeed possible to construct parsing on the basis of the rational theory of memory. The resulting model can furthermore correctly predict qualitative data in psycholinguistics (garden- path phenomena) and its predictions match behavioral measures in a psycholinguistic corpus (Natural Stories Corpus, Futrell et al., 2018). ( ) p   Form students Function SUBJECT Number PLURAL     Form students Function SUBJECT Number PLURAL   Assuming that retrieving a chunk is costly and takes time, retrieval from memory must be constrained. An optimal retrieval system would prioritize those chunks that are more likely needed for the current goal. In general, it should hold that the recall of a piece of information, chunk i, adjusted by the value of the current goal G should not exceed the cost of the retrieval C. (3) P(i) · G < C (3) P(i) · G < C (3) The task of the rational theory of memory is to ﬁnd a reasonable estimation of P(i). In ACT-R, it is assumed that P(i), the probability that i is needed, is conditionalized on two sources of information: (i) the history Hi, that is, the past use of i;and (ii) the current context Q. Citation: Citation: Dotla ˇcil J and de Haan P (2021) Parsing Model and a Rational Theory of Memory. Front. Psychol. 12:657705. doi: 10.3389/fpsyg.2021.657705 June 2021 \| Volume 12 \| Article 657705 Frontiers in Psychology \| www.frontiersin.org Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan (antecedent-reﬂexive) We thus need to estimate P(i\|Hi, Q), which can be easily done using Bayes’ rule. However, rather than expressing the conditional probability directly, it is standard in ACT-R to estimate log-odds. The estimation is expressed in (4) (ic is the complement of i, i.e., P(ic) is the probability that i is not needed; Q, the current context, consists of indices j, which we call cues). The structure of the paper is as follows: in the following section, we brieﬂy introduce the rational theory of memory as part of the cognitive architecture ACT-R. Next, we present transition-based parsers developed in computational linguistics and show how transition-based parsing and cognitive architectures can be combined. The cognitively informed parser is then evaluated on garden-path examples and data from Natural Stories Corpus. Finally, our research is brieﬂy compared to related works in computational psycholinguistics. (4) log( P(i\|Hi,Q) P(ic\|Hi,Q)) = log( P(i\|Hi) P(ic\|Hi) · P(Q\|i) P(Q\|ic)) = log( P(i\|Hi) P(ic\|Hi)) + log(Q j∈Q P(j\|i) P(j\|ic)) The inference in (4) makes the common assumption that while the probability that i is being needed is dependent on Hi and Q, the probabilities of the cues j in the current context Q are mutually independent and not dependent on the history Hi, conditional on i (see Anderson, 1991). The log-odds in (4) have a special status in ACT-R. They are called the activation of i, written as Ai. The activation consists of two parts: the history component [the ﬁrst addend in(4) ] and the context component [the second addend in(4) ]. In ACT-R, the history component is called base- level activation, abbreviated as Bi, and the context component is called spreading activation, which we will abbreviate as Si. We can rewrite the formula as follows2: The inference in (4) makes the common assumption that while the probability that i is being needed is dependent on Hi and Q, the probabilities of the cues j in the current context Q are mutually independent and not dependent on the history Hi, conditional on i (see Anderson, 1991). The log-odds in (4) have a special status in ACT-R. They are called the activation of i, written as Ai. The activation consists of two parts: the history component [the ﬁrst addend in(4) ] and the context component [the second addend in(4) ]. (1) Example of dependencies, signaled by arrows. a. Students rarely know the answer. (subject-verb) b. The man told the woman about himself. (antecedent-reﬂexive) (1) Example of dependencies, signaled by arrows. a. Students rarely know the answer. (subject-verb) b. The man told the woman about himself. (antecedent-reﬂexive) a chunk, representing a simpliﬁed piece of information retrieved in the dependency in (1-a), is shown in (2). In this notation, slot names appear on the left side and their values on the right side. The chunk represents the knowledge that a plural subject of the form students was encountered and stored in memory. 2. MODELING MEMORY RETRIEVAL IN RATIONAL THEORY Adaptive Control of Though-Rational assumes, true to its name, that various cognitive functions should be modeled as a case of rational theory of cognition. Here, we will focus on how memory and memory retrieval are formalized in ACT-R. ACT-R assumes two types of memory: procedural memory and declarative memory. We focus here on the latter, the declarative memory, which is used for the storage of factual knowledge.1 (5) ACT-R activation: log( P(i\|Hi,Q) P(ic\|Hi,Q)) = Ai = Bi + Si (5) “Presentation” in ACT-R means two things. Either it refers to the moment that the chunk was created for the ﬁrst time (i.e., someone learns a particular fact), or the moment when the chunk was successfully recalled from declarative memory to be used in some context, after which it is stored in declarative memory again. Finally, the formula in (9) shows how Ai is related to the time it takes to retrieve a chunk from declarative memory, Ti. The relation between Ai and Ti is modulated by two free parameters, F, latency factor, and f , latency exponent. (9) Retrieval time: Ti = Fe−fAi (F, f – free parameters) When both parameters are set at 1 (their default value), the retrieval time of a chunk i is simply the exponential of its negative activation, which is the reverse odds that the chunk i is needed in the current context [see (4)]: (10) Retrieval time if F, f = 1: Ti = e−Ai = P(ic\|Hi,Q) P(i\|Hi,Q) (10) (6) Base-level activation: Bi = log P k∈H t−d k ! (d – decay, free parameter) It follows from (10) that the more a chunk is needed to achieve the current goal, the faster it will be retrieved. (6) Let us illustrate how all the equations are put together on an example from the introduction, the subject-verb dependency. parameter) The second element in the calculation of activation is given in (7). To keep the calculation manageable, some simplifying assumptions are introduced (see Anderson, 1991; Anderson and Lebiere, 1998). First, it is assumed that the cues j in the current context are independent of each other (and of the history Hi), conditional on i. Second, the denominator, which should be P(j\|ic), is simpliﬁed into P(j) since conditionalizing j on the irrelevant piece of information ic should not aﬀect probabilities signiﬁcantly and can be ignored. The resulting log of probability ratios, log P(j\|i) P(j) is called the associative strength and is standardly abbreviated as Sji. The equation also includes the weight W, which is a free parameter weighing the context component of the activation. Assume we comprehend or produce the verb in (11-a) and want the retrieve the chunk students to resolve the subject-verb dependency. For the purposes of this illustration, we assume that the chunk is represented in memory as shown in (11-b), repeated from (2). (5) Let us see how ACT-R estimates the history and the context components. Before doing so, we want to stress two things. First, the theory we are to discuss is generally and widely accepted by the ACT-R research community. Second, it is important to realize that the estimations of both the history component and the context component are not just arbitrary equations that happen to ﬁt memory data. They should reﬂect the estimations that the The goal of the declarative memory system should be to recall a piece of information i that is needed to achieve the current goal. As is common in ACT-R, we will formalize pieces of information as chunks. These are attribute-value matrices, or, in the terminology of ACT-R, slot-value matrices. An example of June 2021 \| Volume 12 \| Article 657705 Frontiers in Psychology \| www.frontiersin.org 2 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan mind draws from the structure of the environment in order to arrive at the best estimation of P(i) used in (3), just as a rational theory of cognition would make us expect. However, we will not present evidence that the following estimations are generalized from the structure of the environment since this has been done elsewhere (see Anderson, 1991). (2020). It might also help to notice that the formula Sji also expresses the following intuition: the associative strength (and consequently, activation) will be large when j appears only in a few chunks since in that case j is highly predictive for each of those chunks; the associative strength will decrease if there are more chunks in declarative memory that carry j as its value (see Anderson, 1974; Anderson and Lebiere, 1998; Anderson and Reder, 1999 for empirical evidence). The base-level activation Bi of a chunk is given in (6) and captures the fact that the probability that a chunk will be used next time decreases as a power function of the time since the last use, but it is also aﬀected by the number of times that the chunk has been used. The base-level activation is expressed as the log of the sum of t−d k , where tk is the time elapsed between the time of presentation k and the time of retrieval. d is a negative exponent (decay), a free parameter of ACT-R, often set at its default value of 0.5. Frontiers in Psychology \| www.frontiersin.org (5) The dependency needs to be resolved for interpretational purposes since listeners need to know who the agent of know is. It is also necessary for production purposes since speakers need to know what inﬂectional form the verb should have. (11) a. Dependency: Students rarely know the answer. (subject-verb) b. The chunk to be retrieved:   Form students Function SUBJECT Number PLURAL   (11) a. Dependency: Students rarely know the answer. (subject-verb) (11) b. The chunk to be retrieved:   Form students Function SUBJECT Number PLURAL     Form students Function SUBJECT Number PLURAL   (7) Spreading activation: Si = W · log Q j∈Q P(j\|i) P(j) = (7) Spreading activation: Si = W · log Q j∈Q P(j\|i) P(j) = (7) P j∈Q W · log P(j\|i) P(j) = P j∈Q W · Sji The activation of the subject students, its log-odds that the chunk is needed, consists of the base-level activation and the spreading activation. Suppose that 1 s elapsed since storing the chunk in memory and the chunk was not re-used. Then the base-level activation, calculated using the equation in (6), is: The activation of the subject students, its log-odds that the chunk is needed, consists of the base-level activation and the spreading activation. Suppose that 1 s elapsed since storing the chunk in memory and the chunk was not re-used. Then the base-level activation, calculated using the equation in (6), is: (W – weight, free parameter) Finally, the equation in (8) shows how ACT-R estimates the associative strength Sji. This equation is only used if the cue j is predictive of the chunk i. If it is not, Sji is set at 0. Simplifying somewhat, ACT-R assumes that a cue is predictive of a chunk if the cue appears as a value in the chunk. (12) Bstudents = log(1−d) = 0 (12) Bstudents = log(1−d) = 0 3. TRANSITION-BASED PARSING We introduce transition-based parsers and show that they can be, to a large extent, embedded in ACT-R and combined with the memory structures discussed in section 2. Such a combination directly delivers behavioral predictions to be tested in the following sections. • The less often a chunk was used, the lower base-level activation the chunk has. Consequently, chunks that are rarely used will be retrieved slower than chunks used often. • The more a chunk matches cues of the current context, the higher the boost from spreading activation. Consequently, chunks with higher matches with cues should be retrieved faster. Transition-based parsers are parsing systems that predict transitions from one state to another, following decisions made by a classiﬁer. Since the classiﬁer plays a crucial role in this type of parsers, these parsers are also called classiﬁer-based parsers. • Increasing the fan of a cue will increase the time to retrieve an element. For example, imagine that more chunks with the value plural were stored in declarative memory. Then, the associative strength of any chunk with plural would be lower and consequently, it would take more time to retrieve such chunks. Transition-based parsers are most commonly implemented for dependency grammars and arguably, they are most successful and widespread when constructing dependency graphs (Nivre et al., 2007). However, they have also been applied to phrase- structure parsing (Kalt, 2004; Sagae and Lavie, 2005; Liu and Zhang, 2017; Kitaev and Klein, 2018, a.o.). This paper also develops a phrase-structure transition-based parser. We introduce a shift reduce variant of the transition-based parsing algorithm, which is arguably the most common type of transition-based parser for phrase structures, and show how it can be understood in terms of memory systems discussed in the previous section. To the extent that these qualitative predictions are conﬁrmed, we have supporting evidence for the rational theory of memory as implemented in ACT-R. To the extent that quantitative predictions of the model can be well ﬁt to retrieval data, we also have evidence that the estimates of the history and the context component of (4) in ACT-R are on the right track. Various evidence has been collected showing that qualitative as well as quantitative predictions of the retrieval model in ACT- R are justiﬁed. Anderson (1991) and Anderson and Lebiere (1998) present supporting evidence from general cognitive tasks (independent of language). In psycholinguistics, Lewis et al. (2006), Jäger et al. (12) (12) The spreading activation, calculated using the Equations (7) and (8), is given in (13). Note that the cues [subject], [plural] are the cues in the current context, i.e., we assume for this example that these two cues are present in the cognitive context when resolving the subject-verb dependency. (8) Sji = S −log(fanj) (S – maximum associative strength, free parameter) (8) (8) Sji = S −log(fanj) (S – maximum associative strength, free parameter) S is the log of the size of the declarative memory, but commonly, it is hand-selected as a large enough value to ensure that Sji is always positive (see Bothell, 2017). fanj is the number of chunks in memory that have the cue j as its value. For discussion as to why (8) approximates log P(j\|i) P(j) , see Brasoveanu and Dotlaˇcil (13) Sstudents = W · S[subject],students + W · S[plural],students Let us assume that the free parameter S is set at 1 and so is the weight W. Since both cues appear Frontiers in Psychology \| www.frontiersin.org June 2021 \| Volume 12 \| Article 657705 3 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan in the chunk students, we have to calculate both addends as: associated with retrieved parsing steps can then be used to model the eﬀect of context on processing, e.g., investigations that are mainly the domain of psycholinguistic parsing theories, such as the Surprisal Theory (Hale, 2001). To the extent that the resulting model of parsing makes correct quantitative and qualitative predictions, we construct evidence that processing diﬃculties observed during parsing can be approached from the vantage point of the rational theory of memory. The hypothesis explored in this paper is further investigated in Dotlaˇcil (accepted)3, which also studies how individual components of ACT-R retrieval system aﬀect the retrieval of parsing steps and how the retrieval of parsing knowledge interacts with the retrieval of dependencies in processing. (14) Sstudents = 1·(1−log(fan[subject]))+1·(1−log(fan[plural])) (14) The only part that needs to be decided is the value of the fan for two cues. Let us assume that in the memory, there is no other subject and one other plural element. 3. TRANSITION-BASED PARSING (2017); Jäger et al. (2020), among others, summarize evidence that at least some cases of the retrieval of dependencies can be modeled as a case of ACT-R retrieval. (12) Then the calculation proceeds as follows: (15) Sstudents = 1 · (1 −log(1)) + 1 · (1 −log(2)) ≈1.31 Finally, we can calculate retrieval times as follows: Finally, we can calculate retrieval times as follows: (16) Tstudents = F · e−f ·(0+1.31) ≈0.27 s (if F and f set at 1) In section 3, we introduce transition-based parsing and show how such parsers can be built as a case of declarative memory in ACT-R. In section 4, we show how the model can be linked to reaction time data and evaluate its qualitative and quantitative predictions. Based on the discussion of this example, one might note that the ACT-R model of declarative memory makes several predictions regarding retrieval times. Some of those are summarized in the bullet points below: Based on the discussion of this example, one might note that the ACT-R model of declarative memory makes several predictions regarding retrieval times. Some of those are summarized in the bullet points below: • The longer the time elapsed since a chunk was used last time, the lower base-level activation the chunk has. Consequently, chunks that were used a long time ago will be retrieved slower than chunks used recently. Frontiers in Psychology \| www.frontiersin.org 3Dotlaˇcil, J. (accepted). Parsing as a cue-based retrieval model. Cogn. Sci. 3.1. Algorithm of Transition-Based Phrase-Structure Parsing The parsing algorithm works with two databases, a stack of constructed trees S and a stack of upcoming words with their POS (part-of-speech tags) W. When parsing begins, S is empty and W carries the upcoming words as they appear in the sentence, so that the ﬁrst word appears at the beginning of the stack, followed by the second word, etc. The goal of this paper is to apply the retrieval and memory model of ACT-R to a new domain. We will investigate how the rational theory of memory can model parsing knowledge and how the model of parsing can be embedded in ACT-R. We will show that once one thinks of parsing steps as chunks in declarative memory whose retrieval is driven by the same rules as other memory elements, the ACT-R model of memory becomes directly applicable to syntactic parsing. The activation that is Parsing proceeds by selecting actions based on the content of S and W. Every parsing step P is a function from S, W to actions A, that is, P : S × W ; A. In the variant of the parser that we consider, there are three actions that the parser can select: • shift First, no shift can be applied when W is empty. When S is empty, no reduce can be applied and when it has only one tree, reduce binary cannot be applied. Finally, no more than two postulate gaps actions can be applied between two shifts. This last restriction ensures that the system does not fall into the inﬁnite regress of gap postulation. 3.2. Parsing Steps as Memory Retrievals The parsing step has to decide which action (among shift, reduce, and postulate gap) should be taken, and, if reduce is selected, how should the reduction be done: should it be unary or binary and what should the root label of the newly constructed tree be? We investigate the hypothesis that the parsing step can be treated as a case of memory retrieval. The past parsing steps form the declarative memory of the parser. The parser retrieves a parsing step (or parsing steps) from memory that has the highest probability of being needed given the current goal. The current goal, in turn, is to parse the sentence. From this perspective, parsing is just a particular instantiation of rational theory of memory and can be embedded in ACT-R. The activation of a parsing step, i.e., the log-odds that a step is needed, is calculated from the history component and the context component. The former is derived from the time elapsed since the step has been used and re-used, the latter is calculated based on the cues in the current context and the spreading activation from these cues to chunks in declarative memory. We illustrate the steps of the shift-reduce parser on a simple example: parsing of a boy dances. The phrase structure is shown in Figure 1 and the parsing steps are: 1. Starting position: S = [], S = [], 1. Starting position: W = [⟨a, DT⟩, ⟨boy, N⟩, ⟨dances, V⟩] 2. shift S = [⟨ DT a ⟩], W = [⟨boy, N⟩, ⟨dances, V⟩] 3. shift S = [⟨ DT a ⟩, ⟨ N boy ⟩], W = [⟨dances, V⟩⟩] W = [⟨a, DT⟩, ⟨boy, N⟩, ⟨dances, V⟩] S = [⟨ DT a ⟩], W = [⟨boy, N⟩, ⟨dances, V⟩] S = [⟨ DT a ⟩, ⟨ N boy ⟩], W = [⟨dances, V⟩⟩] boy a W = [⟨dances, V⟩] 5. • shift June 2021 \| Volume 12 \| Article 657705 4 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan • reduce • postulate gap FIGURE 1 \| Phrase structure of a boy dances. The ﬁrst action, shift, pops the top element from the stack W and pushes it as a trivial tree onto stack S. An element in W is a pair ⟨word, POS⟩, the tree moved onto the stack is just the POS tag with the terminal the actual word. The ﬁrst action, shift, pops the top element from the stack W and pushes it as a trivial tree onto stack S. An element in W is a pair ⟨word, POS⟩, the tree moved onto the stack is just the POS tag with the terminal the actual word. The second action, reduce, pops the top element (if the reduction is unary) or it pops the top two elements (if the reduction is binary) in the stack of constructed trees S and creates a new tree. If the reduction is unary, the new tree has just one daughter under the root, the tree that was just popped from the stack. If the reduction is binary, the newly created tree has two daughters, the two trees that were just popped from the stack. In either case, the newly constructed tree is pushed on top of the stack S. It is assumed that all trees are at most binary, so no further reductions beyond binary reductions are necessary. FIGURE 1 \| Phrase structure of a boy dances. Finally, the third action, postulate gap, postulates a gap and resolves it to its antecedent. Not every parser in computational linguistics assumes this action, i.e., implemented parsers can proceed just by shifting and reducing (but see Crabbé, 2015; Coavoux and Crabbé, 2017a,b as examples of transition-based parsers that do consider gap resolution). We add gap resolution to our parser since ignoring gaps would make the parser less useful for psycholinguistics, which often studies the eﬀect of gap resolution on processing. In this illustrative example, we assume that the parser knows what the right phrase structure is and parses toward that structure. Of course, the crucial question is what happens when the phrase structure is unknown and the parser needs to predict what action to take. This is discussed in the next section. There are several restrictions on the three actions. 5Using three chunks, rather than a single chunk, to inform about the action, makes the parser less error-prone and sensitive to outliers. Adding more than three chunks does not improve the accuracy of the parser. We brieﬂy discuss the accuracy of the parser in section 5. 4The code for the model is available here: https://github.com/jakdot/parsing- model-and-a-rational-theory-of-memory. • shift antecedent carried (yes or no), i.e., is there an antecedent (like a wh-phrase) that needs to be resolved through a gap and was not resolved yet? The features should be familiar, maybe with the exception of the lexical head. The head is a terminal that projects its phrase (a verb is the head of a verb phrase, a noun is the head of a noun phrase etc.; see Collins, 1997 on head projection in computational parsers, which this work follows). The activation of a chunk is the sum of base-level activation and spreading activation. For base-level activation, we need to estimate how often a parsing step has been used in the past and how much time elapsed. The estimation comes from the frequency of parsing steps, collected from the PTB. The frequencies can be transformed into base-level activation according to the procedure described in Reitter et al. (2011), see also Dotlaˇcil (2018) and Brasoveanu and Dotlaˇcil (2020). The procedure is summarized in Appendix A. p All the features in (17) spread activation to chunks stored in declarative memory, which in turn represent all parsing steps completed in the past. Recalling the right parsing step is a case of memory retrieval that follows the rules in section 2. Consequently, it is predicted that diﬀerent parsing steps might require diﬀerent amounts of time depending on the time it takes to retrieve them. Parsing steps with higher activations will be recalled faster than parsing steps with lower activations. Activations, in turn, are based on the base-level activation and spreading activation, i.e., the ACT-R estimates of the history and the context component in calculating the need log-odds of a chunk. The spreading activation is calculated based on the match between values in chunks and features in the current cognitive context at the moment when the parsing step is recalled. The features are summarized in (17). 4.2. Case 1: Garden-Path Sentences We start the investigations of the predictions of the parser by considering selected garden-path phenomena, taken from previous literature (Bever, 1970; Frazier, 1978; Marcus, 1978; Gibson, 1991; Pritchett, 1992). • shift shift S = [⟨ NP N DT ⟩, ⟨ V dances ⟩] W = [⟨dances, V⟩] NP V While it might be possible to think of the context as complete trees in S and all information in W, we will limit the amount of information in the two databases signiﬁcantly. It will be assumed that S and W carry only some features about the trees and upcoming words, listed in (17). Thus, the parser itself never has a full snapshot of the phrase structure that it is deriving. It only carries some minimal, local information. The phrase structure can always be reconstructed through parsing steps the ACT- R agent (and, arguably, humans) took but there is no single snapshot in which all the information is available to the agent. This position is common in ACT-R parsing, see for example, Lewis and Vasishth (2005). 5. shift , ⟨ VP V dances ⟩] 6. reduce (unary) with label VP 6. reduce (unary) with label VP ⟨ S VP V dances NP N boy DT a ⟩] ⟩] dances boy a June 2021 \| Volume 12 \| Article 657705 Frontiers in Psychology \| www.frontiersin.org 5 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan (17) Features representing context: Parsing novel sentences consist of recalling the needed chunks, i.e., parsing steps collected from the PTB, from declarative memory. The recall is driven by the activation of the chunks. To calculate the activation of each chunk, formulas in section 2 are applied. We assume that the parser will recall the three chunks with the highest activations and choose the action that is the most common one among those three chunks.5 The parser repeats this procedure until it encounters shift. At that moment, the parser is done with integrating word n and can move its attention to word n+1. The activations collected during the parsing are averaged. They can be used to directly predict processing diﬃculties, as in section 4.2, or used to calculate reaction times, as in section 4.3. a. 1 upcoming word with its POS. b. root labels of top 4 elements in S c. lexical head and the POS of the lexical head for top 4 elements in S d. left and right children in top 2 elements in S e. 4. MODELING READING DATA We present an implementation of the model of sentence parsing built on the rational approach to memory and discuss two case studies testing the implementation.4 Section 4.1 introduces the model. Section 4.2 investigates whether the parser can predict processing diﬃculties for selected garden-path phenomena. Section 4.3 investigates whether the parser can be used to model self-paced reading time data from the Natural Stories Corpus (Futrell et al., 2018). We model the predictions for the pairs in (18)–(21). In each pair, the (a) sentence is a classical example of a garden path. The (b) sentence carries the same or almost identical interpretation as the garden path. However, since the disambiguation takes place early in (b) sentences, no garden-path eﬀect is observed. (18) a. The horse raced past the barn fell. b. The horse which raced past the barn fell. (19) a. While she mended the sock fell on the ﬂoor. b. While she mended, the sock fell on the ﬂoor. (20) a. He convinced her tired children are noisy. b. He convinced her that tired children are noisy. (21) a. She gave the boy the dog bit a bandage. b. She gave the boy that the dog bit a bandage. (18) 6The activations are also very low at the beginning of each sentence, irrespective of whether we deal with a garden-path sentence or not. This is an artifact of the selected model. Most cues for spreading activation come from the tree structures already built. Of course, nothing or almost nothing has been built at the beginning of a sentence, hence there are few cues at the start and consequently, spreading activation is low. It is possible to avoid this property of the model, for example, by not counting just matches in built trees, but also matches by the position in a sentence as cues that can boost activations. Frontiers in Psychology \| www.frontiersin.org 4.1. Parsing Model We assume that a declarative memory consists of chunks that represent correct past parsing steps. These chunks are collected from the data in the Penn Treebank (PTB) (Marcus et al., 1993). As is standard, we split the section of the PTB data as follows: all the sections up to and including section 21 are used to train the parser, i.e., to collect the correct parsing steps; section 22 is used for development; section 23 is used to test the accuracy of the parser. Before training we pre-process and prepare the phrase structure by (i) transforming phrases into binary structures in the way described in Roark (2001) (see Roark, 2001; Sagae and Lavie, 2005 on the reasons to do), (ii) annotating phrases with head information, (iii) removing irrelevant information (coreference indices on phrases), (iv) lemmatizing tokens so that lexical heads are stored as lemmas, not as inﬂected tokens. We want to see how the parser parses (18)–(21) and what activation values are predicted for the words in the sentences. We expect that the activation of the retrieved parsing steps should be lower for garden-path cases [(a) examples] compared to the (b) cases. This should happen at the target words, the words at We want to see how the parser parses (18)–(21) and what activation values are predicted for the words in the sentences. We expect that the activation of the retrieved parsing steps should be lower for garden-path cases [(a) examples] compared to the (b) cases. This should happen at the target words, the words at June 2021 \| Volume 12 \| Article 657705 Frontiers in Psychology \| www.frontiersin.org 6 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan FIGURE 2 \| Activations per word for sentence pairs (18)–(21). The yellow bars represent the activations in the sentences that disambiguate early. The blue bars are the activations of the garden-path sentences. The ellipses highlight the activations on the words that trigger the garden-path effect. FIGURE 2 \| Activations per word for sentence pairs (18)–(21). The yellow bars represent the activations in the sentences that disambiguate early. The blue bars are the activations of the garden-path sentences. The ellipses highlight the activations on the words that trigger the garden-path effect. FIGURE 2 \| Activations per word for sentence pairs (18)–(21). The yellow bars represent the activations in the sentences that disa the activations of the garden-path sentences. 4.1. Parsing Model This means that if we restricted our attention to correct parses, the contrast between garden-path sentences and their (b) counterparts would be even larger at the critical words. in the context. This means that if we restricted our attention to correct parses, the contrast between garden-path sentences and their (b) counterparts would be even larger at the critical words. parse is considered again for the next word. That means that the parser will have the correct syntactic structure at every word and will use the correct context for retrieval. Importantly, in a self-paced reading task, readers do much more than just retrieving and applying parsing steps. It seems uncontroversial that a model simulating self-paced reading should, at least, attend visually to word n, retrieve lexical information on that word, parse, press a key (to reveal the next word) and move visual attention to the next word, word n+1. We will add these parts and combine them with the parsing model to construct a more realistic model of reading. The added parts are not created ad hoc, they are based on the (simpliﬁed) models of visual attention and self-paced reading (Anderson and Lebiere, 1998; Brasoveanu and Dotlaˇcil, 2020). One pair in which the contrast between the (a) and the (b) examples goes in the right direction but is so small that the activation contrast is almost irrelevant is the case (18). The fact that the garden-path sentence almost does not diﬀer from the baseline might be caused by the fact that we do not model discourse and semantic phenomena, while Crain and Steedman (1985) showed convincingly that this garden path is sensitive to its context. Since the model does not take context into account, it misses out on discourse eﬀects aﬀecting activations. To conclude, we see that the contrasts in the activation of retrieved parsing steps can be tied to processing diﬃculties and predict cognitive diﬃculties observed in garden-path sentences. The sequential behavior like reading is modeled in ACT-R as a case of procedural knowledge, which sequences processes, such as the ones mentioned above and calls various sub-modules (visual, declarative memory, motor module) to carry out task speciﬁcs. The processes are linked together and controlled by the procedural system. In Figure 3, we represent the processes as boxes, which the procedural system lets ﬁre in the order as signaled by the arrows. 4.1. Parsing Model It is assumed that these processes are repeated on every word. Firing each of these processes takes the same amount of time in the procedural system, speciﬁed in (22). 4.3. Modeling Corpus Reading Data 4.3.1. Introduction We study the predictions of the parsing model for the Natural Stories Corpus (NSC, Futrell et al., 2018). The NSC is a corpus containing 10 English narrative texts with 10,245 lexical tokens in total. The texts were edited to contain various syntactic constructions, including constructions that are very rare. The corpus was read by 181 English speakers using a self-paced reading moving-window paradigm and the self-paced reading data were released along with the texts. Furthermore, all the sentences were annotated according to PTB notational conventions by the Stanford Parser (Klein and Manning, 2003) and checked and hand-corrected. The fact that the NSC has a plethora of syntactic constructions and includes manually controlled PTB-compatible syntactic parses makes the corpus particularly usable for the computational modeling of parsing. (22) Time to start process: r (r – free parameter) In addition to that, submodules involved in a process incur extra processing time based on their own properties. The process attend word visually attends to a word. To keep the model simple, we will assume that visual attention takes a ﬁxed amount of time, in line with basic models of ACT-R (Bothell, 2017). It is assumed that attending takes 50 ms, the default value of process ﬁring in ACT-R. Since visual attention is modeled as a ﬁxed amount of time, any ﬁt of the model to the data must be driven only by retrieval processes: the retrieval of lexical information or the retrieval of syntactic information, which are the only two retrieval processes considered in this paper. 4.1. Parsing Model The ellipses highlight the activations on the words that trigger the garden-path effec with lower activations have higher probability of retrieval failures (Anderson, 1991; Anderson and Lebiere, 1998). Consequently, the decrease in activation can explain processing diﬃculties in general, in particular, the failure to provide a correct parse for garden-path sentences (Pritchett, 1992).6 which processing diﬃculties should be located in garden-path sentences. The target words are fell for (18), fell for (19), are for (20), and bit for (21). We expect the activation to decrease for garden-path sentences at the disambiguation point because the base-level activation of parsing steps should be low (garden- path sentences should not be very frequent in natural data) and because the spreading activation should be low (garden-path sentences move us to the syntactic context that cannot ﬁnd a good match in the past parsing steps hence not many cues will spread activation). The phrase structures built by the parser are correct for all the (b) examples with the exception of (21-b) in which the parser wrongly attaches the noun phrase a bandage inside the relative clause. For the (a) sentences, the parser struggles at the disambiguation point and the parsing steps that it retrieves are not adequate phrase structures. It provides phrase structures that are incorrect but in which locally built phrases are combined in a plausible way. The incorrect parses for the (a) sentences were selected by the parser because they had the highest activations The activations per word are graphically summarized in Figure 2. For this calculation, we assumed default values of free parameters and we set the maximum associative strength, S, from the Equation (8) at 20. As we can see, the (a) examples show lower activations than (b) examples at the target word. Furthermore, with one exception, the classical pair in (18), the diﬀerence not only goes in the predicted direction, but it is large at the critical word (2 points of activations or more). Note also that the contrast in activations usually spills over to the following words. Since lower activations translate into higher retrieval times we see that the model is able to predict increased reading times in garden-path sentences. Furthermore, chunks June 2021 \| Volume 12 \| Article 657705 7 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan in the context. 4.3.2. Reading Model The parser as speciﬁed in sections 2 and 3 and implemented in section 4.1 will be used to model the self-paced reading of sentences in the corpus. However, to make sure that the parser does not go astray, at every word, we collect the correct parse provided by the NSC. This correct parse is used as the context for retrieval: based on this parse, the parser attempts to retrieve a parsing step from declarative memory. The declarative memory consists of parsing steps collected from the PTB, see section 4.1 for details. Then, the average activation of the retrieved chunks is recorded. After the parse for the word is ﬁnished, the correct The processes press key and move visual attention interact with the motor module and the visual module, respectively. Press key is modeled assuming the basic model of motor actions in ACT- R, which is inspired by the EPIC cognitive architecture (Bothell, 2017). It is assumed that readers have their ﬁngers ready on the key to be pressed. In that case, the simple model of motor actions in ACT-R, followed here, postulates that it takes 150 ms to press the key. Crucially, during this time, the procedural system is FIGURE 3 \| Sequential model of reading on one word. Each box represents one process. Arrows show the order in which the processes ﬁre. There are two arrows from retrieve parsing steps because retrieve wh-dependent is only triggered when a gap is postulated by the parser. FIGURE 3 \| Sequential model of reading on one word. Each box represents one process. Arrows show the order in which the processes ﬁre. There are two arrows from retrieve parsing steps because retrieve wh-dependent is only triggered when a gap is postulated by the parser. FIGURE 3 \| Sequential model of reading on one word. Each box represents one process. Arrows show the order in which the pr from retrieve parsing steps because retrieve wh-dependent is only triggered when a gap is postulated by the parser. June 2021 \| Volume 12 \| Article 657705 Frontiers in Psychology \| www.frontiersin.org 8 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan progress beyond the ﬁrst few sentences. Since our model does not represent that, we decided to remove the ﬁrst 10 sentences from each story. 4.3.2. Reading Model Furthermore, we model mRTs only starting at the second word and ending at the second to last word in each sentence since the ﬁrst and last words tend to be outliers due to starting and wrap-up eﬀects. Besides, the starting words are also outliers in our model (see also text footnote 6). free to carry out any other actions in the sequential model. That means that moving visual attention can happen concurrently with key presses. The processes retrieve lex. info, retrieve parsing steps and retrieve wh-dependent are the processes that depend on declarative memory. All processes take at least r amount of time each. Aside from that, they will also take some extra time: the amount of time needed to retrieve a chunk from declarative memory. All relevant equations to calculate retrieval time have been given in section 2. Let us repeat that the retrieval time is a function of activation of a retrieved chunk and modulated by two free parameters (23-a). Activation is calculated as the sum of base-level activation and spreading activation (23-b). The following prior structure for the parameters is assumed: • F ∼Gamma(α = 2, β = 10) • f ∼Gamma(α = 2, β = 4) Given these priors, the values in the range 0–1 are most likely but extremely low values are penalized. The priors for the parameters have the mean values of 0.2 and 0.5, respectively. These priors take into account previous ﬁndings that when F and f are estimated on language studies, including reading data, they are below 1 but usually not exceedingly small and F tends to be smaller than f (Brasoveanu and Dotlaˇcil, 2018, 2020). (23) a. Ti = Fe−fAi (F, f – free parameters) b. Ai = Bi + Si (F, f – free parameters) (23) The base-level activation and spreading activation have been discussed in detail in section 2. Recall that these activations had several free parameters: decay d, weight W, maximum associative strength S. We set the ﬁrst two parameters at their default value 0.5 and 1, respectively (see Anderson and Lebiere, 1998; Bothell, 2017). The maximum associative strength is set at 20 to ensure that associative strength is always positive (see Bothell, 2017). 4.3.2. Reading Model Furthermore, r, the time for the procedural system to ﬁre a process, see (22), is set at 33 ms, as this was found in Dotlaˇcil (accepted)3 to be the median value for an ACT-R model that simulates reading in a self-paced reading experiment. Finally, the time component needed to calculate base-level activation is calculated in the same way for the retrieval of lexical information (words) and the retrieval of parsing steps. It is derived from the frequencies of words and parsing steps, based on the procedure summarized in Appendix A. The estimation of parameters was done using PYMC3 and MCMC-sampling with 1,200 draws, 2 chains and 400 burn-in draws. The sampling chains converged as witnessed by the Rhat value (Rhat for F was 1.036; Rhat for f was 1.028). Frontiers in Psychology \| www.frontiersin.org 4.3.4. Results The mean, median and standard deviation values for the latency factor (F) and latency exponent (f ) of the posterior distributions can be seen in Table 1. FIGURE 4 \| Bayesian model for parameter estimation of Natural Stories Corpus. TABLE 1 \| Estimated parameter values. Mean Median Std F 0.0139 0.0139 0.001 f 0.661 0.655 0.068 June 2021 \| Volume 12 \| Article 657705 FIGURE 4 \| Bayesian model for parameter estimation of Natural Stories This leaves us with two parameters needed to estimate retrieval times from activations: F and f . These will be estimated with a Bayesian modeling procedure. June 2021 \| Volume 12 \| Article 657705 4.3.3. Bayesian Modeling TABLE 2 \| The linear model with Predictive RT as the only independent variable. Estimate SE t-Value p-Value Predictive RT 0.993 0.0024 415.5 p < 0.0001 The mean and median values for F match the estimate in previous Bayesian + ACT-R reading models (Brasoveanu and Dotlaˇcil, 2018, 2020). However, the estimate of f is greater than in previous reading studies. It is possible that this is because the previous reading studies did not take the retrieval of parsing steps into account, focusing only on lexical retrieval and that the previous studies mainly looked at experimental data, while this study models corpus data. The ﬁnding in Table 3 shows that our reading model can capture some aspects of self-paced reading data. However, we want to see that this modeling capability goes beyond what surface features of a text, i.e., position, word length or string frequencies, known to inﬂuence reading times, can account for. For this reason, we consider a more complex model, summarized in Table 4. The confounds we consider are the following: (i) Story (story 1 or story 2, the former being the reference level), (ii) ZONE (the word position in its story, z-transformed), (iii) POSITION (the word position in its sentence, z-transformed), (iv) the interaction of STORY × ZONE, (v) the interaction of ZONE × POSITION, (vi) LOG(FREQ) (log-unigram frequency), (vii) NCHAR (the length of the word in number of characters, z-transformed), (viii) the interaction of NCHAR × LOG(FREQ), (ix) LOG(BIGRAM) (log bigram probability), (x) LOG(TRIGRAM) (log trigram probability). Frequencies and bigram and trigram probabilities are provided in the NSC. Most of the confounds that we input are considered when evaluating computational psycholinguistic models on corpus data (Demberg and Keller, 2008; Boston et al., 2011; Hale, 2014, among others). We see that even after adding the confounds, predicted RTs remain a signiﬁcant predictor and the eﬀect goes in the expected (positive) direction (t = 3.66, p = 0.0003). Thus, our parsing model captures aspects of reading data that are not captured by surface-like factors, e.g., string frequencies, position, number of characters and the interaction of those.7 y To further investigate the model, we check samples from its posterior distribution of predicted RTs (i.e., RTs that the reading model predicts using the posterior distribution of the ﬁtted parameters). We expect that these should correlate with observed meanRTs. 7It might seem surprising that the eﬀect of log-frequency is not signiﬁcant in Table 4. This is because predicted RTs correlate with frequency and because we also include the NCHAR × LOG(FREQ) interaction. In a simpler model lacking the interaction, LOG(FREQ) is signiﬁcant and goes in the expected direction. Frontiers in Psychology \| www.frontiersin.org 4.3.3. Bayesian Modeling There are two parameters that we need to model to ﬁt the reading model to the corpus data: F and f . We will estimate them using Bayesian techniques (see Dotlaˇcil, 2018, Brasoveanu and Dotlaˇcil, 2018, Brasoveanu and Dotlaˇcil, 2019, Brasoveanu and Dotlaˇcil, 2020; Rabe et al., 2021 for other examples of combining Bayesian modeling with ACT-R cognitive models; see Weaver, 2008; Dotlaˇcil, 2018 for arguments why this is necessary). We assume the structure of the model as shown in Figure 4. In this graph, the top layer represents priors, the bottom part is the likelihood. ACT-R(F;f) is the ACT-R cognitive model of reading described in the previous section. When run and supplied with F and f values, it outputs latencies per word. The latencies of the model are then evaluated against the data assuming the likelihood is a normal distribution (measured in milliseconds) with standard deviation 20 ms (the bottom part of the graph). The actual data that we try to model are mean reading times (mRT) per word in the self-paced reading corpus. We select the ﬁrst two (out of 10) stories for the estimation of the parameters. In each story, there is an observable eﬀect of speed-up as readers FIGURE 4 \| Bayesian model for parameter estimation of Natural Stories Corpus. June 2021 \| Volume 12 \| Article 657705 9 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan TABLE 4 \| A full linear model for RTs in the NSC. Estimate SE t-Value p-Value Intercept 258.5 17.2 15 p < 0.0001 Story 7.3 1.3 5.5 p < 0.0001 Zone −3.9 0.87 −4.5 p < 0.0001 Position −2 0.7 −3 0.003 Story:Zone −3.3 1.34 −2.5 0.01 Zone:Position 1.65 0.73 2.25 0.02 Nchar 16.3 3.79 4.3 p < 0.0001 Log(Freq) 0.21 0.52 0.4 0.7 Nchar:log(Freq) −0.68 0.22 −3.1 0.002 Log(Bigram) 0.25 0.63 0.4 0.7 Log(Trigram) −0.88 0.48 −1.82 0.07 Predicted RT 0.15 0.04 3.66 0.0003 TABLE 2 \| The linear model with Predictive RT as the only independent variable. Estimate SE t-Value p-Value Predictive RT 0.993 0.0024 415.5 p < 0.0001 TABLE 3 \| The linear model with Intercept and Predictive RT. Estimate SE t-Value p-Value Intercept 248.4 12.7 19.57 p < 0.0001 Predicted RT 0.220 0.040 5.55 p < 0.0001 TABLE 4 \| A full linear model for RTs in the NSC. 4.3.3. Bayesian Modeling June 2021 \| Volume 12 \| Article 657705 10 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan FIGURE 5 \| Mean and standard deviation summaries of model and data split per trigram, frequency and observed mean RT deciles. The x-axis label shows the upper cut-off point per decile (given in log in case of Frequency). In case of Frequency, only 9 deciles are present. This is because a single word (the) spans the top two deciles. FIGURE 5 \| Mean and standard deviation summaries of model and data split per trigram, frequency and observed mean RT deciles. The x-axis label shows the upper cut-off point per decile (given in log in case of Frequency). In case of Frequency, only 9 deciles are present. This is because a single word (the) spans the top two deciles. sub-corpus that we used for testing (the ﬁrst two stories).9 We tested the ACT-R retrieval model of Boston et al. (2011) with various levels of beam-width k (k = 1, 3, 9, 20, 50, 100), where k speciﬁes the number of syntactic parses built in parallel. It turned out that model predictions with low numbers of k (k ≤20) did not show a signiﬁcant eﬀect on our NSC reading data. For k = 50 and k = 100, the model showed a very wide range of predicted reading times (from 50 to 5,000 ms). When we removed predictions beyond 2,000 ms, the model predictions were signiﬁcant (β = 0.005, t = 3.1). Crucially, the predictions of our model, PREDICTED RT, were also signiﬁcant (β = 0.2, t = 4.1). This supports the position that our model captures the properties of reading missing in an ACT-R model that only simulates the retrieval of dependencies using the ACT-R theory of memory. slight divergence in the 6th and 7th decile of Frequency, for which the model assumes mean RTs faster by 10 and 9 ms). In case of the last graph, in which data are split by observed mean RT deciles, the model copies the linear trend of the data, i.e., predicted mean RTs increase per decile. This trend is also conﬁrmed by a highly signiﬁcant Pearson correlation between predicted mean RT and observed mean RT split by decile (r = 0.88, p < 0.001). 8See also section 5 for comparisons of our model to related works. 9We used the code available at https://conf.ling.cornell.edu/Marisa/. To generate predictions, we made use of the default English training corpus, Brown. We would like to thank an anonymous reviewer, Marisa Boston and John Hale for discussion and help. 4.3.3. Bayesian Modeling However, compared to the actual data, the model has much less extreme values on both ends of the decile spectrum and as the result. While it captures the linear trend in the data, it overestimates RTs in low deciles and underestimates RTs in high deciles. Finally, we compare the predictions of our model to another ACT-R model of reading, presented in Boston et al. (2011). The model of Boston et al. (2011) models the retrieval of dependencies using the assumptions of the ACT-R rational memory. In contrast to our work, Boston et al. (2011) do not model structure building, i.e., the knowledge of parsing steps, using the ACT-R memory.8 For this reason, we would expect that the time predictions of our model remain a signiﬁcant predictor when the predictions of Boston et al. (2011) are included in a linear model of the NSC reading data. To check this, we constructed time predictions of the ACT-R reading model of Boston et al. (2011) for the NSC 4.3.3. Bayesian Modeling This is because the model simulates two steps in processing, namely, lexical retrieval and parsing. Lexical retrieval is aﬀected by the activation of words, which depends on frequency and causes less frequent words take more time to retrieve than more frequent words (see Appendix A for the estimation of base-level activation based on frequency). Syntactic retrieval is aﬀected by the activation of parsing steps, which is the sum of base-level activation and spreading activation. The base- level activation is related to frequency just like word activation and makes less frequent parsing steps take more time to retrieve (see Appendix A). Furthermore, if a reader is in a rare syntactic context (i.e., an uncommon syntactic construction), they are less likely to ﬁnd parsing steps in the past that would provide a good match. This results in a decreased spreading activation, which again aﬀects reading times. Finally, the parser models wh- dependency and retrieving wh-words will increase reading times when the wh-words are far away from the gap site, due to the decrease in their activation. To further inspect the predictions of our Bayesian + ACT-R model and the actual data, we split the predicted and observed data sets into deciles based on trigrams, word frequencies and the actual observed mean RTs. The graphical summaries per decile are given in Figure 5. For trigram probabilities and unigram frequencies, we see that the data predicted by the model follows the trend of the actual data and the mean predicted RT is generally close to the observed mean RT in each decile (with the We now inspect the predictions of the model. First, we run a simple linear model with predicted RTs per word (i.e., RTs that the reading model predicts using the posterior distribution of the ﬁtted parameters) as the independent variable and observed mean RTs as the dependent variable. We see in the summary of the linear model given in Table 2 that the Maximum Likelihood Estimate (MLE) of predicted RT is very close to 1, i.e., in the best linear ﬁt between the predicted and observed RT, the increase of 1 ms in predicted RTs corresponds to the increase of 1 ms in observed RTs. Table 3 shows the ﬁt of the intercept + predicted RT linear model. As we see, predicted RTs are a highly signiﬁcant predictor for observed mean RTs. 5.1. Parsers in Computational Psycholinguistics It is possible to split the computational psycholinguistic approaches to parsing into two types, experience-based theories and memory-based theories. In experience-based theories, it is studied how past experience with syntactic structures aﬀect parsing, most often because of expectations readers form during sentence processing. A popular framework belonging to experience-based approaches is Surprisal Theory (Hale, 2001; Boston et al., 2008, 2011; Levy, 2008, 2011; Smith and Levy, 2013, among others). In memory-based theories, it is studied how the bottleneck of memory aﬀects storage and retrieval during processing. Dependency Locality Theory is an example of a memory-based explanation of processing diﬃculties (Gibson, 1998), and so are theories studying the eﬀect of integration and recall of information from parsing stacks (Van Schijndel and Schuler, 2013; Shain et al., 2016; Rasmussen and Schuler, 2018). Another memory-based theory is the activation-based approach to dependency resolution, often implemented in ACT-R (see Lewis and Vasishth, 2005; Lewis et al., 2006). In works within cognitive architectures, a close aﬃnity can be found between this account and the models of Reitter et al. (2011) and Hale (2014). Unlike Reitter et al. (2011), the current account does not model production, but focuses on comprehension, and it does not study priming of syntactic rules. Furthermore, Reitter et al. (2011) developed a model to generate qualitative eﬀects in priming, while this paper shows that, through the application of ACT-R models in a Bayesian framework, it is possible to model quantitative data patterns. In fact, the presented approach makes it possible to develop a model in which the reading proﬁle of experience-driven processing diﬃculties quantitatively constrains the reading proﬁle of memory-driven processing diﬃculties, since both phenomena are modeled in the same way and modulated by the same free parameters. This has also been assumed in this paper (e.g., the parser for the Natural Stories Corpus assumes the same model for retrieval of wh- dependency, lexical retrieval and the retrieval of parsing steps). However, a close investigation of the interaction of diﬀerent cases of retrieval in the same model goes beyond the scope of this paper. See Dotlaˇcil (accepted)3 for more work in this direction. The two types of approaches oﬀer diﬀerent advantages. While experience-based theories can account for processing diﬃculties tied to construction frequency and local ambiguities (garden- path phenomena), memory-based approaches are used to capture locality eﬀects. 4.4. Summary of the Results We provided empirical evidence for the parsing model that is built on the assumptions of the rational theory of memory proposed in Anderson (1991) and embedded in ACT-R. Two June 2021 \| Volume 12 \| Article 657705 11 Frontiers in Psychology \| www.frontiersin.org Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan The ﬁrst type of diﬃculties has been well-investigated in computational psycholinguistics in general and in the sub-ﬁeld of modeling using cognitive architectures like ACT-R in particular (see Lewis and Vasishth, 2005; Lewis et al., 2006; Dubey et al., 2008; Reitter et al., 2011; Engelmann et al., 2013; Engelmann, 2016; Vogelzang et al., 2017; Brasoveanu and Dotlaˇcil, 2020). Crucially, the second type of diﬃculties has been investigated much less from this perspective. This paper can be seen as an attempt to enhance our understanding on this topic. In this respect, this paper advances current ACT-R analyses of reading, notably Lewis and Vasishth (2005), which do not generalize parsing, relying instead only on hand-coded rules for selected syntactic constructions. An account that oﬀered one framework for both types of processing diﬃculties has been developed in Futrell and Levy (2017), which provides a computational-level analysis (in contrast to the algorithmic- level analysis developed here) and comes to the problem from the opposite direction. Futrell and Levy (2017) provides a single analysis to processing diﬃculties by expanding Surprisal Theory with an extra component (noisy-context) to capture memory-driven diﬃculties. types of evidence were collected. First, processing diﬃculties of garden-path phenomena correspond to activation drop of retrieved parsing steps. Second, the parsing model, combined with some basic assumptions about reading, has been used to model self-paced reading data from the Natural Stories Corpus. After ﬁtting two parameters, the resulting model showed a highly signiﬁcant correlation with observed reading times. The model was able to capture aspects of the reading data that were not captured by other, low-level factors like string frequencies, position or word length. We leave it open which particular aspects of the rational memory might play a dominant role in model ﬁtting, in particular, which of base-level activation and spreading activation was crucial in our ﬁnding. Frontiers in Psychology \| www.frontiersin.org 12The parser could be subsumed under a case of memory-based parsing, see Daelemans et al. (2004). However, unlike the past cases of memory-based parsing, which were inspired by memory structures to deliver the best accuracy on data- driven parsing, the current approach is inspired by memory structures to connect parsing to on-line behavioral measures. Such a link is not considered in the approach of Daelemans et al. (2004). 5.2. Transition-Based Parsing in Computational (Psycho)linguistics Another reason why we see a low accuracy is that the parser assumes a very straightforward relation between memory instances and a parsing step. A parsing step is simply stored in declarative memory.12 This is in contrast to complex training methods commonly assumed in current neural parsers. Relatedly, current computational parsers assume a much richer feature system. They are enriched by vector space models representing lexical information and syntactic information is usually encapsulated in 200 or more features, while our parser has 19 features. p ( y ) g Transition-based parsers were a popular choice of parsers in computational linguistics, especially for dependency grammars (see Nivre et al., 2007; Zhang and Clark, 2008; Kübler et al., 2009). One advantage of transition-based parsers over graph- based parsing and grammar-based parsing is that they are fast, incremental and they allows for rich feature representations (Nivre, 2004; McDonald and Nivre, 2011). Transition-based parsers have also been applied to phrase-structure parsing (Kalt, 2004; Sagae and Lavie, 2005). The recent neural transition-based parsers for phrase-structure building have the F1 value around 95% on the PTB section 23 (Liu and Zhang, 2017; Kitaev and Klein, 2018). Transition-based parsers have also been used in computational psycholinguistics to model EEG data (Recurrent neural network grammars; Dyer et al., 2016; Hale et al., 2018) and reading data (Boston et al., 2008; Rasmussen and Schuler, 2018).10 In any case, it might be worth pointing out that even though the accuracy of the parser is not very high, it suﬃces for the research presented in this paper. The chosen examples in section 4.2 are correctly constructed by the parser when they do not lead to garden path and the parser in section 4.3 was at the end of every step (word) corrected to match the gold standard provided in the corpus, ensuring that the constructed parse is correct. While the high accuracy of the state-of-the-art transition- based parsing is encouraging, as it suggests that this line of parsing can eventually be used to create a very accurate parser, we should note that our parser is nowhere near this accuracy performance. When tested on the section 23 of the Penn Treebank, the parser shows Label Precision as 70.2, Label Recall as 72.4, F1 as 71.3. 5.1. Parsers in Computational Psycholinguistics However, the integration of the two accounts into one framework is arguably still an open issue. In most accounts, two research lines are simply put together as two diﬀerent and separated parts of a model (Demberg and Keller, 2008; Boston et al., 2011; Levy et al., 2013; Van Schijndel and Schuler, 2013). Finally, Hale (2014), Chapters 7 and 8, derives experience- driven processing diﬃculties as a case of (failed, less likely) production compilation/cohesion. This position is not incompatible with the current account, in fact, it complements it. While this work studies the role of declarative memory on parsing, Hale (2014) focuses on the role of procedural memory on parsing. The latter position has arguably been investigated in much more detail in psycholinguistics and in ACT-R than the former position since the seminal works of Lewis (1993) and Lewis and Vasishth (2005). In this respect, the current proposal can be seen as breaking with this tradition. However, both types of memory are crucial for ACT-R as well as other cognitive architectures (see Anderson, 2007) and their interaction is needed to account for complex learning patterns (Lebiere, 1999; Taatgen and Anderson, 2002). It is likely that a highly non-trivial In contrast to the just cited approaches, the current account builds a single analysis of experience-driven and memory- driven processing diﬃculties. It is assumed that both diﬃculties are driven by memory limitations in retrieval, as predicted by rational memory systems. The only diﬀerence is what is being retrieved: memory-driven processing diﬃculties arise when the memory system tries to recall a recently constructed phrase/element to satisfy dependency and encounters problems; experience-driven diﬃculties arise when the same memory system tries to recall a parsing step and encounters problems. June 2021 \| Volume 12 \| Article 657705 12 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan task, such as syntactic structure-building will beneﬁt from investigations that do not limit its investigation to the procedural memory system. but in parsing that is human-like. It is known that a human processor also shows error propagation in parsing, as witnessed by the fact that readers struggle to recover from garden path sentences the longer the wrong interpretation can be held (e.g., Frazier and Rayner, 1982). Thus, it is not a priori clear that error propagation should be avoided. 5.2. Transition-Based Parsing in Computational (Psycho)linguistics When we restrict attention to sentences of 40 words or less, as is common, Label Precision is 73.7, Label Recall is 75.9, and F1 is 74.8.11 The decision to have a simple feature model is driven by the fact that we want to ﬁrst establish that this model of parsing can be useful in predicting reading times. For that, it is preferable to keep the model as comprehensible and simple as possible, otherwise, it would not be clear whether the results reported in section 4 are due to the parsing model or some confound we are not interested in (e.g., meaning similarity present in word vector spaces). For the same reason, we currently made use of the bottom-up parsing algorithm, even though there is a good argument to be made that the bottom-up parsing algorithm is not cognitively adequate. There are well- known issues with bottom-up parsing for psycholinguistics: it accumulates elements on the stack in right-branching structures, suﬀers from disconnectedness and has problems when tied to incremental interpretation (see Resnik, 1992; Crocker, 1999). We assumed the bottom-up parsing algorithm since it is arguably the most common parsing algorithm for transition-based phrase structure parsers and thus, it serves as a very good starting point. We leave it for the future to see whether other parsing algorithms, notably, left-corner parsers, can improve on the current modeling results. There are arguably several reasons for the low performance. First, it has been found that one of the disadvantages of transition-based parsers when compared to another class of data- driven parsers, graph-based parsers, is that they get worse with increase in sentence length and increase in dependence, i.e., error propagation (McDonald and Nivre, 2011). Traditional transition- based parsers, including the parser in this paper, explore just one path. They have to greedily select what path they will follow and stick to it until the end of the sentence. Thus, early mistakes will propagate the error throughout the whole sentence. Better transition-based parsers mitigate this type of mistake through beam search or methods to recover from errors. While the adaptation of these methods could be investigated for psycholinguistics, we are not primarily interested in the best accuracy of the parser on the complex Penn Treebank sentences, 10While the mentioned works in computational psycholinguistics make use of transition-based parsing, they are not closely related to this work. The cited approaches, unlike the current account, do not construct the parsers inside a cognitive architecture and their goal is diﬀerent than developing a single account for experience-based and memory-based processing diﬃculties based on the rational theory of memory. 11Label Precision is calculated as the number of correctly constructed constituents divided by the number of all constituents proposed by the parser. Label Recall is calculated as the number of correctly constructed constituents divided by the number of all constituents present in the gold standard. F1 is the harmonic mean of the two accuracy measures. For the calculation, only non-terminal constituents are used for accuracy (i.e., trivial constituents like ⟨a, DT⟩are ignored so that the accuracy measures are not artiﬁcially inﬂated). Frontiers in Psychology \| www.frontiersin.org REFERENCES Crabbé, B. 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CONCLUSION This paper presented and tested a psycholinguistic parser that has been developed using insights from the rational theory of memory. It has been shown that the rational theory of memory June 2021 \| Volume 12 \| Article 657705 Frontiers in Psychology \| www.frontiersin.org 13 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan AUTHOR CONTRIBUTIONS can be combined with transition-based parsing to produce a data-driven parser that can be embedded in the ACT-R cognitive architecture. The parser has been tested on garden-path sentences and it has been shown that the parser to a large extent predicts processing diﬃculties at correct disambiguation points. The parser has also been evaluated on on-line behavioral data from a self-paced reading corpus and it has been shown that the parser can be ﬁt to data and model quantitative patterns in reading times. JD contributed to the development of the theory, coding, and modeling. PH contributed to coding and modeling. 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(2008). “A tale of two parsers: investigating and combining graph-based and transition-based dependency parsing,” in June 2021 \| Volume 12 \| Article 657705 Frontiers in Psychology \| www.frontiersin.org 16 Parsing Model and a Rational Theory of Memory Dotla ˇcil and de Haan Frontiers in Psychology \| www.frontiersin.org APPENDIX A: CALCULATE BASE-LEVEL ACTIVATION FROM WORD/RULE FREQUENCIES to per year can be found in Hart and Risley (1995). Based on recordings of 42 families, Hart and Risley estimate that children comprehend between 10 million to 35 million words a year, depending to a large extent on the social class of the family, and this amount increases linearly with age. According to the study, a 15-year old has been exposed to anywhere between 50 and 175 million words total. For simplicity, the model will work with the mean of 112.5 million words as the total amount of words a 15-year old speaker has been exposed to. This is a conservative estimate as it ignores production and the linguistic exposure associated with mass media. Furthermore, we assume that each word is accompanied by one parsing step, so there are as many parsing steps as words (again, this is a simpliﬁcation that should not harm modeling). We want to calculate Bi from frequency. d is a free parameter and can be ignored in this discussion. (A1) Bi = log n X k=1 t−d k ! (d- free parameter) (A1) Consider a 15-year old speaker. How can we estimate how often a word/parsing step x was used in language interactions that the speaker participated in? We now know how we get from frequency to the number of usages of x. Simplifying again, we assume that the usages, tk above, are evenly spread during the life span. First, let’s notice that we know the relative frequency of x. We collect that from the British National Corpus (for words) and from the Penn Treebank corpus (for parsing steps). The procedure described here was successfully used in translating frequencies to activations and ultimately reaction times in sentence production (Reitter et al., 2011), eye tracking reading times (Dotlaˇcil, 2018) and reaction times in lexical decision tasks (Brasoveanu and Dotlaˇcil, 2020). We know the lifetime of the speaker (15 years), so if we know the total number of words an average 15-year old speaker has been exposed to, we can easily calculate how many times x was used on average based on the frequency of x. A good approximation of the number of words a speaker is exposed June 2021 \| Volume 12 \| Article 657705 Frontiers in Psychology \| www.frontiersin.org 17
https://openalex.org/W1973030969	https://europepmc.org/articles/pmc2275226?pdf=render	English	null	Er81is a downstream target of Pax6 in cortical progenitors	BMC developmental biology	2,008	cc-by	9,197	Open Research article Er81 is a downstream target of Pax6 in cortical progenitors Tran Cong Tuoc1,2 and Anastassia Stoykova1,2 Address: 1Max-Planck-Institute for Biophysical Chemistry, Göttingen, Germany and 2DFG, Center of Molecular Physiology of the Brain (CMPB), Götingen, Germany Email: Tran Cong Tuoc - tcong@gwdg.de; Anastassia Stoykova - astoyko@gwdg.de * Corresponding author Email: Tran Cong Tuoc - tcong@gwdg.de; Anastassia Stoykova* - astoyko@gwdg.de * Corresponding author Received: 8 October 2007 Accepted: 28 February 2008 Received: 8 October 2007 Accepted: 28 February 2008 Published: 28 February 2008 BMC Developmental Biology 2008, 8:23 doi:10.1186/1471-213X-8-23 BMC Developmental Biology 2008, 8:23 doi:10.1186/1471-213X-8-23 This article is available from: http://www.biomedcentral.com/1471-213X/8/23 © 2008 Tuoc and Stoykova; licensee BioMed Central Ltd. © 2008 Tuoc and Stoykova; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Background: Although the transcription factor Pax6 plays an essential role in neurogenesis, layer formation and arealization in the developing mammalian cortex, the mechanisms by which it accomplishes these regulatory functions are largely unknown. Pax6 and the ETS family transcription factor Er81, which is presumed to play a role in the specification of a sublineage of layer 5 projection neurons, are expressed with a prominent rostrolateral-high to caudomedial-low gradient in cortical progenitors. In the absence of functional Pax6, progenitors do not express Er81 and the rostrolateral cortex lacks Er81-positive layer 5 neurons. In this study, we investigated the transcriptional regulation of Er81 and provide evidence that Er81 is a direct target of Pax6. Results: We identified and analyzed the regulatory function of an evolutionarily conserved upstream DNA sequence in the putative mouse Er81 promoter. Three potential Pax6 binding sites were identified in this region. We found that the presence of one of these sites is necessary and sufficient for full activation of the Er81 promoter in Pax6-transfected HeLa cells, while other still unknown factors appear to contribute to Er81 promoter activity in cortical progenitors and neuronal cells. The results suggest that endogenous Pax6, which is expressed at the highest level in progenitors of the rostrolateral cortex, exerts region-specific control of Er81 activity, thus specifying a subpopulation of layer 5 projection neurons. Conclusion: We conclude that the genetic interplay between the transcription factors, Pax6 and Er81, is responsible, in part, for the regional specification of a distinct sublineage of layer 5 projection neurons. Page 1 of 11 (page number not for citation purposes) BMC Developmental Biology Open Access Pax6 binds to the putative Er81 promoter p p DNA sequences with significant gene regulatory functions are highly conserved during evolution. A comparison of the Er81 locus from mouse, rat, chimpanzee and human revealed the existence of a highly conserved sequence of approximate 2 kb in the 5' region of the putative Er81 pro- moter. To identify potential Pax6 consensus binding sites [16] in this region, we utilized the sequence analysis pack- age GCG [17]. We discovered three potential Pax6 bind- ing sites located at positions -113 to 148, -1190 to 1225, and -1530 to 1565 upstream of the mouse Er81 gene, each of which contained three, four or five mis-matches relative to the consensus Pax6 binding sequence [16]. Using an electrophoretic mobility shift assay (EMSA) and in vitro- translated Pax6 protein, we examined Pax6 binding to these three potential binding sites. The results indicate that Pax6 bound with low affinity to the Pax6 binding site at position -1190 to -1225, but not to any of the other putative sites (Fig. 1B, lanes 3 and 6 and data not shown). To determine whether this low-affinity binding was spe- cific, we pre-incubated binding mixtures with an anti- Pax6 antibody and found that protein-bound, radiola- beled probes were supershifted, confirming the presence of a DNA-Pax6 protein-Pax6 antibody complex (Fig. 1B lane 7). Pax6 binding was also completely abolished after mutating the binding site sequence in the Er81 promoter (Fig. 1A,B lanes 9, 10). Together, these findings indicate that Pax6 interacts specifically with the -1190-1225 regu- latory sequence of the Er81 promoter. To determine whether this binding site is occupied by Pax6 protein in vivo, we performed chromatin immuno- precipitation (ChIP) assays using mouse E15.5 cortical extracts. A 282-bp fragment located at -1322 to -1040 of the Er81 promoter encompassing the Pax6 binding site was precipitated from chromatin by the Pax6 antibody (Fig. 1C, lane 2), but not by pre-immune serum (Pre) or GFP antibody (Fig. 1C, lane 1 and 3). Furthermore, the Pax6 antibody was not able to precipitate a control chro- matin fragment isolated from a region outside of the puta- tive Er81 promoter (Fig. 1C, lane 5). These data indicate that Pax6 protein binds specifically to the putative Er81 promoter both in vitro and in vivo. Background In the absence of Pax6, as exemplified by Pax6/Small eye homozygous (Sey/Sey) embryos, cortical progenitors pro- duce less than half the normal number of neurons; con- versely, retroviral-mediated Pax6 overexpression in cortical progenitors in vitro results in expanded produc- tion of neuronal progeny [11,12]. Pax6 is strongly expressed in early progenitors and, although it directly regulates the activity of the neuronal determination gene, Ngn2 [13]. Ngn2- and Pax6-controlled genetic programs appear to specifically and separately determine the neuro- nal fate of lower and upper neurons [14]. Thus, Ngn2 knock out (KO) and Sey/Sey embryos exhibit selective misspecification of lower and upper cortical layer neu- rons, respectively [14]. Intriguingly, however, in the absence of Pax6, cortical progenitors fail to express the layer 5-specific marker, Er81, which instead exhibits enhanced ectopic expression in the SVZ [15]. We have noted qualitatively similar mispatterning of Er81 expres- sion in the rostrolateral cortex in the cortex-specific condi- tional Pax6 KO mice at juvenile and adult stages (T.C.T., A.S. unpublished observations), prompting us to examine possible genetic interactions between the two transcrip- tion factors, Pax6 and Er81. specification of Er81-positive layer 5 neurons in the rost- rolateral cortex is a Pax6-dependent process. cells in previously established neuronal cell lineages [4]. The projection neurons of the lower (6 and 5) and upper (4–2) layers are generated predominantly from early (E12-E14) or late (E15-E18) progenitors in the two germi- native zones, VZ and SVZ, respectively. Although gener- ated during a specific developmental window, each neuronal layer consists of molecularly distinct neuronal subtypes that arise sequentially [5-7]. For instance, the majority of layer 5 neurons that extend corticospinal pro- jections express the transcription factor, Er81 [8], while another set of layer 5 neurons, marked by the expression of the homeodomain transcription factor, Otx1, make connections with the superior colliculus and pons [5]. Thus, laminar fate is presumably determined not only by the timing of neuronal origin during distinct develop- mental stages, a process controlled by environmental cues [9], but is also critically dependent on intrinsic mecha- nisms that control the molecular phenotype of the neuro- nal sublineages [5]. The mechanisms that control the restricted expression of molecular determinants in distinct classes of neurons during corticogenesis remain unknown. Our previous results, as well as those of other groups, have indicated that neurogenic RG progenitors are intrinsically specified by the expression of Pax6 [10]. Background tors (RG), which divide asymmetrically at the apical surface of the ventricular zone (VZ) and generate both neuronal and glial progeny [3]. After midgestation, RG generate neuronal progenitors, termed intermediate or basal progenitors (BPs), that divide symmetrically at the basal surface of the VZ and in the subventricular zone (SVZ). Thus, while the asymmetric division of RG progen- itors gives rise to progeny with distinct cell fates, the sym- metric division of BPs primarily modulates the number of g In the mammalian neocortex (pallium), neurons with striking morphological and functional diversity are organ- ized radially in six layers, and tangentially into numerous functional domains. Only recently have the molecular and cellular mechanisms that guide the process of cortico- genesis responsible for this organization begun to be resolved [1,2]. The main source of cortical projection neu- rons is the population of pluripotent radial glial progeni- Page 1 of 11 (page number not for citation purposes) BMC Developmental Biology 2008, 8:23 http://www.biomedcentral.com/1471-213X/8/23 cells in previously established neuronal cell lineages [4]. The projection neurons of the lower (6 and 5) and upper (4–2) layers are generated predominantly from early (E12-E14) or late (E15-E18) progenitors in the two germi- native zones, VZ and SVZ, respectively. Although gener- ated during a specific developmental window, each neuronal layer consists of molecularly distinct neuronal subtypes that arise sequentially [5-7]. For instance, the majority of layer 5 neurons that extend corticospinal pro- jections express the transcription factor, Er81 [8], while another set of layer 5 neurons, marked by the expression of the homeodomain transcription factor, Otx1, make connections with the superior colliculus and pons [5]. Thus, laminar fate is presumably determined not only by the timing of neuronal origin during distinct develop- mental stages, a process controlled by environmental cues [9], but is also critically dependent on intrinsic mecha- nisms that control the molecular phenotype of the neuro- nal sublineages [5]. The mechanisms that control the restricted expression of molecular determinants in distinct classes of neurons during corticogenesis remain unknown. Our previous results, as well as those of other groups, have indicated that neurogenic RG progenitors are intrinsically specified by the expression of Pax6 [10]. Background In the absence of Pax6, as exemplified by Pax6/Small eye homozygous (Sey/Sey) embryos, cortical progenitors pro- duce less than half the normal number of neurons; con- versely, retroviral-mediated Pax6 overexpression in cortical progenitors in vitro results in expanded produc- tion of neuronal progeny [11,12]. Pax6 is strongly expressed in early progenitors and, although it directly regulates the activity of the neuronal determination gene, Ngn2 [13]. Ngn2- and Pax6-controlled genetic programs appear to specifically and separately determine the neuro- nal fate of lower and upper neurons [14]. Thus, Ngn2 knock out (KO) and Sey/Sey embryos exhibit selective misspecification of lower and upper cortical layer neu- rons, respectively [14]. Intriguingly, however, in the absence of Pax6, cortical progenitors fail to express the layer 5-specific marker, Er81, which instead exhibits enhanced ectopic expression in the SVZ [15]. We have noted qualitatively similar mispatterning of Er81 expres- sion in the rostrolateral cortex in the cortex-specific condi- tional Pax6 KO mice at juvenile and adult stages (T.C.T., A.S. unpublished observations), prompting us to examine possible genetic interactions between the two transcrip- tion factors, Pax6 and Er81. Page 2 of 11 (page number not for citation purposes) Pax6 binds to the putative Er81 promoter Significantly, the identified Pax6 binding site is also present in the 2-kb upstream-region of the Er81 gene of mouse, rat, chimpanzee and human (data not shown) and in zebrafish [18], suggesting that this binding site is evolutionally conserved. Here we report the identification of a 2-kb promoter sequence of the mouse Er81 gene that drives Er81 expres- sion in a subpopulation of cortical layer 5 neurons. We demonstrate that Pax6 directly controls Er81 activity in both cortical progenitors and in a subset of layer 5 projec- tion neurons. The results further suggest that the cell fate Page 2 of 11 (page number not for citation purposes) Page 2 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/8/23 BMC Developmental Biology 2008, 8:23 Pax6 bind specifically to a putative Er81 promoter Figure 1 Pax6 bind specifically to a putative Er81 promoter. (A) The schematic depicts the putative Er81 promoter, showing the relative positions of a potential Pax6-binding site (BS) and an external control sequence used for ChIP assays (see below). A comparison of the DNA sequences for the potential Pax6-binding site identified in the putative Er81 promoter (at position - 1190-1225), the perfect Pax6-binding site (Cons), and the mutated site (Mut) is also indicated. (B) EMSA analysis of Pax6 bind- ing to 32P-labeled probes corresponding to the potential Pax6 binding site, a perfect Pax6-binding site, and a mutated site. The arrows, open arrowhead and closed arrowhead indicate free probes, probe-protein complexes, and probe-protein-antibody complexes, respectively. Binding of Pax6 to its perfect Pax6-binding site (lane 3) and potential binding site in the putative Er81 promoter (lane 6) is clearly detectable. Probe binding is not observed using the TNT rat reticulocyte lysate (RRL) as a protein control (lanes 2, 5). Binding specificity was confirmed by pre-incubating with an anti-Pax6 antibody, which resulted in a super- shifted DNA-Pax6 protein-Pax6 antibody complex (lanes 4, 7). Binding of Pax6 was completely abolished by mutation of the Pax6 binding-site sequence in the Er81 promoter (lanes 9, 10). (C) ChIP assay. Pax6 antibodies precipitated chromatin contain- ing the Pax6 binding site in the region -1322 to -1040 of the Er81 promoter (lane 2), but were unable to precipitate chromatin isolated from a region outside of the Pax6 binding site (lane 4). Preimmune serum (lane1, Pre) or GFP (lane3) antibodies failed to precipitate chromatin. http://www.biomedcentral.com/1471-213X/8/23 These findings indicate that the Pax6- binding site in the Er81 promoter is an important, but possibly not the sole, cis-acting element responsible for regulating the activity of the Er81 promoter in cortical pri- mary cultures. To determine whether Pax6 is also able to activate the Er81 promoter in vivo, we performed in ovo co-electropo- ration experiments. Er81(wt)-Luc and CMV-Pax6 con- structs (or a CMV-Gfp empty vector control), were electroporated into the hindbrain of Hamburger and Hamilton (HH) stage 11–12 chick embryos. After 2 days, embryos were examined immunohistochemically for expression of Pax6, GFP and luciferase reporter. After the forced expression of Pax6 in VZ progenitors of the hind- brain, a marked expression of the Er81-luciferase reporter was detected as compared to the controls (Fig. 3E/I). In developing neural tube Er81 is expressed endogenously only in differentiated motor and proprioceptive sensory neurons, including a subpopulation of neurons of the inferior olive in the hindbrain [20,21]. Upon electropora- tion, the injection in the forth ventricle, DNA (through the CMV-Pax6 construct) is incorporated in VZ progeni- tors and their descendents, seen as strongly luciferase-pos- itive cells leaving the neural tube (Fig. 3H). Consistent results were also obtained when the forced expression of Pax6 was tagged predominantly into the isthmic region, normally being negative for endogenous Pax6 expression (Fig. 3L/M). Together, these results indicate that forced expression of Pax6 can trans-activate the Er81 promoter in both, a cell culture system and a live embryo. Pax6 is expressed in RG progenitors, but is also present in some differentiated neurons in the adult brain, including some amygdalar nuclei [25]. Upon differentiation, pri- mary cortical cultures contain a mixture of progenitors and different cell types, including neurons and glial cells. To examine the function of the Pax6 binding site identi- fied in the Er81 promoter in a homogeneous neuronal cell population, we performed Er81 reporter assays using the neuronal cell line, Neuro-2A, which expresses little or no Pax6 [26]. Neuro-2A cells were transfected with either Er81(wt)-Luc, Er81(mut)-Luc or pGL3 constructs and cul- tured for 2 days. In agreement with the results obtained in primary cortical cells, transfection of Neuro-2A cells with the Er81(wt)-Luc construct caused almost a four-fold increase in luciferase reporter activity compared to that in pGL3-transfected control cultures (Fig. 3D). http://www.biomedcentral.com/1471-213X/8/23 http://www.biomedcentral.com/1471-213X/8/23 http://www.biomedcentral.com/1471-213X/8/23 http://www.biomedcentral.com/1471-213X/8/23 BMC Developmental Biology 2008, 8:23 Cell type-dependent regulation of Er81 promoter activity We next used deletion analysis to determine the minimal sequence requirements for Er81 promoter activity. A 3.5- kb fragment from the 5' region of the mouse Er81 gene and a series of deletion fragments were subcloned into luciferase reporter plasmids and transfected into mouse primary embryonic (E12.5) cortical cell cultures, as described below (Fig. 2A). The Er81(wt)-Luc construct (p131), containing a 2-kb region of genomic DNA upstream of the Er81 translation initiation codon, was most active in subsequent luciferase assays. fected with Er81(wt)-Luc or Er81(mut)-Luc with or with- out cotransfection of the Pax6 expression construct, CMV- Pax6. As expected, the activity of the wild-type promoter construct was enhanced by Pax6 overexpression, which increased luciferase activity almost 20-fold compared to the reporter activity in control cells. Mutation of the Pax6- binding site in the putative Er81 promoter completely abolished Pax6-dependent activity in the reporter assay (Fig. 3B). These data suggest that, in Pax6-transfected HeLa cells, a single biologically active Pax6-binding site in the Er81 promoter is sufficient to support full promoter activity. To assess the ability of Pax6 to activate the Er81 promoter, we transfected HeLa cells, which lack endogenous Pax6 [19], with Er81(wt)-Luc alone (control) or together with the Pax6-expression plasmid, CMV-Pax6 (Fig. 3A). The control cells exhibited very low basal levels of luciferase activity, whereas co-transfection of Er81(wt)-Luc with increasing amounts of CMV-Pax6 led to robust, concen- tration-dependent increases in luciferase activity (Fig. 3A). To study how the occupation of the binding site by Pax6 in the Er81 promoter is influenced by the presence of endogenous proteins intrinsic to progenitor cells, we eval- uated Er81 promoter activity in primary embryonic (E12.5) cortical cultures. More that 90% of the RC2+ RG progenitors express Pax6 at this stage [10,22]. The cells were electroporated with either Er81(wt)-Luc, Er81(mut)- Luc or empty pGL3 plasmid (control) and cultured for 3 days in vitro (3DIV) in a chemically defined culture medium (see Materials and Methods; [23]). Under these conditions, progenitor proliferation and neuronal differ- entiation in vitro mimic in vivo neurogenesis [10,24]. Notably, in contrast to Pax6-transfected HeLa cells, where transfection of the Er81(mut)-Luc construct completely abolished Er81 promoter activity, approximately 42% of the residual luciferase activity remained in primary corti- cal cultures transfected with this mutant promoter con- struct (Fig. 3C). Page 4 of 11 (page number not for citation purposes) Pax6 binds to the putative Er81 promoter Pax6 bind specifically to a putative Er81 promoter Figure 1 Pax6 bind specifically to a putative Er81 promoter. (A) The schematic depicts the putative Er81 promoter, showing the relative positions of a potential Pax6-binding site (BS) and an external control sequence used for ChIP assays (see below). A comparison of the DNA sequences for the potential Pax6-binding site identified in the putative Er81 promoter (at position - 1190-1225), the perfect Pax6-binding site (Cons), and the mutated site (Mut) is also indicated. (B) EMSA analysis of Pax6 bind- ing to 32P-labeled probes corresponding to the potential Pax6 binding site, a perfect Pax6-binding site, and a mutated site. The arrows, open arrowhead and closed arrowhead indicate free probes, probe-protein complexes, and probe-protein-antibody complexes, respectively. Binding of Pax6 to its perfect Pax6-binding site (lane 3) and potential binding site in the putative Er81 promoter (lane 6) is clearly detectable. Probe binding is not observed using the TNT rat reticulocyte lysate (RRL) as a protein control (lanes 2, 5). Binding specificity was confirmed by pre-incubating with an anti-Pax6 antibody, which resulted in a super- shifted DNA-Pax6 protein-Pax6 antibody complex (lanes 4, 7). Binding of Pax6 was completely abolished by mutation of the Pax6 binding-site sequence in the Er81 promoter (lanes 9, 10). (C) ChIP assay. Pax6 antibodies precipitated chromatin contain- ing the Pax6 binding site in the region -1322 to -1040 of the Er81 promoter (lane 2), but were unable to precipitate chromatin isolated from a region outside of the Pax6 binding site (lane 4). Preimmune serum (lane1, Pre) or GFP (lane3) antibodies failed to precipitate chromatin. Page 3 of 11 (page number not for citation purposes) Characteri Figure 2 Ch Characterization of the Er81 promoter Figure 2 Characterization of the Er81 promoter. (A) Diagram to the left indicates the relative size of deletion constructs used in the reporter assay. The horizontal lines represent deleted fragments of the putative Er81 promoter. Diagram to the right shows the corresponding reporter activities of the indicated deletion constructs in primary cortical cell cultures, expressed as mean ± standard deviation (error bars) in each assay. The asterisk masks the construct, p131 with the highest luciferase activity among other deletion constructs. (B) Multiple sequence alignment of the mouse, rat, chimpanzee, and human Er81 genes was performed using the ClustalW algorithm implemented in the eShadow web application [55]. The x- and y-axes indicate the dis- tance (in base pairs) to the starting codon (0) and percentage variation, respectively. The resulting alignment indicates a highly conserved sequence of approximate 2 kb upstream of the Er81 gene for all species examined. Schematic representation of the vector used to generate a Cre-transgenic mouse line in which the expression of Er81 is driven by the identified 2-kb Er81 pro- moter sequence. The Er81 promoter was subcloned upstream of the β-globin minimal promoter (minPr) in a plasmid contain- ing a DNA fragment encoding Cre [53]. This construct allows the Er81 promoter to simultaneously drive expression of Cre via the β-globin minimal promoter, and a GFP reporter sequence via an IRES sequence. (C) After crossing with Gtrosa26tm1Sho reporter mice [27], LacZ staining of E16.5 forebrains isolated from double-transgenic Er81Cre Gtrosa26 mice showed recom- bination in the L5, and the VZ of cortex, striatum, piriform cortex, and olfactory bulb (C, cross section; D, sagittal section). CP, Cortical plate; Hi, Hippocampus; L5, Layer 5; OB, Olfactory bulb; Pir, Piriform cortex; Str, Striatum; VZ, Ventricular zone. Characterization of the Er81 promoter Figure 2 Characterization of the Er81 promoter. (A) Diagram to the left indicates the relative size of deletion constructs used in the reporter assay. The horizontal lines represent deleted fragments of the putative Er81 promoter. Diagram to the right shows the corresponding reporter activities of the indicated deletion constructs in primary cortical cell cultures, expressed as mean ± standard deviation (error bars) in each assay. The asterisk masks the construct, p131 with the highest luciferase activity among other deletion constructs. http://www.biomedcentral.com/1471-213X/8/23 Remarkably, and in contrast to the experiments with Pax6-transfected HeLa cells and mixed primary cortical cultures, transfec- tion of Neuro-2A cells with Er81(mut)-Luc construct had To further examine the functional significance of the sin- gle Pax6 binding site identified in the Er81 promoter, we mutated the site in the Er81(wt)-Luc plasmid from the original 5' CCCGCT 3' sequence to 5' CTCGAG 3', which does not bind Pax6, generating the Er81(mut)-Luc plas- mid (Fig. 1A,B). We then used a reporter assay to assess the Pax6-dependent transactivation of both wild type and mutated constructs. Accordingly, HeLa cells were trans- Page 4 of 11 (page number not for citation purposes) Page 4 of 11 (page number not for citation purposes) Developmental Biology 2008, 8:23 http://www.biomedcentral.com/1471-213X/8 cterization of the Er81 promoter e 2 acterization of the Er81 promoter. (A) Diagram to the left indicates the relative size of deletion constructs used porter assay. The horizontal lines represent deleted fragments of the putative Er81 promoter. Diagram to the right the corresponding reporter activities of the indicated deletion constructs in primary cortical cell cultures, expressed ± standard deviation (error bars) in each assay. The asterisk masks the construct, p131 with the highest luciferase activ g other deletion constructs. (B) Multiple sequence alignment of the mouse, rat, chimpanzee, and human Er81 genes w med using the ClustalW algorithm implemented in the eShadow web application [55]. The x- and y-axes indicate the (in base pairs) to the starting codon (0) and percentage variation, respectively. The resulting alignment indicates a hig rved sequence of approximate 2 kb upstream of the Er81 gene for all species examined. Schematic representation of t r used to generate a Cre-transgenic mouse line in which the expression of Er81 is driven by the identified 2-kb Er81 p sequence. The Er81 promoter was subcloned upstream of the β-globin minimal promoter (minPr) in a plasmid conta DNA fragment encoding Cre [53]. This construct allows the Er81 promoter to simultaneously drive expression of Cre globin minimal promoter, and a GFP reporter sequence via an IRES sequence. (C) After crossing with Gtrosa26tm1Sho ter mice [27], LacZ staining of E16.5 forebrains isolated from double-transgenic Er81Cre Gtrosa26 mice showed reco on in the L5, and the VZ of cortex, striatum, piriform cortex, and olfactory bulb (C, cross section; D, sagittal section). C http://www.biomedcentral.com/1471-213X/8/23 BMC Developmental Biology 2008, 8:23 Characteri Figure 2 Ch (B) Multiple sequence alignment of the mouse, rat, chimpanzee, and human Er81 genes was performed using the ClustalW algorithm implemented in the eShadow web application [55]. The x- and y-axes indicate the dis- tance (in base pairs) to the starting codon (0) and percentage variation, respectively. The resulting alignment indicates a highly conserved sequence of approximate 2 kb upstream of the Er81 gene for all species examined. Schematic representation of the vector used to generate a Cre-transgenic mouse line in which the expression of Er81 is driven by the identified 2-kb Er81 pro- moter sequence. The Er81 promoter was subcloned upstream of the β-globin minimal promoter (minPr) in a plasmid contain- ing a DNA fragment encoding Cre [53]. This construct allows the Er81 promoter to simultaneously drive expression of Cre via the β-globin minimal promoter, and a GFP reporter sequence via an IRES sequence. (C) After crossing with Gtrosa26tm1Sho reporter mice [27], LacZ staining of E16.5 forebrains isolated from double-transgenic Er81Cre Gtrosa26 mice showed recom- bination in the L5, and the VZ of cortex, striatum, piriform cortex, and olfactory bulb (C, cross section; D, sagittal section). CP, Cortical plate; Hi, Hippocampus; L5, Layer 5; OB, Olfactory bulb; Pir, Piriform cortex; Str, Striatum; VZ, Ventricular zone. Page 5 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/8/23 BMC Developmental Biology 2008, 8:23 The Pax6 binding site is required for full activity of the putative Er81 promoter Figure 3 The Pax6 binding site is required for full activity of the putative Er81 promoter. (A) Exogenously expressed Pax6 activated a co-transfected Er81 promoter-reporter construct in HeLa cells in a dosage-dependent manner. (B) Mutation of the Pax6 binding site completely abolished Pax6-dependent luciferase reporter activity of the putative Er81 promoter in HeLa cells. The plasmid combinations used for transfections are indicated. (C) Mutation of the Pax6-binding site led to a significant decrease in Er81 promoter activity in mouse embryonic (E12.5) primary cortical cultures. (D) The putative Er81 promoter exhibited high activity in Neuro-2A cells (compare Er81(wt)-luc construct and control pGL3). Deletion of the Pax6 binding site did not affect activation of the putative Er81 promoter in Neuro-2A cells (compare Er81(wt)-luc and Er81(mut)-luc constructs). (E-K) Pax6 activated an Er81 promoter-reporter construct in ovo. (E) GFP immunoreactivity demonstrates the high efficiency of the electroporation method (E/F) After co-electroporation of Er81(wt)-Luc and CMV-eGFP no luciferase-positive cells were The 2-kb Er81 promoter sequence drives appropriate expression of Er81 in the cortex of Er81Cre transgenic mice To obtain definitive evidence that the promoter sequences identified in the Er81 gene is capable of correctly driving the endogenous expression of Er81 in the developing brain, we developed an Er81Cre transgenic mouse. This line expresses a DNA construct containing a 2.0-kb frag- ment of the Er81 promoter placed downstream of a human β-globin minimal promoter, followed by a Cre recombinase DNA sequence, and IRES and GFP reporter sequences (Fig. 2B). Er81Cre transgenic mice were crossed with mice from a reporter line, Gtrosa26tm1Sho [27], which contain a loxP-flanked DNA sequence of "stopper" frag- ment positioned upstream of the β-galactosidase-neomy- cin phosphotransferase fusion gene (β-geo). β-geo is expressed only after Cre-mediated excision of loxP- flanked DNA sequences, and thus double-reporter trans- genic Gtrosa26tm1Sho/Cre lines provide a region-specific report of the occurrence of Cre excision by the specific Cre line used [27]. The analysis of LacZ activity at E16.5 in dif- ferent Er81Cre;Gtrosa26tm1Sho double-transgenic founders revealed expression of the reporter in the VZ and layer 5 of the cortex, striatum, piriform cortex and olfactory bulb, a result that is in agreement with the known pattern of Er81 expression in the mouse telencephalon (Fig. 2C,D; [18,28]). We therefore conclude that the 2-kb Er81 pro- moter region is necessary and sufficient for Er81 expres- sion in the mouse telencephalon. Er81 is a member of the Pea3 subfamily of the ETS tran- scription factor family [40] that is expressed in cortical progenitors at the mid- to late stages of neocortical devel- opment in mouse [28], rat and monkey [41]. In the mouse cortex, Er81 transcripts are first detected at E13 in the VZ of the rostrolateral pallium; expression is main- tained in a subset of the pyramidal cells in the lower part of layer 5 in later embryonic stages and in the mature brain. [41]. ETS proteins have been shown to contribute to the specification of various cell types in vertebrates and invertebrates [42]. The documented role of Er81 in the specification of dendritic arborization of proprioceptive sensory neurons in the spinal cord [43] suggests the pos- sibility that Er81 might be involved in the neuronal sub- type specification of projection neurons in the cortex [18,28]. Similar to Pax6, Er81 is also expressed in a prom- inent, graded manner in pallial progenitors, reaching its highest levels in the rostrolateral cortex [28,44,45]. http://www.biomedcentral.com/1471-213X/8/23 http://www.biomedcentral.com/1471-213X/8/23 BMC Developmental Biology 2008, 8:23 no effect on Er81 promoter activity (Fig. 3D). As noted above, Pax6 binds with low affinity to the Er81 promoter, suggesting that high levels of Pax6 expression may be required to exert transcriptional control on Er81. Under the culture conditions used, Pax6 expression in mixed pri- mary cortical cultures is restricted to the RG progenitors cells. Collectively, these findings strongly suggest that the Pax6 binding site identified in the Er81 promoter is essen- tial for activation of Er81 in cortical progenitors, while regulation of Er81 expression in neuronal cells may depend on other factors. intrinsic mechanisms, controlled by the combinatorial expression of transcription factor gradients in the progen- itors, and extrinsic cues provided by the ingrowing tha- lamocortical axons at late developmental stages and after birth [2,29]. In the developing cortex, Pax6 is expressed in RG progen- itors in a rostrolateral-high to caudomedial-low gradient [30,31] and plays essential roles in cortical neurogenesis, and arealization and layer formation [10,12,14,25,30,32- 37]. Accordingly, abolishing Pax6 function leads to defects in cortical molecular regionalization as observed in the Pax6/Small eye brain, where the rostral cortical area shrinks and caudal areas expand [38,39]. Furthermore, there is a decrease in the number of neurons in the Pax6- defficient cortex, in which the neuronal subsets of the upper cortical layers appear to be specifically missing [14,34]. In the current study, we provide the first evidence that Pax6 may determine the neuronal identity of subsets of layer 5 projection neurons by controlling the expres- sion of Er81 in pallial progenitors. The 2-kb Er81 promoter sequence drives appropriate expression of Er81 in the cortex of Er81Cre transgenic mice In the absence of Pax6, Er81 expression in the VZ of the rostrola- teral cortex (where endogenous Pax6 expression is high- est) is essentially undetectable, both in Sey/Sey mutant [15] and in the juvenile cortex of the conditional cortex- specific Pax6KO mutant, (T.C.T and AS, unpublished data). We show here that Pax6 binds with low affinity to a single binding site in the Er81 promoter, and further show that Pax6 protein produces dose-dependent increases in the activity of this promoter. Collectively, these findings suggest that Pax6 controls the generation of Er81+ layer 5 neurons in a dose- and region-specific man- ner, predominantly in the motor and frontoparietal cor- tex. Remarkably, similar region- and dose-dependent regulation of Er81 expression by the neurogenic factor, Ngn2, was demonstrated in Ngn2-KO mice, in which Er81 The Pax6 Figure 3 The Pax6 binding site is required for full activity of the putative Er81 promoter Figure 3 The Pax6 binding site is required for full activity of the putative Er81 promoter. (A) Exogenously expressed Pax6 activated a co-transfected Er81 promoter-reporter construct in HeLa cells in a dosage-dependent manner. (B) Mutation of the Pax6 binding site completely abolished Pax6-dependent luciferase reporter activity of the putative Er81 promoter in HeLa cells. The plasmid combinations used for transfections are indicated. (C) Mutation of the Pax6-binding site led to a significant decrease in Er81 promoter activity in mouse embryonic (E12.5) primary cortical cultures. (D) The putative Er81 promoter exhibited high activity in Neuro-2A cells (compare Er81(wt)-luc construct and control pGL3). Deletion of the Pax6 binding site did not affect activation of the putative Er81 promoter in Neuro-2A cells (compare Er81(wt)-luc and Er81(mut)-luc constructs). (E-K) Pax6 activated an Er81 promoter-reporter construct in ovo. (E) GFP immunoreactivity demonstrates the high efficiency of the electroporation method. (E/F) After co-electroporation of Er81(wt)-Luc and CMV-eGFP, no luciferase-positive cells were detected. (G-I) Co-electroporation of Er81(wt)-Luc and CMV-Pax6 promoted expression from the Er81 promoter-luciferase reporter construct in hindbrain of chick embryos. (K) The table shows the number of embryos used for immunohistochemical analyses and the results obtained with the indicated antibodies. The strength of the immunohistochemical signal is designated as no, weak or strong staining. Note that the images shown in (E-H) represent strong staining for GFP, Luciferase and Pax6 anti- bodies. (L/N) Consistently, co-electroporation of Er81(wt)-Luc and CMV-Pax6 in isthmic region, where Pax6 does not express, led to up-regulation of the Er81 promoter-controlled luciferase reporter. Arrows in images for the whole embryo indicated a region where plasmids were injected. Luciferase activity is expressed as mean ± standard deviation (error bars) in each assay. Er81(wt)-Luc, wild-type putative Er81 promoter-reporter construct; Er81(mut)-Luc, mutated putative Er81 promoter-reporter construct. Page 6 of 11 (page number not for citation purposes) http://www.biomedcentral.com/1471-213X/8/23 Plasmids and antibodies CMV-Pax6 [46] was used for Pax6 expression in mamma- lian cells. pGL3 basic served as the backbone for luciferase reporter constructs. Er81(mut)-Luc was generated by site- direct mutagenesis (Quick Change, Stratagene) using Er81(wt)-Luc as a template. It is interesting to note that both Pax6- and Ngn2-depend- ent expression of Er81 in RG progenitors in vivo is con- fined to progenitors of the rostrolateral cortex. To confirm the functional significance of the Pax6-dependent control sequence identified in the Er81 promoter, we developed a transgenic mouse line in which the 2 kb Er81 promoter region was placed upstream of a Cre recombinase sequence. A detailed analysis of the resulting Er81Cre line is a subject of a separate study. Results obtained thus far indicate, however, that the expression of the LacZ reporter in Er81Cre:Gtrosa26tm1Sho double-transgenic mice faith- fully reproduces the known expression pattern of Er81 in the developing telencephalon, including VZ progenitors and subpopulations of L5 neurons, striatum, piriform cor- tex and olfactory bulb (Fig. 2C,D; also [18,28]. Further- more, the reporter LacZ staining was much fainter in the medial than in the dorsal or lateral pallium. During the process of submitting this paper, Langevin et al [18] reported that a 2-kb region upstream of the zebrafish Er81 is active in the lateral, but not in the medial cortex of the mouse, and identified Pax6-binding sites in a 1.3-kb upstream region [18]. Thus, it is possible that Er81 pro- moter activity in progenitors of the dorsal and lateral pal- The anti-Pax6 monoclonal (1:500, DSHB), anti-Pax6 pol- yclonal (1:500, BABCO), anti-GFP (1:500, Chemicon), anti-firefly Luciferase (1:100, Abcam), Alexa 488 goat anti-mouse, and Alexa 594 goat anti-rabbit (1:400, Invit- rogen) antibodies were used. http://www.biomedcentral.com/1471-213X/8/23 http://www.biomedcentral.com/1471-213X/8/23 http://www.biomedcentral.com/1471-213X/8/23 BMC Developmental Biology 2008, 8:23 lium (rostrally) as well as in the whole caudal pallium depends on combinatorial control by Pax6 and other transcription factors. Such simultaneous binding of Pax6 and the transcription factors, Sox2 and Maf, in the δ-crys- tallin and glucagon promoters, respectively, significantly increases Pax6 transactivation ability [48,49]. Together, these findings strongly suggest that an evolutionarily con- served genetic interplay between Pax6 and Er81 is involved in the regional specification of progenitor iden- tity in the developing cortex. expression is specifically affected only in the rostral cortex [14]. Ngn2 is a direct Pax6 downstream target gene whose expression is regulated by high levels of Pax6 expression only in progenitors of the rostral pallium [13,35]. Addi- tional experiments will be required to determine whether Er81 expression in the rostrolateral cortex is regulated coordinately by Pax6 and Ngn2 or, alternatively, whether Er81 acts as a downstream regulator in the Pax6-Ngn2 pathway. lium (rostrally) as well as in the whole caudal pallium depends on combinatorial control by Pax6 and other transcription factors. Such simultaneous binding of Pax6 and the transcription factors, Sox2 and Maf, in the δ-crys- tallin and glucagon promoters, respectively, significantly increases Pax6 transactivation ability [48,49]. Together, these findings strongly suggest that an evolutionarily con- served genetic interplay between Pax6 and Er81 is involved in the regional specification of progenitor iden- tity in the developing cortex. We found that the Pax6-binding site identified in the 2-kb promoter fragment of Er81 was necessary for full activa- tion of the Er81 promoter by Pax6 in Pax6-transfected- HeLa cells. However, mutation of the Pax6-binding site only partially reduced Er81 promoter activity in primary cortical cell cultures and had no effect in neurons of the Neuro-2A cell line. Given that at 3DIV, cortical primary cultures consist of approximately 45 % progenitors and 55% differentiated neurons [10,22], these findings sug- gest that activation of Er81 depends primarily on Pax6 transcriptional control in cortical progenitors, but activa- tion/maintenance of Er81 expression in differentiated neurons possibly involves other regulatory factors. To identify such potential factors, we performed an in silico search for potential upstream binding sites and identified multiple binding sites for the transcription factors, REST/ NRSF and Brn2, both of which are important in cortical neurogenesis [46,47]. These transcription factors could be important for activation and/or maintenance of Er81 expression in cortical progenitors and subsets of differen- tiated neurons. Conclusion hi In this paper we have shown that a direct genetic interac- tion between the transcription factors, Pax6 and Er81, in cortical VZ may be involved in the regional specification of neuronal subtype identity of a set of layer 5 projection neurons. The low-affinity binding of Pax6 to the 2-kb Er81 promoter suggests that only the high endogenous levels of Pax6 in progenitors of the rostrolateral cortex are capable of regulating Er81 promoter activity, an interpretation that is consistent with the reported regional inhibition of Er81 expression in Pax6-defficient mutants. The expres- sion of Er81 in cortical germinative neuroepithelium in other regions and in mature neurons may involve regula- tion by other molecular determinants acting independ- ently of, or together with, Pax6. Identification of transcription factor binding sites in the Er81 promoter To identify potential Pax6 binding sites within a 2-kb region of the mouse Er81 promoter, we utilized the sequence analysis package, GCG [17], and the previously reported Pax6 consensus-binding site [16]. MatInspector software was used to search for additional transcription factor binding sites in Er81 promoter [50]. Discussion l i Accumulating evidence suggests that the process of corti- cal layering, during which progenitor cells in the VZ and SVZ generate neurons destined to specific cortical layers, depends on both the temporal sequence of progenitor cell origination during a specific developmental stage and the expression of layer-determinant genes. In addition, neu- rons of a specific layer exhibit diversity in number, mor- phology and axonal connectivity across different functional domains of the cortex. Thus, the two funda- mental processes of corticogenesis, layering and arealiza- tion, seem to be closely related. According to the current view, the functional regionalization of the cortex involves Page 7 of 11 (page number not for citation purposes) Page 7 of 11 (page number not for citation purposes) X-Gal staining Embryos from timed mating were fixed and stained over- night with X-Gal at 37°C as described previously [27,53]. Determination of Pax6 DNA binding (EMSA) Determination of Pax6 DNA binding (EMSA) EMSA was performed as described by Bäumer et al [51] with some modifications. Briefly, Pax6 proteins were expressed using the TNT in vitro transcription and transla- tion system (Promega), according to the manufacturer's Page 8 of 11 (page number not for citation purposes) BMC Developmental Biology 2008, 8:23 http://www.biomedcentral.com/1471-213X/8/23 instructions. Double-stranded oligonucleotides (Fig. 1A) were end-labeled using polynucleotide kinase and gramma-P32ATP. The binding reaction was performed for 1 hr on ice in binding buffer (25 mM HEPES pH7.4, 10% glycerol, 75 mM NaCl, 0.25 mM EDTA, 1 mM DTT, 0.1% Nonidet P-40, 1 mM MgCl2, protease inhibitor cocktail) containing 0.5 μg poly-dI-dC, double-stranded oligonu- cleotides (with radial activity at 35000 cpm) and 10 μl of in vitro-translated Pax6 protein. For antibody supershift analyses, 0.5 μl of Pax6 polyclonal rabbit antibody (Babco) was added and samples were incubated for an additional 15 min. Samples were load onto 4% TAE poly- acrylamide gels and electrophoresed at 10 V/cm to resolve complexes. Gels were dried and processed for autoradiog- raphy. otransfection device (Amaxa) and then cultured for 3 days on coverslips coated with poly-D-lysine and Sato medium (DMEM, 100 μg/ml albumin, 100 μg/ml apo-transferrin, 16 μg/ml putrescine, 0.06 ng/ml progesterone, 40 ng/ml selenium, 5 μg/ml insulin, 1 mM sodium pyruvate and 2 mM L-glutamine) [23]. eneration of an Er81Cre transgenic mouse line A 2-kb region upstream from the starting codon of the mouse Er81 gene was amplified as an NheI/Not fragment using the Expand Long Template PCR kit (Roche) and cloned into a Cre-IRES-GFP-Intron-pA plasmid [52,53] in pSL1180 (Pharmacia). Er81Cre mice were generated by pronuclear microinjection. Transgenic mice were identi- fied by PCR analysis or GFP-fluorescence and maintained in a C57BL6/J background. Animals have been handled with permission of the Bezirksregierung Braunschweig in accordance with the German Animal Protection Law. AS and TCT conceived and designed the study. TCT carried out experiments. AS and TCT wrote the manuscript. All authors approved the manuscript Chromatin immunoprecipitation (ChIP) assay Chromatin was extracted from E15.5 mouse cortices. ChIP assays were performed according to the kit manufac- ture's instructions (Upstate Biotechnology) using polyclo- nal Pax6 antibodies (BABCO) to immunoprecipitate Pax6-binding chromatin fragments, with pre-immune serum and polyclonal GFP antibodies (Abcam) as immu- noprecipitation controls (10 μg antibody per immuno- precipitation). Luciferase assay ll HeLa cells or primary cortical cells were lysed and assayed for Luciferase activity according to the assay manufac- turer's instructions (Promega). Acknowledgements We gratefully acknowledge M. Daniel and S. Schlott for outstanding techni- cal assistance and L. Luo for helpful advice and discussions. We thank U. Franke for the generation of the transgenic mouse line. We are very grate- ful to M. Holt for critical comments on the manuscript. This work was sup- ported by the Max Planck Gesellschaft and the DFG- CMPB. p Franke for the generation of the transgenic mouse line. We are very grate- ful to M. Holt for critical comments on the manuscript. This work was sup- ported by the Max Planck Gesellschaft and the DFG- CMPB. In ovo chick electroporation Chick embryos at stage 11–12 (36–48 hr old) were elec- troporated as described in Marquardt et al [54]. Briefly, 2.5 μg/μl of Er81-Luc and 2.5 μg/μl of CMV-Pax6 (or CMV-Gfp) were co-electroporated into the hindbrain or midbrain of chick embryos. After 1–2 days, embryos were collected and analyzed using immunohistochemistry. At least 10 electroporated embryos were analyzed in each experiment. Cell culture and cell transfection 1. Guillemot F, Molnar Z, Tarabykin V, Stoykova A: Molecular mech- anisms of cortical differentiation. Eur J Neurosci 2006, 23(4):857-868. HeLa cells were maintained and cultured in DMEM medium containing 10% fetal calf serum (FCS). 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https://openalex.org/W2539218001	https://www.nature.com/articles/hdy201699.pdf	English	null	What causes mating system shifts in plants? Arabidopsis lyrata as a case study	Heredity	2,016	cc-by	14,240	INTRODUCTION (SRK) protein expressed on the stigma (Stein et al., 1996) that has a matching protein (S-locus cysteine rich or S-locus protein 11 (SCR/SP11); Schopfer et al., 1999) expressed in the pollen coat. Pollen grains that express a variant of SCR matching that of the SRK expressed on the receiving stigma are rejected. The genes encoding these female and male proteins are physically linked and form the S-locus, which is found in a genomic region that shows restricted recombination between a U-box domain protein (At4g21350; B80) and a member of the SRK gene family (ARK3) (Goubet et al., 2012; Roux et al., 2013). There is a complex downstream signalling reaction that is still not completely understood (Goring, 2000; Iwano et al., 2015), but self-compatibility (SC) species typically lack activity of some of these downstream components (for example, Arm-Repeat- Containing Protein 1 (ARC1)) (Indriolo et al., 2012). The SI in Brassicaceae is sporophytic, meaning that expression of both male and female components can be affected by dominance interactions because the protein on the surface of the pollen is deposited by the diploid anther cells (Hatakeyama et al., 1998). Uncovering the mechanisms regulating genetically controlled self- incompatibility (SI) systems in plants and fungi has been of sustained interest to the Genetics Society research community, with articles since the inception of Heredity (see, for example, Lewis, 1947; Bateman, 1952). A search for ‘incompatibility’ in Heredity archives retrieved 969 publications, with 275 related speciﬁcally to reproductive systems. Nevertheless, there is much that we still do not understand. SI is widespread and has multiple independent origins throughout the plant kingdom (see, for example, Raduski et al., 2012). However, it has proven difﬁcult to explain how these recognition systems that require paired speciﬁcity of male and female components evolve and are maintained (Charlesworth, 1988, 1995). A shift from outcrossing to inbreeding is one of the most frequent evolutionary transitions in plants (reviewed in Igic et al., 2008). Nevertheless, what causes breakdown of genetically controlled SI systems and how inbreeding lineages can evolve in the face of inbreeding depression remains poorly understood (reviewed by Vekemans et al., 2014). The rapid techno- logical advances of the past two decades offer new possibilities to address the possible drivers and genetic bases of these transitions. 1Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Glasgow, UK; 2Department of Molecular Biology, Max Planck Institute for Developmental Biology, Tübingen, Germany; 3Computomics GmbH, Tübingen, Germany; 4Centre for Genome Engineering, Institute for Basic Science, Daejeon, South Korea and 5Department of Biology and Ecology, University of Konstanz, Konstanz, Germany Correspondence: Professor BK Mable, Institute of Biodiversity, Animal Health and Comparative Medicine, University of Glasgow, Graham Kerr Building, Glasgow G12 8QQ, UK. E-mail: Barbara.mable@glasgow.ac.uk Received 14 March 2016; revised 12 August 2016; accepted 16 August 2016; published online 2 November 2016 OPEN OPEN Heredity (2017) 118, 52–63 Ofﬁcial journal of the Genetics Society www.nature.com/hdy BK Mable1, J Hagmann2,3, S-T Kim2,4, A Adam1, E Kilbride1, D Weigel2 and M Stift1,5 BK Mable1, J Hagmann2,3, S-T Kim2,4, A Adam1, E Kilbride1, D Weigel2 and M Stift1,5 BK Mable1, J Hagmann2,3, S-T Kim2,4, A Adam1, E Kilbride1, D Weigel2 and M Stift1,5 The genetic breakdown of self-incompatibility (SI) and subsequent mating system shifts to inbreeding has intrigued evolutionary geneticists for decades. Most of our knowledge is derived from interspeciﬁc comparisons between inbreeding species and their outcrossing relatives, where inferences may be confounded by secondary mutations that arose after the initial loss of SI. Here, we study an intraspeciﬁc breakdown of SI and its consequences in North American Arabidopsis lyrata to test whether: (1) particular S-locus haplotypes are associated with the loss of SI and/or the shift to inbreeding; (2) a population bottleneck may have played a role in driving the transition to inbreeding; and (3) the mutation(s) underlying the loss of SI are likely to have occurred at the S-locus. Combining multiple approaches for genotyping, we found that outcrossing populations on average harbour 5 to 9 S-locus receptor kinase (SRK) alleles, but only two, S1 and S19, are shared by most inbreeding populations. Self-compatibility (SC) behaved genetically as a recessive trait, as expected from a loss-of-function mutation. Bulked segregant analysis in SC × SI F2 individuals using deep sequencing conﬁrmed that all SC plants were S1 homozygotes but not all S1 homozygotes were SC. This was also revealed in population surveys, where only a few S1 homozygotes were SC. Together with crossing data, this suggests that there is a recessive factor that causes SC that is physically unlinked to the S-locus. Overall, our results emphasise the value of combining classical genetics with advanced sequencing approaches to resolve long outstanding questions in evolutionary biology. Heredity (2017) 118, 52–63; doi:10.1038/hdy.2016.99; published online 2 November 2016 ORIGINAL ARTICLE What causes mating system shifts in plants? Arabidopsis lyrata as a case study Updated online 7 December 2016: This article was originally published under a standard licence, but has now been made available under a CC BY 4.0 licence. The PDF and HTML versions of the paper have been modiﬁed accordingly. Characterisation of the S-locus in inbreeding and outcrossing populations The purpose of this study was to investigate the cause of loss of SI and subsequent shift to inbreeding within a species where populations that differ in mating system are found in close geographic proximity, using a combination of classical and newer deep sequencing approaches. Speciﬁcally, we compared patterns of S-locus variation in inbreeding and outcrossing populations of A. lyrata from the Great Lakes region and predicted the number of S-haplotypes segregating in these populations. We then performed a bulked segregant analysis using short-read sequencing of pools of individuals segregating for SC in F2 Arabidopsis lyrata provides such a model: it is a largely SI relative of A. thaliana, but in the Great Lakes region of eastern North America, multiple populations have become predominantly inbreeding and a breakdown of SI is observed even in individuals from highly out- crossing populations (Mable et al., 2005; Mable and Adam, 2007). A previous study comparing S-locus genotypes of SC and SI individuals of A. lyrata from this region failed to identify an association with particular S-haplotypes (Mable and Adam, 2007), suggesting that the mechanistic causes of loss of SI might be different in A. lyrata compared with other Brassicaceae (Vekemans et al., 2014). However, the previous study was limited for two reasons. First, only two highly inbreeding populations were included that later turned out to be from different population genetic clusters and thus may represent independent shifts to inbreeding (Foxe et al., 2010). Second, reliable identiﬁcation of S-alleles is challenging because of their high divergence (Mable et al., 2003; Schierup et al., 2006; Mable and Adam, 2007; Schierup et al., 2008), which impeded accurate comparisons of S-locus diversity between inbreeding and outcrossing populations. Advances in sequencing technology and characterisation of S-locus genomic regions from multiple S-haplotypes (Goubet et al., 2012) now make it possible to perform a broader survey of S-haplotype variation and to conduct a detailed assessment of the mechanisms of loss of SI. We then complemented this partial genotyping by cloning and sequencing SRK amplicons from a subset of individuals from each population, using three sets of degenerate forward primers (13FBM, 13-3sF and SRK 497F) with SLGR (see Supplementary Table S2 and Supplementary Information). We also piloted a long-read tagged amplicon approach using MiSeq (Illumina, San Diego, CA, USA) on 24 samples (3 individuals from each of HDC, IND, MAN, PCR, PIN, SBD, TSSA and TSS; see Supplementary Information). Study system g y Unravelling the mechanisms that originally caused functional loss of SI has posed a substantial challenge, partly because most comparisons so far have been made between species where other transitions such as changes in ﬂoral morphology and life history strategies could confound interpretations. Theoretically, loss of SI could be caused by: (1) recombination at the S-locus that breaks up paired speciﬁcity of male and female components; (2) mutations in either the female or male recognition genes that cause a loss of function or lack of recognition; (3) modiﬁers that affect the expression of S-alleles; or (4) mutations in unlinked genes required for the downstream incompatibility response. Conclusions based on transitions occurring in highly selﬁng Arabidopsis thaliana or comparisons with its outcrossing relatives have yielded conﬂicting conclusions (Indriolo et al., 2012; Nasrallah and Nasrallah, 2014; Vekemans et al., 2014; Shimizu and Tsuchimatsu, 2015). Investigating the causes and consequences of loss of SI and a shift to inbreeding within a species that both shows variation in outcrossing rates among populations and still segregates for SC within outcrossing populations could yield new insights. Such an approach should help to disentangle mechanisms for loss of SI from subsequent changes occurring once inbreeding has become established. To screen variation at SRK and ﬂanking genes, we used DNA samples extracted from 192 individuals from 24 populations (8 individuals per population) with known breeding and mating system (Foxe et al., 2010): 16 populations were predominantly SI and outcrossing (0.6oTmo0.99), 7 were predominantly SC and inbreeding (0oTmo0.40) and 1 was classiﬁed as mixed mating, based both on an intermediate outcrossing rate (Tm = 0.41) and the equal presence of both SI and SC individuals (Supplementary Table S1). INTRODUCTION The ancestral state of sporophytic SI (see, for example, Igic et al., 2008) has broken down in several Brassicaceae lineages, and has given rise to highly selﬁng species. This transition involves a two-step process: loss of SI at the level of individuals, followed by a shift to inbreeding at the population level (see Haudry et al., 2012). Theory The Brassicaceae (mustard family) have emerged as a model system for investigating the breakdown of SI. Key to the SI response is the recognition of self-pollen conferred by the S-locus receptor kinase Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 53 predicts that ecological factors such as mate limitation favour inbreeding (Byers and Meagher, 1992; Vekemans et al., 1998); for example, at the front of colonisation waves at range edges (Baker’s law; Baker, 1955; Pannell et al., 2015). S-allele diversity is usually much reduced in inbreeding lineages (reviewed by Vekemans et al., 2014), but it is typically difﬁcult to deduce whether the shift to inbreeding occurred as a result of mate limitation due to a bottleneck in S-alleles, or whether the bottleneck in S-alleles was a result of a selective sweep for self-fertilisation, combined with inherently reduced effective population size in highly inbred populations (Glemin et al., 2006). progeny of experimental crosses, in order to identify the genomic regions that differ between SC and SI pools. This allowed us to test whether: (1) the loss of SI and/or the shift to inbreeding in A. lyrata is associated with particular S-haplotypes; (2) an S-locus bottleneck may have played a role in driving the transition to selﬁng; and (3) loss of SI is due to mutations at the S-locus, modiﬁers of the recognition response or mutations in downstream components of the signalling pathway. Characterisation of the S-locus in inbreeding and outcrossing populations p p SRK genotyping. To compare variation among inbreeding and outcrossing populations at genes directly involved in SI, we focussed on the female component (SRK), because the male component (SCR) has not been sufﬁciently characterised to allow effective screening of large numbers of samples. We initially used allele-speciﬁc forward primers targeting seven SRK alleles previously found in the Great Lakes populations (S1, S3, S13, S19, S20, S23 and S39) with a general reverse primer (SLGR; see Supplementary Table S2). These primers were selected because of their consistent ampliﬁcation of SRK alleles fully linked to the SI response, and with known dominance relationships (Schierup et al., 2001; Prigoda et al., 2005; Mable and Adam, 2007). For clarity, we use ‘allele’ to refer to variants at particular genes within the S-locus and ‘haplotype’ to refer to the speciﬁcity conferred by the combination of male and female components, along with their associated ﬂanking genes. occurring once inbreeding has become established. Arabidopsis lyrata provides such a model: it is a largely SI relative of A. thaliana, but in the Great Lakes region of eastern North America, multiple populations have become predominantly inbreeding and a breakdown of SI is observed even in individuals from highly out- crossing populations (Mable et al., 2005; Mable and Adam, 2007). A previous study comparing S-locus genotypes of SC and SI individuals of A. lyrata from this region failed to identify an association with particular S-haplotypes (Mable and Adam, 2007), suggesting that the mechanistic causes of loss of SI might be different in A. lyrata compared with other Brassicaceae (Vekemans et al., 2014). However, the previous study was limited for two reasons. First, only two highly inbreeding populations were included that later turned out to be from different population genetic clusters and thus may represent independent shifts to inbreeding (Foxe et al., 2010). Second, reliable identiﬁcation of S-alleles is challenging because of their high divergence (Mable et al., 2003; Schierup et al., 2006; Mable and Adam, 2007; Schierup et al., 2008), which impeded accurate comparisons of S-locus diversity between inbreeding and outcrossing populations. Advances in sequencing technology and characterisation of S-locus genomic regions from multiple S-haplotypes (Goubet et al., 2012) now make it possible to perform a broader survey of S-haplotype variation and to conduct a detailed assessment of the mechanisms of loss of SI. Genetic basis of loss of SI Genetic basis of loss of SI Inheritance of selﬁng phenotype in F1 progeny from crosses between SI and SC plants and between SI plants. To study the inheritance of selﬁng phenotypes, we performed several crosses between plants with known selﬁng properties: (1) within population crosses between SI plants from two outcrossing populations (MAN and PIN); (2) between SI plants from the MAN population and SC plants from the predominantly selﬁng PTP population; and (3) between SI plants from the PIN population and SC plants from the predominantly selﬁng RON population. In all cases, an SI individual was used as recipient (mother) to reduce risk of contamination with pollen from the cross recipient. Subse- quently, we determined the selﬁng phenotype of all F1 progeny by performing at least six self-pollinations and scoring fruit set. Plants were considered SC if they produced at least ﬁve full siliques in six replicate self pollinations, SI if at least ﬁve siliques contained no seeds and leaky SI if two or more siliques showed partial development (Stift et al., 2013). Generation of an F2 family that segregates for selﬁng phenotype. To investigate the genetic basis for the loss of SI, we made use of the F1 family derived from the PIN × RON cross, in which all progeny were SI without evidence for leakiness (n = 20) and for which the parents had been genotyped for the S-locus. The PIN parent (PIN 12-3) carried S23 and an unknown SRK allele Sx, whereas the RON plant (RON 19-3) had been inferred to be homozygous S1S1, so that the resulting F1 progeny were either S1Sx or S1S23. Owing to the recessivity of S1 to all other S-haplotypes, the S1Sx and S1S23 siblings express different speciﬁcities (Sx and S23, respectively) and could thus be crossed to generate biparentally inbred F2 progeny (Stift et al., 2013). S-locus characterisation in SC and SI pools. To speciﬁcally determine whether there were differences at the S-locus between the SC and SI pools, we used a sequence- rather than a SNP-based approach, where we could take advantage of the known haplotype structure of S1 based on a previous bacterial artiﬁcial chromosome (BAC) study (Goubet et al., 2012) and our own ﬂanking gene sequencing. Estimating the number of S-haplotypes within populations Estimating the number of S-haplotypes within populations Given previous evidence of strong linkage disequilibrium between B80 and the S-locus in A. lyrata (Hagenblad et al., 2006; Kamau et al., 2007), we used heterozygosity at this locus to predict when we had likely missed alleles at SRK in order to estimate the number of S-alleles in each population. We used the genealogies to predict cases where particular SRK variants were associated with more than one B80 allele or where different SRK variants appeared to share a B80 allele. This was taken into account in the prediction of heterozygosity. Identiﬁcation of SNP sharing among SC and SI pools and SC reference genome sequence. To identify larger genomic regions of different allele frequencies (that is, proportion of reads for each variant found at a single single-nucleotide polymorphism (SNP) site) of genetic variants between the two pools, we employed a strategy similar to the SHOREmap approach (Schneeberger et al., 2009b). Allele frequencies of single positions were then averaged in sliding windows (step size of 10 000 and a window size of 2 00 000 bp) along the genome to yield detectable distinct patterns. The assumption for the SHORE map approach was that genomic regions associated with a particular phenotype should show a depression of heterozygosity in pools of individuals sharing that phenotype as compared with pools of individuals with a different phenotype. To increase the potential strength of this signal, the SNP calls from each pool were compared with a reference genome obtained from a SC individual from the same highly inbreeding population as the SC parent used for the crosses. Given the high heterozygosity expected for A. lyrata, this reference was produced by crossing individuals from RON (sampled from Rondeau Provincial Park in Ontario) to plants raised from seeds from the inbred line of the A. lyrata reference genome, MN47 (Hu et al., 2011), which was from one of the outcrossing populations that we used for our SRK survey (IND; from Indiana Dunes National Lakeshore in Michigan) (see details in Supplementary Information). We predicted that genomic regions associated with the loss of SI would show sharing between the SC pool and the AL4 reference, whereas the SI pool would be polymorphic or show different mutations in these regions. Characterisation of the S-locus in inbreeding and outcrossing populations Brieﬂy, the method allowed sequencing of 900 bp products by shearing of barcoded amplicons. For each sample, CLC Genomics workbench (version 7.5, Qiagen Aarhus, Aarhus, Denmark) was used to assemble contigs de novo and map paired reads back onto them (see details in Supplementary Information). The consensus sequences were then extracted and BLAST was used to identify the most similar sequences available in GenBank. Characterisation of S-locus haplotypes. To assess whether inbreeding and outcrossing populations also differed in broader S-haplotypes, we sequenced several genes ﬂanking the S-locus: B160 (transcription factor; At4g21430) and B120 (S-locus lectin kinase 9; At4g21390) are upstream of the recognition genes SRK/SCR, whereas B80 (U-box domain protein; At4g21350) and B70 (Ethy- lene-responsive protein-like transcription factor; At4g21340) are downstream. For all individuals in our study, B80 and B160 had been sequenced previously (Haudry et al., 2012; Popset accessions: 374282218 and 374282986); here we used primers developed by Kamau and Charlesworth (2005) to sequence and genotype B70 and B120. Strategies for direct sequencing, cloning and haplotype resolution were as described in Haudry et al. (2012). The purpose of this study was to investigate the cause of loss of SI and subsequent shift to inbreeding within a species where populations that differ in mating system are found in close geographic proximity, using a combination of classical and newer deep sequencing approaches. Speciﬁcally, we compared patterns of S-locus variation in inbreeding and outcrossing populations of A. lyrata from the Great Lakes region and predicted the number of S-haplotypes segregating in these populations. We then performed a bulked segregant analysis using short-read sequencing of pools of individuals segregating for SC in F2 We then tested whether different ﬂanking variants associated with the same SRK allele were monophyletic (that is, suggesting common origins) or whether patterns of variation were more consistent with the geographic distribution or mating system of the sampled populations, by reconstructing genealogies for each gene using MEGA 6.0 (Tamura et al., 2013). After applying Model Test (as implemented in MEGA) to choose the most appropriate model of evolution, we performed a maximum likelihood analysis with 1000 bootstrap replicates. We then mapped SRK variants, individual populations and genetic Heredity Heredity Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 54 clusters that had been inferred from STRUCTURE analysis of microsatellite loci (Foxe et al., 2010) onto these trees. Estimating the number of S-haplotypes within populations We used the SHOREmap sliding window analysis to identify broad chromosomal regions showing an excess of homozygosity in the SC pools shared with the AL4 reference and then compared individual SNP calls in these regions to identify particular genes or regions that also showed allele sharing between the SC pools and the AL4 reference but not the SI pool. We then extracted the consensus sequences and used BLAST to ascertain the identity of any genes found in such regions. Here, we used the repeatability index of Stevens and Kay (1989), which provides meaningful estimates for sporophytic SI systems that can have unequal allele frequencies because of dominance (Mable et al., 2003). We calculated the predicted number of S-haplotypes in each population using the formula N = 1[(n2)/(m2)], where N is the number of alleles in the population, n is the number of alleles identiﬁed in the sample and m is the number of gene copies sampled. We calculated the maximum number of alleles assuming every individual within a population had a unique missing haplotype and the minimum assuming they shared a single variant that could not be identiﬁed with the methods used. Characterisation of the S-locus in inbreeding and outcrossing populations In addition, we used individuals that were homozygous at SRK and B80 to assess whether inbreeding populations shared unique S-locus haplotypes (that is, based on SRK and the four ﬂanking genes) or represented a subset of the diversity found in outcrossing populations. within the F2 progeny (see details in Supplementary Information). The SI and SC pools were processed to make sequencing libraries using manufacturer’s protocols for whole-genome sequencing on an Illumina GAII instrument. Three lanes of separate runs (two 150 bp and one 100 bp paired-end read run) were sequenced for each pool, resulting in ∼30 Gb of sequence for each pool. The Illumina quality-ﬁltered reads were mapped against the reference genome sequence MN47 (Hu et al., 2011) using GenomeMapper (Schneeberger et al., 2009a), allowing for up to 10% mismatches/gaps relative to the read length. All alternative alleles relative to the reference base with a minimum frequency within each pool of 10% and a score of at least 25 were called by SHORE, as previously described (Ossowski et al., 2008). within the F2 progeny (see details in Supplementary Information). The SI and SC pools were processed to make sequencing libraries using manufacturer’s protocols for whole-genome sequencing on an Illumina GAII instrument. Three lanes of separate runs (two 150 bp and one 100 bp paired-end read run) were sequenced for each pool, resulting in ∼30 Gb of sequence for each pool. The Illumina quality-ﬁltered reads were mapped against the reference genome sequence MN47 (Hu et al., 2011) using GenomeMapper (Schneeberger et al., 2009a), allowing for up to 10% mismatches/gaps relative to the read length. All alternative alleles relative to the reference base with a minimum frequency within each pool of 10% and a score of at least 25 were called by SHORE, as previously described (Ossowski et al., 2008). Characterisation of S-haplotypes in inbreeding and outcrossing populations Allele-speciﬁc screening revealed all seven alleles previously known to occur in the Great Lakes area among the outcrossing populations, with the inbreeding populations having only three (Table 1 and Supplementary Table S4). Cloning using degenerate primers did not yield further information, as other members of the gene family preferentially ampliﬁed (Supplementary Table S4). However, the Heterozygosity at B80 suggested that an additional unidentiﬁed SRK allele was present in one of the individuals from LPT, with all other individuals from inbreeding populations where only one SRK allele was ampliﬁed being homozygous at B80 (Supplementary Table S4). Characterisation of S-haplotypes in inbreeding and outcrossing populations Table 1 Number of SRK alleles inferred for 8 individuals per population based on direct and indirect genotyping, sorted by genetic cluster predicted by STRUCTURE analysis of microsatellites and population outcrossing rate (both taken from Foxe et al., 2010) indicating the number of individuals containing each of the variants screened in the allele-speciﬁc genotyping, as well as other alleles identiﬁed by cloning or MiSeq analyses Population Tm Cluster SRK alleles inferred Other % Het Homo Min alleles a Max alleles a S1 S3 S13 S19 S20 S23 S39 TC 0.18 1 0 1 0 8 0 0 0 0.14 S19 2 2 TCA 0.48c 1 0 0 0 8 0 0 0 0.00 S19 1 1 TSSA 0.41 1 4 1 3 4 0 0 0 S27 0.63 S1, S19 6 6 TSS 0.91 1 3 4 3 2 0 0 0 S27 0.88 S3 6 11 PTP 0.02 2 8 0 0 0 0 0 0 0.00 S1 1 1 WAS 0.25 2 4 0 0 8 0 0 0 0.50 S19 2 2 RON 0.28 2 8 0 0 0 0 0 0 0.00 S1 1 1 PIN 0.84 2 7 1 0 3 1 0 4 1.00 None 6 6 PCR 0.98 2 8 0 0 0 1 2 0 S27, S45d 0.75 S1 5 8 KTT 0.31 3 0 0 0 8 0 0 0 0.00 S19 1 1 PIR 0.88 3 7 2 0 0 0 1 2 0.75 S1 6 14 LPT 0.13 4 0 0 0 8 0 0 0 0.13 S19 2 2 HDC 0.65 4 8 0 0 0 0 0 0 S45d 0.38 S1 3 3 PRI 0.89 4 8 0 0 1 0 0 1 0.75 S1 5 8 OWB 0.64 5 8 0 1 0 0 0 0 0.13 S1 2 2 PIC 0.77 5 8 2 0 0 0 0 0 S45d 0.50 S1 3 6 LSP 0.94 5 8 0 0 0 0 2 1 0.75 S1 3 8 SBD 0.94 5 5 4 1 1 1 0 0 S45d 0.88 S1 8 14 PUK 0.96 5 2 5 0 5 0 1 2 0.88 S3 6 6 BEI 0.98 5 7 2 1 1 0 0 0 0.50 S1 5 5 IOM 0.94 6 4 4 0 0 0 0 0 0.63 S1 3 14 NCM 0.99 6 6 6 1 0 0 0 1 S45d 0.75 S1, S3 5 5 MAN 0.83 1, 2 7 0 0 0 1 0 1 S27, S52e 0.75 S1 8 11 IND 0.98 2, 5 4 4 1 0 1 0 1 S45d 0.88 S1 5 14 Inbreeding 0.20 0.36 0.02 0.00 0.71 0.00 0.00 0.00 0.11 1.43 1.43 Outcrossingb 0.88 0.79 0.27 0.06 0.10 0.04 0.05 0.10 0.70 5.13 8.63 Abbreviations: Het, heterozygous; Homo, homozygous; Min, minimum; Max, maximum; SRK, S-locus receptor kinase. Genetic basis of loss of SI High-quality DNA extracts were prepared from pools of individuals with the same phenotype (SC or SI) Heredity Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 55 MiSeq analysis of 24 outcrossing individuals appeared promising (see Supplementary Information for more details); alleles identiﬁed through allele-speciﬁc PCR could always be conﬁrmed with the MiSeq analysis and more heterozygotes were resolved using the latter (Supplementary Table S5). This analysis also identiﬁed an additional allele known to be linked to the SI phenotype (S27) and one putatively new allele (named AlySRK52, 80% similar to AlySRK15). MiSeq analysis of 24 outcrossing individuals appeared promising (see Supplementary Information for more details); alleles identiﬁed through allele-speciﬁc PCR could always be conﬁrmed with the MiSeq analysis and more heterozygotes were resolved using the latter (Supplementary Table S5). This analysis also identiﬁed an additional allele known to be linked to the SI phenotype (S27) and one putatively new allele (named AlySRK52, 80% similar to AlySRK15). KF418158.1) (Indriolo et al., 2014), along with another member of that gene family whose relationship to SI remains unclear (PUB17: accession number: XM_002890762.1) (Hu et al., 2011). We used the consensus sequences for each gene targeted to determine whether the SI or SC pools differed in hetero- zygosity or sequence polymorphism. We predicted that if loss of SI was associated with the S-locus itself, then genes at the S-locus should show a difference between SI and SC pools, whereas unlinked members of the SRK gene family should not. We also searched the unmapped and raw reads for each of the reference sequences including the three B80 variants segregating in the crosses in order to predict the SRK alleles present in each of the two pools. KF418158.1) (Indriolo et al., 2014), along with another member of that gene family whose relationship to SI remains unclear (PUB17: accession number: XM_002890762.1) (Hu et al., 2011). We used the consensus sequences for each gene targeted to determine whether the SI or SC pools differed in hetero- zygosity or sequence polymorphism. We predicted that if loss of SI was associated with the S-locus itself, then genes at the S-locus should show a difference between SI and SC pools, whereas unlinked members of the SRK gene family should not. Genetic basis of loss of SI We also searched the unmapped and raw reads for each of the reference sequences including the three B80 variants segregating in the crosses in order to predict the SRK alleles present in each of the two pools. The 48 individuals from predominantly inbreeding populations all had SRK allele S1, S19 or both. Of these, 16 individuals only showed the presence of S1 (all the individuals from the RON and PTP populations), 27 only ampliﬁed S19 (from the remaining inbreeding populations) and 4 were S1S19 heterozygotes (all from the WAS population). The TC population also contained one S3S19 heterozygote. The mixed mating population TSSA contained S1 and S19, but also S3, S13 and S27. Genetic basis of loss of SI The MN47 reference strain is known to have an S13 haplotype (Hu et al., 2011) that shows only 71% similarity (in the extracellular S-domain) to the S1 and S23 haplotypes expected in the pools and hence should be clearly distinguishable. The genomic structure of S23 has not been resolved but we downloaded sequences for ﬁve ﬂanking genes (B160, B120, ARK3, B80 and B70), SRK and SCR from the published BAC sequence for the S1 genomic region (accession numbers: KJ772401–4) to use as references, along with an SRK sequence for S23 obtained from our population survey. We extracted the consensus sequences of the SI and SC pools using SAMtools (Li, 2011), with IUPAC (The International Union of Pure and Applied Chemistry) ambiguity codes used to indicate heterozygous and homozygous sites. To determine segregation of the selﬁng phenotypes, we raised 97 of these individuals from four S1Sx–S1S23 F1 sibling pairs (Supplementary Table S3). Following the procedures described for the F1, most F2 plants could be unambiguously grouped into the previously deﬁned classes SC, SI and leaky SI, but male sterility emerged as a fourth phenotype characterised by shrivelled anthers that produced no visible pollen. To test segregation of SRK in the F2 progeny, we originally screened a subset of individuals using allele-speciﬁc PCR and sequencing of the alleles present in the grandparents. However, as we were not able to identify one of the alleles (Sx), we could not distinguish S1 homozygotes from S1Sx heterozygotes using this approach. We thus exploited the linkage disequilibrium of B80 to SRK to infer segregation of the S-haplotypes in the F2 progeny, which was possible because all four genotypes could be resolved by direct sequencing. We used CLC Genomics Workbench (CLC, Aarhus, Denmark) to map the following to the SI and SC consensus sequences: (1) the reference sequences for SRK, SCR and the ﬂanking genes; and (2) other members of the SRK gene family that are not linked to the SI phenotype (Aly7, Aly9, 13-2, ARK1 and ARK2, Charlesworth et al., 2003; accession numbers: AY186754, AY186756, AY186763, AY186758 and AY186761). We also mapped published sequences from A. lyrata for one of the downstream components of the SI signalling cascade that has been implicated in loss of SI (ARC1; accession number: Bulked segregant analysis (Illumina sequencing). Characterisation of S-haplotypes in inbreeding and outcrossing populations B80 hap75 was found in SI individuals from outcrossing populations but outcrossing populations also had other variants (haps 49, 114 and 122). Although the other ﬂanking genes in S19 homozygotes showed more variation, the LPT population also had unique variants of B120 and B160 that were absent from the other inbreeding populations and the outcrossing populations (Table 2 and Supplementary Table S4). As S19 is a dominant allele, homozygotes were absent from the outcrossing populations but B80 hap75 was also found in an outcrossing population (PUK; Supplementary Table S4). The genealogy suggested that the B80 haplotypes associated with S19 were monophyletic (Figure 1), whereas those associated with other alleles were not (Supplementary Figures S1–S3 and Supplementary Table S6). The recessive allele S1 was found at high frequency among the outcrossing populations and was associated with B80 haplotypes distributed across the genealogy. However, the inbreeding populations contained only two B80 haplotypes (Figure 1 and Supplementary Table S4): hap50 was shared only with outcrossing populations in geographic proximity to inbreeding populations (HDC and PRI), whereas hap43 was widespread among outcrossing populations from different regions. There was also only a single B120 haplotype in RON and PTP that was shared with HDC and PRI. Table 2 Flanking gene variants for individuals that showed ampliﬁcation of only one SRK allele (using direct allele-speciﬁc screening) and were homozygous at B80 SRK B80 B120 B160 B70a Phenotype Populationb N 19 75 25 15 1 SC KTT 6 19 75 25 15 ? SC KTT 2 19 75 59 15 1 SC TCA 1 19 75 ? 15 1 SC TCA 1 19 75 59 15 ? SC TCA 1 19 75 6 15 ? SC, SI TC 5 19 75 24 15 ? SC TC, TCA 4 19 75 ? 15 ? SC TC, TCA 5 19 75 24 15 ? SC TSSA 2 19 75 40 15, 45 ? SC WAS 1 19 75 40 22 ? SC WAS 1 19 75 64 15 ? SC WAS 2 19 75 64 15, 16 ? SC WAS 1 19c 76 26 22 ? SC LPT 7 1 50 7 16 2 PC HDC 1 1 50 7 16 ? PC, SI HDC 2 1 50 7 21 2 PC HDC 1 1 50 7,8 19, 20 2 PC HDC 1 1 50 7 22 17 SC RON 3 1 50 7 22 ? Figure 1 Minimum evolution genealogy of B80 alleles, indicating associations with SRK alleles and geographic distribution. The frequency of each allele is indicated in parentheses after its name. The tree was reconstructed using MEGA 6.0, under a Kimura 2 parameter model of evolution, with rate heterogeneity modelled under a gamma distribution using a rate parameter of 0.45. Numbers on the nodes indicate bootstrap support based on 1000 pseudoreplicates. As low phylogenetic resolution is expected for genes evolving under balancing selection, the main purpose of the tree is for visualisation of relatedness among B80 alleles in relation to their association with SRK alleles. Associated SRK alleles are indicated by name and using coloured branches. Occurrences of each B80 allele in inbreeding and outcrossing populations and in each of the six genetic clusters predicted by STRUCTURE are indicated in the table to the right. Estimating the number of S-haplotypes within populations Estimating the number of S-haplotypes within populations The MiSeq analysis resolved complete heterozygous SRK genotypes for 16/24 of the samples screened, and identiﬁed one new putative SRK allele. Six individuals were predicted to be homozygous and three were predicted to have an unidentiﬁed SRK allele, based on heterozygosity at B80 (Supplementary Tables S4 and S5). ARK3 (Aly8) was not as reliable as B80 for predicting heterozygosity, as in some cases homozygotes for ARK3 had two SRK alleles in the MiSeq analysis (Supplementary Table S5 and Supplementary Information). Some individuals for which only S1 was ampliﬁed were hetero- zygous at B80 for two different alleles associated with S1 (Supplementary Table S4) from disparate parts of the tree (Figure 1). These individuals were thus hypothesised to be homo- zygous for S1 but originating from two different genetic backgrounds; results are also presented assuming that the SRK alleles were not the same (Supplementary Table S7). Based on the repeatability index of Stevens and Kay (1989), outcrossing populations were predicted to have on average between 5 and 9 S-haplotypes per population, whereas inbreeding populations were predicted to have 1.4 (Table 1 and Supplementary Table S7). There was little difference in the number of S-haplotypes predicted in different clusters. Estimating the number of S haplotypes within populations The MiSeq analysis resolved complete heterozygous SRK genotypes for 16/24 of the samples screened, and identiﬁed one new putative SRK allele. Six individuals were predicted to be homozygous and three were predicted to have an unidentiﬁed SRK allele, based on heterozygosity at B80 (Supplementary Tables S4 and S5). ARK3 (Aly8) was not as reliable as B80 for predicting heterozygosity, as in some cases homozygotes for ARK3 had two SRK alleles in the MiSeq analysis (Supplementary Table S5 and Supplementary Information). pp y pp y Some individuals for which only S1 was ampliﬁed were hetero- zygous at B80 for two different alleles associated with S1 (Supplementary Table S4) from disparate parts of the tree (Figure 1). These individuals were thus hypothesised to be homo- zygous for S1 but originating from two different genetic backgrounds; results are also presented assuming that the SRK alleles were not the same (Supplementary Table S7). Based on the repeatability index of Stevens and Kay (1989), outcrossing populations were predicted to have on average between 5 and 9 S-haplotypes per population, whereas inbreeding populations were predicted to have 1.4 (Table 1 and Supplementary Table S7). Characterisation of S-haplotypes in inbreeding and outcrossing populations For each population, the % of individuals predicted to be heterozygous, the alleles predicted to be homozygous and the minimum and maximum number of alleles in the population predicted by the repeatability index of Stevens and Kay (1989) are shown. Rows in bold indicate the average outcrossing rates, proportion of individuals with each SRK allele, heterozygosity and maximum and minimum number of alleles predicted for inbreeding and outcrossing populations. See Supplementary Table S7 for full details. aMinimum number of alleles calculated assuming all missing alleles in a population were the same; maximum assuming all were different. bCalculated excluding the mixed mating population TSSA. cOutcrossing rate was based on only 5 families and 5 individuals per family, and hence it was excluded from calculation of averages. dS45 is unlinked to the S-phenotype and sometimes found with two other SRK alleles but only present in some individuals. eA putatively new allele was allocated the name S52 but phenotypic testing of linkage would be required before ofﬁcial naming as an S-allele. Table 1 Number of SRK alleles inferred for 8 individuals per population based on direct and indirect genotyping, sorted by genetic cluster predicted by STRUCTURE analysis of microsatellites and population outcrossing rate (both taken from Foxe et al., 2010) indicating the number of individuals containing each of the variants screened in the allele-speciﬁc genotyping, as well as other alleles identiﬁed by cloning or MiSeq analyses Heredity Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 56 All of the inbreeding populations with S19 shared one of two B80 variants (which differed at 5 out of 666 bp): hap75, which was found in most of the populations, and hap76, which was only found in LPT (Table 2 and Supplementary Table S4). All individuals from the LPT population shared a single synonymous mutation in the S-domain region of S19. B80 hap75 was found in SI individuals from outcrossing populations but outcrossing populations also had other variants (haps 49, 114 and 122). Although the other ﬂanking genes in S19 All of the inbreeding populations with S19 shared one of two B80 variants (which differed at 5 out of 666 bp): hap75, which was found in most of the populations, and hap76, which was only found in LPT (Table 2 and Supplementary Table S4). All individuals from the LPT population shared a single synonymous mutation in the S-domain region of S19. Characterisation of S-haplotypes in inbreeding and outcrossing populations SC RON, PTP 11 1 50 7 23 ? SC RON 1 1 50 ? 22 ? SC RON 1 1 51 47 38 14 SI PRI 1 1 67 20 16 4 PC IOM 1 1 67 20 16, 24 ? SI IOM 1 1 78 3 15 11, 12 SI LSP 1 1 78 3 15, 22 ? SI LSP 1 1 115 3 15, 41 4 SI SBD 1 1 43 ? 15 ? SI TSSA 1 3 48 6 15 20 SI TSS 1 3 48 ? 40 15 SI PUK 1 Abbreviations: PC, partially self-compatible; SC, self-compatible; SI, self-incompatible; SRK S-locus receptor kinase. Numbers indicate the allele designation at SRK and its ﬂanking genes; unresolved alleles are indicated by ‘?’. For each S-locus haplotype (that is, combination of alleles), the selﬁng phenotype, population and the number of individuals (N) in which it was found are indicated. aB70 showed unreliable ampliﬁcation but some genotypes were resolved. bOutcrossing populations are in bold; the mixed mating population TSSA is in italics. cSingle synonymous mutation in S-domain of SRK compared with other populations. Table 2 Flanking gene variants for individuals that showed ampliﬁcation of only one SRK allele (using direct allele-speciﬁc screening) and were homozygous at B80 Estimating the number of S-haplotypes within populations There was little difference in the number of S-haplotypes predicted in different clusters. Inheritance of the selﬁng phenotype Inheritance of the selﬁng phenotype Although most F1 progeny from the within-population crosses involving SI individuals from the outcrossing population MAN yielded SI individuals, one individual (out of 28 screened) was SC, and leaky SI was found in all of the families (Supplementary Figure S4). Crosses between SI and SC plants (MAN× PTP) yielded a mixture of SC and SI phenotypes (20 SC out of 71 screened). All F1 progeny from crosses involving the outcrossing PIN were SI but the 97 F2 progeny from a cross between an SI individual from PIN and a selﬁng individual from RON segregated for the selﬁng phenotype: 10 were SC; 71 were SI; Figure 1 Minimum evolution genealogy of B80 alleles, indicating associations with SRK alleles and geographic distribution. The frequency of each allele is indicated in parentheses after its name. The tree was reconstructed using MEGA 6.0, under a Kimura 2 parameter model of evolution, with rate heterogeneity modelled under a gamma distribution using a rate parameter of 0.45. Numbers on the nodes indicate bootstrap support based on 1000 pseudoreplicates. As low phylogenetic resolution is expected for genes evolving under balancing selection, the main purpose of the tree is for visualisation of relatedness among B80 alleles in relation to their association with SRK alleles. Associated SRK alleles are indicated by name and using coloured branches. Occurrences of each B80 allele in inbreeding and outcrossing populations and in each of the six genetic clusters predicted by STRUCTURE are indicated in the table to the right. Heredity Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 57 4 were leaky SI; 12 were male sterile; and self-pollinations gave ambiguous results for one. For a balanced comparison in the bulked segregant analysis, we thus combined DNA extracted from all 10 SC individuals for the SC pool and 10 SI individuals for the SI pool. The SI individuals were selected from across the four families (Supplementary Tables S3 and S8). Heredity Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 58 The genetic basis of loss of SI In the F2 progeny segregating for the selﬁng phenotype, S-haplotype segregation based on B80 genotypes revealed that all SC individuals were homozygous for S1, whereas the SI phenotypes included the 3 heterozygous combinations and a single S1 homozygote (Supplementary Table S3). Inheritance of the selﬁng phenotype There was also complete correspondence between genotypes based on direct SRK sequencing and B80 sequen- cing (Supplementary Table S3). However, there was evidence for a segregation bias that differed among the four families pooled for the bulked segregant analysis (Supplementary Table S8). Two families showed a signiﬁcant deﬁcit of S1S1 and S1Sx genotypes and a deﬁcit of S1 alleles overall; one of these families produced exclusively SI individuals, whereas the other showed 10% SC individuals and included 30% of individuals with a male sterile phenotype. The remaining two families showed no bias in terms of genotypes or alleles; one had 14% SC and 21% male sterile individuals, whereas the other had 14% SC individuals but did not include any that were male sterile. we noted that the entire ﬂanking gene region (starting from genes upstream from B70 and continuing downstream from B160) showed extensive homozygosity in the SC pools (with variants shared with the AL4 sequences) but polymorphism in the SI pools (Table 3). Mapping of the raw reads identiﬁed all three parental B80 alleles in the SI pool but only that associated with S1 in the SC pool. Direct B80 genotyping of the F2 individuals conﬁrmed this pattern. Outside of the S-locus region on chromosome 7, we also found evidence for the predicted patterns of association with the SC phenotype based on SNP calls from SHOREmap: out of 55 660 SNPs on the long arm of chromosome 7 (excluding indels), 67 were ﬁxed in the SC pool (based on a threshold of 0.1% polymorphism) but different or polymorphic in the SI pools and shared with AL4 but not the MN47 reference sequence. This pattern was most concentrated in two regions in close proximity to each other but some distance from the S-locus (positions 6 788 674 to 6 788 963 and 7 382 799 to 7 382 948): 15 SNPs in a gene associated with pollen tube development (β-galactosidase) (Rejon et al., 2013) and 16 SNPs in an unidentiﬁed protein adjacent to a gene that has been associated with the SI reaction in Brassica (P-loop containing nucleoside triphosphate hydrolase superfamily protein) (Wang et al., 2014). Inspection of the consensus sequences generated by piling up the short reads conﬁrmed that the SC pool was homozygous, whereas the SI pool was heterozygous for both of these regions; homologues of both genes are located on chromosome 4 of A. Inheritance of the selﬁng phenotype thaliana (positions 13 246 742 to 13 245 999 and 12 809 347 to 12 808 981). Although the SHOREmap output suggested that there also might be candidate genes that differed between the SC and SI pools on chromosome 5, we did not ﬁnd any regions showing a concentration of homozygosity in the SC pool that was shared with AL4 but not with MN47 or the SI pool. Although patterns of polymorphism in each of the pools were very similar (SHOREmap output, Supplementary Figure S5), there were extended regions on both chromosomes 5 and 7 where the SC pool appeared to have low heterozygosity and to be similar to the AL4 reference sequence (that is, dipped towards the 0 side of the graph; Figure 2), whereas the SI pool was polymorphic. The largest region of extended homozygosity was observed between 9 and 10 Mb on the long arm of chromosome 7, the location of the S-locus in A. lyrata (Hu et al., 2011). Inspection of SNP calls across the S-locus region revealed that although variants were clearly present in the ﬂanking genes and in a fragment of SCR, no variants were called at the SRK gene (Table 3). As we know this gene should be highly polymorphic, we concluded that it was too divergent to be mapped to the reference. Nevertheless, A more targeted sequence-based examination of variation at the S-locus conﬁrmed that the S-locus was homozygous in the SC pool Figure 2 SHOREmap output for chromosomes 5, 6 and 7. The trace in red shows comparison of the SC pool with the reference sequence AL4 (from an SC individual from RON) and the trace in blue shows that for the SI pool. The scale at the bottom shows the position along the chromosome. The plots were produced using a step size of 10 000 and a window size of 200 000 bp. For each chromosome plot, the y axis indicates the proportion of reads either matching or showing an alternative to the reference sequence: 0 indicates ﬁxation of variants that match AL4 and 1 indicates ﬁxation for a different variant; the red line in the middle shows 50% heterozygosity. Inheritance of the selﬁng phenotype Note that for most regions, there is no difference between the SI and SC pools, whereas on the short arm of chromosome 5 (and near the centromere) and the long arm of chromosome 7 there are extended regions where the SC pool is more homozygous than the SI pool and skewed towards values near 0 (indicating that it is the same as the AL4 sequence); several examples are shown with arrows on the two chromosomes. The most concentrated region showing this pattern is between 9 and 10 Mb on chromosome 7, the location of the S-locus. Figure 2 SHOREmap output for chromosomes 5, 6 and 7. The trace in red shows comparison of the SC pool with the reference sequence AL4 (from an SC individual from RON) and the trace in blue shows that for the SI pool. The scale at the bottom shows the position along the chromosome. The plots were produced using a step size of 10 000 and a window size of 200 000 bp. For each chromosome plot, the y axis indicates the proportion of reads either matching or showing an alternative to the reference sequence: 0 indicates ﬁxation of variants that match AL4 and 1 indicates ﬁxation for a different variant; the red line in the middle shows 50% heterozygosity. Note that for most regions, there is no difference between the SI and SC pools, whereas on the short arm of chromosome 5 (and near the centromere) and the long arm of chromosome 7 there are extended regions where the SC pool is more homozygous than the SI pool and skewed towards values near 0 (indicating that it is the same as the AL4 sequence); several examples are shown with arrows on the two chromosomes. The most concentrated region showing this pattern is between 9 and 10 Mb on chromosome 7, the location of the S-locus. Figure 2 SHOREmap output for chromosomes 5, 6 and 7. The trace in red shows comparison of the SC pool with the reference sequence AL4 (from an SC individual from RON) and the trace in blue shows that for the SI pool. The scale at the bottom shows the position along the chromosome. The plots were produced using a step size of 10 000 and a window size of 200 000 bp. Inheritance of the selﬁng phenotype For each chromosome plot, the y axis indicates the proportion of reads either matching or showing an alternative to the reference sequence: 0 indicates ﬁxation of variants that match AL4 and 1 indicates ﬁxation for a different variant; the red line in the middle shows 50% heterozygosity. Note that for most regions, there is no difference between the SI and SC pools, whereas on the short arm of chromosome 5 (and near the centromere) and the long arm of chromosome 7 there are extended regions where the SC pool is more homozygous than the SI pool and skewed towards values near 0 (indicating that it is the same as the AL4 sequence); several examples are shown with arrows on the two chromosomes. The most concentrated region showing this pattern is between 9 and 10 Mb on chromosome 7, the location of the S-locus. Inheritance of the selﬁng phenotype Heredity Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 59 Table 3 SNP analysis of the S-locus region (9–10 Mb on chromosome 7) from the SHORE output, indicating the identity of the gene, the number of SNPs called, the number of SNPs in coding regions and the proportion of sites for each gene that: (1) were homozygous in the SC pool; (2) homozygous in both the SC pool and AL4 sequences but not in the SI pool; (3) were ﬁxed for different variants in the SI and SC pools when both were homozygous; (4) shared the same SNP in the SC pool and the AL4 sequences when both were homozygous but the SI pool was heterozygous; (5) showed the same homozygous variant in the SC pool as found in the MN47 reference; (6) showed incomplete coverage in the SC pool, indicated by missing variants because of lack of reads; and (7) had indels in regions that did show read coverage Genea N SNPs N SNPs coding (1) Homo SC (2) AL4 and SC both Homo but SI Het (3) SC ǂ SI when both Homo (4) SC = AL4 when Homo and SI Het (5) SC = MN47 when SC Homo (6) Sites missing in SC (7) Indels T6K22.160 (B160, transcription factor) 25 14 0.88 0.20 0.20 0.20 0.20 0.20 0.12 T6K22.150 4 4 1.00 0.50 0.00 0.50 0.50 0.00 0.00 T6K22.140 19 13 0.47 0.05 0.00 0.05 0.05 0.00 0.00 T6K22.130 145 105 0.96 0.54 0.14 0.45 0.46 0.09 0.03 T6K22.120 (B120, lectin protein kinase) 75 64 1.00 0.49 0.00 0.45 0.45 0.00 0.05 T6K22.110 (ARK3) 207 137 0.88 0.41 0.18 0.34 0.34 0.07 0.12 T6K22.90 (SCRA) 38 0 0.32 0.16 0.11 0.13 0.18 0.13 0.08 T6K22.100 (SRK) 0 T6K22.80 (B80, PUB8) 38 36 1.00 0.53 0.53 0.53 0.53 0.00 0.00 T6K22.70 (B70, ethylene responsive) 9 5 0.78 0.22 0.00 0.22 0.22 0.00 0.11 T6K22.60 (TF dysfunctional tapetum) 28 11 1.00 0.96 0.96 0.96 0.96 0.00 0.14 T6K22.50 (subtilase family protein) 91 53 0.97 0.38 0.00 0.38 0.38 0.00 0.02 T6K22.40 (unknown protein) 34 15 1.00 0.56 0.00 0.56 0.56 0.00 0.03 T6K22.30 (pentatricopeptide repeat-containing protein) 59 59 1.00 0.32 0.00 0.32 0.32 0.00 0.00 T6K22.20 (oxygen-evolving enhancer protein 3-1) 27 1 0.44 0.11 0.00 0.11 0.11 0.00 0.89 T6K22.10 (KatA mRNA for kinesin-like motor protein) 17 1 1.00 0.47 0.00 0.47 0.47 0.00 0.24 Abbreviations: Het, heterozygous; Homo, homozygous; SC, self-compatible; SI, self-incompatible; SNP, single-nucleotide polymorphism; SRK, S-locus receptor kinase; TF, transcription factor. Inheritance of the selﬁng phenotype Flanking genes screened in the population survey are indicated in bold. Note that no SNPs were called in the region where SRK was anticipated to be located. aT6k22 numbers are from the bacterial artiﬁcial chromosome (BAC) clone sequenced by Kusaba et al. (2001). Inheritance of the selﬁng phenotype Although neither SRK nor SCR were mapped to the consensus sequences, the S-locus ﬂanking genes (B70, B80, ARK3, B120 and B160) were found to be complete on scaffold 7 (Supplementary Figure S6) and were homozygous in the SC pools but heterozygous in the SI pools. There was no difference between the two pools for any of the unlinked genes: Aly7 (scaffold 8); Aly9 (scaffold 3); ARK1 and ARK2 (scaffold 2); ARC1 (scaffold 4) and PUB17 (scaffold 1). All but ARC1 were heterozygous in both pools; ARC1 showed no nonsynonymous mutations compared with the published functional allele (which was from the MN47 reference). Although the unlinked 13-2 allele was known to be present in both parents, it did not map to the consensus pools, likely because this locus is absent from the MN47 reference sequence. 2005) was overrepresented in the inbreeding populations but under- represented in the outcrossing populations. Its ﬂanking genes suggest a common origin of S19 in the inbreeding populations, as the associated alleles in the ﬂanking genes are monophyletic and show little variation overall. The same variants are found in some individuals from outcrossing populations but these remain strongly SI; we also did not observe homozygotes in the outcrossing populations that would suggest disruption of S19. One of the inbreeding populations (LPT) has a single bp mutation in both the S-domain (the recognition domain) of SRK and in B80 compared with other populations (and also has a unique variant of B120) but this S-locus haplotype is not found in the other inbreeding populations or in the outcrossing populations. These results could be explained by: (1) disruption of S19 only in the inbreeding populations (for example, by mutations in SCR or recombination between SRK and SCR); (2) presence of a modiﬁer speciﬁc to SC individuals suppressing the expression of S19; or (3) S19 in the inbreeding populations having risen to high frequency because of colonisation history rather than a causal relationship with the breakdown of SI. Enforced selﬁng of SI individuals with S19 could be used to determine whether homozygosity of this haplotype is sufﬁcient for disruption of SI or whether other factors found only in SC individuals are required. Mapping the unassembled reads to the expected SRK alleles in the crosses (S1 and S23) clearly conﬁrmed that SRK was missing from the SHOREmap SNP calls because it had not been mapped to the reference. Inheritance of the selﬁng phenotype Table 3 SNP analysis of the S-locus region (9–10 Mb on chromosome 7) from the SHORE output, indicating the identity of the gene, the number of SNPs called, the number of SNPs in coding regions and the proportion of sites for each gene that: (1) were homozygous in the SC pool; (2) homozygous in both the SC pool and AL4 sequences but not in the SI pool; (3) were ﬁxed for different variants in the SI and SC pools when both were homozygous; (4) shared the same SNP in the SC pool and the AL4 sequences when both were homozygous but the SI pool was heterozygous; (5) showed the same homozygous variant in the SC pool as found in the MN47 reference; (6) showed incomplete coverage in the SC pool, indicated by missing variants because of lack of reads; and (7) had indels in regions that did show read coverage Genea N SNPs N SNPs coding (1) Homo SC (2) AL4 and SC both Homo but SI Het (3) SC ǂ SI when both Homo (4) SC = AL4 when Homo and SI Het (5) SC = MN47 when SC Homo (6) Sites missing in SC (7) Indels 3 SNP analysis of the S-locus region (9–10 Mb on chromosome 7) from the SHORE output, indicating the identity of the gene, the number of SNPs called, the number of SNPs in g regions and the proportion of sites for each gene that: (1) were homozygous in the SC pool; (2) homozygous in both the SC pool and AL4 sequences but not in the SI pool; (3) were for different variants in the SI and SC pools when both were homozygous; (4) shared the same SNP in the SC pool and the AL4 sequences when both were homozygous but the SI pool eterozygous; (5) showed the same homozygous variant in the SC pool as found in the MN47 reference; (6) showed incomplete coverage in the SC pool, indicated by missing variants se of lack of reads; and (7) had indels in regions that did show read coverage N SNPs N SNPs coding (1) Homo SC (2) AL4 and SC both Homo but SI Het (3) SC ǂ SI when both Homo (4) SC = AL4 when Homo and SI Het (5) SC = MN47 when SC Homo (6) Sites missing in SC (7) Indels Heredity Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 60 but heterozygous in the SI pool. Number of S-alleles in outcrossing populations Number of S-alleles in outcrossing populations Also striking from our analyses is the relatively low number of S-haplotypes predicted in most of the North American populations, including those that are highly outcrossing. This is surprising given that it has been estimated that there should be 4100 S-haplotypes in outcrossing species (Castric and Vekemans, 2004) and previous surveys of European A. lyrata have predicted 16–25 haplotypes per population (Mable et al., 2003; Schierup et al., 2008). The lower number of S-haplotypes in the Great Lakes populations is consistent with a predicted bottleneck in North American compared with European populations (Ross-Ibarra et al., 2008). The outcrossing populations with the lowest predicted numbers of S-alleles (HDC and OWB) also had relatively low outcrossing rates (Tm = 0.65 and 0.64, respectively), more SC and leaky SI than SI individuals and high S1 frequency (Supplementary Table S4). These populations thus could be in a transition to inbreeding, possibly because of mate limitation, which has been suggested as a primary driver of transitions from outcrossing to inbreeding (Byers and Meagher, 1992; Vekemans et al., Inheritance of the selﬁng phenotype The unlinked 13-2 sequence was present in the unmapped reads of both the SI and SC pools but none of the other ﬂanking genes were. For SRK, both S1 and S23 alleles were present in the SI pool but only S1 was identiﬁed in the SC pool. Mapping the unassembled reads to the full-length SCR and SRK sequences obtained from the BAC clone for S1 (Goubet et al., 2012) demonstrated that both pools had complete sequences for both recognition genes for this haplotype. Moreover, although there were multiple synonymous SNPs compared with the BAC sequences, there were no nonsynonymous mutations that would indicate disruption of function at either gene. The other main S-haplotype occurring in inbreeding populations (S1) was found in all populations surveyed except for the inbreeding populations KTT, LPT and TC. This SRK allele has been found worldwide, is completely recessive to all other alleles tested and is shared with other Brassicaceae relatives (Schierup et al., 2001; Mable et al., 2004; Paetsch et al., 2006; Castric et al., 2010) but it has not previously been directly associated with loss of SI. We predicted that of the 123 individuals with S1 in the Great Lakes populations, 47% were S1S1 homozygotes. However, 20% of these were found in outcrossing populations, suggesting that it is not just homozygosity for S1 that causes loss of SI. In fact, only six fully SC individuals were found in outcrossing populations; although they all contained S1, four of these were predicted to be heterozygotes (three with S13 and one with an unidentiﬁed haplotype; Supplementary Table S4). Although S1 was associated with at least 18 distinctive B80 haplotypes (Figure 1), only 2 were found in inbreeding populations. Similarly, only three of the wide range of S1 B120 haplotypes were found in the inbreeding populations (Supplementary Figure S1). This is consistent with a bottleneck in S-haplotypes, as has been suggested for all other species compared so far (Vekemans et al., 2014). DISCUSSION Our results demonstrate that the shift to inbreeding in North American Great Lakes populations of A. lyrata is associated with a reduction in the number of SI haplotypes, consistent with theoretical predictions and other experimental studies (reviewed by Vekemans et al., 2014). Based on SRK and its ﬂanking genes, we conclude that two S-locus haplotypes are associated with this transition across multiple genetic backgrounds but that these are also found in SI individuals from outcrossing populations, potentially reﬂecting the very young age of the loss of SI. The bulked segregant analysis of F2 progeny resulting from a cross between SI and SC parents indicated that loss of SI (at least in the genetic background tested) is recessive and may be associated with a modiﬁer of expression of the S1 recognition genes or downstream components of the SI reaction, rather than mutations at the S-locus itself. Although further experi- ments are required to unravel the speciﬁc mechanisms, combining new technologies with classical genetic approaches has revealed new insights into a long-standing question. Characterisation of S-haplotypes in inbreeding and outcrossing populations The multipronged approach to S-locus genotyping used in this study revealed a much clearer pattern of association between S-haplotypes and inbreeding than observed in our previous study (Mable and Adam, 2007). The MiSeq pilot study holds promise for more effective utilisation of short read sequencing approaches as an alternative to previous amplicon-based approaches (for example, using 454 sequen- cing; Jørgensen et al., 2012). We would recommend using the de novo sequencing approach for identifying variants, as assembling to a known database was more error prone. Moreover, the tight association between B80 and SRK variants conﬁrmed in our study provides a useful tool for resolving genotypes with direct Sanger sequencing. Based on our new genotyping, one haplotype (S19) that has been found to be dominant based on segregation analyses (Prigoda et al., Heredity Loss of self-incompatibility in Arabidopsis lyrata BK Mable et al 61 found in the MN47 reference and the S1 and S23 haplotypes expected in the pools. It is thus critical for such highly polymorphic genes to include unassembled reads in comparative analyses. 1998, 2014). If mate limitation occurred during a colonisation bottle- neck (that is, after the last ice age), high levels of biparental inbreeding could have purged deleterious mutations that maintain outcrossing in other parts of the range of A. lyrata (Sletvold et al., 2013). y For both the male and female SI recognition genes (SCR and SRK) and all of the ﬂanking genes tested, comparison of the consensus sequence alignments conﬁrmed that the SC pools were homozygous for S1-associated variants, whereas the SI pools were polymorphic. This was in contrast to other members of the SRK gene family located on other chromosomes, for which there was no difference in heterozygosity between the pools. There were also no differences detected between the pools in genes that have been implicated in the downstream regulation of SI (ARC1 and PUB17) (Indriolo et al., 2012; Goring et al., 2014; Nasrallah and Nasrallah, 2014). ARC1 was homozygous in both pools, and although there were some regions where the pools did not align to the published sequence, these were the same in the SI and SC pools. The role of PUB17 in the SI reaction is less clear (Goring et al., 2014; Nasrallah and Nasrallah, 2014) but it was heterozygous in both pools. Genetic basis of loss of SI Genetic basis of loss of SI Our crossing data demonstrated that all F1 offspring from the SI× SC cross were strongly SI but SC segregated in the F2 generation, indicating that SC as a phenotypic state was recessive. The appearance of male sterile plants (for which the selﬁng phenotype cannot be determined) made it difﬁcult to estimate the exact proportion of SC plants in the F2. Formal testing of genetic models was therefore not possible (Supplementary Table S9). Nevertheless, the fact that all SC plants were homozygous for S1, combined with the ﬁxation of S1 in the predominantly selﬁng RON and PTP populations, strongly suggests that SC is functionally linked to this S-allele. This association with S1 does not appear to be explained by genetic linkage, because S1 homozygosity alone was not sufﬁcient to confer SC. One plant in the F2 progeny (Supplementary Table S3) and 19 plants raised from wild- collected seeds were S1S1 but SI (Supplementary Table S4). S1 homozygotes that are not SC can be explained by a model that invokes a recessive modiﬁer unlinked to the S-locus (scenario A in Supplementary Table S9). Segregation distortion at the S-locus is not unprecedented (Bechsgaard et al., 2004). Still, the observation that there was a bias against S1 in only half of the crosses among F1 plants generated from the same parents (Supplementary Table S8) is also consistent with an unlinked but allele-speciﬁc viability modiﬁer, which could explain the low numbers of SC plants surviving to ﬂowering. We conclude that an unlinked, recessive modiﬁer of the expression of S1 or its associated downstream genes confers SC in S1 homozygotes, at least in the RON and PTP populations. g g The analysis of SNPs that were homozygous in the SC pool, shared with the AL4 reference and heterozygous or different in the SI pool, identiﬁed two candidate genes for unlinked modiﬁers. Both genes have previously been implicated in processes that could affect the SI reaction (that is, prevention of pollen tube development). The P-loop containing nucleoside triphosphate hydrolase superfamily protein has been predicted to inﬂuence indirect interactions between a network of SI-related genes (identiﬁed by comparing SI and SC Chinese cabbage) related to energy metabolism and stress responses in Arabidopsis (Wang et al., 2014). Genetic basis of loss of SI In lilies, β-galactosidase has been hypothesised to contribute to degradation of large polysaccharides secreted by papillae in order to allow them to be incorporated into the growing pollen tube (Rejon et al., 2013). Thus, loss of SI could be associated with regulatory changes that affect the complex network of pathways that normally prevent self-pollen from maturing and producing full-length pollen tubes. Additional experiments to inves- tigate the role that such enzymes play in self- and non-self-pollen reactions in Arabidopsis species would be necessary to conﬁrm their direct involvement, but the bulked segregant analysis even of small pools of 10 individuals provided a useful starting point for narrowing down the search. Results from crosses involving PTP and MAN (Supplementary Figure S4) are similarly consistent with a modiﬁer segregating in outcrossing populations that is only expressed in S1 homozygotes that are also homozygous for the modiﬁer. First, even crosses between SI plants can result in SC offspring. Second, crosses between SI individuals from an outcrossing population (MAN, population S1 frequency 7/8, Supplementary Table S4) and SC individuals from an inbreeding population (PTP, population ﬁxed for S1) yielded variable ratios of SC progeny, generally well below 50%. Although complete SRK genotypes were not resolved for the crosses, SC individuals were only found in cases where the SI parent had S1 (MAN18b, MAN22f but not MAN17f; Supplementary Figure S4). In the MAN population survey, two individuals were S1S1 homo- zygotes but only one was phenotypically SI, even though they shared the same B80 and B120 genotypes (Supplementary Table S4). Further work is needed to identify the exact locus of the recessive modiﬁer of S1 or its associated downstream genes, its exact functioning and to test whether it is the only mechanism for the loss of SI in Arabidopsis lyrata. Characterisation of S-haplotypes in inbreeding and outcrossing populations The S1 BAC clone was obtained from an individual from Iceland (Goubet et al., 2012), and hence some variation would be expected within this recessive speciﬁcity (Castric et al., 2010). However, none of the mutations resulted in amino acid substitutions or altered the reading frame to cause a premature stop codon in SRK or SCR. We thus have no evidence that mutation of the recognition genes themselves has disrupted SI in this cross. ACKNOWLEDGEMENTS Self-incompatibility in the genus Arabidopsis: characterization of the S locus in the outcrossing A. lyrata and its autogamous relative A. thaliana. Plant Cell 13: 627–643. Lewis D (1947). Competition and dominance of incompatibility alleles in diploid pollen. Heredity 1: 85–108. Li H (2011). A statistical framework for SNP calling, mutation discovery, association mapping and population genetical parameter estimation from sequencing data. Bioinformatics 27: 2987–2993. Mable BK, Adam A (2007). Patterns of genetic diversity in outcrossing and selﬁng populations of Arabidopsis lyrata. Mol Ecol 16: 3565–3580. Mable BK, Beland J, Di Berardo C (2004). 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Mate availability in small populations of pl homomorphic sporophytic self-incompatibility. Heredity 68: 353–359. Pannell JR, Auld JR, Brandvain Y, Burd M, Busch JW, Cheptou PO et al. (2015). The scope of Baker's law. New Phyt 208: 656–667. Castric V, Bechsgaard JS, Grenier S, Noureddine R, Schierup MH, Vekemans X (2010). CONCLUSIONS Our results suggest that there has been a bottleneck in S-alleles associated with a transition to inbreeding in A. lyrata but that the mechanisms for loss of SI might be because of modiﬁers of the SI reaction rather than mutations at the S-locus itself. They thus emphasise the importance of considering the two processes separately. The situation found in A. lyrata where SC segregates in all populations but only rises to high frequency in some provides excellent opportu- nities for resolving the speciﬁc mechanisms and selective forces that promote transitions in mating system. Nevertheless, even in this young system, uncovering the genetic basis of this complex transition remains challenging. The SHOREmap analysis helped us to predict genomic regions where there was a difference between SI and SC individuals that could be used to initiate this search for the potential modiﬁers, despite previous concerns that it would only be useful for very large sample sizes (Austin et al., 2011). The bulked segregant analysis indicated that the two pools differed predominantly on chromosomes 5 and 7, with the largest region of homozygosity at the S-locus, between 9 and 10 Mb on the long arm of chromosome 7 (Supplementary Figure S5). 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Sanger sequencing data are available from GenBank, with the following accession numbers: KX923797 for the new AlySRK52 sequence; KX923776-KX923796 for B70, and KX923711-KX923775 for B120. Hatakeyama K, Watanabe M, Takasaki T, Ojima K, Hinata K (1998). Dominance relationships between S-alleles in self-incompatible Brassica campestris L. Heredity 80: 241–247. Haudry A, Zha HG, Stift M, Mable BK (2012). Disentangling the effects of breakdown of self-incompatibility and transition to selﬁng in North American Arabidopsis lyrata. Mol Ecol 21: 1130–1142. Hu TT, Pattyn P, Bakker EG, Cao J, Cheng JF, Clark RM et al. (2011). The Arabidopsis lyrata genome sequence and the basis of rapid genome size change. Nat Genet 43: 476–481. Igic B, Lande R, Kohn JR (2008). Loss of self-incompatibility and its evolutionary consequences. Int J Plant Sci 169: 93–104. Indriolo E, Safavian D, Goring DR (2014). The ARC1 E3 ligase promotes two different self- pollen avoidance traits in Arabidopsis. Plant Cell 26: 1525–1543. pollen avoidance traits in Arabidopsis. Plant Cell 26: 1525–1543. CONFLICT OF INTEREST Indriolo E, Tharmapalan P, Wright SI, Goring DR (2012). The ARC1 E3 ligase gene is frequently deleted in self-compatible Brassicaceae species and has a conserved role in Arabidopsis lyrata self-pollen rejection. Plant Cell 24: 4607–4620. The authors declare no conﬂict of interest. Iwano M, Ito K, Fujii S, Kakita M, Asano-Shimosato H, Igarashi M et al. (2015). Calcium signalling mediates self-incompatibility response in the Brassicaceae. Nat Plants 1: 9. ACKNOWLEDGEMENTS We thank Hong-Guang Zha for performing the ﬂanking gene sequencing; Kirsi Järvi, Laura Murphy and Ryan Carter for assistance with S-locus genotyping; James Buckley and Manikhandan Mudaliar for extracting consensus sequences from the Illumina sequence pools; and Rob Bregman for performing the cross in the parental generation. Original seeds for some populations were collected by Yvonne Willi and David Remington (see Supplementary Table S1). Detailed comments from Xavier Vekemans, Vincent Castric and an anonymous reviewer substantially improved the paper. Anita O Lucaci at the Centre for Genomic Research at Liverpool prepared the libraries and did the MiSeq sequencing and John Kenny developed the pilot long-read tagged amplicon protocol; this work was supported by a Natural Environment Research Council (NERC) We thank Hong-Guang Zha for performing the ﬂanking gene sequencing; Kirsi Järvi, Laura Murphy and Ryan Carter for assistance with S-locus genotyping; James Buckley and Manikhandan Mudaliar for extracting consensus sequences from the Illumina sequence pools; and Rob Bregman for performing the cross in the parental generation. Original seeds for some populations were collected by Yvonne Willi and David Remington (see Supplementary Table S1). 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https://openalex.org/W2580601488	https://digitalcommons.fiu.edu/cgi/viewcontent.cgi?article=1457&context=fce_lter_journal_articles	English	null	Examining gradients in ecosystem novelty: fish assemblage structure in an invaded Everglades canal system	Ecosphere	2,017	cc-by	13,597	1-27-2017 Examining gradients in ecosystem novelty: fish assemblage structure in an invaded Everglades canal system David A. Gandy Earth and Environment Department and Southeast Environmental Research Center, Florida International University; Florida Fish and Wildlife Conservation Commission, dgandy@fiu.edu Jennifer S. Rehage Earth and Environment Department and Southeast Environmental Research Center, Florida International University, rehagej@fiu.edu Jennifer S. Rehage Earth and Environment Department and Southeast Environmental Research Center, Florida International University, rehagej@fiu.edu this and additional works at: https://digitalcommons.fiu.edu/fce_lter_journal_articles of the Life Sciences Commons Follow this and additional works at: https://digitalcommons.fiu.edu/fce_lter_journal_ Part of the Life Sciences Commons Florida International University FIU Digital Commons Florida International University FIU Digital Commons FCE LTER FCE LTER Journal Articles Examining gradients in ecosystem novelty: ﬁsh assemblage structure in an invaded Everglades canal system DAVID A. GANDY1,3, AND JENNIFER S. REHAGE2 1Florida Fish and Wildlife Conservation Commission, Fish and Wildlife Research Institute, Apalachicola Bay Fisheries-Independent Monitoring Field Laboratory, Eastpoint, Florida 32328 USA 2Earth and Environment Department, Southeast Environmental Research Center, Florida International University, Miami, Florida 33199 USA Citation: Gandy, D. A., and J. S. Rehage. 2017. Examining gradients in ecosystem novelty: ﬁsh assemblage structure in an invaded Everglades canal system. Ecosphere 8(1):e01634. 10.1002/ecs2.1634 Abstract. Novel ecosystems result from a combination of altered historical abiotic regimes and new spe- cies assemblages. In freshwater systems, novel environmental conditions often result from large-scale changes to hydrological connectivity as well as species invasions. Novel environmental conditions may affect the survival of aquatic fauna by altering dispersal patterns and resource ﬂuctuations, and/or may impose physiological constraints on native species evolutionarily adapted to particular environments. Fur- ther, novel systems can provide insight into processes driving community structure because re-sorting or ﬁltering of regional biota is a likely consequence of decoupling from historical conditions. Although several studies document the presence of novel conditions, few examine variation or gradients in novelty. The Flor- ida Everglades is a highly invaded and hydrologically altered system characterized by a large network of canals that compartmentalize the ecosystem and act to both increase and decrease connectivity. Little is known about how canals in this region function as habitat for native and nonnative ﬁshes, the extent to which these canals may function as novel habitats, and how these habitat characteristics may inﬂuence dis- tribution, abundance, and assembly patterns. In this study, we examined native and nonnative ﬁsh assem- blages along a gradient of novelty, deﬁned as the loss of wetland connectivity, inﬂuence of the natural hydrological regime, and habitat complexity (well connected to leveed canals). As novelty increased, native species richness and abundance strongly declined and the contribution of nonnatives increased to nearly 50%. Vast differences in community structure across the novelty gradient were strongly inﬂuenced by spa- tial factors and secondarily by hydrological factors, while habitat and abiotic factors were of very low rele- vance. Natives and nonnatives had opposing responses to key hydrological and habitat characteristics. Abundance of native ﬁshes declined with decreased connectivity to adjacent marshes and canal littoral zone width, while nonnative ﬁshes increased signiﬁcantly in the most novel canals. January 2017 ❖Volume 8(1) ❖Article e01634 3 Present address: 350 Carroll Street, East Point, Florida 32328 USA. Recommended Citation Recommended Citation Gandy, D. A., and J. S. Rehage. 2017. Examining gradients in ecosystem novelty: fish assemblage structure in an invaded Everglades canal system. Ecosphere 8(1):e01634. 10.1002/ecs2.1634 This material is based upon work supported by the National Science Foundation through the Florida Coastal Everglades Long-Term Ecological Research program under Cooperative Agreements #DBI-0620409 and #DEB-9910514. Any opinions, findings, conclusions, or recommendations expressed in the material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. This work is brought to you for free and open access by the FCE LTER at FIU Digital Commons. It has been accepted for inclusion in FCE LTER Journal Articles by an authorized administrator of FIU Digital Commons. For more information, please contact dcc@fiu.edu, jkrefft@fiu.edu. 3 Present address: 350 Carroll Street, East Point, Florida 32328 USA. E-mail: David.Gandy@MyFWC.com INTRODUCTION The synergistic effects of anthropogenic distur- bance and species invasions can rapidly alter both ecosystem structure and function (Milton 2003, Root and Schneider 2006, Didham et al. 2007). These changes can profoundly affect all levels of ecological organization and result in the emer- gence of biotic and abiotic conditions that bear lit- tle to no resemblance to their natural counterparts (Fox 2007, Kueffer 2015, Radeloff et al. 2015). Such systems have recently generated much dis- cussion and are often referred to as “no-analog” or “novel” ecosystems (Milton 2003, Hobbs et al. 2006, 2013, Fox 2007, Williams and Jackson 2007). Novel ecosystems are deﬁned by two key charac- teristics which include alterations to historical abi- otic regimes and the presence of new species assemblages, both caused by a combination of varying degrees of environmental degradation (e.g., land use changes) and species invasions (Hobbs et al. 2006, 2009). Novel systems provide an opportunity for insight into community assem- bly processes since re-sorting or ﬁltering of regio- nal biotas is a likely consequence of this decoupling from historical conditions. In freshwater systems, anthropogenic ecosys- tem degradation most often results from disruptions of natural hydrological regimes and spatially widespread disturbances (Pringle 2001, 2003, V€or€osmarty et al. 2010, Arthington 2012). Alterations to the natural ﬂow regime (Poff et al. 1997, Acreman et al. 2014) and aquatic species invasions (Ricciardi and Rasmussen 1999) are recognized as two of the most concerning global threats to aquatic biodiversity (Dudgeon et al. 2006). Altered hydrological connectivity from dams, impoundments, canals, and levees, cre- ated for both water retention and diversion, can result in novel conditions for aquatic fauna that can limit or enhance dispersal abilities, alter resource ﬂuctuations, and impose physiological constraints on native species that are evolutionar- ily adapted to particular natural regimes (Conley et al. 2000, Freeman et al. 2007, Franssen et al. 2013). ) The Everglades is a rain-fed karstic wetland system, which due to drainage and impound- ment, is presently bisected by an extensive net- work of canals and levees. Built for water supply and ﬂood control beginning in the 1880s, nearly 2500 km of canals and levees presently surround and cut through wetlands, impeding sheet ﬂow and compartmentalizing the entire ecosystem (Light and Dineen 1994, Sklar et al. 2002). Examining gradients in ecosystem novelty: ﬁsh assemblage structure in an invaded Everglades canal system Our results suggest that the inherent loss of natural environmental conditions and subsequent replacement by novel ones can lead to extensive changes in ﬁsh community structure. Success or failure at maintaining native assemblages will rely heavily on natural resource manager’s ability to incorporate natural environmental characteristics with ecosystem restoration. Key words: anthropogenic disturbance; canals; community structure; distance-based linear models; ecosystem novelty; Everglades; nonnatives; species invasions. Received 19 June 2016; revised 29 October 2016; accepted 1 November 2016. Corresponding Editor: Debra P. C. Peters. Copyright: © 2017 Gandy and Rehage. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. 3 Present address: 350 Carroll Street, East Point, Florida 32328 USA. E-mail: David.Gandy@MyFWC.com ❖www.esajournals.org ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 1 GANDY AND REHAGE et al. 2008, Lugo 2009, Hobbs et al. 2013). In aquatic and marine systems, examples of novel ecosystems are increasingly being reported (Nils- son and Berggren 2000, Gido et al. 2009, King et al. 2011, Pandolﬁet al. 2011). For instance, algal blooms from non-point source runoff in the Gulf of Mexico have resulted in extensive dead zones and novel species interactions (Rabalais et al. 2002). Similarly, human-induced ocean acid- iﬁcation combined with rising ocean temperatures and pollution has left novel ecosystems in the wake of once thriving coral reefs (Fabricius 2005, Hoegh-Guldberg et al. 2007, Pandolﬁet al. 2011). Although these studies documented ecosystem novelty, they provide little detailed information of variation in novelty, in other words how truly novel these conditions are. This variation can have important implications for resource management, conservation and restoration efforts. Effective resource management requires that we under- stand how novel conditions are, in order to apply appropriate management targets (i.e., protect vs. restore; Woodworth 2013, and Wuerthner et al. 2014). One potentially helpful approach is the identiﬁcation of gradients in novelty in the land- scape as a function of varying degrees of both abi- otic and biotic change (e.g., urbanization; King et al. 2011). January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 INTRODUCTION Canals in this region offer an opportunity to better understand how these structures function as novel habitat for both native and nonnative ﬁshes and how novel conditions inﬂuence the distribution, abundance, and assembly patterns in ﬁsh communities. Canals likely provide per- manent deep-water faunal refuges which were historically rare or absent in the natural Ever- glades ecosystem (Gunderson and Loftus 1993), thus acting as novel aquatic habitats. Canals also vary in their attributes, which we expect will result in a novelty gradient (Fig. 1). Variation in (1) their connectivity to adjacent marshes, (2) in the inﬂuence of the natural seasonal hydrological An estimated 40% of the Earth’s land area may already be covered by novel ecosystems (Ellis et al. 2010, Foley et al. 2011, Barnosky et al. 2012), with many terrestrial examples (Cramer and Hobbs 2002, Lindenmayer et al. 2008, Mascaro January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 2 Fig. 1. (A) Conceptual diagram depicting hypothesized changes in ﬁsh community structure as a function o increasing novelty in Everglades canals. Native species are represented by white symbols, while nonnatives a represented by black symbols, and symbol size corresponds to species abundance. We expect the degree of inv sion to increase with increasing novelty, leading to an increasing number of nonnative species (and higher nonn tive abundances) and a lower number of native species (and lower abundance—at least for some native taxa). A the highest level of novelty, we hypothesize habitat quality to decrease for native species, and for nonnative sp cies to dominate the community. Canals with higher novelty are characterized by lower connectivity to natur habitats, lower inﬂuence of the natural hydrological regime, and lower habitat complexity. Novelty increase from (B) well connected canals to (C) moderately connected canals and to (D) leveed canals. GANDY AND REHAG GANDY AND REHAGE Fig. 1. (A) Conceptual diagram depicting hypothesized changes in ﬁsh community structure as a function of increasing novelty in Everglades canals. Native species are represented by white symbols, while nonnatives are represented by black symbols, and symbol size corresponds to species abundance. We expect the degree of inva- sion to increase with increasing novelty, leading to an increasing number of nonnative species (and higher nonna- tive abundances) and a lower number of native species (and lower abundance—at least for some native taxa). INTRODUCTION At the highest level of novelty, we hypothesize habitat quality to decrease for native species, and for nonnative spe- cies to dominate the community. Canals with higher novelty are characterized by lower connectivity to natural habitats, lower inﬂuence of the natural hydrological regime, and lower habitat complexity. Novelty increases from (B) well connected canals to (C) moderately connected canals and to (D) leveed canals. Fig. 1. (A) Conceptual diagram depicting hypothesized changes in ﬁsh community structure as a function of increasing novelty in Everglades canals. Native species are represented by white symbols, while nonnatives are represented by black symbols, and symbol size corresponds to species abundance. We expect the degree of inva- sion to increase with increasing novelty, leading to an increasing number of nonnative species (and higher nonna- tive abundances) and a lower number of native species (and lower abundance—at least for some native taxa). At the highest level of novelty, we hypothesize habitat quality to decrease for native species, and for nonnative spe- cies to dominate the community. Canals with higher novelty are characterized by lower connectivity to natural habitats, lower inﬂuence of the natural hydrological regime, and lower habitat complexity. Novelty increases from (B) well connected canals to (C) moderately connected canals and to (D) leveed canals. regime, and (3) habitat complexity may drive gradients in novelty that we hypothesize relate to the degree of invasion. connected at high water levels and/or partially leveed), to those with no direct connectivity and virtually no habitat resemblance to Everglades freshwater habitats and thus higher novelty (fully leveed on both sides and isolated from sur- rounding wetlands; Fig. 1). Canals that are well connected to marshes should be more inﬂuenced by the natural wet dry pulsing seasonality of the g Canals exhibit a range in connectivity with surrounding marshes (analogous to ﬂoodplains), ranging from those that are well connected and contain complex littoral zone habitats, to canals with only moderate connectivity (i.e., only ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 3 GANDY AND REHAGE subtropical Everglades (Sklar et al. 2002, McVoy et al. 2011). resemblance to conditions found in Everglades marshes, resulting in a loss of native species and replacement by opportunistic invaders that could establish in these less favorable conditions (Fig. 1). Novelty gradient across Everglades canals Novelty gradient across Everglades canals We sampled ﬁve major canals (L-29, L-31N, L- 31W, C-111, and L-67A) in the central and south- ern Everglades (Fig. 2). Canals were located along the eastern boundary of ENP, bordering agriculture areas to the east, and to the north of ENP and center of Water Conservation Area 3 (WCA 3) bisecting freshwater marshes. To account for variation in canal traits across space and time, we classiﬁed our focal canals into nine canal sampling units (Table 1) on the basis of connectivity within a canal (i.e., presence of water control structures between sections of a canal), and to surrounding marshes (i.e., pres- ence of levees vs. direct connectivity to adjacent marshes; Fig. 1). When a water control structure was present in a canal, we examined 20 years of ﬂow data from each structure in order to demar- cate canal sampling units. For example, the L-29 canal is leveed in the eastern portion but not leveed in the western portion (Fig. 2), and the eastern and western sections of L-29 are sepa- rated by a gravity-fed water control structure (S- 333) which moves water between them, but likely limits the exchange of biota since at times in any given year it is closed. Thus, we delin- eated these two canal sections as separate canal sampling units (Fig. 2, Table 1). ( p ; ) In this study, we examined native and nonna- tive ﬁsh community structure in an Everglades canal network as a function of a gradient in nov- elty, speciﬁcally the loss of wetland connectivity, the natural seasonal hydrology, and habitat com- plexity—conditions not reﬂective of the pre-drai- nage Everglades (McVoy et al. 2011). Our speciﬁc objectives were to (1) examine spatiotemporal variation in both the native and nonnative ﬁsh communities in relation to the degree of novelty in canals, and (2) determine the relative impor- tance of hydrological, habitat, and spatiotemporal factors in driving community structure patterns. We expected that (1) increasing ecosystem novelty would result in a change in community structure driven by a loss in native species and replacement by nonnatives (Fig. 1) and (2) that the relative importance of structuring factors will differ between native and nonnative assemblages. We hypothesized that native species may not be able to cope with increasingly more novel con- ditions, resulting in niche opportunities that allow for increased nonnative species richness and abundance (Fig. 1). INTRODUCTION We also expected hydrological variables to play a stronger role in structuring the native community, given that this community should be pre-adapted and thus responsive to hydrology, particularly the strong signature of seasonal hydrological variation (Trexler et al. 2005, Reh- age and Trexler 2006). We expect then canals with lower connectivity to marshes, lower inﬂuence of the seasonal hydro- logical regime, and lower habitat complexity to be more novel and thus more invaded by nonnative ﬁsh species (Fig. 1). Fish invasions have been a persistent and prominent feature of the Ever- glades ecosystem (Fuller et al. 1999, Kline et al. 2014). This is particularly true in canals, which can act as a source of invasions to protected areas such as Everglades National Park (ENP; Kline et al. 2014). Presently, 34 nonnative ﬁshes are con- sidered established (i.e., they have reproductively viable populations; Shaﬂand et al. 2008) in south Florida, of which 17 are established within ENP (Kline et al. 2014). These numbers are compara- tively large relative to the low native ﬁsh diversity (35 species; Loftus 2000). ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 Novelty gradient across Everglades canals These isolated, deep, and low-com- plexity habitats are unlike any natural aquatic habitat in the Everglades, and thus, we consider January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 5 GANDY AND REHAGE Table 1. Summary of electroﬁshing sampling effort at each of the nine canal sampling units across years: years 1 (2010–2011), 2 (2011–2012), and 3 (2012–2013). (2010–2011), 2 (2011–2012), and 3 (2012–2013). Canal unit name Canal type Available stations (N) Year 1 (2010–2011) Year 2 (2011–2012) Year 3 (2012–2013) Wet Early dry Late dry Wet Early dry Late dry Wet Early dry Late dry L-67A WC 204 5 10 10 8 8 8 8 8 8 L-29 East WC 89 8 10 10 8 8 8 8 8 8 L-29 West WC 97 8 10 10 8 8 8 8 8 8 L-31W North MC 51 8 10 10 8 8 8 8 8 8 L-31W South MC 33 5 10 10 8 8 8 8 8 8 C-111 South MC 53 8 10 10 8 8 8 8 8 8 L-31N North† L 57 0 0 10 8 8 0 8 8 8 L-31N South L 84 8 5 10 8 8 8 8 8 8 C-111 North L 93 8 5 10 8 8 8 8 8 8 Seasonal total 58 80 90 72 72 64 72 72 72 Annual total N = 228 N = 208 N = 216 Notes: Shown are the number of electroﬁshing samples across the three seasonal sampling events per year. Canal units are classiﬁed into three canal types: WC, well connected; MC, moderately connected; L, leveed. † L-31N North was not accessible at times with no samples. Notes: Shown are the number of electroﬁshing samples across the three seasonal sampling events per year. Canal units are classiﬁed into three canal types: WC, well connected; MC, moderately connected; L, leveed. † L-31N North was not accessible at times with no samples. them to exhibit the highest degree of novelty along our deﬁned gradient (Fig. 1). rainfall conditions in years 1 and 2 of sampling (Abtew et al. 2013). Climatic conditions in the dry season of 2010–2011 (year 1) resulted in sev- ere region-wide drought conditions with prolonged drying and a late start to the wet sea- son. In year 3, the system experienced above- average rainfall conditions, and thus the wettest conditions of the 3-year period. Sampling design We used a stratiﬁed random sampling design to allocate replicated electroﬁshing sampling effort across the nine canal sampling units over time (hydrological seasons and years). A stratiﬁed random sampling approach offers a robust design by accounting for variability among strata, incre- asing precision in generating estimates of ﬁsh abundance, and also meets the assumptions of probability statistics (Hansen et al. 2007, Bonar et al. 2009). At each canal sampling unit, we used ArcGIS to designate uniquely identiﬁed ﬁxed sampling stations, each spaced 200 m apart (Table 1). At each seasonal sampling event, we randomly selected 8–10 sampling stations to sam- ple from each canal sampling unit (and in a few rare exceptions only ﬁve stations; Table 1), and re- randomized for each sampling event. We con- ducted a total of nine sampling events (3 sea- sons 9 3 yr) over the course of the study; seasonal sampling events included wet (June– November), early dry (December–February), and late dry (March–May) for each of year of the study. Between 2010 and 2013, sampling totaled 651 samples (9 canal sampling units 9 8–10 repli- cate samples 9 3 seasons 9 3 yr = 651 samples; Table 1). Novelty gradient across Everglades canals One site (L-31N north) was not accessible during the wet, and early dry samples of year 1 and late dry of year 2 (Table 1). ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 Novelty gradient across Everglades canals When resource dynam- ics are altered via environmental disturbances, niche opportunities (e.g., increased resources, escape from enemies) may provide mechanisms by which nonnatives can increase from low den- sities and even become dominant (Shea and Chesson 2002). As ecosystem novelty increases in Everglades canals, we expected environmental conditions to shift to those that share little Since our focus was to understand how a gra- dient in novelty inﬂuences ﬁsh community struc- ture, canal sampling units were classiﬁed into three categories (hereafter CANALTYPE): well- connected (WC), moderately connected (MC), and leveed (L) canals (Table 1). WC canals (n = 3) are connected to longer hydroperiod marshes nearly year-round, experiencing a greater inﬂuence from the natural hydrological January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 4 GANDY AND REHAGE . 2. Map showing the nine canal sampling units spanning across the southern and central portions of the glades and surrounding Everglades National Park. Sites are coded based on their connectivity to marshes: well connected canals; MC, moderately connected canals; L, leveed canals. WC canals are connected to hes year-round, MC canals are connected only at high water conditions, and L canals are completely discon- d from surrounding marsh habitats due to levees on both sides. Fig. 2. Map showing the nine canal sampling units spanning across the southern and central portions of the Everglades and surrounding Everglades National Park. Sites are coded based on their connectivity to marshes: WC, well connected canals; MC, moderately connected canals; L, leveed canals. WC canals are connected to marshes year-round, MC canals are connected only at high water conditions, and L canals are completely discon- nected from surrounding marsh habitats due to levees on both sides. regime (i.e., seasonal rainfall patterns; Fig. 1). MC canals (n = 3) are, on average, only con- nected to adjacent shorter hydroperiod marshes during the wet season and have intermediate habitat complexity in their littoral zones. All WC and MC canals have marsh connectivity on one side of the canal (leveed on only one side). In contrast, L canals (n = 3) have no direct connec- tivity to marsh habitats, receive little inﬂuence from the natural hydrological regime, and have low habitat complexity since they typically lack a littoral zone. Electrofishing sampling effort f g p g ff Fishes were collected using a boat-mounted, generator-powered electroﬁsher (two spherical anodes, one-cathode system, Smith-Root GPP 9.0, Vancouver, Washington, USA). Electroﬁshing is an effective method for sampling ﬁshes in fresh- water habitats, including the Everglades, and elec- troﬁshing catch per unit effort (CPUE) has been shown to provide a reliable index of ﬁsh abun- dance (Chick et al. 1999, 2004, Moulton et al. 2002, Rehage and Trexler 2006, Zale et al. 2013). Boat electroﬁshing uses a ﬂat-bottom aluminum boat to produce a standardized electrical ﬁeld, so that ﬁsh may be electroshocked, immobilized, and easily collected. Each electroﬁshing sample consisted of 300 s of standardized, intermittent power application at 3000 W (Boucek and Rehage 2013). Since canal width is greater than the electric ﬁeld, a shoreline side was targeted for each sam- ple. If a canal had marsh connectivity, then the Across the 3 years of our study, prevailing La Nina conditions resulted in below-average January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 6 GANDY AND REHAGE at the time of each sample as a categorical vari- able (LCON; connected or not connected to adja- cent marsh at the time of sampling). Second, we estimated regional connectivity by calculating the proportion of days each canal unit was con- nected to marshes for the year prior to the date of each sample (DCON) using stage data pro- vided by the Everglades Depth Estimation Net- work (http://www.sofia.usgs.gov/eden/). Data from the closest gauges to each canal unit were used. For both parameters, we deﬁned a canal unit as connected if the average marsh depth was ≥10 cm. Marsh water depths lower than 10 cm in the Everglades have little remaining standing water that is not uniformly distributed across the marsh surface, making conditions unsuitable for many ﬁshes (Chick et al. 2004). marsh side of the canal was always sampled. If both sides were leveed, a shoreline was randomly selected for each sample. To ensure adequate sam- pling across the entire canal shoreline, samples began from two meters out from the littoral zone edge in deep water and crossed the littoral zone in a zigzag fashion at a 45 to 90° angle to the shore (Guy et al. 2009). Electrofishing sampling effort ( y ) Fish were captured by two netters positioned at the front of the boat, and placed in a holding tank to be later identiﬁed and enumerated by species after each sample was completed. Elec- troﬁshing targeted all ﬁsh larger than 3 cm in length (Rehage and Loftus 2007). We used elec- troﬁshing CPUE as an index of abundance. We deﬁned CPUE as the sum of ﬁshes caught and shocked in each sample, adjusted for the length of canal shoreline sampled (measured with a GPS unit; Boucek and Rehage 2013). Shocked ﬁshes included ﬁsh that were not caught by net- ters, but readily identiﬁed and counted while shocking. If ﬁsh identiﬁcation was questionable, ﬁsh were not included in our CPUE, nor were ﬁsh from the opposite shoreline. Thus, elec- troﬁshing CPUE consisted of the number of ﬁsh per 100 m of canal shoreline sampled: ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 Spatiotemporal variation in habitat and abiotic attributes We found marked differences across CANAL- TYPE, SEASON, and YEAR in the littoral habitats of sampled canals (Table 2). We detected a gradi- ent in habitat complexity of canal littoral zones, such that PCOVER, PRICH, LZWIDTH, and LZDEPTH declined as the degree of canal connectivity to surrounding marshes decreased (Appendix S2). Overall, littoral habitats in L canals were markedly less complex and nearly absent, with steep banks and sparse vegetation providing less cover for ﬁsh (Appendix S2). Aver- age LZW and LZD decreased from 2.3 0.1 m and 1.0 0.0 m in WC canals to an average of 0.6 0.1 m and 0.7 0.0 m in L canals, respec- tively. Littoral zone PCOVER and PRICH declined nearly threefold from WC canals to L canals with MC canals showing intermediate conditions (x% COVER = 43.6 1.6 and xPRICH = 4.0 0.2 to x% COVER = 15.3 1.7 and xPRICH = 1.3 0.1, respectively). Seasonally, we detected a general shrinking of the littoral zone, especially between the wet and the late dry, but the magnitude of this effect was not the same across canal types (Table 2). MC canals showed the greatest seasonal reduction in habitat complexity (37% reduction in COVER and 45% reduction in PRICH). pp Models were ﬁtted using a stepwise selection procedure. We used the Akaike’s information cri- terion for selecting the most parsimonious model corrected for small sample size (AICc), and R2 to evaluate the % of variation explained by each variable set (Anderson et al. 2008). We conducted DISTLM separately for the three assemblages: all ﬁshes, natives only, and nonnatives only, using Bray–Curtis resemblance matrices (Faith et al. 1987, Legendre and Gallagher 2001). Prior to anal- ysis, all predictor variables were examined for co- linearity to eliminate redundant variables (r > 0.6) using principal components analysis and draftsman plots (Legendre and Anderson 1999, McArdle and Anderson 2001). Last, we used dbRDA plots to visualize the results of DISTLMs with vectors of predictor variables overlaid. Variation in abiotic conditions was mainly a product of seasonality and sampling YEARS and not CANALTYPE (Table 2). Average DO levels were lowest in the wet season (2.0 mg/L) and highest in the late dry (4.4 mg/L). Statistical analyses We used a two-step approach to examine vari- ation and structure in canal ﬁsh communities in relation to the degree of novelty of canals and hydrological, habitat, and spatiotemporal dri- vers. First, we ﬁtted generalized linear models to examine spatiotemporal variation in abundance and richness of all ﬁshes, and then of natives and nonnatives separately. We then used multivariate tools to test for variation in community structure across space and time, and for the relative contri- bution of predictor variables (hydrological, habi- tat, and abiotic variables). CPUE ¼ Fish netted þ fish shocked distance sampled ðmÞ 100 CPUE ¼ Fish netted þ fish shocked distance sampled ðmÞ 100 Habitat, abiotic, and hydrological attributes At the beginning of each sample, we measured habitat and abiotic conditions to examine their inﬂuence on community structure (Appen- dix S1). Submersed aquatic vegetation (SAV) characteristics were measured using a 0.5-m2 quadrat every meter in a transect perpendicular to the shoreline out to 2 m into deep water. Within each quadrat, we measured SAV percent cover (PCOVER) and species richness (PRICH) as well as the average littoral zone depth (LZDEPTH) and width (LZWIDTH). At each sample, we also measured physicochemical con- ditions including dissolved oxygen (DO), tem- perature (TEMP), and ambient conductivity (COND) with a multisonder YSI unit, and water clarity using a Secchi disk (SECCHI). We examined spatiotemporal variation in ﬁsh abundance and richness of all ﬁshes, natives only, and nonnatives only, as well as habitat and abiotic variables, using univariate two-way nonparametric permutational analysis of vari- ance (PERMANOVA; Anderson 2001) to test for the ﬁxed effects of CANALTYPE (WC, MC, and L) and SEASON (wet, early dry, late dry), the random effect year (YEAR), and the interactions of ﬁxed effects. To examine community structure, we con- ducted two-way nonparametric permutational analysis of variance (PERMANOVA; Anderson 2001, Anderson and Robinson 2003, Anderson et al. 2008) on three assemblage models: all ﬁshes, natives only, and nonnatives only. Each model was based on fourth-root-transformed (to account for rare species) Bray–Curtis similarity matrices (Clarke and Warwick 2001). Models tested for the ﬁxed effects CANALTYPE and To quantify hydrological connectivity between canals and surrounding marshes, we recorded two hydrological parameters (Appendix S1). Statistical analyses First, we measured the local marsh connectivity 7 7 January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org GANDY AND REHAGE Statistical signiﬁcance of predictor sets was assessed at a = 0.05 with 999 random permuta- tions. DISTLMs were conducted using PERMA- NOVA+ for Primer 6.0 (Anderson et al. 2008). We then ﬁtted simple regressions to better under- stand the relationship between native and nonna- tive abundance aggregated across the nine canal sampling units and key predictor variables identi- ﬁed in DISTLM analyses and dbRDAs in Sigma- Plot 11.0, San Jose, California, USA. SEASON, the random effect YEAR, and the inter- actions of ﬁxed effects. We followed signiﬁcant main effects with PERMANOVA post hoc pair- wise comparison tests. All PERMANOVA tests were based on 999 unrestricted permutations of the residuals under a reduced model to obtain a pseudo-F-statistic and subsequent P value using type I sums of squares in PERMANOVA+ for Pri- mer 6.0 (Anderson et al. 2008). SEASON, the random effect YEAR, and the inter- actions of ﬁxed effects. We followed signiﬁcant main effects with PERMANOVA post hoc pair- wise comparison tests. All PERMANOVA tests were based on 999 unrestricted permutations of the residuals under a reduced model to obtain a pseudo-F-statistic and subsequent P value using type I sums of squares in PERMANOVA+ for Pri- mer 6.0 (Anderson et al. 2008). To examine the relationship between ﬁsh com- munity structure and predictor variables, we used distance-based linear models (DISTLMs; Legendre and Anderson 1999). The DISTLM pro- cedure is a distance-based redundancy analysis (dbRDA) that uses multivariate multiple regres- sions and performs a permutation test to model the variability of an assemblage matrix against multiple predictor variables (Anderson et al. 2008). We used DISTLMs to assess the relative contribution of ﬁve predictor variable sets: (1) spatial, (2) temporal, (3) hydrological, (4) habitat, and (5) abiotic factors (Appendix S1). Spatial variables included each canal unit (CANALU- NIT) and CANALTYPE (WC, MC, L), temporal factors included YEAR, and hydrological vari- ables included the annual proportion of days each CANALUNIT was connected to marshes (DCON), the local connectivity of the sampled area to adjacent marshes (LCON), and SEASON (wet, early dry, late dry). Habitat variables included PCOVER, PRICH, LZWIDTH, and LZDEPTH (averaged for each sample), and abi- otic variables included DO, COND, TEMP, and SECCHI (Appendix S1). January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org Spatiotemporal variation in habitat and abiotic attributes Although lower wet season DO values were contrary to what we would expect, previous work has found DO to be highly correlated with phosphorus (McCormick and Laing 2003); we suspect our results here to be driven by phosphorus-enriched January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 8 GANDY AND REHAGE Table 2. Results of PERMANOVAs testing the ﬁxed effects CANALTYPE (WC, MC, and L) and SEASON (wet, early dry, and late dry), the random effect hydrological year (YEAR), and the interactions of ﬁxed effects (CT 9 S) on habitat and abiotic variables. ( ) Effects by variable df F P Effects by variable df F P PCOVER (%) DO (mg/L) CANALTYPE (CT) 2, 622 52.1 0.005 CANALTYPE (CT) 2, 622 1.4 0.403 SEASON (S) 2, 622 3.2 0.159 SEASON (S) 2, 622 7.6 0.048 YEAR 2, 622 17.9 0.001 YEAR 2, 622 5.1 0.005 CT 9 S 4, 622 8.5 0.004 CT 9 S 4, 622 0.6 0.647 PRICH TEMP (°C) CANALTYPE (CT) 2, 622 199.6 0.010 CANALTYPE (CT) 2, 622 2.0 0.248 SEASON (S) 2, 622 5.0 0.084 SEASON (S) 2, 622 7.1 0.043 YEAR 2, 622 12.0 0.001 YEAR 2, 622 13.6 0.001 CT 9 S 4, 622 5.3 0.026 CT 9 S 4, 622 0.8 0.531 LZWIDTH (m) COND (lS/cm) CANALTYPE (CT) 2, 622 75.4 0.004 CANALTYPE (CT) 2, 622 1.4 0.396 SEASON (S) 2, 622 6.1 0.065 SEASON (S) 2, 622 0.4 0.730 YEAR 2, 622 6.3 0.001 YEAR 2, 622 17.3 0.001 CT 9 S 4, 622 1.4 0.291 CT 9 S 4, 622 0.7 0.567 LZDEPTH (m) SECCHI (m) CANALTYPE (CT) 2, 622 6.9 0.077 CANALTYPE (CT) 2, 622 2.7 0.150 SEASON (S) 2, 622 8.3 0.038 SEASON (S) 2, 622 2.6 0.225 YEAR 2, 622 8.4 0.030 YEAR 2, 622 2.3 0.088 CT 9 S 4, 622 0.6 0.660 CT 9 S 4, 622 1.9 0.226 Notes: Signiﬁcant results (P < 0.01) are bolded. Habitat variables include littoral percent vegetation cover (PCOVER), plant species richness (PRICH), littoral zone width (LZWIDTH), and littoral zone depth (LZDEPTH). WC, well connected canals; MC, moderately connected canals; L, leveed canals. Notes: Signiﬁcant results (P < 0.01) are bolded. Habitat variables include littoral percent vegetation cover (PCOVER), plant species richness (PRICH), littoral zone width (LZWIDTH), and littoral zone depth (LZDEPTH). WC, well connected canals; MC, moderately connected canals; L, leveed canals. Spatiotemporal variation in habitat and abiotic attributes represented 99% of total CPUE in WC canals and 68% in MC canals, but only 53% L in canals. Notable is the fact that in L canals, total ﬁsh CPUE was four times lower than in WC canals, and close to half of ﬁsh caught were nonnative (47%). Mean nonnative ﬁsh CPUE was also simi- lar between MC and L canals, an average of 16.7 ﬁsh/100 m (P = 0.730). waters being ﬂushed out of upstream agricul- tural areas into canals during high water periods. For TEMP, mean values were higher in the wet (27.5°C) and lowest in the early dry season (23.7°C, P ≤0.001). January 2017 ❖Volume 8(1) ❖Article e01634 Spatiotemporal variation in fish relative abundance and richness Over 3 years, we collected 28,817 ﬁshes: 23,771 natives (33 spp.) and 5046 nonnatives (16 spp.; Appendix S3). Patterns of ﬁsh abundance and species richness in Everglades canals varied sig- niﬁcantly as a function of both space (CANAL- TYPE) and time (SEASON and YEAR). This was true when considering all taxa combined, as well as native and nonnative ﬁshes separately (Table 3). Total ﬁsh CPUE decreased as canals became more novel (WC to L), particularly as a result of reductions in native ﬁshes (Fig. 3A). On average, we caught 125.6 11.7 ﬁsh/100 m in WC canals, 76.7 7.5 ﬁsh/100 m in MC canals, and only 27.8 4.1 ﬁsh/100 m in L canals. The relative ratio of native to nonnative ﬁshes also decreased with increased novelty. Native ﬁshes Patterns in species richness across CANAL- TYPE generally mirrored those of CPUE (Table 3). Total species richness (mean per 100 m of shoreline sampled) was highest in WC canals (8.9 spp.), intermediate in MC canals (8.1 spp.), and lowest in L canals (5.5 spp.; Fig. 3B). As expected, mean native richness decreased from WC to L canals (WC = 8.3 spp., MC = 5.1 spp., L = 3.1 spp.). But, contrary to expectations, non- native richness was highest in MC canals (3.1 spp.), relative to L (2.4 spp.) vs. <1 spp. on aver- age per 100 m in WC canals. Over time, variation in canal ﬁsh assemblages was detected both as a function of SEASON, YEAR, and a differential effect of SEASON and as a function of CANALTYPE (Table 3). Seasonally January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 9 GANDY AND REHAGE Table 3. Results of PERMANOVAs testing the main ﬁxed effects CANALTYPE (WC, MC, and L), SEASON (wet, early dry, and late dry), the random effect YEAR (HY1, HY2, and HY3), and the CANALTYPE 9 YEAR inter- action on total CPUE, species richness, and ﬁsh composition. Table 3. Results of PERMANOVAs testing the main ﬁxed effects CANALTYPE (WC, MC, and L), SEASON (wet, early dry, and late dry), the random effect YEAR (HY1, HY2, and HY3), and the CANALTYPE 9 YEAR inter- action on total CPUE, species richness, and ﬁsh composition. early dry, and late dry), the random effect YEAR (HY1, HY2, and HY3), and the CANALTYPE 9 YEAR inter- action on total CPUE, species richness, and ﬁsh composition. Spatiotemporal variation in fish relative abundance and richness Temporal patterns in species richness generally followed CPUE patterns (Table 3), with more species caught in the dry season but variation in the timing of peak richness (early vs. late dry season), and minor seasonality in L canals (Fig. 4D–F). Native ﬁsh richness (mean per sam- ple) peaked in the early dry season in MC canals (6.2 vs. 3.9 spp. in the wet season, P < 0.01), but in the late dry in WC canals (9.4 vs. 6.6 spp. in wet season, P = 0.03; Fig. 4E). Nonnative rich- ness showed seasonality only in MC canals, peaking in the early dry season (3.8 in early dry vs. 2.1 in wet, P = 0.01) and matching the behav- ior of native richness in MC canals. In L canals, we observed a signiﬁcant but small-magnitude SEASON effect for native species richness between the early and late dry seasons (2.6–3.6 spp., P = 0.034), and no seasonality in nonnative richness (P > 0.152). ( ) For nonnatives, we detected no seasonality in WC canals where nonnative numbers were very low (<1.8 ﬁsh/100 m; Fig. 4C); however, we did detect seasonality in MC and L canals (P < 0.001; Fig. 4C). In MC canals, nonnative ﬁshes incre- ased sixfold between the wet and early dry, and then decreased by half in the late dry, very simi- lar to the pattern seen in native ﬁshes. In L canals, nonnative numbers increased by nearly twofold between the early dry and late. Spatiotemporal variation in fish relative abundance and richness Effects by assemblage CPUE Species richness Composition (Bray–Curtis dissimilarity) df F P F P df Pseudo-F P (perm) All taxa CANALTYPE (CT) 2, 622 31.9 0.010 23.1 0.024 2, 621 16.5 0.004 SEASON (S) 2, 622 26.1 0.012 34.5 0.038 2, 621 3.5 0.044 YEAR 2, 622 16.8 0.001 13.6 0.001 2, 621 8.7 0.001 CT 9 S 4, 622 9.5 0.004 5.1 0.032 4, 621 2.5 0.004 Natives only CANALTYPE (CT) 2, 622 57.5 0.005 74.5 0.005 2, 610 11.5 0.009 SEASON (S) 2, 622 19.5 0.011 22.6 0.034 2, 610 4.0 0.021 YEAR 2, 622 11.9 0.001 12.8 0.001 2, 610 9.7 0.001 CT 9 S 4, 622 8.4 0.010 4.1 0.045 4, 610 2.7 0.001 Nonnatives only CANALTYPE (CT) 2, 622 47.0 0.025 52.7 0.004 2, 472 13.4 0.002 SEASON (S) 2, 622 9.1 0.036 8.4 0.047 2, 472 1.4 0.318 YEAR 2, 622 10.8 0.001 4.3 0.014 2, 472 1.1 0.362 CT 9 S 4, 622 11.6 0.004 8.9 0.003 4, 472 3.2 0.001 Notes: Signiﬁcant results (P < 0.01) are bolded. PERMANOVA models were run separately for all taxa, native ﬁshes only, and nonnative ﬁshes only. WC, well connected canals; MC, moderately connected canals; L, leveed canals. Notes: Signiﬁcant results (P < 0.01) are bolded. PERMANOVA models were run separately for all taxa, native ﬁshes only, and nonnative ﬁshes only. WC, well connected canals; MC, moderately connected canals; L, leveed canals. and across all CANALTYPEs combined, CPUE generally increased from an average of 33 ﬁsh/ 100 m in the wet season to 83 ﬁsh/100 m in the early dry, and to a peak abundance of 115 ﬁsh/ 100 m in the late dry season (Fig. 4). This three- to fourfold increase in ﬁsh CPUE over the dry sea- son was primarily driven by large increases in the native fauna, and particularly in WC canals (Fig. 4A–C). Thus, the effect of seasonality varied as a function of CANALTYPE (Table 3). In WC canals, native ﬁshes peaked in the late dry season with a nearly 4.5-fold increase relative to the wet season, while increases in MC canals (7.5-fold) occurred earlier in the early dry season (xwet = 14.2 to xearly dry = 105.0 ﬁsh/100 m; Fig. 4B). In contrast, we noted no seasonality in native ﬁsh CPUE in L canals (P = 0.690). ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 Spatiotemporal variation in fish community structure As a whole, ﬁsh community structure showed marked dissimilarity as a function of all effects tested (Table 3). However, the inﬂuence of these structuring drivers varied between the native and nonnative components of the fauna. For January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 10 GANDY AND REHAGE Fig. 3. Total catch per unit effort (CPUE) (no. of ﬁsh/ 100 m; A) and species richness (B) for all ﬁshes (scale pattern bars), natives only (white bars), and non- natives only (gray bars) across the CANALTYPE novelty gradient; WC, well connected canals; MC, moderately connected canals; L, leveed canals. Shown are means SE. and nonnatives only assemblages, respectively (Fig. 5A). For all three groups, spatial factors (CANALUNIT and CANALTYPE) consistently explained a much larger proportion of the vari- ance relative to the other variable sets (32.1% for all ﬁshes, 26.7% for natives, and 26.9% for nonna- tives). Second in importance, although explain- ing a much smaller proportion of the variation, were hydrological variables across the three groups (5.1%, 6.5%, and 3.6%, respectively), while temporal, habitat, and abiotic variables explained a minimal amount of variance (<2%; Fig. 5A). Habitat variables were only important predictors for the all ﬁshes and natives only models (P = 0.001). Abiotic variables had slightly greater explanatory power for nonnatives than for natives (1.9% vs. 1.3%), whereas hydrological variables explained more variance for natives than for nonnatives (6.5% vs. 3.6%). Fig. 3. Total catch per unit effort (CPUE) (no. of ﬁsh/ 100 A) d i i h (B) f ll ﬁh The dbRDA ordinations were considered to be a good representation of ﬁsh community struc- ture since axes 1 and 2 combined represented 63.4%, 67.5%, and 59.3% of the ﬁtted variation in all ﬁshes, natives only, and nonnatives only models (Fig. 5B–D). Three continuous variables were identiﬁed as explaining the most variation in community structure in all three analyses (Fig. 5B–D). The hydrological variable DCON independently explained the most variance consistently for all three models (16.1% all ﬁshes, 13.3% natives only, 8.5% nonnatives only). Among the habitat and abiotic variables, LZW explained the most variation (5.7%) in the natives model, but DO independently explained the most variation (2%) in the nonnatives model (Fig. 5C, D). Regressing CPUE as a function of DCON, LZW, and DO showed varying relation- ships with respect to native vs. nonnative ﬁshes. Spatiotemporal variation in fish community structure Native ﬁshes were more abundant as connec- tivity to surrounding marshes increased and followed a linear relationship, whereas nonna- tives decreased with connectivity in a nonlinear fashion (Fig. 6A, B). Similarly, natives and non- natives also exhibited opposing relationships as a function of habitat complexity (LZW; Fig. 6C, D). Natives were more abundant as the width of the littoral zone increased, while the opposite was true for nonnatives. No signiﬁcant relationship was detected for both native and nonnative abundance as a function of DO (Fig. 6E, F). Fig. 3. Total catch per unit effort (CPUE) (no. of ﬁsh/ 100 m; A) and species richness (B) for all ﬁshes (scale pattern bars), natives only (white bars), and non- natives only (gray bars) across the CANALTYPE novelty gradient; WC, well connected canals; MC, moderately connected canals; L, leveed canals. Shown are means SE. native ﬁshes, community dissimilarity was dri- ven by both spatial and temporal stressors, while nonnative community structure was more spatial than temporally inﬂuenced; only CANALTYPE and the CANALTYPE 9 SEASON interaction were signiﬁcant effects (Table 3). Relative contribution of predictor variable sets The best ﬁtted DISTLMs explained 40.8%, 36.9%, and 33.4% of the variation in commu- nity structure across all ﬁshes, natives only, January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 11 GANDY AND REHAGE Fig. 4. Catch per unit effort (CPUE; no. of ﬁsh/100 m; A–C) and species richness (D–F) by SEASON for all ﬁshes, natives only, and nonnatives only across the CANALTYPE novelty gradient; WC, well connected canals; MC, moderately connected canals; L, leveed canals. Shown are means SE. Fig. 4. Catch per unit effort (CPUE; no. of ﬁsh/100 m; A–C) and species richness (D–F) by SEASON for all ﬁshes, natives only, and nonnatives only across the CANALTYPE novelty gradient; WC, well connected canals; MC, moderately connected canals; L, leveed canals. Shown are means SE. January 2017 ❖Volume 8(1) ❖Article e01634 DISCUSSION variation in canal traits and to the degree of ﬁsh invasion, such that not all man-made canals are created equal. We expected a lower invasion rate in canals with higher connectivity (i.e., year- around) to nearby marshes and higher habitat complexity, and thus lower novelty, and a higher invasion rate associated with canals with low connectivity to marshes (higher novelty). Our ﬁndings matched this prediction well for native taxa, but the pattern deviated for nonnative Human-caused modiﬁcations to both abiotic conditions and biotic composition are increas- ingly leading to novel ecosystems (Milton 2003, Hobbs et al. 2006, 2009, 2013, Williams and Jack- son 2007), and possibly to gradients in such nov- elty that directly relate to the degree of alteration (King et al. 2011). We hypothesized a gradient in novelty in Everglades canals that related to January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 12 GANDY AND REHAGE Fig. 5. Summary of distance-based linear models (A) showing the contribution of spatial, temporal, hydro- logical, habitat, and abiotic predictor variable sets in explaining differences in ﬁsh community structure for each assemblage model. Distance-based redundancy ordinations of the (B) all ﬁshes model, (C) natives only model, and (D) nonnatives only model showing the independent contribution of predictor variables (Appendix S1) from the most parsimonious models (P < 0.01 signiﬁcance). Vectors represent the direction and strength of each pre- dictor variables relationship against the dbRDA axes. WC, well connected canals; MC, moderately connected canals; L, leveed canals; dbRDA, distance-based redundancy analysis F log ass and the Fig. 5. Summary of distance-based linear models (A) showing the contribution of spatial, temporal, hydro- logical, habitat, and abiotic predictor variable sets in explaining differences in ﬁsh community structure for each assemblage model. Distance-based redundancy ordinations of the (B) all ﬁshes model, (C) natives only model, and (D) nonnatives only model showing the independent contribution of predictor variables (Appendix S1) from the most parsimonious models (P < 0.01 signiﬁcance). Vectors represent the direction and strength of each pre- dictor variables relationship against the dbRDA axes. WC, well connected canals; MC, moderately connected canals; L, leveed canals; dbRDA, distance-based redundancy analysis ﬁshes. Native ﬁsh communities were more abun- dant and speciose as novelty decreased (WC > MC > L canals). However, nonnative abun- dance was lowest in WC canals, but similar between MC and L canals. January 2017 ❖Volume 8(1) ❖Article e01634 DISCUSSION In Everglades canals, native ﬁsh richness and abundance declined sharply as hydrological and habitat complexity became more novel with extremely low numbers in the most novel L canals. We suspect this pattern is indicative of poorer habitat quality for natives as canal littoral zones become smaller and less com- plex, and as productivity and prey availability associated with the loss of connectivity to marshes are reduced. Although canals may pro- vide deep, suitable habitat for larger taxa, it is the connectivity to marshes and the presence of littoral zones that enhance ﬁsh numbers (includ- ing prey), as told by the larger increases in ﬁsh abundance in the dry season as surrounding marshes went dry. More complex littoral zones within reservoirs can support a higher diversity in ﬁsh communities and have a greater potential for maintaining native populations, especially juveniles that use these areas to avoid predation (Fernando and Holcık 1991, Matthews et al. 2004). The most salient ﬁnding of our study was that spatial factors appeared to be the most signiﬁ- cant driver of assembly patterns in canal ﬁshes. The location of a canal and the marsh it bisects, as well as the degree of canal connectivity to the marsh habitat, appeared to have a strong inﬂu- ence on ﬁsh assemblages. This ﬁnding agrees with previous work showing that anthropogenic gradients can result in a divergence in ﬁsh com- munities. For instance, Slawski et al. (2008) found that urbanization in the upper Des Plaines River watershed had a strong inﬂuence on ﬁsh species composition, shifting from cool-water riverine specialist to warm-water riverine gener- alist as urbanization in undammed tributaries increased. However, in our study we expected that given the relatively uniform nature of canals as aquatic habitats (i.e., extensive with similar depth and width and relatively low structure except for littoral zones), we would see a high degree of biotic homogenization across the canal domain sampled (e.g., McKinney and Lockwood 1999, Rahel 2002). Gido et al. (2009) found that in the novel habitat of reservoirs, patterns of ﬁsh community structure were homogenous across drainage basins and more so relative to natural stream assemblages. In contrast, our Everglades canal ﬁsh community was markedly spatially segregated, with distinct ﬁsh assemblages along canals and even along sections of the same canal, despite the fact that all sites were part of an inter- connected canal network. DISCUSSION Nonnative richness was lowest in WC canals and, contrary to predic- tions, peaked in MC and was intermediate in L canals. Community structure differed vastly as a function of CANALTYPE, and this structure was primarily inﬂuenced by spatial factors and to a January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 13 GANDY AND REHAGE Fig. 6. Best ﬁt regression models (linear and quadratic) ﬁtted to native (A, C, and E) and nonnative (B, D, and F) ﬁsh abundance (no. of ﬁsh/100 m) against the top hydrological (% days connected to the marsh the year prior), habitat complexity (littoral zone width), and abiotic variables (dissolved oxygen) that explained the most independent variation in community structure from distance-based linear models/redundancy analysis (see Fig. 5). Regressions were ﬁtted to the nine canal sampling unit seasonal means. CPUE, catch per unit effort. ❖www.esajournals.org 14 January 2017 ❖Volume 8(1) ❖Article e01634 Fig. 6. Best ﬁt regression models (linear and quadratic) ﬁtted to native (A, C, and E) and nonnative (B, D, and F) ﬁsh abundance (no. of ﬁsh/100 m) against the top hydrological (% days connected to the marsh the year prior), habitat complexity (littoral zone width), and abiotic variables (dissolved oxygen) that explained the most independent variation in community structure from distance-based linear models/redundancy analysis (see Fig. 5). Regressions were ﬁtted to the nine canal sampling unit seasonal means. CPUE, catch per unit effort. ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 14 GANDY AND REHAGE much lesser extent by other factors (hydrological, habitat, and abiotic). Importantly, we noted con- trasting responses between native and nonnative ﬁsh abundance and key hydrological and habitat factors. Across sampling events, native ﬁsh abundance was positively related to the hydro- logical connectivity of canals to surrounding marshes and size of the canal littoral zones, while the opposite was true for the nonnative ﬁshes. canals comparing movement patterns between native largemouth bass and nonnative Mayan cichlids showed similar and relatively small lin- ear movements within canals (<1 to 4 km), but did detect higher frequencies of movement over greater distances from canals into adjacent marshes in the nonnative Mayan cichlid (Parkos and Trexler 2014). ) Human alterations to aquatic systems can lower habitat quality by disrupting natural geo- morphologic processes, spatial heterogeneity patterns, and the natural ﬂuctuation of resources (Ligon et al. 1995, Humborg et al. 1997, Poff et al. 1997, Rosenberg et al. 2000, Bunn and Arthington 2002). ❖www.esajournals.org DISCUSSION Similar decreases in ﬁsh abundance later in the dry season have been documented in mangrove creeks, which serve as important dry-down habi- tats in the southern Everglades, and which have been attributed to predation by the larger pisci- vores. For example, Boucek and Rehage (2013) found that the arrival of a marsh prey subsidy in southern Everglades mangrove creeks during the onset of the dry season resulted in ﬁtness gains for both marsh (largemouth bass) and estuarine (snook) consumers. Interestingly, nonnative taxa showed a similar increase in MC canals, indicat- ing that they are also likely entering canals from marshes as native ﬁshes do, but their numbers did not experience a decrease later in the season. In WC canals, seasonal increases occurred later in the dry season. Although canals may be lower quality habitats because of the high abundance of predators and low habitat complexity, they could provide better habitat in extreme droughts, playing a greater role in the re-colonization of marshes during these events and under the increased frequency of such events with climate change. Along those lines, differences in native ﬁsh abundance between WC and MC canals likely reﬂect the variation in ﬁsh prey productivity of the marshes they bisect. WC canals connect to longer hydroperiod marshes almost year-round, which have been shown to have higher ﬁsh abundances than the shorter hydroperiod mar- shes that connect to MC canals only during the wet season (Chick et al. 2004, Green et al. 2006). Additionally, canal connectivity to marsh species pools with different community structure may have contributed to the observed patterns in this study. For instance, Parkos et al. (2011) docu- mented differences in ﬁsh community structure in WCA 3A marshes that connect to WC canals compared to ﬁshes within ENP marshes which connect to moderately connected canals. y Variation in the degree of marsh connectivity across canals also likely inﬂuences the role of canals as dry-down refuges. In pulsing systems, seasonal variation in rainfall drives patterns of inundation and thus habitat availability for ﬁshes and other aquatic taxa, such that ﬁsh survival is highly dependent on refuge size, the intensity of the dry-down period, and mobility (Magoulick and Kobza 2003, Robson et al. 2013). DISCUSSION The higher native species richness and abun- dance and lowest degree of invasion in WC canals, and conversely the highest degree of invasion in L canals, are congruent with the bio- tic resistance hypothesis (Elton 1958) and its more recent formulation of the diversity–invasi- bility hypothesis (Tilman 1999). These hypothe- ses postulate that invasive species are expected to fail to invade or have reduced invasion success in more diverse communities. This is due to a lack of open niche opportunities, lower reso- urces, and/or increased competition or other antagonistic interactions as the number of native species and previous invaders increases (Shea and Chesson 2002, Levine et al. 2004). In aquatic This high degree of heterogeneity detected in both the native and nonnative ﬁsh assemblages points to the potential for (1) limited dispersal abilities in ﬁsh species, (2) the ability of water control structures and water management to pre- vent ﬁsh movement and thus increase hetero- geneity, and/or (3) an overwhelming effect of the surrounding marsh characteristics and not canals per se to drive the community structure of canal ﬁsh communities. Recent work in Everglades ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 15 GANDY AND REHAGE systems, studies have found evidence in support of this hypothesis (e.g., Stachowicz et al. 1999, Dunstan and Johnson 2004). However, other mechanisms may also be operating (e.g., Moyle and Marchetti 2006). For instance, Henriksson et al. (2015) found that the presence of one single species (i.e., species identity hypothesis; Crawley et al. 1999, Emery and Gross 2007) better expl- ained Arctic char invasion success into Swedish lakes compared to native species richness or the community saturation level. systems, studies have found evidence in support of this hypothesis (e.g., Stachowicz et al. 1999, Dunstan and Johnson 2004). However, other mechanisms may also be operating (e.g., Moyle and Marchetti 2006). For instance, Henriksson et al. (2015) found that the presence of one single species (i.e., species identity hypothesis; Crawley et al. 1999, Emery and Gross 2007) better expl- ained Arctic char invasion success into Swedish lakes compared to native species richness or the community saturation level. would expect from the hydroperiod of surround- ing marshes. In MC canals, abundance of natives peaked sooner reﬂecting earlier drying of the sur- rounding shorter hydroperiod marshes followed by reductions by ~50% in the late dry season, likely attributed to predation. January 2017 ❖Volume 8(1) ❖Article e01634 DISCUSSION The recur- rent pattern of seasonal drying in Everglades marshes is a major driver of ﬁsh community dynamics as ﬁsh move to both natural (i.e., alliga- tor holes, solution holes, and estuarine mangrove creeks; Loftus and Kushlan 1987, Kobza et al. 2004, Rehage and Loftus 2007, Parkos et al. 2011) and artiﬁcial (i.e., canals; Rehage and Trexler 2006) refuges as water levels recede. In this study, we saw further evidence of the use of canals as dry-down habitats. Fish abundance increased by ﬁvefold in WC and sevenfold in MC in the dry season, with no such increases observed in L canals. The timing of these increases in abundance varied among CANALTYPE, matching what we g For nonnatives, a notable pattern was the increase in their relative contribution as novelty increased, representing nearly 50% of total ﬁsh in L canals. We hypothesize that this ﬁnding relates to the fact that these leveed canals provide lower quality habitats for native ﬁshes, lowering com- petition or biotic resistance (Lowry et al. 2013) and thus favoring the establishment of oppor- tunistic invaders. Otherwise, given the known broad environmental tolerances of nonnative ﬁshes (Moyle and Marchetti 2006), nonnative taxa may be better able to withstand the less favorable conditions of L canals. In lotic systems, novel conditions have often been linked to shifts in assemblages from natives to phenotypically plastic and more tolerant nonnatives (Weaver and Garman 1994, Onorato et al. 1998, Walters et al. 2003). The extremely low contribution of nonnatives in WC canals may relate to variation in the role of canals as thermal refugia. Prior to the beginning of this study in 2010, a severe cold January 2017 ❖Volume 8(1) ❖Article e01634 ❖www.esajournals.org 16 GANDY AND REHAGE snap led to a large mortality event for tempera- ture sensitive taxa (Adams et al. 2012, Matich and Heithaus 2012), including nonnatives such as the Mayan cichlid, blue tilapia, and spotﬁn spiny eel (Boucek and Rehage 2014, Rehage et al. 2016). We suspect nonnatives contributed to a larger although still small part of the ﬁsh com- munity in WC canals prior to the 2010 cold snap. Unpublished records from the Florida Fish and Wildlife Conservation Commission point to non- natives accounting for about 8.1% of ﬁshes caught in the L-67A canal (2006–2009), a WC canal sampled in this study. ACKNOWLEDGMENTS We thank Jeff Kline, and Zach Fratto with Everglades National Park, Kevin Whelan, Raul Urgelles, and the National Park Service, South Florida, and Caribbean Network staff for their contributions to this study. We also thank Kevin Kotun with NPS for help in under- standing how canals function hydrologically. We espe- cially thank all current and past members of Dr. Rehage’s lab for help with sampling and insight, and Dr. Joe Parkos for valuable feedback. This study was made possible by funding provided by the National Parks Service’s Critical Ecosystems Studies Initiative (J5298-10-0011), and an Everglades Foundation Gradu- ate Fellowship and Christina Menendez Fellowship to David Gandy. The project was developed in collabora- tion with the Florida Coastal Everglades Long-Term Ecological Research program DBI-0620409 and DEB- 1237517, and NSF WSC-1204762). This is contribution number 816 from the Southeast Environmental Research Center at Florida International University. p Environmental disturbance is often implicated as a key factor in facilitating species invasions (D’Antonio et al. 1999, Bando 2006, Clark and Johnston 2011). Disturbance can facilitate inva- sion by releasing resource opportunities, escape- ment from natural predators, or indirectly reducing native species abundance and thus resources consumption (Davis et al. 2000, Shea and Chesson 2002). Previous work in aquatic sys- tems also points to the relation and feedback between hydrological disturbance and invasions (Marchetti et al. 2004, Leprieur et al. 2008). Not unlike these studies, we documented opposing relationships between marsh connectivity and the abundance of native vs. nonnative taxa. These relationships suggest, at minimum, that natives and nonnatives are responding to the natural hydrology of the system in different ways. Kiernan et al. (2012) showed that restora- tion of the natural hydrological regime can lead to the recovery of natives in heavily invaded Cal- ifornia streams. Whether Everglades restoration could have the same detrimental effects on non- natives to the beneﬁt of native taxa is not known and merits further work. Regardless, canals are permanent features of the Everglades landscape, since most of this conveyance network that pro- vides water supply and ﬂood control and reroutes water delivery into natural areas will remain in place after ecosystem restoration. Our results suggest that the inherent loss of natural DISCUSSION Temperature records from the cold snap indicated that in WC canal units, the pattern of water ﬂow (from marshes into canals) reached low temperatures in the range of lethal limits of many nonnatives (e.g., Schoﬁeld et al. 2010, Schoﬁeld and Huge 2011), while canals elsewhere remained warmer (J. 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Third edition. American Fisheries Society, Bethesda, Maryland, USA. January 2017 ❖Volume 8(1) ❖Article e01634 SUPPORTING INFORMATION SUPPORTING INFORMATION Additional Supporting Information may be found online at: http://onlinelibrary.wiley.com/doi/10.1002/ecs2. 1634/full nal Supporting Information may be found online at: http://onlinelibrary.wiley.com/doi/10.1002/ecs2 ❖www.esajournals.org January 2017 ❖Volume 8(1) ❖Article e01634 21
https://openalex.org/W3086342930	https://zenodo.org/records/4042613/files/JOR_article_50387.pdf	English	null	New records of exotic crickets in Europe: Homoeogryllus species (Orthoptera: Gryllidea: Phalangopsidae)	Journal of orthoptera research	2,020	cc-by	2,878	Journal of Orthoptera Research 2020, 29(2): 121–125 Journal of Orthoptera Research 2020, 29(2): 121–125 Short Communication Keywords Belgium, identification, Poland, species introductions, spectrogram, tropi cal plants http://zoobank.org/61E24A58-B096-49C7-BC99-830A81153DDF http://zoobank.org/61E24A58-B096-49C7-BC99-830A81153DDF Citation: Z˙urawlew P, Desutter-Grandcolas L, Szymański P, Herman DB (2020) New records of exotic crickets in Europe: Homoeogryllus species (Orthoptera: Gryllidea: Phalangopsidae). Journal of Orthoptera Research 29(2): 121–125. https://doi.org/10.3897/jor.29.50387 the Homoeogryllus genus, 15 can be found in Africa: H. adunctus Gorochov, 1988, H. ambo Gorochov, 2018, H. cavicola Chopard, 1950, H. deviatus Desutter-Grandcolas, 1985, H. gabonensis Des utter-Grandcolas, 1985, H. lyristes Gorochov, 1988, H. maroccanus Desutter-Grandcolas, 1985, H. nigresculus Desutter-Grandcolas, 1985, H. nigripennis Chopard, 1942, H. orientalis Desutter-Grand colas, 1985, H. parvus Chopard, 1936, H. reticulatus (Fabricius, 1781), H. tessellatus (Serville, 1838), H. venosus Saussure, 1878, and H. xanthographus Guérin-Méneville, 1847. The last species, Homoeogryllus longicornis (Walker, 1869) from the Malay Pennin sula (Desutter 1985, Gorochov 2018, Cigliano et al. 2020), actu ally belongs to the genus Meloimorpha Walker, 1870, according to the number of spurs on its hind tibiae (Desutter-Grandcolas and Jaiswara 2012), and, consequently, we propose here the new com bination Meloimorpha longicornis (Walker, 1869), comb. nov. Here, we show data on new records of Homoeogryllus crickets in Europe and provide photos and call spectrograms. the Homoeogryllus genus, 15 can be found in Africa: H. adunctus Gorochov, 1988, H. ambo Gorochov, 2018, H. cavicola Chopard, 1950, H. deviatus Desutter-Grandcolas, 1985, H. gabonensis Des utter-Grandcolas, 1985, H. lyristes Gorochov, 1988, H. maroccanus Desutter-Grandcolas, 1985, H. nigresculus Desutter-Grandcolas, 1985, H. nigripennis Chopard, 1942, H. orientalis Desutter-Grand colas, 1985, H. parvus Chopard, 1936, H. reticulatus (Fabricius, 1781), H. tessellatus (Serville, 1838), H. venosus Saussure, 1878, and H. xanthographus Guérin-Méneville, 1847. The last species, Homoeogryllus longicornis (Walker, 1869) from the Malay Pennin sula (Desutter 1985, Gorochov 2018, Cigliano et al. 2020), actu ally belongs to the genus Meloimorpha Walker, 1870, according to the number of spurs on its hind tibiae (Desutter-Grandcolas and Jaiswara 2012), and, consequently, we propose here the new com bination Meloimorpha longicornis (Walker, 1869), comb. nov. Here, we show data on new records of Homoeogryllus crickets in Europe and provide photos and call spectrograms. Abstract This short note lists new records of exotic crickets belonging to the genus Homoeogryllus (Orthoptera: Gryllidea: Phalangopsidae) in Europe (Poland and Belgium) and discusses the most probable scenarios of their arrival from tropical regions. Photographs and stridulation spectrograms of these crickets are provided. The report concludes that handling termi nals and warehouses with tropical plants are the most common sites in Europe where exotic species of different taxa are recorded. The species Homoeogryllus longicornis (Walker, 1869) is also reclassified to the genus Meloimorpha Walker, 1870. mysław Żurawlew1, Laure Desutter-Grandcolas2, Paweł Szymański3, David Billy Herman4 Przemysław Żurawlew1, Laure Desutter-Grandcolas2, Paweł Szymański3, David Billy Herman4 1 The Orthoptera of Poland Project, Żbiki 45, 63–304 Czermin, Poland. p j 2 Institut de Systématique, Evolution & Biodiversité (ISYEB), Muséum national d’Histoire naturelle, Sorbonne Université, CNRS, EPHE UPMC, UA, 57 rue Cuvier, CP 50, 75231 Paris cedex 05, France. , , , 3 Department of Behavioural Ecology, Faculty of Biology, Adam Mickiewicz University, Uniwersytetu Poznan´skiego 6, 61–114 Poznań, Poland. 4 Dendermondsesteenweg 40, 9000 Gent, Belgium. 3 Department of Behavioural Ecology, Faculty of Biology, Adam Mickiewicz University, Uniwersytetu Poznan´sk 4 Dendermondsesteenweg 40, 9000 Gent, Belgium. Corresponding author: Laure Desutter-Grandcolas (desutter@mnhn.fr) Corresponding author: Laure Desutter-Grandcolas (desutter@mnhn.fr) ademic editor: Diptarup Nandi \| Received 22 January 2020 \| Accepted 27 April 2020 \| Published 14 September 20 Academic editor: Diptarup Nandi \| Received 22 January 2020 \| Accepted 27 April 2020 \| Published 14 Septe Results separated by 30 ms pause and a number of chatter syllables with fundamental frequency ca. 4 kHz and harmonics (Fig. 2c). Dura tion of the first stridulation with 8 syllables (Fig. 2b) was 4.1 s and that with 5 syllables was 2.2 s. Both stridulations were sepa rated by a 7.6 s pause. Homoeogryllus cf. reticulatus (Fabricius, 1781) Figs 1, 2 Observed material.—Belgium, Gent, 8 VIII 2010, 1♂, D. B. Herman leg., specimen not collected, identified based on its calling song. Observed material.—Belgium, Gent, 8 VIII 2010, 1♂, D. B. Herman leg., specimen not collected, identified based on its calling song. Remark.—The specimen was found in a crack of a wall near a port where the wood of tropical trees is stored. The species has been re corded in Egypt, Chad, Senegal, Guinea, Ivory Coast, Benin, Cam eroon, Central African Republic, Equatorial Guinea, Gabon, Re public of the Congo, and Democratic Republic of Congo (= Zaire) (Desutter 1985, Gorochov 2018). Stridulation description.—Only two stridulation series were re corded (Fig. 2a). Each consisted of two fast introductory creaks Fig. 1. Homoeogryllus cf. reticulatus (Fabricius, 1781). Male found in a crack of a wall near the port of Gent (Belgium) in which wood of tropical trees is stored. Homoeogryllus tessellatus (Serville, 1838) Figs 3, 4 P. Z˙URAWLEW, L. DESUTTER-GRANDCOLAS, P. SZYMAŃSKI AND D.B. HERMAN P. Z˙URAWLEW, L. DESUTTER-GRANDCOLAS, P. SZYMAŃSKI AND D.B. HERMAN Results Materials International trade, with its multiple means and routes, may not only speed up the movements of animals, but also allow the spread of non-native animals far beyond their home ranges. Among anthropogenic vectors of nonindigenous species, rising sea transport is now considered to account for the bulk of in troduced species (Sardain et al. 2019). Shipping containers, hull fouling, and ballast waters help small animals, like insects, to reach distant places. Coupled with global warming, introducing non-native species to new habitats as a result of human activi ties may be a significant source of biological invasions and can cause severe harm to native species communities (Wheeler and Hoebeke 2017). Specimens were found in various situations as described in the Results. They were collected when possible, recorded, and identi fied by one of us (LDG), except H. xanthographus (Guérin-Ménev ille, 1847) (see Z˙urawlew 2009). Recordings of spontaneously stridulating crickets were made using Canon Power Shot A570 IS (H. xanthographus), MINT Olympus Digital Dm-1 (H. cf. reticulatus) and a dictaphone (H. tessellatus). Analog recordings were digitized at a sampling rate of 44.1 kHz/16 bits and visualized using seewave package (Sueur et al. 2008) implemented in R (R Core Team 2013) with the following settings: FFT length: 1024, window type: Hann, temporal overlap: 90%. The audio files were uploaded to the Orthoptera Species File Online (Cigliano et al. 2020) and the MNHN Sound Library. However, many adventive or exotic species are introduced to new habitats accidentally and do not establish populations. Crickets within the genus Homoeogryllus Guérin-Méneville, 1847 are one example of this phenomenon. Out of 16 species within Journal of Orthoptera Research 2020, 29(2) 122 Homoeogryllus tessellatus (Serville, 1838) Figs 3, 4 Observed material.—Poland, Warsaw, Łowicka Street, 17–23 VIII 2016, 1♂, M. J. Gorazdowski leg., L. Desutter-Grandcolas det., MNHN. Remark.—The male was found in a snake terrarium. Presumably, the vector was a bromeliad Neoregelia carolinae (Beer) L.B. Smith bought in a garden center. The species is native to Ivory Coast, Guinea, and Sierra Leone (Desutter 1985). Stridulation description.—The stridulation was mostly continuous with repeated creaks of roughly the same length of 0.3 s (Fig. 4a). The beginning of each series consisted of only 3–5 creaks increas ing in length (Fig. 4c). The fundamental frequency of stridulation was around 5 kHz (Fig. 4b). The shortest recorded stridulation was 9.3 s and the longest 35.3 s. Fig. 1. Homoeogryllus cf. reticulatus (Fabricius, 1781). Male found in a crack of a wall near the port of Gent (Belgium) in which wood of tropical trees is stored. Fig. 2. Stridulation of Homoeogryllus cf. reticulatus (Fabricius, 1781) male from Gent (Belgium): a. spectrogram of two recorded stridula tion series; b. spectrogram of one stridulation series in higher resolution of time domain; c. spectrogram of the beginning of stridula tion series with three units (syllables) shown. The oscillograms are shown below and the relative amplitude scales (in dB) on the right of the spectrograms. Abbreviations: dB, decibel; s, second; kHz, kilohertz. Fig. 2. Stridulation of Homoeogryllus cf. reticulatus (Fabricius, 1781) male from Gent (Belgium): a. spectrogram of two recorded stridula tion series; b. spectrogram of one stridulation series in higher resolution of time domain; c. spectrogram of the beginning of stridula tion series with three units (syllables) shown. The oscillograms are shown below and the relative amplitude scales (in dB) on the right of the spectrograms. Abbreviations: dB, decibel; s, second; kHz, kilohertz. Journal of Orthoptera Research 2020, 29(2) 123 P. Z˙URAWLEW, L. DESUTTER-GRANDCOLAS, P. SZYMAŃSKI AND D.B. HERMAN Fig. 3. Homoeogryllus tessellatus (Serville, 1838) male observed in Warsaw (Poland). Homoeogryllus xanthographus (Guérin-Méneville, 1847) Figs 5, 6 Observed material.—Poland, Gołuchów, Pleszew District, 20 I–10 III 2009, 1♂, A. Biernat leg., S. W. Heads det. (see Z˙urawlew 2009). Remark.—The male was found in a wicker basket with potted plants inside a residential building. It had probably been trans ported, as an egg or a nymph, with the coconut bedding into which the plants were potted. Stridulation description.—Recorded stridulations (n = 9) consisted of 4 to 9 elements (Fig. 6a). Two types of elements could be distinguished: shorter creaks repeated 3 to 8 times and a longer, terminating creak (Fig. 6c). One series had two terminating elements (Fig. 2b). The fun damental frequency of stridulation was around 4 kHz. Discussion In this short note, we report new records of two species of Homoeogryllus genus, H. cf. reticulatus and H. tessellatus, in Bel gium and Poland, respectively. In addition, we newly describe the stridulations of H. xanthographus, another Homoeogryllus species which, as an egg or nymph transported in soil substratum, was previously introduced in Poland (Fig. 5; Z˙urawlew 2009). So far, of the species of Homeogryllus, only the stridulations of H. tessella tus, H. cf. reticulatus, H. nigresculus, and H. xanthographus have been described (Desutter 1985, Z˙urawlew 2009). These tropical crickets were probably introduced into Europe by being shipped from Africa, which is a source of tropical plants for Europe. Shipping-mediated introductions of Fig. 3. Homoeogryllus tessellatus (Serville, 1838) male observed in Warsaw (Poland). Fig. 4. Stridulation of Homoeogryllus tessellatus (Serville, 1838) male from Warsaw (Poland): a. spectrogram of recorded stridulation; b. spectrogram of one stridulation series in higher resolution of time domain; c. spectrogram of the beginning of stridulation series with three units (syllables) shown. The oscillograms are shown below and the relative amplitude scales (in dB) on the right of the spectrograms. Abbreviations: dB, decibel; s, second; kHz, kilohertz. Fig. 4. Stridulation of Homoeogryllus tessellatus (Serville, 1838) male from Warsaw (Poland): a. spectrogram of recorded stridulation; b. spectrogram of one stridulation series in higher resolution of time domain; c. spectrogram of the beginning of stridulation series with three units (syllables) shown. The oscillograms are shown below and the relative amplitude scales (in dB) on the right of the spectrograms. Abbreviations: dB, decibel; s, second; kHz, kilohertz. Journal of Orthoptera Research 2020, 29(2) 124 P. Z˙URAWLEW, L. DESUTTER-GRANDCOLAS, P. SZYMAŃSKI AND D.B. HERMAN Fig. 5. Homoeogryllus xanthographus (Guérin-Méneville, 1847) male observed in Gołuchów, Pleszew District (Poland). Eugaster spinulosa (Johansson, 1763) (Bazyluk and Liana 2000) from Africa, Amphiacusta nauta (Desutter-Grandcolas, 1997) from the Caribbean, and that of H. xanthographus support this scenario. In fact, studies show that ports and garden centers in Europe are places where many species of tropical insects and spiders have been recorded (Rozwałka et al. 2016). Apart from the accidental introduction of species associated with woody plants, another pathway that largely contributes to the spread of non-native species is trade and breeding as pets (owing to their pleasant songs) or as food for vertebrate and invertebrate pets. P. Z˙URAWLEW, L. DESUTTER-GRANDCOLAS, P. SZYMAŃSKI AND D.B. HERMAN P. Z˙URAWLEW, L. DESUTTER-GRANDCOLAS, P. SZYMAŃSKI AND D.B. HERMAN Laštůvka 2005). In Poland, 305 species are non-native with insects being most common, followed by molluscs, shellfishes, flatworms, nematodes, arachnids, cnidarians, and annelids (Głowaciński et al. 2012). The difficulty is in evaluating the potential of these tropical species to actually invade and settle, especially in terms of resistance to cold weather and changes in the available resources. Gorochov AV (2018) Taxonomic studies on the cricket subfamilies Pter oplistinae, Phaloriinae and Cacoplistinae (Orthoptera: Gryllidae) from the Old World. Zoosystematica Rossica 27: 40–76. https://doi. org/10.31610/zsr/2018.27.1.40 Hulme PE (2009) Trade, transport and trouble: managing invasive species pathways in an era of globalization. Journal of Applied Ecology 46: 10–18. https://doi.org/10.1111/j.1365-2664.2008.01600.x R Core Team (2013) R: A language and environment for statistical comput d f l h // R Core Team (2013) R: A language and environment for statistical comput ing. R Foundation for Statistical Computing, Vienna. http://www.R- project.org/ Acknowledgements ing. R Foundation for Statistical Computing, Vienna. http://www.R- project.org/ We thank Prof. Bruno Massa (University of Palermo, Italy) for help in preparing this short note. We thank M. J. Gorazdowski for providing H. tessellatus specimens from Warsaw. We also thank Klaus Riede (Research Museum Alexander Koenig, Bonn) and Holger Braun (Museo de La Plata, Argentina) for their review of the manuscript. Rozwałka R, Rutkowski T, Bielak-Bielecki P (2016) New data on in troduced and rare synanthropic spider (Arachnida: Araneae) in Poland (II). Annales UMCS, Biologia 71: 60–85. https://doi. org/10.17951/c.2016.71.1.59 Sardain A, Sardain E, Leung B (2019) Global forecasts of shipping traf fic and biological invasions to 2050. Nature Sustainability 2: 1–274. https://doi.org/10.1038/s41893-019-0245-y Š Journal of Orthoptera Research 2020, 29(2) Discussion Indeed, some Homoeogryllus species are reported to be kept in terrariums, but their true origin and identification have not been confirmed. ii As a consequence of accidental introductions, some species may start new populations, which is a threat to local biodiversity (Hulme 2009). They could also invade hot, human-made places, such as bak eries, houses, or underground electric railroad, as is the case of Ache ta domesticus (Linnaeus, 1758), known as the “grillon du métro” in Paris. Examples of tropical Orthoptera species that were introduced to Europe or North America that can now be found in palm houses, greenhouses, and houses include Tachycines asynamorus Adelung, 1902, and Diestrammena japanica Blatchley, 1920, (Głowaciński et al. 2012, Epps et al. 2014). These species have also been found in Cuba where they are believed to have established new populations (https://www.saltatoria.info/arten%C3%BCbersicht-a-z-species-a- z/homoeogryllus-sp-kuba/), even though they are not listed among the Orthoptera of Cuba (Yong and Perez-Gelabert 2014). Continuous monitoring of all the exotic species found in Europe can contribute greatly to correct identifications in the future, the col lection of new information on their biology, and the identification of new potentially invasive species. For instance, in the Czech Republic, as many as 595 non-native species have been recorded (Šefrová and Fig. 5. Homoeogryllus xanthographus (Guérin-Méneville, 1847) male observed in Gołuchów, Pleszew District (Poland). Fig. 6. Stridulation of Homoeogryllus xanthographus (Guérin–Méneville, 1847), male observed in Gołuchów, Pleszew District (Poland): a. spectrogram of recorded stridulation series; b. spectrogram of one stridulation series in higher resolution of time domain; c. spec trogram of two type units (syllables). The oscillograms are shown below and the relative amplitude scales (in dB) on the right of the spectrograms. Abbreviations: dB, decibel; s, second; kHz, kilohertz. Fig. 6. Stridulation of Homoeogryllus xanthographus (Guérin–Méneville, 1847), male observed in Gołuchów, Pleszew District (Poland): a. spectrogram of recorded stridulation series; b. spectrogram of one stridulation series in higher resolution of time domain; c. spec trogram of two type units (syllables). The oscillograms are shown below and the relative amplitude scales (in dB) on the right of the spectrograms. Abbreviations: dB, decibel; s, second; kHz, kilohertz. 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https://openalex.org/W2163158349	https://europepmc.org/articles/pmc3628490?pdf=render	English	null	A Case of Delayed Myelopathy Caused by Atlantoaxial Subluxation without Fracture	Case reports in orthopedics	2,013	cc-by	2,217	Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2013, Article ID 421087, 4 pages http://dx.doi.org/10.1155/2013/421087 Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2013, Article ID 421087, 4 pages http://dx.doi.org/10.1155/2013/421087 Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2013, Article ID 421087, 4 pages http://dx.doi.org/10.1155/2013/421087 Hindawi Publishing Corporation Case Reports in Orthopedics Volume 2013, Article ID 421087, 4 pages http://dx.doi.org/10.1155/2013/421087 1. Introduction developed and he then visited the Department of Neurology at our hospital. At this visit, intracranial process was denied, but hyperesthesia of the right upper and lower extremities was confirmed. Since no other abnormal findings were observed, he was referred to our department for suspected cervical spine pathology.h Upper cervical spine injuries are rare in the pediatric popula- tion, comprising between 0.6 and 9.5% of all cervical injuries [1], and neurologic disability related to pediatric cervical spine injury is uncommon. Patel et al. found that only one- third of children with these injuries had a neurological deficit and half of these cases showed no initial evidence of a radio- logical anomaly [2]. Many patients with a spinal cord injury without radiographic abnormality show symptoms imme- diately after injury, but some symptoms may also develop after a latency period of several days [3]. Here, we report a rare case of myelopathy caused by atlantoaxial subluxation without fracture, in which symptoms developed after a 24- year latency period. We describe the case in the context of a discussion of the literature. The patient had a history of hospital visits for several weeks for cervical pain after he hit the top of his head severely while diving into a swimming pool at the age of 14 years old. At that time, cervical spine plain X-ray images showed no fracture and he was diagnosed with whiplash injury. After the use of a collar for several weeks, the cervical pain disappeared.it At his first visit to our department, his right and left grip strengths were 47 kg and 42 kg, respectively, and no objective muscular depression was apparent in the extremities. There was also no increased deep tendon reflex or pathological reflex. Cervical spine plain X-ray images showed no fracture but indicated an unstable atlantoaxial joint. The atlantodental interval (ADI) was increased to 8 mm during flexion and reduced to ≤3 mm in extension (Figure 1). Computed tomog- raphy (CT) showed no bone chips at the attachment site of the transverse ligament of the atlas (Figure 2). Magnetic resonance imaging (MRI) showed a high intensity area, which suggested fluid accumulation caused by instability between Ryo Takamatsu, Hiroshi Takahashi, Yuichiro Yokoyama, Fumiaki Terajima, Yasuhiro Inoue, Katsunori Fukutake, and Akihito Wada Correspondence should be addressed to Hiroshi Takahashi; drkan@med.toho-u.ac.jp Received 15 February 2013; Accepted 17 March 2013 Academic Editors: M. Gotoh, M. K. Lyons, and M. H. Zheng Copyright © 2013 Ryo Takamatsu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. We report a case of delayed myelopathy caused by atlantoaxial subluxation without fracture. The patient was a 38-year-old male who became aware of weakness in extremities. The patient had a history of hitting his head severely while diving into a swimming pool at the age of 14 years old. At that time, cervical spine plain X-ray images showed no fracture, and the cervical pain disappeared after use of a collar for several weeks. At his first visit to our department, X-ray images showed an unstable atlantoaxial joint. After surgery, weakness of the extremities gradually improved. At 6 months after surgery, bone union was completed and the symptoms disappeared. This case shows that atlantoaxial ligament injuries are difficult to diagnose and may easily be missed. A high level of suspicion is important in such cases, since neurological compromise or deterioration may occur many years after the injury. 2. Case Report The patient was a 38-year-old male who became aware of weakness in the right side of the body for several months without a specific trigger. He also developed motor skills dis- order in his right hand that made it difficult for him to drive a car. Weakness in his left lower extremity also gradually 2 Case Reports in Orthopedics Case Reports in Orthopedics (a) (b) Figure 3: Cervical spine MRI. In (a) sagittal and (b) axial T2- weighted images, the high intensity area suggested fluid accumula- tion between the anterior arch of the atlas and odontoid process of the epistropheus. (a) (b) ure 1: Plain X-ray images at the first visit under (a) flexio extension. Instability of the atlantoaxial joint was observed I increasing to 8 mm under flexion and reducing to ≤3 m ension. (a) (a) (a) (a) (b) (a) (b) (b) Figure 1: Plain X-ray images at the first visit under (a) flexion and (b) extension. Instability of the atlantoaxial joint was observed, with ADI increasing to 8 mm under flexion and reducing to ≤3 mm in extension. (b) Figure 3: Cervical spine MRI. In (a) sagittal and (b) axial T2- weighted images, the high intensity area suggested fluid accumula- tion between the anterior arch of the atlas and odontoid process of the epistropheus. Figure 2: CT images after myelography. An increased ADI was apparent without evidence of fracture at the attachment site of the transverse ligament. the anterior arch of the atlas and odontoid process of the axis (Figure 3). Based on this information, we diagnosed delayed mye- lopathy due to atlantoaxial subluxation caused by the injury of the transverse ligament of the atlas. Transarticular screw fixation surgery was performed under X-ray fluoroscopic guidance with the atlantoaxial joint in a hat-hook position. After the decortication of the posterior arch of the atlas and lamina of the vertebral arch axis, autologous iliac bone graft- ing was performed. Figure 2: CT images after myelography. An increased ADI was apparent without evidence of fracture at the attachment site of the transverse ligament. 3 Case Reports in Orthopedics Figure 4: Postoperative cervical spine plain X-ray images. Atlanto- axial subluxation was improved with the adhesion of grafted bone. Atlantoaxial subluxation accompanies fracture of the odontoid process of the axis in many cases. Most fractures of the odontoid in younger children occur at the dens-body synchondrosis, the weakest area of C2 [13]. 2. Case Report Traumatic rupture of the transverse ligament as a cause of anatomical feature is extremely rare in children. Mechanisms of injury to the ligament include forced forward flexion and axial loading of the atlas, which opens the ring and causes secondary rupture of the transverse ligament, as in a Jefferson fracture with atlantoaxial subluxation. However, based on the history of injury and imaging findings in our case, we concluded that traumatic injury of the transverse ligament of the atlas occurred after the patient dived into a swimming pool when he was 14 years old. Based on their experience in 4 patients aged 2–9 years old, Floman et al. [14] suggested that conservative treatment is not effective for atlantoaxial subluxation caused by injury of the transverse ligament of the atlas, and that surgical treatment should be performed. Dickman et al. [15] also suggested that early surgery is indicated in children aged ≥14 years old with a type 1 injury of the transverse ligament, whereas conservative treatment with a rigid cervical orthosis is preferred in patients with a type 2 injury with bone fragments in the ligament attachment; however, failure to achieve bone adhesion with such conservative treatment should be corrected with surgi- cal treatment.ii Figure 4: Postoperative cervical spine plain X-ray images. Atlanto- axial subluxation was improved with the adhesion of grafted bone. The postoperative clinical course was uneventful. 4 days after surgery, walking exercises were initiated using a Phila- delphia collar. Weakness of the extremities and motor skills disorder gradually improved. The patient was able to go back to work 3 months after surgery and showed no symptoms 6 months after surgery. Cervical spine plain X-ray images obtained 6 months after surgery showed union of grafted bone and no unstable cervical vertebra during flexion and extension (Figure 4). CT findings obtained at the first visit showed no evidence of fracture at the attachment site of the transverse ligament in our case, indicating a type 1 injury in the Dickman classi- fication. Since the patient showed no neurological symptoms and complained only of cervical pain at the time of the initial trauma, the injury to the transverse ligament of the atlas may have been missed. 2. Case Report There have been no previous reports of cases with such a long latency period, but we suggest that myelopathy in our case developed due to microdamage to the spinal cord caused by an unstable atlantoaxial joint at 24 years after the initial injury. This case shows that atlantoaxial ligament injuries are difficult to diagnose and may easily be missed. A high level of suspicion is important in such cases, since neurological compromise or deterioration may occur many years after the injury. 3. Discussion The incidence of blunt cervical spine injuries in children is 1% to 2% of all trauma victims requiring hospital admission [4]. The injury level of the cervical spine injury differs depending on age, with upper cervical injury being more common in younger children (0 to 8 years old), while lower cervical spine injury tends to occur more in older children (≥9 years old) [5, 6]. Upper cervical spine injuries in children are funda- mentally different from those in adults because of anatomical differences in the developing spine. In children, the mass of the head is disproportionately large and the neck muscles are relatively underdeveloped. Furthermore, the vertebral bodies are wedge-shaped, the articulating facets are angled horizon- tally, the end plates are cartilaginous, and the interspinous ligaments are elastic and lax [7, 8]. These features predispose children to upper cervical spine injuries. Fewer fractures occur in children <8 years old compared to those ≥8 years old because of the greater mobility of the spine and the laxity of the ligaments in younger children. The vertebrae start to ossify and mature at 8 years old. Anteriorly, the vertebral body loses its wedge shape and becomes more rectangular, the orientation of the facets becomes less horizontal and more vertical, and the uncinate process begins to protrude [9–12]. The authors declare that they have no conflict of interests. The authors declare that they have no conflict of interests. References [1] I. Kalfas, J. Wilberger, A. Goldberg, and E. R. Prostko, “Magnetic resonance imaging in acute spinal cord trauma,” Neurosurgery, vol. 23, no. 3, pp. 295–299, 1988. [2] J. C. Patel, J. J. Tepas III, D. L. Mollitt, and P. Pieper, “Pediatric cervical spine injuries: defining the disease,” Journal of Pediatric Surgery, vol. 36, no. 2, pp. 373–376, 2001. [3] D. Pang and J. E. Wilberger Jr., “Spinal cord injury without radiographic abnormalities in children,” Journal of Neurosur- gery, vol. 57, no. 1, pp. 114–129, 1982. [4] A. M. Dietrich, M. E. Ginn-Pease, H. M. Bartkowski, and D. R. King, “Pediatric cervical spine fractures: predominately subtle Case Reports in Orthopedics 4 presentation,” Journal of Pediatric Surgery, vol. 26, no. 8, pp. 995–1000, 1991. [5] R. L. Brown, M. A. Brunn, and V. F. Garcia, “Cervical spine injuries in children: a review of 103 patients treated consecu- tively at a level 1 pediatric trauma center,” Journal of Pediatric Surgery, vol. 36, no. 8, pp. 1107–1114, 2001. [6] E. R. Kokoska, M. S. Keller, M. C. Rallo, and T. R. Weber, “Char- acteristics of pediatric cervical spine injuries,” Journal of Pedi- atric Surgery, vol. 36, no. 1, pp. 100–105, 2001. [7] J. S. Apple, D. R. Kirks, D. F. Merten, and S. Martinez, “Cervical spine fractures and dislocations in children,” Pediatric Radiol- ogy, vol. 17, no. 1, pp. 45–49, 1987. [8] C. A. Dickman, J. M. Zabramski, M. N. Hadley, H. L. Rekate, and V. K. H. Sonntag, “Pediatric spinal cord injury without radiographic abnormalities: report of 26 cases and review of the literature,” Journal of Spinal Disorders, vol. 4, no. 3, pp. 296–305, 1991. [9] F. M. Fesmire and R. C. Luten, “The pediatric cervical spine: developmental anatomy and clinical aspects,” Journal of Emer- gency Medicine, vol. 7, no. 2, pp. 133–142, 1989. [10] M. G. Hamilton and S. T. Myles, “Pediatric spinal injury: review of 61 deaths,” Journal of Neurosurgery, vol. 77, no. 5, pp. 705–708, 1992. [11] L. S. Kewalramani, J. F. Kraus, and H. M. Sterling, “Acute spinal- cord lesions in a pediatric population: epidemiological and clinical features,” Paraplegia, vol. 18, no. 3, pp. 206–219, 1980. [12] M. J. Manary and D. M. Jaffe, “Cervical spine injuries in chil- dren,” Pediatric Annals, vol. 25, no. 8, pp. 423–428, 1996. [13] C. Buhs, M. Cullen, M. Klein, and D. References Farmer, “The pediatric trauma C-spine: is the ’odontoid’ view necessary?” Journal of Pediatric Surgery, vol. 35, no. 6, pp. 994–997, 2000. [14] Y. Floman, L. Kaplan, J. Elidan, and F. Umansky, “Transverse lig- ament rupture and atlanto-axial subluxation in children,” Jour- nal of Bone and Joint Surgery B, vol. 73, no. 4, pp. 640–643, 1991. [15] C. A. Dickman, K. A. Greene, and V. K. H. Sonntag, “Injuries involving the transverse atlantal ligament: classification and treatment guidelines based upon experience with 39 injuries,” Neurosurgery, vol. 38, no. 1, pp. 44–50, 1996.
https://openalex.org/W2979714073	https://www.nature.com/articles/s41598-019-51020-3.pdf	English	null	Low human and murine Mcl-1 expression leads to a pro-apoptotic plaque phenotype enriched in giant-cells	Scientific reports	2,019	cc-by	8,976	Low human and murine Mcl-1 expression leads to a pro-apoptotic plaque phenotype enriched in giant-cells Margaux A. C. Fontaine 1, Marijke M. Westra2, Ilze Bot 2, Han Jin1, Aimée J. P. M. Franssen1, Martine Bot2, Saskia C. A. de Jager2,3, Ivan Dzhagalov4, You-Wen He4, Bart J. M. van Vlijmen5,6, Marion J. J. Gijbels1,7, Chris P. Reutelingsperger 8, Theo J. C. van Berkel2, Judith C. Sluimer 1,9, Lieve Temmerman1 & Erik A. L. Biessen1 Received: 21 February 2019 Accepted: 23 September 2019 Published: xx xx xxxx Received: 21 February 2019 Accepted: 23 September 2019 Published: xx xx xxxx Margaux A. C. Fontaine 1, Marijke M. Westra2, Ilze Bot 2, Han Jin1, Aimée J. P. M. Franssen1, Martine Bot2, Saskia C. A. de Jager2,3, Ivan Dzhagalov4, You-Wen He4, Bart J. M. van Vlijmen5,6, Marion J. J. Gijbels1,7, Chris P. Reutelingsperger 8, Theo J. C. van Berkel2, Judith C. Sluimer 1,9, Lieve Temmerman1 & Erik A. L. Biessen1 The anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) plays an important role in survival and differentiation of leukocytes, more specifically of neutrophils. Here, we investigated the impact of myeloid Mcl-1 deletion in atherosclerosis. Western type diet fed LDL receptor-deficient mice were transplanted with either wild-type (WT) or LysMCre Mcl-1fl/fl (Mcl-1−/−) bone marrow. Mcl-1 myeloid deletion resulted in enhanced apoptosis and lipid accumulation in atherosclerotic plaques. In vitro, Mcl-1 deficient macrophages also showed increased lipid accumulation, resulting in increased sensitivity to lipid-induced cell death. However, plaque size, necrotic core and macrophage content were similar in Mcl-1−/− compared to WT mice, most likely due to decreased circulating and plaque-residing neutrophils. Interestingly, Mcl-1−/− peritoneal foam cells formed up to 45% more multinucleated giant cells (MGCs) in vitro compared to WT, which concurred with an increased MGC presence in atherosclerotic lesions of Mcl-1−/− mice. Moreover, analysis of human unstable atherosclerotic lesions also revealed a significant inverse correlation between MGC lesion content and Mcl-1 gene expression, coinciding with the mouse data. Taken together, these findings suggest that myeloid Mcl-1 deletion leads to a more apoptotic, lipid and MGC-enriched phenotype. These potentially pro-atherogenic effects are however counteracted by neutropenia in circulation and plaque. Antiapoptotic Mcl-1 is a member of the apoptosis regulating Bcl-2 family1. It directly interacts with pro-apoptotic BH3-only proteins Bim and Bid and multidomain proapoptotic Bad2–4, thereby inhibiting apoptosis. Mcl-1 is expressed in various tissues including hematopoietic cells5, in which its overexpression delays cell death in response to various stimuli6. Indeed Mcl-1 is critical for neutrophil survival7–10 at all differentiation stages11. Survival of macrophages lacking Mcl-1 does not seem to be impacted9, albeit that Mcl-1 deficient macrophages were observed to be more sensitive to apoptosis induced either by phagocytosis10, or infection12.i While monocytes and macrophages have been the subject of extensive studies in the atherosclerosis field for years13, neutrophils were only more recently investigated due to their scarce presence in atherosclerotic plaque. www.nature.com/scientificreports www.nature.com/scientificreports www.nature.com/scientificreports Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 Received: 21 February 2019 Accepted: 23 September 2019 Published: xx xx xxxx Low human and murine Mcl-1 expression leads to a pro-apoptotic plaque phenotype enriched in giant-cells Margaux A. C. Fontaine 1, Marijke M. Westra2, Ilze Bot 2, Han Jin1, Aimée J. P. M. Franssen1, Martine Bot2, Saskia C. A. de Jager2,3, Ivan Dzhagalov4, You-Wen He4, Bart J. M. van Vlijmen5,6, Marion J. J. Gijbels1,7, Chris P. Reutelingsperger 8, Theo J. C. van Berkel2, Judith C. Sluimer 1,9, Lieve Temmerman1 & Erik A. L. Biessen1 1Experimental Vascular Pathology Group, Department of Pathology, Cardiovascular Research Institute Maastricht, Maastricht University Medical Center, Maastricht, the Netherlands. 2Division of BioTherapeutics, Leiden Amsterdam Centre for Drug Research, Leiden University, Leiden, the Netherlands. 3Laboratory for Experimental Cardiology, University Medical Center Utrecht, Utrecht, the Netherlands. 4Institue of Microbiology and Immunology, National Yang-Ming University, Taipei, 112, Taiwan. 5Einthoven Laboratory for Vascular and Regenerative Medicine, Leiden University Medical Center, Leiden, The Netherlands. 6Department of Internal Medicine, Division of Thrombosis and Hemostasis, Leiden University Medical Center, Leiden, the Netherlands. 7Department of Molecular Genetics, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands. 8Department of Biochemistry, Cardiovascular Research Institute Maastricht, Maastricht University, Maastricht, the Netherlands. 9Centre for Cardiovascular Science, University of Edinburgh, Edinburgh, UK. Margaux A. C. Fontaine and Marijke M. Westra contributed equally. Correspondence and requests for materials should be addressed to L.T. (email: lieve. temmerman@mumc.nl) www.nature.com/scientificreports/ Nevertheless, it is now believed that neutrophils are instrumental in plaque development and later destabilization, as we and others have shown14–16. Warnatch et al. demonstrated that Neutrophil Extracellular Traps (NETs) prime macrophages to produce inflammatory cytokines, resulting in increased atherogenesis17. Dissection of the contri- bution of neutrophils to atherosclerotic plaque progression has however remained difficult, since other cell pop- ulations are also affected when neutrophils are experimentally manipulated. Deletion of Interferon Regulatory Factor 8 (IRF8) for example results in neutrophilia which exacerbates atherosclerosis in IRF8−/− ApoE−/− chi- meric mice, but was at the same time accompanied by changes in monocytes and several dendritic cell popula- tions18,19. In addition, neutrophils have thus far not been depleted during the complete course of atherosclerosis. Neutrophil depleting antibodies such as the anti–polymorphonuclear leukocyte (PMN) antibody cannot be sus- tained for more than 4 weeks in mice without affecting monocyte counts16.f f g y As Mcl-1 is vital for neutrophil survival, whilst presumably having mild effects on macrophage apoptosis, we hypothesized that myeloid Mcl-1 deletion could serve as an efficient neutropenia model during the full pathogen- esis of atherosclerosis. Moreover, although several Bcl-2 family members have been investigated in the context of atherosclerosis20–23, the role of Mcl-1 in disease progression has not been assessed thus far. To this end, we studied effects of specific deletion of Mcl-1 in the lysozyme M expressing myeloid subsets neutrophils and macrophages on early and advanced atherosclerosis using bone marrow transplantation of Mcl-1fl/fl LysMCre or wild-type (WT) bone marrow into low density lipoprotein receptor-null (LDLr−/−) mice. Results l d Mcl-1 deletion altered neutrophil levels and characteristics. We first quantified Mcl-1 gene expres- sion during atherogenesis. Mcl-1 levels in collar-induced carotid artery lesions of LDLr−/− mice gradually increased during lesion development and in particular in advanced plaques, six weeks after collar induction (Fig. 1A). Of note, this increase was not validated at the protein level. Mcl-1 was mostly expressed in activated macrophages (M1- or M2-macropahges), as compared to other cell types (Fig. 1B). Mcl-1 was also detectable in human atherosclerotic plaques, and its expression did not differ between stable and unstable plaque (Fig. 1C). However, Pearson correlation analysis revealed that Mcl-1 expression did correlate with pathogenic plaque traits, with more traits in unstable plaques, suggesting an involvement in the disease process (Fig. 1D). To ver- ify whether Mcl-1 deletion indeed resulted in an efficient neutropenia model in the context of atherosclerosis, Ldlr−/− recipient mice were transplanted with LysMCre Mcl-1fl/fl (hereafter Mcl-1−/−) or wild type (WT) bone marrow (Fig. 1E). Mcl-1−/− mice showed similar serum cholesterol and triglyceride levels, and body weight (see Supplementary Data Fig. IA–C). Compatible with the notion that Mcl-1 is essential for neutrophil survival7, cir- culating and splenic neutrophil numbers were sharply reduced by 80% and 86%, respectively in Mcl-1fl/fl LysMcre mice9. Circulating neutrophils were depressed in Mcl-1−/− chimeras both at baseline (82% depletion) and even more so under hyperlipidemic conditions (91% depletion) (Fig. 2A). Likewise, neutrophil content in Mcl-1−/− Mcl-1 deletion altered neutrophil levels and characteristics. We first quantified Mcl-1 gene expres- sion during atherogenesis. Mcl-1 levels in collar-induced carotid artery lesions of LDLr−/− mice gradually increased during lesion development and in particular in advanced plaques, six weeks after collar induction (Fig. 1A). Of note, this increase was not validated at the protein level. Mcl-1 was mostly expressed in activated macrophages (M1- or M2-macropahges), as compared to other cell types (Fig. 1B). Mcl-1 was also detectable in human atherosclerotic plaques, and its expression did not differ between stable and unstable plaque (Fig. 1C). However, Pearson correlation analysis revealed that Mcl-1 expression did correlate with pathogenic plaque traits, with more traits in unstable plaques, suggesting an involvement in the disease process (Fig. 1D). To ver- ify whether Mcl-1 deletion indeed resulted in an efficient neutropenia model in the context of atherosclerosis, Ldlr−/− recipient mice were transplanted with LysMCre Mcl-1fl/fl (hereafter Mcl-1−/−) or wild type (WT) bone marrow (Fig. 1E). Low human and murine Mcl-1 expression leads to a pro-apoptotic plaque phenotype enriched in giant-cells Margaux A. C. Fontaine 1, Marijke M. Westra2, Ilze Bot 2, Han Jin1, Aimée J. P. M. Franssen1, Martine Bot2, Saskia C. A. de Jager2,3, Ivan Dzhagalov4, You-Wen He4, Bart J. M. van Vlijmen5,6, Marion J. J. Gijbels1,7, Chris P. Reutelingsperger 8, Theo J. C. van Berkel2, Judith C. Sluimer 1,9, Lieve Temmerman1 & Erik A. L. Biessen1 Our study shows that myeloid Mcl-1 deletion indeed has a profound impact on neutrophil survival. However, Mcl-1 deficient macrophages were seen to accumulate more lipids in vitro and in vivo and were subsequently more sensitive to apoptosis. Moreover, we show that Mcl-1 is implicated in the fusion of macrophages. These combined effects however counteracted each other affecting only viable cell plaque composition, but not plaque growth. Results l d Mcl-1−/− mice showed similar serum cholesterol and triglyceride levels, and body weight (see Supplementary Data Fig. IA–C). Compatible with the notion that Mcl-1 is essential for neutrophil survival7, cir- culating and splenic neutrophil numbers were sharply reduced by 80% and 86%, respectively in Mcl-1fl/fl LysMcre mice9. Circulating neutrophils were depressed in Mcl-1−/− chimeras both at baseline (82% depletion) and even more so under hyperlipidemic conditions (91% depletion) (Fig. 2A). Likewise, neutrophil content in Mcl-1−/− atherosclerotic lesions was decreased, albeit to a lower extent than in blood (Fig. 2B,C), hinting to an enhanced adhesive capacity or faster turnover of residual neutrophils in circulation. Considering that an elevated CXCR4/ CXCR2 balance is associated with regress to the bone marrow and that CXCR4 is an established measure of neu- trophil ageing16, we examined neutrophil phenotype. CXCR4 expression on circulating and peritoneal residual neutrophils was increased (Fig. 2D,E, respectively), suggesting hyperactivation and increased SDF1 migratory capacity. Moreover, responsiveness of remaining neutrophils to the potent neutrophil chemokine CXCL1 was blunted, concordant with the reduced CXCR2 expression by pre-apoptotic neutrophils16. Peritoneal neutrophil influx 2 hours after i.p. injection of CXCL1 was prominent in WT transplanted mice, whereas Mcl-1−/− trans- planted mice only showed a minor, non-significant, increase in peritoneal neutrophils (Fig. 2F,G). Of note, neu- trophil recruitment was paralleled by stromal egress of neutrophils into circulation in WT, but not Mcl-1−/− mice (data not shown). Taken together, these results confirm Mcl-1 as a crucial neutrophil survival factor, also under hyperlipidemic conditions, and demonstrate that Mcl-1 myeloid deletion can be used as a genetic tool to induce a long-lasting, severe neutropenia in atherosclerosis. yp p p g p atherosclerotic lesions was decreased, albeit to a lower extent than in blood (Fig. 2B,C), hinting to an enhanced adhesive capacity or faster turnover of residual neutrophils in circulation. Considering that an elevated CXCR4/ CXCR2 balance is associated with regress to the bone marrow and that CXCR4 is an established measure of neu- trophil ageing16, we examined neutrophil phenotype. CXCR4 expression on circulating and peritoneal residual neutrophils was increased (Fig. 2D,E, respectively), suggesting hyperactivation and increased SDF1 migratory capacity. Moreover, responsiveness of remaining neutrophils to the potent neutrophil chemokine CXCL1 was blunted, concordant with the reduced CXCR2 expression by pre-apoptotic neutrophils16. Peritoneal neutrophil influx 2 hours after i.p. Results l d (C) Mcl-1 gene expression in human atherosclerotic plaques, represented by microarray normalized intensities. (D) Heatmap showing Pearson correlation coefficient (p-values) of Mcl-1 human plaque gene expression correlation with clinical plaque traits. N = 22/23 (stable/unstable). (E) Lethally irradiated LDLr−/− mice were reconstituted with WT or Mcl-1−/− bone marrow, and after 8 weeks of recovery, put on a Western Type Diet (WTD) containing 0.25% cholesterol for 5 weeks (n = 17) or 10 weeks (n = 19). All data is presented as mean ± SEM. p < 0.05. Figure 1. Regulation of Mcl-1 expression in atherosclerosis. (A–C) Mcl-1 gene expression measured by RT- qPCR. (A) Vascular Mcl-1 expression corrected for HPRT housekeeping gene in a model of collar induced carotid artery atherogenesis in LDLr−/− mice. (B) Mcl-1 expression corrected for 18 S housekeeping gene in different mouse cell types. SMCs: smooth muscle cells, MCECs: mouse cardiac endothelial cells. (C) Mcl-1 gene expression in human atherosclerotic plaques, represented by microarray normalized intensities. (D) Heatmap showing Pearson correlation coefficient (p-values) of Mcl-1 human plaque gene expression correlation with clinical plaque traits. N = 22/23 (stable/unstable). (E) Lethally irradiated LDLr−/− mice were reconstituted with WT or Mcl-1−/− bone marrow, and after 8 weeks of recovery, put on a Western Type Diet (WTD) containing 0.25% cholesterol for 5 weeks (n = 17) or 10 weeks (n = 19). All data is presented as mean ± SEM. p < 0.05. macrophage numbers were unchanged (data not shown). This finding led us to examine the lipid loading capac- ity of peritoneal macrophages in vitro. In agreement with the elevated peritoneal foam cell counts in vivo, lipid accumulation in non-stimulated Mcl-1−/− BMDMs in vitro was markedly increased (Fig. 4D). Lipid loading in WT macrophages remained unchanged after incubation with oxLDL for 24 h but was substantially enhanced in Mcl-1−/− macrophages (Fig. 4D). While incubation with VLDL increased lipid content in both WT and Mcl-1−/− macrophages, this increase was considerably higher in the latter cells (Fig. 4D). In keeping with what is observed in peritoneal macrophages, Mcl-1−/− BMDMs also showed an increased lipid uptake capacity (Fig. 4E). Thus, under conditions of hyperlipidemia Mcl-1 appears to be an active regulator of macrophage survival as well as of macrophage lipid loading. Mcl1 deletion induces multinucleated giant cell formation. Results l d injection of CXCL1 was prominent in WT transplanted mice, whereas Mcl-1−/− trans- planted mice only showed a minor, non-significant, increase in peritoneal neutrophils (Fig. 2F,G). Of note, neu- trophil recruitment was paralleled by stromal egress of neutrophils into circulation in WT, but not Mcl-1−/− mice (data not shown). Taken together, these results confirm Mcl-1 as a crucial neutrophil survival factor, also under hyperlipidemic conditions, and demonstrate that Mcl-1 myeloid deletion can be used as a genetic tool to induce a long-lasting, severe neutropenia in atherosclerosis. Myeloid Mcl-1 deletion increased plaque apoptotic cell content but did not affect atheroscle- rotic lesion size. Despite its profound effects on circulating neutrophils, and on plaque neutrophil content, myeloid Mcl-1 deletion did neither alter early nor advanced plaque area, necrotic core size, or plaque macrophage content as compared to controls (Fig. 3A–D). We did observe an increase in plaque apoptosis by 71% and 77% in atherosclerotic lesions of Mcl1−/− mice fed a WTD for 5 and 10 weeks, respectively, compared to WT mice (Fig. 3E), suggesting that Mcl-1 not only plays an important role in the survival of neutrophils, but also of other myeloid plaque-resident cells, such as plaque macrophages and foam cells. Mcl-1 myeloid deletion enhanced lipid-induced apoptosis sensitivity and lipid loading capacity of macrophages. We next investigated the role of Mcl-1 in macrophages and foam cells in atherosclero- sis, reasoning that hyperlipidemia could unleash a role for Mcl-1 in plaque macrophage biology, as has been described for macrophages undergoing infection12. Mcl-1−/− peritoneal macrophages displayed a higher level of apoptosis already at baseline, confirming that the hyperlipidemic environment by itself triggered the Mcl-1 survival program (Fig. 4A). Moreover, Mcl-1−/− peritoneal macrophages, as well as Mcl-1−/− BMDMs (see Supplementary Data Fig. IIA,B), displayed an increased sensitivity towards oxLDL-induced cell death compared to WT macrophages (Fig. 4A-B and Supplementary Data Fig. IIIA–E). Interestingly, peritoneal foam cell numbers were increased by 2.5-fold in Mcl-1−/− compared to WT transplanted mice (Fig. 4C), whereas total peritoneal Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 www.nature.com/scientificreports/ Figure 1. Regulation of Mcl-1 expression in atherosclerosis. (A–C) Mcl-1 gene expression measured by RT- qPCR. (A) Vascular Mcl-1 expression corrected for HPRT housekeeping gene in a model of collar induced carotid artery atherogenesis in LDLr−/− mice. (B) Mcl-1 expression corrected for 18 S housekeeping gene in different mouse cell types. SMCs: smooth muscle cells, MCECs: mouse cardiac endothelial cells. Results l d Much to our surprise, we noticed the abundant presence of multinucleated giant cells (MGCs) in plaques of Mcl-1−/− transplanted animals after 10 weeks on WTD. Indeed, MGC content per plaque was increased by 118% in the Mcl-1−/− transplanted animals as compared to WT (Fig. 5A,B), suggesting that Mcl-1 myeloid deficiency leads to the formation of MGCs. In addition, Mcl-1 myeloid deficiency increased MGCs formation by peritoneal macrophages in vitro both in control conditions and after incubation with oxLDL and VLDL (Fig. 5C,D). To further investigate the Mcl-1-dependent changes underlying MGC formation, we incubated WT BMDMs with IL4 and GM-CSF, two known cytokines to induce giant cells24,25, and compared them to Mcl-1−/− BMDMs. While giant cell formation was unaffected after one week of culture (data not shown), it was significantly increased in Mcl-1−/− BMDMs compared to WT BMDMs at 13 days of culture (P value = 0.002, Fig. 5E). Mechanistically, the effect was partially mediated by hyperlipidemia, as oxLDL or VLDL increased MGC formation in BMDM-derived cells to a larger extent (Fig. 5E). Of note, after 13 days in culture, Mcl-1−/− BMDMs had partially lost their increased capacity of lipid uptake and subsequent lipid-induced apoptosis (Fig. 5F,G, respectively). To assess the relevance of Mcl-1-dependent MGC Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 www.nature.com/scientificreports/ www.nature.com/scientificreports ture.com/scientificreports/ Figure 2. Mcl-1−/− chimeric mice have altered neutrophil levels and characteristics. (A) Circulating neutrophils were defined as Gr1+ and measured by flow cytometry in blood samples obtained from tail vein of WT and Mcl-1−/− bone marrow chimeras. (B) Representative H&E pictures of WT and Mcl-1−/− atherosclerotic plaques (n = 19). Neutrophils are indicated by the arrow. (C) in aortic root atherosclerotic lesions of BM transplanted LDLr−/− after 10 weeks of WTD (n = 19). (D,E) CXCR4 expression in circulating and peritoneal neutrophils respectively assessed by flow cytometry (CXCR4 positive cells within the neutrophil gate of A). (F,G) Circulating and peritoneal neutrophil levels respectively 2 h after CXCL1 injection measured by flow cytometry (CXCR4 positive cells within the neutrophil gate of A). Data is presented as mean ± SEM. *p < 0.001, p < 0.01 and p < 0.05. Figure 2. Mcl-1−/− chimeric mice have altered neutrophil levels and characteristics. (A) Circulating neutrophils were defined as Gr1+ and measured by flow cytometry in blood samples obtained from tail vein of WT and Mcl-1−/− bone marrow chimeras. Results l d (B) Representative H&E pictures of WT and Mcl-1−/− atherosclerotic plaques (n = 19). Neutrophils are indicated by the arrow. (C) in aortic root atherosclerotic lesions of BM transplanted LDLr−/− after 10 weeks of WTD (n = 19). (D,E) CXCR4 expression in circulating and peritoneal neutrophils respectively assessed by flow cytometry (CXCR4 positive cells within the neutrophil gate of A). (F,G) Circulating and peritoneal neutrophil levels respectively 2 h after CXCL1 injection measured by flow cytometry (CXCR4 positive cells within the neutrophil gate of A). Data is presented as mean ± SEM. p < 0.001, p < 0.01 and p < 0.05. formation in human atherosclerosis, we quantified MGCs in human plaques. Cathepsin K+ multinucleated cells were frequently found in both stable and unstable plaques (Fig. 6A,B). Interestingly, their presence inversely correlated with Mcl-1 gene expression in unstable plaque, and only in unstable lesions associated significantly with lesion hemorrhages and calcifications (P value = 0.044 and 0.055, respectively, Fig. 6C,D). Thus, our results unveil a hitherto unknown link between Mcl-1 and MGC formation, which is influenced by hyperlipidemia, an association potentially preserved in human atherosclerotic lesions as well. Discussion In this study, we evaluated the effects of myeloid Mcl-1 deletion and its accompanying neutropenia on athero- sclerosis progression. In addition to extreme neutropenia, Mcl-1 deficiency resulted in increased macrophage apoptosis and lipid handling, and triggered multinucleated giant cell formation. First, we found that myeloid Mcl-1 deletion dramatically reduced neutrophil numbers both in circulation and in atherosclerotic lesions. This is in keeping with extensive data on the vital role of Mcl-1 in neutrophil survival9,10,26,27 which as we now Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 www.nature.com/scientificreports/ Figure 3. Effect of myeloid Mcl-1 deficiency on atherosclerotic lesion size and composition. (A) Representative micrographs of Oil Red O stained aortic root sections in WT and Mcl1−/− mice after 5 and 10 weeks of WTD. (B) Atherosclerotic lesion size after 5 weeks or 10 weeks of WTD quantified on Oil Red O staining using Leica image analysis system. (C) Plaque necrotic core size after 10 weeks of WTD. (D) Plaque macrophage content after 10 weeks of WTD assessed on Moma-2 positive staining. (E) Plaque apoptotic cell content after 5 weeks or 10 weeks of WTD, measured by TUNEL staining. Data is presented as mean ± SEM. p < 0.01. Figure 3. Effect of myeloid Mcl-1 deficiency on atherosclerotic lesion size and composition. (A) Representative micrographs of Oil Red O stained aortic root sections in WT and Mcl1−/− mice after 5 and 10 weeks of WTD. (B) Atherosclerotic lesion size after 5 weeks or 10 weeks of WTD quantified on Oil Red O staining using Leica image analysis system. (C) Plaque necrotic core size after 10 weeks of WTD. (D) Plaque macrophage content after 10 weeks of WTD assessed on Moma-2 positive staining. (E) Plaque apoptotic cell content after 5 weeks or 10 weeks of WTD, measured by TUNEL staining. Data is presented as mean ± SEM. p < 0.01. igure 3. Effect of myeloid Mcl-1 deficiency on atherosclerotic lesion size and composition. (A) Representative /t demonstrated remains valid in a hyperlipidemic environment. On the other hand, Mcl-1 overexpression28,29 and Bim deletion23, a pro-apoptotic Bcl-2 family member and Mcl-1 antagonist, do not affect circulating myeloid cell numbers, suggesting that physiological Mcl-1 levels are sufficient for normal cell function. Our work thus provides a mouse model for continuous neutropenia, an important advantage to models used in earlier studies addressing neutrophil contribution to atherogenesis. Though Zernecke et al. Discussion demonstrated that CXCR4 blockade aggravated atherosclerosis due to increased neutrophil recruitment to the plaque, they were unable to extend the neutrophil depletion beyond a 4 week period16. They did show that plaques of neutrophil depleted mice were smaller and had a lower neutrophil and macrophage content, but did not evaluate plaque apoptosis or necrotic core size. Similarly so, CCL3−/− LDLr−/− bone marrow chimeras with 50% less neutrophils, developed smaller atherosclerotic lesions30. Consequently, we were highly surprised by the unaffected atherosclerotic lesion burden in neutropenic LysMCre Mcl-1−/− mice.f p y Apparently the atheroprotective effects of Mcl-1 deletion in neutrophils in LDLr−/− mice are counteracted by potentially pro-atherogenic effects on other cell types targeted by the LysM conditional Mcl-1 deletion, we there- fore examined in greater detail the role of Mcl-1 in atherosclerotic LysM+ monocytes and macrophages.f g y y p g Although Mcl-1 loss was previously shown to have no effect on monocyte and macrophage development in wild type mice9,10, Mcl-1−/− macrophages were more sensitive to apoptosis upon an infection12 or phagocytic challenge10. We found that the apoptotic cell content in advanced aortic root lesions (10 weeks of WTD) was increased by 44% in mice with myeloid Mcl-1 deficiency. As neutrophils are only scarcely present in advanced lesions14 and most apoptotic cells were located in the central atheroma (data not shown), the high apoptotic cell density is likely to reflect dying LysM+ plaque macrophages. Our work thus identifies Mcl-1 as a major survival protein in atherosclerotic lesions. Atherosclerotic lesion burden was however unaltered in Mcl-1−/− BM recipi- ents, as were necrotic core, macrophage and collagen content. Similar results were obtained when studying plaque initiation five weeks after WTD. Our results correspond with those from Thorp et al.21, who showed increased macrophage apoptosis, but unchanged lesion burden in Bcl-2flox-LysMCre ApoE−/− mice that are deficient in macrophage and neutrophil Bcl-221. In turn, hematopoietic Bim deficiency, a pro-apoptotic Bcl-2-family member, had no impact on macrophage apoptosis and lesion burden in LDLr−/− mice23. Thus, Mcl-1−/− deletion in mac- rophages led to higher apoptosis level in advanced plaques, however it is clear from the above that this not always translates into a pro-atherogenic plaque progression. p g p q p g In addition to an increased sensitivity to oxLDL induced cell death, Mcl-1−/− macrophages showed augmented lipid accumulation after incubation with oxLDL and VLDL. Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 Discussion Data is presented as mean ± SEM. p < 0.001, p < 0.01 and p < 0.05. those of Halvorsen et al.31, who reported reduced IL-10 induced oxLDL loading by THP-1 macrophages in vitro after siRNA mediated silencing of Mcl-1 and Bfl-1 expression. The authors did not assess effects of Mcl-1 inhibi- tion alone, without IL-10 stimulation. Based on our data we hypothesize that the apoptosis-prone phenotype of Mcl-1−/− macrophages is at least partly caused by the increased uptake of lipids. p g p y y p p Another remarkable characteristic was the high propensity of Mcl-1−/− cells to form multinucleated giant cells (MGCs). MGCs, a hallmark of several chronic inflammatory diseases32,33, originate from monocyte-macrophage lineage and result from cell fusion32,34. Giao et al. have illustrated the presence of TRAP-positive (osteoclast like giant) cells in close relation to calcified regions and TRAP-negative MGCs in advanced human atherosclerotic plaques35. In addition, Samokhin et al.36 showed that mice fed a Paigen diet displayed a 4-fold increase in MGC number in atherosclerotic lesions36. In our study, we observed a higher macrophage fusion capacity in both Mcl- 1−/− peritoneal and BMDMs, which increased even more upon oxLDL or VLDL stimulation. Furthermore, Mcl- 1−/− deficiency in BMDMs seemed to promote MGC formation, independently of lipid uptake. In line with our in vitro findings, Mcl-1−/− atherosclerotic plaques had a 4-fold increase in MGC presence as compared to WT lesions. Additionally, the presence of MGCs in human unstable plaques correlated negatively with Mcl-1 gene expression and significantly associated with hemorrhages and calcifications in the lesion. The exact role of MGCs in atherosclerosis is not yet fully understood, however it was previously shown that MGCs facilitate vascular smooth muscle cell migration in the context of atherosclerosis by producing cathepsin K and destroying the elastin fibers36. Although not providing conclusive evidence, our findings support such pro-atherogenic role of MGCs. To our knowledge, we are the first to implicate Mcl-1 in the fusion of macrophages. Possibly, this is related to increased oxidative phosphorylation capacity for energy production in Mcl-1−/− deficient BMDMs (data not shown), however the exact mechanism by which Mcl-1 induces MGC formation remains to be investigated. ) y g In summary, myeloid Mcl-1 deficiency led to a profound and sustained neutropenia in hyperlipidemic LDLr−/− mice accompanied by enhanced oxLDL induced macrophage death in vitro, as well as increased ath- erosclerotic lesion apoptosis. Discussion In keeping, we observed elevated foam cell levels in vivo in the peritoneal cavity of Mcl-1−/− BM compared to WT BM recipients. These findings seem to contrast with Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 www.nature.com/scientificreports/ Figure 4. Effect of Mcl-1 myeloid deletion on macrophage apoptosis and lipid loading. (A) PS exposure of unstimulated or oxLDL (40 µg/ml) stimulated peritoneal macrophages measured by Annexin-V-OG staining. (B) PS exposure of unstimulated or oxLDL/VLDL stimulated bone marrow derived macrophages (BMDMs) measured by Annexin-V-OG staining. (C) Peritoneal foam cell presence of WT and Mcl-1−/− mice after 10 weeks of WTD assessed by Oil Red O staining in vitro. (D) Lipid loading capacity of peritoneal macrophages after oxLDL (20 µg/ml) and vLDL (50 µg/ml) exposure. (E) Lipid uptake capacity of BMDMs after oxLDL (20 µg/ml) and VLDL (50 µg/ml) incubation. Data is presented as mean ± SEM. p < 0.001, p < 0.01 and p < 0.05. Figure 4. Effect of Mcl-1 myeloid deletion on macrophage apoptosis and lipid loading. (A) PS exposure of unstimulated or oxLDL (40 µg/ml) stimulated peritoneal macrophages measured by Annexin-V-OG staining. (B) PS exposure of unstimulated or oxLDL/VLDL stimulated bone marrow derived macrophages (BMDMs) measured by Annexin-V-OG staining. (C) Peritoneal foam cell presence of WT and Mcl-1−/− mice after 10 weeks of WTD assessed by Oil Red O staining in vitro. (D) Lipid loading capacity of peritoneal macrophages after oxLDL (20 µg/ml) and vLDL (50 µg/ml) exposure. (E) Lipid uptake capacity of BMDMs after oxLDL (20 µg/ml) and VLDL (50 µg/ml) incubation. Data is presented as mean ± SEM. p < 0.001, p < 0.01 and p < 0.05. Figure 4. Effect of Mcl-1 myeloid deletion on macrophage apoptosis and lipid loading. (A) PS exposure of l d ( l) l d l h d b Figure 4. Effect of Mcl-1 myeloid deletion on macrophage apoptosis and lipid loading. (A) PS exposure of unstimulated or oxLDL (40 µg/ml) stimulated peritoneal macrophages measured by Annexin-V-OG staining. (B) PS exposure of unstimulated or oxLDL/VLDL stimulated bone marrow derived macrophages (BMDMs) measured by Annexin-V-OG staining. (C) Peritoneal foam cell presence of WT and Mcl-1−/− mice after 10 weeks of WTD assessed by Oil Red O staining in vitro. (D) Lipid loading capacity of peritoneal macrophages after oxLDL (20 µg/ml) and vLDL (50 µg/ml) exposure. (E) Lipid uptake capacity of BMDMs after oxLDL (20 µg/ml) and VLDL (50 µg/ml) incubation. Discussion p < 0.001, p < 0.01 and p < 0.05. Figure 5. Effect of Mcl-1 deletion on macrophage fusion. (A) Representative pictures of mouse aortic root sections stained with H&E. Multinucleated Giant Cells are indicated by the arrow. MGCs were quantified as cells containing 2 or more round nuclei. (B) MGCs quantification in the atherosclerotic lesions of WT and Mcl1−/− chimeras after 10 weeks of WTD. (C) Representative pictures of Oil Red O stained unstimulated, oxLDL or VLDL stimulated peritoneal macrophages. Multinucleated Giant Cells are indicated by the arrow (D) Quantification of Giant Cell population in peritoneal macrophages depicted in (C). (E) MGCs in BMDMs after 6d of culture in the presence of either oxLDL, VLDL or nothing. (F) Lipid loading capacity of MGCs after 13 d in culture. (G) PS exposure in MGC population after 13 d in culture. Data is presented as mean ± SEM. Figure 5. Effect of Mcl-1 deletion on macrophage fusion. (A) Representative pictures of mouse aortic root sections stained with H&E. Multinucleated Giant Cells are indicated by the arrow. MGCs were quantified as cells containing 2 or more round nuclei. (B) MGCs quantification in the atherosclerotic lesions of WT and Mcl1−/− chimeras after 10 weeks of WTD. (C) Representative pictures of Oil Red O stained unstimulated, oxLDL or VLDL stimulated peritoneal macrophages. Multinucleated Giant Cells are indicated by the arrow (D) Quantification of Giant Cell population in peritoneal macrophages depicted in (C). (E) MGCs in BMDMs after 6d of culture in the presence of either oxLDL, VLDL or nothing. (F) Lipid loading capacity of MGCs after 13 d in culture. (G) PS exposure in MGC population after 13 d in culture. Data is presented as mean ± SEM. p < 0.001, p < 0.01 and p < 0.05. Figure 5. Effect of Mcl-1 deletion on macrophage fusion. (A) Representative pictures of mouse aortic root i Discussion Furthermore, Mcl-1 deficiency was shown to enhance lipid uptake and induce the formation of MGCs in vitro and in vivo. In line with the observation in mice, Mcl-1 gene expression negatively correlated with the presence of MGCs in human unstable plaque. Taken together, the markedly lower neutro- phil numbers mask the combined effects of a more lipid rich and apoptotic plaque in myeloid Mcl-1 deficient animals, and result in an unchanged lesion development and progression. Our results clearly identify Mcl-1 as a macrophage survival protein under hyperlipidemia and uncover a hitherto unknown role for Mcl-1 in MGC formation. Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 www.nature.com/scientificreports/ Material and Methods Figure 5. Effect of Mcl-1 deletion on macrophage fusion. (A) Representative pictures of mouse aortic root sections stained with H&E. Multinucleated Giant Cells are indicated by the arrow. MGCs were quantified as cells containing 2 or more round nuclei. (B) MGCs quantification in the atherosclerotic lesions of WT and Mcl1−/− chimeras after 10 weeks of WTD. (C) Representative pictures of Oil Red O stained unstimulated, oxLDL or VLDL stimulated peritoneal macrophages. Multinucleated Giant Cells are indicated by the arrow (D) Quantification of Giant Cell population in peritoneal macrophages depicted in (C). (E) MGCs in BMDMs after 6d of culture in the presence of either oxLDL, VLDL or nothing. (F) Lipid loading capacity of MGCs after 13 d in culture. (G) PS exposure in MGC population after 13 d in culture. Data is presented as mean ± SEM. p < 0.001, p < 0.01 and p < 0.05. Figure 5. Effect of Mcl-1 deletion on macrophage fusion. (A) Representative pictures of mouse aortic root sections stained with H&E. Multinucleated Giant Cells are indicated by the arrow. MGCs were quantified as cells containing 2 or more round nuclei. (B) MGCs quantification in the atherosclerotic lesions of WT and Mcl1−/− chimeras after 10 weeks of WTD. (C) Representative pictures of Oil Red O stained unstimulated, oxLDL or VLDL stimulated peritoneal macrophages. Multinucleated Giant Cells are indicated by the arrow (D) Quantification of Giant Cell population in peritoneal macrophages depicted in (C). (E) MGCs in BMDMs after 6d of culture in the presence of either oxLDL, VLDL or nothing. (F) Lipid loading capacity of MGCs after 13 d in culture. (G) PS exposure in MGC population after 13 d in culture. Data is presented as mean ± SEM. Material and Methods Mcl-1 gene expression in different cell types. Murine 3T3 fibroblasts, smooth muscle cells (SMCs), cardiac endothelial cells (MCECs), and BMDMs stimulated with either lipopolysaccharide (10 ng/ml) and Interferon-γ (100 U/ml) (M1 macrophage) or Interleukin-4 (20 ng/ml) (M2 macrophage) were cultured. Total RNA was extracted and gene expression was analyzed by qPCR. Mcl-1 and housekeeping gene 18 S primers used are listed in Supplementary Table 1. Bone marrow transplantation and atherosclerosis induction. Male recipient LDLr−/− mice were housed in sterile ventilated cages with food (RM3, Special Diet Services) and water ad libitum. Antibiotics (83 mg/l ciprofloxacin, 67 mg/l Polymixin B and 5 g/l sugar) were supplied in the drinking water. Mice were exposed to a single dose of 9 Gy total body irradiation (0.19 Gy/min, 200 kV, 4 mA) using an Andrex Smart 225 Röntgen source (YXLON International) one day before transplantation. Bone marrow was extracted from femur and tibia of male Mcl-1fl/fl LysMcre (hereafter Mcl-1−/−) donors and wild type (WT) littermates. Irradiated LDLr−/− mice received 2.5 × 106 BM cells of either Mcl-1−/− (n = 17) or WT (n = 15) via tail vein injection. After a recovery period of eight weeks mice were put on a WTD containing 0.25% cholesterol and 15% cacao butter (Diet W, Special Diet Services) for an additional five (plaque initiation) or ten weeks (advanced plaque). Blood cell analysis and flow cytometry. Blood samples were taken by tail bleeding immediately before BMT, prior to (week 0) and after four weeks of WTD feeding (week 4) and at the time of sacrifice (week 5 or week 10). Peritoneal leukocytes were isolated at the time of sacrifice by peritoneal lavage with 10 ml PBS. Whole blood and peritoneal lavage samples were analyzed using a Sysmex blood cell analyzer (XT-2000i). For flow cytometry, WBC and peritoneal leukocytes were stained with fluorescently labelled antibodies against F4/80, CD19, CD4, CD71 and CD11b (eBioscience) and Gr1, CD8 and CXCR4 (BD Pharmingen). Fluorescence-activated cell sorting (FACS) analysis was performed on a FACSCalibur with CellQuest software (BD Biosciences). Tissue harvesting and analysis. Two hours before sacrifice, Mcl-1−/− or WT mice (n = 5) received intra- peritoneal injections of CXCL1 (200 ng/ml in 1 ml PBS) or PBS control. The mice were anesthetized and perfused with PBS. Cryosections of the aortic root tissue were stained with hematoxylin and eosin (HE) or Oil Red O. Material and Methods Animals. All animal work was approved by animal regulatory authority of Leiden University and performed in compliance with Dutch national guidelines. Low density lipoprotein receptor-null (LDLr−/−) mice were obtained from the local animal breeding facility. Mcl-1fl/fl LysMcre mice were obtained from the Department of Immunology, Duke University Medical Center, USA and were backcrossed to C57BL/6J. Mcl-1 gene expression during atherogenesis. Twenty male LDLr−/− mice were fed a Western-type diet (WTD) two weeks prior to surgery and throughout the experiment. Atherosclerotic carotid artery lesions were induced by perivascular collar placement37. Carotid Mcl-1 gene expression was analyzed prior to and two, four, six and eight weeks after collar placement (n = 4, per timepoint).The mice were anaesthetized and perfused with phosphate buffered saline (PBS) after which both common carotid arteries were isolated. After dissection of the adventitia, plaque containing segments were excised based on macroscopic examination, snapfrozen and stored at −80°c. Two to three atherosclerotic plaques were pooled per sample and total RNA was isolated using Trizol reagent (Invitrogen). Gene expression was analyzed by real time PCR (qPCR) using ABI PRISM 7700 Sequence Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 www.nature.com/scientificreports/ Figure 6. Presence of Multinucleated Giant Cells in human atherosclerotic plaques. (A) Representative pictures of cathepsin K stained human unstable plaques. Multinucleated Giant Cells are indicated by the arrow (B) MGCs are quantified as cells positive for cathepsin K and containing 2 or more round nuclei. (C) Pearson correlation analysis showing coefficient (p-values) between MGCs presence and MCL-1 gene expression levels in human unstable plaque segments (n = 18). (D) Heatmap showing Pearson’s correlation coefficient/p- values between the presence of MGCs and other clinical plaque traits. N = 22/23 (stable/unstable). Indicates significant correlation. Figure 6. Presence of Multinucleated Giant Cells in human atherosclerotic plaques. (A) Representative pictures of cathepsin K stained human unstable plaques. Multinucleated Giant Cells are indicated by the arrow (B) MGCs are quantified as cells positive for cathepsin K and containing 2 or more round nuclei. (C) Pearson correlation analysis showing coefficient (p-values) between MGCs presence and MCL-1 gene expression levels in human unstable plaque segments (n = 18). (D) Heatmap showing Pearson’s correlation coefficient/p- values between the presence of MGCs and other clinical plaque traits. N = 22/23 (stable/unstable). Indicates significant correlation. Detector (Applied Biosystems) with SYBR-Green technology. Mcl-1 and housekeeping gene Hprt primers used are listed in Supplementary Table 1. Material and Methods Lesion size was quantified using a Leica DMRE microscope with camera and Leica Qwin Imaging software (Leica Ltd). MGCs were defined as macrophages with two or more round nuclei on the HE slides and quantified by an animal pathologist. Immunohistochemical stainings were performed for macrophage (MOMA-2, Sigma) and vSMC (α-smooth muscle actin, Sigma) content. Apoptotic cell content was quantified using terminal deoxytrans- ferase dUTP nick-end labeling (TUNEL) kit (Roche Diagnostics). Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 www.nature.com/scientificreports/ LDL and VLDL isolation and oxLDL preparation. LDL and very low-density lipoprotein (VLDL) were isolated from human plasma by density gradient ultracentrifugation for 20 h at 4 °C38. LDL concentration was adjusted to 0.5 mg/ml with PBS and oxidized by incubation with 0.32 mM CuSO4 overnight at 37 °C after which the oxidation reaction was terminated by addition of 50 µM EDTA. oxLDL was then dialyzed against PBS con- taining 10 µM EDTA for 24 h. Final oxLDL and VLDL concentration were measured using the bicinchonic acid protein kit (Thermo Fisher Scientific). BMDMs and peritoneal macrophages. Bone marrow cells were isolated by flushing femurs and tibia with PBS and single cell suspensions were obtained by passing the suspension through a 70 µm nylon cell strainer (BD Falcon). Bone marrow cells were differentiated into macrophages by culturing in RPMI-1640 medium (GIBCO Invitrogen), supplemented with 10% (vol/vol) heat inactivated fetal calf serum (FCS) (GIBCO, Invitrogen), glu- tamine (2 mM), penicillin (100 U/ml), streptomycin (100 μg/ml) and 15% (vol/vol) L929-conditioned medium for 7 days. After differentiation, BMDMs were harvested with lidocaine and replated at 0.5 × 105 cells/well in a 96 well imaging plate (BD #353219, Corning Life Sciences) for apoptosis, lipid uptake and fusion assay. Peritoneal leukocytes were isolated from Mcl-1−/− and WT mice following an i.p. injection of PBS before sacrifice. Cells were pooled from 2–3 mice per genotype and plated at 0.25 × 106 cells/well in 8 chamber culture slides or 24-well plates (BD Falcon) and non-adherent peritoneal macrophages were removed. Lipid loading of BMDMs and peritoneal macrophages. Adherent peritoneal macrophages were stim- ulated with 20 µg/ml oxLDL or 50 µg/ml VLDL for 24 hours, after which slides were washed with PBS and stained with Oil Red O (ORO). Lipid loading was quantified as the ratio between the ORO stained area and total cell surface. Material and Methods BMDMs were loaded with 20 µg/ml oxLDL or 50 µg/ml VLDL combined with 5 µg/ml and 12.5 µg/ml Topfluor cholesterol (Avanti Polar Lipids) respectively for 2.5 h (D0) and 120 h (D5). Cell nuclei were stained with Hoechst (Sigma Aldrich) at 15 µg/ml. Apoptosis of peritoneal macrophages and BMDMs. Adherent peritoneal macrophages were stim- ulated with 40 µg/ml oxLDL for 24 hours. The macrophages were detached with Accutase (PAA Laboratories GmbH), stained with FITC-labeled Annexin-V (ImmunoTools) and propidium iodide (Sigma Aldrich) and sub- sequently analyzed by flow cytometry (FACSCalibur, BD Biosciences). For the BMDM apoptosis assay, cells were loaded with 20 µg/ml oxLDL or 50 µg/ml VLDL for 6 h (D0) or 120 h (D5) and subsequently stained with Hoechst and Oregon-green or Alexa-647 labelled Annexin-V for 15 minutes39. Fusion assay. BMDMs were loaded with 20 µg/ml oxLDL or 50 µg/ml VLDL for either 24 h (D1) or 120 h (D5). Macrophage fusion was measured by co-staining with calcein-AM (ThermoFisher Scientific) at 1 µg/ml and Hoechst. MGCs were defined as having two or more nuclei per calcein-segmented cell. Image analysis. For all 96-well plate immunofluorescence assays (apoptosis, lipid loading and cell fusion), nine images per well were taken using the high content analyzer (HCA) BD pathway 855 (BD Biosciences) with 10x objective and further analyzed using Attovision and DIVA software (BD Biosciences). Human atherosclerotic plaque collection. Stable and unstable human carotid atherosclerotic plaques segments (classified according to Virmani et al.40) were collected from the same symptomatic patient (n = 22/23) undergoing carotid endarterectomy in Maastricht University Medical Centre (MUMC, The Netherlands) and Zuyderland Medical Center (Sittard, the Netherlands). Collection, storage, and use in the Maastricht Pathology Tissue Collection (MPTC) were approved by medical ethical committee (16-4-181) and in accordance with the “Code for Proper Secondary Use of Human Tissue in the Netherlands” (http://www.fmwv.nl). Atherosclerotic plaque segments were alternatively snapfrozen for RNA and microarray analysis, or formalin-fixed for paraffin-embedding. Human atherosclerotic plaque histology. The human plaque sections adjacent to the snapfrozen segment were classified to determine plaque type according to Virmani et al.40. HE staining was used to quan- tify plaque size, lipid core size and hemorrhage. Alizarin red staining was done to measure the percentage of calcification. Human atherosclerotic plaque immunohistochemistry. All stainings were performed on adjacent plaque sections for vascular endothelial marker CD31 (Dako), macrophage marker CD68 (Dako), T-cell marker CD3 (Dako) and MGC marker (cathepsin K41). References 1. Kozopas, K. M., Yang, T., Buchan, H. L., Zhou, P. & Craig, R. W. MCL1, a gene expressed in programmed myeloid cell differentiation has sequence similarity to BCL2. Proc. Natl. Acad. Sci. USA 90, 3516–20 (1993). q y 2. Opferman, J. T. et al. Development and maintenance of B and T lymphocytes requires antiapoptotic MCL-1. Nature 426, 671–676 (2003). ( ) 3. Clohessy, J. G., Zhuang, J., de Boer, J., Gil-Gómez, G. & Brady, H. J. M. Mcl-1 Interacts with Truncated Bid and Inhibits Its Induction of Cytochrome c Release and Its Role in Receptor-mediated Apoptosis. J. Biol. Chem. 281, 5750–5759 (2006). 4. Willis, S. N. et al. 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Human atherosclerotic plaque RNA extraction and transcriptomics. RNA was isolated by Guanidium Thiocyanate lysis followed by Cesium Chloride gradient centrifugation, and then purified using the Nucleospin RNAII kit. 750 ng of biotinylated cRNA per sample was hybridized to Illumina Human Sentrix-8 V2.0 BeadChip® and washed according to the Illumina standard procedure. Scanning was performed on the Illumina BeadStation 500. Raw expression data were extracted from the images using default settings and without normalization. Statistics. Values are expressed as mean ± standard error of the mean (SEM). All statistical analyses were performed using Prism (GraphPad Software). Statistically significant differences (p < 0.05) were evaluated using the Student’s t-test unless stated otherwise. Pearson correlation analysis was performed to assess the Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 www.nature.com/scientificreports/ association between Mcl-1 gene expression or MGCs presence and clinical plaque traits. p < 0.05, p < 0.01 and p < 0.001. association between Mcl-1 gene expression or MGCs presence and clinical plaque traits. p < 0.05, p < 0.01 and p < 0.001. association between Mcl-1 gene expression or MGCs presence and clinical plaque traits. p < 0.05, p < 0.01 and *p < 0.001. References K. & West, C. E. Separation of plasma lipoproteins by density-gradient ultracentrifugation. Anal. Biochem. 65, 42–49 (1975).fi ( ) 39. van Genderen, H. et al. In vitro measurement of cell death with the annexin A5 affinity assay. Nat. Protoc. 1, 363–367 (2006). Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3 10 www.nature.com/scientificreports/ 0. Otsuka, F. et al. Natural progression of atherosclerosis from pathologic intimal thickening to late fibroatheroma in human coronary arteries: A pathology study. Atherosclerosis 241, 772–782 (2015).i p gy y 1. Donners, M. M. P. C. et al. Cathepsin K Deficiency Prevents the Aggravated Vascular Remodeling Response to Flow Cessation in ApoE−/− Mice. PLoS One 11, e0162595 (2016). Acknowledgementsh g This work is part of the research programme #912.02.037 to EB and BV, which is financed by the Netherlands Organization for Scientific Research (NWO). Author Contributions M.F., L.T. and M.W. designed and performed experiments and wrote the manuscript, I.B., A.F., M.B., H.J., S.J. performed experiments. M.G. quantified giant cells in murine and human atherosclerotic plaques. Y.-W. H. and I.D. kindly provided the animals. C.R. kindly provided Oregon Green and Alexa-647 labelled Annexin-V. B.V., T.B. and E.B. provided funding and coordinated the study, E.B. and J.S. gave critical input to experiments and the manuscript. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019-51020-3.h Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-019- Competing Interests: The authors declare no competing interests. Competing Interests: The authors declare no competing interests. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2019 Scientific Reports \| (2019) 9:14547 \| https://doi.org/10.1038/s41598-019-51020-3
https://openalex.org/W2592498323	https://europepmc.org/articles/pmc5328967?pdf=render	English	null	Strength of Excitation Is Negatively Associated with Aggressive Behavior after Interpersonal Rejection	Frontiers in psychology	2,017	cc-by	5,573	ORIGINAL RESEARCH published: 28 February 2017 doi: 10.3389/fpsyg.2017.00296 Strength of Excitation Is Negatively Associated with Aggressive Behavior after Interpersonal Rejection Joanna Rajchert1* and Mikołaj Winiewski2 1 Institute of Psychology, The Maria Grzegorzewska University, Warsaw, Poland, 2 Faculty of Psychology, University of Warsaw, Warsaw, Poland 1 Institute of Psychology, The Maria Grzegorzewska University, Warsaw, Poland, 2 Faculty of Psychology, University of Warsaw, Warsaw, Poland This study explored how the Pavlovian temperamental traits strength of excitation (SE) and strength of inhibition (SI) were related to rejection and aggression. We predicted that rejection would increase aggression, but that higher SE and SI would mitigate this effect. Participants (n = 117) completed Strelau and Zawadzki’s (1998) Pavlovian Temperament Survey. A week later they were told that a peer wanted (acceptance) or did not want (rejection) to work with them and they were given a chance to react aggressively by damaging that person’s chance of getting a job. We found that only high SE was negatively related to rejected individuals’ aggression. The results are related to the diathesis-stress and catalyst models’ accounts of the role of temperament in shaping experience of social stress. Edited by: Edited by: Mark Hallahan, College of the Holy Cross, USA Correspondence: Joanna Rajchert Correspondence: Joanna Rajchert jrajchert@aps.edu.pl Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology Received: 03 June 2016 Accepted: 16 February 2017 Published: 28 February 2017 Citation: Rajchert J and Winiewski M (2017) Strength of Excitation Is Negatively Associated with Aggressive Behavior after Interpersonal Rejection. Front. Psychol. 8:296. doi: 10.3389/fpsyg.2017.00296 Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology Specialty section: This article was submitted to Personality and Social Psychology, a section of the journal Frontiers in Psychology Received: 03 June 2016 Accepted: 16 February 2017 Published: 28 February 2017 j Research (Rajchert and Winiewski, 2016) showed that also temperamental characteristics (strength of Behavioral Activation and Inhibition System) play a role in rejection – aggression relationship. Having a sensitive Behavioral Activation System (BAS) increased displaced aggression after ostracism, whilst having a strong Behavioral Inhibition System (BIS) limited direct aggression (negative evaluation of the interaction partner) after rejection. Other research highlights the role of vulnerability to environmental stressors, expressed in temperamental, physiologic or genetic make-up of individuals (for review see Belsky and Pluess, 2009). Vulnerable individuals Keywords: temperament, strength of inhibition, strength of excitation, aggressive behavior, interpersonal rejection INTRODUCTION Reviewed by: Lisabeth Fisher DiLalla, Southern Illinois University Carbondale, USA Ya-tang Li, Lisabeth Fisher DiLalla, Southern Illinois University Carbondale, USA Ya-tang Li, California Institute of Technology, USA Interpersonal rejection is a major threat to human well-being and so could be considered an intense stressor. Even moderately intensive interpersonal rejection, such as telling someone that he or she has not been chosen for group work, is followed by symptoms of psychological stress, such as negative emotions (anger, fear, anxiety, distress, and hostility) (Smart Richman and Leary, 2009) and physiological reactions such as the cortical and cardiovascular changes associated with high arousal (Stroud et al., 2000; Gunnar et al., 2003; Blackhart et al., 2007; Ford and Collins, 2010). There is evidence that rejection also leads to aggression in laboratory contexts, where it is variously operationalized as the administration of a higher intensity of noise to the target, the allocation of more hot sauce to the target or a more negative evaluation of an interaction partner which might adversely aﬀect his or her job prospects (e.g., Baumeister et al., 2007; DeWall et al., 2010). The eﬀect of rejection on aggression may, however, be mitigated by particular individual characteristics, such as narcissism, trait self-control, trait-anger, trait-aggressiveness, readiness for aggression and impulsivity (Bushman and Baumeister, 1998; Twenge and Campbell, 2003; Tangney et al., 2004; Bettencourt et al., 2006; Rajchert et al., 2016). California Institute of Technology, USA Correspondence: Joanna Rajchert jrajchert@aps.edu.pl Correspondence: Joanna Rajchert jrajchert@aps.edu.pl Citation: Rajchert J and Winiewski M (2017) Strength of Excitation Is Negatively Associated with Aggressive Behavior after Interpersonal Rejection. Front. Psychol. 8:296. doi: 10.3389/fpsyg.2017.00296 February 2017 \| Volume 8 \| Article 296 1 Frontiers in Psychology \| www.frontiersin.org Temperament and Rejection Effects on Aggression Rajchert and Winiewski tend to react more negatively to adverse conditions, such as neglectful or insensitive parenting (e.g., Caspi et al., 2002) and other negative life events. Moreover, research conducted with infants and toddlers indicates that parental rejection increased externalizing symptoms in children, particularly highly irritable children and children with a diﬃcult temperament (e.g., Kochanska, 1993; Belsky et al., 1998; Morris et al., 2002). The present study adds to the growing literature on individual diﬀerences in reactivity to rejection by focusing on experimentally induced rejection and another temperamental traits, namely strength of excitation (SE) and strength of inhibition (SI). SD = 0.95) were included in analysis. The data from 23 other participants were dropped from the sample because they indicated the disbelief in the cover story of rejection manipulation or the aggression measurement. Students were told that participation in the study would allow them “to get to know how the psychological studies look like and help them to design their own studies in the future.” This was the only encouragement they received to participate. Students were not given grades or points for participation and were allowed to withdraw from participation in the study at any time for any reason. All students agreed to participate and none of them withdrew. The sex distribution in the sample was typical of the population of social science students. The study was carried out in accordance with the recommendations of Academic Ethical Review Board and all participants provided written, informed consent in accordance with the Declaration of Helsinki. Pavlov’s (1952) theory of the basic propensities of the nervous system - developed further by Strelau et al. (1999) - posits that the central nervous system has two main propensities: SE and SI. SE and SI are theoretical constructs that predict inter-individual variability in the dynamic of behaviors, which is reﬂected in tempo and intensity of reaction. SE is also referred to as the ‘strength’ or ‘endurance’ of the nervous system, and high SE is characterized by chronically low arousal and low activation even in threatening situations. The stronger the nervous system, the lower the excitation elicited by stressful stimuli. Aggressive Behavior Measurement gg We measured aggressive behavior using a negative evaluation procedure similar to that used in other studies (Rothaus and Worchel, 1960; Wingrove and Bond, 1998; Twenge et al., 2001); negative evaluation has been recognized as a valid operationalization of aggressive behavior (Bettencourt et al., 2006). The experimenter told the participant that the peer who had assessed his or her video was applying for a job as the experimenter’s assistant. To apply, she needed an assessment from her peers, which the participant could provide. All participants agreed and completed a 10-items questionnaire about her suitability for the job (e.g., ‘You can rely on this person’; ‘This person is friendly’) using a 10-point Likert scale ranging from 1 (completely disagree) to 10 (completely agree). The scores were reverse coded before being summed so that the index would represent a negative evaluation. It should be noted that part of the data used in the second study described by Rajchert and Winiewski (2016) were also used in this study, namely index of aggression after rejection, although with reference to diﬀerent moderators of the rejection - aggression relationship. Procedure The ﬁrst part of the study was conducted during an introductory psychology class and presented as a separate activity (not connected to the later experimental procedure). The students completed the PTS and the BIS/BAS scale (Carver and White, Citation: SE is the individual’s ability to endure intense or long-lasting stimulation without passing into protective inhibition (apathy). The second propensity is deﬁned by the ease with which the nervous system creates conditional reactions (extinction, diﬀerentiation, and delay) and manifests when it is necessary to adapt a response to environmental requirements. Measurement of Strength of Excitation and Strength of Inhibition Participants completed the two scales from the Polish version of the Pavlovian Temperamental Survey (PTS; Strelau et al., 1999; Polish version by Strelau and Zawadzki, 1998) that measure SE and SI. Both scales consist of 19 items to which responses are given using a four-point Likert scale ranging from 1 (completely agree) to 4 (completely disagree). The original Polish version of the questionnaire has good internal validity (Cronbach’s alpha was 0.75 for SI and 0.85 for SE) and reliability. Ruch et al. (1991) showed that PTS variables correlated with the corresponding dimensions of Eysenck’s Personality Questionnaire (Eysenck et al., 1985): SE was positively correlated with extraversion and negatively correlated with neuroticism, whereas SI was negatively correlated with psychoticism, neuroticism and extraversion. These temperamental features, that is SE and SI, may inhibit aggressive reactions to rejection in two distinct ways. Endurance (arousability, SE) moderates the state of stress which occurs after rejection whereas inhibition (SI) gives an individual voluntary control over his or her aggressive urges. Developmental studies have shown that high arousability, which is theoretically related to low levels of SE and is also referred to as ineﬃcient involuntary or automatic control is positively associated with aggressive tendencies, aggression and conduct problems in children and adolescents. Whereas eﬀortful control, which is theoretically related to SI and encompasses processes such as attention, activation and inhibitory control is negatively related aggression and antisocial behavior (Capaldi and Rothbart, 1992; Kochanska, 1993; Rothbart et al., 1994; Derryberry and Rothbart, 1997; Eisenberg et al., 2000). In accordance to this, we propose that the temperamental traits SE and SI are independently negatively related to aggressive behavior following rejection. Manipulation of Rejection The procedure was inspired by Twenge et al. (2001), who showed that social exclusion caused people to retaliate aggressively toward those who had excluded them. Similar procedures have been used in other studies of exclusion (e.g., Leary et al., 1995; Nezlek et al., 1997). Participants were told that they had been randomly paired with another participant, a ‘peer’ (actually a female confederate) whom they would never meet in person, although they would get to know each other through a video interview. First, participants watched a video clip of the female confederate answering questions such as ‘What is your favorite food in the cafeteria?’ All participants watched the same pre-recorded video. The participants were asked to rate the interview and decide whether they would like to work with the person who had appeared in the video. After participants had done this and their ratings had been collected they were video- recorded answering similar questions. Their feedback and video was delivered to the female peer by the experimenter, who reassured participants that the peer would watch and rate their video before receiving the feedback they had given on her interview (we did not want participants to think that ratings made by the peer had been biased by reading their feedback). When the experimenter returned participants were told whether their ‘peer’ had liked their interview and wanted to work with them in the future. Participants were randomly assigned to experimental conditions. In the rejection condition (n = 56) the feedback was: ‘The interview was poor! I would not like to work with this person’ and in the acceptance condition (n = 61) it was ‘The interview was cool. I would like to work with this person.’ After this information the aggressive behavior was measured and all participants were debriefed. presented here are original and have not been presented elsewhere). SE was negatively related to aggression and men had higher SE scores. We used hierarchical multiple regression to investigate the impact of social rejection on aggressive behavior and the moderating role of Pavlovian temperamental features (SI and SE); the two interaction terms (experimental condition with SE and SI) were introduced in the second step (Table 2). Analyses showed that, after controlling for rejection - which was the strongest predictor of aggressive behavior - SE was negatively related to aggressive reaction. DISCUSSION Numerous studies have indicated that rejection or exclusion conditions cause more aggressive behavior than an acceptance condition (for review, see Leary et al., 2006; Baumeister et al., 2007), especially when rejected individuals have no chance of regaining their included status. Our study provided further conﬁrmation of this ﬁnding as rejected participants were more aggressive than accepted participants (see also Rajchert and Winiewski, 2016). Manipulation of Rejection The interaction terms were added in the next step, which showed that only the interaction of SE with rejection explained aggressive reaction. To explain the interaction we calculated a simple slope for high and low (±SD) values of SE. Analyses revealed that the eﬀect of rejection on aggression was high and negative for low values of SE (B = 3.47, SE = 0.43, p < 0.001) but was smaller at high values of SE (B = 1.69, SE = 0.42, p < 0.001) (see Figure 1). The interaction shows that in high SE participants, social exclusion prompted a less aggressive response toward a peer who had rejected them earlier than it did in low SE participants. Participants Data from 117 ﬁrst-year pedagogy and psychology students (102 women and 15 men), aged from 19 to 24 years (M = 19.5, February 2017 \| Volume 8 \| Article 296 Frontiers in Psychology \| www.frontiersin.org 2 Temperament and Rejection Effects on Aggression Rajchert and Winiewski TABLE 1 \| Zero-order correlations. 1 2 3 4 (1) Aggressive behavior (2) Strength of excitation (SE) −0.22∗ (3) Strength of inhibition (SI) −0.09 0.14 (4) Gender (0 – female) −10 0.21∗ −0.05 (5) Rejection (0 – acceptance) 0.61∗∗ 0.02 −0.13 −0.06a M 4.66 2.32 2.64 SD 2.09 0.45 0.38 Minimum 1 1.47 1.27 Maximum 10 3.58 3.47 Cronbach’s alpha 0.97 0.86 0.71 ∗p < 0.05, ∗∗p < 0.001; aPhi coefﬁcient. TABLE 1 \| Zero-order correlations. 1994). The results of analyses of BIS/BAS scales as moderators of the association between rejection and aggression were described by Rajchert and Winiewski (2016) and so they are not presented here. One week after they had completed the assessment of temperament the same students were individually invited to participate in a study that was ostensibly about ‘social networks and social media.’ The recruitment procedure of volunteers was the same as the procedure of questionnaires completion. All participants who completed the PTS scale agreed to take part in the ostensibly second study and signed separate informed consent. During the subsequent procedure we induced a feeling of rejection or acceptance and measured aggressive behavior. Frontiers in Psychology \| www.frontiersin.org RESULTS First, we calculated zero-order correlations (Table 1). Point- biserial correlations indicated that there was a strong association between rejection (experimental condition) and aggressive behavior. In the rejection condition, responses to the partner were substantially more aggressive (this is the only result described in Rajchert and Winiewski, 2016; all other results There are also studies showing that individual diﬀerences inﬂuence the magnitude of the aggressive response to rejection (Twenge and Campbell, 2003; Buckley et al., 2004; Ayduk et al., 2008; Rajchert, 2015; Rajchert et al., 2016). However, although the role of temperament in response to stress and coping behaviors is well-recognized, we are not aware of February 2017 \| Volume 8 \| Article 296 3 Temperament and Rejection Effects on Aggression Rajchert and Winiewski TABLE 2 \| Hierarchical multiple regressions models with Pavlovian temperamental dimensions as predictors of aggressive behavior. Step 1 Step 2 B SE B B SE B Constant 3.43∗∗∗ 0.22 3.45∗∗∗ 0.21 Rejection (0 – acceptance) 2.59∗∗∗ 0.30 2.60∗∗∗ 0.29 SE −1.07∗∗ 0.35 −0.05 0.49 SI 0.13 0.40 0.34 0.79 Gender (0 – female) −0.12 0.46 −0.23 0.44 SI × Rejection −0.31 1.02 SE × Rejection −1.84∗∗ 0.67 R2 0.43 0.48 F 21.23∗∗∗ 16.33∗∗∗ 1R2 0.04 1F 4.14∗ ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. FIGURE 1 \| Interaction effect of rejection and strength of excitation on aggression. Slopes are plotted at the ±1 SD. any research that has looked at the temperamental features low on SE. The exact mechanism through which SE reduces TABLE 2 \| Hierarchical multiple regressions models with Pavlovian temperamental dimensions as predictors of aggressive behavior. Step 1 Step 2 B SE B B SE B Constant 3.43∗∗∗ 0.22 3.45∗∗∗ 0.21 Rejection (0 – acceptance) 2.59∗∗∗ 0.30 2.60∗∗∗ 0.29 SE −1.07∗∗ 0.35 −0.05 0.49 SI 0.13 0.40 0.34 0.79 Gender (0 – female) −0.12 0.46 −0.23 0.44 SI × Rejection −0.31 1.02 SE × Rejection −1.84∗∗ 0.67 R2 0.43 0.48 F 21.23∗∗∗ 16.33∗∗∗ 1R2 0.04 1F 4.14∗ ressions models with Pavlovian temperamental dimensions as predictors of aggressive behavior. TABLE 2 \| Hierarchical multiple regressions models with Pavlovian temperamental dimensions as predictors of aggressive behavior. FIGURE 1 \| Interaction effect of rejection and strength of excitation on aggression. Slopes are plotted at the ±1 SD. FIGURE 1 \| Interaction effect of rejection and strength of excitation on aggression. Slopes are plotted at the ±1 SD. Frontiers in Psychology \| www.frontiersin.org RESULTS FIGURE 1 \| Interaction effect of rejection and strength of excitation on aggression. Slopes are plotted at the ±1 SD. low on SE. The exact mechanism through which SE reduces aggression remains to be determined. At this point we can only cite research on correlates of SE which showed that arousability, which is the foundation of SE, is also related to other lower-order temperamental traits such as emotional reactivity, positive and negative aﬀect, behavioral activation and inhibition systems, neuroticism and introversion (Strelau, 1994). Most of these features have, in turn, been related to the perceived intensity of stress and to coping responses (e.g., Watson et al., 1988; Heponiemi et al., 2003; Strelau and Zawadzki, 2005; Boyes and French, 2009). Our study adds to these results by showing that a common neurophysiologically and genetically grounded feature, namely arousability (manifested as SE) may underlie many of the ﬁndings linking personality variables with reductions in aggressive, retaliatory responses to rejection. any research that has looked at the temperamental features that moderate the rejection–aggression association other than recently published results from the same study which we discuss here (Rajchert and Winiewski, 2016) dealing with how BIS/BAS strength inﬂuences the rejection–aggression relationship. This research explored the role played by two temperamental traits, SE and SI, in the relationship between rejection and direct, retaliatory aggression. Higher central nervous system strength (SE) - manifested as the ability to function eﬀectively even during intense stimulation - contributed to lower aggression toward a partner after rejection. We also observed a main eﬀect of SE, with higher SE resulting in less aggression, when controlling for rejection. We conclude, like Chess and Thomas (1991) and Strelau (1995), that high SE participants either experienced the rejection episode as less stressful or were able to cope with the situation better than individuals February 2017 \| Volume 8 \| Article 296 Frontiers in Psychology \| www.frontiersin.org 4 Temperament and Rejection Effects on Aggression Rajchert and Winiewski We also predicted that high inhibition would restrain aggressive behavior after exclusion, but this prediction was not fulﬁlled. The most plausible explanation for this is that participants in our experiment perceived retaliatory aggression as justiﬁed and fair under the circumstances and therefore did not inhibit their aggression. SI represents the capacity to delay or refrain from action when it is considered appropriate to do so. It is not a blanket tendency to inhibit impulses. RESULTS In Pavlov’s theory and experiments, SI was deﬁned as the ease with which dogs learned conditional reactions and in this sense inhibition is observable only in particular situations. In our experiment the partner evaluated rejected individuals very harshly and wrongfully on the basis of scant evidence; it is therefore unsurprising that when given the opportunity, they reciprocated. Other research has shown that when individuals are convinced that rejection is unfair and undeserved it elicits a more intense aggressive reaction (Smart Richman and Leary, 2009). The second explanation is that participants’ negative evaluation of the target who had rejected them, although aggressive toward the target, could be perceived as prosocial in the sense that it potentially protected other people from being exposed to an experimenter with a tendency to unwarranted insulting behavior. The third explanation refers to the I3 theory. Denson et al. (2012) explained aggressive responses as the sum of the interaction of three components: self-regulatory strength (Inhibition), provocation strength (Instigation) and Impellance. This would imply that in our experiment the instigation to aggression was not fully counterbalanced fully by the inhibitory factors. 2001) it is open to criticism. Anderson and Bushman’s (2002) deﬁnition of aggression speciﬁes that the target of aggression must be motivated to avoid being hurt and the aggressor must be motivated to hurt the target and must be convinced that the intended behavior will hurt the target. It is conceivable that the peer evaluation might not be considered hurtful by the target and there are various motivations for giving a negative evaluation other than aggression (for example providing reliable feedback on the interaction partner to the future employer). In future studies other measures of aggression should be used to check whether the interaction eﬀect is robust. In summary, our study shows that although rejection contributes to aggressive behavior, the intensity of the aggression elicited by regression also depends on core temperamental traits. A very basic trait, the strength of nervous system, which, for example, determines an individual’s capacity to work eﬀectively in over-stimulating environments or dangerous situations, also inﬂuences how bearable rejection is. On the other hand, the a strong inhibitory capacity, which manifests in the covering of feelings and the ability to delay or refrain from initiating behavior, does not always diminish aggression. CONCLUSION The following limitations should be taken into consideration when interpreting the results of the study. First of all, the majority of the sample was female. Studies show that there are sex diﬀerences in direct, physical aggression in real-world settings (Archer, 2004). There has been much less research on the issue of sex diﬀerences in the context of laboratory studies of the rejection – aggression relationship, which measure mild forms of aggression. Other studies which have used similar rejection manipulations and measures of aggression did not even analyze sex diﬀerences (Twenge et al., 2001; Buckley et al., 2004). We controlled for the eﬀects of sex statistically, nevertheless our results should be replicated in a sample with a more even sex distribution, possibly not made up of college students. FUNDING Preparation of this manuscript was supported by Grant DEC- 2011/01/D/HS6/05486 form National Science Centre (Narodowe Centrum nauki). Another potential limitation is the use of peer evaluation as an index of aggression. Although this measure has been used in other studies of aggressive behavior (e.g., Twenge et al., ETHICS STATEMENT This study was carried out in accordance with the recommendations of the Academic Ethical Review Board (Scientiﬁc Research Ethics Committee of The Maria Grzegorzewska University). All subjects gave written informed consent in accordance with the Declaration of Helsinki. Archer, J. (2004). Sex diﬀerences in aggression in real-world settings: a meta- analytic review. Rev. Gen. Psychol. 8, 291–322. doi: 10.1037/1089-2680.8.4.291 Ayduk, Ö, Gyurak, A., and Luerssen, A. (2008). Individual diﬀerences in the rejection–aggression link in the hot sauce paradigm: the case of AUTHOR CONTRIBUTIONS JR prepared the manuscript (wrote introduction, method and discussion, formated and submitted), planned and conducted the research, interpreted the results. MW analyzed the data prepared the result section of the manuscript; he also took part in planning the content of manuscript parts and in interpretation of results. RESULTS In some instances, behaving aggressively might serve a higher cause or be regarded as appropriate, for example aggression intended to ‘teach someone a lesson’ or to protect others (Ferguson and Beaver, 2009). In such cases one would not necessarily observe an angry outburst of aggression related to low self-regulation; one might also (or instead) observe a cool, intentional act of aggression. Anderson, C. A., and Bushman, B. J. (2002). Human aggression. Annu. Rev. Psychol. 53, 27–51. doi: 10.1146/annurev.psych.53.100901.135231 REFERENCES Anderson, C. A., and Bushman, B. J. (2002). Human aggression. Annu. Rev. Psychol. 53, 27–51. doi: 10.1146/annurev.psych.53.100901.135231 Anderson, C. A., and Bushman, B. J. (2002). Human aggression. Annu. Rev. 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(1988). Development and validation of brief measures of positive and negative aﬀect: the PANAS scales. J. Pers. Soc. Psychol. 54, 1063–1070. doi: 10.1037/0022-3514.54.6.1063 Copyright © 2017 Rajchert and Winiewski. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. Wingrove, J., and Bond, A. J. (1998). Angry reactions to failure on a cooperative computer game. Aggress. Behav. 24, 27–36. doi: 10.1002/(SICI)1098-2337(1998) 24:1<27::AID-AB3>3.0.CO;2-P February 2017 \| Volume 8 \| Article 296 Frontiers in Psychology \| www.frontiersin.org 7
https://openalex.org/W2992408906	https://www.iiste.org/Journals/index.php/JEDS/article/download/50323/51980	English	null	HIV/AIDS Collaborative Governance and the Role of an Annual Retreat in Fostering Health Services: The Palm Beach County Experience	Journal of Economics and Sustainable Development	2,019	cc-by	13,899	1. Introduction The growing complexity of problems that demand public attention also necessitate joint engagement of stakeholders from various sectors. The collective engagement of stakeholders allows for creative solutions to complex problems. Collaborative governance which usually involves stakeholders from, at least, two sectors working together to address public concerns has become an acceptable approach for tackling problems in the public sphere. Such levels of engagement whether for health, environment, law enforcement, and social services among others (Freeman 1997; Choi and Robertson 2014; Daniel, Pinel, & Brooks 2013; Metze and Levelt 2012; Agbodzakey 2015) enable collective decision-making, the pooling of resources, and commitment that foster sustainable solutions. One such complex public problem requiring collaborative governance is the Human Immunodeficiency Virus (HIV) and Acquired Immune Deficiency Syndrome (AIDS) hereafter referred to as HIV/AIDS. For decades, HIV/AIDS has been and is still a complex problem with devastating impacts on various segments of the population. The complexity of the HIV/AIDS problem continues despite medical advances to curb its impact. It is currently estimated that globally, about 36.7 million people are infected and living with HIV/AIDS and of this amount, an estimated 1.1 million people are living in the United States (UNAIDS, 2017; CDC, 2017). The gravity of the devastating impacts on individuals and communities call for innovative approaches to tackle the epidemic and associated challenges. In the U.S., the enactment of the Ryan White Comprehensive AIDS Resources Emergency Act (hereafter referred to as the CARE Act) in 1990 marked a political acknowledgement of the epidemic and the commitment of resources to areas that have been disproportionally impacted by the epidemic. In fact, the enactment of the CARE Act that was somewhat necessitated by fierce advocacy work, the glaring loss of lives, and a change in the national mood, demonstrated more willingness of politicians to deal with the HIV/AIDS epidemic. As part of the implementation strategy, the CARE Act mandates establishment or designation of HIV Health Services Planning Councils to provide care and treatment in areas that are disproportionately impacted [Eligible Metropolitan Areas (EMAs)]. For instance, the CARE Act states, “to be eligible for assistance…, the chief elected official described in subsection (a) (l) shall establish or designate an HIV health services planning council…” and identifies key stakeholders to be part of the Council (CARE Act, 1990, Sec. 2602). www.iiste.org www.iiste.org Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 www.iiste.org www.iiste.org HIV/AIDS Collaborative Governance and the Role of an Annual Retreat in Fostering Health Services: The Palm Beach County Experience James K. Agbodzakey1* Nicholas Bolden2 Sandra Schrouder3 James K. Agbodzakey1* Nicholas Bolden2 Sandra Schrouder3 1.SERCH/Public Leadership, University of North Texas at Dallas, Dallas, TX, U.S.A. 2.Department of Politics, Philosophy, and Public Administration, Columbus State University, Columbus, GA, U.S.A. 3.Sandra Schrouder, School of Professional and Career Education (PACE), Barry University, Miami Shores, FL, U.S.A. James K. Agbodzakey1* Nicholas Bolden2 Sandra Schrouder3 1.SERCH/Public Leadership, University of North Texas at Dallas, Dallas, TX, U.S.A. ndra Schrouder, School of Professional and Career Education (PACE), Barry University, Miami Shores, F U.S.A. Abstract The complex HIV/AIDS conundrum calls for innovative approaches to tackle associated challenges. The multifaceted nature of the problem and the enormous burden on various sectors of a country’s economy warrant a cross-sector stakeholders’ engagement for effective and sustainable solutions. This research examines the use of an annual retreat involving interviews of key stakeholders involved in health services CARE Council as an avenue for collaborative governance within a local system context in the fight against the epidemic. The results highlight how inclusive and strategic preparation can result in judicious allocation priorities for various service categories. The results also convey how relevant capacities and competencies are jointly developed through creative inputs, outputs and/or outcomes to target populations. The apparent role of facilitative leadership and the embrace of cultural humility promote mutual commitment among the stakeholders toward purposeful ends. The case represents the viability of collaborative governance as a feasible postmodern alternative to mainstream governance and service delivery to various publics, especially in this contemporary era of complexity and volatility. Keywords: Health Services, HIV/AIDS, Annual Retreat, Target Populations, CARE Act, Collaborative Governance y p , p y p y p y y Keywords: Health Services, HIV/AIDS, Annual Retreat, Target Populations, CARE Act, Collaborative Governance. DOI: 10.7176/JESD/10-22-08 DOI: 10.7176/JESD/10-22-08 Publication date: November 30th 2019 Publication date: November 30th 2019 1. Introduction The councils at the various EMAs are thereby tasked to make allocation priorities to various service categories for those infected and affected through collaborative engagement of cross-sector stakeholders. The efforts of the councils at the various local 64 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 www.iiste.org levels, collectively represent U.S. government’s commitment to address the HIV/AIDS conundrum within its borders and territories even as the country is equally involved in helping address the epidemic at the global level. levels, collectively represent U.S. government’s commitment to address the HIV/AIDS conundrum within its borders and territories even as the country is equally involved in helping address the epidemic at the global level. Collaborative governance has thus become a commonly utilized approach by health services planning councils in the U.S. as part of the implementation of the CARE Act to provide care and treatment to target populations. Collaborative governance thereby brings together stakeholders who are mostly service providers, non-elected community leaders, and HIV/AIDS infected and affected individuals as council members to effectuate the intents and purpose of the CARE Act. Currently, the twenty-four (24) EMAs in the U.S. and its territories utilize collaborative governance to promote care and treatment of target populations. g p g p p The council in each EMA utilizes leadership and institutional design mechanisms that help fulfil its purpose. Usually, such arrangements include various committees, workshops, retreats, and other training programs to foster effective performance. For instance, workshops and other training programs are geared toward building council members’ capacity in various areas on a regular basis. A retreat is usually a whole-day annual gathering of council members and other relevant stakeholders. This has become an essential forum to engage various stakeholders to promote the work of the councils. This research will focus on one of such councils, Palm Beach HIV CARE Council (hereafter referred to as CARE Council), particularly, its use of an annual retreat as an avenue to foster collaborative governance. The CARE Council has consistently utilized retreats since the early 1990s to assemble various stakeholders to discuss various themes and issues pertinent to the overall collaborative governance efforts, more so than some of the other councils, and is duly recognized for its creative approach to the annual retreat (Agbodzakey, 2015). 2. Conceptualizing Collaborative Governance Collaborative governance as an emergent approach to collective problem solving has become an acceptable governing arrangement in the public sector at various levels. The opportunity that collaborative governance provides to engage stakeholders from public, private and nonprofit/civic sectors to collectively work together to address a common problem makes it very appealing (Emerson & Nabatchi, 2015; Gray, 1989). For instance, the use of collaborative governance by HIV Health Services Planning Councils in Eligible Metropolitan Areas (EMAs) in the U.S. is a viable strategy for care and treatment of target populations (Agbodzakey, 2017). The viability of collaborative governance as a framework for collective problem solving thereby demands some conceptual explication to help relate to its core elements from theory, policy and practical perspectives. Many scholars explain the concept of collaborative governance in ways to promote knowledge and understanding of what it is and how it works. The next segment of the paper highlights the various conceptual explanations of collaborative governance; its essence, benefits and challenges. g Research by Barbara Gray (1989) serves as a seminal work on collaborative governance. The extent to which she influenced scholarship on collaborative governance is evidenced by the numerous references to her work by other scholars. According to Gray (1989), collaborative governance “is a process through which parties who see different aspects of a problem can constructively explore their differences and search for solutions that go beyond their own limited vision of what is possible” (p.5). Gray’s conceptualization highlights the intentional engagement of stakeholders across various sectors to fulfil a common purpose which in this case is geared toward finding workable solutions to a problem that bedevil multiple stakeholders and to some extent society at large. The nature of the problem and the urgency for collective action by all affected and/or caring parties render sectional interests secondary as finding creative solutions to the problem. Furthermore, Gray’s conceptual explanation reveals how the process of engagement by the various stakeholders in collaborative governance can set the stage for constructive deliberations that foster effective output and/or outcomes for the greater good. The perspective of Bryson, Crosby, and Stone (2006) on collaborative governance is very related to that of Gray (1989) and further underscores the collective stakeholder engagements in collaborative arrangements. In their publication on collaborative governance which is otherwise termed cross-sector collaboration, Bryson et al. 1. Introduction As an avenue for collaborative governance, the annual retreat is important to the CARE Council’s efforts to realize legislative intents as it relates to care and treatment of target populations. To achieve the objectives of the study, the paper will be guided by this research question: How does the Palm Beach County’s HIV CARE Council annual retreat enables HIV/AIDS collaborative governance? The research question seeks to explore how Palm Beach County’s HIV CARE Council’s annual retreat enables HIV/AIDS collaborative governance. The paper will highlight how the retreat enables collaborative governance of the CARE Council by relying on data garnered through observations, interviews, and review of documents. The analysis and discussion of the evidence will help draw conclusions and implications for collaborative governance in a local system context as part of efforts to collectively address the HIV/AIDS problem. The remaining sections of the paper are organized into six (6) segments. These include conceptualizing collaborative governance, excerpts on the CARE Council and the annual retreat, methodology, analysis and discussion, impact, conclusion and implication. 2. Conceptualizing Collaborative Governance (2006) define the concept “as the linking or sharing of information, resources, activities, and capabilities by organizations in two or more sectors to achieve jointly an outcome that could not be achieved by organizations in 65 Journal of Economics and Sustainable Development www.iiste.org ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 one sector separately” (p. 44). This definition points to the obvious realization of the advantages of joint efforts of stakeholders from various sectors in finding effective solutions to a common problem. By pooling resources and capabilities these cross-sector stakeholders jointly create an opportunity for workable and lasting solutions that maximizes benefits to entities and society as a whole. Such level of cross-sector collaboration when appropriately designed has the likelihood to create sustainable solutions to various problems/challenges. Other scholars such as Thomson and Perry (2006), Zurba (2014), Emerson, Nabatchi & Balogh (2012), Ansell and Gash (2008), Huxham and Vangen (2005), Ring and Van de Ven (1994), Johnston, Hicks, Nan, & Auer (2011), Healy (1997), Beierle (2000), Rene, & Tharsi (2004), Provan and Kenis (2008), and Sirianni (2009) share their perspectives on collaborative governance in relation to finding solutions to common and complex problems. l ( ) ll b i i f / l Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 www.iiste.org one sector separately” (p. 44). This definition points to the obvious realization of the advantages of joint efforts of stakeholders from various sectors in finding effective solutions to a common problem. By pooling resources and capabilities these cross-sector stakeholders jointly create an opportunity for workable and lasting solutions that maximizes benefits to entities and society as a whole. Such level of cross-sector collaboration when appropriately designed has the likelihood to create sustainable solutions to various problems/challenges. Other scholars such as Thomson and Perry (2006), Zurba (2014), Emerson, Nabatchi & Balogh (2012), Ansell and Gash (2008), Huxham and Vangen (2005), Ring and Van de Ven (1994), Johnston, Hicks, Nan, & Auer (2011), Healy (1997), Beierle (2000), Rene, & Tharsi (2004), Provan and Kenis (2008), and Sirianni (2009) share their perspectives on collaborative governance in relation to finding solutions to common and complex problems. For Emerson et al. (2012), collaborative governance accentuate constructive engagement of actors/players from various public agencies, civic, nonprofit and private entities with established framework to collectively and responsively fulfil a public purpose. 2. Conceptualizing Collaborative Governance Thus, the joint efforts of cross-sector stakeholders in collaborative governance enable the addressing of a public concern which otherwise could remain unresolved if the stakeholders elect to act alone. The shared responsibility on the part of the stakeholders from cross sectors is guided by a shared understanding of the gravity of a public situation or problem and the need to act collectively create synergy for success. In fact, the exposition by Emerson et al. (2012) of collaborative governance reflects the perspective of Ansell and Gash’s (2008) work which highlights multi-stakeholder engagements for decision making and/or implementation. This approach sees communication, shared understanding, commitment, consensus, leadership, and design mechanisms as essential variables that enable outputs and/or outcomes that promote problem solving for societal benefits. The stakeholders in this case are united by a common resolve to collectively address a problem whether mandated or not, despite sectional differences that they may have prior to working together or even during the course of their collective efforts. Relatedly, Agbodzakey (2017) explication of collaborative governance as a joint undertaking by cross-sector stakeholders in making and/or implementing decisions as part of efforts to address a complex problem, further points to how a glaring challenge unites stakeholders for collective action. Specifically, the reference to HIV/AIDS collaborative governance experience of Health Services Planning Councils convey understanding of how representation and participation of relevant stakeholders in an inclusive process enable allocation priorities for care and treatment of those infected and affected by the epidemic. While the Councils’ experience is within the context of mandated collaboration, the process of engagement, critical variables, and attendant outputs bear semblance to collaborative governance in dealing with any other complex problem regardless of the context. g g y p p g The various conceptual explication of collaborative governance as referenced above provide an insight into how complex problems bring together stakeholders from various sectors to work assiduously to address the problem. By coming together and by establishing mechanisms for engagement, collaborative governance promotes creative and sustainable solutions with a high probability that the stakeholders will own the likely outcomes of their joint efforts. Furthermore, the conceptual expositions reveal that while the stakeholders may have obvious divergent interests, differences, and persuasions, the willingness to act collectively to address a complex problem for the most part helps dissuade unnecessary antagonism that could derail joint efforts. 2. Conceptualizing Collaborative Governance As an emergent viable approach, collaborative governance is now commonly used at the local, state, national and international levels to tackle complex issues or concerns. Similarly, scholarly work on collaborative governance is on the ascendancy. While there is a general understanding of collaborative governance as a multi- stakeholder engagement across sectors for collective problem solving, scholars in their respective capacities sometimes frame the concept differently. Terms such as collaborative management, interagency coordination, participatory management, public-private partnerships, deliberative democracy, deliberative governance, participatory governance, interactive policy making, stakeholder governance, collaborative democracy, collaborative innovation, and network governance are used to connote collaborative governance (Gray, 1989; Ansell & Gash, 2008; Anonymous, 2006; Sørensen & Torfing, 2011; McGuire, 2006; Bryson et al., 2006; 2015; Sirianni, 2009; Sørensen & Torfing, 2011; Provan & Kenis, 2008; Agbodzakey, 2012). This study uses the term collaborative governance to indicate joint efforts of stakeholders across sectors to help address the complex HIV/AIDS problem with a particular focus on the experience of HIV CARE Council in Palm Beach County. As a concept, collaborative governance has governance as a key component and scholars such as Ansell and Gash (2008); Scott and Thomas (2017) explain the nexus and variation between both terms. Governance in this context pertains to the exercise of power, authority, laws, rules, practices as it relates to delivery of public goods and services and/or conduct of public affairs in general, in either democratic or non-democratic context (Michalski, Miller & Stevens, 2001; Fukuyama, 2013; Lynn, Heinrich & Hill, 2000). Collaborative governance is a unique arrangement by stakeholders across sectors to address a specific complex problem, with the hope of finding sustainable solutions (Gray, 1989; Ansell & Gash, 2008; Emerson et al., 2012). Whether mandated or not, collaborative governance promotes joint actions of various stakeholders from different spheres unlike governance which remains in the domain of the political sphere and can be exercised without direct participation of stakeholders from other sectors. One other issue relative to collaborative governance that is worth pointing out is its relatedness and variation to concepts such as networks, cooperation, and coordination. Networks which could require multi-stakeholder engagement does not need to have a complex problem, regular face-to-face interactions, and broad participation like collaborative governance (Agbodzakey, 2012; Freeman, 1997; McGuire, 2006). 2. Conceptualizing Collaborative Governance In fact, scholars identify likely benefits and challenges of collaborative governance which helps to highlight the realities of multi- stakeholder engagements for collective problem solving. The notable benefits of collaborative governance as enumerated by scholars include collective resolve and responsibility to tackle common challenges, creative and sustainable solutions to problems, achieve economies of scale, collective resource pool to promote effectiveness, jointly structured design protocols of engagement, regular joint capacity development efforts for collective action, responsive representation and participation of relevant community and organizational stakeholders, promotion of deliberative decision making, communication, shared understanding, consensus, and legitimacy among stakeholders, and joint ownership of outputs and/or outcomes for the common good (Ansell & Gash, 2008; Emerson et al., 2012; Bryson et al., 2015; Scott & Thomas, 2017; Sabatier et al., 2005; Choi & Robertson 2014; Sirianni 2009; Agbodzakey, 2012; Susskind & Cruikshank, 1987; McGuire, 2006; Donahue & Zeckhauser, 2011; Bel & Warner, 2008 among others). On the other hand, observations regarding notable challenges of collaborative governance include the following: power differentiation and utilization, time and other resource constraints, turf battles, intentional holdout by some stakeholders, uncertain outcomes, occasional conflicts and ambiguity, delayed decisions, and succession challenges (Memon & Kirk 2010; Hageman & Bogue 1998; Scott & Thomas, 2017; Agbodzakey, 2015; Metze & Levelt 2012; Sabatier et al., 2005; Margerum, 2011; Emerson et al. 2012; Booher, 2004; Berardo, Heikkila, & Gerlak 2014; Vangen & Huxham 2012). ) The enumerated benefits and challenges above point to the realities of collaborative governance in addressing complex societal problems. Nonetheless, the opportunity to bring various stakeholders from different sectors to jointly make decisions and/or implement decisions, make collaborative governance more appealing compared to adversarial management as it offers better prospects for viable solutions (Freeman, 1997; Ansell & Gash, 2008; Gray, 1989; McGuire, 2006; Bingham and O’Leary, 2008; Healey, 1997). The next few paragraphs will highlight 66 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 iste.org related and interchangeable terms associated with collaborative governance, the concept of governance and its connectedness to collaborative governance and how networks, cooperation, and coordination are related but different from collaborative governance. related and interchangeable terms associated with collaborative governance, the concept of governance and its connectedness to collaborative governance and how networks, cooperation, and coordination are related but different from collaborative governance. 2. Conceptualizing Collaborative Governance Similarly, cooperation that involves different stakeholders working toward purposeful ends, and coordination which requires established protocols of engagement for joint efforts are less intense and less inclusive when compared with collaborative governance (Margerum, 2011; Bertelli & Smith, 2010). In essence, collaborative governance promotes cooperation and coordination among stakeholders in the performance of various joint endeavours (Scott & Thomas, 2017; Gerlak, Lubell, & Heikkila, 2012; Karkkainen, 2002). In the case of the Palm Beach County’s HIV CARE Council for care and treatment of HIV/AIDS infected and affected persons, collaborative governance has been used for decades. The joint efforts of target populations, service providers, and non-elected community leaders with support from county, state and federal governments enabled allocation of resources for various service categories as part of the implementation of the CARE Act. The use of annual retreats as a point of convergence, deliberation, education, and strategic planning complement the Council’s yearlong multi-stakeholder engagements to enable various category of services to target populations. While studies have been done on collaborative governance in various settings, the use of retreat as an avenue for collaboration and the attendant contributions has not been a focus in the scholarly literature. This study will thereby highlight the case of CARE Council’s use of an annual retreat as a component of collaborative governance and will draw some implications for policy, research, and practice. 3. Retreat as an Avenue for Collaborative Governance This regular annual event extends representation and participation in collaborative governance beyond the established three (3) core groups of the CARE Act and local policies and procedures (CARE Council Bylaws, 2005; CARE Act, 1990), and enables perspectives of various stakeholders for creative solutions for care and treatment. To some extent, the retreat exemplifies an inclusive participation forum of cross-sector stakeholders in collaborative governance. The retreat provides an opportunity for interaction among various stakeholders in seven-to-eight (7-8) hour time frame and consists of multiple sessions. This once-in-a-year event provides a unique opportunity to some stakeholders to make meaningful contributions to the CARE Council’s overall efforts in each grant year. In the past few years, some of the themes addressed at the annual retreat include cultural competency, role of advocacy on HIV/AIDS, collaborative engagement and collaborative leadership, care and treatment regimes, continuum of care, system of care, future of the CARE Act, pertinent emergent issues on HIV/AIDS, code of ethics on health services, role of the Council in the fight against HIV/AIDS, building collaborative capacity for care and treatment, and health systems challenges (CARE Council, 2016; CARE Council Comprehensive Plan, 2012). At the retreat, professionals and/or experts facilitate discussions and group activities among the stakeholders to help formulate strategies for the way forward for pertinent care and treatment services. The strategies are further developed and acted on by the CARE Council. Thus, the retreat outputs are discussed by the various committees of the CARE Council with specific assigned tasks to committees to enhance care and treatment efforts. The retreat efforts usually set the stage for the rest of CARE Council’s undertakings in each grant year. To some extent, the annual retreat of the CARE Council helps promote outputs based on inputs from cross- sector stakeholders. By bringing together various stakeholders including council members, agency officials, community advocates, key individuals and other relevant stakeholders in a structured, but flexible forum of engagement, encourages dialogue and formulation of workable solutions for the benefits of target populations. The benefits emanating from the annual retreat would fall into both process, and output/outcome categories, and thereby help relate the extant literature suppositions on collaborative engagement in addressing complex problems (Carlson, 2007; Fung 2006; Bryson et al., 2006). 3. Retreat as an Avenue for Collaborative Governance This regular annual event extends representation and participation in collaborative governance beyond the established three (3) core groups of the CARE Act and local policies and procedures (CARE Council Bylaws, 2005; CARE Act, 1990), and enables perspectives of various stakeholders for creative solutions for care and treatment. To some extent, the retreat exemplifies an inclusive participation forum Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 Vol.10, No.22, 2019 realization of the intents and purposes of the CARE Council. Furthermore, the CARE Council occasionally create ad hoc committees as needed to attend to pertinent issues to enhance performance of its roles and responsibilities. For instance, the CARE Council sometimes uses an ad hoc committee to assist in the planning of the annual retreat. The annual retreat of the CARE Council serves as a viable avenue for collaborative governance As a forum realization of the intents and purposes of the CARE Council. Furthermore, the CARE Council occasionally create ad hoc committees as needed to attend to pertinent issues to enhance performance of its roles and responsibilities. For instance, the CARE Council sometimes uses an ad hoc committee to assist in the planning of the annual retreat. realization of the intents and purposes of the CARE Council. Furthermore, the CARE Council occasionally create ad hoc committees as needed to attend to pertinent issues to enhance performance of its roles and responsibilities. For instance, the CARE Council sometimes uses an ad hoc committee to assist in the planning of the annual retreat. The annual retreat of the CARE Council serves as a viable avenue for collaborative governance. As a forum for inclusive representation, education, deliberation, and strategic planning, the retreat provides a unique space for divergent and convergent thinking on various issues pertinent to care and treatment of target populations in the county. The consistency with which the CARE Council and other key stakeholders organize and execute the retreat over the years attests to its unique contributions to health services collaborative governance for care and treatment of target populations. The retreat represents one of the key aspects of ensuring participation in collaborative governance at an Eligible Metropolitan Area (EMA). 4. Methodology gy This study uses a case study approach to examine the phenomenon of an annual retreat by the Palm Beach County’s HIV CARE Council (CARE Council) as part of collaborative governance for care and treatment of target populations. For decades, the CARE Council has successfully organized the annual retreat to generate relevant outputs and/or outcomes to effectively complement care and treatment efforts through collaborative engagement of various stakeholders. The focus on the annual retreat helps to ascertain the nature of multi-stakeholder engagement for collective problem solving. Many scholars highlight how the use of a case study approach to examine phenomenon of interest enable discoveries (Yin, 2003; Stake, 1998; Bennett & George, 2005 Gerring, 2004; Thomas, 2003; Hamel et al., 1998; Jensen & Rogers, 2001; Baxter & Jack, 2008; Ospina & Dodge2005). The contributions of case study to scholarly inquiry in public administration cannot be overemphasized. Stake (1998) points out how case study enables using multiple ways to obtain answers in relation to a phenomenon of interest; Yin (2003), Thomas, (2003), and Hamel et al. (1993) underscore answers to ‘how’ and ‘why’ questions because of a case study; and Jensen and Rogers (2001) accentuate uniqueness of conditional findings and deciphering of cause and effect relationships through case study, which can be hardly achieved when using other methodologies. Generally, case study promotes understanding of phenomenon and theory building through essential discoveries (Bennett & George, 2005; Saldana & Omasta, 2017). This study uses a case study approach to examine the phenomenon of an annual retreat by the Palm Beach County’s HIV CARE Council (CARE Council) as part of collaborative governance for care and treatment of target populations. For decades, the CARE Council has successfully organized the annual retreat to generate relevant outputs and/or outcomes to effectively complement care and treatment efforts through collaborative engagement of various stakeholders. The focus on the annual retreat helps to ascertain the nature of multi-stakeholder engagement for collective problem solving. Many scholars highlight how the use of a case study approach to examine phenomenon of interest enable discoveries (Yin, 2003; Stake, 1998; Bennett & George, 2005 Gerring, 2004; Thomas, 2003; Hamel et al., 1998; Jensen & Rogers, 2001; Baxter & Jack, 2008; Ospina & Dodge2005). The contributions of case study to scholarly inquiry in public administration cannot be overemphasized. 3. Retreat as an Avenue for Collaborative Governance This study which aims to examine the role of an annual retreat to CARE Council’s collaborative governance will rely on data from interviews, observations, and review of documents to draw some insightful conclusions in an attempt to provide possible answers to the question: how does Palm Beach County’s HIV CARE Council’s annual retreat enables HIV/AIDS collaborative governance? The next section of this paper will focus on methods used in the data collection and will be followed by analysis and discussion of findings, conclusions, and implications. 3. Retreat as an Avenue for Collaborative Governance The Palm Beach County’s HIV CARE Council (hereafter referred to as the CARE Council) is the health services collaborative governance entity dedicated to care and treatment of HIV/AIDS infected and affected persons in the county. The CARE Council is one of twenty-four (24) health services councils in the U.S. with a mandate to provide category of services for the target populations. Thus, as an Eligible Metropolitan Areas (EMA), the county established the CARE Council in the early 1990s to make decisions and allocate priorities as part of the implementation of the Ryan White Comprehensive AIDS Resources Emergency Care Act (CARE Act, 1990). Eligible Metropolitan Areas (EMAs) are areas disproportionately impacted by HIV/AIDS and are eligible for federal funds to provide care and treatment to those in need of service (CARE Act, 1990). As part of the use of collaborative governance for collective problem solving, the CARE Council has various committees that enable the performance of its various roles and responsibilities. These committees are executive committee, planning committee, priorities and allocation committee, membership committee, support services committee, medical services committee, community awareness committee, and quality assurance and evaluations committee (CARE Council Bylaws, 2013, p.9). Each of these committees perform unique functions that promote 67 Journal of Economics and Sustainable Development www.iiste.org ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 realization of the intents and purposes of the CARE Council. Furthermore, the CARE Council occasionally create ad hoc committees as needed to attend to pertinent issues to enhance performance of its roles and responsibilities. For instance, the CARE Council sometimes uses an ad hoc committee to assist in the planning of the annual retreat. The annual retreat of the CARE Council serves as a viable avenue for collaborative governance. As a forum for inclusive representation, education, deliberation, and strategic planning, the retreat provides a unique space for divergent and convergent thinking on various issues pertinent to care and treatment of target populations in the county. The consistency with which the CARE Council and other key stakeholders organize and execute the retreat over the years attests to its unique contributions to health services collaborative governance for care and treatment of target populations. The retreat represents one of the key aspects of ensuring participation in collaborative governance at an Eligible Metropolitan Area (EMA). 4. Methodology Stake (1998) points out how case study enables using multiple ways to obtain answers in relation to a phenomenon of interest; Yin (2003), Thomas, (2003), and Hamel et al. (1993) underscore answers to ‘how’ and ‘why’ questions because of a case study; and Jensen and Rogers (2001) accentuate uniqueness of conditional findings and deciphering of cause and effect relationships through case study, which can be hardly achieved when using other methodologies. Generally, case study promotes understanding of phenomenon and theory building through essential discoveries (Bennett & George, 2005; Saldana & Omasta, 2017). This case study relies on interview of CARE Council members, the Grantee and other key stakeh collaborative governance and is triangulated with review of documents and observations. The participan 68 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 www.iiste.org unique roles at the Council, agencies and communities in Palm Beach County. Most of the participants have participated in the annual retreat for at least a decade and perceived their participation as an enriching experience. These participants’ perspectives are essential to knowledge and understanding of the use of an annual retreat as an avenue to foster performance of CARE Council’s roles and responsibilities through strategic engagement and strategic planning involving relevant cross-sector stakeholders. There were a total of ten (10) in-depth interviews conducted by the authors from June 2016 to May 2017. The participants were mostly CARE Council and committee members. Six of the participants were female and four were male, and on average, each participant has been part of the Council for at least ten years. Each participant was asked a series of questions that revolved around the annual retreat and its role to the CARE Council’s collaborative governance. The questions were semi-structured and presented each participant an opportunity to provide additional useful details to help achieve the objectives of the study. The interviews were conducted at the convenience of the participants, and relevant research protocols were followed to promote confidentiality and the integrity of the study (DiCicco-Bloom & Crabtree, 2006). The in-depth nature of the interviews (at least, an hour and half per each interview session) enabled rich conversations with participants which provided needed data on the participants’ retreat experiences and thereby created meaning as expected of qualitative interviews (Yin, 1984; Rubin & Rubin, 1995; Hostein & Gubrium, 1999; Saldaña & Omasta, 2017). 4. Methodology In particular, the interviews provided useful information regarding how participation, representation, and outputs of the annual retreat complement the work of the Council as it relates to making various decisions and allocation priorities for various service categories to promote health and general wellbeing of the target populations. p g g g p p In an attempt to construct a narrative that better represents the annual retreat experience of the participants, data from the interviews are triangulated with observation of various CARE Council annual retreats by the authors, at least 6 within a period of 10 years (2009; 2014; 2015; 2016; 2018; & 2019). The authors closely observed the various presentations and attendant discussions among stakeholders, stakeholder participation in the various group activities, facilitation of group activities, interactions at various tables and other activities aimed at effectively engaging participants. Furthermore, documents related to the annual retreat such as the agenda, group activity reports, and presentation handouts were reviewed for additional insights. Scholars such as Greene et al. (1989), Flick (2004), and Jick (1979) articulate how the use of different methods enable generation of data that complement and help triangulate perspectives on the issue or phenomenon of study. For instance, Salkind (2009) highlights how observation of a study’s context and participants help create meaning about the phenomenon of interest. For the analysis and reporting of findings, the study will mostly utilize interview data with complementary themes from review of documents and authors’ observation notes. Overall, the data and analysis which involve coding, developing themes and categories, and integration would promote knowledge and understanding of the role that the CARE Council’s annual retreat plays in collaborative governance to address the HIV/AIDS conundrum in the county. 5. Analysis and Discussion The rationale for this research is to ascertain the role of an annual retreat as a key aspect of HIV/AIDS collaborative governance in South Florida, specifically in Palm Beach County. The annual retreat serves as a strategic forum of engagement involving various key stakeholders in formulating strategies to help provide needed care and treatment to HIV/AIDS infected and affected persons. The extent of participation by various groups including service providers, non-elected community leaders, target populations, policy entrepreneurs, key individuals, other relevant publics, private and nonprofit entities among others, promotes robust discussions, deliberations and measures in a way that complement the work of the CARE Council. Data that was collected through interviews, observations, and review of documents were coded and developed into themes, categories and concepts in compliance with qualitative research expectations (Saldaña & Omasta, 2017; Hostein & Gubrium, 1999; Yin 2003; DiCicco-Bloom & Crabtree, 2006). The analysis thereby represents synthesis of the derived categories, concepts and themes that emerged from the data which helps to provide insights into the role of an annual retreat to CARE Council’s collaborative governance. Thus, the following segments of the paper highlights themes such as strategic preparation, inclusive participation, retreat as an educational moment, evolution of the retreat, and cultural humility in a quest to decipher retreat’s strategic impact on CARE Council’s collaborative governance as it relates to care and treatment of HIV/AIDS infected and affected populations. These themes would help answer the research question: how does Palm Beach County’s HIV CARE Council’s annual retreat enables HIV/AIDS collaborative governance? The expectation is that answers to the question would promote knowledge and understanding of the role of an annual retreat as a key forum for collaborative governance in the fight against HIV/AIDS. 5.1 Strategic Preparation Preparation is key to effective engagement, especially if it is well thought out and involves relevant stakeholders in the planning process for a forum such as an annual retreat. The process of soliciting insights and participation of critical stakeholders through a series of face-to-face deliberations and other mediums of information exchange 69 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 help to generate an agenda that enables realization of goals and objectives of the retreat. Scholars such as Bryson et al. (2006), Ansell and Gash (2008), Crosby and Bryson (2012), Quick and Feldman (2014), Bryson et al. 5.2 Inclusive participation Agencies that know about AIDS also participate. Leaders and politicians, bureaucrats etc. are occasionally part of it. Key staff also attend. Participants provide better perspectives on issues including sharing perspective on HIV/AIDS services with providers and non-providers of HIV/AIDS services. Clients who are not members also get to learn what is being done, learn about services and what they can do to contribute to the process. The retreat is inclusive not exclusive as all relevant parties are invited each year. This response highlights the extent of stakeholders’ engagement at the annual retreat and the diverse perspectives that are shared to help formulate care and treatment strategies. Observation of various retreats to a large extend attests to their inclusive and integrated nature. The respondent’s assertion on inclusiveness points to openness in including other groups in the retreat as emergent and needed for effective collective action on HIV/AIDS. 5.3 Evolution of the retreat 5.2 Inclusive participation Inclusive participation in collaborative governance, particularly as it relates to engagements at a forum such as an annual retreat is likely to promote creative ideas, commitment, and sustainable solutions to a complex problem which in this case is the HIV/AIDS conundrum. Bringing together stakeholders from public, private, civic/nonprofit sectors who are directly or indirectly involved in the provision of health services in a collaborative process encourages formulation of strategies which could translate into allocation priorities for various service categories. The literature on inclusive participation in collaborative governance highlights how stakeholders build and draw on their collective strengths to navigate challenges and generate workable solutions in collective problem solving (Huxham and Vangen 2005; Bryson et al., 2015; Berardo, Heikkila, and Gerlak 2014; Saz-Carranza and Ospina 2011; Cheng & Daniels, 2005; Ansell & Gash, 2008; Emerson et al., 2015; Margerum, 2011) p g g ) Responses by participants to questions gauging the extent of participation suggest organizers are intentional in reaching out to various relevant stakeholders i.e. groups, individuals, including consumers of services, non- Ryan White service providers, CARE Council members, and other interested parties. Furthermore, the responses seem to suggest an inclusive retreat promotes formulation of strategies and solutions for care and treatment, encourages open conversation and divergent viewpoints among stakeholders, serves as a reunion among stakeholders to renew their commitment to the fight against HIV/AIDS, fosters teamwork, and enhances understanding of the CARE Council’s roles and responsibilities, achievements and challenges. The response below by one participant epitomizes perspectives on inclusive participation in the annual retreat: g p g by one participant epitomizes perspectives on inclusive participation in the annual retreat: Agencies that do not know much about AIDS but provide services participate to increase awareness and could join CARE Council as a result. Agencies that know about AIDS also participate. Leaders and politicians, bureaucrats etc. are occasionally part of it. Key staff also attend. Participants provide better perspectives on issues including sharing perspective on HIV/AIDS services with providers and non-providers of HIV/AIDS services. Clients who are not members also get to learn what is being done, learn about services and what they can do to contribute to the process. The retreat is inclusive not exclusive as all relevant parties are invited each year. Agencies that do not know much about AIDS but provide services participate to increase awareness and could join CARE Council as a result. 5. Analysis and Discussion (2008), Simo and Bies (2007), and Clarke and Fuller (2010) highlight how strategic preparation promotes realization of goals and objectives of collaborative governance in addressing complex problems. help to generate an agenda that enables realization of goals and objectives of the retreat. Scholars such as Bryson et al. (2006), Ansell and Gash (2008), Crosby and Bryson (2012), Quick and Feldman (2014), Bryson et al. (2008), Simo and Bies (2007), and Clarke and Fuller (2010) highlight how strategic preparation promotes realization of goals and objectives of collaborative governance in addressing complex problems. The responses of participants to questions related to preparation for the retreat point to intense deliberations involving various key stakeholders, generation of relevant themes for the retreat, strategy formulation for problem solving, logistics and agenda setting, learning and commitment for collective action, team building exercise, and innovation in care coordination. For instance, one participant points out: County, health council staff, CARE Council representatives on committees County, health council staff, CARE Council representatives on committees and other volunteers review what was done previously and what emergent issues they are dealing with currently and decide various details of what needs to be addressed at the retreat. These days, clients are involved in planning for the retreat and others are asked to participate based on expertise, interest, and everyone is accommodated as no one is turned away. y y This response appears to suggest preparation for the retreat as a team effort involving various stakeholders who help set the agenda in a way that would promote effective engagement, learning and formulation of solutions to problems in the context of collaborative governance. 5.4 Retreat as an educational moment The nature of multi-stakeholder engagements at the annual retreat appears to serve as an educational moment for all participants including CARE Council members. The diverse representation of stakeholders and perspectives, the various topics of presentation and deliberation, and group activities seem to promote knowledge and understanding of collaborative governance for care and treatment of target populations. Scholars such as Plotnikof (2016), Bryson et al. (2015), Leach, Weible, Vince, Siddiki, & Calanni (2013), Kooiman (1993), Sirianni (2009), and Beierle & Cayford (2002) point out how multi-stakeholder engagement in established forums help grow knowledge and understanding of inclusive structures and processes, and enable viable solutions for change and collective problem solving, especially complex ones. Responses from participants to questions geared toward the educational benefits of participation in the annual retreat seem to suggest consensus among participants on the influence of the various topics of presentation and discussion over the years i.e. diversity and inclusion, collaboration and leadership, code of ethics, cultural competency, effective deliberation, resilience through peer support, efforts to stop HIV and find cure, how to fight stigma, care and treatment expectations, management of continuum of care, and the benefits and challenges related to members’ knowledge and understanding of HIV/AIDS collaborative governance. Furthermore, the participants appear to acknowledge how the annual retreat is empowering, promotes networking among stakeholders, exposes various stakeholders to the roles and responsibilities of the CARE Council, helps develop key competencies, and encourages participation and decision making. A comment by one respondent suggests: g p p g y p gg The annual retreat helps grow knowledge of decision makers, complement training workshops that are provided to members throughout the year and helps training workshops that are provided to members throughout the year and helps Council members make data driven decisions. g p p g y p Council members make data driven decisions. The above statement highlights the educational benefits of the annual retreat to collaborative governance, which seems to indicate fostering care and treatment of HIV/AIDS infected and affected at the county. 5.3 Evolution of the retreat Evolution in collaborative governance whether in the areas of annual forum such as a retreat or otherwise shows that evidence-based changes to establish goals and objectives can be better achieved for the common good. This means that changing dynamics of HIV/AIDS epidemic over the years needs adaptive approaches even in the process of engagement associated with care and treatment to ensure acceptable outputs and/or outcomes. Scholars such as Provan and Kenis (2008), Thomson and Perry (2006), Plotnikof (2016), Bryson et al., (2015), Purdy (2012), Vangen & Winchester (2013) underscore organic or necessitated evolution and/or changes associated with multi- stakeholder engagement for collective problem solving. 70 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 iiste.org Responses of participants to questions related to progression of the retreat suggest evolution in terms of category of participants, consistency of stakeholder engagement, focus on trending and emergent issues important to care and treatment, evolution of intensity and hours/duration of stakeholder engagement at the retreat, and progression from focus on medication needs to holistic and communal needs of infected and infected populations. The responses seem to suggest there is now a focus on divergent and tailored presentations, expertise, concentrated changes in programmatic areas and system of care while remaining consistent in putting the event together, evolution in importance and setting priorities, and overall changes in a positive direction. Relatedly, observation of the retreat over the years point to incremental progression in participation in ways that foster common resolve for collective action on HIV/AIDS. The response below shows how the CARE Council retreat as an annual forum of stakeholders’ engagement has evolved over the years. There used to be a handful of people at the retreat but now more people are getting involved. In the beginning folks were dying of HIV/AIDS and we were helping them to die with dignity. The retreat has grown with knowledge and expertise. We are no longer focusing on helping clients get services and die with dignity, we do more than that. We now focus on services that meet essential needs in view of growth and maturity of HIV/AIDS and associated issues and services. 5.3 Evolution of the retreat There used to be a handful of people at the retreat but now more people are getting involved. In the beginning folks were dying of HIV/AIDS and we were helping them to die with dignity. The retreat has grown with knowledge and expertise. We are no longer focusing on helping clients get services and die with dignity, we do more than that. We now focus on services that meet essential needs in view of growth and maturity of HIV/AIDS and associated issues and services. g y The statement above appears to reiterate the versatility of the CARE Council when it comes to the annual retreat, particularly the aspect of intentional changes to ensure adaptability to emergent issues on HIV/AIDS as a conduit to promote care and treatment of target populations through various service categories. 5.4 Retreat as an educational moment 6. The Annual Retreat’s Impact on the Work of the Care Council 0 a; C Cou c Cou t o C e ts & Se v ces epo t, 009; 0 b). Figure 1: The CARE Council Retreat Model Source: Model designed by the authors The role of collaborative governance in the CARE Council’s annual retreat in the HIV/AIDS arena shows (a) for retreats, strategic preparation enable constructive multi-stakeholder engagements; (b) the inclusive nature of the forum consisting of service providers, target populations, non-elected community stakeholders and other critical partners in the fight against HIV/AIDS; (c) the evolving nature of events that embodies progress at various HIV/AIDS fronts through various intentional and strategic engagements; (d) an educational moment that help to grow stakeholders’ capacity, knowledge and task performance; and (e) a gathering that promote tolerance and understanding of the “other” in a quest for meaningful and sustainable solutions to the complex HIV/AIDS problem. In summary, the role of collaborative governance in the CARE Council’s annual retreat highlights its essential role in generating resource pool, commitment, networking, shared understanding, communication, Figure 1: The CARE Council Retreat Model Figure 1: The CARE Council Retreat Model Figure 1: The CARE Council Retreat Model S d l d i d b h h Source: Model designed by the authors Source: Model designed by the authors The role of collaborative governance in the CARE Council’s annual retreat in the HIV/AIDS arena shows (a) for retreats, strategic preparation enable constructive multi-stakeholder engagements; (b) the inclusive nature of the forum consisting of service providers, target populations, non-elected community stakeholders and other critical partners in the fight against HIV/AIDS; (c) the evolving nature of events that embodies progress at various HIV/AIDS fronts through various intentional and strategic engagements; (d) an educational moment that help to grow stakeholders’ capacity, knowledge and task performance; and (e) a gathering that promote tolerance and understanding of the “other” in a quest for meaningful and sustainable solutions to the complex HIV/AIDS problem. 6. The Annual Retreat’s Impact on the Work of the Care Council The role of an annual retreat to the CARE Council’s collaborative governance is critical for care and treatment efforts in Palm Beach County. The use of the retreat as an avenue for collaborative governance seems to promote purposeful multi-stakeholder engagement around established agenda in a way that enables formulation of goals and strategies for delivery of various category of services to target populations. For instance, the use of group activities involving various stakeholders at the 2016 CARE Council Annual Retreat to collectively formulate strategies for care and treatment served as a critical component of the CARE Council’s 5-Year Integrated Plan (CARE Council 2016) and laid the foundation for most of the tasks of the various committees of the CARE Council and the CARE Council itself in the grant year. Similarly, presentation and discussion of key issues and topics during past retreats, i.e. cultural competency, diversity and inclusion, leadership and collaboration, management of continuum of care, future of Ryan White CARE Act, health systems challenges serve as an invaluable educational moments and complement existing capacities of members and the CARE Council. p g p The diagram below depicts the role of the annual retreat in collaborative governance of the CARE Council. It relates participation of state and non-sate stakeholders including target populations such as healthcare providers, social service providers, local agencies, community-based organizations, affected communities, individuals with AIDS, Grantee, and non-elected community leaders. These stakeholders’ engagement at the retreat and CARE Council enable outputs such as allocation priorities for various service categories, integrated plans, and needs assessment reports. For instance, the outputs of the 2016 retreat, especially generated documents based on group activities by the various stakeholders, allowed the various CARE Council committees to develop strategies and performed tasks relevant to the completion of the 5-Year Integrated Plan. Thus, there is some connectedness between stakeholders’ involvement in planning for the retreat, eventual engagement at the retreat and CARE Council, and attendant outputs which usually translate into services to those infected and affected. For example, from 2000-2006 timeframe, the CARE Council allocated $63,830,957 and $43,151,318 in 2009-2013 for the provision of various category of services annually to about 5,143 and 3,049 people respectively in need of care (Agbodzakey, 2017; CARE Council Comprehensive Plan, 2006; CARE Council Compiled Allocation Priorities, 2014a; CARE Council Count of Clients & Services Report, 2009; 2014b). 5.5 Cultural humility 5.5 Cultural humility Observation of multi-stakeholder engagement at the annual retreat and comments from the participants during the interviews seem to suggest intentional efforts to understand the ‘other’ as a conduit to promote collaborative governance for HIV/AIDS care and treatment. Cultural humility which is defined as “having an interpersonal stance that is other-oriented rather than self-focused, characterized by respect and lack of superiority toward an individual’s cultural background and experience” (Hook, Davis, Owen, Worthington, & Utsey, 2013, p.353) appears to be palpable in how various stakeholders engage each other at the annual retreat. Over the years, efforts have been made to listen to key populations and participants in order to better understand their narratives, perspectives, issues, and concerns to help formulate strategies for care and treatment. Furthermore, the retreat provides the opportunity for service providers, non-elected community leaders, and county officials to interact with HIV/AIDS infected and affected individuals in an environment that is safe, encouraging and embracing of a difference in a collaborating setting geared toward formulating solutions for health and general wellbeing of target populations. A statement by one of the respondents’ notes that the success of the retreat is based on respect for consumers of services and the desire to empower them to be effective leaders in HIV/AIDS arena, seems to convey the extent to which cultural humility is embraced at the retreat. 71 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 Research by Yeager and Bauer-Wu (2013), Alsharif (2012), Foronda and Baptiste (2016) Chang, Simon, and Dong (2012), Foster (2009), Cruess & Cruess (2010), and Isaacson (2014) points to how operating from a standpoint of cultural humility nurtures effective engagement of relevant stakeholders. These scholars allude to how cultural humility serves as a better alternative to cultural competency in helping to effectively engage key constituencies in collective problem solving. The experience of the annual retreat as it relates to engagement of key populations and the consistency of intentional efforts to harness various expertise and experiences for sustainable solutions within the context of collaborative governance for care and treatment, appears to be helpful. 7. Conclusion and Implication The complex nature of the HIV/AIDS epidemic calls for innovative approaches to address associated challenges, one of which is care and treatment of infected and affected populations. The multifaceted nature of the problem and the domino impact on various sectors of a country’s economy necessitated collective action from all relevant stakeholders. Collaborative governance which promotes intentional engagement of cross-sector stakeholders for creative solutions to complex problems, has become a commonly utilized approach in the past few decades. In the case of HIV/AIDS in the U.S., the enactment of the Ryan White Comprehensive AIDS Resource Emergency Act (CARE Act) in 1990 with a key aspect that focuses on care and treatment of target populations enabled collaborative governance for allocation priorities to various service categories for those infected and affected. The HIV/AIDS CARE Council of Palm Beach County which is a health services planning council, embraced the use of collaborative governance over the years to foster effective and constructive decisions for care and treatment with annual retreat as one of the key components. This study examines the use of an annual retreat by the CARE Council as an avenue for collaborative governance to ascertain its role in enabling the fulfilment of legislative intents to benefit target populations. The results suggest that annual retreat serves as a unique opportunity for collaborative governance in promoting strategic preparation that enables strategic stakeholder engagement for innovative health services delivery to target populations through enhanced CARE Council roles and responsibilities. Furthermore, the results highlight how the annual retreat helps to develop needed capacity, mutual commitment, and competencies for creative inputs, outputs and/or outcomes related to the HIV/AIDS problem. It is equally apparent that openness and intentional engagement of all relevant stakeholders, especially otherwise usually excluded groups, promote common resolve and sustainable solutions with facilitative leadership serving as a lynchpin to all of the integrated efforts to purposeful ends. As such, collaborative governance represents a viable alternative approach to the usually mainstream top-down management when it comes to addressing community crises and/or complex problems with or without an established legislative mandate. The efforts of the CARE Council within a local system context represents an essential part of the U.S. government’s commitment to tackle the HIV/AIDS problem and to promote health and general wellbeing of infected and affected populations. While the study’s findings cannot be generalized to other Eligible Metropolitan Areas (EMAs) in the U.S. 6. The Annual Retreat’s Impact on the Work of the Care Council In summary, the role of collaborative governance in the CARE Council’s annual retreat highlights its essential role in generating resource pool, commitment, networking, shared understanding, communication, The role of collaborative governance in the CARE Council’s annual retreat in the HIV/AIDS arena shows (a) for retreats, strategic preparation enable constructive multi-stakeholder engagements; (b) the inclusive nature of the forum consisting of service providers, target populations, non-elected community stakeholders and other critical partners in the fight against HIV/AIDS; (c) the evolving nature of events that embodies progress at various HIV/AIDS fronts through various intentional and strategic engagements; (d) an educational moment that help to grow stakeholders’ capacity, knowledge and task performance; and (e) a gathering that promote tolerance and understanding of the “other” in a quest for meaningful and sustainable solutions to the complex HIV/AIDS problem. In summary, the role of collaborative governance in the CARE Council’s annual retreat highlights its essential role in generating resource pool, commitment, networking, shared understanding, communication, 72 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 www.iiste.org consensus and strategic plans among actual and some potential players in the HIV/AIDS arena for the common good. While the annual retreat represents a whole day (seven-to-eight hour) event in a year, it also helps to shape the focus of other key partners working directly or indirectly to promote care and treatment. consensus and strategic plans among actual and some potential players in the HIV/AIDS arena for the common good. While the annual retreat represents a whole day (seven-to-eight hour) event in a year, it also helps to shape the focus of other key partners working directly or indirectly to promote care and treatment. To some extent, the retreat helps to renew the collaborative spirit among stakeholders, fosters new initiatives and engagements, instigates provision of needed services to target populations who are not currently in the continuum of care, and broadens overall efforts to keep the focus on HIV/AIDS. This appears to be a conduit to sustain preventative and remedial, if not curative measures. The retreat is more of a strategic engagement that enables strategic planning by relevant stakeholders to vigorously tackle current and emergent challenges associated with the HIV/AIDS epidemic. 6. The Annual Retreat’s Impact on the Work of the Care Council Evidently, there is somewhat consensus among scholars on the strategic use of collaborative governance by cross-sector stakeholders to develop capacity, workable measures, and competencies to foster effective solutions to complex problems, which in this case, is the HIV/AIDS conundrum (Weber & Khademian 2008; Quick and Feldman 2014; Simo 2009; Simo & Bies 2007; Ritvala, Salmi & Andersson, 2014; Bryson et al. 2008; Babiak and Thibault 2009; Crosby and Bryson 2012, and Agranoff, 2012). The case of the CARE Council and the well-intended annual retreat appears to be a truly progressive undertaking to shape current and future efforts in the fight against HIV/AIDS in Palm Beach County. Undoubtedly, leadership is equally key in the progress and success of the annual retreat. A closer examination of the CARE Council’s annual retreat attests to the key role of facilitative leadership in ensuring collaborative success. Facilitative leadership in this context implies catalytic efforts of leaders from the initial planning of the retreat to post retreat activities by the CARE Council. The nonconventional nature of leadership in the CARE Council’s collaborative governance accentuated the viability of cross-sector and multi-stakeholder engagements as it used a lateral leadership mechanism highlighted in research by Freeman 1997; Margerum 2002; Lasker and Weiss 2003; Murdock, Wiessner, and Sexton 2005; Ansell & Gash, 2008; Huxham and Vangen 2000; and Imperial 2005, to formulate workable solutions. Facilitative leadership in this case also ensured representation, participation, and empowerment of otherwise usually excluded groups and thus provides useful insights for emergent multi- stakeholder arrangements geared toward solving complex problems (Agbodzakey, 2012; Vangen & Huxham, 2003, and Ansell and Gash, 2008). 7. Conclusion and Implication and its territories because of the uniqueness of the experience at Palm Beach County, it does offer insights into the likely benefits of embracing intentionally inclusive multi-stakeholder engagement event on a consistent basis as a conduit to collective problem solving. Future research could examine the use of an annual retreat in a comparative perspective within the context of collaborative governance and other complementary workshops/training efforts at EMAs to help establish some connectedness to care and treatment outputs and/or outcomes. 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Future Studies, OECD Publication. Retrieved December, 2008 http://www.oecd.org/dataoecd/15/0/17394484.pdf. Murdock, B. S., Wiessner, C., & Sexton, K. (2005). References Stakeholder participation in voluntary environmental agreements: Analysis of 10 Project XL case studies. Science, technology, & human values, 30(2), pp.223- 250. Ospina, S. M., & Dodge, J. (2005). Narrative inquiry and the search for connectedness: Practitioners and academics developing public administration scholarship. Public Administration Review, 65(4), 409-423. Palm Beach County CARE Council. (2005). Byelaws. Palm Beach County, Florida Palm Beach County CARE Council. (2006). Palm Beach County EMA comprehensive plan. Palm Beach County, Florida Palm Beach County CARE Council. (2009). Count of clients and service report. Palm Beach County, Florida Palm Beach County CARE Council (2012) Palm Beach County EMA comprehensive plan Palm Beach County Palm Beach County CARE Council. (2009). Count of clients and service report. Palm Beach County, Fl Palm Beach County CARE Council. (2012). Palm Beach County EMA comprehensive plan. Palm Beach Florida Palm Beach County CARE Council. (2013). Byelaws. Palm Beach County, Florida Palm Beach County CARE Council. (2014a). Compiled allocation priorities, FY 2009-2013. West Palm Beach EMA. Palm Beach County, Florida y Palm Beach Country CARE Council. (2014b). Ryan White part A count of clients and service report. P l B h C t Fl id y ( ) y p p Palm Beach County, Florida Palm Beach County, Florida y, Palm Beach County CARE Council. (2016). Palm Beach County integrated prevention and patient care plan. Palm Beach County, Florida. Plotnikof, M. (2016). Letting go of managing? Struggles over managerial roles in collaborative governanc Journal of Working Life Studies, 6, 109 an, K. G., & Kenis, P. (2008). Modes of network governance: Structure, management, and effectiveness JPART 18(2), pp.229-252. ( ), pp Purdy, J. M. (2012). A framework for assessing power in collaborative governance processes. Public Administration Review 72(3), pp.409-417 ( ) pp Quick, K.S., & Feldman, M. S. (2014). Boundaries as junctures: collaborative boundary work for building efficient resilience. Journal of Public Administration Research and Theory 24(3), pp.673-695 René, B., & Tharsi, T. (2004). Multi-party collaboration as social learning for interdependence: developing relational knowing for sustainable natural resource management. Journal of Community & Applied Social Psychology, 14(3), pp.137-153. y gy ( ) pp Ring, P. S, and Van de Ven, A. H. (1994). Developmental processes of cooperative interorganizational relationships. Academy of Management Review, 19(1), pp.90-118. Ritvala, T., Salmi, A., & Andersson, P. (2014). MNCs and local cross-sector partnerships: The case of a smarter Baltic Sea. References International Business Review, 23(5), pp.942-951 ( ) pp Rubin, I. S., & Rubin, H. J. (1995). Qualitative interviewing: The art of hearing data. Thousand Oaks, CA Ryan White comprehensive aids and resource emergency (CARE) Act (1990). Retrieved October 1, 2017,http://hab.hrsa.gov/livinghistory/timeline\1990.html ( ) Q g g Ryan White comprehensive aids and resource emergency (CARE) Act (1990). Retrieved October 1, 2017,http://hab.hrsa.gov/livinghistory/timeline\1990.html Sabatier, P. A., Focht, W., Lubell, M., Trachtenberg, Z., Vedlitz, A., & Matlock, M. (2005). Swimming u Collaborative approaches to watershed management. MIT press pp g p Saldana, J., & Omasta, M. (2017). Qualitative research: Analyzing life. SAGE Publications. aldana, J., & Omasta, M. (2017). Qualitative research Salkind, N. (2009). Exploring research.7th eds. Upper Saddle River, NJ: Prentice Hall Saz-Carranza, A, & Ospina, S. M. (2011). Behavioral dimensions of governing Salkind, N. (2009). Exploring research.7th eds. Upper Sa S. M. (2011). Behavioral dimensions of governing ( ) g g interorganizational goal-directed networks—managing unity–diversity tension. Journal of Public Administration and Research Theory 21(2), pp.327-365. rganizational goal-directed networks—managing unity–diversity tension. Journal of Public Administrati and Research Theory 21(2), pp.327-365. y ( ) pp cott, T. A., & Thomas, C. W. (2017). Unpacking the collaborative toolbox: Why and when do public man Scott, T. A., & Thomas, C. W. (2017). Unpacking the collaborative toolbox: Why and when do public managers 76 Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 www.iiste.org www.iiste.org choose collaborative governance strategies? Policy studies journal, 45(1), pp.191-214 Simo, G., & Bies. A. L. (2007). The role of nonprofits in disaster response: an expanded model of cro collaboration. Special issue, Public Administration Review 67(s1), pp.125-142. Simo, G. (2009). Sustaining cross-sector collaborations: Lessons from New Orleans. Public Organization Review 9(4), p.367. Sirianni, C. (2009). Investing in democracy: Engaging citizens in collaborative governance. Washington, DC: Brookings Institution Press Sørensen, E., & Torfing, J. (2011). Enhancing collaborative innovation in the public sector. Administration & Society, 43(8), pp.842-868. Stake, R. (1998). “Cases studies” (pp.86-109), in Denzin, N.K., Lincoln, Y.S.eds, Strategies of Qualitative Inquiry. Thousand Oaks, CA Susskind, L, & Cruikshank. J. (1987). Breaking the impasse: Consensual approaches to resolving public New York: Basic Books. Thomson, A. M., & Perry, J. L. (2006). Collaboration processes: Inside the black box. Public Administration Review, 66(s1), pp.20-32. ( ) pp Thomas, R. M. (2003). Blending Qualitative and Quantitative Research Methods in Theses And Dissertations. Thousand Oaks, CA: Corwin Press UNAIDS (2017). First Author Dr. James Korku Agbodzakey is an Associate Professor of Public Leadership and the Director of the Urban SERCH Institute at UNT Dallas. He has extensive work experience in public, private, civic/nonprofit sectors in Africa, Caribbean, and the United States of America. His principal research focuses on HIV/AIDS care and treatment, particularly how the use of collaborative governance framework impacts categories of services to target populations. Dr. Agbodzakey served as a delegate for AIDS 2012 in Washington, D.C.; AIDS 2014 in Melbourne Australia; AIDS 2016 in Durban, South Africa; AIDS 2018 in Amsterdam, Netherlands, and recently as a delegate at the 10th IAS Conference on HIV Science in Mexico City, Mexico in 2019. Dr. Agbodzakey is committed to working with various stakeholders to help address contemporary complex urban challenges for citizens’ benefits. References The global HIV/AIDS epidemic. Retrieved on October 4, 2017 from https://www.hiv.gov/hiv- basics/overview/data-and-trends/global-statistics Vangen, S., & Huxham, C. (2003). Nurturing collaborative relations: Building trust in interorganizational collaboration. The Journal of Applied Behavioral Science, 39(1), pp.5-31 Vangen, S., and Huxham, C. (2012). The tangled web: unraveling the principle of common goals in collaborations. JPART 22, 22(4), pp.731-760. en, S. & Winchester, N. (2013) ‘Managing cultural diversity in collaborations’, Public Management Revie 16(5), pp.686-707 ( ) pp Weber, E. P., and Khademian, A. M. (2008). Wicked problems, knowledge challenges, and collaborative capacity builders in network settings. Public Administration Review 68(2), pp.334-349 g ( ) pp Yeager, K. A., & Bauer-Wu, S. (2013). Cultural humility: Essential foundation for clinical researchers. Applied Nursing Research, 26(4), pp.251-256 K. (1984). “Introduction” (pp. 1–18), selection from conducting case studies (pp. 83–97) and “reporting studies” (pp. 141–166). In: Case study research: Design and methods. 3rd ed. Hills CA: Sage Yin, R. K. (1984). “Introduction” (pp. 1–18), selection from conducting case studies (pp. 83–97) and “reporting case studies” (pp. 141–166). In: Case study research: Design and methods. 3rd ed. Beverly Hills CA: Sage Yin, R. K. (2003). Case study research: design and methods. Thousand Oaks, CA: Sage Publications Zurba, M. (2014). Leveling the playing field: fostering collaborative governance towards on‐going reconciliation. Environmental Policy and Governance, 24(2), pp.134-146 choose collaborative governance strategies? Policy studies journal, 45(1), pp.191-214 Second Author Dr. Nicholas Bolden is an Assistant Professor of Public Administration and Policy at Columbus State University. His major teaching and research interest focus on state and local policy and public management, specifically in the areas of economic development, workforce development, and state and local government finance. Dr. Bolden has taught extensively in the field of public administration and provides training and policy consultant services to state and local agencies. Recently, Dr. Bolden served as a Work-Based Learning policy consultant to the Alabama Department of Commerce and The Office of the Governor, Kay Ivey. His recent publication he co-authored Do Political Parties Matter for the Funding Status of State Pension Plans appeared in the Journal of Regional and Policy Analysis. Nicholas Bolden received a Master of Public Administration from Troy University with a specialization in Adult Education and Training, and a Ph.D. in Public Administration and Policy from Auburn University. 77 Journal of Economics and Sustainable Development www.iiste.org ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 Third Author Dr. Sandra Schrouder is an Associate Professor of Public Administration at Barry University in the School of Professional and Career Education (PACE). Dr. Schrouder has over 20 years teaching experience at both the graduate and undergraduate level and has taught in Jamaica, the Bahamas and the United States. Dr. Schrouder is committed to community development and engagement through her years of involvement in Parkland City Government Advisory Board, Broward Health Community Relations Board among other community organizations. Her research interests are in the areas of management, public policy analysis, public finance, education policies, organizational ethics, bureaucratic discretion, and juvenile justice. She frequently reviews articles for peer-reviewed academic journals as well as new and prospective textbooks in her field. She is the co- author of Issues in the Development of Contemporary Caribbean Economies, along with Rupert Rhodd and Donna Cooke (2007). Journal of Economics and Sustainable Development ISSN 2222-1700 (Paper) ISSN 2222-2855 (Online) Vol.10, No.22, 2019 g Third Author Third Author Dr. Sandra Schrouder is an Associate Professor of Public Administration at Barry University in the School of Professional and Career Education (PACE). Dr. Schrouder has over 20 years teaching experience at both the graduate and undergraduate level and has taught in Jamaica, the Bahamas and the United States. Dr. Schrouder is committed to community development and engagement through her years of involvement in Parkland City Government Advisory Board, Broward Health Community Relations Board among other community organizations. Her research interests are in the areas of management, public policy analysis, public finance, education policies, organizational ethics, bureaucratic discretion, and juvenile justice. She frequently reviews articles for peer-reviewed academic journals as well as new and prospective textbooks in her field. She is the co- author of Issues in the Development of Contemporary Caribbean Economies, along with Rupert Rhodd and Donna Cooke (2007). Dr. Sandra Schrouder is an Associate Professor of Public Administration at Barry University in the School of Professional and Career Education (PACE). Dr. Schrouder has over 20 years teaching experience at both the graduate and undergraduate level and has taught in Jamaica, the Bahamas and the United States. Dr. Schrouder is committed to community development and engagement through her years of involvement in Parkland City Government Advisory Board, Broward Health Community Relations Board among other community organizations. Her research interests are in the areas of management, public policy analysis, public finance, education policies, organizational ethics, bureaucratic discretion, and juvenile justice. She frequently reviews articles for peer-reviewed academic journals as well as new and prospective textbooks in her field. She is the co- author of Issues in the Development of Contemporary Caribbean Economies, along with Rupert Rhodd and Donna Cooke (2007). 78
https://openalex.org/W2527247137	https://aura.abdn.ac.uk/bitstream/2164/7943/3/Inch_et_al_2016_International_Journal_of_Pharmacy_Practice.pdf	English	null	It’s not what you do it’s the way that it’s measured: quality assessment of minor ailment management in community pharmacies	International journal of pharmacy practice	2,016	cc-by	7,453	Keywords Keywords community pharmacy services; health care; non-prescription medicines; quality indicators; self-care Research Paper Research Paper y International Journal of Pharmacy Practice 2017, 25, pp. 253–262 y International Journal of Pharmacy Practice 2017, 25, pp. 253–262 Correspondence Correspondence Dr M. C. Watson, Health Services Research Unit, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK. E-mail: m.c.watson@abdn.ac.uk Setting Prospective, observational study of simulated patient (SP) visits to community pharmacies in Grampian (Scotland) and East Anglia (England). Setting Prospective, observational study of simulated patient (SP) visits to community pharmacies in Grampian (Scotland) and East Anglia (England). Method Eighteen pharmacies (nine per centre) were recruited within a 25-mile radius of Aberdeen or Norwich. Consultations for four minor ailments were evaluated: back pain; vomiting/diarrhoea; sore throat; and eye discomfort. Each pharmacy received one SP visit per ailment (four visits/pharmacy; 72 visits total). Visits were audio-recorded and SPs completed a data collection form Method Eighteen pharmacies (nine per centre) were recruited within a 25-mile radius of Aberdeen or Norwich. Consultations for four minor ailments were evaluated: back pain; vomiting/diarrhoea; sore throat; and eye discomfort. Each pharmacy received one SP visit per ailment (four visits/pharmacy; 72 visits total). Visits were audio-recorded and SPs completed a data collection form immediately after each visit. Primary Outcome Measure Each SP consultation was assessed for appropriate- ness against product licence, practice guidelines and study-speciﬁc consensus standards developed by a multi-disciplinary consensus panel. Results Evaluable data were available for 68/72 (94.4%) visits. Most (96%) vis- its resulted in the sale of a product; advice alone was the outcome of three vis- its. All product sales complied with the product licence, 52 (76%) visits complied with practice guidelines and seven visits achieved a ‘basic’ standard according to the consensus standard. Conclusion Appropriateness of care varied according to the standard used. Pharmacy-speciﬁc quality standards are needed which are realistic and relevant to the pharmacy context and which reﬂect legal and clinical guidelines to pro- mote the safe and effective management of minor ailments in this setting. © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Internatio It’s not what you do it’s the way that it’s measured: quality assessment of minor ailment management in community pharmacies Jackie Incha, Terry Porteousb, Vivienne Maskreyc, Annie Blythc, Jackie Burra, Jennifer Clelandd, David J. Wrighte, Richard Hollandf, Christine M. Bonda and Margaret C. Watsonb aCentre of Academic Primary Care, University of Aberdeen, Aberdeen, UK, bHealth Services Research Unit, University of Aberdeen, Aberdeen, UK, cFaculty of Medicine and Health, Norwich Medical School, Norwich Research Park, University of East Anglia, Norwich, UK, dDivision of Medical and Dental Education, John Simpson Chair of Medical Education, University of Aberdeen, Aberdeen, UK, eSchool of Pharmacy, University of East Anglia, Norwich, UK and fSchool of Medicine, Health Policy Practice, University of East Anglia, Norwich, UK Keywords community pharmacy services; health care; non-prescription medicines; quality indicators; self-care Correspondence Dr M. C. Watson, Health Services Research Unit, University of Aberdeen, Foresterhill, Aberdeen AB25 2ZD, UK. E-mail: m.c.watson@abdn.ac.uk Received January 8, 2016 Accepted August 3, 2016 doi: 10.1111/ijpp.12305 Keywords community pharmacy services; health care; non-prescription medicines; quality indicators; self-care Abstract Background Effective management of minor ailments in community pharma- cies could reduce the burden on alternative high-cost services (general prac- tices, Emergency Departments). Evidence is needed regarding the appropriateness of management of these conditions in community pharmacies. Objective To explore the appropriateness of minor ailment management in community pharmacies. International Journal of Pharmacy Practice 2017, 25, pp. 253--262 se, which permits use, . International Journal of Pharmacy Practice 2017, 25, pp. 253--262 l of Pharmacy Practice half of Royal Pharmaceutical Society rms of the Creative Commons Attribution License, which permits use, um, provided the original work is properly cited. International Journal of Pharmacy Practice 2017, 25, pp. 253--262 Introduction visits and 13% of general practice visits in the UK are for minor ailments that could have been managed in commu- nity pharmacies.[3,4] At the same time, there is evidence from academic[5,6] and commercial investigations[7] of low-quality management of these consultations in Emergency Departments (EDs) and general practices are overwhelmed by demand, due in part to patients present- ing with conditions suitable for management by other ser- vice providers, including community pharmacists and their teams.[1,2] Recent estimates suggest that 5% of ED 254 Quality assessment of minor ailment management community pharmacies involving suboptimal advice or inappropriate sales. These ﬁndings are of concern at a time when there is increasing interest in promoting the advisory role of the community pharmacist in addition to the technical supply function.[8] Design and setting A prospective, observational design was used and included community pharmacies in Grampian (Scotland) and East Anglia (England). SP visits tested the management of four minor ailments. Aim The aim of this study was to explore the appropriateness (process and outcome) of minor ailment management in community pharmacies using simulated patients (SPs). Primary outcome measure The appropriateness of the content and outcome of each SP visit was assessed against three standards: the summary of product characteristics (SPC) if a pro- duct was sold/supplied; clinical guidelines; and a consensus standard (comprising consultation pro- cess and outcome) (developed by the multi-disciplinary consensus panel). Manufacturers’ SPCs describe medicinal product prop- erties and the conditions for their use. Existing clinical guidelines were identiﬁed for the management of the con- ditions presented in each of the four scenarios. These included guidelines from SIGN (Scottish Intercollegiate Guidelines Network), NICE (National Institute for Health and Clinical Excellence), NHS Inform (a national health information service in Scotland), and the Royal Pharma- ceutical Society (Table 2). Recruitment of community pharmacies Eighteen pharmacies were required (nine each from Grampian and East Anglia), to give a wide variation in terms of type (independent, multiple) and location (urban, rural). All community pharmacies within a 25- mile radius of the main city in each centre (Aberdeen (n = 35), Norwich (n = 30)) were identiﬁed from Health Board (Scotland) and local lists and study invitation packs were mailed. Pharmacies were excluded if they had participated in earlier phases of the MINA programme.[4] Interested pharmacies were stratiﬁed by type and location and randomly selected to generate the sample. Signed informed consent was sought from the lead pharmacist within each participating pharmacy. Non-pharmacist staff could opt out of participation if preferred. Simulated patients and consultations Ten SPs (one per scenario plus one reserve per centre) were recruited from existing SP groups in the Medical Schools at the Universities of Aberdeen and East Anglia. A 4-h training session, informed by previous studies,[10] was delivered at each centre. Training focused on deliver- ing a standardised performance while responding natu- rally to pharmacy personnel questions.[11] Each SP was trained to perform one scenario and wore a hidden microphone to digitally record their consultations. To minimise the risk of detection and any resulting Hawthorne effect,[12] pharmacy staff were blind to the timing and number of SP visits, as well as the ailments that would be tested. The order of SP visits to pharmacies was randomised. Participating pharmacists and pharmacy assistants wore study badges throughout the study period to ensure that SPs did not consult non-participating staff. SPs distinguished pharmacy staff from pharmacists by uniforms worn and company name badges. Each phar- macy was supplied with reply-paid postcards to complete (date/time/visit details) and return to the research team, if they suspected receiving an SP visit. Participating phar- macies were scheduled to receive one SP visit per minor ailment, i.e. four visits per pharmacy (72 visits in total) over an 8-week period (February–March 2013). This study was part of the 2-year MINA research pro- gramme,[4] which derived evidence to inform the future delivery of minor ailment services in community pharma- cies in the UK. © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society International Journal of Pharmacy Practice 2017, 25, pp. 253--262 © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society Scenario development Table 1 Simulated patient scenario Table 1 Simulated patient scenario Scenario 1 Scenario 2 Scenario 3 Scenario 4 Back pain Sore throat Vomiting & diarrhoea Eye discomfort Presentation: I need something for my back (female) Presentation: I need something for a sore throat (male) Presentation: I need something for vomiting and diarrhoea (female) Presentation: I need something for my eye (female) Symptoms, If asked; Pain in her lower back The pain began this morning when she bent over to pick up her trousers The pain is on both sides of her lower back, and has continued on and off since, when bending and coughing No other symptoms; feels well otherwise Symptoms, If asked; Had sore throat for four days Yellow/green coloured phlegm Not able to eat anything, but trying to drink Also has headache Feels a bit hot Symptoms, If asked; Had vomiting and diarrhoea for two days Not able to eat anything, but drinking Don’t have a temperature No blood in stools Symptoms, If asked; Discomfort in eye No visual disturbance Awoke today with a red, crusty eye, but it seems to have improved a bit No idea what could have caused this No other symptoms; otherwise well History, if asked; 38 years old Doesn’t usually have a problem back No previous trauma No sudden weight loss No relevant lifestyle issues Current job doesn’t involve physical work Nothing alleviates or worsens the symptoms No other medical conditions History, if asked; 61 year old Doesn’t smoke Gets quite a lot of sore throats Doesn’t have any chronic respiratory illnesses, like asthma, sinusitis, bronchitis, COPD or emphysema History, if asked; 55 year old Son was vomiting at the weekend, but nobody else in the family has it Doesn’t get this sort of thing often Hasn’t been abroad recently No other medical conditions History, if asked; In her sixties No previous eye problems Nobody else in the household has got it Doesn’t wear contact lenses No other medical conditions Treatment, if asked; Not using any medicines No known drug allergies No action taken No simple analgesia at home Treatment, if asked; Not using any medicines No known drug allergies Has taken paracetamol for headache and sucking cough sweets to ease throat Treatment, if asked; Not using any medicines No known drug allergies No action taken Treatment, if asked; Not using any medicines No known drug allergies No action taken Table 2 Medication suggested by professional guidelines Minor ailment Guideline Medication suggested by guideline Back pain NICE Guideline CG88[31] & NHS Inform Regular paracetamol, if insufﬁcient advice NSAIDS or weak opioids Sore throat NHS Inform & SIGN guidelines[16] Recommend ibuprofen and paracetamol as a ﬁrst line of treatment for sore throats Vomiting and diarrhoea NHS Inform[17] Keep hydrated. Scenario development The four minor ailments that occur with the highest fre- quency in EDs and general practices[3,4,9] were chosen for scenario development; they were back pain, vomiting and diarrhoea, sore throat and eye discomfort. A standardised scenario for each minor ailment (Table 1) was developed for the SP visits by a 28-person multi-disciplinary consen- sus panel[4] (see later). Each scenario was derived from actual consultations which occurred during an earlier phase of the MINA programme.[9] Scenario-speciﬁc consensus standards (which included process and outcome) were developed by the multi-disci- plinary consensus panel which comprised community pharmacists (n = 8), ED consultants (n = 4), ED nurses (n = 4), GPs (n = 4), practice nurses (n = 4) and lay members (n = 4) from Scotland, England, Northern Ireland and Wales. Panel members operated independently and 255 Jackie Inch et al. © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society International Journal of Pharmacy Practice 2017, 25, pp. 253--262 Appropriateness of consultation process and outcome Outcome of visit (medication/advice supplied) Perceived professionalism of staff involved (5-point scale) All product sales (outcome) complied with the product licence, 52 (76%) visits complied with practice guideli- nes and seven visits achieved a ‘basic’ standard accord- ing to the consensus standard for process (Tables 5 and 6). No consultation achieved a ‘good’ standard of prac- tice for process. In terms of the content of SP consul- tations, the extent to which WWHAM[15] components were elicited varied substantially across scenarios (Fig- ure 1). Overall satisfaction with visit (5-point scale) Number of other customers on premises (as a marker of busyness) Duration of the visit (from digital recorder) Data collection There were two components to the data collection; the digital recordings of the consultation (see above) and a data collection form completed after every visit by the SP. This included: This included: Staff involved (pharmacist/counter staff) Questions asked Results Twenty pharmacies (31% (20/65)) consented to partici- pate; 18 were selected (independent (6), small chain (4) and large chain (8)). Pharmacies were located in urban (5) and suburban (7) areas and small towns (6). All planned SP visits (n = 72) were completed. Two pharmacies reported ‘suspected’ SP visits, one of which corresponded to an actual visit; data from this visit were excluded from analysis. Digital data were unavail- able for three visits (due to recording problems), so these were also excluded from the analysis. Evaluable data were available for 68/72 (94.4%) visits. Most (96% n = 65) visits resulted in the sale of a product; advice Scenario development Advocates the use of anti-diarrhoea medication Eye discomfort RPS[18] Indicates ‘marginal beneﬁt with chloramphenicol’, but does not discourage it’s use Table 2 Medication suggested by professional guidelines ‘WWHAM’[13] (Who is it for?; What are the symptoms?; How long have the symptoms been present?; Any medica- tion tried already?; what Medication used currently?), and likely outcomes (product sale, advice provision). Panel members were asked to add any items that they perceived would be relevant or important to include in a pharmacy consultation for each scenario. The responses were collated and then fed back to panel members who were asked to indicate which components they considered represented ‘basic’ or ‘good’ practice standards for the management of the four conditions in a community phar- macy (Table 3) in terms of process and outcome. Con- sensus was deﬁned as ≥60% agreement between panel members (i.e. ≥17/28) for both ‘basic’ or ‘good’ consulta- tion management. Only items where the consensus threshold was met were included in the ﬁnal standard. When using these standards to assess SP consultations, all components had to be delivered for the consultation to meet the consensus standard for process. the process was conducted by email. An outline scenario was developed for each of the four minor ailments which included a list of possible consultation components in terms of content (information gathering/provision), including questions from the aide memoire known as Quality assessment of minor ailment management 256 alone was the outcome of three visits. One-third of vis- its involved interaction with a pharmacist only. Most visits were conducted when the pharmacies had <3 cus- tomers and most SPs waited between 1 and 5 min for their consultation. Three visits lasted more than ﬁve minutes (Table 4). Data management and analysis All data were entered into SPSS version 20.[14] Indepen- dent accuracy checks were performed on 10% of the data. The results are presented as descriptive statistics. Vomiting and diarrhoea (n = 17): Ten consultations complied with NHS Inform guidelines[17] which advocate the use of an anti-diarrhoeal product. Six SP visits resulted in the sale of anti-nausea or anti-emetic prod- ucts, the use of which was not supported by the guideline. Only one consultation fulﬁlled the basic consensus stan- dard for outcome which advocated advice only. Ethical approval Ethical approval was granted by the College of Life Sciences and Medicine Ethics Review Board, University of Aberdeen. Eye discomfort (n = 17): Eleven consultations resulted in the sale of chloramphenicol products (suggested as having marginal beneﬁt in the RPSGB guideline[18]). One visit resulted in advice only and was therefore compliant with the ‘basic’ outcome standard advocated by the con- sensus panel. Five other consultations resulted in other eye products being sold. © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society Assessment of the management of minor ailments presented during SP visits Three researchers (JI, JC and JB) independently validated the data from the data collection forms against digital recordings for quality assurance purposes. Each researcher listened to each digital recording and compared their interpretations with the SP data collection forms. There were few discrepancies and consensus was deﬁned as when two out of three researchers were in agreement. Back pain (n = 17): In terms of products sold, all the back pain scenario visits resulted in appropriate outcomes when assessed against both the clinical guidelines and consensus panel standard (Table 5). [ ] Sore throat (n = 17): The SIGN guideline[16] states that there is no good quality evidence for non-prescription throat sprays, lozenges or gargles. No visit complied with the clinical guidelines because they all resulted in the sale of at least one of these products. In contrast, however, all sales complied with the ‘basic’ consensus standard in terms of outcome, i.e. products sold. International Journal of Pharmacy Practice 2017, 25, pp. 253--262 General professionalism and satisfaction SPs rated the visits highly in terms of the general pro- fessionalism of pharmacy staff involved in their consul- tation (Table 6). High levels of satisfaction (very satisﬁed/satisﬁed) were also reported. Of the eight visits where the SP was not satisﬁed, six involved the same SP. International Journal of Pharmacy Practice 2017, 25, pp. 253--262 257 Jackie Inch et al. Table 3 Consultation components to represent ‘basic’ and ‘good’ practice as recommended by the multidisciplinary consensus panel Scenario 1: Back pain Scenario 2: Sore throat Scenario 3: Vomiting & diarrhoea Scenario: 4 Eye discomfort What are the symptoms? What are the symptoms? What are the symptoms? What are the symptoms? Who is the patient? How long have the symptoms been present? Who is the patient? How long have the symptoms been present? How long have the symptoms been present? Are any other medications being used (for other conditions)? How long have the symptoms been present? Is this patient a contact lens wearer? Are any other medications being used (for other conditions)? Advise re. symptoms and action if symptoms continue Are any other medications being used (for other conditions)? Ensure advice re no contact lenses until condition resolved and ensure good cleaning of lenses if not disposable Does the patient have other medical conditions? Timescale deﬁned (no improvement after 3 period referral advised) Does the patient have other medical conditions? Who is the patient? Dosage instructions (if medication has been recommended) Who is the patient? Has the patient stopped taking oral ﬂuids? Dosage instructions (if medication has been recommended) Advise re. symptoms and action if symptoms continue How old is the patient? Advise re hydration/ﬂuids Advise re. symptoms and action if symptoms continue Does the patient have any other symptoms? Has any action been taken? Advise re. symptoms and action if symptoms continue Timescale deﬁned (no improvement after 9 period referral advised) Has any action been taken? Does the patient have other medical conditions? Dosage instructions (if medication has been recommended) Has any action been taken? Does anything alleviate or worsen symptoms Is there any associated temperature? Has any action been taken? Are any other medications being used (for other conditions)? Check understanding Does the patient have a history of chronic or recurrent sinusitis? Have they tried non-prescribed over the counter, herbal or home remedies? Does the patient have other medical conditions? Ask if already have simple analgesia at home? © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society Summary The appropriateness of the SP visits in terms of process and outcome varied according to the standard used. All SP con- sultations complied with the SPCs. The majority (n = 76%) of visits also complied with clinical guidelines. Few consul- tations reﬂected the standards deﬁned by the consensus panel, particularly in terms of information gathering. Strengths and limitations Discrepancies have previously been shown to exist between self-reported and actual recorded data,[19] but in this study, the digital recordings provided objective data for analysis. Non-verbal communication is an important component of healthcare consultations,[20] but was not assessed in this study. Duplicate, independent data extrac- tion and assessment reduced the risk of assessor bias. The inclusion of different types and locations of phar- macies in two countries with different pharmacy service contracts increased the generalisability of the results. Pharmacies self-selected to participate in the study. No data were collected from the non-participants. It is possi- ble that the participating pharmacies may have had higher levels of appropriate practice compared with non-partici- pants and as such, the results may overestimate appropri- ate practice across all pharmacies. Furthermore, the inclusion of pharmacies within a 25-mile radius of the study centre may not be comparable with national data due to the omission of rural pharmacies. Female cus- tomers are higher users of pharmacies than males, there- fore, to maximise face validity, most of the SPs in this study were female. However, there is evidence to suggest that patient gender can inﬂuence the type of information given by pharmacists during counselling.[21] Pharmacist/ counter assistant gender may also inﬂuence communica- tion during consultations[22], but pharmacy personnel gender was not recorded in this current study. Discussion Table 4 Summary of consultation characteristics (%n (derived from data collection form) Characteristic % (n) % (n) % (n) Members of staff involved in consultation Pharmacist Non-pharmacist Unknow 38.9 (28) 66.7 (48) 18.0 (13 Duration of consultation (minutes) ≤1 2–5 >5 33.3 (24) 58.5 (45) 4.2 (3) Duration of wait prior to consultation (minutes) ≤1 – >5 72.2 (52) 26.4 (19) 1.4 (1) Busyness of pharmacy (number of customers waiting at time of SP consultation) 0 1–2 ≥3 General professionalism and satisfaction Does the patient have a chronic respiratory illness? Any other members of the household affected? Have they had previous eye problems e.g. scleritis/iritis? Relevant product information (if a product recommended) Does the patient have any other symptoms? Does the patient have any other symptoms? Ensure there is no pain from within the eye, and that there exists no disturbances from bright lights. (Rule out iritis) If anti-inﬂammatories suggested check contra indications Check understanding Has there been any recent travel e.g. Overseas? Is there a history of possible foreign body/eye abrasion Encourage early mobilisation for muscular back pain Relevant product information (if a product recommended) Is there any blood present in the stools (faeces)? Are there any other members of the household affected? Refer to GP ‘if symptoms persist’ Dosage instructions (if medication has been recommended) Do you have a temperature? Is the patient using the children’s medication?=112) Ask if there are any questions Blood in stools - if so refer to GP Advise about actions to prevent spread of infection Are there any clinical signs of dehydration? - reduced urine output, tachycardia? Advice about hygiene within the household and not re-infecting family members (cross infection) Check understanding Check understanding Timescale deﬁned (no improvement after 9 period referral advised) Advice about self limiting nature of viral or bacterial conjunctivitis in otherwise healthy individuals Advise about actions to prevent spread of infection Relevant product information (if a product recommended) Advise about handwashing/food contact etc. Bold text represents ‘basic’ consultation requirements of the Multi-disciplinary Consensus Panel. Normal print represents additional requirements to reﬂect a ‘good’ consultation Bold text represents ‘basic’ consultation requirements of the Multi-disciplinary Consensus Panel. Normal print represents additional requirements to reﬂect a ‘good’ consultation. International Journal of Pharmacy Practice 2017, 25, pp. 253--262 258 Quality assessment of minor ailment management Quality assessment of minor ailment management Table 4 Summary of consultation characteristics (%n (derived from data collection form) Characteristic % (n) % (n) % (n) Members of staff involved in consultation Pharmacist Non-pharmacist Unknown 38.9 (28) 66.7 (48) 18.0 (13) Duration of consultation (minutes) ≤1 2–5 >5 33.3 (24) 58.5 (45) 4.2 (3) Duration of wait prior to consultation (minutes) ≤1 – >5 72.2 (52) 26.4 (19) 1.4 (1) Busyness of pharmacy (number of customers waiting at time of SP consultation) 0 1–2 ≥3 hors. International Journal of Pharmacy Pra hn Wiley & Sons Ltd on behalf of Royal Pha International Journal of Pharmacy Practice 2017, 25, pp. 253--262 General discussion Table 5 Assessment of SP consultation outcomes against the summary of product characteristics, guidelines and consensus list proposed Table 5 Assessment of SP consultation outcomes against the summary of product characteristics, guidelines and consensus list proposed Scenario Appropriateness of outcome compared with: Back pain (N = 17) % (n) Sore throat (N = 17) % (n) Vomiting & diarrhoea (N = 17) % (n) Eye discomfort (N = 17) % (n) Consensus list proposed outcome 100 (17) 100 (17) 15.8 (1) 15.8 (1) Summaries of product characteristics 100 (17) 100 (17) 100 (17) 100 (17) NICE Guideline[30] 100 (17) NHS Inform (Scotland)[17] 100 (17) 82.3 (14) 58.8 (10) SIGN guideline (117), April 2010[16] 0 (0) Royal pharmaceutical society reference guide[18] 64.7 (11) Outcome of Consultation Appropriate outcome 100 (17) 100 (17) 5.8 (1) 5.8 (1) Product sold 94.1 (16) 100 (17) 94.1 (16) 94.1 (16) Ibuprofen + Curaheat pad (2) Difﬂam/Covonia/ Chloraseptic throat sprays 9 7 Dioralyte + Loperamide 9 2 Brolene 9 1 Ibuprofen 9 12 Dry cough linctus 9 3 Pepto-Bismol 9 4 Tubilux 9 1 Ibuprofen + Paracetamol 9 2 Strepsils/Tyrozets lozenges 9 4 Dioralyte + Buccastem 9 1 Chloramphenical 9 11 Strefen 9 1 Loperamide 9 8 Optrex eye drops 9 2 Strepsils + Ultrachloraseptic throat spray 9 1 Motilium 9 1 Golden eye ointment 9 1 Tyrozets + Benylin dry cough 9 1 No sale 5.8 (1) 0 5.8 (1) 5.8 (1) No sale Table 6 Assessment of SP consultation content against the consensus list Table 6 Assessment of SP consultation content against the consensus list Back pain N = 17 Eye discomfort N = 17 Vomiting and diarrhoea N = 17 Sore throat N = 17 Consultation content (Basic) n (sp) n (sp) n (sp) n (sp) What are the symptoms? 16 (8) 16 (5) 17 (12) 16 (5) Who is the patient? 16 (16) – 15 – How long have the symptoms been present? 14 (9) 16 (10) 14 (1) 3 (2) Are any other medications being used (for other conditions)? 13 – 12 13 Does the patient have other medical conditions? 9 – 2 – Advise re. symptoms and action if symptoms continue 7 9 9 2 Is this patient a contact lens wearer? General discussion 13 – 12 13 Does the patient have other medical conditions? 9 – 2 – Advise re. symptoms and action if symptoms continue 7 9 9 2 Is this patient a contact lens wearer? – 5 – – Ensure advice re no contact lenses until condition resolved and ensure good cleaning of lenses if not disposable – 0 – – Has the patient stopped taking oral ﬂuids? – – 0 – Advise re hydration/ﬂuids – – 15 (1) – Dosage instructions (if medication has been recommended) 14 12 6 – Timescale deﬁned (no improvement after a speciﬁed period referral advised) – 6 – 2 General professionalism (SP rating) n n n n Exceptional interaction with member of staff 7 7 5 6 Member of Staff helpful, polite and competent 10 8 12 10 Non-professional approach, lacked conﬁdence 0 2 0 1 Satisfaction with Consultation (SP rating) % (n) % (n) % (n) % (n) Satisﬁed 94.1 (16) 64.7 (11) 94.1 (16) 100.0 (17) Not satisﬁed including uncertain 5.9 (1) 35.3 (6) 5.9 (1) 0 (SP) Indicates where the SP provided unsolicited information. Questions/advice that was conditional on answers of other questions. Bold items represent WWHAM[15] items. International Journal of Pharmacy Practice 2017, 25, pp. 253--262 © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society Jackie Inch et al. 259 259 Jackie Inch et al. Jackie Inch et al. General discussion – 5 – – Ensure advice re no contact lenses until condition resolved and ensure good cleaning of lenses if not disposable – 0 – – Has the patient stopped taking oral ﬂuids? – – 0 – Advise re hydration/ﬂuids – – 15 (1) – Dosage instructions (if medication has been recommended) 14 12 6 – Timescale deﬁned (no improvement after a speciﬁed period referral advised) – 6 – 2 General professionalism (SP rating) n n n n Exceptional interaction with member of staff 7 7 5 6 Member of Staff helpful, polite and competent 10 8 12 10 Non-professional approach, lacked conﬁdence 0 2 0 1 Satisfaction with Consultation (SP rating) % (n) % (n) % (n) % (n) Satisﬁed 94.1 (16) 64.7 (11) 94.1 (16) 100.0 (17) Not satisﬁed including uncertain 5.9 (1) 35.3 (6) 5.9 (1) 0 (SP) Indicates where the SP provided unsolicited information. Questions/advice that was conditional on answers of other questions. Bold items represent WWHAM[15] items. General discussion This study clearly demonstrates that the choice of stan- dard impacts substantially on the assessment of appropri- ateness. To the best of our knowledge, this has not been reported previously and is a novel ﬁnding of our study. Some of the clinical guidelines and consensus standards (for outcome) conﬂicted with the broader SPC indica- tions. It is unclear to what extent the clinical guidelines used in this study are referred to routinely in community pharmacy practice. No condition-speciﬁc clinical guideli- nes were identiﬁed that had been developed for use in Table 5 Assessment of SP consultation outcomes against the summary of product characteristics, guidelines and consensus list proposed outcome Scenario Appropriateness of outcome compared with: Back pain (N = 17) % (n) Sore throat (N = 17) % (n) Vomiting & diarrhoea (N = 17) % (n) Eye discomfort (N = 17) % (n) Consensus list proposed outcome 100 (17) 100 (17) 15.8 (1) 15.8 (1) Summaries of product characteristics 100 (17) 100 (17) 100 (17) 100 (17) NICE Guideline[30] 100 (17) NHS Inform (Scotland)[17] 100 (17) 82.3 (14) 58.8 (10) SIGN guideline (117), April 2010[16] 0 (0) Royal pharmaceutical society reference guide[18] 64.7 (11) Outcome of Consultation Appropriate outcome 100 (17) 100 (17) 5.8 (1) 5.8 (1) Product sold 94.1 (16) 100 (17) 94.1 (16) 94.1 (16) Ibuprofen + Curaheat pad (2) Difﬂam/Covonia/ Chloraseptic throat sprays 9 7 Dioralyte + Loperamide 9 2 Brolene 9 1 Ibuprofen 9 12 Dry cough linctus 9 3 Pepto-Bismol 9 4 Tubilux 9 1 Ibuprofen + Paracetamol 9 2 Strepsils/Tyrozets lozenges 9 4 Dioralyte + Buccastem 9 1 Chloramphenical 9 11 Strefen 9 1 Loperamide 9 8 Optrex eye drops 9 2 Strepsils + Ultrachloraseptic throat spray 9 1 Motilium 9 1 Golden eye ointment 9 1 Tyrozets + Benylin dry cough 9 1 No sale 5.8 (1) 0 5.8 (1) 5.8 (1) Table 6 Assessment of SP consultation content against the consensus list Back pain N = 17 Eye discomfort N = 17 Vomiting and diarrhoea N = 17 Sore throat N = 17 Consultation content (Basic) n (sp) n (sp) n (sp) n (sp) What are the symptoms? 16 (8) 16 (5) 17 (12) 16 (5) Who is the patient? 16 (16) – 15 – How long have the symptoms been present? 14 (9) 16 (10) 14 (1) 3 (2) Are any other medications being used (for other conditions)? International Journal of Pharmacy Practice 2017, 25, pp. 253--262 © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society Consultation process While community pharmacists and staff in this study asked questions (information gathering) and gave advice in the majority of consultations, some items central to safe supply were asked infrequently. For example, while SPs were often asked about current medication, they were rarely asked about co-morbidities. Similarly, information about how to use medicines was commonly provided, but advice about what to do should symptoms continue was rarely given. Consultations that focus on directions for medication use and dose, rather than on side effects and adverse events, were also identiﬁed in a review of counselling practices on prescription medicines in community pharmacies.[24] However, the extent and type of communication between SPs and pharmacists in our study was similar to other stud- ies of OTC medicines in the UK.[11,25,26] The multidisciplinary consensus panel generated ‘basic’ and ‘good’ standards for this study. Although the panel included eight community pharmacists, the components of some standards could be considered unrealistic for the management of minor ailments in the community phar- macy context. For example, the vomiting and diarrhoea standard included eliciting whether there were any clinical signs of dehydration including ‘reduced urine output, tachycardia’ which lack face validity in a community phar- macy setting. Other components of the ‘good’ standard were also problematic. For example, few ‘sore eye’ scenario consultations included elicitation of whether the SP was using other medication; there are, however, no major drug interactions associated with the topical use of chloram- phenicol which calls into question the validity of this com- ponent. These examples demonstrate not only the challenge of measuring appropriateness of healthcare management in this setting but also the potential lack of validity of using standard generic guidance, e.g. WWHAM, to assess a range of conditions when all items may not be relevant. Not satisﬁed including uncertain (SP) Indicates where the SP provided unsolicited information. (SP) Indicates where the SP provided unsolicited information. *Questions/advice that was conditional on answers of other questions. Bold items represent WWHAM[15] items. International Journal of Pharmacy Practice 2017, 25, pp. 253--262 © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society International Journal of Pharmacy Practice 2017, 25, pp. 253--262 © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society 260 Quality assessment of minor ailment management Figure 1 Content of simulated patient consultations (in relation to WWHAM REF protocol) (%(n): by scenario. [Colour ﬁgure can be viewed at wileyonlinelibrary.com] Figure 1 Content of simulated patient consultations (in relation to WWHAM REF protocol) (%(n): by scenario. [Colour ﬁgure can be viewed at wileyonlinelibrary.com] mulated patient consultations (in relation to WWHAM REF protocol) (%(n): by scenario. [Colour ﬁgure can be viewed a community pharmacies; only product-speciﬁc guidelines were identiﬁed, e.g. chloramphenicol.[18] Our study high- lights a need for clinical guidance to be tailored for use by community pharmacists and their teams and which does not conﬂict with other accepted guidance. The cur- rent lack of standardised tools will not only contribute to a lack of consistency between pharmacists and other health care providers, but may lead to variation in clinical practice and appropriateness of care that is so often reported in the wider international literature.[6] In the UK, the General Pharmaceutical Council is developing standards for the sale or supply of Pharmacy medici- nes,[23] but the content is as yet unspeciﬁed and unlikely to be condition-speciﬁc. © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society International Journal of Pharmacy Practice 2017, 25, pp. 253--262 Comparison with existing literature Effective consultations between pharmacy staff and their patients/customers are crucial to ensuring appropriate medication use and desired patient outcomes in terms of increased patient knowledge, adherence and/or decreased medication errors.[6,27,28] Hence, training in information gathering and provision (consultation skills) is now a core part of the pharmacy undergraduate and post-qualiﬁca- tion training programmes.[29] The relatively recent Medi- cation Related Consultation Framework (MRCF)[30] is promoted by the Royal Pharmaceutical Society in the UK as a tool for pharmacists to develop their consultation skills in general. However, elements of the framework lack face validity in relation to the management of over-the- counter consultations, e.g. conﬁrmation of patient’s Jackie Inch et al. 261 identify, documentation of full medical history. Further- more, most consultations for minor ailments or for OTC medicine requests are dealt with by medicine counter assistants for whom there is no organisation currently responsible (in the UK at least) for providing ongoing training (although it is the responsibility of the pharma- cist-in-charge to ensure that staff are competent to deliver their tasks). Indeed, in some countries, e.g. Australia, there is no requirement for support staff to have under- taken any training prior to their involvement in selling or recommending medicines (Pers. Commun, L. Seubert, 2014). The need for tighter regulatory control for phar- macy support staff is paramount, as is the provision of effective and ongoing training for these vital members of the pharmacy workforce. strategy to improve practice and achieve safe and effective patient care. The use of formal standards would also pro- vide pharmacists and their staff the criteria against which they could expect to be assessed. Authors’ contributions JI and JB contributed to acquisition of data, analysis and interpretation of data. TP contributed to conception and study design and critically revising the ﬁnal document. VM and AB contributed towards study design and acqui- sition of data. JC contributed towards conception, design, interpretation of data and critically revising the ﬁnal doc- ument. DJW and RH contributed to conception, design and interpretation of data. MCW and CMB contributed to conception, design and analysis, interpretation of data, drafting, critically revising and ﬁnal approval of paper and of report. The management of minor ailments was appropriate when assessed by two of the three standards used. Lower rates of appropriateness were achieved with the more aspirational consensus standards. There is a need for pharmacists and their staff to enhance their consultation skills not only through improved communication perfor- mance, but also by expanding their knowledge of health conditions. This would maximise the effectiveness of the management of minor ailments in the community phar- macy setting. We suggest that the development of quality standards for the management of such conditions is one 2. Hippisley-Cox J, Vinogradova Y. Trends in Consultation Rates in Gen- eral Practice 1995/1996 to 2008/9: Analysis of the QResearch Database. Final report to NHS Information Centre and Department of Health. Nottingham: The NHS Information Centre for Health and Social Care, 2009. Research implications Our study highlights the difﬁculty of measuring the appropriateness of minor ailment management in the community pharmacy setting. Quality standards are needed by which practice can be consistently, fairly and accurately assessed. The effect on practice of applying such standards also needs to be evaluated. Effective train- ing methods are needed to enhance consultation manage- ment in general and communication skills in particular, e.g. information gathering. While there has been minimal evaluation of this to date, there is evidence to suggest that communication behaviour can be enhanced by training.[5] Conﬂict of interest The authors declare that they have no conﬂicts of interest to disclose. Acknowledgments We thank the patients, health professionals and other NHS staff who contributed to this programme of research; members of the consensus panels and the advi- sory group; members of the PPRT Steering Group and to PRUK for funding this research. Funding This independent research was supported by the Phar- macy Practice Research Trust (PPRT) which merged with the Pharmaceutical Trust for Educational and Charitable Objects in December 2012 to form Pharmacy Research UK (PRUK). The views expressed in this publication are those of the authors and not necessarily those of the PPRT or of PRUK. © 2016 The Authors. International Journal of Pharmacy Practice published by John Wiley & Sons Ltd on behalf of Royal Pharmaceutical Society International Journal of Pharmacy Practice 2017, 25, pp. 253--262 References 3. Watson MC et al. A cohort study of inﬂuences, health outcomes and costs of patients’ health-seeking behaviour for minor ailments from primary and emergency care settings. 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https://openalex.org/W2104329193	https://www.scielo.br/j/bjps/a/WsGVXLwcXV4SyxD39PLnBcC/?lang=en&format=pdf	English	null	Medication errors: classification of seriousness, type, and of medications involved in the reports from a university teaching hospital	Brazilian Journal of Pharmaceutical Sciences	2,013	cc-by	6,427	Gabriella Rejane dos Santos Dalmolin1,, Eloni Terezinha Rotta2, José Roberto Goldim1,3 a Rejane dos Santos Dalmolin1,, Eloni Terezinha Rotta2, José Roberto Goldim1 1Research Laboratory in Bioethics and Research Ethics, Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil, 2Pharmaceutical Service/Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil, 3Bioethics Service/Hospital de Clínicas de Porto Alegre, Porto Alegre, RS, Brazil Medication errors can be frequent in hospitals; these errors are multidisciplinary and occur at various stages of the drug therapy. The present study evaluated the seriousness, the type and the drugs involved in medication errors reported at the Hospital de Clínicas de Porto Alegre. We analyzed written error reports for 2010-2011. The sample consisted of 165 reports. The errors identified were classified according to seriousness, type and pharmacological class. 114 reports were categorized as actual errors (medication errors) and 51 reports were categorized as potential errors. There were more medication error reports in 2011 compared to 2010, but there was no significant change in the seriousness of the reports. The most common type of error was prescribing error (48.25%). Errors that occurred during the process of drug therapy sometimes generated additional medication errors. In 114 reports of medication errors identified, 122 drugs were cited. The reflection on medication errors, the possibility of harm resulting from these errors, and the methods for error identification and evaluation should include a broad perspective of the aspects involved in the occurrence of errors. Patient safety depends on the process of communication involving errors, on the proper recording of information, and on the monitoring itself. Uniterms: Medication/errors. Drug therapy. Patients/safety. Erros envolvendo medicamentos ocorrem frequentemente em hospitais, possuem natureza multidisciplinar e podem ocorrer nas várias etapas da terapia medicamentosa. O estudo avaliou a seriedade, o tipo e os medicamentos envolvidos nos erros de medicação notificados no Hospital de Clínicas de Porto Alegre. Foram analisadas notificações de erros realizadas por escrito em 2010-2011. A amostra foi composta por 165 notificações. Cento e catorze notificações foram classificadas como erros de fato (erros de medicação) e 51 notificações, como erros potenciais. Apesar de se ter realizado maior número de notificações de erros de medicação em 2011, comparativamente a 2010, não houve alteração significativa no perfil de seriedade destes eventos. O tipo de erro mais frequente foi o de prescrição (48,25%). Os erros ocorridos ao longo do processo geraram, em algumas situações, novos erros de medicação associados. Nas 114 notificações de erros de medicação identificadas citaram-se 122 medicamentos. Correspondence: G. R. S. Dalmolin. LAPEBEC/HCPA, Laboratório 12213. Rua Ramiro Barcelos, 2350, 90035-903 - Porto Alegre - RS, Brasil. E-mail: dalmolingabriella@gmail.com Article Brazilian Journal of Pharmaceutical Sciences vol. 49, n. 4, oct./dec., 2013 Unitermos: Medicação/erros. Terapia medicamentosa. Pacientes/segurança. INTRODUCTION action they carry out, but this does not imply in guilt by association. In this sense, Bioethics may help develop an adequate understanding of the need to establish contingency measures for predictable risks. The healthcare system is extremely complex. It involves critical situations of risk, an interaction between multiple professionals and institutions, and depends on significant support from technology. The characteristics of the system may heighten the risk of mistakes and worsen the consequences of these mistakes. In this sense, it is important to assess risk and damage to patients in the search for ultimate patient safety (Rosa, Perini, 2003). The World Health Organization definition of patient safety establishes that unnecessary harm or potential harm associated with healthcare should be reduced to an acceptable minimum (WHO, 2009).i The implementation of systems for the detection and prevention of medication errors must be one of the pharmacovigilance goals at healthcare institutions. Nunes and colleagues (2008) underscored the need for systematic and continuous evaluation to decrease the incidence of errors and contribute to the identification and report of new error possibilities, which, in turn may have been mistakenly categorized as adverse drug reactions. These systems may also contribute to the identification of occurrences that would normally be kept secret (Nunes et al., 2008). Medication errors may be defined as any preventable event that may cause or lead to inappropriate medication use or patient harm while the medication is in the control of the healthcare professional, patient, or consumer. The error may be related to professional practice, to healthcare products, to procedures, to communication problems (including prescribing, product labeling, packaging, and nomenclature), to compounding, to dispensing, to distribution, to administration, to education, to monitoring, and to the proper use of medications (NCCMERP, 2001a). Spontaneous reporting, chart review, review of medical prescriptions, and direct observation are the main methods for identification and evaluation of medication errors (Coimbra, 2006). Each method has its advantages and disadvantages; the methods adopted should be adapted to institutional goals and used as management tools for healthcare quality improvement (Bohomol, Ramos, 2007). During the process of implementation of an institutional program of pharmacovigilance, the methods may be implemented progressively. Spontaneous reporting may be the method of choice for an initial stage; it allows for the participation of all sectors of the institution and for the implementation of a more autonomous culture of error checking and evaluation. Gabriella Rejane dos Santos Dalmolin1,, Eloni Terezinha Rotta2, José Roberto Goldim1,3 A reflexão sobre os erros de medicação e a possibilidade de danos decorrentes dos mesmos, assim como dos métodos para a sua identificação e avaliação, deve incluir uma ampla perspectiva dos aspectos envolvidos na sua ocorrência. A segurança dos pacientes depende deste processo de comunicação, do registro adequado das informações e do monitoramento propriamente dito. Unitermos: Medicação/erros. Terapia medicamentosa. Pacientes/segurança. 794 G. R. S. Dalmolin, E. T. Rotta, J. R. Goldim METHOD The study was carried out at Hospital de Clínicas de Porto Alegre (HCPA). The HCPA is a general teaching hospital with a hospital bed capacity of 795, which are divided in 18 conventional inpatient units, five intensive care units, and the adults and children emergency units. The reports categorized as errors that actually occurred (category B-I) were also categorized as for the type of error according to the American Society of Health- System Pharmacists (ASHP). The ASHP classifies errors as prescribing error, dispensing error, omission error, wrong time error, unauthorized drug error, improper dose error, wrong dosage-form error, wrong drug-preparation error, wrong administration-technique error, deteriorated drug error, monitoring error, compliance error, and other medication error (ASHP, 1993).i g y We carried out a retrospective, cross-sectional study to evaluate the medication error reports, submitted in written form to the Group for Safe Use of Medications (GUS, in Portuguese) at the HCPA, from January 2010 to December 2011. The Group is constituted by representatives from the Medications Committee, Pharmaceutical Service, and Nursing Group. This group analyzes the error reports submitted through the voluntary reporting system at the HCPA. We excluded reports that did not have enough information for their correct categorization, the reports that were duplicated, and the reports that were made after the period investigated (even though they refer to an event that happened during the period). The study sample included 165 spontaneous error reports.i We carried out the pharmacological classification of drugs involved in errors. We used the Anatomical Therapeutic Chemical Classification System of the WHO Collaborating Centre for Drug Statistics Methodology. The drugs can be classified into five different levels according to: anatomical main group (first level), therapeutic main group (second level), therapeutic/ pharmacological subgroup (third level), chemical/ therapeutic/pharmacological subgroup (fourth level), and the chemical substance (fifth level) (WHO, 2012). Statistical analyses were carried out to verify the frequency and association distributions between the variables. We used the chi-square test to evaluate the associations (using EPI INFO software, version 3.5.2). The level of significance was set at 5% (P=0.05). We carried out a survey of the GUS files with the aim of identifying in reports actual errors (medication errors) and potential errors (circumstances or events that may lead to errors). The medication errors identified were classified according to seriousness, type, and pharmacological class. INTRODUCTION Other more direct methods may be interpreted by healthcare professionals as external evaluation measures. International studies have established that the medication error rate varies from 1.5 to 35.0% depending on several factors. These factors include type of classification, method of error identification, and the common denominator adopted (Otero et al., 2008). According to Wannmacher (2005), recognizing errors is the best way to improve the quality and safety of healthcare activities. In Brazil, there has been increasing interest and discussions about medication errors. However, the studies that have been published have issues that need to be addressed; these issues involve conceptual clarity, geographical distribution, professional perspective, and the focus of ethical aspects involved. In relation to conceptual clarity, first, there were studies that reported adverse events, adverse drug reactions, and medication errors indistinctly (Rissato et al., 2008). In relation to geographical distribution, there is a predominance of studies carried out in southeastern Brazil, especially São Paulo (Optiz, 2006). Almost all studies adopt the point of view of nursing professionals (Telles Filho, Praxedes, 2009). Also, few Brazilian studies have addressed either the ethical aspects involved in medication errors or the most adequate way to address these situations (Coli et al., 2010). Usually, medication errors are detected only if there are clinical consequences manifested by the patient, such as the presence of symptoms sometime after the administration of the medication, thus alerting the healthcare professionals (Carvalho, Cassiani, 2002). The clinical consequences of the error may be confounded with adverse reactions to medication, but medication errors are different from adverse drug reactions. According to the World Health Organization (WHO, 1972), an adverse reaction is any harmful or undesirable effect presented after administration of medication at doses normally used for prophylaxis, diagnosis, or treatment of diseases. It is not possible to prevent adverse reactions, even though the possibility of an adverse reaction is known. The possibility that predictable harm may occur results in the need for establishing adequate prevention measures. The Precautionary Principle established by Hans Jonas (1994) is based on the Ethics of Responsibility. Healthcare professionals are always responsible for any The aim of the present study was to evaluate the seriousness, the type, and the medications involved in medication errors reported at the Hospital de Clínicas de Porto Alegre in 2010 and 2011. INTRODUCTION 795 Medication errors: classification of seriousness, type, and of medications involved in the reports from a University Teaching Hospital Error Reporting and Prevention Error Category Index (NCCMERP, 2001b). FIGURE 1 - Classification of seriousness of medication errors according to the National Coordinating Council for Medication Error Reporting and Prevention (NCCMERP). RESULTS AND DISCUSSION The sample included 165 error reports with similar number of occurrences for each year: 84 in 2010 and 81 in 2011. The reports were spontaneous and submitted by healthcare professionals from conventional inpatient units, intensive care units, and emergency units. According to NCCMERP taxonomy (NCCMERP, 2001b), harm is defined as temporary or permanent impairment of the physical, emotional, or psychological function or structure of the body and/or pain resulting therefrom requiring intervention. All reports associated with harm resulted in temporary harm only (categories E and F). There were no reports involving permanent harm or fatal outcomes (categories G, H, and I) (Table I).i The seriousness of the reports showed that 114 reports were actual errors (medication errors) and 51 reports were categorized as potential errors. For 2010, 34 of the 84 reports (40.48%) were evaluated as potential errors (category A). In 2011, 17 of the 81 reports (20.99%) were classified as category A. There was a significant association between the year of report and category A reports (X2=7.33; P=0.007) (Table I). The results did not show a statistically significant association in relation to harm (X2=0.06; P>0.05). This result was obtained by the comparison of the categories that are not associated with harm (categories B, C, and D) with the categories that are associated with harm (categories E to I).i A possible explanation for the decrease in potential error situations may be the educational efforts of the GUS. The aim of these efforts was to better inform HCPA professionals about medication error situations; they included events and direct efforts of improvement of the potential error situations reported. There was no significant association between the need to implement a measure (categories E to I) or at most monitor the patient (categories B to D) (X2=0.06; P>0.05). The necessary measures included medical intervention (category E), which occurred in 7.88% of reports, and prolonging hospital stay (category F), in 1.21% of cases. The reports of circumstances or events potentially associated with errors are of great importance for the implementation of prevention measures. The Precautionary Principle (Jonas, 1994) states that the existence of risk of serious harm, or of irreversible harm requires the implementation of measures to predict the occurrence of the damage. The implementation of educational measures is a current and necessary proposition for hospitals. METHOD In order to evaluate mistakes in the process of reporting medication errors, we also evaluated the reports of adverse drug reactions submitted to the Pharmaceutical Service at the HCPA in 2011. Seriousness was categorized using nine different categories, from A to I. The classification includes occurrence of an error; if the error reached the patient, or not; the harm associated with the error; and the necessary measures (Figure 1). The classification was based on the National Coordinating Council for Medication The study was approved by the Ethics Committee of the HCPA (project number 10-0445). The researchers Error category Error occurrence Reached patient Associated harm Necessary measures A Potential No No No B Yes No No No C Yes Yes No No D Yes Yes No Monitoring E Yes Yes Temporary Medical intervention F Yes Yes Temporary Hospitalization or prolonging hospital stay G Yes Yes Permanent Variable H Yes Yes Risk of death Vital support intervention I Yes Yes Death - G. R. S. Dalmolin, E. T. Rotta, J. R. Goldim 796 that reached the patient but did not cause harm. This result suggests that professionals already adopted the practice of reporting error situations, but may still have difficulties informing situations in which errors reached the patient. This difficulty may be caused by several reasons that have been reported in previous studies; the reasons include shame, guilt, and fear (Santos et al., 2007). filled out and signed a form indicating the Terms of Use for the Data, which aimed to preserve the privacy of the information contained in the reports. RESULTS AND DISCUSSION These measures are a way of safeguarding the legitimate interests of those (that is, every admitted patient and of the society as a whole) who may need hospital services. Healthcare professionals face a great challenge of recognizing the possibility of harm and the need to evaluate harm based on the knowledge available (Goldim, 2002). In relation to seriousness, though there was an increase in medication error reports in 2011 in comparison to 2010, there was no significant change in the profile of seriousness of these events. The results indicate, however, that educational measures that address medication errors are fostering the reports of more serious situations. The NCCMERP Medication Error Index was proposed in 1996 and revised in 2001. Several authors advocate it is the most adequate method (Santell et al., 2003; Forrey et al., 2007). However, more data from different institutions need to be collected (in methodologically similar fashion) so that comparisons can be made with the data presently available (Lacasa et al., 2012; Menendez et al., 2012). We identified two Brazilian studies that applied this classification. One investigated prescription errors (Néri et al., 2011), and the other investigated errors in the preparation of intravenous medications (Camerini, Silva, 2011). However, the two studies do not provide the same comprehensive approach of the present study.i Evaluation of the second criterion of seriousness showed that 109 (66.06%) of the reports did not reach the patients (categories A and B), out of which 59 happened in 2010 and 50 in 2010. Comparison of the years 2010 and 2011 did not show a significant association between not reaching (categories A and B) and reaching (categories C to I) the patient (X2=1.33; P>0.05) (Table 1). In 2010, the most frequent category was A (40.48%); but in 2011, the most frequent category was B (40.74%). The follow-up of reports during the two years showed that category B errors were the most frequently reported. In 2011, there was a 5.21% increase in the error reports From a Bioethics standpoint, the classification of seriousness raises some issues. RESULTS AND DISCUSSION If harm was inflicted upon a patient due to medication error, there may be doubts among the healthcare team about the communication to the 797 ation errors: classification of seriousness, type, and of medications involved in the reports from a University Teaching Hospit TABLE I - Seriousness classification for medication errors according to the National Coordinating Council for Medication Error Reporting and Prevention (NCCMERP), HCPA, 2010 - 2011 (N=165) Error category N (2010) Fr (%) N (2011) Fr (%) Total (2010-1) Fr (%) A 34 40.48 17 20.99 51 30.91 B 25 29.76 33 40.74 58 35.15 C 6 7.14 10 12.35 16 9.7 D 12 14.29 13 16.05 25 15.15 E 7 8.33 6 7.41 13 7.88 F 0 0 2 2.47 2 1.21 G 0 0 0 0 0 0 H 0 0 0 0 0 0 I 0 0 0 0 0 0 TOTAL 84 100 81 100 165 100 Notes: N= number of error reports Fr= percentage of error reports /total of error reports problems with understanding handwriting). However, if not used properly, electronic prescriptions may not help to ensure safer prescription of medications. The electronic prescription alone does not eliminate the possibility of medication errors (Koppel et al., 2005). The system adopted for electronic prescriptions should be constantly reevaluated. patients or their relatives. The ethical issue is not whether to communicate or not, but rather how to communicate to the patient and relatives, and when. The patient medical records, which belong to the patient, report this type of situation. In this sense, the adequate report of an error helps to build a relationship of trust between patient and healthcare staff.i Dispensing errors were reported only in 2010. A previous study developed in the HCPA (Almeida, 2010) indicated that the bar code reading system associated with changes in the infrastructure and work processes are the major factors behind the reduction in error rates for dispensing drugs in the hospital. After exclusion of the situations classified as potential error (category A), we carried out the classification of type of medication error (categories B to I) according to the ASHP taxonomy. The type of medication errors may be classified from prescription errors to compliance to the treatment itself (Table II). The frequency of spontaneous reports of errors of omission (2.63%) and of wrong time error (1.75%) was low. RESULTS AND DISCUSSION This suggests these cases may be underreported in the spontaneous reports. In studies that carried out direct observation of the medication administration stage, the errors of omission and of wrong time were above 10.0% and 8.0%, respectively (Costa et al., 2006). These results corroborate Bohomol and Ramos (2007), who showed that professionals involved with the administration of medications do not consider omissions (which do reach the patient) an error. Similarly, these professionals characterize the wrong time in medication administration as a justifiable error due to the high number of patients attended or by the lack of sufficient medications. The most frequent type of medication error associated with the process was prescribing error: 48.25% of cases during the two years of the study. Most cases of prescribing error did not reach the patient because they were intercepted by the nursing staff. Prescribing errors involved ambiguous information, duplicate medications, and mistakes associated incorrect information in the computer system. Ambiguous information is identified when the prescription defines that the medication should be used “as necessary” or “according to medical orientation.” Duplicate medications are identified when the same medication is included twice in the same prescription (at times with different dosages). Mistakes involving incorrect information are usually associated with units of measure, such as indicating milliliters instead of milligrams. The administration of non-authorized medications was reported in 13 events (11.40%). In some of these situations the medication prescribed was administered to the wrong patient. This suggests that the first goal for patient safety (JCI, 2006), that is, the correct identification of patients, was not adequately met in these cases. Electronic prescription of medication is internationally accepted as a tool that ensures safer use of medications; it reduces the medication error rates by facilitating the reading of the prescription (it avoids G. R. S. Dalmolin, E. T. Rotta, J. R. RESULTS AND DISCUSSION Goldim 798 TABLE II - Types of medication errors according to the American Society of Health-System Pharmacists (ASHP), HCPA, 2010 - 2011 (N=114) Type of error N (2010) Fr (%) N (2011) Fr (%) Total (2010-1) Fr (%) Prescribing 24 48 31 48.44 55 48.25 Dispensing 5 10 0 0 5 4.39 Omission 2 4 1 1.56 3 2.63 Wrong time 2 4 0 0 2 1.75 Unauthorized drug 5 10 8 12.5 13 11.40 Improper dose 5 10 8 12.5 13 11.40 Wrong dosage-form 0 0 0 0 0 0 Wrong drug-preparation 4 8 9 14.06 13 11.40 Wrong administration-technique 3 6 5 7.81 8 7.02 Deteriorated drug 0 0 0 0 0 0 Monitoring 0 0 0 0 0 0 Compliance 0 0 0 0 0 0 Other 0 0 2 3.13 2 1.75 TOTAL 50 100 64 100 114 100 Notes: N=number of medication error reports Fr=percentage of medication error reports/total of medication error reports TABLE II - Types of medication errors according to the American Society of Health-System Pharmacists (ASHP), HCPA, 2010 - 2011 (N=114) on errors according to the American Society of Health-System Pharmacists (ASHP), HCPA, 2010 - medication error reports Fr=percentage of medication error reports/total of medication error reports The classification of type of medication error helps to identify which stages of the process of administering medications should be improved. In this sense, better communication between the professionals is a factor that deserves more attention. A culture of infallibility, which is present in the healthcare field, may lead to a lack of communication about mistakes between professionals. Spontaneous reporting may be inversely associated with the seriousness and the type of error that occur. Though it is a difficult task, it is necessary to separate errors caused by human error from errors caused by the system. The aim is not to find a culprit, but rather to establish personal and institutional accountability. Improper dose errors (11.40%) were related to administration of drugs in doses higher or lower than prescribed, administration of an extra dose of the drug, and duplicate administration of a dose of the drug. They were mainly caused by poor understanding of prescriptions and failure of nursing staff to control the time of drug administration. RESULTS AND DISCUSSION In another Brazilian study (Teixeira, Cassiani, 2010), dose errors in drug administration were also among the three most common types of medication errors, accounting for 24.3% of medication errors related to drug doses administered differently than prescribed. Thirteen events (11.40%) were assessed as wrong drug-preparation errors and eight events (7.02%) as wrong administration-technique errors. Miasso et al. (2006) analyzed the medication preparation and administration process in medical units at four Brazilian hospitals. Among the problems identified as contributing to the occurrence of medication errors, wrong drug-preparation errors and wrong administration-technique errors were observed in all hospitals. More than one medication may be included in the same error report. In the 114 reports there were 122 drugs cited, which were classified according to the ATC classification system (Table III) using only the first level of the classification, which groups drugs according to anatomical main group. Drugs in code N (nervous system) were the most frequent medications associated with reports (30.33%). For example, morphine and the antiepileptics carbamazepine and clonazepam are among the medications reported in the two years of study. Medication errors may generate additional associated errors, which occur exactly because there are successive actions, or actions that are associated with a chain of events. For example, there were prescribing errors and preparation errors that led to other errors associated with drug administration (omission error, improper dose error, wrong time error, and unauthorized drug error) in nine cases (7.89%), out of which five (10.0%) occurred in 2010 and four (6.25%) in 2011. Code B drugs (blood and blood-forming organs) and code J drugs (anti-infectives for systemic use) were next in the reports; 15.57% and 14.75% frequency in the reports. In 2011 there was a 12.31% increase in reports of category J medications. RESULTS AND DISCUSSION The reports included, for example, ation errors: classification of seriousness, type, and of medications involved in the reports from a University Teaching Hospit 799 TABLE III - Drugs involved in medication error reports according to the Anatomical Therapeutic Chemical Classification System, HCPA, 2010 - 2011 (N=122) ATC level 1 N (2010) Fr (%) N (2011) Fr (%) Total (2010-1) Fr (%) A – Alimentary tract and metabolism 7 13.46 7 10 14 11.48 B – Blood and blood-forming organs 9 17.31 10 14.29 19 15.57 C – Cardiovascular system 5 9.62 4 5.71 9 7.38 D – Dermatologicals 0 0 0 0 0 0 G – Genitourinary system and sex hormones 1 1.92 0 0 1 0.82 H – Systemic hormonal preparations, excluding sex hormones and insulins 0 0 3 4.29 3 2.46 J – Anti-infectives for systemic use 4 7.69 14 20 18 14.75 L – Antineoplastic and immunomodulating agents 3 5.77 2 2.86 5 4.09 M – Musculoskeletal system 1 1.92 2 2.86 3 2.46 N – Nervous system 18 34.62 19 27.14 37 30.33 P – Antiparasitic products, insecticides and repellents 0 0 0 0 0 0 R – Respiratory system 2 3.85 4 5.71 6 4.92 S – Sensory organs 0 0 0 0 0 0 V – Various 0 0 0 0 0 0 Insufficient information for ATC classification 2 3.85 5 7.14 7 5.74 TOTAL 52a 100 70b 100 122 100 Notes: N= number of drugs cited in medication error reports Fr=percentage of drugs cited in medication error reports /total of drugs cited in medication error reports a In 2 reports more than one medication was cited. b In 6 reports more than one medication was cited. the following antibacterial drugs in more than one occasion: metronidazole, meropenem, and clarithromycin. Franciscatto et al. (2011) had already reported the need to implement actions for improving this specific goal. Once more, considering the classification of the medications involved in errors, patient medical records is an important document for evaluation and recording of information about prescription of medications and nursing reports of administration of the medication. Information about medication errors and about the medications themselves may be lost if not properly recorded. All healthcare professionals should be well-informed about the importance of recording possible errors and the drugs involved. The type of information necessary and the proper section for the description should be well-established. FINANCIAL SUPPORT The study received financial support from the Research and Events Incentive Fund (FIPE/HCPA). RESULTS AND DISCUSSION medication errors in 334 reports of adverse drug reactions. Eleven of these errors were code E, and one was code F. The types of error reported included prescribing errors, wrong drug-preparation error, and wrong administration- technique error. The drugs involved in adverse reaction reports included amphotericin b, with more than one occurrence, and vancomycin. The rate of adverse drug reactions mistakenly reported as medication errors was 3.59%; all had associated harm. The occurrence of associated harm may have been a confounding factor in the reporting of medication errors as an adverse drug reaction. The reflection on medication errors and the possible harm that may ensue should follow a broad investigation of aspects involved in the occurrence of the error. The safety of patients depends on the process of communication of errors, on the adequate report and recording of information, and on the monitoring itself. All of these steps depend on the adequate understanding of the importance of each step, and on the compliance, on the part of all healthcare professionals. Spontaneous reports of errors or potential error situations may help identify failures and weaknesses in the medication system in an Institution. If the spontaneous report system is well-structured and adequately managed, it may gradually ensure the participation of all collaborators in an Institution. As the data obtained from reports are converted into information through categorization and evaluation, a process of decision-making begins to take place. This process allows for actions to be carried out in the sense of correcting, whenever possible, these situations. The authors suggest that further research be conducted in order to study how the discussion of medication errors is flowing among professionals that act on the different stages of the medication system, seeking to verify the barriers and incentives that may prevent or encourage the reporting of medication errors. ACKNOWLEDGMENTS This study had some limitations regarding the quality of error reports. The voluntary reporting system at the HCPA was made available to staff at the end of 2009. Medication errors can be reported spontaneously through free text or standardized form. In most reports received during the study period the events were described by free text.Some reports did not have enough information for their correct categorization. Thus, the GUS meeting minutes were also used to search for additional information. Nevertheless, some reports (9) could not be used in the study. The authors would like to thank the GUS members for their availability and the student Frederico Maciel for the organization of the data collected. RESULTS AND DISCUSSION The report and recording of medication errors, including the description of the involved drug, ensure the monitoring of appropriate medication use. A study carried out in the state of Goiás, Brazil (Silva et al., 2011) evaluated 230 reports from the nursing staff and that contained medication errors. The main drugs associated with errors were antineoplastic and immunomodulating agents (code L; 24.3%), anti- infectives for systemic use (code J; 20.9%), and blood and blood-forming organs (code B; 15.3%). High-alert medications such as NPH insulin (code A), regular insulin (code A), and enoxaparine (code B) were associated with several error reports. These medications have an inherent risk for harming patients if there is a mistake in the process of drug administration. The seriousness of the errors associated with these drugs is high; the drugs may lead to permanent or fatal outcomes (Rosa et al., 2009). The present study suggests that there is still room at the HCPA for improvement in the third international goal for patient safety, that is, improvement of high-alert medications safety. A previous study by In order to evaluate mistakes in the process of reporting medication errors, we also evaluated the reports of adverse drug reactions at the Pharmaceutical Service of the HCPA. There were only data for 2011. There were 12 800 G. R. S. Dalmolin, E. T. Rotta, J. R. Goldim C) C) In relation to the drugs involved, including high-alert medications, there was a predominance (60.65%) of medications for the nervous system (code N), blood and blood-forming organs (code B), and anti -infectives for systemic use (code J). medication errors in 334 reports of adverse drug reactions. Eleven of these errors were code E, and one was code F. The types of error reported included prescribing errors, wrong drug-preparation error, and wrong administration- technique error. The drugs involved in adverse reaction reports included amphotericin b, with more than one occurrence, and vancomycin. The rate of adverse drug reactions mistakenly reported as medication errors was 3.59%; all had associated harm. The occurrence of associated harm may have been a confounding factor in the reporting of medication errors as an adverse drug reaction. C) In relation to the drugs involved, including high-alert medications, there was a predominance (60.65%) of medications for the nervous system (code N), blood and blood-forming organs (code B), and anti -infectives for systemic use (code J). CONCLUSION ALMEIDA, S.H.O. Incorporação de novas tecnologias de informação em um sistema de distribuição de medicamentos: avaliação quanto ao aumento da segurança de pacientes. Porto Alegre, 2010. 129 p. [Dissertion of master degree. Faculty of Pharmacy, Federal University of Rio Grande do Sul]. Based on the results for the medication error reports in 2010 and 2011 at the HCPA: A) In relation to seriousness, 30.91% of reports were about potential error (category A). In 60.0% of reports, medication errors did not reach the patient (category B) or reached the patient but did not cause harm (C and D). In the remaining reports, there was temporary harm associated (E and F); there were no reports associated with permanent harm or death (G, H, and I). AMERICAN SOCIETY OF HOSPITAL PHARMACISTS. ASHP guidelines on preventing medication errors in hospitals. Am. J. Hosp. Pharm., v.50, n.2, p.305-314, 1993. B) BOHOMOL, E.; RAMOS, L.H. Erro de medicação: importância da notificação no gerenciamento da segurança do paciente. Rev. Bras. Enferm., v.60, n.1, p.32-36, 2007. B) In relation to type of error, 48.25% of reports were associated with prescription. There were prescribing errors and preparation errors that led to other errors associated with drug administration in nine cases (7.89%). 801 of seriousness, type, and of medications involved in the reports from a University Teaching Hospital Medication errors: classification of seriousness, type, and of medications involved in the reports from a University Teaching Hospital CAMERINI, F.G.; SILVA, L. Segurança do paciente: análise do preparo de medicação intravenosa em hospital da rede sentinela. Texto & Contexto - Enferm., v.20, n.1, p.41-49, 2011. 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https://openalex.org/W4317512874	https://www.frontiersin.org/articles/10.3389/fnmol.2023.1073723/pdf	English	null	T-cell infiltration in the central nervous system and their association with brain calcification in Slc20a2-deficient mice	Frontiers in molecular neuroscience	2,023	cc-by	10,373	OPEN ACCESS OPEN ACCESS EDITED BY Giulia Abate, University of Brescia, Italy REVIEWED BY Huang Weiyi, Wuxi People's Hospital, China Jian-Huan Chen, Jiangnan University, China CORRESPONDENCE Lei Shi shilei_0328@hrbmu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Brain Disease Mechanisms, a section of the journal Frontiers in Molecular Neuroscience RECEIVED 18 October 2022 ACCEPTED 03 January 2023 PUBLISHED 20 January 2023 CITATION Zhang Y, Ren Y, Zhang Y, Li Y, Xu C, Peng Z, Jia Y, Qiao S, Zhang Z and Shi L (2023) T-cell infiltration in the central nervous system and their association with brain calcification in Slc20a2-deficient mice. Front. Mol. Neurosci. 16:1073723. doi: 10.3389/fnmol.2023.1073723 OPEN ACCESS EDITED BY Giulia Abate, University of Brescia, Italy REVIEWED BY Huang Weiyi, Wuxi People's Hospital, China Jian-Huan Chen, Jiangnan University, China CORRESPONDENCE Lei Shi shilei_0328@hrbmu.edu.cn †These authors have contributed equally to this work SPECIALTY SECTION This article was submitted to Brain Disease Mechanisms, a section of the journal Yi Zhang 1,2†, Yaqiong Ren 1†, Yueni Zhang 1,2, Ying Li 1,3, Chao Xu 1,4, Ziyue Peng 1,4, Ying Jia 2,3, Shupei Qiao 1,3, Zitong Zhang 1,2 and Lei Shi 1,2* 1 Human Molecular Genetics Group, NHC Key Laboratory of Molecular Probes and Targeted Diagnosis and Therapy, The Fourth Affiliated Hospital of Harbin Medical University, Harbin, China, 2 Department of Medical Genetics, College of Basic Medical Sciences, Harbin Medical University, Harbin, China, 3 Department of Child and Adolescent Health, School of Public Health, Harbin Medical University, Harbin, China, 4 Department of Pediatrics, The Second Affiliated Hospital of Harbin Medical University, Harbin, China Primary familial brain calcification (PFBC) is a rare neurodegenerative and neuropsychiatric disorder characterized by bilateral symmetric intracranial calcification along the microvessels or inside neuronal cells in the basal ganglia, thalamus, and cerebellum. Slc20a2 homozygous (HO) knockout mice are the most commonly used model to simulate the brain calcification phenotype observed in human patients. However, the cellular and molecular mechanisms related to brain calcification, particularly at the early stage much prior to the emergence of brain calcification, remain largely unknown. In this study, we quantified the central nervous system (CNS)-infiltrating T-cells of different age groups of Slc20a2-HO and matched wild type mice and found CD45+CD3+ T-cells to be significantly increased in the brain parenchyma, even in the pre-calcification stage of 1-month-old -HO mice. TYPE Original Research PUBLISHED 20 January 2023 DOI 10.3389/fnmol.2023.1073723 TYPE Original Research PUBLISHED 20 January 2023 DOI 10.3389/fnmol.2023.1073723 TYPE Original Research PUBLISHED 20 January 2023 DOI 10.3389/fnmol.2023.1073723 COPYRIGHT © 2023 Zhang, Ren, Zhang, Li, Xu, Peng, Jia, Qiao, Zhang and Shi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. brain calcification, Slc20a2, T-cell, blood–brain barrier, permeability, transcytosis, FTY720 Mouse model Slc20a2 encodes a multi-transmembrane type III sodium-dependent phosphate (Pi) cotransporter 2 (Pit-2), which is essential for regulating Pi homeostasis in the cerebrospinal fluid (CSF) and brain parenchyma. Its functional deficiency impedes inward Pi transport into brain endothelial cells (ECs) or smooth muscle cells (SMCs), leading to paracellular Pi accumulation (Wallingford et al., 2017). CSF-Pi levels are significantly elevated in patients with SLC20A2-related PFBC (Paucar et al., 2017; Hozumi et al., 2018) and in Slc20a2 homozygous (HO) knockout mice (Jensen et al., 2016; Wallingford et al., 2017). Excess extracellular Pi is known to cause tissue toxicity (Razzaque, 2011; Hong et al., 2015) and is associated with neuroinflammation (Brown, 2020), so as the accumulation of intracellular Pi (Legati et al., 2015; Zhao et al., 2022). The central nervous system (CNS)-resident immune cells, particularly microglia, macrophages, and astrocytes, are important sources of neuroinflammatory cytokines. Among these, CD45+ microglia (Keller et al., 2013), PDPN+, LCN2+, or C3+ neurotoxic reactive astrocytes (Zarb et al., 2019), along with novel calcification- associated microglia (Zarb et al., 2021), are known to significantly increase and cluster around the perivascular spaces of calcified microvessels in PFBC mice. Slc20a2tm1a(EUCOMM)Wtsi mice were purchased from the European Mouse Mutant Archive (Munich, Germany; Skarnes et al., 2011). The mice were maintained under specific pathogen-free conditions and allowed free access to water and chow under a light–dark cycle of 12 h. Heterozygous Slc20a2 mice were cross-mated to generate wild-type (WT) and HO mice. Littermates were used in subsequent experiments. All research procedures related to mouse models were scrutinized and approved by the Ethics Committee of the Fourth Affiliated Hospital of Harbin Medical University. OPEN ACCESS The accumulation of the CD3+ T-cells appeared to be associated with the severity of brain calcification. Further immunophenotyping revealed that the two main subtypes that had increased in the brain were CD3+ CD4− CD8– and CD3+ CD4+ T-cells. The expression of endothelial cell (EC) adhesion molecules increased, while that of tight and adherents junction proteins decreased, providing the molecular precondition for T-cell recruitment to ECs and paracellular migration into the brain. The fusion of lymphocytes and EC membranes and transcellular migration of CD3-related gold particles were captured, suggesting enhancement of transcytosis in the brain ECs. Exogenous fluorescent tracers and endogenous IgG and albumin leakage also revealed an impairment of transcellular pathway in the ECs. FTY720 significantly alleviated brain calcification, probably by reducing T-cell infiltration, modulating neuroinflammation and ossification process, and enhancing the autophagy and phagocytosis of CNS-resident immune cells. This study clearly demonstrated CNS-infiltrating T-cells to be associated with the progression of brain calcification. Impairment of blood–brain barrier (BBB) permeability, which was closely related to T-cell invasion into the CNS, could be explained by the BBB alterations of an increase in the paracellular and transcellular pathways of brain ECs. FTY720 was found to be a potential drug to protect patients from PFBC-related lesions in the future. Zhang Y, Ren Y, Zhang Y, Li Y, Xu C, Peng Z, Jia Y, Qiao S, Zhang Z and Shi L (2023) T-cell infiltration in the central nervous system and their association with brain calcification in Slc20a2-deficient mice. Front. Mol. Neurosci. 16:1073723. doi: 10.3389/fnmol.2023.1073723 COPYRIGHT © 2023 Zhang, Ren, Zhang, Li, Xu, Peng, Jia, Qiao, Zhang and Shi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. KEYWORDS brain calcification, Slc20a2, T-cell, blood–brain barrier, permeability, transcytosis, FTY720 01 Frontiers in Molecular Neuroscience Frontiers in Molecular Neuroscience frontiersin.org frontiersin.org Zhang et al. 10.3389/fnmol.2023.1073723 Immunofluorescence Brain tissues embedded in optimal cutting temperature media were cut into 5 μm sections and stored at –80°C. Brain sections were fixed with 4% paraformaldehyde (PFA) for 10 min on ice and blocked with blocking buffer [5% normal goat/donkey serum, 1% bovine serum albumin (BSA), 0.3% Triton X-100, and 0.3% glycine in 0.01 M phosphate-buffered saline (PBS)] for at least 1 h at 25°C. The sections were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies conjugated to Alexa Fluor 488 or 594. Images were obtained using a confocal microscope (C2 confocal system, Nikon, Japan). All the antibodies and immunofluorescence parameters are listed in Supplementary Table S1. The blood–brain barrier (BBB) serves as a highly selective, restrictive, and dynamic monolayer barrier for peripheral immune cells and foreign immunogens while also overseeing the CNS immune surveillance and brain homeostasis and maintaining the normal physiological roles of the CNS (Zlokovic, 2008; Abbott et al., 2010). Microvascular ECs, the core components of the BBB, express cell adhesion molecules (CAMs) to restrict peripheral immune cell crawling and adherence and possess specific cell junction proteins to prevent harmful immunogen extravasation and maintain beneficial exchange through paracellular and transcellular pathways under physiological conditions (Wilson et al., 2010; Engelhardt and Ransohoff, 2012; Pulgar, 2018). Maintaining a low rate of endocytosis and transcytosis in the BBB also contributes to normal CNS function. Transcellular pathway could be mediated by clathrin or caveolin-1 (Cav-1) expressed on ECs (Andreone et al., 2017; Ayloo and Gu, 2019). The proto-oncogene tyrosine-protein kinases, Src and Cav-1, participate in the transcytosis process, where Src regulates the formation of endocytic vesicles by phosphorylating Cav-1, the primary structural component of caveolae (Parton et al., 1994; Kim et al., 2009). Introduction Peripheral immune cell infiltration in the CNS has been reported as coinciding with neurological disorders, such as Alzheimer’s disease, multiple sclerosis, and stroke (Ellwardt et al., 2016; Liebner et al., 2018), among which CD4+ and CD8+ T-cells account for the main subpopulations. FTY720 (Fingolimod) is a sphingosine-1-phosphate receptor modulator, which inhibits egress of lymphocytes from lymph nodes, potentially reducing trafficking of T-cells into the CNS (Mandala et al., 2002; Cohen and Chun, 2011), and was utilized to inhibit the circulation of peripheral T-cells to treat CNS autoimmune diseases such as multiple sclerosis (Chun et al., 2019). In Pdgfbret/ret PFBC mice, CD45hi leukocyte infiltration modified the course of neuroinflammation, and the infiltration and neuroinflammation could be alleviated by FTY720 (Torok et al., 2021). However, in Slc20a2-PFBC mice, the cellular and molecular mechanisms related to brain calcification, particularly in a high-Pi microenvironment or areas of microcalcification, remain largely unknown. Primary familial brain calcification (PFBC; OMIM#213600), also known as Fahr’s disease, is a rare neurodegenerative disorder characterized by bilateral and symmetric calcification along the microvessels or inside neuronal cells in the basal ganglia, thalamus, and cerebellum. This disease is accompanied by multiple neurological manifestations such as movement disorders, cognitive impairment, and psychiatric signs, which commence after the age of 40 years (Tadic et al., 2015; Grangeon et al., 2019; Xu et al., 2022). Primary familial brain calcification is caused by the loss-of-function (LOF) variants, either in a dominant or recessive inheritance pattern, in one of seven genes in humans: solute carrier family 20 member 2 (SLC20A2; Wang et al., 2012), platelet derived growth factor receptor beta (PDGFRB; Nicolas et al., 2013), platelet derived growth factor subunit B (PDGFB; Keller et al., 2013), xenotropic and polytropic retrovirus receptor 1 (XPR1; Legati et al., 2015), myogenesis regulating glycosidase (putative) (MYORG; Yao et al., 2018), junctional adhesion molecule 2 (JAM2; Cen et al., 2020; Schottlaender et al., 2020) and cytidine/uridine monophosphate kinase 2 (CMPK2; Zhao et al., 2022). frontiersin.org Frontiers in Molecular Neuroscience Immunoelectron microscopy Mice were transcardially perfused with a mixture of 0.2% glutaraldehyde and 4% PFA in 0.1 M phosphate buffer under deep anesthesia. The brain’s basal ganglia, thalamus, and hypothalamus regions were quickly removed and cut into 1-mm3 pieces, placed into 0.2% glutaraldehyde in 0.1 M phosphate buffer, and fixed for 2 h at 25°C. After dehydration, embedding, and sectioning, the sections were incubated with 1% BSA blocking buffer for 30 min at 25°C and then incubated with rabbit anti-CD3 antibody (1:50; Abcam, ab5690) in 1% BSA overnight at 4°C. After washing, the sections were incubated with 10 nm gold-labeled goat anti-rabbit IgG (1:50; Sigma-Aldrich, G7402) and stained with 1% aqueous uranyl acetate for 20 min, followed by lead citrate for 30 s. The sections were photographed under a HITACHI HT7800 electron microscope. Western blotting Total protein was extracted from brain microvessels and quantified using a BCA Protein Assay Kit (Solarbio, PC0020). After adjusting to the consistent concentration, proteins were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (Bio-Rad, 161-0183), followed by the transfer of proteins from the gels to polyvinylidene difluoride membranes. 5% non-fat milk was used to avoid non-specific Blood–brain barrier (BBB) permeability/ leakage detection Micro-computed tomography (IRIS PET/CT imaging system, Inviscan, France) was applied to show the distribution of calcifications in the brains of the mice. Mice were deeply anesthetized with isoflurane for approximately 5 min before the experiment. They were then placed on the imaging stage under anesthesia with isoflurane gas during the entire imaging process. A total of 1,945 coronal plane images were obtained from each mouse. Approximately 70 images showed calcification in the hypothalamus and midbrain of the brains of 12-month-old Slc20a2-HO mice. Mice received an intraperitoneal injection of 2% Evans Blue (Sigma- Aldrich, E2129) in normal saline (4 ml/kg body weight) 2 h before sacrifice or a tail vein injection of 100 μl of 3 or 70 kDa tetramethylrhodamine-dextran (2 and 10 mg/ml, Invitrogen, D7162 and D1818, respectively) 1 h before sacrifice. Subsequently, the mice were transcardially perfused with 30 ml of PBS, followed by 60 ml of ice-cold 4% PFA. Brain tissue was post-fixed in 4% PFA for 4 h, dehydrated in 30% sucrose overnight at 4°C, and embedded in an optimal cutting temperature medium on dry ice. Brain sections were cut into 10- or 30-μm-thick sections for Evans Blue or tetramethylrhodamine-dextran staining and visualized using a Nikon C2 confocal microscope at 555- or 580-nm emission, respectively. FTY720 administration Slc20a2-HO mice, aged 1.5 months, were given 0.5 mg/kg FTY720 (Selleck, S5002) diluted in dimethyl sulfoxide or dimethyl sulfoxide alone via intraperitoneal administration three times a week, once every 2 days. After 12 weeks, the mice were transcardially perfused with 30 ml of PBS, and half of the brain was extracted for RNA using TRIzol™ Reagent (Invitrogen, 15596018), while the other half was fixed in ice-cold 4% PFA for 16 h. After dehydration in 30% sucrose at 4°C overnight, the brains were collected for further experiments. Leukocyte isolation and flow cytometry The mice were anesthetized and transcardially perfused with ice-cold Hank’s balanced salt solution (Ca/Mg-free). The brain was immediately isolated and homogenized mechanically through a 100 μm cell strainer, followed by digestion with Liberase (Roche, 5401020001) for 1 h at 37°C. The cell suspension was passed through a 70 μm cell strainer and rinsed with Hank’s balanced salt solution (Ca/Mg-free) containing 10% fetal bovine serum with or without DNase I (Roche, 10104159001). After density gradient centrifugation using 25% Percoll to remove myelin and cell debris, the cell pellet was resuspended in 02 Frontiers in Molecular Neuroscience frontiersin.org Zhang et al. 10.3389/fnmol.2023.1073723 Hank’s balanced salt solution (Ca/Mg-free) containing 10% fetal bovine serum. A single-cell suspension was prepared for flow cytometry with a BD Accuri C6 Plus instrument. All antibodies and additional information for flow cytometric analysis are listed in Supplementary Table S1. protein binding for 1 h at 25°C, followed by overnight incubation with primary antibodies diluted in 5% non-fat milk or 5% BSA at 4°C. After incubation with secondary antibodies, an enhanced chemiluminescence substrate was added to the membranes to highlight the target bands. All the antibodies used and additional information for western blotting are listed in Supplementary Table S1. Quantitative real-time polymerase chain reaction (qRT-PCR) Total RNA was extracted from brain microvessels using TRIzol™ Reagent (Invitrogen, 15596018) and then reverse-transcribed into first- strand complementary DNA using the SuperScript™ IV RT-PCR System (Invitrogen, 12594025). qRT-PCR was performed utilizing LightCycler® 480 SYBR Green I Master Mix (Roche, 04887352001) on a Roche 480 instrument. All assays were performed in triplicate, and the levels of each gene were normalized to those of the housekeeping gene Hprt. All primer sequences and additional information for qRT-PCR are listed in Supplementary Table S2. Brain microvessel isolation Mice were anesthetized with an intraperitoneal injection of 20% Ulatan (70 μl/10 g body weight). The entire brain was isolated from the skull and washed in cold isotonic sucrose buffer (0.32 mol/l sucrose, 3 mmol/l HEPES in distilled water, pH = 7.40). Each brain sample was homogenized in 5 ml sucrose buffer using a Dounce homogenizer, followed by centrifugation at 4500 × g for 10 min at 4°C. After removing the supernatant, the precipitate was resuspended in sucrose buffer and centrifuged again. The final pellet was resuspended in 2 ml sucrose buffer and passed through a 40 μm nylon mesh. Brain microvessels were retained on the mesh and eluted with ice-cold 0.01 M PBS (pH = 7.40) for collection. Tissue was obtained by centrifugation, and RIPA (YEASEN, 20115ES60), cell lysis buffer (CST, 9803), or TRIzol™ Reagent (Invitrogen, 15596018) were added to extract protein or RNA. Frontiers in Molecular Neuroscience frontiersin.org RNA sequencing bioinformatic analysis Brain RNA samples from mice administered the placebo/DMSO (n = 4), and FTY720 (n = 6) were collected for RNA sequencing. RNA libraries for RNA-seq were prepared using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® following manufacturer’s protocol. Illumina Casava 1.8 software was applied for basecalling. Raw data of fastq format were firstly processed through in-house Perl scripts. Reference genome GRCm38/mm10 and gene model annotation files were downloaded and then built and paired-end clean reads were aligned to the reference genome using Hisat2 v2.0.5. Differentially expressed genes (DEGs) were defined as the criterion p < 0.05 and protein-coding function. A volcano plot was constructed to map all DEGs using the Ggplot2 package (version 3.0.3). Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were applied to identify the DEG function, with the cut-off criteria of p < 0.01 and 0.05. Results were presented using the clusterProfiler package (version 3.0.3). Statistical analysis All data are presented as individual mean ± SD or SEM. CD45+CD3+ cells in the brains of 1-, 3.5-, and 8-month-old mice were counted in ten equally spaced sections covering whole brain with at least three biological replicates. The composition and percentage of T-cells in the brain parenchyma of five groups of 12-month-old mice were analyzed. For the quantitative analysis of Evans Blue leakage, under the identical conditions of sample processing and capture parameters, the average fluorescence intensity was calculated on three whole brain slices of each mouse using ImageJ (version 1.53c). For the evaluation of BBB integrity related with astrocytes and pericytes, AQP4/PDGF-Rβ and CD31 double positive areas were determined using the ImageJ area measurement tool as the percentage of AQP4-or PDGF-Rβ-positive fluorescent areas covering CD31-positive areas with five equally spaced sections in the 1- and 3.5-month-old mice. For statistical analysis, three visual fields were randomly selected in the cortex, basal ganglia, and midbrain regions of each brain slice. Simultaneously, the fluorescence intensity of AQP4- and PDGF-Rβ-positive areas was statistically analyzed. Shapiro–Wilk test and Levene’s test were used to examine the normality of the data and equality of variance, respectively. All data were compared using unpaired t-test except for the data of the expression of proteins from brain microvessels using paired t-test. Statistical significance was set at p < 0.05. , , , , and n.s. represent p < 0.05, p < 0.01, p < 0.001, p < 0.0001, and “not statistically significant,” respectively. Results To explore whether T-cells have the possibility of entry into the CNS through the paracellular pathway, we evaluated the expression of EC permeability-associated proteins including selectins, CAMs and cell junctions. In isolated brain microvessels of 3.5-month-old Slc20a2-HO mice, the expression levels of selectin (E-selectin and P-selectin) and CAMs (Icam-1, Icam-2, and Vcam-1) were significantly higher than those Central nervous system (CNS)-infiltrating T-cells mainly consisted of CD4+ and CD4−CD8-T-cell subsets Consistent with other neuroinflammatory diseases, both CD4+ and CD8+ cells, two classic subsets of CD3+ T-cells, were present in the brain parenchyma of 1-month-old Slc20a2-HO mice (Figure 1D), and these were hardly observed in -WT mice of the same age. To further precisely determine the subpopulation and proportion of infiltrated T-cells associated with brain calcification, flow cytometric analysis was performed on the brains of 12-month-old mouse pairs, as well as 14- and 21-month-old -HO mice. Firstly, micro-computed tomography had demonstrated the presence of calcified plaques in the hypothalamus, basal ganglia, and midbrain of 12-month-old -HO mice (Supplementary Figure S1B), which was consistent with previous reports (Wallingford et al., 2017; Jensen et al., 2018; Ren et al., 2021). Flow cytometric analysis confirmed an increase in CD45hi and CD3+ cells in the brains of 12-month-old -HO, compared with -WT mice (proportion of all single cells = CD45hi: WT: 0.980%, HO: 2.020%, p < 0.0001; CD3+ cells = WT: 0.672%, HO: 1.532%, p < 0.0001). In the CD3+ T-cell subpopulation, CD4+ and CD4–CD8 double-negative T (DNT) cells significantly increased in the brains of -HO mice (proportion of all single cells = CD4+ cells: WT: 0.218%, HO: 0.434%, p = 0.0086; DNT cells = WT: 0.314%, HO: 0.770%, p = 0.0003). However, CD8+ and CD4– CD8 double-positive T (DPT) subsets were not significantly different between the -HO and -WT groups (proportion of all single cells = CD8+ cells: WT: 0.070%, HO: 0.240%, p = 0.0541; DPT cells = WT: 0.056%, HO: 0.094%, p = 0.2438; Figure 1E). Furthermore, CD3+CD4+ and DNT subpopulations were increased in the older mouse groups (14- and 21-month-old), and CD45hi cells were also noted to increase with age (Figure 1E; Supplementary Figure S1C). von Kossa staining von Kossa staining was applied to detect phosphate ions (PO4 3−). Following the staining protocol (Abcam, ab150687), the sections were dewaxed, rehydrated, and incubated with 5% silver nitrate for 45 min 03 frontiersin.org Zhang et al. 10.3389/fnmol.2023.1073723 under ultraviolet rays. After rinsing several times with fresh distilled water, the sections were incubated with 5% sodium thiosulfate solution for 10 min to wash the unreacted silver ions, followed by a nuclear fast red solution. and CD3 markers were used to label T-cells by immunofluorescence. CD45+CD3+ T-cells emerged in the brain parenchyma of -HO and -WT mice at the age of 1 month (Figure 1A). To explore the relationship between CNS-infiltrating T-cells and calcification severity, T-cells were quantified in 3.5- and 8-month-old mice (Figures 1B,C), representing the pathological stages of slight and moderate calcification (Wallingford et al., 2017; Jensen et al., 2018; Ren et al., 2021). There was a significant increase in the number of CD45+CD3+ T-cells in -HO mice among the three age groups, with approximately a 2-, 3-, and 4-fold increase, compared to that in -WT mice, respectively (Figure 1C). More T-cells appeared in the brains of mice with severe brain calcification and older age, suggesting that T-cells infiltrated persistently with increasing calcification and age (Figure 1C; Supplementary Figure S1A). Notably, most infiltrating T-cells clustered around the calcified regions (Figure 1B; Supplementary Figure S1A). frontiersin.org T-cell infiltration positively correlated with brain calcification and aging To determine the presence of T-cell infiltration in the brain parenchyma of Slc20a2-HO mice at the pre-calcification stage, CD45 04 Frontiers in Molecular Neuroscience frontiersin.org Zhang et al. 10.3389/fnmol.2023.1073723 A C E B D FIGURE 1 Increased CNS Infiltration of T-Cells and Their Subpopulations in Slc20a2-HO Mice. (A) CD45+ CD3+ T-cells in the brain parenchyma of 1-month-old Slc20a2-WT and -HO mice. (B) Accumulation of T-cells around the calcification in 8-month-old -HO mice. Dashed lines indicate the outline of the calcified spots. (C) Absolute number of T-cells in the brain of -WT and -HO mice at 1-, 3.5-, and 8-month-old (Data are shown as mean ± SEM; 1 month, n = 6 pairs; 3.5 months, n = 3 pairs; 8 months, n = 3 pairs; 10 equally spaced sections covering entire brain with three biological replicates). (D) CD4+ and CD8+ T-cells in brain parenchyma of 1-month-old -HO mice. (E) Leukocyte immunophenotyping in 12-month-old -WT and -HO mice. Scale bar, 40 μm. p < 0.0001; p < 0.001; p < 0.01; n.s., not statistically significant. D A B B D A C E E C E Increased CNS Infiltration of T-Cells and Their Subpopulations in Slc20a2-HO Mice. (A) CD45+ CD3+ T-cells in the brain parenchyma of 1-month-old Slc20a2-WT and -HO mice. (B) Accumulation of T-cells around the calcification in 8-month-old -HO mice. Dashed lines indicate the outline of the calcified spots. (C) Absolute number of T-cells in the brain of -WT and -HO mice at 1-, 3.5-, and 8-month-old (Data are shown as mean ± SEM; 1 month, n = 6 pairs; 3.5 months, n = 3 pairs; 8 months, n = 3 pairs; 10 equally spaced sections covering entire brain with three biological replicates). (D) CD4+ and CD8+ T-cells in brain parenchyma of 1-month-old -HO mice. (E) Leukocyte immunophenotyping in 12-month-old -WT and -HO mice. Scale bar, 40 μm. *p < 0.0001; p < 0.001; p < 0.01; n.s., not statistically significant. in -WT mice (Figure 2A), which is a prerequisite for T-cell infiltration. The endothelium of the brain vessels forms an integral and tightly sealed monolayer, which mainly depends on a series of tight junctions (TJs: occludin, claudin-5, and ZO-1) and adherents junction (AJ: VE-cadherin) expressed on the lateral and contact surfaces of each ECs. Further, they maintain the integrity of the paracellular pathways (Bazzoni and Dejana, 2004; Wallez and Huber, 2008; Tietz and Engelhardt, 2015). Transcytosis of brain endothelium increased in Slc20a2-HO mice The expression of clathrin and phosphorylated and total Src was not altered, but that of phosphorylated and total Cav-1 was decreased in 3.5-month-old Slc20a2-HO mice. Mfsd2a, a key suppressor of caveolae formation and Cav-1 expression, showed slightly increased, coinciding with the decrease of Cav-1 (Figure 3A). The distribution of total Cav-1 remained unchanged in 1-month-old -HO mice (Figure 3B). Transmission electron microscopy (TEM) and immunoelectron microscopy were implemented to investigate whether T-cells traveled Astrocyte and pericyte density and coverage of microvessels remained unchanged Both astrocytes and pericytes play an essential role in the maintenance of BBB integrity (Zlokovic, 2008). Thus, the distribution pattern and coverage of astrocytes and pericytes in blood vessels were explored using AQP4, PDGF-Rβ, and CD31 antibodies to label astrocytes, pericytes, and vessels, respectively. There were no significant differences in the localization, morphology, polarization, and coverage of astrocytes or pericytes in Slc20a2-HO mice compared with -WT mice at the age of either 1 or 3.5 months (Supplementary Figure S3). T-cell infiltration positively correlated with brain calcification and aging In our study, the expression of cell junctions decreased in the brain microvessels of 3.5-month-old -HO mice (Figure 2B); however, the distribution patterns of them, such as localization, morphology, and polarization, remained unchanged in 1-month-old -HO mice (Figure 2C). Exogenous tracers (Evans Blue, 3 kDa, and 70 kDa TMR-dextran) were utilized to directly evaluate BBB permeability in the pre-calcification stage of -HO mice. Under the consistent parameters of fluorescence intensity and exposure time, a slight but significantly increased permeability was observed in the cortex, basal ganglia, and midbrain of the 1.5-month-old pre-calcification -HO mice, compared to -WT (Figure 2D; Supplementary Figure S2). These findings indicate that the increased paracellular permeability made the entry of T-cells into the brain parenchyma more feasible, which was also the cause of neuroinflammation. directly across BBB-ECs. The fusion of lymphocytes and EC membranes was observed by TEM in the brains of 6-month-old -HO mice; furthermore, immunoelectron microscopy with CD3 antibodies to label T-cells demonstrated that colloidal gold particles were present within brain ECs (Figure 3C), suggesting that CD3+ T-cells or endogenous plasma proteins entered the brain parenchyma via the transmembrane pathway. Endogenous IgG and albumin (small molecules) could be physically transported across the intact BBB; they were substantially increased in the perivascular spaces of the neurovascular unit (NVU) of 1-month-old pre-calcification -HO mice; macromolecules, such as blood-borne fragments of fibronectin, were undetectable in the brain parenchyma of mice of the same age (Figure 3D). Frontiers in Molecular Neuroscience FTY720 Alleviated brain calcification probably By multiple mechanisms in Slc20a2-HO mice To explore the relationship between T-cell infiltration and calcification, FTY720 was utilized to inhibit the circulation of peripheral 05 frontiersin.org Zhang et al. 10.3389/fnmol.2023.1073723 T-cells to reduce their CNS infiltration. The procedure for FTY720 administration is shown in Figure 4A. Three months after intraperitoneal administration, brain calcification was significantly reduced in FTY720- the key molecular and biological mechanisms involved in FTY720’s effects on the brain samples in the two groups. A total of 482 DEGs were identified using a filtering criterion of p < 0.05, among which 259 and A B C D FIGURE 2 Expression and distribution pattern of core adhesion molecules and junction proteins and the blood–brain barrier (BBB) permeability of paracellular pathway. (A) The relatively increased expression of selectin and cell adhesion molecules (CAMs) in brain microvessels of 3.5-month-old Slc20a2-HO compared to that of -WT mice (n = 3 pairs). (B) Decreased protein expression of TJs (occludin, claudin-5, and ZO-1) and AJ (VE-cadherin) in brain microvessels of 3.5-month-old -HO compared to that of -WT mice. (C) No difference in localization, morphology, and polarization of TJs and AJ proteins in 1-month-old -WT and -HO mice. Brain microvessels visualized by CD31. (D) Evans blue have slightly increased entry into the brain parenchyma of -HO compared to -WT mice at the age of 1.5 months. Scale bars, 40 μm (C), 80 μm (D). *p < 0.0001; p < 0.001; p < 0.01; p < 0.05. A B C D FIGURE 3 Transcellular Pathway Alterations in The brain endothelium of Slc20a2-HO mice. (A) Expression of endocytosis- and transcytosis-related proteins in brain microvessels of 3.5-month-old Slc20a2-WT and -HO mice. *p < 0.01; p < 0.05; n.s., not statistically significant. (B) Distribution pattern (localization, morphology, and polarization) of total caveolin-1 in 1-month-old mice. (C) The fusion of endothelial and lymphocyte cell membranes (red arrows) in 6-month-old -HO mice (C, Left). CD3+ colloidal gold particles in the endothelial cell membrane of the midbrain in 5-month-old -HO mice. Black triangles indicate gold particles (C, Right). EC, endothelial cell; L, vascular lumen. Scale bars, 6 and 1.5 μm (C, left); 0.5 μm (C, right). (D) Endogenous IgG and albumin, rather than fibronectin, leaked into the brains of 1-month-old -HO mice. Scale bar, 40 μm (B,D). A B C D FIGURE 2 Expression and distribution pattern of core adhesion molecules and junction proteins and the blood–brain barrier (BBB) permeability of paracellular pathway. FTY720 Alleviated brain calcification probably By multiple mechanisms in Slc20a2-HO mice (A) The relatively increased expression of selectin and cell adhesion molecules (CAMs) in brain microvessels of 3.5-month-old Slc20a2-HO compared to that of -WT mice (n = 3 pairs). (B) Decreased protein expression of TJs (occludin, claudin-5, and ZO-1) and AJ (VE-cadherin) in brain microvessels of 3.5-month-old -HO compared to that of -WT mice. (C) No difference in localization, morphology, and polarization of TJs and AJ proteins in 1-month-old -WT and -HO mice. Brain microvessels visualized by CD31. (D) Evans blue have slightly increased entry into the brain parenchyma of -HO compared to -WT mice at the age of 1.5 months. Scale bars, 40 μm (C), 80 μm (D). **p < 0.0001; p < 0.001; p < 0.01; p < 0.05. A B A C D D C D FIGURE 2 Expression and distribution pattern of core adhesion molecules and junction proteins and the blood–brain barrier (BBB) permeability of paracellular pathway. (A) The relatively increased expression of selectin and cell adhesion molecules (CAMs) in brain microvessels of 3.5-month-old Slc20a2-HO compared to that of -WT mice (n = 3 pairs). (B) Decreased protein expression of TJs (occludin, claudin-5, and ZO-1) and AJ (VE-cadherin) in brain microvessels of 3.5-month-old -HO compared to that of -WT mice. (C) No difference in localization, morphology, and polarization of TJs and AJ proteins in 1-month-old -WT and -HO mice. Brain microvessels visualized by CD31. (D) Evans blue have slightly increased entry into the brain parenchyma of -HO compared to -WT mice at the age of 1.5 months. Scale bars, 40 μm (C), 80 μm (D). **p < 0.0001; p < 0.001; p < 0.01; p < 0.05. A B C D FIGURE 3 Transcellular Pathway Alterations in The brain endothelium of Slc20a2-HO mice. (A) Expression of endocytosis- and transcytosis-related proteins in brain microvessels of 3.5-month-old Slc20a2-WT and -HO mice. *p < 0.01; p < 0.05; n.s., not statistically significant. (B) Distribution pattern (localization, morphology, and polarization) of total caveolin-1 in 1-month-old mice. (C) The fusion of endothelial and lymphocyte cell membranes (red arrows) in 6-month-old -HO mice (C, Left). CD3+ colloidal gold particles in the endothelial cell membrane of the midbrain in 5-month-old -HO mice. Black triangles indicate gold particles (C, Right). EC, endothelial cell; L, vascular lumen. Scale bars, 6 and 1.5 μm (C, left); 0.5 μm (C, right). (D) Endogenous IgG and albumin, rather than fibronectin, leaked into the brains of 1-month-old -HO mice. Frontiers in Molecular Neuroscience FTY720 Alleviated brain calcification probably By multiple mechanisms in Slc20a2-HO mice Scale bar, 40 μm (B,D). B B A A D D D C C FIGURE 3 Transcellular Pathway Alterations in The brain endothelium of Slc20a2-HO mice. (A) Expression of endocytosis- and transcytosis-related proteins in brain microvessels of 3.5-month-old Slc20a2-WT and -HO mice. *p < 0.01; p < 0.05; n.s., not statistically significant. (B) Distribution pattern (localization, morphology, and polarization) of total caveolin-1 in 1-month-old mice. (C) The fusion of endothelial and lymphocyte cell membranes (red arrows) in 6-month-old -HO mice (C, Left). CD3+ colloidal gold particles in the endothelial cell membrane of the midbrain in 5-month-old -HO mice. Black triangles indicate gold particles (C, Right). EC, endothelial cell; L, vascular lumen. Scale bars, 6 and 1.5 μm (C, left); 0.5 μm (C, right). (D) Endogenous IgG and albumin, rather than fibronectin, leaked into the brains of 1-month-old -HO mice. Scale bar, 40 μm (B,D). T-cells to reduce their CNS infiltration. The procedure for FTY720 administration is shown in Figure 4A. Three months after intraperitoneal administration, brain calcification was significantly reduced in FTY720- treated mice compared with that in the placebo group (FTY720: placebo = 6:4; p = 0.0181; Figure 4B). RNA sequencing helped determine the key molecular and biological mechanisms involved in FTY720’s effects on the brain samples in the two groups. A total of 482 DEGs were identified using a filtering criterion of p < 0.05, among which 259 and 223 genes were upregulated and downregulated, respectively (Figure 4C; Supplementary Table S3, and GSE218335). GO and KEGG enrichment 06 Frontiers in Molecular Neuroscience frontiersin.org 10.3389/fnmol.2023.1073723 Zhang et al. A C D E B FIGURE 4 FTY720 alleviated brain calcification and mRNA profile analysis. (A) A schematic diagram of the FTY720 administration process. (B) Representative images of brain calcification between placebo- and FTY720-treated mice. Area of decreased brain calcification in FTY720-treated mice compared to the placebo group. Scale bars, 1,000 μm, 150 μm. p < 0.05. (C) A volcano plot for the DEGs revealed by RNA-seq, in which 259 and 223 genes were upregulated and downregulated, respectively, with p < 0.05 as the cut-off criteria. (D,E) GO and KEGG enrichment analyses of the DEGs, with p < 0.01 and 0.05 as the cut-off criteria, respectively. A B B A C D E D E C FIGURE 4 FTY720 alleviated brain calcification and mRNA profile analysis. (A) A schematic diagram of the FTY720 administration process. FTY720 Alleviated brain calcification probably By multiple mechanisms in Slc20a2-HO mice (B) Representative images of brain calcification between placebo- and FTY720-treated mice. Area of decreased brain calcification in FTY720-treated mice compared to the placebo group. Scale bars, 1,000 μm, 150 μm. p < 0.05. (C) A volcano plot for the DEGs revealed by RNA-seq, in which 259 and 223 genes were upregulated and downregulated, respectively, with p < 0.05 as the cut-off criteria. (D,E) GO and KEGG enrichment analyses of the DEGs, with p < 0.01 and 0.05 as the cut-off criteria, respectively. analyses were performed to ascertain the functional changes in FTY720 treatment, and all terms were divided into the biological process (BP), cellular component (CC), and molecular function (MF) ontologies, as well as KEGG pathways. The DEGs highly enriched in GO_BP were mainly related to the “Ossification” (5 genes) and “Negative Regulation of Cell Cycle” (14 genes) terms; in the GO_CC subset, DEGs were enriched in the “Myelin” (4 genes) and various “Cell Junctions” (24 genes) terms; further, from the GO_MF perspective, the items related to transporter and channel activity (33 genes) constituted most of the enriched terms (Figure 4D; Supplementary Table S3). The “PI3K-AKT Signaling Pathway” was the top one enriched pathway in the KEGG analysis, with eight downregulated and nine upregulated genes. It is worth noting that seven genes in the autophagy pathway were also enriched in KEGG analysis (Figure 4E; Supplementary Table S3). interferon-gamma (IFN-γ) and interleukin-17-related cytokines, which contribute to chronic inflammation. In contrast, Th2 and T regulatory cells are more inclined to modulate the inflammatory responses of brain-resident immune cells (Gonzalez and Pacheco, 2014; Sonar and Lal, 2017; Solleiro-Villavicencio and Rivas-Arancibia, 2018). CNS-infiltrating DNTs were markedly increased in patients and mice with ischemic stroke and positively associated with brain injury (Meng et al., 2019; Kim et al., 2021). In Pdgfbret/ret PFBC mice, the expression of CAMs was upregulated in the cerebral vessels, which was accompanied by more lymphocytes entering the brain parenchyma. Infiltration of peripheral leukocytes into the CNS induces susceptibility to autoimmune inflammation (Torok et al., 2021). Zarb demonstrated that in Pdgfbret/ret mice, a novel microglial subset, calcification-associated microglia, accumulated around vascular calcified regions or in the perivascular space and effectively inhibited brain calcification (Zarb et al., 2021). Brain calcification induces the formation of neurotoxic astrocytes, although the exact functions of these cells remain unknown (Zarb et al., 2019). Frontiers in Molecular Neuroscience frontiersin.org Discussion Slc20a2-deficient mice exhibit the clinical symptoms of premature aging that progressively become more severe with age (Beck-Cormier et al., 2019; Ren et al., 2021). Human aging results in physiological brain calcification (Yamada et al., 2013) and a precipitous decline in the adult neural stem or progenitor cells with concomitant cognitive impairment (van Praag et al., 2005; Villeda et al., 2011), similar to the phenotypes of Slc20a2-HO mice (Ren et al., 2021). In aging brains, CNS-resident immune cells, such as microglia, increase, and peripheral blood-derived T-cells get recruited (Gemechu and Bentivoglio, 2012). CNS-infiltrating T-cells have been reported to play important roles in neurological diseases and are associated with neuroinflammation. CD4+ T-cells can be classified into several functional subgroups based on their surface markers. The T helper (Th)1 and Th17 subgroups secrete high levels of The aging brain has a marked susceptibility to circulatory proteins (Conboy et al., 2005; Yang et al., 2020). Endogenous plasma proteins can pass through the BBB via a ligand-specific receptor-mediated pathway during adolescence, which changes into non-specific caveolin-mediated transcytosis in the aging brain (Yang et al., 2020). Similarly, increased endocytosis or transcytosis is regarded as an early event of BBB injury in migraine, brain injury, and ischemic stroke (Knowland et al., 2014; Sadeghian et al., 2018). In the brain parenchyma of Slc20a2-HO mice, endogenous IgG and albumin gets transported across an impaired BBB and is presented in the perivascular spaces of the NVU (Jensen et al., 2018), suggesting the enhancement of endocytosis or transcytosis. Cav-1 expression is necessary for the formation and transcytosis of caveolae as a structural protein; otherwise, it acts as a potent inhibitor of endothelial 07 frontiersin.org Zhang et al. 10.3389/fnmol.2023.1073723 nitric oxide synthase activity to negatively regulate paracellular permeability (Schubert et al., 2002). In an ischemic stroke mouse model, Cav-1 deficiency correlated with the increase in the degradation of TJs and the hydrolytic activity of matrix metalloproteinases, thereby enhancing BBB permeability (Choi et al., 2016). functional defects also cause brain calcification (Vanlandewijck et al., 2018; Yao et al., 2018; Meek et al., 2022). This evidence suggested that pericytes and astrocytes, or the differences in protein glycosylation in their supporting BBB-ECs, might affect the pathogenesis of brain calcification by regulating endocytosis or transcytosis functions (Keller et al., 2013; Villasenor et al., 2017). Discussion Slc20a2 mainly expresses in the core components of the NVU, such as SMCs, ECs, pericytes, and the end-processes of astrocytes (Inden et al., 2016; Wallingford et al., 2017; Jensen et al., 2018; Vanlandewijck et al., 2018). Intracellular microcalcifications or tiny calcified granules were detected in the pericytes and astrocytes of SLC20A2-related patients (Miklossy et al., 2005) and mice (Jensen et al., 2018); however, neuronal and EC cell death only appeared in severely calcified areas (Kobayashi et al., 1987; Miklossy et al., 2005; Wszolek et al., 2006; Kimura et al., 2016). These findings suggest that, in the Slc20a2-HO mice, pericytes and astrocytes have functional defects due to intracellular calcification at an early stage, which then activates endocytosis or transcytosis of BBB-ECs (Alvarez et al., 2013; Keller et al., 2013; Villasenor et al., 2016; Jensen et al., 2018), accompanied by increased BBB permeability from the paracellular pathway. FTY720 significantly alleviated autoinflammatory lesions in a Pdgfbret/ret experimental autoimmune encephalomyelitis model and improved the survival rate of the mice (Terry et al., 2016; Torok et al., 2021). In our experiment, FTY720 partially inhibited brain calcification in Slc20a2-HO mice, suggesting that CNS-infiltrating T-cells could promote the pathogenesis of brain calcification. Inhibition of the PI3K-AKT pathway can activate autophagy, relieving vascular calcification phenotype in vitro and in vivo. In CD73-deficient fibroblasts, the increased activation of AKT blocked autophagy and resulted in arterial calcification (Moorhead 3rd et al., 2020). Dexamethasone accelerated the extracellular matrix calcification through activation of AKT signaling and the inhibition of autophagy in osteoarthritis mouse model (Chen et al., 2021). In the db/db mice, 9-PAHSA (a novel endogenous fatty acid) treatment down-regulated Akt–mTOR and activates autophagy in diabetic myocardium, ameliorating carotid vascular calcification (Wang et al., 2021). FTY720 was also reported to play a critical regulatory role in the bone remodeling, particularly in the regulation of osteoclasts, which were closely related to vascular calcification (Ishii et al., 2009; Xiao et al., 2018). Slc20a2 deficiency disturbed brain Pi homeostasis, forming a high-Pi microenvironment (Wallingford et al., 2017) and enhancing the sensitivity of arteriolar SMCs to induce calcification. Pi toxicity is also associated with cellular stress and neuroinflammation (Brown, 2020). Pdgfbret/ret and Slc20a2-HO mice exhibited persistent retinitis (Lindblom et al., 2003; Park et al., 2017) and ocular degeneration (Ren et al., 2021), suggesting high-Pi-induced neuroinflammation. Data availability statement The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found at: https://www.ncbi.nlm.nih.gov/geo/, GSE218335 Furthermore, there were no apparent impairments in pericyte coverage and BBB integrity in humans (Paucar et al., 2017) and mice (Wallingford et al., 2017; Jensen et al., 2018; Nahar et al., 2020) with SLC20A2-related PFBC, which is partially consistent with our results. PDGF-B and its receptor PDGF-Rβ, two more culprits for PFBC (Keller et al., 2013; Nicolas et al., 2013), are mainly and separately expressed in brain ECs and pericytes, and they play an essential role in regulating the functions of BBB-ECs. Only Pdgfbret/ret mice, in which the tissue distribution of PDGF-B is altered, probably due to the loss of a proteoglycan-binding motif, develop brain calcification (Keller et al., 2013; Vanlandewijck et al., 2015; Nikolakopoulou et al., 2017; Nahar et al., 2020). Inexplicably, the EC permeability was higher in the noncalcification-prone than in the calcification-prone brain areas; this region-specific heterogeneity in permeability was not associated with cell junction proteins or pericyte loss (Moura et al., 2017; Villasenor et al., 2017). MYORG encodes an endoplasmic reticulum-localized α-glucosidase that is specifically expressed in astrocytes, and its Discussion XPR1 is the only known Pi exporter in humans, its LOF mutations cause brain calcification, suggesting the essential roles of intracellular Pi homeostasis for maintaining brain health (Legati et al., 2015). CMPK2 biallelic LOF mutations were linked to neuron mitochondrial defects and the elevated intracellular Pi levels; Cmpk2-KO, as well as KI mice bearing patient-derived variants developed brain calcification (Zhao et al., 2022). LOF mutations in cell junctions impair brain ECs, disrupt their integrity, enhance BBB permeability (Saitou et al., 2000; Nitta et al., 2003; Argaw et al., 2009), and cause brain calcification only in humans (Mochida et al., 2010; O'Driscoll et al., 2010; Cen et al., 2020; Schottlaender et al., 2020). Jam2-HO mice developed widespread prominent vacuolation instead of brain calcification in the midbrain, cerebral, and cerebellar cortexes (Schottlaender et al., 2020), suggesting that LOF mutations in cell junctions alone are insufficient to cause brain calcification in mice. We demonstrated that T-cells infiltrated the CNS of Slc20a2-HO mice and that this infiltration rate increased with age. 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YR, YZ, YuZ, and LS checked and revised the grammar, syntax, and 08 Frontiers in Molecular Neuroscience frontiersin.org Zhang et al. 10.3389/fnmol.2023.1073723 Funding This study was funded by the National Key R&D Program of China (2020YFA0804000) and the Tou-Yan Innovation Team Program of Heilongjiang Province (2019-15). Supplementary material The Supplementary material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fnmol.2023.1073723/full# supplementary-material The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest. Publisher’s note logical errors. All authors contributed to the article and approved the submitted version. All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. 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W4300451032.txt	https://zenodo.org/record/1219818/files/Verstka_36_1_-285-293.pdf	en		Management by financial processes of decentralization and forming of territorial communities	DOAJ (DOAJ: Directory of Open Access Journals)	2,018	cc-by	4,638	ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ ДАННЫЕ ОБ АВТОРАХ Гордей Оксана Дмитриевна, доктор экономических наук, профессор, профессор кафедры финансов e-mail: oksana_@ukr.net Пацай Богдан Дмитриевич, кандидат физико-математических наук, доцент, доцент кафедры экономической кибернетики e-mail: pacaj@ukr.net Университет государственной фискальной службы Украины ул. Университетская, 31, м. Ирпень Киевская обл., 08201, Украина DATA ABOUT THE AUTHORS Hordei Oksana, Doctor of Economics, Professor, Professor Department of the Finance e-mail: oksana_@ukr.net Patsai Bogdan, Candidate of Physical and Mathematical Sciences, Associate Pro-fessor, Department of Economic Cybernetics e-mail: pacaj@ukr.net University State Fiscal Service of Ukraine University’s street, 31, Irpin, Kyiv region, 08201, Ukraine УДК 330.33.012:669(477) УПРАВЛІННЯ ФІНАНСОВИМИ ПРОЦЕСАМИ ДЕЦЕНТРАЛІЗАЦІЇ ТА ФОРМУВАННЯ ТЕРИТОРІАЛЬНИХ ГРОМАД Горошкова Л.А., Волков В.П. У роботі проведене моделювання умов стабільного функціонування фінансової системи територіальної громади. Реформування міжбюджетних відносин є стимулом для місцевих громад ефективніше формувати дохідну частини бюджету з подальшим переходом на самозабезпечення та більш раціональне планування своїх видатків, що у майбутні фінансові періоди дозволять зробити територіальні громади економічно та фінансово спроможними. Показано, що унаслідок прийняття змін до Податкового кодексу України та Бюджетного кодексу України суттєво розширені фінансові можливості місцевого самоврядування. На сьогодні держава здійснює суттєву фінансову підтримку процесу децентралізації. Доведено, що фінансова підтримка реформ адміністративно-територіального устрою України не повинна перетворитись на перерозподіл коштів Державного бюджету на користь окремих територіальних громад та їх об’єднань. Тобто кінцевою метою реформ повинно стати створення фінансово спроможних і самодостатніх територіальних громад.. В роботі проведене моделювання етапів та умов забезпечення сталого розвитку територіальних громад. Доведено, що необхідно виокремити три стадії: 1) мінімізація збитків, досягнення беззбитковості та формування умов самодостатності у фінансовому забезпеченні; 2) досягнення прибутковості та рентабельності; 3) досягнення рівня фінансової стійкості, достатнього для сталого розвитку територій. Доведено, що найбільшу фінансову допомогу з боку держави територіальні громади потребують на перших двох стадіях. У зв’язку з цим, запропоновано на перших двох стадіях планувати експоненційне зростання обсягів державної підтримки. На третій стадії, залишається необхідною потреба у зовнішньому фінансуванні з боку держави. Але на цьому етапі більшість територій вже повинна знаходиться на тому рівні фінансової стійкості, на якому більшу частину її прибутковості і рентабельності можливо забезпечити за рахунок внутрішньогалузевих ресурсів та можливостей. Доведено, що на третій стадії, зникає необхідність стрімкого зростання видатків, їх можливо зафіксувати на певному достатньому для підтримки сталого розвитку рівні. Таку ситуацію найкраще можливо описати логістичною кривою. Ключові слова: територіальна громада, бюджет, доходи бюджету, видатки бюджету, бюджетна політика, стабільний розвиток © Горошкова Л.А., Волков В.П., 2018 Економічний вісник університету \| Випуск № 36/1 285 ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ УПРАВЛЕНИЕ ФИНАНСОВЫМИ ПРОЦЕССАМИ ДЕЦЕНТРАЛИЗАЦИИ И ФОРМИРОВАНИЯ ТЕРРИТОРИАЛЬНЫХ ОБЩИН Горошкова Л.А., Волков В.П. В работе проведенное моделирование условий стабильного функционирования финансовой системы территориальных общин. Реформирование межбюджетных отношений является стимулом для местных общин эффективнее формировать доходную части бюджета с дальнейшим переходом на самообеспечение и более рациональное планирование своих расходов, что в будущие финансовые периоды позволят сделать территориальные общины экономически и финансово состоятельным. Показано, что вследствие принятия изменений в Налоговый кодекс Украины и Бюджетный кодекс Украины существенно расширены финансовые возможности местного самоуправления. На сегодня государство осуществляет существенную финансовую поддержку процесса децентрализации. Доказано, что финансовая поддержка реформ административно-территориального устройства Украины не должна превратиться в перераспределение средств Государственного бюджета в пользу отдельных территориальных общин и их объединений. То есть конечной целью реформ должно стать создание финансово состоятельных и самодостаточных территориальных общин. В работе проведенное моделирование этапов и условий обеспечения устойчивого развития территориальных общин. Доказано, что необходимо выделить три стадии: 1) минимизация убытков, достижения безубыточности и формирования условий самодостаточности в финансовом обеспечении; 2) достижения прибыльности и рентабельности; 3) достижения уровня финансовой стойкости, достаточного для устойчивого развития территорий. Доказано, что наибольшую финансовую помощь со стороны государства территориальные общины нуждаются на первых двух стадиях. В связи с этим, предложено на первых двух стадиях планировать экспоненциальный рост объемов государственной поддержки. На третьей стадии, остается необходимой потребность во внешнем финансировании со стороны государства. Но на этом этапе большинство территорий уже должно находится на том уровне финансовой стойкости, на котором большую часть ее прибыльности и рентабельности возможно обеспечить за счет внутриотраслевых ресурсов и возможностей. Доказано, что на третьей стадии, исчезает необходимость стремительного роста расходов, их возможно зафиксировать на определенном достаточном для поддержки устойчивого развития уровни. Такую ситуацию лучше всего описать логистической кривой. Ключевые слова: территориальная община, бюджет, доходы бюджета, расходы бюджета, бюджетная политика, стабильное развитие MANAGEMENT BY FINANCIAL PROCESSES OF DECENTRALIZATION AND FORMING OF TERRITORIAL COMMUNITIES Goroshkova L., Volkov V. In work the carried out modeling of conditions of stable functioning of a financial system of a territorial community. The reforming between budget of the attitudes is by stimulus for local communities on the one hand, namely, more effectively to form profitable parts of the budget, and from another - to pass to self-maintenance and more rational planning of the charges, which in the future financial periods will allow to make territorial communities economically self-sufficient and financial capable. Is shown, that owing to acceptance of changes in the Tax code of Ukraine and Budget code of Ukraine the financial opportunities of local self-management are essentially extended. On the today state carries out essential financial support of process of decentralization. Is proved, that the financial support of reforms of the administrative - territorial device of Ukraine should not turn to redistribution of a means of the State budget for the benefit of separate territorial communities and their associations. That is a ultimate goal of reforms should become creation financial of capable and self-sufficient territorial communities. In work modeling stages and conditions maintenance of constant development of territorial communities is carried out. Is proved, that is necessary to allocate three stages: 1) minimization of the losses, achievement without the losses and formation of conditions of self-sufficiency in financial maintenance; 2) achievement profit and profitability; 3) achievement of a level of financial stability, sufficient for steady development of territories. 286 Економічний вісник університету \| Випуск № 36/1 ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ Is proved, that the greatest financial help on the part of the state the territorial communities demand at first two stages. In this connection, is offered at first two stages to plan exponential growth of volumes of state support. At the third stage, there is necessary a need for external financing on the part of the state. But at this stage the majority of territories already owe is at that level of financial stability, on which it is possible to supply the most part it profit and profitability for the account inside branch of resources and opportunities. Is proved, that at the third stage, the necessity of prompt growth of the charges disappears, they are possible for fixing on determined sufficient for support of constant development a level. Such situation best is possible for describing by a logistical curve. Key words: territorial community, budget, incomes of the budget, charges of the budget, budget politics, stable development. Постановка проблеми у загальному вигляді та її зв’язок із важливими науковими чи практичними завданнями. Реформування міжбюджетних відносин є нагальною потребою для місцевих громад з подвійною метою: з одного боку – ефективніше формувати дохідну частини бюджету, з іншого – переходити на самозабезпечення та раціональне планування своїх видатків, що у майбутньому дозволять зробити територіальні громади економічно самодостатніми та фінансово спроможними. Саме тому у сучасних умовах децентралізації особливої актуальності набуває проблема формування ефективної системи управління фінансами територіальних громад. Аналіз останніх досліджень і публікацій, у яких започатковано розв’язання даної проблеми і на які спирається автор. Аналізу проблем управління грошовими потоками та місцевими фінансами присвячені роботи таких вітчизняних вчених, як В.Вакуленко, Н.Гринчук, Ю.Ганущак, В.Ковальов, О.Синцова та ін. Результати власних досліджень проблеми наведені в [1-12]. Виокремлення невирішених проблем, яким присвячена стаття. На сьогодні на законодавчому рівні вже створені умови для децентралізації влади та підвищення ролі і значення територіальної громади у забезпеченні ефективності функціонування територій і держави загалом. Стаття 143 Конституції України містить перелік повноважень територіальної громади як первинного суб’єкта місцевого самоврядування, які здійснюються безпосередньо або через утворені ними органи місцевого самоврядування. Органам місцевого самоврядування можуть надаватися законом окремі повноваження органів виконавчої влади. Держава фінансує здійснення цих повноважень у повному обсязі за рахунок коштів Державного бюджету України або шляхом віднесення до місцевого бюджету у встановленому законом порядку окремих загальнодержавних податків, передає органам місцевого самоврядування відповідні об'єкти державної власності. Унаслідок прийняття змін до Податкового кодексу України та Бюджетного кодексу України суттєво розширені фінансові можливості місцевого самоврядування [2]. Але, проведені нами дослідження показали, що поряд з розширенням дохідної частини місцевих бюджетів, відбувся перерозподіл і видатків, перш за все соціального характеру. За таких умов існує необхідність пошуку ефективної моделі управління фінансовими ресурсами територіальної громади на початкових стадіях функціонування. Формулювання цілей статті. Основним завданням роботи є моделювання системи управління фінансовими ресурсами територіальної громади з метою створення механізму, що дозволять зробити територіальні громади економічно самодостатніми та фінансово спроможними. Виклад основного матеріалу дослідження з повним обґрунтуванням отриманих наукових результатів. У Конституції України територіальна громада визначена як первинний суб’єкт місцевого самоврядування. Відповідно до статті 140, місцеве самоврядування є правом територіальної громади − жителів села чи добровільного об'єднання у сільську громаду жителів кількох сіл, селища та міста − самостійно вирішувати питання місцевого значення в межах Конституції і законів України. Закон «Про місцеве самоврядування в Україні» містить таке визначення: територіальна громада − жителі, об'єднані постійним проживанням у межах села, селища, міста, що є самостійними адміністративнотериторіальними одиницями, або добровільне об'єднання жителів кількох сіл, що мають єдиний адміністративний центр. Стаття 143 Конституції України містить перелік повноважень територіальної громади як первинного суб’єкта місцевого самоврядування, які здійснюються безпосередньо або через утворені ними органи місцевого самоврядування. Це управління майном, що є в комунальній власності; затвердження програм соціально-економічного та культурного розвитку і контроль їх виконання; затвердження бюджетів відповідних адміністративно-територіальних одиниць і контроль їх виконання; встановлення місцевих податків і зборів відповідно до закону; забезпечення проведення місцевих референдумів та реалізації їх результатів; утворення, реорганізація та ліквідація комунальних підприємств, організацій і установ, а також здійснення контролю за їх діяльністю; вирішення інших питань місцевого значення, віднесених законом до їхньої компетенції. Економічний вісник університету \| Випуск № 36/1 287 ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ Органам місцевого самоврядування можуть надаватися законом окремі повноваження органів виконавчої влади. Держава фінансує здійснення цих повноважень у повному обсязі за рахунок коштів Державного бюджету України або шляхом віднесення до місцевого бюджету у встановленому законом порядку окремих загальнодержавних податків, передає органам місцевого самоврядування відповідні об'єкти державної власності. Унаслідок прийняття змін до Податкового кодексу України та Бюджетного кодексу України суттєво розширені фінансові можливості місцевого самоврядування [2]. На сьогодні держава здійснює суттєву фінансову підтримку процесу децентралізації. Так, у Державному бюджеті на 2016 році із загального фонду державного бюджету місцевим бюджетам були надані субвенції на формування інфраструктури об’єднаних територіальних громад на суму 1 млрд. грн. Ці кошти були спрямовані на підтримку об’єднаних територіальних громад, розвиток інфраструктури та адміністративних центрів в громадах. Обсяг фінансування із загального фонду Державного бюджету Державного фонду регіонального розвитку склав 3 млрд. грн. Додатково було передбачено 1,94 млрд. грн. субвенції місцевим бюджетам на соціально-економічний розвиток окремих територій та 0,95 млрд. грн. коштів для капітальних видатків за програмою «Підтримка державних та регіональних інвестиційних проектів». Загалом, у Державному бюджеті на 2016 рік було закладено кошти для 159 об’єднаних територіальних громад. За час реформ державна підтримка регіонального розвитку та розвитку інфраструктури громад за час реформи зросла у 30 разів: з 0,5 млрд в 2014 до 14,9 млрд грн у 2017 році. На розвиток територій у 2018 році з Державного бюджету планують направити близько 37,8 млрд грн. Ця сума не включає в себе освітню та медичну субвенції, додаткові дотації, субвенції і дотації соціального захисту населення. Заплановані такі напрями та програми, на фінансування яких у 2018 році виділятимуться кошти з державного бюджету: 1) За бюджетною програмою «Загальнодержавні заходи у сферах культури та мистецтв, охорони культурної спадщини, вивезення, ввезення і повернення культурних цінностей, державної мовної політики, міжнаціональних відносин, релігії та захисту прав національних меншин» на підтримку та розвиток петриківського розпису передбачено 5 млн грн та на розвиток української пісні – 15 млн грн. 2) Субвенція на здійснення заходів щодо соціально-економічного розвитку окремих територій – 5 млрд грн. 3) Субвенція на реалізацію заходів, спрямованих на розвиток системи охорони здоров'я у сільській місцевості – 1 млрд грн. 4) Державна підтримка будівництва (придбання) доступного житла – 100 млн грн (головний розпорядник коштів (ГРК) - Мінрегіон). 5) Підтримка регіональної політики – 449,5 млн грн (ГРК - Мінрегіон). 6) Фонд енергоефективності - 1,6 млрд грн (ГРК - Мінрегіон). 7) Будівництво футбольних полів зі штучним покриттям в регіонах України – 370 млн грн (ГРКМінрегіон). 8) Фінансова підтримка Державного фонду сприяння молодіжному житловому будівництву – 6,85 млн грн (ГРК - Мінрегіон). 9) Реалізація Загальнодержавної цільової програми «Питна вода України» – 200 млн грн (ГРК - Мінрегіон). 10) Впровадження та координація заходів проекту розвитку міської інфраструктури, заходів в секторі централізованого теплопостачання України, надзвичайної кредитної програми для України, програми розвитку муніципальної інфраструктури України та заходів з відновлення сходу України – 88,9 млн грн (ГРК - Мінрегіон). 11) Реалізація надзвичайної кредитної програми для відновлення України – 50 млн грн (ГРК Мінрегіон). 12) Реалізація Державної цільової економічної програми енергоефективності – 400 млн грн (ГРК - Державне агентство з енергоефективності та енергозбереження України). 13) Державний фонд регіонального розвитку (ДФРР) – 6 млрд грн. 14) Субвенція з державного бюджету місцевим бюджетам на формування інфраструктури об'єднаних територіальних громад – 1,9 млрд грн. 15) Субвенція з державного бюджету місцевим бюджетам на реалізацію проектів в рамках Надзвичайної кредитної програми для відновлення України – 1,5 млрд грн. 16) Фінансова підтримка заходів в агропромисловому комплексі шляхом здешевлення кредитів – 66 млн грн (ГРК – Мінагропром) 288 Економічний вісник університету \| Випуск № 36/1 ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ 17) Підвищення кваліфікації фахівців агропромислового комплексу – 21,5 млн грн (ГРК – Мінагропром). 18) Фінансова підтримка заходів в агропромисловому комплексі – 5 млн грн (ГРК – Мінагропром). 19) Фінансова підтримка розвитку фермерських господарств – 1 млрд грн (ГРК – Мінагропром). 20) Державна підтримка розвитку хмелярства, закладення молодих садів, виноградників та ягідників і нагляд за ними – 300 млн грн (ГРК – Мінагропром). 21) Державна підтримка галузі тваринництва – 4 млрд грн (ГРК – Мінагропром). 22) Фінансова підтримка сільгосптоваровиробників – 945 млн грн (ГРК – Мінагропром). 23) Субвенція з державного бюджету місцевим бюджетам на фінансове забезпечення будівництва, реконструкції, ремонту і утримання автомобільних доріг загального користування місцевого значення, вулиць і доріг комунальної власності у населених пунктах – 11,5 млрд грн (Укравтодор). 24) Субвенція з державного бюджету місцевим бюджетам на будівництво/реконструкцію палаців спорту - 150 млн грн (Мінмолодьспорт). 25) Субвенція з державного бюджету місцевим бюджетам на здійснення заходів щодо підтримки територій, що зазнали негативного впливу внаслідок збройного конфлікту на сході України – 34 млн грн. 26) Державна підтримка заходів, спрямованих на зменшення обсягів викидів (збільшення абсорбції) парникових газів, у тому числі на утеплення приміщень закладів соціального забезпечення, розвиток міжнародного співробітництва з питань зміни клімату – 1 млрд грн. 27) Першочергове забезпечення сільських населених пунктів централізованим водопостачанням – 125 млн грн. Але, на нашу думку, фінансова підтримка реформ адміністративно-територіального устрою України не повинна перетворитись на перерозподіл коштів Державного бюджету на користь окремих територіальних громад та їх об’єднань. Схожий механізм вже існував у державі – здійснювався перерозподіл коштів Державного бюджету на користь певних регіонів, але вин виявив свою неспроможність. Тобто кінцевою метою реформ повинно стати створення фінансово спроможних і самодостатніх територіальних громад. А держава повинна сприяти цьому, а не постійно забезпечувати процес функціонування громад. Таким чином, створення умов стабільного розвитку територіальних громад, на нашу думку, проходить певні стадії: 1) Мінімізація збитків, досягнення беззбитковості та формування умов самодостатності у фінансовому забезпеченні. 2) Досягнення прибутковості та рентабельності. 3) Досягнення рівня фінансової стійкості, достатнього для сталого розвитку територій. З врахуванням зазначених стадій доцільно регулювати обсяги державних видатків на розвиток територіальних громад. Найбільшу фінансову допомогу з боку держави територіальні громади потребують на перших двох стадіях. Безумовно, що перш за все території необхідно вивести з кризового стану, забезпечити, як мінімум можливість досягнення беззбитковості (перша стадія). На другій стадії роль держави набуває особливого значення, оскільки отримання прибутку та досягнення рентабельності територіальних громад не гарантує переходу територій до сталого розвитку. На третій стадії, залишається необхідною потреба у зовнішньому фінансуванні з боку держави. Але на цьому етапі більшість територій вже повинна знаходиться на тому рівні фінансової стійкості, на якому більшу частину її прибутковості і рентабельності можливо забезпечити за рахунок внутрішньогалузевих ресурсів та можливостей. Відсутність у подальшому підвищення рівня фінансування на розвиток територіальних громад державою зумовлена тим, що основною метою процесу децентралізації та оптимізації фінансового механізму управління, на нашу думку, є не принцип максимізації прибутку, а принцип створення умов сталого розвитку за рахунок оптимального використання власних фінансових і матеріальних ресурсів. З урахуванням зазначених особливостей, можливо запропонувати таку схему управління державним фінансуванням розвитку територіальних громад. На перших двох стадіях доцільним є експоненційне зростання обсягів державної підтримки. На третій стадії, зникає необхідність стрімкого зростання видатків, їх можливо зафіксувати на певному достатньому для підтримки сталого розвитку рівні. Таку ситуацію найкраще можливо описати логістичною кривою. На нашу думку для того, щоб описати динаміку зміни обсягів фінансування територіальних громад на перших двох стадіях доцільно використати математичний апарат опису динамічних систем – диференційні рівняння. Існує потреба у моделюванні динаміки та обсягів, по-перше, Економічний вісник університету \| Випуск № 36/1 289 ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ додаткового фінансування для досягнення беззбитковості діяльності громад; по-друге – загального обсягу необхідних фінансових ресурсів. Приріст обсягів додаткового фінансування dN громад в деякому інтервалі часу t буде пропорційним величині обсягів фінансування – N. Цей приріст пропорційний довжині малого інтервалу. Приписуючи цю властивість функції, що розглядається як безперервна, одержуємо: dN = εN dt, (1) де ε – постійний коефіцієнт пропорційності, що виражає відношення швидкості приросту обсягів фінансування З рівняння dN до N. Назвемо його коефіцієнтом приросту обсягів фінансування. dt dN = εN dt інтегруванням одержимо N = N0 е ε (t − t 0) , (2) де Т = t – t0 Це експоненціальний закон, сутність якого полягає в тому, що при зростанні часу в арифметичній прогресії, обсяги фінансування змінюються в геометричній прогресії. Практично можливо визначити число ε, що характеризує темп зміни обсягів додаткового фінансування громад. Дійсно, за час Т = t – t0 обсяги фінансування збільшуються в еεТ раз, що більше, дорівнює чи менше одиниці, у залежності від того, збільшується, зменшується чи залишається незмінним обсяг фінансування. При цьому ε не залежить від початкового моменту часу. Таким чином, для опису динаміки зміни обсягів додаткового фінансування для досягнення беззбитковості громад можливо запропонувати функцію (2). Динаміку зміни обсягів державного фінансування також можливо описати функцією (2). Модель, описана формулою (2) є певною мірою ідеалізованою, оскільки ми вважали, що зовнішнє середовище незмінне. Якщо ж вважати, що це середовище повільно змінюється, то для короткого відрізку часу можливо вважати, що задача залишається незмінною. Але тоді коефіцієнт приросту 1 dN повільно мінявся б зі зміною зовнішніх умов. Якби був відомий цей закон змін, то N dt мало би місце диференціальне рівняння: dN = ε(t) N (t). dt (3) Таким чином, варто сформулювати гіпотезу про залежність коефіцієнтів приросту від функції N і часу t. Цю гіпотезу математично запишемо у вигляді функцій від N, відкіля одержуємо диференціальні рівняння: 1 dN i = f ( N 1 , N 2 ,... N p ). N i dt (4) dN ε 2 = εN – N , dt К (5) Якщо ж коефіцієнти приросту залежать не тільки від миттєвої величини N1, але і від значень у попередні періоди, їх не можливо розглядати як функції від N1. У цьому випадку виникає необхідність розглядати складні інтегро-диференціальні рівняння, що дає можливість при модулюванні фінансових ресурсів територіальних громад врахувати зовнішні можливості стабілізації її фінансового стану. Ними, на нашу думку, можуть бути пошук додаткових інвестиційних ресурсів, поряд з видатками бюджету; впровадження заходів щодо зменшення ресурсо- та енергомісткості та ін. На третій стадії більш оптимальною моделлю динаміки бюджетного фінансування буде не експоненційна, а логістична модель. Таким чином, динаміку обсягів фінансових ресурсів, що формують достатній рівень фінансово-економічної безпеки територіальної громади, можливо описати логістичним рівнянням: де ε – постійний коефіцієнт пропорційності, що є відношенням швидкості приросту обсягів фінансування 290 dN до N; dt Економічний вісник університету \| Випуск № 36/1 ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ К = Nmax – максимально можливий і безпечний обсяг додаткового бюджетного фінансування територіальної громади. Рівняння (5) на відміну від (1) є нелінійним диференціальним рівнянням. Відомо, що нелінійні залежності краще описують реальні економічні системи. Рівняння (5) описує динаміку зміни обсягів необхідного додаткового бюджетного фінансування територіальних громад за умови відсутності зовнішнього впливу на систему, тобто дає можливості моделювати вплив внутрішніх чинників. Серед основних внутрішніх резервів зниження обсягів необхідного фінансування відзначимо, в першу чергу, оптимізація системи управління власними фінансовими ресурсами, необхідність зниження собівартості товарів, робіт та послуг за рахунок зменшення ресурсо- та енергомісткості, непродуктивних втрат ресурсів і т. ін. Окрім внутрішніх резервів необхідне зовнішнє втручання, що забезпечить можливість зменшення обсягів необхідного фінансування. Для того, щоб описати динаміку процесу додаткового фінансування територіальних громад з урахуванням зовнішніх чинників, пропонуємо використати модель: ε 2 dN = εN – N – q, К dt (6) де q – складова, що враховує інтенсивність впливу. Основним фактором зовнішнього впливу є неконкретизованість економічних прав територіальних громад України та невизначеність механізмів їх реалізації. Так в Україні наявні територіальні громади, що позбавлені реальної можливості управління земельними ресурсами, що є основою їхнього розвитку; не завжди територіальні громади мають можливість реалізувати свої права на комунальне майно, оскільки відсутні публічні реєстри об’єктів комунальної власності, які належать окремим громадам; для більшості поселень досі не визначені територіальні межі, не забезпечено реалізацію механізму правонаступництва на об’єкти комунальної власності та ін. У зв’язку з цим, зазначена проблема потребує додаткового дослідження. На початку процесу реформування адміністративно-територіального устрою на принципах децентралізації було визначено, що державна підтримка розвитку територіальних громад буде тривати п’ять років. З них три – вже пройшли. Крім того, як бачимо з наведених у другому розділі даних, загальна сума підтримки дійсно щорічно зростає, але одночасно зростає і кількість територіальних громад. Виникає важливе питання – що відбудеться після 2019 року, коли буде припинена державна підтримка? На нього є одна відповідь – в межах територіальної громади повинні бути створений механізм беззбиткового її функціонування. Висновки з даного дослідження і перспективи подальших розвідок у даному напрямку. Показано, що унаслідок прийняття змін до Податкового кодексу України та Бюджетного кодексу України суттєво розширені фінансові можливості місцевого самоврядування. На сьогодні держава здійснює суттєву фінансову підтримку процесу децентралізації. Доведено, що фінансова підтримка реформ адміністративно-територіального устрою України не повинна перетворитись на перерозподіл коштів Державного бюджету на користь окремих територіальних громад та їх об’єднань. Тобто кінцевою метою реформ повинно стати створення фінансово спроможних і самодостатніх територіальних громад.. В роботі проведене моделювання етапів та умов забезпечення сталого розвитку територіальних громад. Доведено, що необхідно виокремити три стадії: 1) мінімізація збитків, досягнення беззбитковості та формування умов самодостатності у фінансовому забезпеченні; 2) досягнення прибутковості та рентабельності; 3) досягнення рівня фінансової стійкості, достатнього для сталого розвитку територій. Найбільшу фінансову допомогу з боку держави територіальні громади потребують на перших двох стадіях. На третій стадії, залишається необхідною потреба у зовнішньому фінансуванні з боку держави. Але на цьому етапі більшість територій вже повинна знаходиться на тому рівні фінансової стійкості, на якому більшу частину її прибутковості і рентабельності можливо забезпечити за рахунок внутрішньогалузевих ресурсів та можливостей. У зв’язку з цим, запропоновано на перших двох стадіях планувати експоненційне зростання обсягів державної підтримки. На третій стадії, зникає необхідність стрімкого зростання видатків, їх можливо зафіксувати на певному достатньому для підтримки сталого розвитку рівні. Таку ситуацію найкраще можливо описати логістичною кривою. Економічний вісник університету \| Випуск № 36/1 291 ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ Література 1. Територіальна громада як базова ланка адміністративно-територіального устрою України: проблеми та перспективи реформування. − К.: НІСД, 2016. − 61 с. 2. Батанов О.В. Територіальна громада – первинний суб’єкт муніципальної влади в Україні: поняття та ознаки / О.В. Батанов : [Електронний ресурс]. – Режим доступу: http://www.cvk.gov.ua/visnyk/pdf/2008_2/visnik_st_13.pdf. 3. Муркович Л. Територіальна громада як суб’єкт місцевого самоврядування в Україні: теоретичні аспекти / Л. Муркович : [Електронний ресурс]. – Режим доступу: http://www.dbuapa.dp.ua/vidavnictvo/2010/2010_02%285%29/10mliuta.pdf. 4. Молодожен Ю.Б. Поняття і сутність територіальної громади в системі місцевого самоврядування України / Ю.Б. Молодожен // Університетські наукові записки: Часопис Хмельницького університету. – 2006. – № 2. – С. 128-136. 5. Горошкова Л. А. Управління фінансовими потоками програм створення територіальних громад / Л. А. Горошкова, В. П. Волков, Г. В. Коваленко // Управління проектами та розвиток виробництва. – 2017. - №1 (61). – С.93-100. 6. Горошкова Л. А. Створення та управління продовольчими ресурсами територіальної громади / Л. А.Горошкова, В. П. Волков, Р. О. Карбівничий // Управління проектами та розвиток виробництва. – 2017. - №1 (61). – С.100-108. 7. Горошкова Л. А. Технологія якісного і кількісного управління територіальними громадами / Л. А. Горошкова, В. П. Волков, Г. В. Коваленко, Р. О. Карбівничий // Управління проектами та розвиток виробництва. – 2017. - №2 (62). – С. 29-41. References 1. Territorial community as a base part of the administrative - territorial device of Ukraine: problems and prospects of reforming. The analytical report. - К.: NISI, 2016. - 61 p. 2. Batanov О.V. A territorial community - primary subject of municipal authority in Ukraine: concept and attribute : http://www.cvk.gov.ua/visnyk/pdf/2008_2/visnik_st_13.pdf. 3. Murkovich L.A. territorial community as the subject of local self-management in Ukraine: theoretical aspects : http://www.dbuapa.dp.ua/vidavnictvo/2010/2010_02%285%29/10mliuta.pdf. 4. Molodozhen Y.B. Concept and essence of a territorial community in system of local selfmanagement of Ukraine // University scientific slip: a magazine of Khmelnitskiy university. - 2006. - № 2. - P. 128-136. 5. Horoshkova L., Volkov V., Kovalenko G. Management of financial flows of the programs of creation of territorial communities // Management of the projects and development of manufacture. - 2017. - №1 (61). – P. 93-100. 6. Horoshkova L., Volkov V., Karbivnychyi Р. Creation and management of food resources of a territorial community // Management of the projects and development of manufacture. - 2017. - №1 (61). – P. 100-108. 7. Horoshkova L., Volkov V., Kovalenko G., Karbivnychyi Р. Technology of qualitative and quantitative management of territorial communities // Management of the projects and development of manufacture. - 2017. - №2 (62). – P. 29-41. ДАНІ ПРО АВТОРІВ Горошкова Лідія Анатоліївна, д.е.н., доцент, академік Академії економічних наук України, професор кафедри підприємництва, менеджменту організацій та логістики e-mail: goroshkova69@gmail.com Волков Володимир Петрович, д.т.н., професор, академік Академії економічних наук України, професор кафедри підприємництва, менеджменту організацій та логістики e-mail: volkovvp49@gmail.com Запорізький національний університет, вул.Жуковського, 66, Запоріжжя 69600, Україна ДАННЫЕ ОБ АВТОРАХ Горошкова Лидия Анатольевна, д.э.н., доцент, академик Академии экономических наук Украины, профессор кафедры предпринимательства, менеджмента организаций и логистики e-mail: goroshkova69@gmail.com Волков Владимир Петрович, д.т.н., профессор, академик Академии экономических наук Украины, профессор кафедры предпринимательства, менеджмента организаций и логистики 292 Економічний вісник університету \| Випуск № 36/1 ФІНАНСИ, ГРОШОВИЙ ОБІГ ТА КРЕДИТ e-mail: volkovvp49@gmail.com Запорожский национальный университет, ул.Жуковского, 66, Запорожье 69600, Украина DATA ABOUT THE AUTHORS Goroshkova Lidiia, D. Sc. in Economics, Professor of the Chair of entrepreneurship, management of organizations and logistics, e-mail: goroshkova69@gmail.com Volkov Vladimir, D. Sc. in technical, professor, Professor of the Chair of entrepreneurship, management of organizations and logistics, e-mail: volkovvp49@gmail.com Zaporizhzhya National University, Zhukovskogo str., 66 Zaporizhzhya 69600 Ukraine УДК 338.242.2 ВДОСКОНАЛЕННЯ ДЕРЖАВНОЇ ФІНАНСОВОЇ ПОЛІТИКИ ПІДТРИМКИ ПУБЛІЧНОПРИВАТНОГО ПАРТНЕРСТВА ЯК ІНСТРУМЕНТУ СОЦІАЛЬНОГО РОЗВИТКУ Качула С.В. Предметом дослідження є теоретичні та практичні питання державної фінансової політики розвитку публічно-приватного партнерства в Україні. Мета дослідження: аналіз впливу державної фінансової політики на становлення публічноприватного партнерства (ППП) в Україні як інструменту реалізації соціальних цілей держави/органів місцевого самоврядування і окреслення напрямів удосконалення фінансового інструментарію підтримки пріоритетних соціальних проектів ППП. Методи дослідження. У роботі застосовано сукупність наукових методів і підходів, у тому числі аналізу, системний, порівняльний, що дозволило реалізувати концептуальну єдність дослідження. Результати роботи. У статті розглянуто питання розвитку публічно-приватного партнерства (ППП) як інструменту соціального розвитку. Показано, що сьогодні важливим завданням державної фінансової політики є подальший розвиток публічно-приватного партнерства, як інструменту забезпечення ефективного фінансування суспільного розвитку. Проаналізовано нормативно-правову базу у аспекті регулювання державної фінансової підтримки проектів ППП в Україні. Визначено основні чинники, що впливають на дієвість інструментарію державної фінансової політики підтримки ППП в сучасних умовах функціонування національної соціально-економічної системи. Галузь застосування результатів: система державного фінансового регулювання, державна фінансова політика. Висновки. Удосконалення напрямів фінансування проектів ППП повинне здійснюватися у руслі підвищення результативності використання бюджетних коштів, удосконалення середньо- та довгострокового бюджетного прогнозування та планування, наукового обгрунтування пріоритетів суспільного розвитку. Вдосконалення інструментарію державної фінансової політики в Україні для вирішення пріоритетних цілей суспільного розвитку необхідно здійснювати з урахуванням сформованого інституційного середовища та можливостей його покращення, поглиблення процесів децентралізації та стану державних і місцевих фінансів, дотриманням вимоги бюджетної стійкості та боргової безпеки. Ключові слова: публічно-приватне партнерство (ППП), соціальний розвиток, бюджетні кошти, державна фінансова політика. IMPROVEMENT OF THE STATE FINANCIAL POLICY OF SUPPORT OF PUBLIC-PRIVATE PARTNERSHIPS AS AN INSTRUMENT FOR SOCIAL DEVELOPMENT Kachula S.V. The subject of research are theoretical and practical questions of the state financial policy of the development of Public-Private Partnership in Ukraine as an instrument of social development. © Качула С.В., 2018 Економічний вісник університету \| Випуск № 36/1 293
https://openalex.org/W2140341406	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Figure_Legend_from_Personalizing_the_Treatment_of_Pediatric_Medulloblastoma_Polo-like_Kinase_1_as_a_Molecular_Target_in_High-Risk_Children/22400492/1/files/39846257.pdf	English	null	Personalizing the Treatment of Pediatric Medulloblastoma: Polo-like Kinase 1 as a Molecular Target in High-Risk Children	Cancer research	2,013	cc-by	859	Supplementary Table S2. Summary of the pediatric MB patients included in the study Validation cohort. All of the children were referred to The Hospital for Sick Children (Sick Kids), Toronto, ON. Supplementary Table S3. PAM class prediction validation for MB subgroup assignment of Discovery cohort and cultured cells. Supplementary Figure Legends Supplementary Table S2. Summary of the pediatric MB patients included in the study Validation cohort. All of the children were referred to The Hospital for Sick Children (Sick Kids), Toronto, ON. Supplementary Figure Legends Supplementary Table S1. Summary of the pediatric MB patients included in the study Discovery cohort. All of the children were referred to the British Columbia Children’s Hospital between 1986-2011. Supplementary Figure Legends Supplementary Table S1. Summary of the pediatric MB patients included in the study Discovery cohort. All of the children were referred to the British Columbia Children’s Hospital between 1986-2011. Supplementary Table S4. PAM class prediction validation for MB subgroup assignment of Validation cohort. Supplementary Figure S1. MB survival in Validation cohort is stratified by molecular subtype. Subtype is a prognostic marker for (A) progression-free survival (n=55, Breslow P=0.186) and (B) overall survival (n=58, Breslow P=0.005) in the Validation cohort. SHH and Group 3 MB did not statistically vary in outcome (Progression-free survival, n=55, P=0.526; Overall Survival, n=58, P=0.814). (C) Patients as well as four MB cell lines were subtyped into the different categories based on gene expression and represented using a heatmap (Red= high expression, Green= low Supplementary Table S3. PAM class prediction validation for MB subgroup assignment of Discovery cohort and cultured cells. Supplementary Table S4. PAM class prediction validation for MB subgroup assignment of Validation cohort. Supplementary Table S4. PAM class prediction validation for MB subgroup assignment of Validation cohort. plementary Figure S2. PLK1 is invariably expressed between patient cohorts Supplementary Figure S2. PLK1 is invariably expressed between patient cohorts and between MB subgroups. Using NanoString nCounter analysis, (A) the average of all MB patient samples were significantly higher then the normal cerebellum and found at similar levels between Discovery (n=63) and Validation cohorts (n=58) (T-test, P=0.625). (B) The Grubb’s test was used to remove single outliers from both data sets. PLK1 was invariably expressed across all four MB subgroups in both the Discovery cohort (Kruskal-Wallis, P=0.071) and Validation cohort (Kruskal-Wallis, P=0.743). (C) Progression or relapse free survival (Log rank P=0.457, Breslow P=0.666) and overall survival (Log rank P=0.158, Breslow P=0.164) between cohorts is not significantly different. Supplementary Figure S1. MB survival in Validation cohort is stratified by Supplementary Figure S1. MB survival in Validation cohort is stratified by molecular subtype. Subtype is a prognostic marker for (A) progression-free survival (n=55, Breslow P=0.186) and (B) overall survival (n=58, Breslow P=0.005) in the Validation cohort. SHH and Group 3 MB did not statistically vary in outcome (Progression-free survival, n=55, P=0.526; Overall Survival, n=58, P=0.814). (C) Patients as well as four MB cell lines were subtyped into the different categories based on gene expression and represented using a heatmap (Red= high expression, Green= low expression). Cell lines (ONS76, UW228, UW426, DAOY) were all statistically classified as SHH (PAM= 1.0E+00). Supplementary Figure S5. MB cell PLK1 inhibition morphology using Supplementary Figure S6. MB cell lines treated with anti-cancer drugs. (A) BI2536 inhibited the self-renewal of Daoy tumorspheres when treated with 5 or 10nM BI2536 for 6 days. (B) The ONS76 and UW426 cells responded to chemotherapeutic agents- vincristine, cisplatin and etoposide in 72hr cell growth assays. Supplementary Figure S5. MB cell PLK1 inhibition morphology using Supplementary Figure S5. MB cell PLK1 inhibition morphology using immunofluorescence. PLK1 was inhibited in Daoy cells using siPLK1 or 10nM BI2536 for 24hrs the stained for alpha-tubulin (green), pericentrin (red), and DAPI nuclear staining (blue). Supplementary Table S5. Univariate and multivariate analyses of clinical, pathological and biological endpoints in the Validation cohort. Supplementary Table S5. Univariate and multivariate analyses of clinical, pathological and biological endpoints in the Validation cohort. Supplementary Figure S3. BI2536 sensitivity depends on PLK1 expression in primary MB cells. (A) Primary cultured Group 4 MB BT025 retained gene expression characteristics of tumor of origin as demonstrated using NanoString analysis of mRNA extracted from primary cultured cells and FFPE tissue from matched patient tumor. (B) qRT-PCR analysis of PLK1 mRNA confirms low expression in BT025 relative to hNSC and Daoy. (C) BT014 were not responsive to 10nM BI2536 (6 day treatment) (scale bar=140μm). (D) PLK1 mRNA levels in freshly isolated patient-derived MB samples (BTX001, BT006, BT007, BT008, BT274), relative to normal human astrocytes (HA). All mRNA levels were quantified by qRT-PCR. Supplementary Figure S4. MB cells lines expressing PLK1 experience cell cycle arrest and reduced proliferation with PLK1 inhibition. (A) PLK1 transcript levels were measured in normal cerebellum, MB cell lines, and patient samples corresponding to culture samples BTX001 and BT014 using NanoString nCounter analysis. (B) PLK1 protein was assessed in Daoy, ONS76 and UW426 MB cell lines. (C) The ONS76 and UW426 cells were sensitive to BI2536 as it inhibited growth in a time and dose- dependent manner. (D) The percent of Ser10 phosphorylated histone H3 positive Daoy cells was measured using high content screening over a 72hr timecourse to examine cell cycle G2/M arrest.
W2017171592.txt	https://journals.plos.org/plosone/article/file?id=10.1371/journal.pone.0067759&type=printable	en		Association of Serum Uric Acid with 2-Hour Postload Glucose in Chinese with Impaired Fasting Plasma Glucose and/or HbA1c	PloS one	2,013	cc-by	5,937	Association of Serum Uric Acid with 2-Hour Postload Glucose in Chinese with Impaired Fasting Plasma Glucose and/or HbA1c Hong-Qi Fan1., Wei Tang2., Zhi-Xiao Wang1., Su-Juan Wang1, Yue-Hua Qin1, Qi Fu1, Yuan Gao1, Min Sun1, Mei Zhang1, Hong-Wen Zhou1, Tao Yang1* 1 Department of Endocrinology, The First Affiliated Hospital of Nanjing Medical University, Nanjing, Jiangsu, China, 2 Department of Endocrinology, The Affiliated Jiangyin Hospital of Southeast University Medical College, Jiangyin, Jiangsu, China Abstract Objective: To examine whether serum uric acid (SUA) is associated with 2-hour postload glucose (2-h PG) in Chinese with impaired fasting plasma glucose (IFG) and/or HbA1c (IA1C). Research Design and Methods: Anthropometric and biochemical examinations, such as SUA concentration, were performed in 3763 individuals from all the villages in Baqiao County, China. A 75-g oral glucose tolerance test (OGTT) was conducted in 1197 Chinese with prediabetes as having IFG (110# fasting plasma glucose [FPG] ,126 mg/dl and HbA1c ,6.5%), IA1C (5.7% # HbA1c ,6.5% and FPG ,126 mg/dl), or both. Results: The present study included 1197 participants with IFG and/or IA1C (mean age 56.5610.3 years; 50.6% men). In multivariate linear regression, after adjustment for gender, age, smoking and drinking, body mass index (BMI), systolic and diastolic blood pressure (SBP, DBP), lipid profiles, logarithmic transformed C-reactive protein (log-CRP), estimated glomerular filtration rate (e-GFR), FPG and HbA1c, with a 1-mg/dl increment of SUA, 2-h PG increased by 5.0460.72 (P,0.001), 3.0661.08 (P = 0.001), 5.4061.26 (P,0.001), and 2.3462.16 mg/dl (P = 0.056) in all participants, in participants with normal glucose tolerance (NGT), with impaired glucose tolerance (IGT), and with 2-h newly diagnosed diabetes (2-h NDM, with 2-h PG $200 mg/dl), respectively. In both men and women, 2-h PG increased progressively and significantly from the lower to the upper SUA tertiles (P,0.001). Moreover, in multivariate logistic regression, 1-standard deviation (SD; 1.53 mg/dl) increment of SUA was significantly associated with a 36% higher risk for 2-h NDM (Odds ratio [CI 95%]: 1.36 [1.09–1.99]; P = 0.03). Conclusions: SUA is significantly associated with 2-h PG in Chinese with IFG and/or IA1C. Citation: Fan H-Q, Tang W, Wang Z-X, Wang S-J, Qin Y-H, et al. (2013) Association of Serum Uric Acid with 2-Hour Postload Glucose in Chinese with Impaired Fasting Plasma Glucose and/or HbA1c. PLoS ONE 8(7): e67759. doi:10.1371/journal.pone.0067759 Editor: Yiqing Song, Brigham & Women’s Hospital, and Harvard Medical School, United States of America Received March 2, 2013; Accepted May 22, 2013; Published July 3, 2013 Copyright: ß 2013 Fan et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This study was supported by grants from the National Natural Science Foundation of China (81100578), the National Key Technologies R&D Program of China, during the 11th Five-Year Plan Period (2007BAI07A10) and the Jiangsu Province Science and Technology Support Program (BE2009613). Competing Interests: The authors have declared that no competing interests exist. * E-mail: yangt@njmu.edu.cn . These authors contributed equally to this work. [IA1C] of 5.7–6.4%) are at high risk of future type 2 diabetes, with 70% of them developing type 2 diabetes within 10 years [10]. More importantly, patients with prediabetes seem to share the similar concomitant damage to end target organs, as patients with diabetes [11]. High blood glucose concentration or elevated HbA1c was shown to be a risk factor for cardiovascular complications, even in nondiabetic individuals [12]. Although the underlying mechanism is still controversial, fasting plasma glucose (FPG) and HbA1c, the most common glycemic indexes, could not completely explain the observed risk. Recently, in the Diabetes Epidemiology: Collaborative Analysis of Diagnostic Criteria in Europe (DECODE) study, it was demonstrated that FPG concentrations alone could not identify individuals at increased risk of cardiovascular complications associated with hyperglycemia, and the oral glucose tolerance Introduction Serum uric acid (SUA), the end product of purine metabolism, possesses both antioxidant and pro-oxidant properties, which depend on its chemical microenvironment. In clinical investigations, SUA was reported to be associated with gout, hypertension, atherosclerosis, metabolic syndrome, diabetes and prediabetes [1– 3]. More recently, hyperuricemia was documented in subjects with cardiovascular diseases [4] and recognized as an independent predictor of myocardial infarction and stroke [5]. On the other hand, type 2 diabetes is a recognized and independent risk factor for cardiovascular disease [6–8], even in the absence of coronary artery disease or hypertension [9]. Patients with prediabetes (based on impaired fasting glucose [IFG], impaired glucose tolerance [IGT], or impaired HbA1c PLOS ONE \| www.plosone.org 1 July 2013 \| Volume 8 \| Issue 7 \| e67759 Uric Acid and 2-Hour Postload Glucose test (OGTT) could provide additional prognostic information [13]. In addition, in the Diabetes Control and Complications Trial, it was reported that the degree of glucose load, which was not completely reflected by mean HbA1c, was more strongly associated with the observed risk of cardiovascular diseases [14]. Although many studies have indicated a critical role of postload glucose in the development of complications, the postchallenge values are still frequently being neglected [15]. This is probably due to the inconvenience and costs of an OGTT measurement. Considering the importance of postload hyperglycemia in the DECODE study [13] and others [14,16,17], and the crucial role of SUA in the development of cardiovascular diseases [2–5], we therefore aimed to investigate the association of SUA and 2-h PG in patients with IFG and/or IA1C. Laboratory Determinations All laboratory measurements were performed after a fasting of at least 12 h. Plasma glucose (with an intra-assay coefficient of variation [CV] of 2.4% and interassay CV of 3.5%), triglyceride, total and low-density lipoprotein (LDL) and high-density lipoprotein (HDL) cholesterol concentrations, serum creatinine, and SUA were measured by enzymatic methods (Chemistry Analyzer Au2700, Olympus Medical Engineering Company, Japan). Hyperuricemia was defined as $7 mg/dl (in men) or $6 mg/dl (in women). Serum insulin was assessed in duplicate by a highly specific radioimmunoassay using two monoclonal antibodies (intra-assay CV, 2.5%; interassay CV, 3.7%). C-reactive protein (CRP) was measured by a high-sensitivity turbidimetric immunoassay (Behring, Marburg, Germany). Values of estimated glomerular filtration rate (e-GFR; mL/min/1.73 m2) were calculated by using the equation proposed by investigators in the Chronic Kidney Disease Epidemiology (CKD-EPI) Collaboration [19]. A simplified 75-g OGTT was performed with 0-, 30-, and 120min sampling for plasma glucose and insulin after a fasting of at least 12 h. Then, 1197 participants were classified into three groups according to the OGTT results (normal glucose tolerance [NGT], 2-h PG ,140 mg/dl; impaired glucose tolerance [IGT], 140#2-h PG ,200 mg/dl; 2-h newly diabetes mellitus [2-h NDM], 2-h PG $200 mg/dl). Hepatic insulin sensitivity was evaluated by HOMA with the equation: (fasting glucose [mmol/L] 6fasting insulin [mU/L])/22.5. Whole body Insulin sensitivity index was evaluated by the Matsuda index with the equation: 10,000/square root of (fasting glucose6fasting insulin) 6 (mean glucose6mean insulin during OGTT). The Matsuda index is strongly related to the euglycemic hyperinsulinemic clamp, the gold standard assessment of insulin sensitivity [20]. Methods Study Population The present study was conducted from January to May in 2010 in the framework of routine health examinations in all the villages in Baqiao County in Gaoyou, a newly established rural residential area about 299 kilometers northwest to Shanghai, China. Study protocol was approved by the Ethics Committee of the First Affiliated Hospital of Nanjing Medical University. From the local authorities, we obtained the population data of 10008 inhabitants aged from 18 to 74 years. Proportionately stratified random sampling was used to select a representative sample from the total population, and the population size was set at 5000 participants. 3918 individuals, out of the 5000 (78.4%), participated after having given informed written consent. 155 individuals were excluded from the present study, because they had established diabetes (n = 82; defined as the use of glucose-lowering agents, or diagnosed diabetes, or both), missing information on history of diabetes (n = 17), FPG (n = 18) or HbA1c (n = 8), selfreported gout or using of allopurinol or uricosuric agents (n = 30). After further exclusion of individuals with normal FPG (,110 mg/dl) and HbA1c (,5.7%) (n = 1831) and newly diabetes (FPG$126 mg/dl, or HbA1c$6.5% or both [n = 272]), 1660 individuals, out of the 3763, with IFG (110#FPG,126 mg/dl and HbA1c,6.5%), or IA1C (HbA1c: 5.7% # HbA1c,6.5% and FPG,126 mg/dl), or both, were selected and invited for further OGTT, and eventually 1197 individuals with prediabetes as having IFG and/or IA1C participated in the present study. Statistics Analysis SAS software, version 9.1 (SAS Institute Inc), was applied for database management and statistical analyses. Skewed variables were log-transformed. Means and proportions were compared by ANOVA and Fisher’s exact test, respectively. Multivariate linear regression was applied to investigate the determinants of 2-h PG in patients with different glucose tolerance (NGT, IGT, 2-h NDM) in a full adjustment model including covariables of age, BMI, current smoking and drinking habits, SBP and DBP, lipid parameters, eGFR, FPG, HbA1c and logarithmic transformed CRP (log-CRP). Multivariate logistic regression was used to study risk factors of 2-h NDM (0, 1) in a stepwise model, with age, FPG and HbA1C forced in the model. We stratified the participants into three groups by the tertiles of their SUA in men and women, respectively, and investigated their association with 2-h PG by general linear model analysis. Gender interaction analysis was performed by general linear model analysis with gender, SUA and gender*SUA put in the model. Correlations of log-CRP and insulin sensitivity with SUA were studied with Pearson’s method. P,0.05 was considered statistically significant. Field Work Blood pressure (BP) was measured by trained physician using a mercury sphygmomanometer according to the guidelines of the British Hypertension Society [18]. After at least 5 min rest, BP was measured five times with 2-minute interval on the right arm of each participant in the sitting position. These five readings were averaged for further analysis. Hypertension was defined as systolic BP (SBP) at least 140 mmHg and/or diastolic BP (DBP) at least 90 mmHg, and/or use of antihypertensive medication. The same observer also administered a standardized questionnaire to collect information on medical history, smoking habits, alcohol consumption, and the use of medications. Smoking and drinking were defined as at least smoking one cigarette per day or drinking once per week in the past year, respectively. Body weight and body height were measured in each participant, and BMI was calculated as a ratio of the body weight in kilograms to the square of the height in meters. PLOS ONE \| www.plosone.org Results Study Population Characteristics of the 1197 patients with IFG and/or IA1C are presented in Table 1 according to their glucose tolerance, with NGT (n = 837), IGT (n = 309), and 2-h NDM (n = 51). There was no significant difference among the three groups in drinking (P = 0.10), DBP (P = 0.05), HDL (P = 0.05), LDL (P = 0.05), creatinine (P = 0.19), and e-GFR (P = 0.52), but a significant difference in gender distribution (P = 0.01). Furthermore, from NGT to 2-h NDM group, a gradual and significant increasing 2 July 2013 \| Volume 8 \| Issue 7 \| e67759 Uric Acid and 2-Hour Postload Glucose In Table 3, multivariate stepwise logistic regression was performed to determine the influential factors of 2-h NDM, with adjustment for age, FPG and HbA1C forced in the model and gender, smoking and drinking, BMI, SBP, DBP, total cholesterol, HDL and LDL, triglycerides, SUA, e-GFR and log-CRP. In addition to age, FPG and HbA1c, SUA was the only factor significantly associated with 2-h NDM (OR = 1.36 [1.09–1.99]; P = 0.03). Of note, SUA was also significantly associated with log-CRP (r = 0.25, P = 0.001) and whole body insulin sensitivity calculated by Matsuda index (r = 20.28, P,0.001) (Fig. 2) but not hepatic insulin sensitivity calculated by HOMA (r = 20.06, P = 0.084). trend was observed in age (P = 0.001), BMI (P,0.001), SBP (P,0.001), total cholesterol (P = 0.04), triglycerides (P,0.001), Log-CRP (P = 0.007), FPG (P,0.001), HbA1c (P,0.001), SUA (P = 0.002) and proportion of hyperuricemia (P = 0.002) and hypertension (P,0.001), but a decreasing trend in the proportion of smoking (P = 0.03). Multivariate Analyses Multiple lineal regression analysis was performed to test the independent association between SUA and 2-h PG, with adjustment for gender, age, smoking and drinking, BMI, SBP, DBP, total cholesterol, HDL and LDL, triglycerides, log-CRP, eGFR, FPG and HbA1c. With a 1-mg/dl increment of SUA, 2-h PG increased by 5.0460.72 (P,0.001), 3.0661.08 (P = 0.001), 5.4061.26 (P,0.001), and 2.3462.16 mg/dl (P = 0.056) in all participants, in participants with NGT, with IGT, and with 2-h NDM, respectively (Table 2). Other independent predictors of 2-h PG were age, BMI, SBP, FPG and HbA1c in all participants; SBP and FPG in participants with NGT; smoking, LDL and HbA1c in participants with IGT; and smoking in participants with 2-h NDM. In Figure 1, 2-h PG levels across gender-specific tertiles of SUA were present. In multivariate general linear regression, 2-h PG gradually and significantly increased from the lower to the upper SUA tertiles in both genders. Although women had a higher 2-h PG than men at each tertile of SUA levels, there was no significant interaction between the gender and the SUA level (P for interaction = 0.89). Discussion The main finding of this study was that elevated SUA in individuals with prediabetes, defined as IFG and/or IA1c, was significantly associated with higher 2-h PG and risk of type 2 diabetes, independent of FPG, HbA1c and other recognized risk factors. Currently, gout and renal disorders were considered as the main consequences of hyperuricemia, and SUA was recognized as a potential risk factor for hypertension [1,21], stroke [5,22], and cardiovascular diseases [23] More recently, some studies suggested that type 2 diabetes was strongly associated with hyperuricemia, and lowering SUA was related to the decreased incidence of diabetes [24]. Some epidemiological studies also indicated that mild-to-moderate elevated FPG was positively associated with SUA, whereas modest or extremely elevated FPG was inversely Table 1. Characteristics in patients with prediabetes according to glucose tolerance. Variables All (n = 1197) NGT (n = 837) IGT (n = 309) 2-h NDM (n = 51) P Male gender, n (%) 606 (50.6) 447 (53.4) 136 (44.0) 23 (45.1) 0.01 Age, years 56.5610.3 55.8610.6 57.569.4 60.868.8 0.001 Smoking (%) 430 (35.9) 321 (38.4) 95 (30.7) 14 (27.5) 0.03 Drinking (%) 285 (23.7) 211 (25.2) 67 (21.7) 7 (13.7) 0.10 ,0.001 BMI, kg/m 2 24.963.2 24.563.1 25.563.3 25.763.5 SBP, mm Hg 141.4620.1 139.8619.7 145.1621.1 146.6620.7 ,0.001 DBP, mm Hg 87.669.8 87.269.8 88.769.7 88.268.9 0.05 Hypertension (%) 320 (26.7) 189 (22.6) 105 (34.0) 26 (51.0) ,0.001 Total cholesterol, mmol/l 5.1460.99 5.0961.00 5.2460.96 5.3161.01 0.04 HDL cholesterol, mmol/l 1.3060.31 1.3160.30 1.2760.31 1.2660.30 0.05 LDL cholesterol, mmol/l 3.0660.71 3.0360.72 3.1460.69 3.1560.72 0.05 Triglycerides, mmol/l 1.6461.53 1.5161.36 1.9161.88 2.0461.76 ,0.001 Log-CRP, mmol/l 0.9260.91 0.8660.95 1.0360.86 1.1960.78 0.007 Creatinine, mmol/l 68.1616.3 68.7616.4 66.7616.3 68.9614.9 0.19 FPG, mg/dl 104.40612.60 102.62610.87 108.04612.72 111.62610.86 ,0.001 2-h PG, mg/dl 7.061.3 5.961.2 9.060.9 12.361.2 ,0.001 HbA1c, % 5.960.3 5.960.3 6.060.3 6.260.4 ,0.001 SUA, mg/dl 5.161.5 5.061.5 5.361.6 5.661.8 0.001 Hyperuricemia (%) 170 (14.2) 103 (12.3) 53 (17.2) 14 (27.5) 0.002 e-GFR, ml/min per 1.73 m2 97.8616.0 98.4616.1 98.0616.2 97.8614.2 0.52 Data were presented as mean (6 SD) or number (%). Means and proportions were compared by ANOVA and Fisher’s exact test, respectively. P values testing the overall difference among NGT, IGT and 2-h NDM groups. Definitions of hypertension, hyperuricemia, 2-h NDM (2-h newly diagnosed diabetes) were in methods. BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high density lipoprotein; LDL, low density lipoprotein; log-CRP, logarithmic transformed Creactive protein; FPG, fasting plasma glucose; 2-h PG, 2 hour postload glucose; SUA, serum uric acid; e-GFR, estimated glomerular filtration rate. International system of units (SI) conversion: plasma glucose 1 mg/dl = 1/18 mmol/l; SUA 1 mg/dl = 59.5 mmol/l. doi:10.1371/journal.pone.0067759.t001 PLOS ONE \| www.plosone.org 3 July 2013 \| Volume 8 \| Issue 7 \| e67759 Uric Acid and 2-Hour Postload Glucose Table 2. Muiltiple linear regression analysis between 2-h PG and different covariates in the entire study population and in groups with different glucose tolerance. All (n = 1197) NGT (n = 837) IGT (n = 309) b ± SE P b ± SE SUA, mg/dl 5.0460.72 ,0.001 3.0661.08 0.001 Age, +10 years 0.2060.06 0.001 0.0760.04 0.10 P b ± SE 2-h NDM (n = 51) P b ± SE 5.4061.26 ,0.001 2.3462.16 0.056 20.0160.01 0.87 0.1960.29 0.53 P Smoking (1 = yes, 2 = no) 0.2160.15 0.18 0.1060.11 0.38 0.3160.14 0.03 0.8260.76 0.04 Drinking (1 = yes, 2 = no) 0.0960.15 0.55 0.0760.11 0.50 0.0360.15 0.82 0.4660.78 0.56 BMI, kg/m2 0.0560.02 0.006 0.0260.01 0.18 0.0260.02 0.27 0.0260.06 0.76 0.41 SBP, +10 mm Hg 0.1160.04 0.06 0.0660.03 0.05 0.0460.03 0.17 0.160.1 DBP, +10 mm Hg 0.0260.08 0.75 0.0860.06 0.88 0.0260.07 0.74 0.260.3 0.62 Total cholesterol, mmol/l 0.2360.24 0.33 0.4260.16 0.80 0.4260.24 0.09 1.7360.99 0.09 HDL cholesterol, mmol/l 20.1560.32 0.64 20.3160.23 0.16 20.5660.31 0.07 21.1061.22 0.37 LDL cholesterol, mmol/l 0.2460.29 0.41 0.0960.20 0.63 0.5960.30 0.05 1.7661.27 0.17 Triglycerides, mmol/l 0.1060.07 0.13 0.0160.05 0.83 0.0960.06 0.14 0.1460.19 0.45 Log-CRP, mmol/l 0.0360.05 0.59 0.0360.04 0.43 0.0260.05 0.75 0.1360.31 0.67 FPG, mg/dl 0.6460.08 ,0.001 0.2260.07 0.001 0.1360.07 0.06 0.4360.29 0.14 HbA1c, % 1.2460.21 ,0.001 0.0860.16 0.60 0.4960.19 0.01 1.1060.64 0.09 0.00260.002 0.32 20.00160.002 0.39 0.00260.003 0.49 0.00160.013 0.93 e-GFR, ml/min per 1.73 m 2 Multiple linear regression analysis was performed to investigate the determinants of 2-h postload glucose, after adjustment for gender, age, smoking and drinking, BMI, SBP, DBP, total cholesterol, HDL and LDL, triglycerides, log-CRP, e-GFR, FPG and HbA1c. Values were regression coefficient (b) 6 standard error (SE). Definitions of NGT (normal glucose tolerance), IGT (impaired glucose tolerance) and 2-h NDM (2-h newly diagnosed diabetes) were in methods. SUA, serum uric acid; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high density lipoprotein; LDL, low density lipoprotein; log-CRP, logarithmic transformed C-reactive protein; FPG, fasting plasma glucose; e-GFR, estimated glomerular filtration rate. International system of units (SI) conversion: plasma glucose 1 mg/dl = 1/18 mmol/l; SUA 1 mg/dl = 59.5 mmol/l. doi:10.1371/journal.pone.0067759.t002 associated with SUA [25–27]. To data, there were limited studies on the relation between SUA and postload plasma glucose, particularly in pre-diabetes, and even the available data were controversial [26,28]. For instance, in the Qingdao study [27], Table 3. Multivariate logistic analysis of the risk factors for 2h NDM (n = 1197). OR (95% CI) P Age, years 1.63 (1.14–2.30) 0.007 HbA1c, % 1.89 (1.44–2.48) ,0.001 FPG, mg/dl 1.70 (1.28–2.25) ,0.001 1.36(1.09–1.99) 0.03 Forced variables Selected SUA, mg/dl Figure 1. 2-h PG levels across the gender-specific tertiles of SUA in men (&) and women (m). Mean 2-h PG levels across genderspecific tertiles of SUA resulting from a general linear model were present, with adjustment for age, smoking and drinking, BMI, SBP, DBP, total cholesterol, HDL and LDL, triglycerides, log-CRP, FPG, HbA1c and e-GFR. Vertical lines denoted standard error (SE). Reference values of 2-h PG were111.78 mg/dl in men and 125.10 mg/dl in women. 2-h PG, 2 hour postload glucose; SUA, serum uric acid; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; HDL, high density lipoprotein; LDL, low density lipoprotein; log-CRP, logarithmic transformed C-reactive protein; FPG, fasting plasma glucose; e-GFR, estimated glomerular filtration rate. International system of units (SI) conversion: plasma glucose 1 mg/dl = 1/18 mmol/l; SUA 1 mg/ dl = 59.5 mmol/l. doi:10.1371/journal.pone.0067759.g001 PLOS ONE \| www.plosone.org Multivariate stepwise logistic regression was performed to determine the influential factors of 2-h NDM (0, 1), in which age, FPG and HbA1C were forced in the model and gender, smoking and drinking, BMI, SBP, DBP, total cholesterol, HDL and LDL, triglycerides, SUA, log-CRP and e-GFR were considered as potential influential factors. OR (odds ration) and 95% CI (confidence interval) was calculated per 10 years in age and per 1-SD in other quantitative variables and presence against absence in qualitative variables. Only significant influential factors were shown in the table. 2-h NDM, 2-h newly diabetes mellitus; BMI, body mass index; SBP, systolic blood pressure; DBP, diastolic blood pressure; FPG, fasting plasma glucose; HDL, high density lipoprotein; LDL, low density lipoprotein; SUA, serum uric acid; log-CRP, logarithmic transformed C-reactive protein; e-GFR, estimated glomerular filtration rate. International system of units (SI) conversion: plasma glucose 1 mg/dl = 1/18 mmol/l; SUA 1 mg/dl = 59.5 mmol/l. doi:10.1371/journal.pone.0067759.t003 4 July 2013 \| Volume 8 \| Issue 7 \| e67759 Uric Acid and 2-Hour Postload Glucose Figure 2. Relation between SUA and log-CRP and insulin sensitivity. Insulin sensitivity was calculated by Matsuda index. SUA, serum uric acid; log-CRP, logarithmic transformed C-reactive protein. International system of units (SI) conversion: plasma glucose 1 mg/dl = 1/18 mmol/l; SUA 1 mg/dl = 59.5 mmol/l. doi:10.1371/journal.pone.0067759.g002 a 36% higher risk for type 2 diabetes. Therefore, if confirmed, this simple and inexpensive measurement of SUA levels in individuals with IFG and/or IA1C would help to screen patients at relatively low risk of future type 2 diabetes, who would not need for further OGTT. This finding would be important, considering the heavy economic burden and expected increasing incidence of type 2 diabetes in rural areas of China. On the other hand, our study also add to the growing body of evidence suggesting that SUA control would be beneficial in the prevention or delay of the deterioration of glucose tolerance in populations at high risk of diabetes. In consistence with our findings, previous studies also indicated the significant association between SUA and insulin resistance predominantly in skeletal muscles [39,40], and the elevated postload glucose would be attributed to the skeletal muscles insulin sensitivity [41], the primary contributor to the whole body insulin sensitivity. This association of SUA with skeletal muscles insulin resistance could be explained by the proinflammatory effects of SUA, namely interfering with postload glucose uptake and impairing blood flow to skeletal muscles [39,40]. In addition, SUA reduces endothelial NO bioavailability in humans [42], animals and cell [43,44], increases CRP and oxygen free radicals production [44], and exerts a direct scavenging effect [45,46]. All factors would interfere with skeletal muscles insulin sensitivity and eventually result in the elevated 2-h PG. In keeping with this, the present study and others showed that CRP was significantly related to SUA (Fig. 2) even after adjustment for other potential confounders (data not shown), confirming the role of SUA-induced inflammation in skeletal insulin resistance. Of note, our findings also showed that whole body insulin sensitivity, but not hepatic insulin sensitivity was significantly related to SUA. Since skeletal muscles insulin sensitivity is the primary contributor to the whole body insulin sensitivity, it seems reasonable to suppose that the underlying mechanism, which links SUA to 2-h PG, might be the SUA-mediated chronic low-grade inflammation and whole body insulin resistance primarily in skeletal muscles. Moreover, we also found that the association of SUA with 2-h PG persisted even after additional adjustment for CRP and whole body insulin sensitivity (data not shown), indicating the exist of the inflammation- and insulin resistance-independent pathways that links SUA to 2-h PG, which need to be investigated in the future studies. The strength of the present study includes: the simultaneous measurement of FPG and HbA1c, no usage of any glucoselowering or SUA-lowering agents, and the population-based design with relatively large sample size and comprehensive SUA was negatively and significantly associated with 2-h PG at the higher range of the 2-h PG distribution (2-h PG $144 mg/dl), but not at the normal range of 2-h PG. However, in another clinical study, SUA was significantly and positively correlated with 2-h PG at the normal range of 2-h PG in nondiabetic Mauritian subjects [28]. Our study, for the first time, demonstrated that SUA in individuals with prediabetes (IFG and/or IA1C) was positively and significantly associated with 2-h PG, independent of FPG, HbA1c and additional recognized risk factors. Furthermore, we also demonstrated that SUA levels had significant impact on the development of diabetes in patients with IFG and/or IA1c, since 1-SD (1.53 mg/dl) increment in SUA signaled a 36% higher risk for type 2 diabetes. Interestingly, a recent study also showed that SUA was strongly associated with 1-h PG in hypertensive NGT individuals, and similar finding could also be observed in hypertensive IGT and diabetes [29]. Our study, together with above mentioned studies, indicated that SUA might be a major determinant of 2-h PG in patients with prediabetes, suggesting the crucial role of SUA in the deterioration of glucose tolerance. A recent study showed that the prevalence of prediabetes was approximately 15% in China [30] and many of them would eventually develop type 2 diabetes [10,31]. Moreover, the proportion of undiagnosed diabetes is also high in China, accounting for 50–80% of the diabetic population [32–34]. Considering the growing burden of prediabetes in China, it is practically important to find a simple and portable measurement, especially in rural areas, to screen patients (IFG and/or IA1C) at relatively high risk of future type 2 diabetes, in terms of early prevention and intervention. Indeed, about half of undiagnosed diabetes in Asia actually met the criteria of diabetes by their elevated 2-h PG in an OGTT, but with their fasting glucose in normal range [35], and previous studies demonstrated that the risk of future diabetes in prediabetes was, at least partly, dependent on their postload glucose level or glucose tolerance status [36,37]. Therefore, the current recommendation is to perform an OGTT in individuals with IFG and/or IA1C to better assess their risk of future type 2 diabetes [38]. However, because of some traditional superstition in China, examination with frequent blood sampling, like OGTT, is thought to be harmful to people’s health, so in many areas in China, especially in the rural area, OGTT is not only a time-consuming and expensive test, but also an ‘‘invasive’’ one. For this reason, OGTT as a mass screening tool may not be suitable in low-income rural areas with limited resources for medical care. In our study, 1-SD increment in SUA levels signaled PLOS ONE \| www.plosone.org 5 July 2013 \| Volume 8 \| Issue 7 \| e67759 Uric Acid and 2-Hour Postload Glucose adjustment (including HbA1c and FPG). Several limitations of our study need to be considered. First, the cross-sectional design does not allow any causality analysis. Second, selective bias can be introduced, because individuals with relatively lower glucose or HbA1c levels might have a lower participation rate than those with higher glucose or HbA1c levels. Third, some parameters in the present study were estimated by formula or well-accepted methods, such as estimating GFR by the newly CKD-EPI formula and the whole body insulin sensitivity by the OGTT. In conclusion, SUA in individuals with IFG and/or IA1c is strongly associated with 2-h PG, independent of FPG and HbA1c and other established risk factors, suggesting a significant role of SUA in the deterioration of glucose toleration. Interventional studies are warranted to further investigate the nature of this association. Acknowledgments We appreciate the collaboration from all participants of the Gaoyou study and the support from the local health bureau. Author Contributions Conceived and designed the experiments: TY HF. Performed the experiments: HF WT ZW SW YQ QF YG MS MZ HZ TY. Analyzed the data: HF WT ZW. Contributed reagents/materials/analysis tools: TY HF. Wrote the paper: HF. References 22. Bos MJ, Koudstaal PJ, Hofman A, Witteman JCM, Breteler MMB (2006) Uric acid is a risk factor for myocardial infarction and stroke: the Rotterdam Study. Stroke 37: 1503–1507. 23. Baker JF, Krishnan E, Chen L, Schumacher HR (2005) Serum uric acid and cardiovascular disease: recent developments, and where do they leave us? Am J Med 118: 816–826. 24. Dehghan A, van Hoek M, Sijbrands EJ, Hofman A, Witteman JC (2008) High serum uric acid as a novel risk factor for type 2 diabetes. Diabetes Care 31: 361– 362. 25. Tuomilehto J, Zimmet P, Wolf E, Taylor R, Ram P, et al. (1988) Plasma uric acid level and its association with diabetes mellitus and some biological parameters in a biracial population of Fiji. Am J Epidemiol 127: 321–336. 26. Herman JB, Medalie JH, Goldbourt U (1976) Diabetes, prediabetes and uricaemia. Diabetologia 12: 47–52. 27. Nan H, Dong Y, Gao W, Tuomilehto J, Qiao Q (2007) Diabetes associated with a low serum uric acid level in a general Chinese population. Diabetes Res Clin Pract 76: 68–74. 28. Hodge AM, Boyko EJ, de Courten M, Zimmet PZ, Chitson P, et al. (2001) Leptin and other components of the Metabolic Syndrome in Mauritius-a factor analysis. Int J Obes Relat Metab Disord 25: 126–131. 29. Perticone F, Sciacqua A, Perticone M (2012) Serum uric acid and 1-h postload glucose in essential hypertension. http://www.ncbi.nlm.nih.gov/ pubmed?term = Arturi%20F%5BAuthor%5D&cauthor = true&cauthor_ uid = 22011411Diabetes Care 35: 153–157. 30. Yang W, Lu J, Weng J, Jia WChina National Diabetes and Metabolic Disorders Study Group, et al. (2010) Prevalence of diabetes among men and women in China. N Engl J Med 362: 1090–1101. 31. Li G, Zhang P, Wang J, Gregg EW, Yang W, et al. (2008) The long-term effect of lifestyle interventions to prevent diabetes in the China Da Qing Diabetes Prevention Study: a 20-year follow-up study. Lancet 371: 1783–1789. 32. Gao W, Dong Y, Nan H, Tuomilehto J, Qiao Q (2008) The likelihood of diabetes based on the proposed definitions for impaired fasting glucose. Diabetes Res Clin Pract 79: 151–155. 33. Gu D, Reynolds K, Duan X, Xin X, Chen J, et al. (2003) Prevalence of diabetes and impaired fasting glucose in the Chinese adult population: International Collaborative Study of Cardiovascular Disease in Asia (InterASIA). Diabetologia 46: 190–1198. 34. Jia WP, Pang C, Chen L, Bao YQ, Lu JX, et al (2007) Epidemiological characteristics of diabetes mellitus and impaired glucose regulation in a Chinese adult population: the Shanghai Diabetes Studies, a cross-sectional 3-year followup study in Shanghai urban communities. Diabetologia 50: 286–292. 35. Qiao Q, Nakagami T, Tuomilehto J, Borch-Johnsen K, Balkau B, et al. (2000) Comparison of the fasting and the 2-h glucose criteria for diabetes in different Asian cohorts. Diabetologia 43: 1470–1475. 36. Abdul-Ghani MA, Abdul-Ghani T, Ali N, Defronzo RA (2008) One-hour plasma glucose concentration and the metabolic syndrome identify subjects at high risk for future type 2 diabetes. Diabetes Care 31: 1650–1655. 37. Unwin N, Shaw J, Zimmet P, Alberti KG (2002) Impaired glucose tolerance and impaired fasting glycaemia: the current status on definition and intervention. Diabet Med 19: 708–723. 38. American Diabetes Association (2009) Standards of medical care in diabetes– 2009. Diabetes Care 32: S13–S61. 39. Roy D, Perreault M, Marette A (1998) Insulin stimulation of glucose uptake in skeletal muscles and adipose tissues in vivo is NO dependent. Am J Physiol 274: E692–E699. 40. Zoccali C, Maio R, Tripepi G, Mallamaci F, Perticone F (2006) Inflammation as a mediator of the link between mild to moderate renal insufficiency and endothelial dysfunction in essential hypertension. J Am Soc Nephrol 17: S64– S68. 41. Abdul-Ghani MA, Matsuda M, Balas B, DeFronzo RA (2007) Muscle and liver insulin resistance indexes derived from the oral glucose tolerance test. Diabetes Care 30: 89–94. 1. So A, Thorens B (2010) Uric acid transport and disease. J Clin Invest 120: 1791– 1799. 2. Fang J, Alderman MH (2000) Serum uric acid and cardiovascular mortality the NHANES I epidermiologic follow-up study, 1971–1992. National Health and Nutrition Examination Survey. JAMA 283: 2404–2410. 3. Culleton B, Larson M, Kannel W, Levy D (1999) Serum uric acid and risk for cardiovascular diasease and death: the Framingham heart study. Ann Intern Med 131: 7–13. 4. Verdecchia P, Schillaci G, Reboldi G, Santeusanio F, Porcellati C, et al. (2000) Relation between serum uric acid and risk of cardiovascular disease in essential hypertension. The PIUMA study. Hypertension 36: 1072–1078. 5. Bos MJ, Koudstaal PJ, Hofman A, Witteman JC, BretelerMM (2006) Uric acid is a risk factor for myocardial infarction and stroke: the Rotterdam study. Stroke 37: 1503–1507. 6. Haffner SM (2000) Coronary heart disease in patients with diabetes. N Engl J Med 342: 1040–1042. 7. Huxley R, Barzi F, Woodward M (2006) Excess risk of fatal coronary heart disease associated with diabetes in men and women: meta-analysis of 37 prospective cohort studies. BMJ 332: 73–78. 8. Jansson SP, Andersson DK, Svärdsudd K (2010) Mortality trends in subjects with and without diabetes during 33 years of follow-up. Diabetes Care 33: 551– 556. 9. Kannel WB, Hjortland M, Castelli WP (1974) Role of diabetes in congestive heart failure: the Framingham study. Am J Cardiol 34: 29–34. 10. Pour OR, Dagogo-Jack S (2011) Prediabetes as a therapeutic target. Clin Chem 57: 215–220. 11. Tabák AG, Herder C, Rathmann W, Brunner EJ, Kivimäki M (2012) Prediabetes: a high-risk state for diabetes development. Lancet 379: 2279–2290. 12. Balkau B, Shipley M, Jarrett RJ, Pyörälä K, Pyörälä M, et al. (1998) High blood glucose concentration is a risk factor for mortality in middle-aged nondiabetic men: 20-year follow-up in the Whitehall Study, the Paris Prospective Study, and the Helsinki Policemen Study. Diabetes Care 21: 360–367. 13. The DECODE Study Group (1999) Glucose tolerance and mortality: comparison of WHO and American Diabetes Association diagnostic criteria. Lancet 354: 617–621. 14. The Diabetes Control and Complications Trial Research Group (1995) The relationship of glycemic exposure (HbA1c) to the risk of development and progression of retinopathy in the Diabetes Control and Complications Trial. Diabetes 44: 968–983. 15. Groeneveld Y, Petri H, Hermans J, Springer MP (1999) Relationship between blood glucose level and mortality in type 2 diabetes mellitus: a systematic review. Diabet Med 16: 2–13. 16. Barrett-Connor E, Ferrara A (1998) Isolated postchallenge hyperglycemia and the risk of fatal cardiovascular disease in older women and men. Diabetes Care 21: 1236–1239. 17. Temelkova-Kurktschiev TS, Koehler C, Henkel E, Leonhardt W, Fuecker K, et al. (2000) Postchallenge Plasma Glucose and Glycemic Spikes Are More Strongly Associated With Atherosclerosis Than Fasting Glucose or HbA1c Level. Diabetes Care 23: 1830–1834. 18. Petrie JC, O’Brien ET, Littler WA, de Swiet M (1989) Recommendations on blood pressure measurement by a working party of the British Hypertension Society. Brit Med J 293: 611–615. 19. Levey AS, Stevens LA, Schmid CH, http://www.ncbi.nlm.nih.gov/ pubmed?term = Schmid%20CH%5BAuthor%5D&cauthor = true&cauthor_ uid = 19414839Zhang YL, Castro AF 3rd, et al; CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) (2009) A new equation to estimate glomerular filtration rate. Ann Intern Med 150: 604–612. 20. Matsuda M, DeFronzo R (1999) Insulin sensitivity indices obtained from oral glucose tolerance testing: comparison with the euglycemic insulin clamp. Diabetes Care 22: 1462–1470. 21. Johnson RJ, Feig DI, Herrera-Acosta J, Kang DH (2005) Resurrection of uric acid as a causal risk factor in essential hypertension. Hypertension 45: 18–20. PLOS ONE \| www.plosone.org 6 July 2013 \| Volume 8 \| Issue 7 \| e67759 Uric Acid and 2-Hour Postload Glucose 42. Zoccali C, Maio R, Mallamaci F, Sesti G, Perticone F (2006) Uric acid and endothelial dysfunction in essential hypertension. J Am Soc Nephrol 17: 1466– 1471. 43. Khosla UM, Zharikov S, Finch JL, Nakagawa T, Roncal C, et al. (2005) Hyperuricemia induces endothelial dysfunction. Kidney Int 6: 1739–1742. 44. Kang DH, Park SK, Lee IK, Johnson RJ (2005) Uric acid-induced C-reactive protein expression: implication on cell proliferation and nitric oxide production of human vascular cells. J Am Soc Nephrol 16: 3553–3562. PLOS ONE \| www.plosone.org 45. Corry DB, Eslami P, Yamamoto K, Nyby MD, Makino H, et al. (2008) Uric acid stimulates vascular smooth muscle cell proliferation and oxidative stress via the vascular reninangiotensin system. J Hypertens 26: 269–275. 46. Mercuro G, Vitale C, Cerquetani E, Zoncu S, Deidda M, http://www.ncbi.nlm. nih.gov/pubmed?term = Fini%20 M%5BAuthor%5D&cauthor = true&cauthor_ uid = 15464681et al. (2004) Effect of hyperuricemia upon endothelial function in patients at increased cardiovascular risk. Am J Cardiol 94: 932–935. 7 July 2013 \| Volume 8 \| Issue 7 \| e67759
https://openalex.org/W4362481944	https://aacr.figshare.com/articles/journal_contribution/Supplementary_Material_for_Talbert_and_Yang_et_al_from_Dual_Inhibition_of_MEK_and_PI3K_Akt_Rescues_Cancer_Cachexia_through_both_Tumor-Extrinsic_and_-Intrinsic_Activities/22503772/1/files/39963223.pdf	English	null	Supplementary Material for Talbert and Yang et al. from Dual Inhibition of MEK and PI3K/Akt Rescues Cancer Cachexia through both Tumor-Extrinsic and -Intrinsic Activities	null	2,023	cc-by	1,794	Talbert et al Figure S1. Talbert et al Figure S1. g G. A. B. F. C. E. D. MEK162 Vehicle Supplementary Figure 1. MEK162 treatment prevents cancer-induced weight loss and muscle wasting. (A) Mice bearing C-26 tumors and treated with MEK162 have significantly larger average GAST muscle fiber sizes compared to vehicle-treated mice (A log transformation was performed to eliminate heteroscedasticity). (B) Representative cross-sections demonstrating muscle fiber size from vehicle and MEK162-treated mice bearing LLC tumors. Scale bar = 20 μm (C) Treatment of mice bearing LLC tumors with MEK162 results in a right shift of GAST muscle fiber cross-sectional areas. (D) Mice bearing LLC tumors treated with MEK162 have significantly larger average GAST muscle fiber sizes compared to vehicle-treated mice. (E) Treatment of control, non-tumor-bearing CD2F1 mice with MEK162 does not alter mouse body weight. (F) Treatment of control, non-tumor-bearing CD2F1 mice with MEK162 does not increase muscle mass. (G) Treatment of C-26 bearing mice with MEK162 results in muscle masses that are similar to those of control mice without tumors. Data are presented as mean ± standard error of the mean (SEM) n=5-7/group * = p<0 05 compared to vehicle-treated mice A. B. MEK162 Vehicle A. B. G. F. C. E. D. Supplementary Figure 1. MEK162 treatment prevents cancer-induced weight loss and muscle wasting. (A) Mice bearing C-26 tumors and treated with MEK162 have significantly larger average GAST muscle fiber sizes compared to vehicle-treated mice (A log transformation was performed to eliminate heteroscedasticity). (B) Representative cross-sections demonstrating muscle fiber size from vehicle and MEK162-treated mice bearing LLC tumors. Scale bar = 20 μm (C) Treatment of mice bearing LLC tumors with MEK162 results in a right shift of GAST muscle fiber cross-sectional areas. (D) Mice bearing LLC tumors treated with MEK162 have significantly larger average GAST muscle fiber sizes compared to vehicle-treated mice. (E) Treatment of control, non-tumor-bearing CD2F1 mice with MEK162 does not alter mouse body weight (F) Treatment of control non tumor bearing CD2F1 mice with MEK162 does not D. E. D. C. E. D. C. E. G. G. F. F. Supplementary Figure 1. MEK162 treatment prevents cancer-induced weight loss and muscle wasting. (A) Mice bearing C-26 tumors and treated with MEK162 have significantly larger average GAST muscle fiber sizes compared to vehicle-treated mice (A log transformation was performed to eliminate heteroscedasticity). (B) Representative cross-sections demonstrating muscle fiber size from vehicle and MEK162-treated mice bearing LLC tumors. Talbert et al Figure S1. Scale bar = 20 μm (C) Treatment of mice bearing LLC tumors with MEK162 results in a right shift of GAST muscle fiber cross-sectional areas. (D) Mice bearing LLC tumors treated with MEK162 have significantly larger average GAST muscle fiber sizes compared to vehicle-treated mice. (E) Treatment of control, non-tumor-bearing CD2F1 mice with MEK162 does not alter mouse body weight. (F) Treatment of control, non-tumor-bearing CD2F1 mice with MEK162 does not increase muscle mass. (G) Treatment of C-26 bearing mice with MEK162 results in muscle masses that are similar to those of control mice without tumors. Data are presented as mean ± standard error of the mean (SEM).n=5-7/group, * = p<0.05 compared to vehicle-treated mice. Supplementary Figure 1. MEK162 treatment prevents cancer-induced weight loss Talbert et al Figure S2. E. A. B. D. C. Vehicle MEK162 Supplementary Figure 2. Development of a MEK162 resistant C-26 tumor cell line. (A) MEK162 treatment reduces serum IL-6 levels of C-26 tumor-bearing mice. (B) C-26 induced-cachexia activates ERK signaling in QUAD muscle compared to control, non-tu- mor-bearing mice, and treatment with MEK162 decreases ERK activation in muscle. (C) MEK162 treatment has no impact on C-26R tumor weight at the 19 day time point. (D) MEK162 treatment leads to an increase in serum IL-6 in C-26R tumor-bearing mice, supporting that C-26R cells are resistant to MEK162 in vivo. (E) C-26R cells appear morpho- logically different that C-26 cells, with C-26 cells exhibiting a rounded phenotype, while C-26R cells are elongated and display cellular projections. Data are presented as mean ± standard error of the mean (SEM). n=5-11/group, * = p<0.05 compared to vehicle-treated mice. A. A. B. B. B. A. E. D. C. Vehicle MEK162 E. Vehicle MEK162 D. E. C. MEK162 Supplementary Figure 2. Development of a MEK162 resistant C-26 tumor cell line. (A) MEK162 treatment reduces serum IL-6 levels of C-26 tumor-bearing mice. (B) C-26 induced-cachexia activates ERK signaling in QUAD muscle compared to control, non-tu- mor-bearing mice, and treatment with MEK162 decreases ERK activation in muscle. (C) MEK162 treatment has no impact on C-26R tumor weight at the 19 day time point. (D) MEK162 treatment leads to an increase in serum IL-6 in C-26R tumor-bearing mice, supporting that C-26R cells are resistant to MEK162 in vivo. (E) C-26R cells appear morpho- logically different that C-26 cells, with C-26 cells exhibiting a rounded phenotype, while C-26R cells are elongated and display cellular projections. Talbert et al Figure S1. Data are presented as mean ± standard error of the mean (SEM). n=5-11/group, * = p<0.05 compared to vehicle-treated mice. Talbert et al Figure S3. A. Supplementary Figure 3. MEK162 prevents cancer-induced weight loss and muscle wasting in part via a tumor extrinsic mechanism. (A) C-26R tumor-bearing mice have increased average GAST fiber cross-sectional area. Data are presented as mean ± standard error of the mean (SEM). n= 5/group, * = p<0.05 compared to vehicle-treated mice. A. Supplementary Figure 3. MEK162 prevents cancer-induced weight loss and muscle wasting in part via a tumor extrinsic mechanism. (A) C-26R tumor-bearing mice have increased average GAST fiber cross-sectional area. Data are presented as mean ± standard error of the mean (SEM). n= 5/group, * = p<0.05 compared to vehicle-treated mice. Talbert et al Figure S4. D. A. B. C. Supplementary Figure 4. MEK162 prevents cancer-induced weight loss and muscle wasting when used in combination with buparlisib. (A) Buparlisib was sufficient to decrease tumor weight, with MEK162+ buparlisib further decreasing tumor weight (A log transformation was performed to eliminate heteroscedasticity). (B) MEK162 and buparlisib have on-target effects, and on-target effects are maintained when MEK162 and buparlisib are dosed in combination. (C) Vehicle-treated mice lost more body mass than mice treated with MEK162 alone or in combination with buparlisib. (D) Treatment of C-26 tumor-bearing mice with MEK162 either alone or in combi- nation with buparlisib results in increased average GAST muscle fiber cross-sectional area (CSA) compared to vehicle-treated mice (A log transformation was performed to eliminate heteroscedas- ticity). Data are presented as mean ± standard error of the mean (SEM). n=5-6/group. * = p<0.05 compared to vehicle-treated mice. # = p<0.05 versus both single agent MEK162 and single agent A. B. A. B. D. C. Supplementary Figure 4. MEK162 prevents cancer-induced weight loss and muscle wasting when used in combination with buparlisib. (A) Buparlisib was sufficient to decrease tumor weight, with MEK162+ buparlisib further decreasing tumor weight (A log transformation was performed to eliminate heteroscedasticity). (B) MEK162 and buparlisib have on-target effects, and on-target effects are maintained when MEK162 and buparlisib are dosed in combination. (C) Vehicle-treated mice lost more body mass than mice treated with MEK162 alone or in combination with buparlisib. Talbert et al Figure S1. (D) Treatment of C-26 tumor-bearing mice with MEK162 either alone or in combi- nation with buparlisib results in increased average GAST muscle fiber cross-sectional area (CSA) compared to vehicle-treated mice (A log transformation was performed to eliminate heteroscedas- ticity). Data are presented as mean ± standard error of the mean (SEM). n=5-6/group. * = p<0.05 compared to vehicle-treated mice. # = p<0.05 versus both single agent MEK162 and single agent buparlisib. Ψ = p<0.05 versus single agent buparlisib. D. C. D. C. D. C. Supplementary Figure 4. MEK162 prevents cancer-induced weight loss and muscle wasting when used in combination with buparlisib. (A) Buparlisib was sufficient to decrease tumor weight, with MEK162+ buparlisib further decreasing tumor weight (A log transformation was performed to eliminate heteroscedasticity). (B) MEK162 and buparlisib have on-target effects, and on-target effects are maintained when MEK162 and buparlisib are dosed in combination. (C) Vehicle-treated mice lost more body mass than mice treated with MEK162 alone or in combination with buparlisib. (D) Treatment of C-26 tumor-bearing mice with MEK162 either alone or in combi- nation with buparlisib results in increased average GAST muscle fiber cross-sectional area (CSA) compared to vehicle-treated mice (A log transformation was performed to eliminate heteroscedas- ticity). Data are presented as mean ± standard error of the mean (SEM). n=5-6/group. * = p<0.05 compared to vehicle-treated mice. # = p<0.05 versus both single agent MEK162 and single agent buparlisib. Ψ = p<0.05 versus single agent buparlisib. Talbert et al Figure S5. D. A. B. C. Supplementary Figure 5. MEK162 and buparlisib modulate immune biomarkers. (A) CD4+ cell depletion was effective, as the number of splenic CD4+ T cells is reduced in mice treated with anti-CD4 antibody. (B) CD8+ cell depletion was effective, as the number of CD8+ T cells was decreased in mice treated with anti-CD8 antibody. (C) Representative images of CD3+ cells in C-26 tumors. (D) Infiltration of CD3+ cells in tumors does not differ between groups. Data are presented as mean ± standard error of the mean (SEM). n=5-6/group. * = p<0.05 compared to vehicle-treated mice. € = p<0.05 versus MEK162 + buparlisib. $ = p<0.05 versus Anti-CD8 + MEK162 + buparlisib. A. B. A. B. A. B. D. C. Supplementary Figure 5. MEK162 and buparlisib modulate immune biomarkers. (A) CD4+ cell depletion was effective, as the number of splenic CD4+ T cells is reduced in mice treated D. C. D. C. Talbert et al Figure S1. Supplementary Figure 5. MEK162 and buparlisib modulate immune biomarkers. (A) CD4+ cell depletion was effective, as the number of splenic CD4+ T cells is reduced in mice treated with anti-CD4 antibody. (B) CD8+ cell depletion was effective, as the number of CD8+ T cells was decreased in mice treated with anti-CD8 antibody. (C) Representative images of CD3+ cells in C-26 tumors. (D) Infiltration of CD3+ cells in tumors does not differ between groups. Data are presented as mean ± standard error of the mean (SEM). n=5-6/group. * = p<0.05 compared to vehicle-treated mice. € = p<0.05 versus MEK162 + buparlisib. $ = p<0.05 versus Anti-CD8 + MEK162 + buparlisib. Gene Primer Pairs Size (bp) Atrogin-1 5’ - AGATTCGCAAGCGTTTGATC - 3’ 5’ - GGGAAAGTGAGACGGAGCAG - 3’ 204 MuRF1 5’ - TGGCGATTGTCACAAAGTGG - 3’ 5’ - CCCTCTCTAGGCCACCGAGT - 3’ 100 Mul1 5’ - AGGGCATTCTTTCAGAAGCA - 3’ 5’ - GGGGTGGAACTTCTCGTACA - 3’ 329 Musa1 5’ - CAGAGGATTGGGACGGGACG - 3’ 5’ - GTGAGTGCTGCTGTACCTCTT - 3’ 167 Atg5 5’ - ATCAGACCACGACGGAGCGG - 3’ 5’ - GGCGACTGCGGAAGGACAGA - 3’ 116 Bnip3 5’ - CAGAGCGGGGAGGAGAAC - 3’ 5’ - GAGGCTGGAACGCTGCTC - 3’ 80 GAPDH 5’ - AGCCTCGTCCCGTAGACAAAA - 3’ 5’ - GCCTTGACTGTGCCGTTGATT - 3’ 199 Supplementary Table 1. Primer sequences used for real-time RT-PCR.
https://openalex.org/W3005389897	https://www.biorxiv.org/content/biorxiv/early/2019/10/01/789404.full.pdf	English	null	Estimating the power of sequence covariation for detecting conserved RNA structure	Bioinformatics	2,020	cc-by	8,904	conserved RNA structure Elena Rivas1, Jody Clements2 and Sean R. Eddy1,3,4 1 Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA, 2 Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia, USA, 3 Howard Hughes Medical Institute, 4 John A. Paulson School of Engineering and Applied Sciences, Harvard University, Cambridge, Massachusetts 02138, USA . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint Abstract Pairwise sequence covariations are a signal of conserved RNA secondary structure. We de- scribe a method for distinguishing when lack of covariation signal can be taken as evidence against a conserved RNA structure, as opposed to when a sequence alignment merely has in- sufficient variation to detect covariations. We find that alignments for several long noncoding RNAs previously shown to lack covariation support do have adequate covariation detection power, providing additional evidence against their proposed conserved structures. Comparative analyses of pairwise covariations in RNA sequence alignments have a successful history in consensus RNA secondary structure prediction, where the existence of a conserved struc- ture is assumed a priori 1–7. A statistically different question arises when covariation analysis is used to infer whether or not a genomic region is constrained by an evolutionarily conserved RNA secondary structure, as evidence for a structure-dependent function. For example, this question arises in analysis of long noncoding RNAs (lncRNAs) of uncertain mechanism. For this, one wants to determine if the covariation signal is distinguishable from a null hypothesis of primary sequence conservation patterns alone. We previously introduced R-scape (RNA structural covariation above phylogenetic expecta- tion), a method for evaluating the statistical significance of covariation support for conserved RNA 1 1 . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint basepairs8. R-scape analyses found that the covariation evidence for proposed conserved structures of several long noncoding RNAs including HOTAIR9, SRA10, and the RepA region of Xist11;12 is not statistically significant8. Lack of significant covariation signal does not necessarily mean there is no conserved RNA structure. Abstract It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: ioRxiv preprint enables estimation of power(s), the expected sensitivity for detecting any proposed basepair with s total substitutions. For an alignment with B total proposed basepairs, the expected fraction of basepairs with significant covariation support is 1 B ∑B b=1 power(sb). We use this number, which we call alignment power, to compare covariation support across different alignments with different numbers of proposed conserved basepairs. We define an arbitrary threshold of 10% alignment power to distinguish low-power from high-power alignments. Only low-power alignments of conserved structural RNAs should lack significant covariation support. We analyzed all 3,016 seed alignments for known conserved structural RNAs in Rfam v14.1 and compared the fraction of basepairs with significant covariation support versus estimated alignment power (Figure 2a; Supplemental Table S1). Many Rfam alignments (66%, 1,985/3,016) have no statistically significant covariation support for any annotated consensus basepair, and al- most all of these (98%, 1,945/1,985) are low-power alignments. Only 1% (40/3,016) are high-power alignments with no significant detected covariations (shaded in red in Figure 2a; Supplemental Table S2). Rfam, though curated, is a large compendium with a nonzero error rate. Upon ex- amination, we believe these 40 families are enriched for inaccuracies. For example, the miR-1937 family (RF01942) (66% alignment power) is annotated in miRbase15 as a tRNA sequence fragment unlikely to be a bona fide miRNA. Previous analysis of several long noncoding RNAs including HOTAIR9, SRA10, and the Xist RepA region11;12 found no significant covariation support for their proposed structures, but left open the possibility that the existing alignments lacked sufficient variation8. We reanalyzed the four HOTAIR lncRNA domain alignments and consensus structures proposed by ref. 9, and the SRA alignment and consensus structure in ref. 10. All five alignments are high-power, estimated to be able to detect 23-50 significant basepair covariations each (Figure 2b; Supplemental Table S3). Abstract An alignment could merely have too little sequence variation to detect significant co- variation (Figure 1a). To know when an alignment has sufficient variation, we want to estimate the statistical power (the expected sensitivity) of detecting significant covariations. In a “low-power” alignment, covariation analysis is inconclusive because a conserved RNA secondary structure could be present without inducing sufficient covariation signal. In a high-power alignment, observing no supporting covariations does provide evidence against a conserved structure. Many details of an alignment affect covariation analysis, but we hypothesized that detection power should depend primarily on the total number of single residue substitutions si,j in two align- ment columns i and j in a proposed consensus pair. We take the sequence phylogeny into account in inferring si,j by inferring a maximum likelihood tree, using the Fitch parsimony algorithm13 to estimate a number of substitutions si at each column independently, and taking si,j = si + sj (Methods). We tested this idea using synthetic RNA alignments evolved under simulated pairwise con- straints. Figure 1b-d show simulations based on a cobalamin riboswitch alignment (Rfam RF00174) of 430 sequences and 42 annotated consensus basepairs. We choose a random sequence as the root and evolve it down a sub-sampled and rescaled phylogenetic tree, using an evolutionary model that includes basepair substitutions, insertions, and deletions14, to generate a synthetic alignment with a desired number of taxa and average percentage identity (Methods). We repeat this to create synthetic alignments over a wide range of sequence number and diversity. We use R-scape on each alignment to determine the number of basepairs with significant covariation support (E-value < 0.05). Neither the number nor the diversity of sequences in the alignment alone suffices to esti- mate detection power (Figure 1b,c), whereas si,j does have a good relationship to power (Figure 1d). Using either deeper or more diverse alignments increases si,j and detection power (Figure 1e). We empirically fit the relationship between substitutions and detection power (at a significance threshold of E < 0.05) on a dataset of alignments of 87 RNA families in Rfam v14.0 with known three dimensional structures, consisting of 7,012 annotated basepairs (Figure 1f). The fitted curve 2 2 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. Abstract Although the covariation analysis of these lncRNAs has been a subject of disagreement16;17, these results provide new evidence for the view that HOTAIR and SRA do not have evolutionarily conserved RNA structures. Xist RNA is perhaps the best studied lncRNA, but it remains unclear whether Xist’s role in X dosage compensation depends on any conserved RNA structure, as opposed to its sequence alone. Several different conserved structures have been proposed for the conserved 5’ RepA region of Xist11;12;18, two of which are based on covariation analysis of alignments of 10-13 sequences11;12. 3 3 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: ioRxiv preprint Although R-scape finds no significant covariation support for the proposed RepA structures, our method finds that these are low-power alignments, so the R-scape covariation analyses are incon- clusive (Figure 2b; Supplemental Table S3). A conserved structure for the ∼1.3 kb Xist RepA lncRNA (including the conserved repeat A and F regions) has been proposed recently18 from a deeper and more diverse alignment of 57 sequences. Although the R2R visualization program used by Liu et al. highlighted many potential covariations18, statistical analysis by R-scape identifies only one significant covarying basepair with an E-value of 0.005, out of 334 proposed pairs. Our method judges this alignment to be high-power, estimated to be sufficient to detect about 110/334 basepairs. The repeat A+F region is the most conserved region of Xist, but Xist is a large RNA and it is possible that other Xist regions could show covariation support for conserved RNA structure (Figure 2c). Starting with the human XIST genomic sequence, we used the nhmmer homology search program19 to identify 21 regions of significant sequence similarity with mouse Xist (E-value < 10−5). Eleven regions correspond to insertions of well-studied ancient transposons according to Dfam analysis20. Abstract For the remaining 10 unique sequence conserved regions, we iteratively built up alignments of homologs from 47-65 vertebrate species. All of these are high-power alignments; none show significant covariation support for any basepair (Figure 2b; Supplemental Table S3). In order to test for long range base pairing, we created a concatenated alignment of all ten XIST homology regions for 32 species. This concatenated alignment also has sufficient power but not covariations are observed. Experimental evidence from chemical probing and crosslinking has been used in making struc- ture predictions for the HOTAIR, SRA, and Xist lncRNAs. However, essentially any RNA, even a random RNA sequence, folds into some secondary structure. Lack of covariation signal in high- power RNA sequence alignments for these lncRNAs suggests that whatever structure they adopt is not detectably constraining their evolution, and thus may not be relevant for their function. An important caveat in covariation analysis is that the input sequence alignment is assumed to be reasonably correct. Spurious apparent covariations can be created artifactually by sliding conserved primary sequence regions under proposed stems. We identified an example of this arti- fact in a proposed conserved structure for COOLAIR, an Arabidopsis lncRNA21. The COOLAIR alignment is a low-power alignment of only 6 aligned sequences, yet R-scape identifies 6 significant 4 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: ioRxiv preprint covarying basepairs, 4 of them in one proposed helix (Figure 2d). Upon inspection, it appears that misalignment introduced artifactual covariations (Figure 2e). We realigned the COOLAIR sequences using Infernal22 (Methods), which brought regions of strong primary sequence identity back into alignment (Figure 2f). The revised COOLAIR alignment is still low-power, and has only one significant supported basepair with a marginal E-value of 0.048. The R-scape software now reports estimated statistical power calculations along with observed pairwise correlations. Abstract We expect that one important future use of covariation power analysis is to enable quantitative use of negative information by excluding pairs that are unlikely to be conserved basepairs because they have high-power and no significant covariation. 5 5 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: ioRxiv preprint Estimating substitution number sij Given an input RNA sequence alignment, R-scape infers a maximum likelihood phylogenetic tree using FastTree (version 2.1.10)23, then infers a maximum parsimony assignment of substitutions at each independent column i to each branch of the tree using our implementation of the Fitch algorithm13. For phylogeny-aware statistical significance testing as described in ref. 8, R-scape then uses this information in tree-based simulations to construct synthetic negative control alignments that preserve average identity, composition, and phylogenetic relationships of the original alignment, while randomizing pairwise correlations. An empirical null distribution for a pairwise covariation statistic (default is the G-test statistic, related to mutual information) is then obtained from all pairs of columns i, j in these simulated alignments. In the new method for statistical power estimation, the same tree and inferred maximum par- simony substitutions are used to obtain the total substitutions si (summed over branches) at each individual column i. For a proposed basepair involving two columns i, j, we use sij = si + sj, the sum of the independent variation at each column. We also tested a more expensive variation where we parsimoniously infer pairwise substitutions jointly at all column pairs i, j to obtain sij, rather than assuming column independence, which gave similar results (data not shown). Simulations Simulated alignments were produced with the program R-scape-sim. Given an input sequence alignment with a consensus RNA secondary structure, R-scape-sim calculates a maximum likelihood phylogenetic tree with branch lengths. It sub-samples the original phylogenetic tree to a desired number of taxa, and linearly scales branch lengths to achieve a desired average percentage identity amongst the aligned sequences. One sequence is selected at random as a root and its evolution is simulated down the tree branches using a probabilistic evolutionary model. The evolutionary model consists of rate matrices for single (unpaired) residue substitution, pairwise (basepair) substitution, insertion, and deletion events8;14. We used this simulation procedure on the Rfam Cobalamin riboswitch alignment (RF00174) to generate 29,976 synthetic alignments with sequence number 6 6 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint ranging from five to 190, and average percentage identity ranging from 30% to 100%. The simulated alignments were randomly downsampled to 3, 000 in order to produce the scatter plots of Figure 1. Alignment power The expected sensitivity for detecting a basepair b with sb substitutions is power(sb), from the empirical fitted curve shown in Figure 1f. In addition to reporting the pairs that significantly covary with their corresponding E-value, R-scape now also reports for each pair the inferred number of substitutions sb and the estimated power(sb). As an overall summary statistic for an alignment with a proposed structure, R-scape reports the total number of basepairs expected to have significant covariation support, cov-bp-exp = B ∑ b=1 power(sb), cov-bp-exp = ∑ b=1 power(sb), and the alignment power, defined as the fraction of base pairs expected to have significant support, and the alignment power, defined as the fraction of base pairs expected to have significant support, alignment power = cov-bp-exp B = 1 B B ∑ b=1 power(sb). In this work, alignments with > 10% power are considered to have sufficient power. In this work, alignments with > 10% power are considered to have sufficient power. Empirical power(s) curve It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: ioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint Empirical power(s) curve For each of 7,012 annotated consensus basepairs in 87 RNA families Rfam v14.0 with known three dimensional structures, we use R-scape to calculate the statistical significance (expectation value, E-value) and estimate sij for each proposed pair in Rfam “seed” alignments. We binned proposed pairs with identical sij and calculated the frequency of pairs with significant support E < 0.05 in each bin. For 1, 653 such points for bins s = 0 to 1, 652, we fitted a polynomial of degree 10 by minimizing least-square-error. The choice of degree 10 was arbitrary, and simpler functions such as 1 −e−λs did not fit as well. In binning the data by integer s, the number of basepairs per bin is variable. For large values of s there are few basepairs per bin (often 0-2), leading to noisy data, which is why we fit all point for s ≤150, and only those with at least 80% power for s > 150. For Figure 1f we plotted the Rfam data differently, in equal-size bins, by ranking all 7,012 basepairs by increasing E-values, dividing them into 70 equal bins, and calculating the means s and power(s) in each bin. This plot is less noisy at high s. We did not reestimate the fitted curve when we replotted. Fitted power(s) values starting from s = 0.012 are hardcoded in the R-scape source code; for s > 226 we set power(s) = 1. Our approach treats power(s) as a function solely of s. This approximates away an important additional dependency on alignment length. R-scape E-values are multiple-test-corrected; the num- ber of potential basepairs depends on the input alignment length. Detecting significant support for a basepair in a longer alignment requires more signal because the background of non-pairs is higher. We considered fitting power(s, p) to a range of different p-value thresholds p (i.e. before multiple test correction to E-values) but decided this was impractical. Instead the fitted power curve treats all alignments as approximately the same length. The actual lengths of the Rfam seed alignments used in Figure 1f range from 40 consensus columns (HIV retroviral Psi packing element) to 3,680 consensus columns (eukarya LSU rRNA). 7 7 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. R-scape statistical test modes R-scape has two statistical modes to test the presence of a conserved RNA structure. By default, R-scape considers all pairs as equivalent and performs an statistical test as to which of the all possible L(L −1)/2 pairs (for an alignment of length L) are significantly covarying. This is R- scape’s default one-set test. Alternatively, if a consensus secondary structure is provided, R-scape allows an optional two-set test consisting of two independent tests on two different sets. One test is on the proposed structure (the set of basepairs); the other parallel test is on all other possible pairs in the alignment (the set of non basepairs). On the set of basepairs, R-scape extracts the alignment’s support for the annotated structure. On the set of non basepairs, R-scape identifies other possible covarying basepairs not present in the given structure. Estimating the alignment power requires a proposed structure, thus the use of R-scape’s two-set mode. Under the one-set mode, R-scape still reports the power for each of the significantly covarying basepairs, assuming that those could be part of a structure. The covariation and covariation power analyses provided in this manuscript for all lncRNAs have been obtained with R-scape’s two-set mode on the proposed secondary structures. 8 8 . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a ertified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: ioRxiv preprint lncRNA alignment sources CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint mentary material in Stockholm format. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint lncRNA alignment sources HOTAIR domain 1-4 alignments (D1-D4) and proposed consensus structure used in ref. 9 were kindly provided to us by S. Somarowthu. HOTAIR domain 1-4 alignments (D1-D4) and proposed consensus structure used in ref. 9 were kindly provided to us by S. Somarowthu. The SRA alignment and proposed consensus structure used in ref. 10 were unavailable to us. The proposed secondary structure of the human ncSRA was reproduced by hand from Supplementary Figure 1 of ref. 10. A SRA alignment was produced by imposing the human ncSRA proposed structure in the Multiz100way alignment of the ncSRA region obtained from the UCSC human genome browser (http://genome.ucsc.edu). This alignment includes 76 mammalian species. The Xist repeat A region alignment used in ref. 11 and four alternative proposed consensus structures that we call RepA.S0 through RepA.S3 were reproduced from Supplemental Fig. 5 in ref. 11. The Xist repeat A region alignment and proposed consensus structure in ref. 12 were kindly provided to us by W. Moss. The RepA lncRNA alignment (spanning repeat A and repeat F regions) with a proposed con- sensus structure in ref. 18 was kindly provided to us by the authors. We produced our own RepA lncRNA alignment of 65 sequences and got similar results: a high- power alignment sufficient to detect about 254 basepairs, but no significantly supported covarying pairs. The proposed structures for all ten XIST conserved regions were produced using R-scape. As described in the main text, we used nhmmer19 to identify 21 significant local alignments (at E < 10−5) between human XIST and mouse Xist, covering 79% of the XIST RNA sequence. We used nhmmer and Dfam20 to determine that 11 of these conserved regions correspond to known transposable elements including retroposons (L2d 3end), DNA transposons (Charlie29a, Charlie29b), SINEs (FLAM C), and retroviral LTRs (LTR78). For the remaining 10 conserved regions we used an nhmmer profile of the mouse/human pairwise alignment to search a database of vertebrate genome sequences, resulting in 10 alignments consisting of 47-65 homologous sequences, which we name XIST h1 through XIST h10. We used the A. thaliana COOLAIR lncRNA sequence and the consensus structure proposed in ref. 21 to construct a single-sequence Infernal profile22, then used Infernal to align all six COOLAIR homologs to this profile. All alignments (with consensus structure annotation, where applicable) are included in supple- 9 . References [1] R. W. Holley, J. Apgar, G. A. Everett, J. T. Madison, M. Marquisee, S. H. Merrill, J. R. Penswick, and A. Zamir, “Structure of a ribonucleic acid,” Science, vol. 14, pp. 1462–1465, 1965. [2] H. F. Noller, J. Kop, V. Wheaton, J. Brosius, R. R. Gutell, A. M. Kopylov, F. Dohme, W. Herr, D. A. Stahl, R. Gupta, and C. R. Woese, “Secondary structure model for 23S ribosomal RNA,” Nucl. Acids Res., vol. 9, pp. 6167–6189, 1981. [3] R. R. Gutell, B. Weiser, C. R. Woese, and H. F. Noller, “Comparative anatomy of 16S-like ribosomal RNA,” Prog. Nucl. Acids Res. Mol. Biol., vol. 32, pp. 155–216, 1985. [4] N. R. Pace, D. K. Smith, G. J. Olsen, and B. D. 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Arkin, “FastTree 2 - approximately maximum-likelihood trees for large alignments,” PLOS ONE, vol. 5, p. e9490, 2010. Supplementary information is linked to the online version of the paper. Supplementary information is linked to the online version of the paper. Acknowledgment We thank the Centro de Ciencias de Benasque Pedro Pascual, Spain, where ideas for this manuscript were developed. Authors contribution E.R. and S.R.E. designed the method and wrote the manuscript. E.R. wrote the code, designed and carried out the experiments. Author information An R-scape web server is at eddylab.org/R-scape, with a link to down- load source code. The authors declare that they have no competing financial interests. Corre- spondence and requests for materials should be addressed to E.R. (elenarivas@fas.harvard.edu). Author information An R-scape web server is at eddylab.org/R-scape, with a link to down- load source code. The authors declare that they have no competing financial interests. Corre- spondence and requests for materials should be addressed to E.R. (elenarivas@fas.harvard.edu). 12 a d b e f c covariation? variation? evidence for a A U C G A U C G A U C G A U C U A G C G A U C U A U A U A U A U A U A U ? conserved basepair? 0 20 40 60 80 0 50 100 150 200 % basepairs signifcantly covarying number of sequences 100 30 40 50 60 70 80 90 100 % basepairs signifcantly covarying average pairwise % identity 20 40 60 80 100 0 0.1 1 10 100 % basepairs signifcantly covarying average # of substitutions 20 40 60 80 100 0 7,012 basepairs (from 83 RNAs with crystal structures) 1.0 0.8 0.6 0.4 0.0 0.2 0 50 100 150 200 250 300 Number of substitutions in basepair (s) P( Evalue(s) < 0.05) 0 20 40 60 80 100 20 40 60 80 100 120 140 160 180 average percentage identity number of sequences % id # seqs ~30 ~50 ~75 65 65 65 10 22 68 % cov bps % id # seqs ~30 ~50 ~75 83 70 57 40 40 40 medium sensitivity high sensitivity low sensitivity % cov bps (~75 %) (~50 %) (~30 %) % of covarying basepairs Figure 1 a covariation? variation? evidence for a A U C G A U C G A U C G A U C U A G C G A U C U A U A U A U A U A U A U ? conserved basepair? . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint Supplementary information is linked to the online version of the paper. b 0 20 40 60 80 0 50 100 150 200 % basepairs signifcantly covarying number of sequences 100 f c 30 40 50 60 70 80 90 100 % basepairs signifcantly covarying average pairwise % identity 20 40 60 80 100 0 c g d e 0.1 1 10 100 % basepairs signifcantly covarying average # of substitutions 20 40 60 80 100 0 f 0 20 40 60 80 100 20 40 60 80 100 120 140 160 180 average percentage identity number of sequences % id # seqs ~30 ~50 ~75 65 65 65 10 22 68 % cov bps % id # seqs ~30 ~50 ~75 83 70 57 40 40 40 medium sensitivity high sensitivity low sensitivity % cov bps (~75 %) (~50 %) (~30 %) % of covarying basepairs e f 7,012 basepairs (from 83 RNAs with crystal structures) 1.0 0.8 0.6 0.4 0.0 0.2 0 50 100 150 200 250 300 Number of substitutions in basepair (s) P( Evalue(s) < 0.05) 7,012 basepairs (from 83 RNAs with crystal structures) 0 0 0 0 P( Evalue(s) < 0.05) 50 100 150 200 250 Number of substitutions in basepair (s) Figure 1 13 Figure 1: (a) Three different patterns for two alignment columns proposed to form a consensus basepair. (a, left) The two columns have variation and covariation (mutual infor- mation is 1 bit). This pattern is consistent with a basepair conserved throughout evolution. (a, middle) The two columns have variation but not covariation (mutual information is 0.0). These two positions are unlikely to form a basepair. (a, right) Two columns with no covariation and no variation. This pattern is consistent with a A-U basepair, but there is no evolutionary evidence for it. (b,c,d) Scatter plots of power (% sensitivity) for detecting basepairs in simulated alignments. Each point represents the fraction of 42 consensus basepairs in a simulated Cobalamin alignment detected with an R-scape E-value < 0.05, as a function of sequence number (b), average pairwise % identity (c), or inferred number of substitutions in two columns sij (d). (e) The same simulated alignments binned by low (yellow ∼27 −32%), medium (blue ∼47 −52%) or high (red ∼74 −76%) sensitivity and scatter plotted, illustrating how detection power increases either by increasing sequence number or sequence diversity. (f) Power of covariation as a function of the total number of substitutions. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint Supplementary information is linked to the online version of the paper. The orange line is the fitted power(s) curve. Each blue dot represents the empirical data we fit to: the mean fraction of significantly covarying basepairs and mean sij in a set of 100 annotated basepairs from Rfam seed alignments, out of 7,012 total basepairs ordered by increasing number of substitutions. 14 a c b f e d A.thaliana UUUGUGCCCUAAUUUG.AUCCUCAGGUUUGGGUU.C.AAGUCGCCGGA A.lyrata UUUAUUCCCUAAUUUG.AUCCUCAGGUU-GGGUU.CgAAGUCGCCGGA C.rubella CUUGUGCCCUAAUUUG.AUCCUCGGCUUUGGGUU.C.AAGUCGCCGGA A.alpina UUUGUGCCCUAAUUUGgAUCCUCAGGUUCGGUCUuU.AAGUCGCCUGA E.salsugineum UCCGUGCCCUAAUUUG.AUCCUCAGGUACGG-UU.C.AAGUCGCCAGA B.rapa UCUGUGCCCUAAUUUG.AUCCAGAGGUUCGG-UU.C.AAGUGGCCGGA #=GC SS_cons <<<<-<<----<<<<<.<<<<________>>>->.>.>>>>->>>>>> Infernal alignment reproduction: Figure 2A, Hawkes et al. NAR 2016 C U Y G U G C C C Y U C C C A G G U U Y G G R G C C G A 5´ Arabidopsis COOLAIR 90% 97% 75% 50% nucleotide present nucleotide 75% N N 97% N 90% identity A.thaliana UU.UGUGC.CCUAAUUUGA..U.CC.UCAGGUUUGG..G.UUCAAGUCGCC....GGA A.lyrata UU.UA.UUCCCUAAUUUGAU.C.CU..CAGGUU.GG..G.UUCGA...AGUCGCCGGA C.rubella UU..GUGCCCUA...AUUU.GAUCC.UCGGCUUUGGGUUC.AAGU..CGCC.....GG A.alpina UU..GUGCCCUA..AUUUG.GAUCC.UCAGGUUCGGU.UCUUAAG.UCGCCU....GA E.salsugineum UCC.GUGCCCUA...AUUU.GA.UCCUCAGGUACGGU.UC.AAGU..CGCCA....GA B.rapa UC.UGUGCCCUA...AUUU.GAUCC.AGAGGUUCGGU.UC.AAGU..GGCC....GGA #=GC SS_cons <<-<<-<<-------<<<<-<<-<<_________>>-->>->>>>--->>>---->>> * ** * * ** * Figure S2C, Hawkes et al. NAR 2016 0 20 40 80 60 100 0 20 40 60 80 100 fraction of basepairs that covary (%) alignment power (%) SECIS-1 PhotoRC-II tRNA manA ZMP-ZTP Bacteria-large-SRP mir-154 Fungi-SRP ykoK CyVA-1 int-alpA IMES-1 gut-2 cow-rumen-2 LOOT RAGATH-18 group-II-D1D4-7 Bacteria-small-SRP tmRNA IsrR mir-1937 Prion-pknot Plant-SR K-chan-RES MIR480 msiK Clostridiales-2 Metazoa-SRP Telomerase-vert Telomerase-cil ROOL SNORD96 > 10% power but not covariations (% of basepairs expected to covary) c4-2 5S-rRNA human XIST 73852753 73820615 chrX rev XIST 1 11373 24770 32103 exon1 exon2 exon3 exon4 exon5 exon6 repeats A F B C fragment D E mouse homol. TEs non-TE homol. Supplementary information is linked to the online version of the paper. # aligned FLAM_C Charlie29a LTR52 MIRc L2d_3end Charlie29b FLAM_C LTR78 MIRb LTR78 L2d_3end L2d_3end (linx3) (linx3) 65 52 55 61 55 56 73 60 59 47 h1 h2 h3 h4 h5 h6 h7 h8 h9 h10 40 60 80 100 alignment power (%) XIST h2 XIST h4 HOTAIR D2 XIST h6 ncSRA HOTAIR D1 HOTAIR D4 repA lncRNA 0 HOTAIR D3 0 20 40 60 80 100 fraction of basepairs that covary (%) COOLAIR XIST h8 XIST h7 XIST h1 # % bp exp obs tot HOTAIR D1 37 74 149 48.4 0 HOTAIR D2 31 74 134 23.0 0 HOTAIR D3 34 68 125 41.4 0 HOTAIR D4 31 69 165 38.6 0 ncSRA 76 78 234 49.7 0 Alignment RepA lncRNA 57 68 334 108.6 1 XIST h1 (repA,F) 65 68 254 100.7 0 XIST h2 (repB,C) 52 68 41 25.1 0 XIST h5 55 70 63 18.4 0 XIST h6 56 79 112 20.7 0 XIST h7 73 61 123 50.4 0 COOLAIR 6 80 166 0.8 1 XIST h8 (repE) 60 66 89 34.7 0 seqs ID XIST h9 47 70 32 7.2 0 XIST h10 59 73 61 15.9 0 XIST h3 55 68 53 16.2 0 XIST h4 61 60 64 28.6 0 cov bp XIST h1-h10 32 72 980 141.3 0 Figure 2 a 0 20 40 80 60 100 0 20 40 60 80 100 fraction of basepairs that covary (%) alignment power (%) SECIS-1 PhotoRC-II tRNA manA ZMP-ZTP Bacteria-large-SRP mir-154 Fungi-SRP ykoK CyVA-1 int-alpA IMES-1 gut-2 cow-rumen-2 LOOT RAGATH-18 group-II-D1D4-7 Bacteria-small-SRP tmRNA IsrR mir-1937 Prion-pknot Plant-SR K-chan-RES MIR480 msiK Clostridiales-2 Metazoa-SRP Telomerase-vert Telomerase-cil ROOL SNORD96 > 10% power but not covariations (% of basepairs expected to covary) c4-2 5S-rRNA b 40 60 80 100 alignment power (%) XIST h2 XIST h4 HOTAIR D2 XIST h6 ncSRA HOTAIR D1 HOTAIR D4 repA lncRNA 0 HOTAIR D3 0 20 40 60 80 100 fraction of basepairs that covary (%) COOLAIR XIST h8 XIST h7 XIST h1 # % bp exp obs tot HOTAIR D1 37 74 149 48.4 0 HOTAIR D2 31 74 134 23.0 0 HOTAIR D3 34 68 125 41.4 0 HOTAIR D4 31 69 165 38.6 0 ncSRA 76 78 234 49.7 0 Alignment RepA lncRNA 57 68 334 108.6 1 XIST h1 (repA,F) 65 68 254 100.7 0 XIST h2 (repB,C) 52 68 41 25.1 0 XIST h5 55 70 63 18.4 0 XIST h6 56 79 112 20.7 0 XIST h7 73 61 123 50.4 0 COOLAIR 6 80 166 0.8 1 XIST h8 (repE) 60 66 89 34.7 0 seqs ID XIST h9 47 70 32 7.2 0 XIST h10 59 73 61 15.9 0 XIST h3 55 68 53 16.2 0 XIST h4 61 60 64 28.6 0 cov bp XIST h1-h10 32 72 980 141.3 0 c human XIST 73852753 73820615 chrX rev XIST 1 11373 24770 32103 exon1 exon2 exon3 exon4 exon5 exon6 repeats A F B C fragment D E mouse homol. . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint . CC-BY 4.0 International license a certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under The copyright holder for this preprint (which was not this version posted October 1, 2019. ; https://doi.org/10.1101/789404 doi: bioRxiv preprint Supplementary information is linked to the online version of the paper. TEs non-TE homol. # aligned FLAM_C Charlie29a LTR52 MIRc L2d_3end Charlie29b FLAM_C LTR78 MIRb LTR78 L2d_3end L2d_3end (linx3) (linx3) 65 52 55 61 55 56 73 60 59 47 h1 h2 h3 h4 h5 h6 h7 h8 h9 h10 f Figure 2 Figure 2 15 Figure 2: (a) Power of covariation for structural RNAs. Each point represents one of 2,209 Rfam families (seed alignments) with at least 10 annotated consensus basepairs, plotting the frac- tion of annotated basepairs that show a significant covariation signal (R-scape E< 0.05) versus “alignment power”, the fraction expected to show a significant signal. Points are color coded by positive predictive value (PPV): blue are PPV > 95%, yellow 50 −95%, red < 50%. Red shaded region along the bottom indicates alignments with sufficient power, defined as > 10%, but no signif- icant detected covariations. (b) Results for HOTAIR, SRA, Xist, and COOLAIR lncRNA alignments. Inset table shows details for each alignment, including the total number of annotated basepairs, the expected number that should show significant covariation (i.e. alignment power times total bp), and the number observed with significant covariation. Supplemental Table S3 describes all lncRNA alignments and proposed structures tested. (c) Human XIST RNA conservation. Representation of the locations of ten human/mouse unique sequence conserved regions h1-h10 in XIST/Xist, relative to XIST repeat regions A-F and to other human/mouse conserved sequence corresponding to ancient transposable elements (TEs). (d) The proposed COOLAIR helix H10, redrawn from ref. 21 displaying R-scape’s covariation annotation. (e) The proposed align- ment of six homologous sequences for COOLAIR helix 10 resulting in the apparent covariations in (d). (f) A revised COOLAIR H10 alignment more consistent with primary sequence conservation. The apparent R-scape covariations from the original alignment in (e) disappear, while the proposed structure in (d) is maintained. Identical sets of residues in both alignments are shaded in the same color. 16 b e f c on? on? or a A U C G A U C G A U C G A U C U A G C G A U C U A U A U A U A U A U A U ? air? Supplementary information is linked to the online version of the paper. 0 20 40 60 80 0 50 100 150 200 % basepairs signifcantly covarying number of sequences 100 30 40 50 60 70 80 90 100 % basepairs signifcantly covarying average pairwise % identity 20 40 60 80 100 0 10 100 ge # of substitutions 7,012 basepairs (from 83 RNAs with crystal structures) 1.0 0.8 0.6 0.4 0.0 0.2 0 50 100 150 200 250 300 Number of substitutions in basepair (s) P( Evalue(s) < 0.05) 0 20 40 60 80 100 20 40 60 80 100 120 140 160 180 average percentage identity number of sequences % id # seqs ~30 ~50 ~75 65 65 65 10 22 68 % cov bps % id # seqs ~30 ~50 ~75 83 70 57 40 40 40 medium sensitivity high sensitivity low sensitivity % cov bps (~75 %) (~50 %) (~30 %) % of covarying basepairs a d b e f c covariation? variation? evidence for a A U C G A U C G A U C G A U C U A G C G A U C U A U A U A U A U A U A U ? conserved basepair? 0 20 40 60 80 0 50 100 150 200 % basepairs signifcantly covarying number of sequences 100 30 40 50 6 % basepairs signifcantly covarying average pa 20 40 60 80 100 0 0.1 1 10 100 % basepairs signifcantly covarying average # of substitutions 20 40 60 80 100 0 7,012 basepairs (from 83 1.0 0.8 0.6 0.4 0.0 0.2 0 50 100 Number of subst P( Evalue(s) < 0.05) 0 20 40 60 80 100 20 40 60 80 100 120 140 160 180 average percentage identity number of sequences % id # seqs ~30 ~50 ~75 65 65 65 10 22 68 % cov bps % id # seqs ~30 ~50 ~75 83 70 57 40 40 40 medium sensitivity high sensitivity low sensitivity % cov bps (~75 %) (~50 %) (~30 %) % of covarying basepairs b 0 20 40 60 80 0 50 100 150 200 % basepairs signifcantly covarying number of sequences 100 f c 30 40 50 60 70 80 90 100 % basepairs signifcantly covarying average pairwise % identity 20 40 60 80 100 0 d covariation? variation? Supplementary information is linked to the online version of the paper. evidence for a A U C G A U C G A U C G A U C U A G C G A U C U A U A U A U A U A U A U ? conserved basepair? e q 0 20 40 60 80 100 20 40 60 80 100 120 140 160 180 average percentage identity number of sequences % id # seqs ~30 ~50 ~75 65 65 65 10 22 68 % cov bps % id # seqs ~30 ~50 ~75 83 70 57 40 40 40 medium sensitivity high sensitivity low sensitivity % cov bps (~75 %) (~50 %) (~30 %) % of covarying basepairs f e f 7,012 basepairs (from 83 RNAs with crystal structures) 1.0 0.8 0.6 0.4 0.0 0.2 0 50 100 150 200 250 300 Number of substitutions in basepair (s) P( Evalue(s) < 0.05) 7,012 basepairs (from 83 RNAs with crystal structures) 1 0 0.1 1 10 100 % basepairs signifcantly covarying average # of substitutions 20 40 60 80 100 0 50 100 150 200 250 Number of substitutions in basepair (s) b f e d A.thaliana UUUGUGCCCUAAUUUG.AUCCUCAGGUUUGGGUU.C.AAGUCGCCGGA A.lyrata UUUAUUCCCUAAUUUG.AUCCUCAGGUU-GGGUU.CgAAGUCGCCGGA C.rubella CUUGUGCCCUAAUUUG.AUCCUCGGCUUUGGGUU.C.AAGUCGCCGGA A.alpina UUUGUGCCCUAAUUUGgAUCCUCAGGUUCGGUCUuU.AAGUCGCCUGA E.salsugineum UCCGUGCCCUAAUUUG.AUCCUCAGGUACGG-UU.C.AAGUCGCCAGA B.rapa UCUGUGCCCUAAUUUG.AUCCAGAGGUUCGG-UU.C.AAGUGGCCGGA #=GC SS_cons <<<<-<<----<<<<<.<<<<________>>>->.>.>>>>->>>>>> Infernal alignment reproduction: Figure 2A, Hawkes et al. NAR 2016 C U Y G U G C C C Y U C C C A G G U U Y G G R G C C G A 5´ Arabidopsis COOLAIR 90% 97% 75% 50% nucleotide present nucleotide 75% N N 97% N 90% identity A.thaliana UU.UGUGC.CCUAAUUUGA..U.CC.UCAGGUUUGG..G.UUCAAGUCGCC....GGA A.lyrata UU.UA.UUCCCUAAUUUGAU.C.CU..CAGGUU.GG..G.UUCGA...AGUCGCCGGA C.rubella UU..GUGCCCUA...AUUU.GAUCC.UCGGCUUUGGGUUC.AAGU..CGCC.....GG A.alpina UU..GUGCCCUA..AUUUG.GAUCC.UCAGGUUCGGU.UCUUAAG.UCGCCU....GA E.salsugineum UCC.GUGCCCUA...AUUU.GA.UCCUCAGGUACGGU.UC.AAGU..CGCCA....GA B.rapa UC.UGUGCCCUA...AUUU.GAUCC.AGAGGUUCGGU.UC.AAGU..GGCC....GGA #=GC SS_cons <<-<<-<<-------<<<<-<<-<<_________>>-->>->>>>--->>>---->>> * ** * * ** * Figure S2C, Hawkes et al. Supplementary information is linked to the online version of the paper. NAR 2016 20 40 60 80 100 alignment power (%) SECIS-1 PhotoRC-II tRNA manA ZMP-ZTP Bacteria-large-SRP mir-154 Fungi-SRP ykoK CyVA-1 int-alpA IMES-1 gut-2 cow-rumen-2 LOOT RAGATH-18 group-II-D1D4-7 Bacteria-small-SRP tmRNA IsrR mir-1937 Prion-pknot Plant-SR K-chan-RES MIR480 msiK tridiales-2 Metazoa-SRP rase-vert Telomerase-cil ROOL D96 > 10% power but not covariations (% of basepairs expected to covary) c4-2 5S-rRNA human XIST 753 73820615 11373 24770 32103 exon1 exon2 exon3 exon4 exon5 exon6 B C fragment D E FLAM_C Charlie29a LTR52 MIRc L2d_3end Charlie29b FLAM_C LTR78 MIRb LTR78 L2d_3end L2d_3end (linx3) (linx3) 52 55 61 55 56 73 60 59 47 h2 h3 h4 h5 h6 h7 h8 h9 h10 40 60 80 100 alignment power (%) XIST h2 XIST h4 HOTAIR D2 XIST h6 ncSRA HOTAIR D1 HOTAIR D4 repA lncRNA 0 HOTAIR D3 0 20 40 60 80 100 fraction of basepairs that covary (%) COOLAIR XIST h8 XIST h7 XIST h1 # % bp exp obs tot HOTAIR D1 37 74 149 48.4 0 HOTAIR D2 31 74 134 23.0 0 HOTAIR D3 34 68 125 41.4 0 HOTAIR D4 31 69 165 38.6 0 ncSRA 76 78 234 49.7 0 Alignment RepA lncRNA 57 68 334 108.6 1 XIST h1 (repA,F) 65 68 254 100.7 0 XIST h2 (repB,C) 52 68 41 25.1 0 XIST h5 55 70 63 18.4 0 XIST h6 56 79 112 20.7 0 XIST h7 73 61 123 50.4 0 COOLAIR 6 80 166 0.8 1 XIST h8 (repE) 60 66 89 34.7 0 seqs ID XIST h9 47 70 32 7.2 0 XIST h10 59 73 61 15.9 0 XIST h3 55 68 53 16.2 0 XIST h4 61 60 64 28.6 0 cov bp XIST h1-h10 32 72 980 141.3 0 a 0 20 40 80 60 100 0 20 40 60 80 100 fraction of basepairs that covary (%) alignment power (%) SECIS-1 PhotoRC-II tRNA manA ZMP-ZTP Bacteria-large-SRP mir-154 Fungi-SRP ykoK CyVA-1 int-alpA IMES-1 gut-2 cow-rumen-2 LOOT RAGATH-18 group-II-D1D4- Bacteria-small- tmRNA IsrR mir-1937 Prion-pknot Plant-SR K-chan-RES MIR480 msiK Clostridiales-2 Metazoa-SRP Telomerase-vert Telomerase-cil ROOL SNORD96 > 10% power but not covariations (% of basepairs expected to covary) c4-2 5S-rRNA 40 60 80 100 alignment power (%) XIST h2 XIST h4 HOTAIR D2 XIST h6 ncSRA HOTAIR D1 HOTAIR D repA lncRNA 0 HOTAIR D3 0 20 40 60 80 100 fraction of basepairs that covary (%) COOLAIR XIST h8 XIST h7 XIST h1 # % bp exp obs tot HOTAIR D1 37 74 149 48.4 0 HOTAIR D2 31 74 134 23.0 0 HOTAIR D3 34 68 125 41.4 0 HOTAIR D4 31 69 165 38.6 0 ncSRA 76 78 234 49.7 0 Alignment RepA lncRNA 57 68 334 108.6 1 XIST h1 (repA,F) 65 68 254 100.7 0 XIST h2 (repB,C) 52 68 41 25.1 0 XIST h5 55 70 63 18.4 0 XIST h6 56 79 112 20.7 0 XIST h7 73 61 123 50.4 0 COOLAIR 6 80 166 0.8 1 XIST h8 (repE) 60 66 89 34.7 0 seqs ID XIST h9 47 70 32 7.2 0 XIST h10 59 73 61 15.9 0 XIST h3 55 68 53 16.2 0 XIST h4 61 60 64 28.6 0 cov bp XIST h1-h10 32 72 980 141.3 0 2 4 6 fraction of basepairs that covary (%) 0 20 40 60 fraction of basepairs that covary (%) f e d A.thaliana UUUGUGCCCUAAUUUG.AUCCUCAGGUUUGGGUU.C.AAGUCGCCGGA A.lyrata UUUAUUCCCUAAUUUG.AUCCUCAGGUU-GGGUU.CgAAGUCGCCGGA C.rubella CUUGUGCCCUAAUUUG.AUCCUCGGCUUUGGGUU.C.AAGUCGCCGGA A.alpina UUUGUGCCCUAAUUUGgAUCCUCAGGUUCGGUCUuU.AAGUCGCCUGA Infernal alignment reproduction: Figure 2A, Hawkes et al. Supplementary information is linked to the online version of the paper. NAR 2016 C U Y G U G C C C Y U C C C A G G U U Y G G R G C C G A 5´ Arabidopsis COOLAIR 90% 97% 75% 50% nucleotide present nucleotide 75% N N 97% N 90% identity A.thaliana UU.UGUGC.CCUAAUUUGA..U.CC.UCAGGUUUGG..G.UUCAAGUCGCC....GGA A.lyrata UU.UA.UUCCCUAAUUUGAU.C.CU..CAGGUU.GG..G.UUCGA...AGUCGCCGGA C.rubella UU..GUGCCCUA...AUUU.GAUCC.UCGGCUUUGGGUUC.AAGU..CGCC.....GG A.alpina UU..GUGCCCUA..AUUUG.GAUCC.UCAGGUUCGGU.UCUUAAG.UCGCCU....GA E.salsugineum UCC.GUGCCCUA...AUUU.GA.UCCUCAGGUACGGU.UC.AAGU..CGCCA....GA B.rapa UC.UGUGCCCUA...AUUU.GAUCC.AGAGGUUCGGU.UC.AAGU..GGCC....GGA #=GC SS_cons <<-<<-<<-------<<<<-<<-<<_________>>-->>->>>>--->>>---->>> * ** * * ** * Figure S2C, Hawkes et al. NAR 2016 40 60 80 100 alignment power (%) XIST h2 XIST h4 HOTAIR D2 XIST h6 ncSRA HOTAIR D1 HOTAIR D4 repA lncRNA 0 HOTAIR D3 0 20 COOLAIR XIST h8 XIST h7 XIST h1 c ( p p y) human XIST 73852753 73820615 chrX rev XIST 1 11373 24770 32103 exon1 exon2 exon3 exon4 exon5 exon6 repeats A F B C fragment D E mouse homol. TEs non-TE homol. # aligned FLAM_C Charlie29a LTR52 MIRc L2d_3end Charlie29b FLAM_C LTR78 MIRb LTR78 L2d_3end L2d_3end (linx3) (linx3) 65 52 55 61 55 56 73 60 59 47 h1 h2 h3 h4 h5 h6 h7 h8 h9 h10 e F f
https://openalex.org/W2971230652	https://europepmc.org/articles/pmc6780899?pdf=render	English	null	Rheumatoid Arthritis-Associated Mechanisms of Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans	Journal of clinical medicine	2,019	cc-by	16,337	Review Received: 30 July 2019; Accepted: 21 August 2019; Published: 26 August 2019 Received: 30 July 2019; Accepted: 21 August 2019; Published: 26 August 2019 Abstract: Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology characterized by immune-mediated damage of synovial joints and antibodies to citrullinated antigens. Periodontal disease, a bacterial-induced inﬂammatory disease of the periodontium, is commonly observed in RA and has implicated periodontal pathogens as potential triggers of the disease. In particular, Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans have gained interest as microbial candidates involved in RA pathogenesis by inducing the production of citrullinated antigens. Here, we will discuss the clinical and mechanistic evidence surrounding the role of these periodontal bacteria in RA pathogenesis, which highlights a key area for the treatment and preventive interventions in RA. Keywords: Rheumatoid arthritis; Porphyromonas gingivalis; Aggregatibacter actinomycetemcomitans; periodontitis; periodontal disease; citrullination; peptidylarginine deiminase; ACPA; anti-CCP Journal of Clinical Medicine Journal of Clinical Medicine J. Clin. Med. 2019, 8, 1309; doi:10.3390/jcm8091309 2. The Focal Infection Theory, Oral Sepsis, and RA In order to discuss the potential role of periodontal pathogens in the etiology of RA, it is important to ﬁrst review the historical background leading to the link between the mouth and RA. In contrast to other arthritides known for several centuries, the ﬁrst description of RA (initially termed “primary asthenic gout”) was not made until 1800 by Augustin Jacob Landré-Beauvais [17]. Later, Alfred Garrod in 1859 proposed the name “rheumatoid arthritis” for diseases previously known as chronic rheumatic arthritis and rheumatic gout, and provided a detailed clinical description to distinguish RA from gout [18]. Once RA was established as an independent entity, numerous investigators tried to identify the etiology of this disease. Due to the inﬂammatory nature of RA, it was thought that the etiology was infectious, leading to the search for microbial organisms in the joints of patients with RA. Between 1887 and the early 1900s, several investigators claimed to identify diﬀerent bacteria in RA synovial ﬂuid and tissue [3–6]. Although the results were inconsistent and not reproducible [19–22], these initial studies reinforced the idea that RA had an infectious origin, either due to bacteria within the joint itself or to a toxin produced by microorganisms in some other part of the body [23]. During the late 1800s, it was widely accepted that an infection in one part of the body may have eﬀects on a diﬀerent anatomical site (e.g., the syphilitic chancre is followed by systemic infection [24]). Thus, this notion fueled speculation that similar to other chronic diseases [25], RA was the result of microbial dissemination from distant sites of chronic focus of infection, including nasal sinuses, gut, lungs, genitourinary tract and the mouth [26–30]. This idea, known as the focal infection theory, deﬁned the cause and treatment of a broad range of diseases, including RA, for several decades in the early 1900s [25,31,32]. In 1891, Willoughy D. Miller ﬁrst conjectured that oral pathogens play a signiﬁcant role in the production of local and systemic diseases [33,34]. This notion was further promoted by William Hunter in 1900, who was responsible for highlighting that the mouth was a major focus of infection driving diseases in other organs of the body. In particular, he reached the incorrect conclusion that pernicious anemia was the result of infective gastritis caused by oral sepsis [35–38]. 1. Introduction Rheumatoid arthritis (RA) is an autoimmune disease of unknown etiology characterized by synovial inﬂammation, joint destruction, and high titer autoantibodies [1]. The disease aﬀects 0.5–1% of the adult population worldwide and is associated with increased mortality rates compared with the general population [2]. A microbial origin in RA has been hypothesized for more than a century [3–6], yet, diﬀerent to chronic diseases that have an infectious origin, a causal agent for RA has not been identiﬁed. Instead, numerous studies support the idea that the etiology of RA is multifactorial with a complex interplay between genetic and environmental factors [7]. Multiple risk factors have been associated with RA, including speciﬁc HLA (human leukocyte antigen) alleles, female sex, smoking, obesity, infections, and menopause, among others [8]. Nevertheless, these factors are also common in the general population, which makes it diﬃcult to understand why only few individuals develop RA and not others with similar risk. Even in monozygotic twins, the disease concordance is rather low, varying between 8.8 and 21% [9–12]. Although stochastic events may explain the discordance in disease development among individuals at risk, the current model of RA relies on the contribution of an environmental factor, most likely a microbial agent, which may be responsible for triggering the disease in susceptible individuals. Assuming that a microorganism is involved in RA pathogenesis, at least two hypotheses may explain why this causal agent has not yet been found. First, RA may be triggered by a rare pathogen that is exclusive to patients with RA, which has not yet been discovered because of the lack of proper technologies. Second, diﬀerent to other chronic infectious diseases that are linked to a single pathogen, RA may result from the individual or interacting eﬀects of diﬀerent microbial agents likely uniﬁed by triggering common arthritogenic pathways. Moreover, like other risk factors in RA, microbial species J. Clin. Med. 2019, 8, 1309; doi:10.3390/jcm8091309 www.mdpi.com/journal/jcm J. Clin. Med. 2019, 8, 1309 2 of 24 with arthritogenic potential may also be common in the general population, but only drive RA in the perfect setting of other predisposing elements. While it cannot be excluded that a single unknown pathogen may cause RA, current studies suggest that dysbiotic microbiomes may play a role in the pathogenesis of the disease [13–15]. 2. The Focal Infection Theory, Oral Sepsis, and RA Following these observations, oral sepsis (including caries, gingivitis, stomatitis, periodontitis, and tonsillitis) was considered a major focus of infection causing systemic diseases in which the etiology was unknown, such as RA [25,30,39,40]. This notion was further supported by anecdotal reports of patients with “rheumatism” cured after the removal of carious teeth [41], leading to the use of tonsillectomies and tooth extractions as the standard of treatment [29,31]. In the 1930s, this theory was refuted by the demonstration that neither tooth extraction nor tonsillectomy provided clinical beneﬁt to patients with RA [42–44]. 1. Introduction In this regard, several bacterial candidates have been mechanistically linked to RA either by creating a pro-inﬂammatory environment or by inducing the production of autoantibodies [13–15]. Here, we will discuss the evidence surrounding the potential role of two of these microbial agents, Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (Aa), in the mechanistic model of RA. Importantly, these pathogens are not exclusive to RA. Indeed, they are causal agents of periodontitis, a common disease in the adult population initially associated with RA in the late 1800s [16]. 3. Periodontitis and RA Among the diﬀerent causes of oral sepsis that were linked to RA, an association with “pyorrhea alveolaris” was noticed in 1895 [16]. Pyorrhea alveolaris, also known as Riggs’ disease and currently termed periodontal disease (PD) or periodontitis, was described by John W. Riggs in 1875 [45]. Periodontitis is a bacterial-induced chronic inﬂammatory disease aﬀecting the tissues that support 3 of 24 J. Clin. Med. 2019, 8, 1309 the teeth, including the alveolar bone. The disease results from dysbiosis of the oral microbiota and is associated with destruction of periodontal tissue, potentially leading to tooth loss [46]. Since the implantation site of the teeth is also an articulation, it was initially thought that the destruction of the periodontal membrane and alveolar bone in patients with RA and pyorrhea alveolaris was part of same RA-induced damage aﬀecting the dentoalveolar joint [16]. Nevertheless, the establishment of the focal infection theory tilted the paradigm to underscore periodontitis as a cause (not as a consequence) of RA in the early 1900s [26–28]. y [ ] However, after the downfall of the focal infection theory as the cause of RA in the 1930s, the study of periodontitis in the pathogenesis of RA was left behind. Later, at least two major advances renewed interest in periodontitis as a component of the mechanistic model of RA. The ﬁrst occurred between the 1960s–1990s, when signiﬁcant advances were made in the immunopathogenesis of periodontitis, which suggested important mechanistic similarities with autoimmune diseases [47], in particular RA [48]. These include common mechanisms of immune-mediated tissue damage, patterns of pro-inﬂammatory cytokines (e.g., interleukin (IL)-1, IL-6 and tumor necrosis factor α), immune complex deposition and complement activation [48–50]. Rapidly progressive periodontitis is also associated with HLA-DRB1 alleles linked to RA [51,52]. Moreover, B cells and plasma cells predominate in the aﬀected sites in chronic periodontitis [53–55], autoantibodies such as rheumatoid factor (RF) and anti-collagen antibodies are found in the periodontal lesion [56–59], and RF can be detected in dental periapical lesions from patients with RA [60]. The second advance was in 1999, when it was discovered that an important periodontal pathogen (P. gingivalis) secretes a peptidylarginine deiminase (PAD)-like enzyme [61], which was incorrectly thought to be equivalent to human PADs at the time [62]. 3. Periodontitis and RA As PAD enzymes are responsible for generating citrullinated proteins, major targets of autoantibodies in RA [63], it was hypothesized that periodontitis drives RA via the production of citrullinated antigens by P. gingivalis [62]. Together, these ﬁndings have provided the basis for the renewed theory that periodontitis and RA may be mechanistically related and potentially linked by a common etiologic factor. In the last 10 years, numerous epidemiological studies, extensively reviewed elsewhere [64–66], have reported a positive association of RA with PD when compared to healthy (non-RA) controls. Overall, a recent meta-analysis found that patients with RA had a 13% greater risk of periodontitis compared to healthy controls, ranging from 4 to 23% (RR: 1.13; 95% CI: 1.04, 1.23; p = 0.006) [65]. In addition, a case-control study from the Medical Biobank of Northern Sweden found that periodontitis, characterized as marginal jawbone loss, precedes the clinical onset of RA [67], supporting a potential role for PD in RA pathogenesis. Not every study, however, has conﬁrmed this association either by comparing RA with healthy controls [68,69] or with patients with osteoarthritis (OA) [65]. Although these studies have methodological diﬀerences that may explain their discrepancies, a causal relation between RA and periodontitis may be diﬃcult to sustain based purely on association studies. A major caveat in the epidemiological association between RA and periodontitis is that PD is likely the most frequent chronic infectious disease in humans worldwide. The overall rate of PD in the adult US population is 47%, with 38% over age 30 and 64% over age 65 having either severe or moderate periodontitis [70]. Moreover, severe forms of periodontitis aﬀect 11.2% of the global adult population [71]. Considering that almost half of the adult population has some form of PD, it may be hard to demonstrate a causal relationship with RA, since its prevalence is only 0.5–1% of the adult population [2]. Indeed, the relative risk of periodontitis in patients with RA is only 1.13 when compared to healthy controls, and of 1.10 compared to OA [65]. Despite these potential shortcomings, additional studies have been centered on addressing whether periodontitis, and in particular periodontal pathogens, may have a mechanistic role in RA through the production of citrullinated antigens. J. Clin. Med. 2019, 8, 1309 4 of 24 4. Citrullination and RA The discovery that the majority of patients with RA have antibodies to citrullinated proteins (known as ACPAs) [63,72,73] marked an important advance in understanding potential pathogenic mechanisms in RA [1]. Citrullination is an enzymatic process mediated by the peptidylarginine deiminases (PADs) in which arginine residues are deiminated to generate citrulline residues [74]. Five PADs have been identiﬁed in humans (PAD1–4 and 6) [1], but only PAD1–4 have citrullinating activity [75]. PAD2 and PAD4 have gained prominence as potential candidates that drive citrullination of self-antigens in RA due to their increased expression in rheumatoid synovial tissue and ﬂuid [76–78]. PADs are calcium dependent enzymes. Four, ﬁve, and six calcium-binding sites were identiﬁed in the structure of PAD1, PAD4, and PAD2, respectively, with calcium binding inducing conformational changes required to generate the active site cleft [79–81]. PADs are highly speciﬁc for peptidylarginine residues, requiring at least one additional amino acid residue N-terminal to the site of modiﬁcation [74,82]. Thus, these enzymes can only citrullinate arginine residues within polypeptide chains but not at their termini (i.e., they are endodeiminases). Diﬀerent from arginine deiminases (ADI), which catalyze the deimination of free L-arginine, PADs cannot generate citrulline from free L-arginine [74]. PADs 2, 3, and 4 form homodimers, whereas PAD1 is monomeric in solution [79–81]. Each PAD monomer contains a C-terminal catalytic domain and an N-terminal domain involved in substrate binding and protein–protein interactions [79–81]. The PADs are highly conserved and share 50%–55% sequence identity [79], but exhibit distinct substrate preferences and tissue expression [83,84]. Citrullination is a normal process across multiple tissues in humans [85]. More than 200 proteins are citrullinated in diﬀerent healthy human tissues, with the highest levels found in the brain and lungs [85]. Together, this set of proteins is referred to as the citrullinome. Large amounts of citrullinated proteins are found in RA synovial ﬂuid, including more than 100 proteins that are normally citrullinated among diﬀerent normal tissues [85–91]. This unique pattern of citrullination that includes proteins spanning the range of molecular weights is termed hypercitrullination [87]. Similar to the RA joint, it is noteworthy that periodontitis is also characterized by the accumulation of large amounts of citrullinated proteins with similar patterns of hypercitrullination found in RA [92,93], supporting the notion that PD is a potential source of citrullinated autoantigens. Understanding which components in periodontitis are responsible for driving hypercitrullination may, therefore, provide important mechanistic insights into RA pathogenesis. 5. P. gingivalis in RA Pathogenesis gingivalis, but not anti-RgpB antibodies, were positively associated with RA [129], highlighting that the anti-P. gingivalis antibody assays are not comparable. Interestingly, one study reported that anti-RgpB antibody concentrations increase before the onset of RA symptoms [123], suggesting a temporal relationship between P. gingivalis infection and the clinical onset of RA. Unlike the detection of antibodies to P. gingivalis, which has shown some positive association with RA, a number of studies detecting P. gingivalis by PCR and metagenomic sequencing in saliva, subgingival plaque and/or GCF failed to conﬁrm such an association [103–111]. In one of these studies, however, P. gingivalis was detected more frequently in a small subgroup of patients with recently diagnosed, never-treated RA, in comparison with patients with established RA or healthy controls; although this ﬁnding could be explained by a higher prevalence of severe periodontitis in those patients [111]. More recently, an increase in the relative abundance of P. gingivalis was found at healthy periodontal sites in at risk anti-CCP antibody positive individuals compared with anti-CCP negative healthy controls, though the signiﬁcance of this ﬁnding is unclear [112]. From these studies, it is diﬃcult to reach a deﬁnitive conclusion as to the relationship between RA and P. ginigvalis. A complicating factor is that every assay has diﬀerent implications with regard to the status of P. gingivalis in the patient (e.g., carrier, active infection, or past and present exposure), and their signiﬁcance is likely inﬂuenced by the source of antigen (in the case of antibodies), periodontal sample collection, and the control group used for comparison (e.g., OA, non-RA, healthy controls with or without PD). Overall, it is likely that positive associations with P. gingivalis may reﬂect PD severity rather than RA status, which importantly, neither supports nor excludes a pathogenic role for P. gingivalis in RA. 5. P. gingivalis in RA Pathogenesis P. gingivalis was linked to periodontal disease in the early 1960s, initially named Bacteroides melaninogenicus (B. melaninogenicus) [94], which was later found to include two species, B. melaninogenicus (later Prevotela melaninogenica) and B. asaccharolyticus [95]. The name B. gingivalis was later proposed to distinguish oral from non-oral B. asaccharolyticus [96,97], and in the late 1980s, B. gingivalis was further reclassiﬁed in a new genus, the Porphyromonas [98]. P. gingivalis has been implicated in the pathogenesis of periodontitis by subverting host immune defenses, leading to overgrowth of oral commensal bacteria, which causes inﬂammatory tissue destruction [99,100]. Diﬀerent approaches have been used to address a potential association between P. gingivalis and RA, many of which have shown inconsistent or inconclusive results. These include: (1) bacterial detection in gingival tissue, subgingival plaque and/or gingival crevicular ﬂuid (GCF) either by staining using anti-P. gingivalis antibodies, bacterial culture, and/or DNA ampliﬁcation (PCR) [101–107]; (2) metagenomic sequencing in saliva and subgingival plaque, which surveys complex microbial communities [103,106,108–112]; and (3) measuring antibodies to P. gingivalis in serum [101,113–125], which in contrast to the other assays that detect the existing bacteria in the mouth, it is an indirect test that determines past or present exposure to P. gingivalis. Due to its convenience, the detection of antibodies to P. gingivalis (mainly IgG) has been the most widely used assay to study the relationship between this pathogen and RA. However, it is important to highlight that there is not a standard assay to measure such antibodies. All studies used in house 5 of 24 J. Clin. Med. 2019, 8, 1309 ELISAs with diﬀerent types of P. gingivalis antigens. These include bacterial cells either as intact bacteria (not ﬁxed or ﬁxed with formalin) or bacterial extracts generated by sonication [101,113–119], puriﬁed recombinant proteins (e.g., bacterial PAD, arginine gingipain (RgpB), or hemin binding protein 35 (HBP35)] [120–124,126], HtpG peptides (HSP90 homologue) [111], P. gingivalis-speciﬁc lipopolysaccharide or outer membrane antigens [125,127]. In regard to the bacterial strains, studies have included either laboratory strains or clinical isolates, which may have some antigenic diﬀerences [128]. Considering conﬂicting conclusions from these studies [101,113–125] and others not cited in this review, an association between anti-P. gingivalis antibodies and RA is diﬃcult to sustain. However, based on a selected number of publications, two meta-analysis have reported a signiﬁcant association between higher antibody titers to P. 5. P. gingivalis in RA Pathogenesis gingivalis and RA when compared to healthy controls with unknown PD status [129], and to healthy controls with and without PD [130]. Anti-P. gingivalis antibody levels also positively correlated with ACPAs in one study [129]. Nevertheless, a signiﬁcant association was not found between antibodies to P. gingivalis and RA when compared to non-RA controls (e.g., population-based case-controls) [129]. This ﬁnding may be explained by the presence of individuals with chronic diseases and high prevalence of severe PD in the non-RA population, supporting the idea that the relative abundance of P. gingivalis is associated with PD severity regardless of RA [111]. Moreover, one meta-analysis showed that only antibodies to whole P. gingivalis, but not anti-RgpB antibodies, were positively associated with RA [129], highlighting that the anti-P. gingivalis antibody assays are not comparable. Interestingly, one study reported that anti-RgpB antibody concentrations increase before the onset of RA symptoms [123], suggesting a temporal relationship between P. gingivalis infection and the clinical onset of RA. ELISAs with diﬀerent types of P. gingivalis antigens. These include bacterial cells either as intact bacteria (not ﬁxed or ﬁxed with formalin) or bacterial extracts generated by sonication [101,113–119], puriﬁed recombinant proteins (e.g., bacterial PAD, arginine gingipain (RgpB), or hemin binding protein 35 (HBP35)] [120–124,126], HtpG peptides (HSP90 homologue) [111], P. gingivalis-speciﬁc lipopolysaccharide or outer membrane antigens [125,127]. In regard to the bacterial strains, studies have included either laboratory strains or clinical isolates, which may have some antigenic diﬀerences [128]. Considering conﬂicting conclusions from these studies [101,113–125] and others not cited in this review, an association between anti-P. gingivalis antibodies and RA is diﬃcult to sustain. However, based on a selected number of publications, two meta-analysis have reported a signiﬁcant association between higher antibody titers to P. gingivalis and RA when compared to healthy controls with unknown PD status [129], and to healthy controls with and without PD [130]. Anti-P. gingivalis antibody levels also positively correlated with ACPAs in one study [129]. Nevertheless, a signiﬁcant association was not found between antibodies to P. gingivalis and RA when compared to non-RA controls (e.g., population-based case-controls) [129]. This ﬁnding may be explained by the presence of individuals with chronic diseases and high prevalence of severe PD in the non-RA population, supporting the idea that the relative abundance of P. gingivalis is associated with PD severity regardless of RA [111]. Moreover, one meta-analysis showed that only antibodies to whole P. 5.1. P. gingivalis in Experimental Models of Arthritis In addition to epidemiological data, experimental studies using diﬀerent animal models of immune-mediated arthritis (e.g., collagen-induced arthritis (CIA), methylated bovine serum albumin-induced arthritis, and the SKG mouse model) and diﬀerent routes of bacterial inoculation (including oral, intraperitoneal and subcutaneous chambers) reached the conclusion that P. gingivalis can increase the incidence and/or exacerbate the disease [131–141]. However, many of these studies have important caveats. Several studies have reported that DBA/1 mice (a susceptible strain to induce CIA) are resistant to oral colonization by P. gingivalis and therefore, have recommended the use of non-oral routes of inoculation or diﬀerent strains of mice to study the eﬀect of P. gingivalis in experimental arthritis [131,136,142]. These ﬁndings contrast with others that have successfully induced PD with P. gingivalis in DBA/1 mice [132,134,135]. Among DBA/1 J. Clin. Med. 2019, 8, 1309 6 of 24 mice that developed periodontitis, however, there are also diﬀerences observed, with some studies showing that PD exacerbates CIA [132,134], but not others [135]. Using the SKG model of spontaneous arthritis, there are also discrepancies in the induction of PD by P. gingivalis, and whether this process aggravates arthritis [141,142]. Although these conﬂicting data may be explained by diﬀerences in mouse strains or environmental conditions that may aﬀect the immune system in mice, the data certainly highlights the lack of reproducible mouse models to study the eﬀect of periodontitis and P. gingivalis in autoimmune arthritis. g g Using models of arthritis in rats, periodontitis induced by P. gingivalis had no eﬀect in the development or severity of pristane-induced arthritis in Dark Agouti rats [143]. More recently, however, one study showed that P. gingivalis-associated periodontitis was suﬃcient to induce erosive arthritis in Lewis rats, providing the ﬁrst direct evidence that P. gingivalis may have the capacity to induce arthritis [144]. Several mechanisms have been proposed by which P. gingivalis may promote autoimmune arthritis in humans and in experimental animals. These include the induction of a systemic Th17 cell response [132–134], induction of autoantibodies [131,138,143,144], cross-reactivity between bacterial and host antigens [145], by promoting C5a generation [141], through direct dissemination of P. gingivalis into the joints [136], and by swallowing the bacteria, which may alter the gut microbiota and gut immune system [137]. Nevertheless, the production of citrullinated autoantigens by a PAD enzyme released from P. gingivalis is considered the most important mechanistic evidence to support a role of P. 5.1. P. gingivalis in Experimental Models of Arthritis gingivalis in RA pathogenesis [14,146]. 5.2. P. gingivalis PAD (PPAD) The existence of an arginine deiminase-like enzyme in P. gingivalis was suggested in the early 1990’s [147], and the enzyme was puriﬁed and characterized in 1999 [61]. PPAD is not evolutionarily related to mammalian PADs. Structurally, it is a close relative of agmatine deiminases, which are found across bacteria [148]. Diﬀerent to mammalian PADs, PPAD does not require calcium for catalysis, it is only composed of a ~40 kDa catalytic domain, and it can convert free L-arginine to free L-citrulline [61,148,149]. In addition, PPAD only modiﬁes C-terminal arginine residues (i.e., an exodeiminase), as those generated after peptide cleavage by arginine gingipain (Rgp) [149], which is also secreted by P. gingivalis. Several PPAD variants have been identiﬁed through the analysis of P. gingivalis genomes and clinical isolates [150]. One of these variants (termed PPAD-T2) has two-fold higher catalytic activity compared with PPAD from the reference strain (PPAD-T1) [150]. Whether PPAD variants are relevant for P. gingivalis pathogenesis is unknown. PPAD is detected in the outer membrane (OM) fractions of P. gingivalis and as a secreted enzyme, which is found in a soluble form and in association with outer membrane vesicles (OMVs) [120,151,152]. In most clinical isolates (termed type I isolates), extracellular PPAD is mainly found in secreted OMVs and to a minor extent in a soluble form. In contrast, a small subset of clinical isolates (termed type II isolates) showed minimal levels of PPAD in OMVs and most of the enzyme in the soluble form [152]. Type II isolates are associated with a lysine residue at position 373 in PPAD [152]. The clinical signiﬁcance of P. gingivalis type I and type II subsets in the pathogenesis of PD and RA is unknown. Secreted PPAD is believed to be a major virulence factor of P. gingivalis due to its capacity to generate ammonia during deimination of arginine to citrulline. Ammonia may protect P. gingivalis during acidic cleansing in the mouth [153], and promote periodontal infection by inhibiting neutrophil function [154,155]. 5.3. PPAD-Mediated Citrullination of Bacterial and Host Proteins 5.3. PPAD-Mediated Citrullination of Bacterial and Host Proteins A potential association between PPAD and citrullination in RA was initially suggested as a hypothesis in 2004 [62]. The ﬁrst experimental evidence, published in 2010, provided two major ﬁndings [156]. The ﬁrst was that clinical isolates of P. gingivalis were highly enriched in citrullinated proteins, suggesting that Rgp and PPAD are actively cleaving and citrullinating a large number of J. Clin. Med. 2019, 8, 1309 7 of 24 substrates in P. gingivalis. The second was that after cleavage by Rgp, PPAD citrullinates C-terminal arginine residues in ﬁbrinogen and α-enolase, two important targets of ACPAs in RA. Together, these data provided initial evidence to suggest that bacterial and host proteins citrullinated by PPAD might initiate the loss of tolerance to citrullinated autoantigens in RA [156]. Subsequent studies, however, did not conﬁrm the abundant citrullination initially found in P. gingivalis [120,128]. Indeed, although PPAD may citrullinate some proteins in P. gingivalis, this feature appears to be exclusive to a few bacterial strains (it was only detectable in reference strain W83 and the clinical isolate MDS45), and potential citrullination seems to be limited to only six bacterial proteins [128]. Whether C-terminal citrullinated peptides derived from these bacterial proteins are speciﬁc targets of ACPAs is unknown. Interestingly, PPAD is stable at low pH, resistant to limited proteolysis, and retains signiﬁcant activity after boiling [61]. Therefore, depending on the processing of P. gingivalis for protein analysis, it is possible that citrullination of bacterial proteins may occur in vitro following cell lysis [120]. As an artifact, this may explain the reports of abundant citrullination (including endocitrullination) in lysates from P. gingivalis [121,157], as well as the ﬁnding that some monoclonal ACPAs cross-react with citrullinated outer membrane antigens (OMAs) from P. gingivalis lysates [157]. Regarding autoantigen citrullination by P. gingivalis [156], it is important to highlight that neither Rgp nor PPAD have substrate speciﬁcity. The ﬁnding that ﬁbrinogen and α-enolase are cleaved and citrullinated by these enzymes is, therefore, not surprising, as any other protein would be predicted to undergo the same processing when exposed to these enzymes. Since cleavage and citrullination by Rgp and PPAD is not speciﬁc for RA autoantigens, additional evidence is required to support a role of PPAD in the lack of tolerance to citrullinated autoantigens. For example, this could be achieved by demonstrating that C-terminal citrullination mediated by Rgp and PPAD enhances immunoreactivity to autoantigens in RA. 5.5. PPAD in Experimental Models of Arthritis To establish a direct role of PPAD in the production of ACPAs in RA, several experimental models of arthritis have been used to demonstrate that P. gingivalis induces or exacerbates arthritis via PPAD-mediated autoantigen citrullination and ACPA production. However, while several studies appear to conﬁrm this hypothesis [131,138,144], these studies also highlight important misunderstandings in the study of protein citrullination and ACPAs in RA. For example, in a model of P. gingivalis and CIA, citrullinated proteins were mistakenly quantiﬁed by a colorimetric method, which measures both free citrulline and peptidylcitrulline [131]. In addition, unconventional methods have been used to detect or deﬁne ACPAs. This include the quantiﬁcation of IgG levels against ACPA (i.e., anti-ACPA antibodies) rather than antibodies to the citrullinated substrates themselves [138]. More recently, one study showed that P. gingivalis drives ACPAs (detected by the anti-CCP2 assay) and erosive arthritis in Lewis rats, providing the ﬁrst evidence that this microbial agent may be arthritogenic [144]. However, it also demonstrated that the antibody response was not speciﬁc to the citrullinated forms of antigens [144]. The failure to demonstrate a role of PPAD in autoantigen citrullination and ACPA production in mice and rats is not surprising. Although these animal models express synovial citrullinated proteins and recapitulate several features of the human disease [140,165], they do not produce antibodies speciﬁc for citrullinated proteins as observed in RA [165–169]. Moreover, they have a high amount of false-positive reactivity toward citrullinated peptides [166], which is frequently misinterpreted as ACPA positivity when non-citrullinated peptide controls are not included to conﬁrm speciﬁcity [131,139,140]. Considering these important shortcomings, even if PPAD plays a critical role in the induction of ACPAs in RA, this hypothesis will be hard to demonstrate using current models of immune-mediated arthritis. Indeed, the data from some animal models of arthritis strongly suggest that P. gingivalis may exacerbate or induce disease by mechanisms independent of ACPA production, which may involve the activity of PPAD as a virulence factor targeting either free L-citrulline or protein substrates [131]. Thus, although epidemiological and experimental data may suggest a potential role of P. gingivalis in RA pathogenesis, there is no experimental evidence to support that this process is mediated by the induction of ACPAs against host or bacterial proteins citrullinated by PPAD. J. Clin. Med. 2019, 8, 1309 J. Clin. Med. 2019, 8, 1309 8 of 24 Although some RA sera appear to react with cyclic citrullinated peptides derived from pro-PPAD (i.e., CPPs) [121,162], it is unclear whether these peptides are recognized by antibodies to native PPAD or if similar to commercial CCPs, CPPs may function as non-speciﬁc targets to detect ACPAs [161]. In this regard, although it was justiﬁed that CPPs were generated without knowledge that endocitrullination of pro-PPAD in E. coli was an artifact, there is no scientiﬁc support to continue their use in the study of RA. Nevertheless, antibodies to CPPs are still being used as biomarkers to link PPAD self-endocitrullination and RA pathogenesis. In particular, serum reactivity against the peptide termed CPP3 has been used to deﬁne clinical associations between P. gingivalis and RA [122,123,163,164], and to demonstrate that P. gingivalis can induce antibodies to citrullinated PPAD in rats [143]. Since the signiﬁcance of CPPs is unclear, the ﬁndings generated with these peptides should be taken with caution. More importantly, serum reactivity to CPPs should not be considered as a maker of citrullination induced by PPAD. 5.4. Self-Endocitrullination of Pro-PPAD PPAD is expressed as a pro-enzyme (thereafter pro-PPAD) that contains four domains: an N-terminal pro-peptide, a catalytic domain, an immunoglobulin-superfamily (IgSF) domain and a C-terminal domain (CTD) [148,149]. The mature enzyme only contains the catalytic and IgSF domains [148]. N-terminal processing appears to maintain enzyme activity and stability [120,158], while C-terminal cleavage is required for cell surface translocation of PPAD [159,160]. During the production and immunogenic analysis of recombinant pro-PPAD, it was noticed that the pro-enzyme undergoes self-endocitrullination when expressed in E. coli, and that this modiﬁed protein was preferentially recognized by RA sera when compared with the native pro-enzyme [121]. Using 13 cyclic PPAD peptides (termed CPP1-CPP13, which should not be confused with cyclic citrullinated peptides or CCPs) encompassing 18 potential citrullination sites in pro-PPAD, the study further identiﬁed immunodominant endocitrullinated peptides recognized by RA antibodies [121]. Although these results were surprising, because PPAD has no endocitrullination activity, it was assumed that these ﬁndings were analogous to PPAD from P. gingivalis. Thus, the data were interpreted to suggest that loss of tolerance to citrullinated proteins in RA may originate from an antimicrobial immune response directed against citrullinated PPAD [121]. Nevertheless, further studies demonstrated that self-endocitrullination of pro-PPAD was an artifact that resulted from the lack of N-terminal processing of the enzyme expressed in E. coli [120]. The absence of endocitrullination in PPAD has been additionally conﬁrmed in structural studies using processed PPAD expressed in E. coli or generated in P. gingivalis [148,149]. In accordance with these ﬁndings, it was also demonstrated that although patients with RA have antibodies to PPAD, these antibodies have no preferential reactivity to the citrullinated pro-enzyme made in E. coli [120]. Together, these data demonstrated that self-endocitrullination of pro-PPAD in E. coli and the serum reactivity to this modiﬁed protein are unlikely to reﬂect the function and immunogenicity of PPAD from P. gingivalis [14,120,161]. 6.1. Infection Due to Aa Aa, initially named Bacterium actinomycetem comitans, is a Gram-negative oral pathobiont ﬁrst described in 1912 as a co-isolate from actinomycosis lesions [170]. It was reclassiﬁed as Actinobacillus actinomycetemcomitans and Haemophilus actinomycetemcomitans [171], and ﬁnally transferred in 2006 to a new genus, the Aggregatibacter [172]. For many years, it was thought that Aa was unable to cause infection, except in conjunction with Actinomyces [173,174]. This idea was initially supported by the ﬁnding that injection of a pure culture of Aa in the skin of a normal individual produced no J. Clin. Med. 2019, 8, 1309 9 of 24 infection [174]. In the 1960’s, however, several cases of Aa infection not associated with actinomycosis were reported, which were mainly associated with endocarditis [175–177]. Aa is now considered part of the HACEK (Haemophilus, Aggregatibacter (previously Actinobacillus), Cardiobacterium, Eikenella, Kingella) group of bacteria that are an unusual cause of infective endocarditis [178]. In addition, it is recognized as a cause of serious infections that can aﬀect almost any organ, although these are very uncommon [179,180]. Although Aa is a rare cause of infection, this bacterium has attracted special attention since the 1980s because of its strong association with severe forms of periodontitis [181–185], both localized aggressive periodontitis (LAP) and chronic periodontitis [181,182,186–195]. Aa expresses several virulence factors [196]. Among these, the production of leukotoxin A (LtxA) is considered the major pathogenic component in the progression of aggressive periodontitis [195]. LtxA is a member of the repeats-in-toxin (RTX) family of pore-forming proteins [197]. Lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18), Mac-1 (CD11b/CD18) and αXβ2 (CD11c/CD18) act as receptors for LtxA (CD18 harbors the major binding site for LtxA), which accounts for the selective killing of human leukocytes [198]. By secreting LtxA, Aa induces cytolysis of target cells, thereby disabling host immune defenses and permitting escape from immune surveillance. Aa isolates exhibit variable virulence potential [195]. Strains of the serotype b JP2 genotype, characterized by a 530-bp deletion in the promoter region of the leukotoxin operon, has been shown to be highly virulent [199,200]. This genotype, which expresses 10- to 20-fold-higher levels of LtxA due to deletion of a transcriptional terminator [201], is associated with LAP primarily in subjects of African descent [202]. A more detailed analysis of the pathogenic mechanisms associated with the induction of periodontitis by Aa have been reviewed recently. [203] 6.3. Aa Exposure and RA Pathogenesis Similar to P. gingivalis, epidemiological studies to identify an association between Aa and RA have been done by detecting antibodies in serum. In particular, IgG antibodies to LtxA (used as markers of Aa infection) have been measured in two large cohorts of patients with RA [92,210]. In one study that included patients with established RA, anti-LtxA antibodies were signiﬁcantly associated with RA when compared to healthy individuals without PD [92]. Similarly, anti-LtxA antibodies were associated with chronic periodontitis in patients without RA, and the strongest association was observed in individuals with severe periodontitis [92]. Antibodies to LtxA may, therefore, identify a subgroup of RA patients with moderate to severe PD. Interestingly, this study also found that the association between HLA-DRB1 susceptibility alleles (known as shared epitope alleles) and RA autoantibodies was restricted to patients who had evidence of Aa exposure. This suggested that in susceptible individuals, LtxA-induced hypercitrullination may play a role in ACPA production [92]. In support of this hypothesis, ACPA production and RA-like symptoms were recently reported in a patient with Aa endocarditis who had strong a genetic susceptibility to RA conferred by three HLA alleles linked to ACPA-positive RA [211]. In a diﬀerent study using a large cohort of patients with early RA, anti-LtxA antibodies were also signiﬁcantly enriched in RA compared to healthy controls and patients with other inﬂammatory arthritides (in both groups PD status was unknown) [210], supporting the high prevalence of Aa exposure in RA, both in early and established disease. However, this study found that the interaction between HLA-DRB1-SE and anti-CCP positivity was not exclusive to anti-LtxA-positive patients with RA [210]. Major diﬀerences among these cohorts may explain this discrepancy [212]. Analogous to P. gingivalis, metagenomic sequencing and PCR analysis in subgingival plaque have not found a signiﬁcant association between the presence of Aa and RA [104,105,108,110]. In one study, however, analysis of GCF using commercial DNA probes (micro-Ident) identiﬁed Aa as the only periodontal pathogen showing signiﬁcant diﬀerences between RA and non-RA controls (both with and without PD) [107]. Thus, similar to P. gingivalis, positive associations with Aa likely indicate PD severity irrespective of RA status. Few studies have explored the potential role of Aa in experimental arthritis [142,213]. However, deﬁning the causal eﬀect of Aa in animal models of arthritis has the same limitations as those described for the study of P. gingivalis. 6.2. Aa-Induced Hypercitrullination and the Production Citrullinated RA Autoantigens A role of Aa in the pathogenesis of RA was suggested in 2016, during the search for periodontal pathogens that may explain the presence of hypercitrullination in periodontitis [92]. The study of gingival tissue from patients with PD using anti-citrulline antibodies provided initial evidence that protein citrullination is increased in PD [204,205], supporting the idea that periodontitis is a potential source of citrullinated autoantigens. In addition, the data suggested the possibility that this process was linked to the activity of PPAD [205]. However, although further analysis by mass spectrometry (MS) of GCF conﬁrmed that protein citrullination is increased in PD, this approach also provided two novel ﬁndings [92]. First, GCF from patients with PD is highly enriched in citrullinated proteins, mimicking patterns of cellular hypercitrullination found in the rheumatoid joint [92]. Second, peptide spectra of citrullinated proteins in periodontitis were consistent with endocitrullination [92], suggesting that an abnormal activation of host PADs, rather than PPAD, may be responsible for this process. Importantly, the presence of endocitrullination in periodontitis has been further conﬁrmed by MS in GCF and periodontal tissue from patients with PD, both with and without RA [93]. Among diﬀerent microbial species associated with severe periodontitis, in vitro studies identiﬁed Aa as the only pathogen that could reproduce the citrullinome found in periodontitis and RA, including the production of well-known targets of ACPAs [92]. In contrast to P. gingivalis, however, Aa does not encode a PAD-like enzyme. Instead, Aa drives citrullination by hyperactivating host PADs through the activity of LtxA. This toxin targets neutrophils, which are enriched in PAD enzymes and constitute the major immune cells in PD and the RA joint [206–208]. Since PADs are calcium dependent, the prominent calcium inﬂux generated by the lytic eﬀect of LtxA hyperactivates these enzymes, driving global hypercitrullination of a wide range of cellular proteins [92]. During this process, cell membrane integrity is eventually lost and membrane rupture occurs with release of citrullinated contents into the extracellular space [92]. Interestingly, this process is similar to the induction of hypercitrullination by host immune-eﬀector pathways (i.e., perforin and complement) in the RA joint [87], suggesting that membranolytic damage by pore-forming proteins may be a unifying mechanism in the production of citrullinated autoantigens [209]. The form of cell death induced by these pore-forming mechanisms J. Clin. Med. 6.2. Aa-Induced Hypercitrullination and the Production Citrullinated RA Autoantigens 2019, 8, 1309 10 of 24 has recently been named leukotoxic hypercitrullination (LTH) [209], to distinguish it from other forms of neutrophil death that do not induce hypercitrullination. 6.3. Aa Exposure and RA Pathogenesis Moreover, LtxA is known to have activity against leukocytes in primates, with no toxicity on rodent cells [214]. Therefore, diﬀerent to the human model, any eﬀect that Aa may have in the induction of experimental arthritis in rodents is unlikely to be driven by LtxA-induced hypercitrullination and the production of ACPAs. 7. P. gingivalis and Aa in the Mechanistic Model of RA: Causal Agents, Risk Factors, Disease Modulators, or Research Distractors Assuming that periodontitis has a mechanistic role in RA, P. gingivalis and Aa may inﬂuence disease pathogenesis at diﬀerent phases of RA development. The production of autoantibodies is an early and asymptomatic event that precedes the clinical onset of RA by several years [215,216]. The presence of a chronic and amplifying immune response against bacterial-induced RA autoantigens, in particular citrullinated antigens is, therefore, one of the most attractive hypothesis to explain disease initiation and potentially a causal association. In this context, this model explains why there is so much interest in establishing a causal relationship between P. gingivalis and the production of ACPAs in either experimental arthritis or RA. Although the current evidence neither demonstrate nor exclude the possibility that PPAD generates citrullinated autoantigens, other possibilities may explain a potential role of P. gingivalis in RA pathogenesis. Interestingly, protein citrullination is a process that is increased during inﬂammation [217]. However, while transient inﬂammation may not be suﬃcient to initiate J. Clin. Med. 2019, 8, 1309 11 of 24 an immune response to citrullinated proteins, periodontitis is a chronic non-fatal illnesses that can start during adolescence and progress over decades [218]. During this time, it is possible that some individuals at risk may develop autoantibodies to citrullinated proteins. P. gingivalis, a keystone microorganism in periodontitis, is certainly a strong candidate for promoting chronic inﬂammation [100], which may indirectly drive the production of autoantibodies in susceptible individuals. In contrast to P. gingivalis, a potential role of Aa in the lack of tolerance to RA-associated autoantigens involves the induction of lytic hypercitrullination [92]. In this regard, diﬀerent factors such as Aa virulence (i.e., LtxA production) and bacterial load may deﬁne the risk of developing autoantibodies in the context of Aa infection in susceptible individuals [92,211]. Importantly, as other bacterial species also produce pore-forming toxins [219], it is possible that other pathogens may induce hypercitrullination and ACPAs at other mucosal sites, including the lung and gut [1,209]. LTH as a mechanism of autoantigen production by diﬀerent pathogens may explain why every patient with seropositive RA does not have exposure to Aa and/or PD. p p Interestingly, patients with RA show abnormalities in central and peripheral B cell tolerance checkpoints [220–222], likely due to an intrinsic genetic predisposition [223,224]. This results in the accumulation of autoreactive and polyreactive B cells that can recognize immunoglobulins and citrullinated peptides [220,221]. 7. P. gingivalis and Aa in the Mechanistic Model of RA: Causal Agents, Risk Factors, Disease Modulators, or Research Distractors Depending on genetic and environmental risk factors, autoreactive or polyreactive naïve B cells may have a selective advantage to develop into high aﬃnity autoreactive memory B cells through somatic mutations and aﬃnity maturation. In this scenario, autoantigens generated by arthritogenic bacteria may promote the expansion and maturation of already existing autoreactive cells, rather than initiating the loss of tolerance to host antigens. A prerequisite of having autoreactive/polyreactive B cells to induce pathogenic autoantibodies by P. gingivalis or Aa may explain why only a limited number of individuals with PD may develop RA. Although the hypothesis that periodontal pathogens (e.g., P. gingivalis or Aa) are causal agents of RA is an attractive idea, it is also possible that these bacteria may only be relevant as risk factors for the development of ACPA-positive RA. Alternatively, once RA has been established, persistent low-grade inﬂammation associated with PD may modulate RA progression and severity. Lastly, despite the enormous enthusiasm of demonstrating that periodontitis is relevant for RA pathogenesis, it cannot yet be excluded that patients with RA may have a higher risk of developing PD (even during the pre-clinical phase of RA), but neither periodontitis, P. gingivalis, nor Aa may play a pathogenic role in the disease. 9. Conclusions Although some epidemiological and mechanistic data supports a role for P. gingivalis and Aa in the pathogenic model of RA, it is important to underscore that these oral pathobionts do not fulﬁll the Henle-Koch’s postulates of causation [230–232]. These microbial agents neither occur in every case of the disease, nor are speciﬁc for patients with RA. In the context of inducing ACPA production in vivo to satisfy the third postulate, it will require the use of animal models that can truly reproduce the autoantibody response observed in RA. Considering these shortcomings, demonstrating or discarding a role of P. gingivalis and Aa in the pathogenesis of RA may require a diﬀerent view of how to deﬁne disease causality by these microbial agents [212]. In the case of P. gingivalis, it is imperative to recognize that its potential role in RA involves elucidation of at least three diﬀerent questions. The ﬁrst is whether P. gingivalis is relevant for RA pathogenesis, either by initiating or exacerbating the disease. The second is whether P. gingivalis is important for RA because of the production of PPAD or because it plays a critical role in the induction of periodontitis by inciting inﬂammation. Indeed, it appears that ligation-induced periodontitis (without additional infection with P. gingivalis) is suﬃcient to exacerbate CIA in Wistar rats [233]. The third is whether PPAD drives RA due to the production of citrullinated antigens and the induction of ACPAs in the host or because this enzyme is a virulence factor that facilitates bacterial growth and the establishment of PD. Although these questions have been somewhat addressed, deﬁnitive conclusions cannot be reached from the available data. Deﬁning the role of PPAD in the production of citrullinated antigens and whether C-terminal deimination oﬀers an advantage for peptide immunogenicity remains a high priority. Importantly, other potential arthritogenic mechanisms induced by P. gingivalis, besides autoantigen citrullination, should be kept in consideration. In the case of Aa, this model oﬀers an advantage over other periodontal bacteria because of its capacity to induce neutrophil hypercitrullination and the release of citrullinated autoantigens through osmotic lysis [92]. However, the number of studies linking Aa with RA are too limited to truly determine its pathogenic signiﬁcance for the disease. Similar to P. gingivalis, rigorous studies are necessary to support or refute the role of Aa in the etiology of RA. 8. Therapeutic Implications The possibility of identifying a factor that triggers a multifactorial disease, such as RA, has critical implications for treatment and preventive interventions. The success of any therapy, however, may depend on targeting such a factor at the correct time during the evolution of the disease [225]. In this regard, although a systematic review and meta-analysis suggested that periodontal treatment may decrease some markers of disease activity in RA [226], these ﬁndings remain controversial [227]. The lack of an eﬃcient response to PD treatment on RA disease activity may suggest that periodontitis has no pathogenic role in RA, or that it is most important for disease initiation, during the pre-clinical phase, and that treatment during established disease is no longer eﬀective. If a periodontal pathogen is relevant for the initial breech of tolerance to arthritogenic autoantigens, aiming to treat asymptomatic autoantibody positive (e.g., anti-CCP, RF, or any other autoantibody) individuals may still be too late to stop the progression to symptomatic RA. In this scenario, preventive therapies (e.g., oral hygiene and potentially vaccines against periodontal pathogens) should be initiated in at-risk individuals prior to the development of these serologies. In contrast, if periodontitis is important for the ampliﬁcation of the autoimmune response in preclinical RA, there is a window of opportunity to target seropositive asymptomatic individuals. Indeed, to date, this is the most feasible hypothesis that could be therapeutically addressed. However, if periodontal pathogens only play a role in the induction of speciﬁc subtypes of autoantibodies (e.g., ACPAs), eradication of these agents during the pre-clinical 12 of 24 J. Clin. Med. 2019, 8, 1309 phase of the disease may only decrease the risk of developing certain autoantibodies (and potentially further RA severity), but may not change the course of the disease, including the production of antibodies to other autoantigens. Interestingly, recent evidence suggest that periodontitis is a potential source of proteins modiﬁed by malondialdehyde-acetaldehyde adducts and carbamylation [228], which are also common targets of autoantibodies in RA. [229] Thus, independent of the periodontal pathogen, preventing, or targeting periodontitis may be the best exploratory option to prevent RA. Conﬂicts of Interest: F.A. and E.D. are authors on issued patent no. 8,975,033, entitled “Human autoantibodies speciﬁc for PAD3 which are cross-reactive with PAD4 and their use in the diagnosis and treatment of rheumatoid arthritis and related diseases” and on provisional patent no. 62/481,158 entitled “Anti-PAD2 antibody for treating and evaluating rheumatoid arthritis”. F.A. serves as consultant for Bristol-Myers Squibb. E.D. received a research grant from Pﬁzer and from Bristol-Myers Squibb. E.D. previously served on the scientiﬁc advisory board for Padlock Therapeutics, Inc. The remaining authors declare that they have no conﬂicts of interests. Funding: This research was funded by the Jerome L. Greene Foundation, Rheumatology Research Foundation, and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) at the National Institutes of Health (NIH) grant number R01 AR069569. The content of this paper is solely the responsibility of the author and does not represent the oﬃcial views of the NIAMS or the NIH. Author Contributions: Conceptualization and writing—original draft preparation: F.A.; writing—review and editing: E.G.-B., A.M., and E.D. 9. Conclusions Together, these oral pathobionts pose an opportunity to understand whether bacterial-associated citrullination is a mechanism involved in RA pathogenesis. This has important implications for the implementation of preventive interventions in RA. Author Contributions: Conceptualization and writing—original draft preparation: F.A.; writing—review and editing: E.G.-B., A.M., and E.D. Funding: This research was funded by the Jerome L. Greene Foundation, Rheumatology Research Foundation, and National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS) at the National Institutes of Health (NIH) grant number R01 AR069569. The content of this paper is solely the responsibility of the author and does not represent the oﬃcial views of the NIAMS or the NIH. Conﬂicts of Interest: F.A. and E.D. are authors on issued patent no. 8,975,033, entitled “Human autoantibodies speciﬁc for PAD3 which are cross-reactive with PAD4 and their use in the diagnosis and treatment of rheumatoid arthritis and related diseases” and on provisional patent no. 62/481,158 entitled “Anti-PAD2 antibody for treating and evaluating rheumatoid arthritis”. F.A. serves as consultant for Bristol-Myers Squibb. E.D. received a research grant from Pﬁzer and from Bristol-Myers Squibb. E.D. previously served on the scientiﬁc advisory board for Padlock Therapeutics, Inc. The remaining authors declare that they have no conﬂicts of interests. 13 of 24 J. Clin. Med. 2019, 8, 1309 References 1. Darrah, E.; Andrade, F. Rheumatoid arthritis and citrullination. Curr. Opin. Rheumatol. 2018, 30, 72–78. [CrossRef] 2. 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https://openalex.org/W4281916910	https://link.springer.com/content/pdf/10.1007/s00454-022-00396-7.pdf	English	null	Efficient Quantisation and Weak Covering of High Dimensional Cubes	Discrete and computational geometry/Discrete & computational geometry	2,022	cc-by	11,808	Discrete & Computational Geometry (2022) 68:540–565 https://doi.org/10.1007/s00454-022-00396-7 Discrete & Computational Geometry (2022) 68:540–565 https://doi.org/10.1007/s00454-022-00396-7 Jack Noonan1 · Anatoly Zhigljavsky1 Received: 22 May 2020 / Revised: 22 January 2022 / Accepted: 2 February 2022 / Published online: 3 June 2022 © The Author(s) 2022 Received: 22 May 2020 / Revised: 22 January 2022 / Accepted: 2 February 2022 / Published online: 3 June 2022 © The Author(s) 2022 Abstract Let Zn = {Z1, . . . , Zn} be a design; that is, a collection of n points Z j ∈[−1, 1]d. We study the quality of quantisation of [−1, 1]d by the points of Zn and the problem of quality of coverage of [−1, 1]d by Bd(Zn,r), the union of balls centred at Z j ∈Zn. We concentrate on the cases where the dimension d is not small, d ≥5, and n is not too large, n ≤2d. We deﬁne the design Dn,δ as a 2d−1 design deﬁned on vertices of the cube [−δ, δ]d, 0 ≤δ ≤1. For this design, we derive a closed-form expression for the quantisation error and very accurate approximations for the coverage area vol ([−1, 1]d ∩Bd(Zn,r)). We provide results of a large-scale numerical investigation conﬁrming the accuracy of the developed approximations and the efﬁciency of the designs Dn,δ. Keywords Covering · Quantisation · Facility location · Space-ﬁlling · Computer experiments · High dimension · Voronoi set Mathematics Subject Classiﬁcation 11H31 · 05B40 · 52C17 1.2 Main Problems of Interest We will study the following two main characteristics of designs Zn = {Z1, . . . , Zn} ⊂Rd. 1. Quantization error. Let X = (x1, . . . , xd) be uniform random vector on [−1, 1]d. The mean squared quantisation error for a design Zn is deﬁned by 1. Quantization error. Let X = (x1, . . . , xd) be uniform random vector on [−1, 1]d. The mean squared quantisation error for a design Zn is deﬁned by θ(Zn) = EXϱ2(X, Zn), where ϱ2(X, Zn) = min Zi∈Zn ∥X −Zi∥2. (1) (1) 2. Weak covering. Denote the proportion of the cube [−1, 1]d covered by the union of n balls Bd(Zn,r) = n j=1 Bd(Z j,r) by 2. Weak covering. Denote the proportion of the cube [−1, 1]d covered by the union of n balls Bd(Zn,r) = n j=1 Bd(Z j,r) by Cd(Zn,r) := vol ([−1, 1]d ∩Bd(Zn,r)) 2d . Cd(Zn,r) := vol ([−1, 1]d ∩Bd(Zn,r)) 2d . For given radius r > 0, the union of n balls Bd(Zn,r) makes the (1−γ )-coverage of the cube [−1, 1]d if For given radius r > 0, the union of n balls Bd(Zn,r) makes the (1−γ )-coverage of the cube [−1, 1]d if Cd(Zn,r) = 1 −γ. (2) (2) Complete coverage corresponds to γ = 0. In this paper, the complete coverage of [−1, 1]d will not be enforced and we will mostly be interested in weak covering, that is, achieving (2) with some small γ > 0. Complete coverage corresponds to γ = 0. In this paper, the complete coverage of [−1, 1]d will not be enforced and we will mostly be interested in weak covering, that is, achieving (2) with some small γ > 0. Two n-point designs Zn and Z′ n will be differentiated in terms of performance as follows: (a) Zn dominates Z′ n for quantisation if θ(Zn) < θ(Z′ n); (b) if for a given γ ≥0, Cd(Zn,r1) = Cd(Z′ n,r2) = 1 −γ and r1 < r2, then the design Zn provides a more efﬁcient (1 −γ )-coverage than Z′ n and is therefore preferable. In Sect. 1.4 we extend these deﬁnitions by allowing the two designs to have different number of points and, moreover, to have different dimensions. 1.1 Main Notation – ∥· ∥: the Euclidean norm; d – ∥· ∥: the Euclidean norm; d – ∥· ∥: the Euclidean norm; – Bd(Z,r) = {Y ∈Rd : ∥Y −Z∥≤r}: d-dimensional ball of radius r centered at Z ∈Rd; – Bd(Z,r) = {Y ∈Rd : ∥Y −Z∥≤r}: d-dimensional ball of radius r centered at Z ∈Rd; Editor in Charge: Kenneth Clarkson Jack Noonan Noonanj1@cardiff.ac.uk Anatoly Zhigljavsky ZhigljavskyAA@cardiff.ac.uk 1 School of Mathematics, Cardiff University, Cardiff, CF244AG, UK 123 541 Discrete & Computational Geometry (2022) 68:540–565 – Zn = {Z1, . . . , Zn}: a design; that is, a collection of n points Z j ∈Rd; – Zn = {Z1, . . . , Zn}: a design; that is, a collection of n points Z j ∈Rd; – Bd(Zn,r) = n j=1 Bd(Z j,r); j – Bd(Zn,r) = n j=1 Bd(Z j,r); j 1 j – Cd(Zn,r) = vol ([−1, 1]d ∩Bd(Zn,r))/2d: the proportion of the cube [−1, 1]d covered by Bd(Zn,r); j – Cd(Zn,r) = vol ([−1, 1]d ∩Bd(Zn,r))/2d: the proportion of the cube [−1, 1]d covered by Bd(Zn,r); – vectors in Rd are row-vectors; – for any a ∈R, a = (a, a, . . . , a) ∈Rd. – for any a ∈R, a = (a, a, . . . , a) ∈Rd. 1.2 Main Problems of Interest Numerical construction of n-point designs with moderate values of n with good quantisation and coverage properties has recently attracted much attention in view of diverse applications in several ﬁelds including computer experiments [7, 8, 11], global optimization [15], function approximation [12, 13], and numerical integration [9]. Such designs are often referred to as space-ﬁlling designs. Readers can ﬁnd many additional references in the citations above. Unlike the exiting literature on space- ﬁlling, we concentrate on theoretical properties of a family of very efﬁcient designs and derivation of accurate approximations for the characteristics of interest. 123 123 542 Discrete & Computational Geometry (2022) 68:540–565 1.3 Relation Between Quantisation and Weak Coverage The two characteristics, Cd(Zn,r) and θ(Zn), are related: Cd(Zn,r), as a function of r ≥0, is the c.d.f. of the r.v. ϱ(X, Zn) while θ(Zn) is the second moment of the distribution with this c.d.f.: θ(Zn) = r≥0 r2dCd(Zn,r). (3) (3) In particular, this yields that if an n-point design Z∗ n maximises, in the set of all n- point designs, Cd(Zn,r) for all r > 0, then it also minimises θ(Zn). Moreover, if r.v. ϱ(X, Zn) stochastically dominates ϱ(X, Z′ n), so that Cd(Z′ n,r) ≤Cd(Zn,r) for all r ≥0 and the inequality is strict for at least one r, then θ(Zn) < θ(Z′ n). The relation (3) can alternatively be written as θ(Zn) = r≥0 r dCd(Zn, √r), (4) (4) where Cd(Zn, √r), considered as a function of r, is the c.d.f. of the r.v. ϱ2(X, Zn) and hence θ(Zn) is the mean of this r.v. Relation (4) is simply another form of (1). where Cd(Zn, √r), considered as a function of r, is the c.d.f. of the r.v. ϱ2(X, Zn) and hence θ(Zn) is the mean of this r.v. Relation (4) is simply another form of (1). 1.5 Thickness of Covering Let γ = 0 in Deﬁnition 1.2. Then r1(Zn) is the covering radius associated with Zn so that the union of the balls Bd(Zn,r) with r = r1(Zn) makes a coverage of [−1, 1]d. Let us tile up the whole space Rd with the translations of the cube [−1, 1]d and corresponding translations of the balls Bd(Zn,r). This would make a full coverage of the whole space; denote this space coverage by Bd(Z(n),r). The thickness Θ of any space covering is deﬁned, see [1, (1), Chap.2], as the average number of balls containing a point of the whole space. In our case of Bd(Z(n),r), the thickness is Θ(Bd(Z(n),r)) = n vol(Bd(0,r)) vol([−1, 1]d) = nrd vol(Bd(0, 1)) 2d . The normalised thickness, θ, is the thickness Θ divided by vol(Bd(0, 1)), the volume of the unit ball, see [1, (2), Chap. 2]. In the case of Bd(Z(n),r), the normalised thickness is The normalised thickness, θ, is the thickness Θ divided by vol(Bd(0, 1)), the volume of the unit ball, see [1, (2), Chap. 2]. In the case of Bd(Z(n),r), the normalised thickness is is θ(Bd(Z(n),r)) = nrd 2d = dd/2[R1(Z(n))]d, where we have recalled that r = r1(Zn) and R1−γ (Zn) = n1/dr1−γ (Zn)/(2 √ d) for any 0 ≤γ ≤1. We can thus deﬁne the normalised thickness of the covering of the cube by the same formula and extend it to any 0 ≤γ ≤1: where we have recalled that r = r1(Zn) and R1−γ (Zn) = n1/dr1−γ (Zn)/(2 √ d) for any 0 ≤γ ≤1. We can thus deﬁne the normalised thickness of the covering of the cube by the same formula and extend it to any 0 ≤γ ≤1: Deﬁnition 1.3 Let Bd(Zn,r) be a (1 −γ )-coverage of the cube [−1, 1]d with 0 ≤ γ ≤1. Its normalised thickness is deﬁned by θ(Bd(Zn,r)) = ( √ d R)d, (9) (9) where R = n1/dr/(2 √ d), see (5). In view of (9), we can reformulate the deﬁnition (7) of the (1 −γ )-covering optimal design by saying that this design minimises (normalised) thickness in the set of all (1 −γ )-covering designs. 1.4 Re-Normalised Versions and Formulation of Optimal Design Problems In view of (13), the naturally deﬁned re-normalised version of θ(Zn) is Qd(Zn) = n2/dθ(Zn)/(4d). From (4) and (3), Qd(Zn) is the expectation of n2/dϱ2(X, Zn)/(4d) and the second moment of the r.v. n1/dϱ(X, Zn)/(2 √ d) respectively. This suggests the following re-normalization of the radius r with respect to n and d: R = n1/dr 2 √ d . (5) (5) (5) We can then deﬁne optimal designs as follows. Let d be ﬁxed, Zn = {Zn} be the set of all n-point designs and Z = ∞ n=1 Zn be the set of all designs. Deﬁnition 1.1 The design Z∗ m with some m is optimal for quantisation in [−1, 1]d, i Qd(Z∗ m) = min n min Zn∈Zn Qd(Zn) = min Z∈Z Qd(Z). (6) (6) Deﬁnition 1.2 The design Z∗ m with some m is optimal for (1−γ )-coverage of [−1, 1]d, if Deﬁnition 1.2 The design Z∗ m with some m is optimal for (1−γ )-coverage of [−1, 1]d, if R1−γ (Z∗ m) = min n min Zn∈Zn R1−γ (Zn) = min Z∈Z R1−γ (Z). (7) (7) Here 0 ≤γ ≤1 and for a given design Zn ∈Zn, Here 0 ≤γ ≤1 and for a given design Zn ∈Zn, R1−γ (Zn) = n1/dr1−γ (Zn) 2 √ d , (8) (8) 123 12 Discrete & Computational Geometry (2022) 68:540–565 Discrete & Computational Geometry (2022) 68:540–565 543 Importance of the factor √ d in (5) will be seen in Sect. 3.5 where we shall study the asymptotical behaviour of (1 −γ )-coverings for large d. 1.6 The Design of the Main Interest We will be mostly interested in the following n-point design Zn = Dn,δ deﬁned only for n = 2d−1: Design Dn,δ: a 2d−1 design deﬁned on vertices of the cube [−δ, δ]d, 0 ≤δ ≤1. 123 544 Discrete & Computational Geometry (2022) 68:540–565 For theoretical comparison with design Dn,δ, we shall consider the following simple design, which extends to the integer point lattice Zd (shifted by 1/2) in the whole space Rd: For theoretical comparison with design Dn,δ, we shall consider the following simple design, which extends to the integer point lattice Zd (shifted by 1/2) in the whole space Rd: Design D(0) n : the collection of 2d points (±1/2, . . . , ±1/2), all vertices of the cube [−1/2, 1/2]d. Without loss of generality, while considering the design Dn,δ we assume that the point Z1 ∈Dn,δ = {Z1, . . . , Zn} is Z1 = δ = (δ, . . . , δ). Similarly, the ﬁrst point in D(0) n is Z1 = 1/2 = (1/2, . . . , 1/2). Note also that for numerical comparisons, in Sect. 4 we shall introduce one more design. The design Dn,1/2 extends to the lattice Dd (shifted by 1/2) containing points X = (x1, . . . , xd) with integer components satisfying x1 + . . . + xd = 0 (mod 2), see [1, Sect. 7.1, Chap. 4]; this lattice is sometimes called “checkerboard lattice”. The motivation to theoretically study the design Dn,δ is a consequence of numerical results reported in [14] and [4], where the present authors have considered n-point designs in d-dimensional cubes providing good coverage and quantisation and have shown that for all dimensions d ≥7, the design Dn,δ with suitable δ provides the best quantisation and coverage per point among all other designs considered. Aiming at practical applications mentioned in Sect. 1.2, our aim was to consider the designs with n which is not too large and in any case does not exceed 2d. If the number of points n in a design is much larger than 2d, then we may use the following scheme of construction of efﬁcient quantisers in the cube [−1, 1]d: (a) con- struct one of the very efﬁcient lattice space quantisers, see [1, Sect. 3, Chap. 1.6 The Design of the Main Interest 2], (b) take the lattice points belonging to a very large cube, and (c) scale the chosen large cube to [−1, 1]d. In view of [2, Thm. 8.9], as n →∞, the normalised quantisation error Qd(Zn) of the sequence of resulting designs Zn converges to the respective quan- tisation error of the lattice space quantiser. However, for any given n the study of quantisation error of such designs is difﬁcult (both, numerically and theoretically) as there could be several non-congruent types of Voronoi cells due to boundary condi- tions. Note also that the boundary conditions make signiﬁcant difference in relative efﬁciencies of the resulting designs. In particular, the checkerboard lattice Dd is better than the integer-point lattice Zd for all d ≥3 as a space quantiser and becomes the best lattice space quantiser for d = 4 but in the case of cube [−1, 1]d, the design Dn,δ (with optimal δ) makes a better quantiser than D(0) n for d ≥7 only; see Sect. 2.4 for theoretical and numerical comparison of the two designs. 1.7 Structure of the Rest of the Paper and the Main Results In Sect. 2 we study Qd(Dn,δ), the normalised mean squared quantisation error for the design Dn,δ. There are two important results, Theorems 2.2 and 2.3. In Theorem 2.2, we derive the explicit form for the Voronoi cells for the points of the design Dn,δ and in Theorem 2.3 we derive a closed-form expression for Qd(Dn,δ) for any δ > 0. As a consequence, in Corollary 2.4 we determine the optimal value of δ. The main result of Sect. 3 is Theorem 3.2, where we derive closed-form expressions (in terms of Cd,Z,r, the fraction of the cube [−1, 1]d covered by a ball Bd(Z,r)) for the Discrete & Computational Geometry (2022) 68:540–565 545 coverage area with vol ([−1, 1]d ∩Bd(Zn,r)). Then, using accurate approximations for Cd,Z,r, we derive approximations for vol ([−1, 1]d ∩Bd(Zn,r)). In Theorem 3.4 we derive asymptotic expressions for the (1−γ )-coverage radius for the design Dd,1/2 and show that for any γ > 0, the ratio of the (1−γ )-coverage radius to the 1-coverage radius tends to 1/ √ 3 as d →∞. Numerical results of Sect. 3.5 conﬁrm that even for rather small d, the 0.999-coverage radius is much smaller than the 1-coverage radius providing the full coverage. coverage area with vol ([−1, 1]d ∩Bd(Zn,r)). Then, using accurate approximations for Cd,Z,r, we derive approximations for vol ([−1, 1]d ∩Bd(Zn,r)). In Theorem 3.4 we derive asymptotic expressions for the (1−γ )-coverage radius for the design Dd,1/2 and show that for any γ > 0, the ratio of the (1−γ )-coverage radius to the 1-coverage radius tends to 1/ √ 3 as d →∞. Numerical results of Sect. 3.5 conﬁrm that even for rather small d, the 0.999-coverage radius is much smaller than the 1-coverage radius providing the full coverage. In Sect. 4 we demonstrate that the approximations developed in Sect. 3 are very accurate and make a comparative study of selected designs used for quantisation and covering. In Appendices A–C, we provide proofs of the most technical results. In Appendix D, for completeness, we brieﬂy derive an approximation for Cd,Z,r with arbitrary d, Z, and r. The two most important contributions of this paper are: a) derivation of the closed- form expression for the quantisation error for the design Dn,δ, and b) derivation of accurate approximations for the coverage area vol ([−1, 1]d ∩Bd(Zn,r)) for the design Dn,δ. 2.2 Re-Normalisation of the Quantisation Error To compare efﬁciency of n-point designs Zn with different values of n, one must suitably normalise θ(Zn) with respect to n. Specialising a classical characteristic for quantisation in space, as formulated in [1, (86), Chap. 2], we obtain Qd(Zn) = 1 d · (1/n) n i=1 V (Zi) ∥X −Zi∥2 d X (1/n) n i=1 vol(V (Zi)) 1+2/d . (12) (12) Note that Qd(Zn) is re-normalised with respect to dimension d too, not only with respectton.Normalization1/d withrespecttod isverynaturalinviewofthedeﬁnition of the Euclidean norm. Using (10), for the cube [−1, 1]d, (12) can be expressed as Qd(Zn) = n2/dθ(Zn) d n i=1 vol(V (Zi)) 2/d = n2/dθ(Zn) d vol([−1, 1]d)2/d = n2/d 4d θ(Zn). (13) (13) 2.1 Reformulation in Terms of the Voronoi Cells Consider any n-point design Zn = {Z1, . . . , Zn}. The Voronoi cell V (Zi) for Zi ∈Zn is deﬁned as Consider any n-point design Zn = {Z1, . . . , Zn}. The Voronoi cell V (Zi) for Zi ∈Z is deﬁned as V (Zi) = {x ∈[−1, 1]d : ∥Zi −x∥≤∥Z j −x∥for j ̸= i}. The mean squared quantisation error θ(Zn) introduced in (1) can be written in terms of the Voronoi cells as follows: The mean squared quantisation error θ(Zn) introduced in (1) can be written in terms of the Voronoi cells as follows: θ(Zn) = EX min i=1,...,n ∥X −Zi∥2 = 1 vol([−1, 1]d) n i=1 V (Zi) ∥X −Zi∥2d X, (10) here X = (x1, . . . , xd) and d X = dx1dx2 . . . dxd. where X = (x1, . . . , xd) and d X = dx1dx2 . . . dxd. This reformulation has signiﬁcant beneﬁt when the design Zn has certain structure. In particular, if all of the Voronoi cells V (Zi), i = 1, . . . , n, are congruent, then we can simplify (10) to θ(Zn) = 1 vol(V (Z1)) V (Z1) ∥X −Z1∥2d X. (11) (11) In Sect. 2.4, this formula will be the starting point for derivation of the closed-form expression for θ(Zn) for the design Dn,δ. In Sect. 2.4, this formula will be the starting point for derivation of the closed-form expression for θ(Zn) for the design Dn,δ. Discrete & Computational Geometry (2022) 68:540–565 546 2.3 Voronoi Cells for Dn,ı Proposition 2.1 ConsiderthedesignD(0) n,δ,thecollectionofn =2d points(±δ, . . . , ±δ), 0 < δ <1. The Voronoi cells for this design are all congruent. The Voronoi cell for the point δ = (δ, δ, . . . , δ) is the cube Proposition 2.1 ConsiderthedesignD(0) n,δ,thecollectionofn =2d points(±δ, . . . , ±δ), 0 < δ <1. The Voronoi cells for this design are all congruent. The Voronoi cell for the point δ = (δ, δ, . . . , δ) is the cube Proposition 2.1 ConsiderthedesignD(0) n,δ,thecollectionofn =2d points(±δ, . . . , ±δ), 0 < δ <1. The Voronoi cells for this design are all congruent. The Voronoi cell for the point δ = (δ, δ, . . . , δ) is the cube C0 = {X = (x1, . . . , xd) ∈Rd : 0 ≤xi ≤1, i = 1, 2, . . . , d}. (14) (14) Proof Consider the Voronoi cells created by the design D(0) n,δ in the whole space Rd. For the point δ = (δ, δ, . . . , δ), the Voronoi cell is clearly {X = (x1, . . . , xd) : xi ≥0}. By intersecting this set with the cube [−1, 1]d we obtain (14). ⊓⊔ Theorem 2.2 The Voronoi cells of the design Dn,δ = {Z1, . . . , Zn} are all congruent. The Voronoi cell for the point Z1 = δ = (δ, δ, . . . , δ) ∈Rd is Theorem 2.2 The Voronoi cells of the design Dn,δ = {Z1, . . . , Zn} are all congruent. The Voronoi cell for the point Z1 = δ = (δ, δ, . . . , δ) ∈Rd is V (Z1) = C0 ∪ d j=1 U j, (15) (15) where C0 is the cube (14) and where C0 is the cube (14) and U j = {X = (x1, x2, . . . , xd) ∈Rd : −1 ≤x j ≤0, \|x j\| ≤xk ≤1 for all k ̸= j}. (16 U j = {X = (x1, x2, . . . , xd) ∈Rd : −1 ≤x j ≤0, \|x j\| ≤xk ≤1 for all k ̸= j}. (16) (16) he volume of V (Z1) is vol(V (Z1)) = 2. The volume of V (Z1) is vol(V (Z1)) = 2. Proof The design Dn,δ is symmetric with respect to all components implying that all n = 2d−1 Voronoi cells are congruent immediately yielding that their volumes equal 2. 123 123 Discrete & Computational Geometry (2022) 68:540–565 547 Consider V (Z1) with Z1 = δ. Since Dn,δ ⊂D(0) n,δ, where design D(0) n,δ is introduced in Proposition 2.1, and C0 is the Voronoi set of δ for design D(0) n,δ, C0 ⊂V (δ) for design Dn,δ too. Consider the d cubes adjacent to C0: Consider V (Z1) with Z1 = δ. Since Dn,δ ⊂D(0) n,δ, where design D(0) n,δ is introduced in Proposition 2.1, and C0 is the Voronoi set of δ for design D(0) n,δ, C0 ⊂V (δ) for design Dn,δ too. Consider the d cubes adjacent to C0: C j = {X = (x1, x2, . . . , xd) ∈Rd : −1 ≤x j ≤0, 0 ≤xi ≤1 for all i ̸= j}; (17 C j = {X = (x1, x2, . . . , xd) ∈Rd : −1 ≤x j ≤0, 0 ≤xi ≤1 for all i ̸= j}; (17) (17) (17) j = 1, . . . , d. A part of each cube C j belongs to V (Z1). This part is exactly the set U j deﬁned by (16). This can be seen as follows. A part of C j also belongs to the Voronoi set of the point X jk = δ −2δe j −2δek, where el = (0, . . . , 0, 1, 0, . . . , 0) with 1 placed at l-th place; all components of X jk are δ except j-th and k-th components which are −δ. We have to have \|x j\| ≤xk, for a point X ∈C j to be closer to Z1 than to X jk. Joining all constraints for X = (x1, x2, . . . where C0 is the cube (14) and , xd) ∈C j (k = 1, . . . , d, k ̸= j) we obtain (16) and hence (15). ⊓⊔ 2.4 Explicit Formulae for the Quantisation Error heorem 2.3 For the design Dn,δ with 0 ≤δ ≤1, we obtain Theorem 2.3 For the design Dn,δ with 0 ≤δ ≤1, we obtain θ(Dn,δ) = d δ2 −δ + 1 3 + 2δ d + 1, (18) Qd(Dn,δ) = 2−2/d δ2 −δ + 1 3 + 2δ d(d + 1) . (19) (18) (19) Proof To compute θ(Dn,δ), we use (11), where, in view of Theorem 2.2, vol(V (Z1)) = 2. Using the expression (15) for V (Z1) with Z1 = δ, we obtain Proof To compute θ(Dn,δ), we use (11), where, in view of Theorem 2.2, vol(V (Z1)) = 2. Using the expression (15) for V (Z1) with Z1 = δ, we obtain θ(Zn) = 1 2 V (Z1) ∥X −Z1∥2 d X = 1 2 C0 ∥X −Z1∥2 d X + d U1 ∥X −Z1∥2 d X . (20) (20) Consider the two terms in (20) separately. The ﬁrst term is easy: C0 ∥X −Z1∥2 d X = C0 d i=1 (xi −δ)2dx1 . . . dxd = d 1 0 (x −δ)2dx = d δ2 −δ + 1 3 . (21) (21) 123 Discrete & Computational Geometry (2022) 68:540–565 548 For the second term we have U1 ∥X −Z1∥2 d X = 0 −1 1 \|x1\| . . . 1 \|x1\| d i=1 (xi −δ)2dx2 . . . dxd dx1 = 0 −1 (x1 −δ)2(1 + x1)d−1dx1 + (d −1) 0 −1 (1 + x1)d−2 1 \|x1\| (x2 −δ)2dx2 dx1 = δ2 −δ + 1 3 + 4δ d(d + 1) . (22) (22) Inserting the obtained expressions into (20) we obtain (18). The expression (19) is a consequence of (13), (18), and n = 2d−1. ⊓⊔ Inserting the obtained expressions into (20) we obtain (18). The expression (19) is a consequence of (13), (18), and n = 2d−1. ⊓⊔ A simple consequence of Theorem 2.3 is the following corollary. Corollary 2.4 The optimal value of δ minimising θ(Dn,δ) and Qd(Dn,δ) is δ∗= 1 2 − 1 d(d + 1) ; (23) (23) for this value, for this value, Qd(Dn,δ∗) = min δ Qd(Dn,δ) = 2−2/d 1 12 + d2 + d −1 (d + 1)2d2 . (24) (24) Let us make several remarks. Let us make several remarks. Let us make several remarks. Let us make several remarks. 1. 2.4 Explicit Formulae for the Quantisation Error . . , 50 0.20 0.10 0.30 0.2 0.0 Qd 0.4 δ 0.6 0.8 1.0 Fig. 2 Qd(Dn,δ) as a function of δ and Qd(D(0) n ) = 1/12; d = 10 0.20 0.10 0.30 0.2 0.0 Qd 0.4 δ 0.6 0.8 1.0 Fig. 2 Qd(Dn,δ) as a function of δ and Qd(D(0) n ) = 1/12; d = 10 Fig. 2 Qd(Dn,δ) as a function of δ and Qd(D(0) n ) = 1/12; d = 10 the quantisation error Qd for Dd in Rd is slightly smaller than Qd for Dn,1/2 in [−1, 1]d. the quantisation error Qd for Dd in Rd is slightly smaller than Qd for Dn,1/2 in [−1, 1]d. the quantisation error Qd for Dd in Rd is slightly smaller than Qd for Dn,1/2 in [−1, 1]d. 5. The optimal value of δ in (23) is smaller than 1/2. This is caused by a non- symmetrical shape of the Voronoi cells V (Z j) for designs Dn,δ, which is clearly visible in (15). 5. The optimal value of δ in (23) is smaller than 1/2. This is caused by a non- symmetrical shape of the Voronoi cells V (Z j) for designs Dn,δ, which is clearly visible in (15). 6. The minimal value of Qd(Dn,δ∗) with respect to d is attained at d = 15. 7. Formulae (23) and (24) are in agreement with numerical results presented in Table 4 of [14] and Table 5 of [4]. 7. Formulae (23) and (24) are in agreement with numerical results presented in Table 4 of [14] and Table 5 of [4]. Let us now brieﬂy illustrate the results above. In Fig. 1, the black circles depict the quantity Qd(Dn,δ∗) as a function of d. The quantity Qd(D(0) n ) = 1/12 is shown with the solid red line. We conclude that from dimension seven onwards, the design Dn,δ∗ provides better quantisation per points than the design D(0) n . Moreover, for d > 15, the quantity Qd(Dn,δ∗) slowly increases and converges to 1/12. Typical behaviour of Qd(Dn,δ) as a function of δ is shown in Fig. 2. This ﬁgure demonstrates the signiﬁcance of choosing δ optimally. 2.4 Explicit Formulae for the Quantisation Error The value δ∗can be alternatively characterised by the well-known optimality con- dition of a general design saying that each design point of an optimal quantiser must be a centroid of the related Voronoi cell; see e.g. [10]. Speciﬁcally, each design point Zi ∈Dn,δ is the centroid of V (Zi) if and only if δ = δ∗. 1. The value δ∗can be alternatively characterised by the well-known optimality con- dition of a general design saying that each design point of an optimal quantiser must be a centroid of the related Voronoi cell; see e.g. [10]. Speciﬁcally, each design point Zi ∈Dn,δ is the centroid of V (Zi) if and only if δ = δ∗. 2. From (19), for the design Dn,1/2 we get 2. From (19), for the design Dn,1/2 we get 2. From (19), for the design Dn,1/2 we get Qd(Dn,1/2) = 2−2/d 1 12 + 1 (d + 1)d ; (25) (25) this value is always slightly larger than (24). this value is always slightly larger than (24). this value is always slightly larger than (24). 3. For the one-point design D(0) = {0} with the single point 0 and the design D(0) n with n = 2d points (±1/2, . . . , ±1/2) we have Qd(D(0)) = Qd(D(0) n ) = 1/12, which coincides with the value of Qd in the case of space quantisation by the integer-point lattice Zd, see [1, Chaps. 2 and 21]. 4. The quantisation error (25) for the design Dn,1/2 have almost exactly the same form as the quantisation error for the ‘checkerboard lattice’ Dd in Rd; the difference is in the factor 1/2 in the last term in (25), see [1, (27), Chap. 21]. Naturally, 4. The quantisation error (25) for the design Dn,1/2 have almost exactly the same form as the quantisation error for the ‘checkerboard lattice’ Dd in Rd; the difference is in the factor 1/2 in the last term in (25), see [1, (27), Chap. 21]. Naturally, Discrete & Computational Geometry (2022) 68:540–565 549 0.085 0.075 0.095 0.105 Qd 20 10 d 30 40 50 Fig. 1 Qd(Dn,δ∗) and Qd(Dn,1/2) as functions of d and Qd(D(0) n ) = 1/12; d = 3, . . . , 50 0.085 0.075 0.095 0.105 Qd 20 10 d 30 40 50 Fig. 1 Qd(Dn,δ∗) and Qd(Dn,1/2) as functions of d and Qd(D(0) n ) = 1/12; d = 3, . The following lemma is straightforward. Lemma 3.1 Consider a design Zn = {Z1, . . . , Zn} such that all Voronoi cells V (Zi) are congruent. Then for any Zi ∈Zn, Cd(Zn,r) = Vd,Zi,r. In view of Theorem 2.2, for design Dn,δ all Voronoi cells V (Zi) are congruent and vol(V (Zi)) = 2; recall that n = 2d−1. By then applying Lemma 3.1 and without loss of generality we have chosen Z1 = δ = (δ, δ, . . . , δ) ∈Rd, we have for any r > 0 Vd,δ,r = vol (V (δ) ∩Bd(δ,r)) 2 = Cd(Dn,δ,r). (26) (26) In order to formulate explicit expressions for Vd,δ,r, we need the important quantity, proportion of intersection of [−1, 1]d with one ball. Take the cube [−1, 1]d and a ball Bd(Z,r) = {Y ∈Rd : ∥Y −Z∥≤r} centred at a point Z = (z1, . . . , zd) ∈Rd; this point Z could be outside [−1, 1]d. The fraction of the cube [−1, 1]d covered by the ball Bd(Z,r) is denoted by In order to formulate explicit expressions for Vd,δ,r, we need the important quantity, proportion of intersection of [−1, 1]d with one ball. Take the cube [−1, 1]d and a ball Bd(Z,r) = {Y ∈Rd : ∥Y −Z∥≤r} centred at a point Z = (z1, . . . , zd) ∈Rd; this point Z could be outside [−1, 1]d. The fraction of the cube [−1, 1]d covered by the ball Bd(Z,r) is denoted by Cd,Z,r = vol ([−1, 1]d ∩Bd(Z,r)) 2d . Cd,Z,r = vol ([−1, 1]d ∩Bd(Z,r)) 2d . 3 Closed-Form Expressions for the Coverage Area with Dn,ı and Approximations In this section, we will derive explicit expressions for the coverage area of the cube [−1, 1]d by the union of the balls Bd(Dn,δ,r) associated with the design Dn,δ intro- duced in Sect. 1.2. That is, we will derive expressions for the quantity Cd(Dn,δ,r) for 123 550 Discrete & Computational Geometry (2022) 68:540–565 all values of r. Then, in Sect. 3.3, we shall obtain approximations for Cd(Dn,δ,r). The accuracy of the approximations will be assessed in Sect. 4.2. 3.1 Reduction to Voronoi Cells For an n-point design Zn = {Z1, . . . , Zn}, denote the proportion of the Voronoi cell around Zi covered by the ball Bd(Zi,r) as Vd,Zi,r := vol (V (Zi) ∩Bd(Zi,r)) vol(V (Zi)) . Vd,Zi,r := vol (V (Zi) ∩Bd(Zi,r)) vol(V (Zi)) . The following lemma is straightforward. 3.3 Approximation for Cd(Dn,ı, r) Accurate approximations for Cd,Z,r for arbitrary d, Z, and r were developed in [14]. By using the general expansion in the central limit theorem for sums of independent non-identical r.v., the following approximation was developed: Cd,Z,r ∼= Φ(t) + ∥Z∥2 + d/63 5 √ 3(∥Z∥2 + d/15)3/2 (1 −t2)ϕ(t), (30) (30) where where t = √ 3(r2 −∥Z∥2 −d/3) 2 ∥Z∥2 + d/15 . A short derivation of this approximation is included in Appendix D. Using (30), we formulate the following approximation for Cd(Dn,δ,r). Approximations for Cd(Dn,δ,r). Approximate the values C·,·,· in formulae (27), (28), and (29) with corresponding approximations (30). 3.2 Expressing Cd(Dn,ı, r) Through Cd,Z,r Theorem 3.2 Depending on the values of r and δ, the quantity Cd(Dn,δ,r) can be expressed through Cd,Z,r for suitable Z as follows. – For r ≤δ: – For r ≤δ: Cd(Dn,δ,r) = Cd,2δ−1,2r 2 . (27) (27) – For δ ≤r ≤1 + δ: Cd(Dn,δ,r) = 1 2 Cd,2δ−1,2r + d r−δ 0 C d−1, 2δ−1−t 1−t , 2 √ r2−(t+δ)2 1−t (1 −t)d−1 dt . (28) (28) 123 123 Discrete & Computational Geometry (2022) 68:540–565 551 – For r ≥1 + δ: Cd(Dn,δ,r) = 1 2 Cd,2δ−1,2r + d 1 0 C d−1, 2δ−1−t 1−t , 2 √ r2−(t+δ)2 1−t (1 −t)d−1dt . (29) – For r ≥1 + δ: Cd(Dn,δ,r) = 1 2 Cd,2δ−1,2r + d 1 0 C d−1, 2δ−1−t 1−t , 2 √ r2−(t+δ)2 1−t (1 −t)d−1dt . (29 (29) The proof of Theorem 3.2 is given in Appendix A. 3.4 Simple Bounds for Cd(Dn,ı, r) Lemma 3.3 For any r ≥0, 0 < δ < 1, and δ = (δ, δ, . . . , δ) ∈Rd, the quantit Cd(Dn,δ,r) can be bounded as follows: Lemma 3.3 For any r ≥0, 0 < δ < 1, and δ = (δ, δ, . . . , δ) ∈Rd, the quantity Cd(Dn,δ,r) can be bounded as follows: Cd,2δ−1,2r + Cd,A,2r 2 ≤Cd(Dn,δ,r) ≤Cd,2δ−1,2r, (31) (31) where A = (2δ + 1, 2δ −1, . . . , 2δ −1) ∈Rd. The proof of Lemma 3.3 is given in Appendix B. In Figs. 3 and 4, using the approxima- tion given in (30) we study the tightness of the bounds given in (31). In these ﬁgures, the dashed red line, dashed blue line and solid black line depict the upper bound, the lower bound and the approximation for Cd(Dn,δ,r) respectively. We see that the upper bound is very sharp across r and d; this behaviour is not seen with the lower bound. Discrete & Computational Geometry (2022) 68:540–565 552 r 0.2 0.0 0.4 0.6 0.8 1.0 1.5 1.0 2.0 Fig. 3 Cd(Dn,δ,r) with upper and lower bounds: d = 20 r 0.2 0.0 0.4 0.6 0.8 1.0 2.6 2.8 3.0 3.2 3.4 Fig. 4 Cd(Dn,δ,r) with upper and lower bounds: d = 100 r 0.2 0.0 0.4 0.6 0.8 1.0 1.5 1.0 2.0 Fig. 3 Cd(Dn,δ,r) with upper and lower bounds: d = 20 r 0.2 0.0 0.4 0.6 0.8 1.0 1.5 1.0 2.0 Fig. 3 Cd(Dn,δ,r) with upper and lower bounds: d = 20 r 0.2 0.0 0.4 0.6 0.8 1.0 2.6 2.8 3.0 3.2 3.4 Fig. 4 Cd(Dn,δ,r) with upper and lower bounds: d = 100 r 0.2 0.0 0.4 0.6 0.8 1.0 2.6 2.8 3.0 3.2 3.4 Fig. 4 Cd(Dn,δ,r) with upper and lower bounds: d = 100 3.5 ‘Do Not Try to Cover the Vertices’ In this section, we theoretically support the recommendation ‘do not try to cover the vertices’ which was ﬁrst stated in [14] and supported in [4] on the basis of numerical evidence. In other words, we will show on the example of the design Dn,1/2 that in large dimensions the attempt to cover the whole cube rather than 0.999 of it leads to a dramatic increase of the required radius of the balls. Theorem 3.4 Let γ be ﬁxed, 0 ≤γ ≤1. Consider (1 −γ )-coverings of [−1, 1]d generated by the designs Dn,δ and the associated normalised radii R1−γ (Dn,δ), see (8). For any 0 < γ < 1 and 0 ≤δ ≤1, the limit of R1−γ (Dn,δ), as d →∞, exists and achieves minimal value for δ = 1/2. Moreover, R1−γ (Dn,1/2)/R1(Dn,1/2) →1/ √ 3 as d →∞, for any 0 < γ < 1. Theorem 3.4 Let γ be ﬁxed, 0 ≤γ ≤1. Consider (1 −γ )-coverings of [−1, 1]d generated by the designs Dn,δ and the associated normalised radii R1−γ (Dn,δ), see (8). For any 0 < γ < 1 and 0 ≤δ ≤1, the limit of R1−γ (Dn,δ), as d →∞, exists and achieves minimal value for δ = 1/2. Moreover, R1−γ (Dn,1/2)/R1(Dn,1/2) →1/ √ 3 as d →∞, for any 0 < γ < 1. Proof is given in Appendix C. Proof is given in Appendix C. In Figs. 5 and 6 using a solid red line we depict the approximation of Cd(Dn,1/2,r) as a function of R = n1/dr/(2 √ d), see (5). The vertical green line illustrates the value of R0.999 and the vertical blue line depicts R1 = n1/d√d + 8/(4 √ d). These ﬁgures illustrate that as d increases, for all γ we have R1−γ /R1 slowly tending to 1/ √ 3. From the proof of Theorem 3.4, it transpires that Cd(Dn,δ,r) as a function of R converges to the jump function with the jump at 1/(2 √ 3). Discrete & Computational Geometry (2022) 68:540–565 553 0.1 0.2 0.2 0.0 0.3 0.4 0.4 R 0.5 0.6 0.6 0.8 1.0 0.7 Fig. 5 Cd(Dn,1/2,r) with R0.999 and R1: d = 5 0.2 0.0 0.4 0.6 0.8 1.0 0.1 0.2 0.3 R 0.4 0.5 Fig. 6 Cd(Dn,1/2,r) with R0.999 and R1: d = 50 0.1 0.2 0.2 0.0 0.3 0.4 0.4 R 0.5 0.6 0.6 0.8 1.0 0.7 Fig. 5 Cd(Dn,1/2,r) with R0.999 and R1: d = 5 0.1 0.2 0.2 0.0 0.3 0.4 0.4 R 0.5 0.6 0.6 0.8 1.0 0.7 Fig. 5 Cd(Dn,1/2,r) with R0.999 and R1: d = 5 0.2 0.0 0.4 0.6 0.8 1.0 0.1 0.2 0.3 R 0.4 0.5 Fig. 6 Cd(Dn,1/2,r) with R0.999 and R1: d = 50 4 Numerical Studies For comparative purposes, we introduce another design which is one of the most popular designs (both, for quantisation and covering) considered in applications. Design Sn: Z1, . . . , Zn are taken from a low-discrepancy Sobol’s sequence on the cube [−1, 1]d. For constructing the design Sn, we use the R-implementation provided in the well- known ‘SobolSequence’ package [3]. For Sn, we have set n = 1024 and F2 = 10 (an input parameter for the Sobol sequence function). Sobol sequences Sn attain their best space-ﬁlling properties when n is a power of 2; that is, when n = 2ℓfor some integer ℓ. We have chosen ℓ= 10. As we study renormalised characteristics Qd( · ) and R1−γ ( · ) of designs, exact value of ℓfor Sn with n = 2ℓis almost irrelevant: in particular, numerically computed values Qd(S2ℓ) and R1−γ (S2ℓ) for ℓ= 8, 9, 11, 12 are almost indistinguishable from the corresponding values for ℓ= 10 provided below in Tables 1 and 2. By varying values of ℓ, we are not improving space-ﬁlling properties of S2ℓ. In fact, increase of ℓgenerally leads to a slight deterioration of normalised space-ﬁlling characteristics (including Qd( · ) and R1−γ ( · )) of Sobol sequences. 123 123 123 Discrete & Computational Geometry (2022) 68:540–565 554 4.1 Quantisation and Weak Covering Comparisons In Table 1, we compare the normalised mean squared quantisation error Qd(Zn) deﬁned in (13) across three designs: Dn,δ∗with δ∗given in (23), D(0) n and Sn. In Table 2, we compare the normalised statistic R1−γ introduced in (7), where we have ﬁxed γ = 0.01. For designs Dn,δ (with the optimal value of δ), Dn,1/2 and D(0) n we have also included R1, the smallest normalised radius that ensures the full coverage. Let us make some remarks concerning Tables 1 and 2. 1 Let us make some remarks concerning Tables 1 and 2. – In conjunction with Fig. 1, Table 1 shows that for d ≥7, the quantisation for design Dn,δ∗is superior over all other designs considered. – For the weak coverage statistic R1−γ , the superiority of Dn,δ with optimal δ over all other designs considered is seen for d ≥10. g – For the designs Dn,δ, the optimal value of δ minimising R1−γ depends on γ . – From remark 6 of Sect. 2.4, the minimal value of Qd(Dn,δ∗) with respect to d is attained at d = 15. For d > 15, the quantity Qd(Dn,δ∗) increases with d, slowly converging to Qd(D(0) n ) = 1/12. This non-monotonic behaviour can be seen in Table 1. – Unlike the case of Qd(Dn,δ∗), such non-monotonic behaviour is not seen for the quantity R1−γ and R1−γ (Dn,δ) monotonically decreases as d increases. Also, Theorem 3.4 implies that for any γ ∈(0, 1), R1−γ (Dn,δ) →1/(2 √ 3) ∼= 0.289 as d →∞. 4.1 Quantisation and Weak Covering Comparisons Table 1 Normalised mean squared quantisation error Qd for three designs and different d d = 5 d = 7 d = 10 d = 15 d = 20 Qd(Dn,δ∗) 0.0876 0.0827 0.0804 0.0798 0.0800 Qd(D(0) n ) 0.0833 0.0833 0.0833 0.0833 0.0833 Qd(Sn) 0.0988 0.1003 0.1022 0.1060 0.1086 Table 2 Normalised statistic R1−γ across d with γ = 0.01 (value in brackets corresponds to optimal δ) d = 5 d = 7 d = 10 d = 15 d = 20 R1−γ (Dn,δ) 0.4750 (0.54) 0.3992 (0.53) 0.3635 (0.52) 0.3483 (0.51) 0.3417 (0.50) R1−γ (Dn,1/2) 0.4765 0.4039 0.3649 0.3484 0.3417 R1−γ (D(0) n ) 0.4092 0.3923 0.3766 0.3612 0.3522 R1−γ (Sn) 0.4714 0.4528 0.4256 0.4074 0.3967 R1(Dn,δ) 0.6984 (0.54) 0.6555 (0.53) 0.6178 (0.52) 0.5856 (0.51) 0.5714 (0.50) R1(Dn,1/2) 0.7019 0.6629 0.6259 0.5912 0.5714 R1(D(0) n ) 0.5000 0.5000 0.5000 0.5000 0.5000 Table 2 Normalised statistic R1−γ across d with γ = 0.01 (value in brackets corresponds to optimal δ) Discrete & Computational Geometry (2022) 68:540–565 555 0.2 0.0 0.4 0.6 0.8 1.0 δ 0.2 0.4 0.6 0.8 1.0 Fig. 7 Cd(Dn,δ,r) and its approximation: d = 5, r from 0.7 to 1.1 increasing by 0.1 0.2 0.0 0.4 0.6 0.8 1.0 δ 0.2 0.4 0.6 0.8 1.0 Fig. 7 Cd(Dn,δ,r) and its approximation: d = 5, r from 0.7 to 1.1 increasing by 0.1 0.2 0.0 0.4 0.6 0.8 1.0 0.2 0.4 0.6 δ 0.8 1.0 Fig. 8 Cd(Dn,δ,r) and its approximation: d = 10, r from 0.95 to 1.15 increasing by 0.05 0.2 0.0 0.4 0.6 0.8 1.0 0.2 0.4 0.6 δ 0.8 1.0 Fig. 8 Cd(Dn,δ,r) and its approximation: d = 10, r from 0.95 to 1.15 increasing by 0.05 4.2 Accuracy of Covering Approximation and Dependence on ı In this section, we assess the accuracy of the approximation of Cd(Dn,δ,r) developed in Sect. 3.3 and the behaviour of Cd(Dn,δ,r) as a function of δ. In Figs. 7, 8, 9, and 10, the thick dashed black lines depict Cd(Dn,δ,r) for several different choices of r; these values are obtained via Monte Carlo simulations. The thinner solid lines depict its approximation of Sect. 3.3. These ﬁgures show that the approximation is extremely accurate for all r, δ, and d; we emphasise that the approximation remains accurate even for very small dimensions like d = 3. These ﬁgures also clearly demonstrate the δ-effect saying that a signiﬁcantly more efﬁcient weak coverage can be achieved with a suitable choice of δ. This is particularly evident in higher dimensions, see Figs. 9 and 10. Figs. 11 and 12 illustrate Theorem 3.4 and show the rate of convergence of the covering radii as d increases. Let the probability density function f (r) be deﬁned by dCd(Dn,δ,r) = f (r) dr, where Cd(Dn,δ,r) as a function of r is viewed as the c.d.f. of the r.v. r = ϱ(X, Zn), see Sect. 1.3. Trivial calculations yield that the density for the normalised radius R expressed by (5) is pd(R) := 2 √ d n−1/d f (2 √ d n−1/d R). In Fig. 11, we depict the density pd( · ) for d = 5, 10, 20 with blue, red, and black lines respectively. The respective c.d.f.’s R 0 pd(τ) dτ are shown in Fig. 12 under the same colouring scheme. 12 123 3 Discrete & Computational Geometry (2022) 68:540–565 556 0.2 0.0 0.4 0.6 0.8 1.0 δ 0.2 0.4 0.6 0.8 1.0 Fig. 9 Cd(Dn,δ,r) and its approximation: d = 15, r from 1.15 to 1.35 increasing by 0.05 0.2 0.0 0.4 0.6 0.8 1.0 δ 0.2 0.4 0.6 0.8 1.0 Fig. 9 Cd(Dn,δ,r) and its approximation: d = 15, r from 1.15 to 1.35 increasing by 0.05 0.2 0.0 0.4 0.6 0.8 1.0 δ 0.2 0.4 0.6 0.8 1.0 Fig. 10 Cd(Dn,δ,r) and its approximation: d = 50, r from 2.05 to 2.35 increasing by 0.075 0.2 0.0 0.4 0.6 0.8 1.0 δ 0.2 0.4 0.6 0.8 1.0 Fig. 4.2 Accuracy of Covering Approximation and Dependence on ı 10 Cd(Dn,δ,r) and its approximation: d = 50, r from 2.05 to 2.35 increasing by 0.075 0 5 10 15 0.0 0.1 0.2 0.3 R 0.4 0.5 0.6 0 5 10 15 0.0 0.1 0.2 0.3 R 0.4 0.5 0.6 Fig. 11 Densities fd(R) for the design Dn,δ∗; d = 5, 10, 20 Fig. 11 Densities fd(R) for the design Dn,δ∗; d = 5, 10, 20 4.3 Stochastic Dominance In Figs. 13 and 14, we depict the c.d.f.’s for the normalised distance n1/dϱ(X, Zn)/ (2 √ d) for two designs: Dn,δ∗in red, and D(0) n in black. We can see that the design Dn,δ∗stochastically dominates the design D(0) n for d = 10 but for d = 5 the design D(0) n is preferable to the design Dn,δ∗although there is no clear domination; this is in line with ﬁndings from Sects. 2.4 and 4.1, see e.g. Fig. 1, Tables 1 and 2. g g g In Fig. 15, we depict the c.d.f.’s for the normalised distance n1/dϱ(X, Zn)/(2 √ d) foresign D(0) n (in red) and design Sn (in black). We can see that for d = 5, the design D(0) n stochastically dominates the design Sn. The style of Fig. 16 is the same as Fig. 15, In Fig. 15, we depict the c.d.f.’s for the normalised distance n1/dϱ(X, Zn)/(2 √ d) foresign D(0) n (in red) and design Sn (in black). We can see that for d = 5, the design D(0) n stochastically dominates the design Sn. The style of Fig. 16 is the same as Fig. 15, 123 Discrete & Computational Geometry (2022) 68:540–565 557 0.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 R 0.5 0.6 Fig. 12 c.d.f.’s of R for the design Dn,δ∗; d = 5, 10, 20 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.2 0.4 0.6 0.8 1.0 R Fig. 13 d = 5: design D(0) n is preferable to design Dn,δ∗ 0.0 0.2 0.4 0.6 0.8 1.0 R 0.1 0.2 0.3 0.4 0.5 Fig. 14 d = 10: design Dn,δ∗stochastically dominates design D(0) n 0.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 R 0.5 0.6 Fig. 12 c.d.f.’s of R for the design Dn,δ∗; d = 5, 10, 20 0.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 R 0.5 0.6 Fig. 12 c.d.f.’s of R for the design Dn,δ∗; d = 5, 10, 20 0.1 0.2 0.3 0.4 0.5 0.6 0.0 0.2 0.4 0.6 0.8 1.0 R Fig. 13 d = 5: design D(0) n is preferable to design Dn,δ∗ 0.0 0.2 0.4 0.6 0.8 1.0 R 0.1 0.2 0.3 0.4 0.5 Fig. 14 d = 10: design Dn,δ∗stochastically dominates design D(0) n however we set d = 10 and the design D(0) n is replaced with the design Dn,δ∗. 4.3 Stochastic Dominance Here we see a very clear stochastic dominance of the design Dn,δ∗over the design Sn. All ﬁndings are consistent with ﬁndings from Sect. 4.1, see Tables 1 and 2. Acknowledgements The authors are grateful to our colleague Iskander Aliev for numerous intelligent discussions. The authors are very grateful to the referee for their comments that greatly improved the presentation of the paper. Furthermore, we are grateful for the concise proof of Theorem 3.2 suggested by the reviewer which has been included in this manuscript. OpenAccess ThisarticleislicensedunderaCreativeCommonsAttribution4.0InternationalLicense,which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included 12 3 Discrete & Computational Geometry (2022) 68:540–565 558 0.6 0.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 0.5 R Fig. 15 d = 5: design D(0) n stochastically dominates design Sn 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 R Fig. 16 d = 10: design Dn,δ∗stochastically dominates design Sn 0.6 0.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 0.5 R Fig. 15 d = 5: design D(0) n stochastically dominates design Sn 0.6 0.0 0.2 0.4 0.6 0.8 1.0 0.1 0.2 0.3 0.4 0.5 R Fig. 15 d = 5: design D(0) n stochastically dominates design Sn 0.1 0.2 0.3 0.4 0.5 0.0 0.2 0.4 0.6 0.8 1.0 R Fig. 16 d = 10: design Dn,δ∗stochastically dominates design Sn in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Appendix A: Proof of Theorem 3.2 In view of (26), Cd(Dn,δ,r) = Vd,δ,r for all 0 ≤δ ≤1 and r ≥0 and we shall derive expressions for Vd,δ,r rather than Cd(Dn,δ,r). Case (a): r ≤δ. To prove this case, we observe i) for this range of r, Bd(δ,r) ⊂ [0, 1]d; ii) the fraction of a cube covered by a ball is preserved under invertible afﬁne transformations; iii) the afﬁne transformation x →2x −1 maps the ball Bd(δ,r) and cube [0, 1]d to Bd(2δ −1, 2r) and [−1, 1]d, respectively. This leads to Vd,δ,r = vol (Bd(δ,r)) 2 vol([0, 1]d) = vol (Bd(2δ −1, 2r)) 2 vol([−1, 1]d) = Cd,2δ−1,2r 2 . Case (b): δ ≤r ≤1 + δ. Using (15) we obtain Case (b): δ ≤r ≤1 + δ. Using (15) we obtain Vd,δ,r = vol (Bd(δ,r) ∩C0) + d vol (Bd(δ,r) ∩U1) 2 . 123 559 Discrete & Computational Geometry (2022) 68:540–565 The ﬁrst quantity in the numerator has been considered in case (a) and it is simply Cd,2δ−1,2r. Therefore we aim to reformulate the second quantity within the brackets, vol (Bd(δ,r) ∩U1). Denote by P(t) = {(x1, x2, . . . , xd) : x1 = t}, the (d −1)- dimensional hyperplane. Then vol (Bd(δ,r) ∩U1) = 0 δ−r vold−1(P(t) ∩Bd(δ,r) ∩U1) dt. Notice further that U1 ∩P(t) = {t} × [\|t\|, 1]d−1, for −1 ≤t ≤0, Bd(δ,r) ∩P(t) = {t} × Bd−1 δ, r2 −(t −δ)2 for δ −r ≤t ≤0,r ≥δ, (32) (32) where δ = (δ, . . . , δ) ∈Rd−1 and the natural identiﬁcation of P(t) with Rd−1 is used. The r.h.s. in (32) are a (d −1)-dimensional cube and ball respectively. Since covered fraction is preserved under afﬁne transformations in Rd−1, it sufﬁces to construct one, denote by φ, for which φ([\|t\|, 1]d−1) = [−1, 1]d. In P(t), such φ maps the cube from (32) to the standard cube [−1, 1]d. Clearly, φ can be taken as φ : x →x −(1 + \|t\|)/2 (1 −\|t\|)/2 = 2x −(1 + \|t\|) 1 −\|t\| , where 1 = (1, . . . , 1) and \|t\| = (\|t\|, . . . , \|t\|) are constant vectors in Rd−1. Note that where 1 = (1, . . . , 1) and \|t\| = (\|t\|, . . . , \|t\|) are constant vectors in Rd−1. Appendix A: Proof of Theorem 3.2 Note that φ Bd−1 δ, r2 −(t −δ)2 = Bd−1 2δ −(1 + \|t\|) 1 −\|t\| , 2 r2 −(t −δ)2 1 −\|t\| . Finally, by the preservation of covered fraction, we obtain vold−1(P(t) ∩Bd(δ,r) ∩U1) = vold−1([\|t\|, 1]d−1) · C d−1, 2δ−1−\|t\| 1−\|t\| , 2 √ r2−(t−δ)2 1−\|t\| . vold−1(P(t) ∩Bd(δ,r) ∩U1) = vold−1([\|t\|, 1]d−1) · C d−1, 2δ−1−\|t\| 1−\|t\| , 2 √ r2−(t−δ)2 1−\|t\| . As a result, Vd,δ,r = 1 2 Cd,2δ−1,2r + d 0 δ−r C d−1, 2δ−1−\|t\| 1−\|t\| , 2 √ r2−(t−δ)2 1−\|t\| (1 −\|t\|)d−1 dt = 1 2 Cd,2δ−1,2r + d r−δ 0 C d−1, 2δ−1−t 1−t , 2 √ r2−(t+δ)2 1−t (1 −t)d−1 dt . (33) (33) Case (c): r ≥1 + δ. Case (c) is almost identical to case (b), with the only change occurring within the lower limit of integration in (33); the lower limit of the integral remains at −1 for all r ≥1 + δ. Since the steps are almost identical to case (b), they are omitted and we simply conclude: Case (c): r ≥1 + δ. Case (c) is almost identical to case (b), with the only change occurring within the lower limit of integration in (33); the lower limit of the integral remains at −1 for all r ≥1 + δ. Since the steps are almost identical to case (b), they are omitted and we simply conclude: Vd,δ,r = 1 2 Cd,2δ−1,2r + d 1 0 C d−1, 2δ−1−t 1−t , 2 √ r2−(t+δ)2 1−t (1 −t)d−1 dt . 123 123 Discrete & Computational Geometry (2022) 68:540–565 560 Appendix B: Proof of Lemma 3.3 (a) Let us ﬁrst prove the upper bound in (31). Consider the set U j deﬁned in (16) and the associated set U ′ j = X = (x1, x2, . . . , xd) ∈[0, 1]d : \|x j\| ≤xk ≤1 for all k ̸= j ⊂C0. We have vol(U j) = vol(U ′ j) = 1/d and V (δ) = C0 ∪ d j=1 U j, d j=1 U ′ j = C0 = [0, 1]d. Let us prove that for any r ≥0 we have Let us prove that for any r ≥0 we have vol (U j ∩Bd(δ,r)) ≤vol (U ′ j ∩Bd(δ,r)). (34) (34) With any X = (x1, x2, . . . , xd) ∈U ′ 1, we associate the point X′ = (−x1, x2, . . . , xd) ∈U1 by simply changing the sign in the ﬁrst component. For these two points, we have ∥δ −X∥2 = (x1 −δ)2 + d k=2 (xk −δ)2 < (−x1 −δ)2 + d k=2 (xk −δ)2 = ∥δ −X′∥2. Therefore, ∥δ −X∥2 ≤r implies ∥δ −X′∥2 ≤r yielding (34). To prove the upper bound in (31) for all r we must consider two cases: r ≤δ and r ≥δ. For r ≤δ, we clearly have Vd,δ,r = Cd,2δ−1,2r 2 ≤Cd,2δ−1,2r. For r ≥δ, using (34) we have Vd,δ,r = vol (Bd(δ,r) ∩C0) + d vol (Bd(δ,r) ∩U1) 2 ≤vol (Bd(δ,r) ∩C0) + d vol (Bd(δ,r) ∩U ′ 1) 2 = vol (Bd(δ,r) ∩C0) = Cd,2δ−1,2r and hence the upper bound in (31). (b) Consider now the lower bound in (31). For j ≥2, with the set U j we now associate the set Vj = ˜X = (x1, . . . , xd) : −1 ≤x1 ≤0, 0 ≤xm ≤1 (for m > 1), \|x j\| ≤\|xk\| ≤1 for k ̸= j . 123 123 Discrete & Computational Geometry (2022) 68:540–565 561 With any point X = (x1, x2, . . . , xd) ∈U j (here x j is negative and \|x j\| ≤\|xk\| ≤1 for k ̸= j) we associate point ˜X = (−x1, x2, . . . , x j−1, −x j, x j+1, . . . , xd) ∈Vj by changing sign in the ﬁrst and j-the component of X ∈U j. Appendix B: Proof of Lemma 3.3 2 123 Discrete & Computational Geometry (2022) 68:540–565 562 For r ≤δ, since vol (Bd(δ,r) ∩C1) = Cd,A,2r = 0, we have Vd,δ,r = Cd,2δ−1,2r + Cd,A,2r 2 For r ≤δ, since vol (Bd(δ,r) ∩C1) = Cd,A,2r = 0, we have Vd,δ,r = Cd,2δ−1,2r + Cd,A,2r 2 and hence the lower bound in (31). and hence the lower bound in (31). Appendix B: Proof of Lemma 3.3 Setting without loss of generality j = 2, we have for these two points: ∥δ −X∥2 = (x1 −δ)2 + (x2 −δ)2 + d k=3 (xk −δ)2 ≤(−x1 −δ)2 + (−x2 −δ)2 + d k=3 (xk −δ)2 = ∥δ −˜X∥2, ∥δ −X∥2 = (x1 −δ)2 + (x2 −δ)2 + d k=3 (xk −δ)2 ≤(−x1 −δ)2 + (−x2 −δ)2 + d k=3 (xk −δ)2 = ∥δ −˜X∥2, where the inequality follows from the inequalities x1 ≥0, x2 < 0, and \|x2\| < x1 containing in the deﬁnition of U2. Therefore, ∥δ −˜X∥2 ≤r implies ∥δ −X∥2 ≤r, where from where the inequality follows from the inequalities x1 ≥0, x2 < 0, and \|x2\| < x1 containing in the deﬁnition of U2. Therefore, ∥δ −˜X∥2 ≤r implies ∥δ −X∥2 ≤r, where from vol (U j ∩Bd(δ,r)) ≥vol (Vj ∩Bd(δ,r)). (35) (35) To prove the lower bound in (31) for all r we must consider two cases: r ≤δ and r ≥δ. For r ≥δ, using (35) we have To prove the lower bound in (31) for all r we must consider two cases: r ≤δ and r ≥δ. For r ≥δ, using (35) we have Vd,δ,r = 1 2 vol (Bd(δ,r) ∩C0) + d i=1 vol ((Bd(δ,r) ∩Ui)) ≥1 2 vol (Bd(δ,r) ∩C0) + vol (Bd(δ,r) ∩U1) + d i=2 vol (Bd(δ,r) ∩Vi) = vol (Bd(δ,r) ∩C0) + vol (Bd(δ,r) ∩C1) 2 , where C1 is given in (17). To compute vol (Bd(δ,r) ∩C1), we shall use a similar technique to the proof of Theorem 3.2. The afﬁne transformation x →2x + (1, −1, −1, . . . , −1) maps the ball Bd(δ,r) and the cube C1 to Bd(A, 2r) and [−1, 1]d respectively, where A = (2δ + 1, 2δ −1, . . . , 2δ −1). Since the fraction of covered volume is preserved under invertible afﬁne transformations, one has vol (Bd(δ,r) ∩C1) vol(C1) = Cd,A,2r and hence we can conclude: Vd,δ,r ≥vol (Bd(δ,r) ∩C0) + vol (Bd(δ,r) ∩C1) 2 = Cd,2δ−1,2r + Cd,A,2r 2 . Vd,δ,r ≥vol (Bd(δ,r) ∩C0) + vol (Bd(δ,r) ∩C1) 2 = Cd,2δ−1,2r + Cd,A,2r 2 . Appendix C: Proof of Theorem 3.4 Before proving Theorem 3.4, we prove three auxiliary lemmas. Lemma C.1 Let r = rα,d = α √ d with α ≥0 and Za,b;d = (a, b, b, . . . , b) ∈Rd. Then the limit limd→∞Cd,Za,b;d,2r exists and Lemma C.1 Let r = rα,d = α √ d with α ≥0 and Za,b;d = (a, b, b, . . . , b) ∈Rd. Then the limit limd→∞Cd,Za,b;d,2r exists and lim d→∞Cd,Za,b;d,2r = ⎧ ⎪⎨ ⎪⎩ 0 if α < 1/3 + b2/2, 1/2 if α = 1/3 + b2/2, 1 if α > 1/3 + b2. Proof Deﬁne Proof Deﬁne tα = √ 3(d(4α2 −b2 −1/3) + b2 −a2) 2 a2 + (d −1)b2 + d/15 . As the r.v. ηz introduced in Appendix D are concentrated on a ﬁnite interval, for ﬁnite a and b the quantities of ρa := E(\|ηa −a2 −1/3\|3) and ρb := E(\|ηb −b2 −1/3\|3) are bounded. By applying the Berry–Esseen theorem (see [5, Sect. 2, Chap. 5]) to Cd,Za,b,2r, there exists some constant C such that −C max {ρa/σ 2 a , ρb/σ 2 b } (σ 2a + (d −1)σ 2 b )1/2 + Φ(tα) ≤Cd,Za,b,2r ≤Φ(tα) + C max {ρa/σ 2 a , ρb/σ 2 b } (σ 2a + (d −1)σ 2 b )1/2 , where σ 2 a = var(ηa) and σ 2 b = var(ηb). By the squeeze theorem, it is clear that if 4α2 −b2 −1/3 > 0 and hence α > 1/3 + b2/2, then Cd,Za,b,2r →1 as d →∞. If α < 1/3 + b2/2, then Cd,Za,b,2r →0 as d →∞. If α = 1/3 + b2/2, then Cd,Za,b,2r →1/2 as d →∞. ⊓⊔ where σ 2 a = var(ηa) and σ 2 b = var(ηb). By the squeeze theorem, it is clear that if 4α2 −b2 −1/3 > 0 and hence α > 1/3 + b2/2, then Cd,Za,b,2r →1 as d →∞. If α < 1/3 + b2/2, then Cd,Za,b,2r →0 as d →∞. If α = 1/3 + b2/2, then Cd,Za,b,2r →1/2 as d →∞. ⊓⊔ Lemma C.2 Let r = α √ d. Then for δ = (δ, δ, . . . , δ), we have Lemma C.2 Let r = α √ d. Then for δ = (δ, δ, . . . , δ), we have lim d→∞Vd,δ,r = lim d→∞Cd,2δ−1,2r = ⎧ ⎪⎨ ⎪⎩ 0 if α < 1/3 + (2δ −1)2/2, 1/2 if α = 1/3 + (2δ −1)2/2, 1 if α > 1/3 + (2δ −1)2/2. 123 123 123 12 Discrete & Computational Geometry (2022) 68:540–565 563 oof Using Lemma C.1 with Za,b = A = (2δ + 1, 2δ −1, . . . , 2δ −1), we obtain Proof Using Lemma C.1 with Za,b = A = (2δ + 1, 2δ −1, . . . Proof Deﬁne , 2δ −1), we obtain lim d→∞Cd,A,2r = lim d→∞Cd,2δ−1,2r = ⎧ ⎪⎨ ⎪⎩ 0 if α < 1/3 + (2δ −1)2/2, 1/2 if α = 1/3 + (2δ −1)2/2, 1 if α > 1/3 + (2δ −1)2/2. By then applying the squeeze theorem to the bounds in Lemma 3.3 using the fact from Lemma 3.1 we have Vd,δ,r = Cd(Zn,r), we obtain the result. ⊓⊔ To determine the value of r that leads to the full coverage, we utilise the following simple lemma. To determine the value of r that leads to the full coverage, we utilise the following simple lemma. Lemma C.3 For design Dn,δ, the smallest value of r that ensures a complete coverage of [−1, 1]d satisﬁes Lemma C.3 For design Dn,δ, the smallest value of r that ensures a complete coverage of [−1, 1]d satisﬁes lim d→∞ r1 √ d = 1 −δ if δ ≤1/2, δ if δ > 1/2. Proof of Theorem 3.4 From Lemma C.2, it is clear that the smallest α and hence r is attained with δ = 1/2. Moreover, Lemma C.2 provides lim d→∞Vd,1/2,r = lim d→∞Cd,0,2r = ⎧ ⎪⎨ ⎪⎩ 0 if α < 1/(2 √ 3), 1/2 if α = 1/(2 √ 3), 1 if α > 1/(2 √ 3), meaning for any 0 < γ < 1, r1−γ = √ d/(2 √ 3). By then applying Lemma C.3 with δ = 1/2, we obtain r1 = √ d/2 and hence r1−γ /r1 →1/ √ 3 as d →∞. ⊓⊔ Appendix D: Derivation of Approximation (30) Let U = (u1, . . . , ud) be a random vector with uniform distribution on [−1, 1]d so that u1, . . . , ud are i.i.d.r.v. uniformly distributed on [−1, 1]. Then for given Z = (z1, . . . , zd) ∈Rd and any r > 0, Cd,Z,r = P{∥U −Z∥≤r} = P{∥U −Z∥2 ≤r2} = P ⎧ ⎨ ⎩ d j=1 (u j −z j)2 ≤r2 ⎫ ⎬ ⎭. That is, Cd,Z,r, as a function of r, is the c.d.f. of the r.v. ∥U −Z∥. Let u have the uniform distribution on [−1, 1] and z ∈R. The ﬁrst three central moments of the r.v. ηz = (u −z)2 can be easily computed: Eηz = z2 + 1 3, var(ηz) = 4 3 z2 + 1 15 , 123 Discrete & Computational Geometry (2022) 68:540–565 564 μ(3) z = E [ηz −Eηz]3 = 16 15 z2 + 1 63 . (36) (36) onsider the r.v. ∥U −Z∥2 = d i=1 ηz j = d j=1(u j −z j)2. From (36) and indepen- ence of u1, . . . , ud, we obtain Consider the r.v. ∥U −Z∥2 = d i=1 ηz j = d j=1(u j −z j)2. From (36) and indepen- dence of u1, . . . , ud, we obtain μd,Z = E∥U −Z∥2 = ∥Z∥2 + d 3 , σ 2 d,Z = var(∥U −Z∥2) = 4 3 ∥Z∥2 + d 15 , and μ(3) d,Z = E[∥U −Z∥2 −μd,Z]3 = d j=1 μ(3) z j = 16 15 ∥Z∥2 + d 63 . If d is large enough then the conditions of the CLT for ∥U −Z∥2 are approximately met and the distribution of ∥U −Z∥2 is approximately normal with mean μd,Z and variance σ 2 d,Z. That is, we can approximate Cd,Z,r by Cd,Z,r ∼= Φ r2 −μd,Z σd,Z , (37) (37) where Φ( · ) is the c.d.f. of the standard normal distribution: where Φ( · ) is the c.d.f. of the standard normal distribution: where Φ( · ) is the c.d.f. of the standard normal distribution: Φ(t) = t −∞ ϕ(v) dv with ϕ(v) = e−v2/2 √ 2π . The approximation (37) can be improved by using an Edgeworth-type expansion in the CLT for sums of independent non-identically distributed r.v. Appendix D: Derivation of Approximation (30) The approximation (37) can be improved by using an Edgeworth-type expansion in the CLT for sums of independent non-identically distributed r.v. General expansion in the central limit theorem for sums of independent non- identical r.v. has been derived by Petrov, see [5, Thm. 7, Chap. 6]; the ﬁrst three terms of this expansion have been specialised in [6, Sect. 5.6]. By using only the ﬁrst term in this expansion, we obtain the following approximation for the distribution function of ∥U −Z∥2: P ∥U −Z∥2 −μd,Z σd,Z ≤x ∼= Φ(x) + μ(3) d,Z 6(σ 2 d,Z)3/2 (1 −x2)ϕ(x), leading to the following improved form of (37): leading to the following improved form of (37): Cd,Z,r ∼= Φ(t) + ∥Z∥2 + d/63 5 √ 3(∥Z∥2 + d/15)3/2 (1 −t2)ϕ(t), 123 123 12 565 Discrete & Computational Geometry (2022) 68:540–565 where t = td,∥Z∥,r = r2 −μd,Z σd,Z = √ 3(r2 −∥Z∥2 −d/3) 2 ∥Z∥2 + d/15 . References 1. Conway, J.H., Sloane, N.J.A.: Sphere Packings, Lattices and Groups. Grundlehren der Mathematischen Wissenschaften, vol. 290. Springer, New York (1999) 1. Conway, J.H., Sloane, N.J.A.: Sphere Packings, Lattices and Groups. Grundlehren der Mathematischen Wissenschaften, vol. 290. Springer, New York (1999) 2. Graf, S., Luschgy, H.: Foundations of Quantization for Probability Distributions. Lecture Notes in Mathematics, vol. 1730. Springer, Berlin (2000) 2. Graf, S., Luschgy, H.: Foundations of Quantization for Probability Distributions. Lecture Notes in Mathematics, vol. 1730. Springer, Berlin (2000) . Kuo, F., Joe, S., Matsumoto, M., Mori, S., Saito, M.: Sobol sequences with better two-dimensional projections (2017). https://CRAN.R-project.org/package=SobolSequence 4. Noonan, J., Zhigljavsky, A.: Non-lattice covering and quantization of high dimensional sets. In: Black Box Optimization, Machine Learning, and No-Free Lunch Theorems, vol. 170, pp. 273–318. Springer, Cham (2021) 4. Noonan, J., Zhigljavsky, A.: Non-lattice covering and quantization of high dimensional sets. In: Black Box Optimization, Machine Learning, and No-Free Lunch Theorems, vol. 170, pp. 273–318. Springer, Cham (2021) 5. Petrov, V.V.: Sums of Independent Random Variables. Ergebnisse der Mathematik und ihrer Grenzge- biete, vol. 82. Springer, New York (1975) 5. Petrov, V.V.: Sums of Independent Random Variables. Ergebnisse der Mathematik und ihrer Grenzge- biete, vol. 82. Springer, New York (1975) 6. Petrov, V.V.: Limit Theorems of Probability Theory. Sequences of Independent Random Vari- ables. Oxford Studies in Probability, vol. 4. Oxford Science Publications. Oxford University Press, New York (1995) 6. Petrov, V.V.: Limit Theorems of Probability Theory. Sequences of Independent Random Vari- ables. Oxford Studies in Probability, vol. 4. Oxford Science Publications. Oxford University Press, New York (1995) 7. Pronzato, L.: Minimax and maximin space-ﬁlling designs: some properties and methods for construc- tion. J. Société Française de Stat. 158(1), 7–36 (2017) 7. Pronzato, L.: Minimax and maximin space-ﬁlling designs: some properties and methods for construc- tion. J. Société Française de Stat. 158(1), 7–36 (2017) 8. Pronzato, L., Müller, W.G.: Design of computer experiments: space ﬁlling and beyond. Stat. Compu 22(3), 681–701 (2012) 9. Pronzato, L., Zhigljavsky, A.: Bayesian quadrature, energy minimization, and space-ﬁlling desig SIAM/ASA J. Uncertain. Quantif. 8(3), 959–1011 (2020) 10. Saka, Y., Gunzburger, M., Burkardt, J.: Latinized, improved LHS, and CVT point sets in hypercubes. Int. J. Numer. Anal. Model. 4(3–4), 729–743 (2007) 10. Saka, Y., Gunzburger, M., Burkardt, J.: Latinized, improved LHS, and CVT point sets in hypercubes. Int. J. Numer. Anal. Model. 4(3–4), 729–743 (2007) 11. References Santner, T.J., Williams, B.J., Notz, W.I.: The Design and Analysis of Computer Experiments. Spring Series in Statistics. Springer, New York (2003) 12. Schaback, R., Wendland, H.: Kernel techniques: from machine learning to meshless methods. Ac Numer. 15, 543–639 (2006) 13. Wendland, H.: Scattered Data Approximation. Cambridge Monographs on Applied and Computational Mathematics, vol. 17. Cambridge University Press, Cambridge (2005) 14. Zhigljavsky, A., Noonan, J.: Covering of high-dimensional cubes and quantization. Oper. Res. Forum 1(3), # 18 (2020) Ž 15. Zhigljavsky, A., Žilinskas, A.: Bayesian and High-Dimensional Global Optimization. SpringerBrie in Optimization. Springer, Cham (2021) Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. 123
https://openalex.org/W4284680268	https://f1000research.com/articles/11-755/v1/pdf	English	null	Performance of Malnutrition Universal Screening Tool and Patient-Generated Global Subjective Assessment in screening for cancer-related malnutrition in Nairobi, Kenya	F1000Research	2,022	cc-by	11,257	F1000Research 2022, 11:755 Last updated: 14 OCT 2024 RESEARCH ARTICLE Performance of Malnutrition Universal Screening Tool and Patient-Generated Global Subjective Assessment in screening for cancer-related malnutrition in Nairobi, Kenya [version 1; peer review: 1 approved, 1 approved with reservations] Caroline M.N. Auma , Marshal M. Mweu , Rose O. Opiyo Open Peer Review Approval Status 1 2 version 2 (revision) 07 Nov 2023 version 1 07 Jul 2022 view view Oscar Ngesa, Taita Taveta University, Voi, Kenya 1. Mariana Kruger , Stellenbosch University, Stellenbosch, South Africa 2. Any reports and responses or comments on the article can be found at the end of the article. First published: 07 Jul 2022, 11:755 https://doi.org/10.12688/f1000research.123059.1 Latest published: 07 Nov 2023, 11:755 https://doi.org/10.12688/f1000research.123059.2 v1 Abstract Background: Malnutrition is a common feature among oncology patients. It is responsible for poor response and tolerance to anticancer therapy, increased morbidity, and mortality. More than half of malnourished cancer patients remain undetected owing to lack of effective screening. Body mass index is the main indicator for assessing malnutrition in Kenyan public hospitals. However, it underestimates weight loss in patients with chronic illnesses. The Malnutrition Universal Screening Tool and Patient-Generated Subjective Global Assessment have been widely used in research and clinical practice and have both reported good validity and reliability. However, their diagnostic evaluation has not been performed in Kenya. y Methods: A cross-sectional study was conducted among 138 and 76 cancer outpatients from Kenyatta National Hospital and Texas cancer treatment centres, respectively. Participants had a confirmed disease, stage 1-4 cancer, and aged 18 years and above. They were screened for malnutrition using both Malnutrition Universal Screening Tool and Patient Generated-Subjective Global Assessment. A separate study questionnaire was utilized to gather participant’s socio-demographic and clinical characteristics. A Bayesian latent class modelling framework was employed to infer the tests’ estimates based on participants ‘cumulative scores from the two tests. p p Results: The cut-off value of ≥ 1 and ≥ 4 gave the best combination of sensitivity and specificity of Malnutrition Universal Screening Tool and Patient Generated-Subjective Global Assessment. Both tests yielded statistically similar sensitivities and specificities. Predictive values were comparable between the tests and across the two populations. The posterior median true prevalences of malnutrition were high (˃ 54%) and numerically similar between the studied populations. Conclusions: The performance of both tests among patients with Page 1 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 cancer is similar. Healthcare workers are therefore at liberty to use either of them to inform treatment. Given the high true prevalence of cancer-related malnutrition, routine screening is critical and should be included as part of cancer care. cancer is similar. Healthcare workers are therefore at liberty to use either of them to inform treatment. Given the high true prevalence of cancer-related malnutrition, routine screening is critical and should be included as part of cancer care. Keywords Bayesian Latent Class Model, Malnutrition, Malnutrition Universal Screening Tool, Patient Generated Global Subjective Assessment, Test accuracy, Cancer patients. This article is included in the Agriculture, Food and Nutrition gateway. This article is included in the Agriculture, Food and Nutrition gateway. Introduction Malnutrition is a common feature in oncology patients.1 Globally, malnutrition affects about 39 to 87% of patients with cancer2 depending on the assessment method3 and the type of cancer involved.4 For example, while breast cancer patients have a low risk of weight loss and subsequent malnutrition, patients with gastrointestinal, lung, and head and neck tumours have a high risk.5 In Kenya, 31% of patients with cancer are malnourished and the greatest burden lies among patients with gastrointestinal tumours at 49.1%.6 Cancer-induced malnutrition has significant clinical and economic burden. The malnutrition in these patients is associated with reduced responsiveness and tolerance to treatment, impaired immune function, increased risk of complications, reduced survival and quality of life, and high healthcare expenditures.4,7,8 Globally, 50% of patients dying from cancer are malnourished.9 About 20% of deaths in patients with cancer occur due to malnutrition-related complications rather than the direct effect of malignancy.10 Despite its high prevalence and adverse sequelae in cancer patients, malnutrition in more than half of these patients remains undetected and untreated owing to lack of effective screening.11–13 complications, reduced survival and quality of life, and high healthcare expenditures.4,7,8 Globally, 50% of patients dying from cancer are malnourished.9 About 20% of deaths in patients with cancer occur due to malnutrition-related complications rather than the direct effect of malignancy.10 Despite its high prevalence and adverse sequelae in cancer patients, malnutrition in more than half of these patients remains undetected and untreated owing to lack of effective screening.11–13 Timely recognition of malnutrition is critical for appropriate and intensive nutrition support12 to stop or counteract weight loss and subsequent malnutrition.14,15 Currently, body mass index (BMI) is the major indicator for the assessment of malnutrition in Kenyan public hospitals.16,17 However, BMI when used independently is a less sensitive parameter in predicting malnutrition due to large tumours9 and short term fluid imbalance.18,19 Therefore, patients can suffer malnutrition regardless of BMI value because loss in lean body mass is masked by ascites and body fat.20,21 The commonly available screening tools for use in oncology setting are Patient Generated-Subjective Global Assessment (PG-SGA) and Malnutrition Universal Screening Tool (MUST). Introduction In addition, PG-SGA contains more nutrition impact symptoms that are vital to cancer patients.18 Lastly, PG-SGA assess nutrition status on a continuous scale, which allows it to detect subtle changes in nutritional status occurring over a short period and triage patients for appropriate nutrition intervention.25,31,36 Overall, PG-SGA allows malnourished patients to be quickly identified and prioritized for care, thus a suitable screening tool.22,25 The MUST is designed to categorize adults as either malnourished, obese, or at risk of malnutrition and identify those who would benefit from appropriate nutrition intervention.37 It does not incorporate subjective/clinical parameters reflecting changes in nutritional risk and status and neither does it require time-consuming calculations.23 It is also quick, inexpensive, easy to apply and interpret, and hence acceptable to both patients and healthcare workers.23,38,39 MUST combines weight loss and BMI, which reduces misclassification errors, thus improving Se and Sp.19 Additionally, a patient can screen him/herself without the help of a health worker with a time response as low as 5 minutes.40 Although these tools have been extensively validated in various countries, their performance has not been evaluated in Kenya. Additionally, in countries where they have been evaluated, evaluations were done against reference standards such as Subjective Global Assessment,25,26 Mini-Nutritional Assessment,27 Nutritional-Risk Screening-2002,41 Albu- min and BMI.42 This may have plagued the estimates with information and selection bias.43 Nevertheless, the accuracy of two or more tests can be quantified without prior knowledge of the true disease status by using latent class models.43,44 Thus, the objective of the present study was to derive the Se and Sp together with the positive and negative predictive values of PG-SGA and MUST for malnutrition in head and neck, respiratory, and gastrointestinal cancer patients using the Bayesian Latent Class Model (BLCM). Corresponding author: Caroline M.N. Auma (carolinemuseka@gmail.com) Corresponding author: Caroline M.N. Auma (carolinemuseka@gmail.com) How to cite this article: Auma CMN, Mweu MM and Opiyo RO. Performance of Malnutrition Universal Screening Tool and Patient- Generated Global Subjective Assessment in screening for cancer-related malnutrition in Nairobi, Kenya [version 1; peer review: 1 approved, 1 approved with reservations] F1000Research 2022, 11:755 https://doi.org/10.12688/f1000research.123059.1 First published: 07 Jul 2022, 11:755 https://doi.org/10.12688/f1000research.123059.1 Page 2 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 Introduction These screening tools assess patients’ nutrition status using a cluster of two or more nutrition-related parameters, thus are superior to BMI.4,19,20 PG-SGA and MUST have been widely used in both clinical practice22,23 and research.24 They have reported good sensitivity (Se) and specificity (Sp) in identifying the nutrition status of patients with cancer.25–30 The PG-SGA is a simple method employed in assessment of nutritional risk and identification of those who would benefit from nutritional support, thus considered the most appropriate tool for detecting malnutrition in cancer patients.31 The PG-SGA is a 4-in-1 instrument to screen, assess, and monitor malnutrition and its risk factors and to triage for interdisciplinary intervention.25,32 It is inexpensive, quick to complete33 and user-friendly.34,35 More to its merits, the first four boxes can be completed by a patient, which empowers them on matters related to their health and reduces the total time involved In addition PG SGA contains more nutrition impact symptoms that are vital to cancer patients 18 Lastly The PG-SGA is a simple method employed in assessment of nutritional risk and identification of those who would benefit from nutritional support, thus considered the most appropriate tool for detecting malnutrition in cancer patients.31 The PG-SGA is a 4-in-1 instrument to screen, assess, and monitor malnutrition and its risk factors and to triage for interdisciplinary intervention.25,32 It is inexpensive, quick to complete33 and user-friendly.34,35 More to its merits, the first four boxes can be completed by a patient, which empowers them on matters related to their health and reduces the total time involved. In addition, PG-SGA contains more nutrition impact symptoms that are vital to cancer patients.18 Lastly, PG-SGA assess nutrition status on a continuous scale, which allows it to detect subtle changes in nutritional status occurring over a short period and triage patients for appropriate nutrition intervention.25,31,36 Overall, PG-SGA allows malnourished patients to be quickly identified and prioritized for care, thus a suitable screening tool.22,25 interdisciplinary intervention. It is inexpensive, quick to complete and user-friendly. More to its merits, the first four boxes can be completed by a patient, which empowers them on matters related to their health and reduces the total time involved. Sample size determination The required sample size was estimated using McNemar’s sample size formula for paired proportions.46 nper test ¼ Zα=2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pdisc p þZβ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pdisc P2diff q Pdiff 0 @ 1 A 2 Pdisc ¼ 1Se1 ð Þþ 1Se2 ð Þ Pdiff ¼ 1Se1 ð Þ 1Se2 ð Þ nper test ¼ Zα=2 ﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pdisc p þZβ ﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃﬃ Pdisc P2diff q Pdiff 0 @ 1 A 2 Pdisc ¼ 1Se1 ð Þþ 1Se2 ð Þ Pdiff ¼ 1Se1 ð Þ 1Se2 ð Þ where: nper test was the sample size required for each test, Zα=2 (1.96) was the critical value for specifying the two tailed 95% confidence level, Zβ (-0.84) was the critical value specifying the statistical power of 80% desired, Se1and Se2 were sensitivity estimates of the MUST and PG-SGA, respectively, from literature. Notably, Se1 was set at 0.828 and Se2 set at 0.98.25 Based on the specified figures above, a total sample size of 214 was reached after adjusting upwards by 5% to account for non-response. where: nper test was the sample size required for each test, Zα=2 (1.96) was the critical value for specifying the two tailed 95% confidence level, Zβ (-0.84) was the critical value specifying the statistical power of 80% desired, Se1and Se2 were sensitivity estimates of the MUST and PG-SGA, respectively, from literature. Notably, Se1 was set at 0.828 and Se2 set at 0.98.25 Based on the specified figures above, a total sample size of 214 was reached after adjusting upwards by 5% to account for non-response. Study procedures and outcome assessment Study procedures and outcome assessment Two research assistants with a medical background who had previously been trained on interviewing techniques recruited and interviewed participants. Upon obtaining informed consent from the participants, the MUST and PG-SGA (either English or Kiswahili version depending on the individual’s preference) were administered through a face-to-face interview in a private room within the Cancer Clinic. The MUST and PG-SGA tools were completed just before provision of oncology services. Aside from the nutrition status, the patients’ socio-demographic characteristics (age, sex, marital status, level of education, area of residence and employment status) and clinical characteristics (tumour site and cancer stage) were also recorded. Sample size determination Ethical consideration The study participants provided written informed consent prior to engaging in the study. Approval to conduct the study was granted by Kenyatta National Hospital and University of Nairobi joint Ethics and Research Committee (KNH-ERC/ A/315). Target condition Study design and setting A facility-based cross-sectional study was conducted to evaluate the performance of the MUST and PG-SGA in screening for malnutrition among cancer patients. This study design was most suitable due to the ease of recruitment of cancer patients presenting to the cancer clinic and its suitability for the descriptive nature of the study. Page 3 of 17 Page 3 of 17 Page 3 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 The study was conducted at Kenyatta National Hospital-Cancer Treatment Centre (KNH-CTC) and Texas Cancer Centre (TCC) in Nairobi, Kenya. KNH is the largest national public referral hospital in Kenya. It registers 80,000 inpatients and 500,000 outpatients annually. It offers comprehensive cancer services, which include diagnosis and treatment (chemo- therapy, radiotherapy, surgical and hormonal therapy). The three most commonly diagnosed carcinomas are cervical, breast, and oesophageal cancer in that order. KNH-CTC provides both inpatient and outpatient services. Although nutrition screening of cancer patients is not conducted routinely, there is an in-house nutritionist whose services are sought under certain circumstances. The TCC is the leading private cancer care and treatment centre in Kenya. It receives approximately 11000 patients annually. Apart from cancer diagnosis and treatment, TCC offers pain and palliative care services, counselling, physiotherapy, and rehabilitation services. With regards to nutrition services, currently, TCC does not have an in-house nutritionist, but under certain circumstances, one is outsourced. Consequently, nurses and doctors offering oncological services provide nutrition support. Weight and height are the measurements taken for BMI scoring but not routinely. Study population, eligibility and selection of participants y p p g y p p The study population consisted of all patients with head and neck, respiratory and gastrointestinal cancers attending outpatient oncology clinic at KNH-CTC and TCC. On eligibility, a patient had to have established disease, that is, stage 1-4 cancer, aged 18 years and above, physically stable, and have given informed consent. To obtain the study sample, a simple random sampling technique was applied to a sampling frame comprising of patients booked for evaluation in each facility on a given day. To ascertain the number of patients to be sampled from each facility, a probability proportional to size sampling45 was employed guided by the number of patients seen in a given facility in a particular month. As such, 138 and 76 cancer patients from KNH-CTC and TCC, respectively, were recruited. g This referred to the latent (unobserved) malnutrition status regardless of the severity level among head and neck, respiratory and gastrointestinal cancer patients that is targeted by MUST and PG-SGA. Patient-Generated Subjective Global Assessment The PG-SGA relies on weight history, changes in patient’s dietary intake, presence of gastrointestinal symptoms, functionality, diagnosis, age, metabolic stress, and physical examination (subcutaneous fat loss, muscle wasting, and fluid status) to assess a patient’s nutritional risk. For each of the above-mentioned components, 0-4 points are awarded based on the relative impact on nutritional status. The overall total scores of PG-SGA range from 0-52. Based on the scores, patients were divided into three categories: (PG-SGA < 2, well-nourished/no risk), (PG-SGA ≥2 and < 9 suspected/moderate risk), (PG-SGA ≥9, severe risk). Three potential cut-offs were examined: individuals being categorised as malnourished if they had scores ≥2,9,26 ≥4,42,47 ≥9.25,48 Target condition g This referred to the latent (unobserved) malnutrition status regardless of the severity level among head and neck, respiratory and gastrointestinal cancer patients that is targeted by MUST and PG-SGA. Page 4 of 17 Page 4 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 Malnutrition Universal Screening Tool g The MUST employs three stand-alone criteria to classify patients’ nutritional status: BMI score (BMI ˃20 kg/m2 = 0, BMI 18.5-20 kg/m2 = 1, BMI <18.5 kg/m2 = 2), unintentional weight loss score in the last 3 to 6 months (weight loss < 5% = 0, weight loss 5-10% =1, weight loss ˃ 10% = 2) and cute disease effect score (add a score of 2 if there has been or is likely to be no nutrition intake for > 5 days). Each parameter is scored on a scale of 0, 1, or 2 giving an overall total score of six. Overall risk of malnutrition is classified as low risk if the score = 0, moderate risk if the score = 1, and a score of ≥2 signifies high risk.23 In this study, a patient with a total score of ≥1 was considered malnourished. Statistical analysis A Bayesian modelling framework was used to derive the prevalence, Se and Sp estimates along with the predictive values of the tests. The BLCM was fitted in OpenBUGS software (v 3.2.2)49 but called from R (v. 3.6.2) via the Brugs package (v0.9-0).50 Importantly, the model design and reporting were guided by the standards for reporting of diagnostic accuracy studies that use BLCMs (STARD-BLCM).51 The Bayesian code is available as Underlying data.52 According to Hui and Walter,44 the BLCM has the following assumptions: (i) there must be two or more populations with differing prevalences. For this study, two populations of patients attending KNH-CTC and TCC facilities were established. Owing to the inherent socio-economic differences between the facilities’ catchment populations, the prevalences of cancer-related malnutrition were presumed to be distinct. (ii) The Se and Sp of both tools should be consistent across the populations. (iii) Given the disease status, there was conditional independence between the two tests. This was a reasonable assumption considering the two tools have separate symptom targets: MUST targets changes in body composition thus, assesses chronic malnutrition whereas PG-SGA assesses gastrointestinal symptoms preventing food intake thus, targets acute malnutrition.53 Consequently, the probability of a patient testing either positive or negative to one test is not affected by the result of the other test. It was assumed that the different combinations of test results, for each population, observed as counts Ok ð Þ follow a multinomial distribution: OkjSeiSpipk mulitinomial probknk OkjSeiSpipk mulitinomial probknk Where Sei andSpi are the Se and Sp measures for the ith test (i=1, 2) and pk represent the kth population’s prevalence. Probk, represents a vector of probabilities of observing the tests’ results’ combinations (such as +, +), while nk is the sample size used in population k. The probabilities are defined using the specific test characteristics (Se and Sp) and prevalence (p) of each population. For example, prob1 for a person who tests positive to both tests is given by: prob1 ¼ Pr T1 þT2 þjDþ ð ÞþPr T1 þT2 þjD ð Þ ¼ Se1Se2P1 þ 1Sp1 ½ 1Sp2 ½ 1P1 ½ Since there were two populations, the Latent Class Model contained six parameters: each test’s Se and Sp, as well as two population-specific prevalences. These six parameters were estimated from six degrees of freedom obtained from each of the two populations. As there was no reliable prior information on any of the parameters, uninformative priors (beta (1, 1)) were used. The Positive Predictive Value (PPV) and Negative Predictive Value (NPV) for each test within population k were derived using the following formula: PPV ¼ PKSei= PkSei þ 1Pk ½ 1SPi ½ ð Þ NPV ¼ 1PK ½ Spi= Pk 1Sei ½ þ 1Pk ½ Spi ð Þ Page 5 of 17 Page 5 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 To select the most optimal cut-off values, Youden indices54 were computed using the Se and Sp obtained at each of the following pairs of cut-off values: ≥1 for MUST paired with each of the following PG-SGA cut-off values (≥2, ≥4, and ≥9). The pair of cut-off points with the highest Youden index was chosen. Two Markov Chain Monte Carlo (MCMC) chains with varying values were used to initialize the model. We ran 6000 iterations of the model with the initial 3500 discarded as the burn-in phase. Convergence of the MCMC chain was assessed by visual appraisal of the density plots and Gelman-Rubin Diagnostic plots. The posterior distribution of prevalence of each of the two populations, the test estimates, and their respective predictive values were reported as the median and their associated 95% posterior credible intervals (PCI). Results Socio-demographic and clinical characteristics of study participants A total of 214 participants were selected, and 202 consented to participate, giving a response rate of 94.4%. However, of the 202 participants, 14 were excluded from analysis due to lack of records on previous anthropometric measurements, which rendered their total scores from MUST and PG-SGA unreliable. The socio-demographic and clinical features of 188 participants are displayed in Table 1. Notably, the participants’ median age was 56.5 years (range: 18-81 years). Majority of the participants were males (64.36%). Only 46% of the Table 1. Summary statistics of socio-demographic and clinical characteristics of study participants from Kenyatta National Hospital-Cancer Treatment Centre (KNH-CTC) and Texas Cancer Centre (TCC), Kenya, (n = 188). (n = 188). Variable Values Median Range Frequency n (%) Age (in completed years) Overall (N = 188) - 56.5 18-81 - KNH-CTC (n = 126) - 56 19-80 - TCC (n = 62) - 57.5 18-81 - Sex Overall Male - - 121 (64.36) Female - - 67 (35.64) KNH-CTC Male - - 81 (64.21) Female - - 45 (35.71) TCC Male - - 40 (64.52) Female - - 22 (35.48) Marital status Overall Single - - 17 (9.04) Married - - 145 (77.13) Separated/divorced - - 8 (4.26) Widowed - - 18 (9.57) KNH-CTC Single - - 10 (7.94) Married - - 99 (78.57) Separated/divorced - - 7 (5.56) Widowed - - 10 (7.93) TCC Single - - 7 (11.29) Married - - 46 (74.19) Separated/divorced - - 1 (1.61) Widowed - - 8 (12.90) Page 6 of 17 Page 6 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 Table 1. Results Continued Variable Values Median Range Frequency n (%) Level of education Overall No formal education - - 15 (7.98) Primary - - 86 (45.74) Secondary - - 63 (33.51) Tertiary - - 24 (12.77) KNH-CTC No formal education - - 9 (7.14) Primary - - 60 (47.62) Secondary - - 44 (34.92) Tertiary - - 13 (10.32) TCC No formal education - - 6 (9.68) Primary - - 26 (41.94) Secondary - - 19 (30.65) Tertiary - - 11 (17.74) Area of residence Overall Nairobi - 29 (15.43) Other - 97 (84.57) Employment status Overall Employed - - 64 (34.04) Unemployed - - 124 (65.96) KNH-CTC Employed - - 39 (30.95) Unemployed - - 87 (69.05) TCC Employed - - 25 (40.32) Unemployed - - 37 (59.68) Tumour location Overall Gastrointestinal 102 (54.26) Head and neck - - 67 (35.64) Respiratory - - 19 (10.11) KNH-CTC Gastrointestinal - - 71 (56.35) Head and neck - - 43 (34.13) Respiratory - - 12 (9.52) TCC Gastrointestinal - - 31 (50.00) Head and neck - - 24 (38.71) Respiratory - - 7 (11.29) Stage of cancer disease Overall Stage I - - 23 (12.23) Stage II - - 46 (24.47) Stage III - - 55 (29.26) Stage IV - - 64 (34.04) KNH-CTC Stage I - - 19 (15.07) Stage II - - 35 (27.78) Stage III - - 35 (27.78) Stage IV - - 37 (29.37) TCC Stage I - - 4 (6.45) Stage II - - 11 (17.74) Stage III - - 20 (32.36) Stage IV - - 27 (43.55) Pa F1000Research 2022, 11:755 Last updated: 14 OCT 2024 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 participants had attained at least a secondary school certification. Clinically, patients with gastrointestinal cancers formed the largest proportion of the sample at 54.26%. Approximately 34% of the participants had stage 4 cancer. participants had attained at least a secondary school certification. Clinically, patients with gastrointestinal cancers formed the largest proportion of the sample at 54.26%. Approximately 34% of the participants had stage 4 cancer. Test outcomes, sensitivity and specificity Test outcomes, sensitivity and specificity y p y The cross-classified counts of the two test results at various cut-off points are presented in Table 2. The cut-off value of ≥1 and ≥4 gave the best combination of the Se and Sp of MUST and PG-SGA (see Table 3). Thus, this cut-off was used to infer subsequent parameters. The PG-SGA registered a Se of (92.4; 95% PCI [81.2; 99.6]) and Sp of (72.5% 95% PCI [54; 97.2]) while MUST had a Se of (83.1 95% PCI [67.4; 98.9]) and a Sp (85.7; 95% PCI [71.4; 99.6]). Although the Se and Sp of the two tests differed numerically, they were statistically similar based on 95% PCIs. Discussion Thus, inaccurate weight in patients with oedema or ascites could lead to underestimation of nutritional risk in some patients.33 Sensitivity and specificity of the MUST and PG SGA in screening for malnutrition In this study, MUST registered a Se of 83.1% supported by finding from other studies28,29 and a Sp of 85.7% in agreement with findings from previous studies.28,30 However, in the literature, the Se of MUST has been shown to vary from 29%55 to 97%41 similar to its Sp from 48.9%56 to 94.5%.29 This inconsistency can be logically explained by the percentage weight loss, which is a crucial parameter in the MUST tool.28 For example, patients may have lost weight in the past, but their present weight recovery was not calculated since MUST does not give this provision.56 In addition, contrary to this study, in those studies, estimation of the test accuracy was based on the assumption that a perfect reference standard existed. This could have led to biased estimates. More so, MUST in its categorization of nutrition status, relies on the percentage of weight lost between 3 and 6 months. As a result, where patients were required to self-report the involuntary weight loss, there was a possibility of under-estimation hence affecting Sp. Ramboer, Verhamme57 in their study reflected the propensity for underestimation of weight loss in oncology patients. In this study, all patients with no previous records of anthropometric measurements were excluded from the analysis. Although MUST was found to be a suitable screening tool, it requires that patients be weighed for the determination of BMI and weight loss. Thus, inaccurate weight in patients with oedema or ascites could lead to underestimation of nutritional risk in some patients.33 The PG-SGA yielded a Se and Sp of 92.4% and 72.5%, respectively. The findings demonstrate that the PG-SGA is a highly sensitive malnutrition screening tool in the outpatient oncology population, consistent with findings from previous studies.27,25,26,42 An ideal screening tool would be 100% sensitive and specific. But, the necessity to categorize all malnourished patients (Se) correctly takes precedence over misclassification of well-nourished individuals (Sp).58 The Sp of PG-SGA varies greatly in published studies, with estimates ranging from 2.3%1 to 88.1%.27 The Sp of 72.5% observed in this study falls within the above range. Predictive values and prevalences Predictive values and prevalences On predictive values, NPVs and PPVs were statistically similar between the tests and across the two populations (Table 4). In KNH, MUST had a NPV of 77.9% and a PPV of 89.7% while PG-SGA had a NPV of 87.2% and a PPV of 82.9%. In TCC, MUST had a NPV of 81.2% and a PPV of 87.8% while PG-SGA a NPV of 89.2% and a PPV of 80.1%. The posterior median true prevalences of malnutrition were high (˃54%) and numerically similar between the studied populations (Table 4). Table 2. Cross-classified counts by population for MUST and PG-SGA tests for screening for malnutrition among cancer patients seeking outpatient oncological care at Kenyatta National Hospital-Cancer Treatment Centre (KNH-CTC) and Texas Cancer Centre (TCC), Kenya during the period October-November 2021. Population Cut point Test outcome (MUST/PG-SGA) MUST/PG-SGA (+a/+) (+/-b) (-/+) (-/-) Total (%) KNH-CTC ≥1, ≥2 65 3 40 18 126 (67%) TCC 32 1 25 4 62 (33%) KNH-CTC ≥1, ≥4 61 7 23 35 126 (67%) TCC 26 7 12 17 62 (33%) KNH-CTC ≥1, ≥9 33 35 6 52 126 (67%) TCC 16 17 4 25 62 (33%) aPositive. bNegative. Table 3. Estimates of sensitivity and specificity of MUST and PG-SGA at different pair of cut-off values and Youden indices for the best cut-off point. Table 3. Estimates of sensitivity and specificity of MUST and PG-SGA at different pair of cut-off values and Youden indices for the best cut-off point. Cut-off Value Test parameter Estimate (95% PCI) Y.indexMUST Y.indexPG-SGA MUST PG-SGA ≥1 ≥2 SeMUST 11.7 (0.7; 32.6) -55.38 -49.9 SpMUST 46.6 (3.4; 77.6) SePG-SGA 33.7(5.6; 45) SpPG-SGA 2.8 (0.15; 8.7) ≥1 ≥4 SeMUST 83.1 (67.4; 98.9) 69.2 65.0 SpMUST 85.7 (71.4; 99.6) SePG-SGA 92.4 (81.2; 99.6) SpPG-SGA 72.5 (54; 97.2) ≥1 ≥9 SeMUST 88.9 (74.9; 99.3) 61.07 58.7 SpMUST 71.3 (55.5; 97.5) SePG-SGA 66.2 (43.9; 97.1) SpPG-SGA 92.9 (82.9; 99.6) Page 8 of 17 Page 8 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 Table 4. Estimates of positive and negative predictive values of MUST and PG-SGA and Prevalences across Kenyatta National Hospital-Cancer Treatment Centre (KNH-CTC) and Texas Cancer Centre (TCC). PPV=Positive Predictive Value; NPV=Negative Predictive Value. Discussion The variability could be due to three things: First, use of different reference standards in deriving test estimates, which was not the case in this study, for example, specificities of 2.3% and 88.1% were based on albumin and Mini-Nutrition Assessment tool, respectively. Secondly, different administration methods, since the score from participant-versus researcher administered may not be comparable. This is because, the authors of PG-SGA speculate that patients may over-report symptoms based solely on their presence, regardless of their impact on food intake,58 thus underestimating Sp. For example, Sp of 21.8%59 and 82%25 were derived from patient and researcher administered studies, respectively. This study was researcher administered thus a good Sp was registered. Lastly, the scores derived from component Number Three of PG-SGA questionnaire (nutrition impact symptoms and other factors). In this section, any symptom impairing food intake reported by the patient is scored, and all points are cumulative (maximum 24 points).22 This could also rise the number of false positives thus underestimating Sp. However, this wide range of symptoms may help detect more patients at risk of malnutrition. As the detection of symptoms that impair nutritional intake in the early stages of the disease may be advantageous for proactively preventing cancer-related malnutrition.58 The observed high Se and the ability to determine what elements are influencing nutrition status make PG-SGA a suitable nutritional screening tool. However, its accuracy is dependent upon the experience of an observer.60 Therefore, it requires a trained healthcare professional to complete the assessment and score the tool.58 Predictive values and prevalences Location Test parameter Estimate (95% PCI MUST PG-SGA KNH NPV 77.9 (49.8; 98.9) 87.2 (65.2; 99.3) PPV 89.7 (73.7; 99.4) 82.9 (62.5; 99.1) Prevalence 59.1 (41.9; 75.6) TCC NPV 81.2 (50.9; 99.1) 89.2 (64.2; 99.6) PPV 87.8 (65.6; 99.4) 80.1 (54.2; 98.9) Prevalence 54.6 (32.8; 75.7) Table 4. Estimates of positive and negative predictive values of MUST and PG-SGA and Prevalences across Kenyatta National Hospital-Cancer Treatment Centre (KNH-CTC) and Texas Cancer Centre (TCC). PPV=Positive Predictive Value; NPV=Negative Predictive Value. Discussion Discussion Sensitivity and specificity of the MUST and PG-SGA in screening for malnutrition 28 29 Sensitivity and specificity of the MUST and PG SGA in screening for malnutrition In this study, MUST registered a Se of 83.1% supported by finding from other studies28,29 and a Sp of 85.7% in agreement with findings from previous studies.28,30 However, in the literature, the Se of MUST has been shown to vary from 29%55 to 97%41 similar to its Sp from 48.9%56 to 94.5%.29 This inconsistency can be logically explained by the percentage weight loss, which is a crucial parameter in the MUST tool.28 For example, patients may have lost weight in the past, but their present weight recovery was not calculated since MUST does not give this provision.56 In addition, contrary to this study, in those studies, estimation of the test accuracy was based on the assumption that a perfect reference standard existed. This could have led to biased estimates. More so, MUST in its categorization of nutrition status, relies on the percentage of weight lost between 3 and 6 months. As a result, where patients were required to self-report the involuntary weight loss, there was a possibility of under-estimation hence affecting Sp. Ramboer, Verhamme57 in their study reflected the propensity for underestimation of weight loss in oncology patients. In this study, all patients with no previous records of anthropometric measurements were excluded from the analysis. Although MUST was found to be a suitable screening tool, it requires that patients be weighed for the determination of BMI and weight loss. Predictive values of the MUST and PG-SGA in screening for malnutrition In this study, the two tests yielded comparable estimates of PPV and NPV across the two populations based on the observed 95% PCIs. Therefore, if each test is used singly to screen for malnutrition, it is reliable and can inform treatment. Hence, the tool of choice is dependent on the purpose of the assessment, prognosis or even on the response to nutritional intervention33 bearing in mind the inherent shortcomings of each. Page 9 of 17 Page 9 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 True prevalence of malnutrition True prevalence of malnutrition The prevalence of malnutrition among patients with cancer was high (˃ 54%). The findings are not unusual since cancer patients, particularly those suffering from head and neck, lung, and gastrointestinal cancer, carry the greatest burden of malnutrition among hospital patients.61 Although the findings of this study are similar to those observed in Spain (50%)62 and Italy (51%),63 higher prevalences have been reported elsewhere. For example, malnutrition was found to be present in 76% of ambulant cancer patients getting radiation therapy in Brazil25; In London 71% of cancer patients were malnourished,64 and in Ethiopia, a prevalence of 90.6% in ambulant cancer patients on treatment was reported.65 The prevalence of cancer-induced malnutrition is often cited as 40-80%,5,66 which largely depends on assessment method, clinical setting, and case-mix of patients. Notably, the lower and upper PCI limits in this study fall within the quoted range (40-80%). With respect to the parameters of this study, the study focused on specific malignancies, there was no restriction on cancer stage, and the analysis did not take into account whether or not patients received treatment. Moreover, this study used a combination of MUST and PG-SGA to estimate prevalence, unlike the above studies that employed one tool. The above factors could have been the cause of the disparity between the results of this study and the above-mentioned studies. In Kenya, in contrast to this study, lower prevalences of 31%6 and 13.4%17 have been obtained using the same population. Although lower, 31% falls within the lower PCI limit obtained at TCC in this study. The disparity could be ascribable to the different PG-SGA classification methods used. In their study, Opanga, Kaduka6 utilized the categorical classification, while this study employed the scoring method. Predictive values of the MUST and PG-SGA in screening for malnutrition The scoring method is more sensitive than the categorical classification, thus capturing more patients and leading to a higher prevalence.47 For example, Ræder, Henriksen47 recorded a Se of 50.0% and 60.7% for PG-SGA categorical and scoring classification methods, respectively. With regards to Kaduka, Bukania,17 the disparity could be explained by the different assessment methods used. A prevalence of 13.4%17 was based on BMI < 18.5 kg/m2 while in this study it was based on both the PG-SGA and MUST, which are superior to BMI. In Kenya, in contrast to this study, lower prevalences of 31%6 and 13.4%17 have been obtained using the same population. Although lower, 31% falls within the lower PCI limit obtained at TCC in this study. The disparity could be ascribable to the different PG-SGA classification methods used. In their study, Opanga, Kaduka6 utilized the categorical classification, while this study employed the scoring method. The scoring method is more sensitive than the categorical classification, thus capturing more patients and leading to a higher prevalence.47 For example, Ræder, Henriksen47 recorded a Se of 50.0% and 60.7% for PG-SGA categorical and scoring classification methods, respectively. With regards to Kaduka, Bukania,17 the disparity could be explained by the different assessment methods used. A prevalence of 13.4%17 was based on BMI < 18.5 kg/m2 while in this study it was based on both the PG-SGA and MUST, which are superior to BMI. The major strength of this study was using a Bayesian Latent Class Modelling framework, which minimized bias in test estimates, as it does not rely on a reference standard. A few limitations are inherent in this study that readers should be aware of while interpreting the findings. First, the PG-SGA is in form of a questionnaire targeting symptoms occurring within two weeks or one month of the time of screening. Therefore, the study participants may have failed to recall accurately, leading to either over-reporting or under-reporting of their symptoms. These may have biased the tool’s Se and Sp. Second, due to the study’s focus on specific cancers, findings may not be generalizable to other ambulant cancer populations. Third, patients who rejected taking part in this study did so due to pain, nausea, and weakness or because they thought, the study was too burdensome. Since these patients were more likely to be malnourished, this could have resulted in sample bias. Conclusions Using the Latent Class analysis, we have estimated the Se, Sp, and predictive values of both PG-SGA and MUST. The two tests achieved an accepted professional standard for Se (≥80%) and yielded good specificities. In respect to predictive values, the two tests produced comparable estimates. As such, healthcare workers are at liberty to use any of the two screening tools. Considering the high true prevalence observed in the two study populations, malnutrition screening among cancer patients should be done at diagnosis, during treatment and follow-up. Acknowledgements Acknowledgements The authors wish to express their sincere gratitude to the study participants for contributing to the research. References Elia M: ‘MUST’REPORT Nutritional screening of adults: a multidisciplinary responsibility 2003; p. 1–95. 24. Zhang Z, Wan Z, Zhu Y, et al.: Prevalence of malnutrition comparing NRS2002, MUST, and PG-SGA with the GLIM criteria in adults with cancer: A multi-center study. Nutrition. 2021 Mar; 83: 111072. Epub 2020/12/29. eng. PubMed Abstract\|Publisher Full Text 25. Bauer J, Capra S, Ferguson M: Use of the scored Patient- Generated Subjective Global Assessment (PG-SGA) as a nutrition assessment tool in patients with cancer. Eur. J. Clin. Nutr. 2002; 56(8): 779–785. Publisher Full Text 26. Nitichai N, Angkatavanich J, Somlaw N, et al.: Validation of the Scored Patient-Generated Subjective Global Assessment (PG-SGA) in Thai setting and association with nutritional parameters in cancer patients. Asian Pac. J. Cancer Prev. 2019; 20(4): 1249–1255. PubMed Abstract\|Publisher Full Text 27. Dubhashi S, Kayal A: Preoperative nutritional assessment in elderly cancer patients undergoing elective surgery: MNA or PG-SGA? Indian J. Surg. 2015; 77(2): 232–235. PubMed Abstract\|Publisher Full Text 28. Boléo-Tomé C, Monteiro-Grillo I, Camilo M, et al.: Validation of the Malnutrition Universal Screening Tool (MUST) in cancer. Br. J. Nutr. 2012; 108(2): 343–348. PubMed Abstract\|Publisher Full Text 29. Hettiarachchi J, Madubhashini P, Miller M: Agreement between the Malnutrition Universal Screening Tool and the Patient- Generated Subjective Global Assessment for Cancer Outpatients Receiving Chemotherapy: A Cross-Sectional Study. Nutr. Cancer. 2018; 70(8): 1275–1282. PubMed Abstract\|Publisher Full Text Pa 20. Davies M: Nutritional screening and assessment in cancer- associated malnutrition. Eur. J. Oncol. Nurs. 2005; 9(SUPPL. 2): S64–S73. Publisher Full Text 21. White JV, Guenter P, Jensen G, et al.: Consensus statement: Academy of Nutrition and Dietetics and American Society for Parenteral and Enteral Nutrition: characteristics recommended for the identification and documentation of adult malnutrition (undernutrition). JPEN J. Parenter. Enteral Nutr. 2012 May; 36(3): 275–283. P bM d Ab t t\|P bli h F ll T t 6. Opanga Y, Kaduka L, Bukania Z, et al.: Nutritional status of cancer outpatients using scored patient generated subjective global assessment in two cancer treatment centers, Nairobi, Kenya. BMC Nutrition. 2017; 3(1): 1–7. Publisher Full Text 7. Dewys WD, Begg C, Lavin PT, et al.: Prognostic effect of weight loss prior to chemotherapy in cancer patients. Am. J. Med. 1980; 69(4): 491–497. Publisher Full Text 22. Jager-Wittenaar H, Ottery FD: Assessing nutritional status in cancer: Role of the Patient-Generated Subjective Global Assessment. Curr. Opin. Clin. Nutr. Metab. Care. 2017; 20(5): 322–329. Publisher Full Text 8. Data availability Underlying data y g Raw dataset for the study is kept under restricted access since it contains sensitive participant information. Access to the raw data is possible upon placing a formal request to the corresponding author (carolinemuseka@gmail.com). The replication data and analysis scripts for this manuscript are available from the Harvard Dataverse. Harvard Dataverse: Performance of Malnutrition Universal Screening Tool and Patient-Generated Global Subjective Assessment in screening for cancer-related malnutrition in Nairobi County, Kenya. https://doi.org/10.7910/DVN/ HS5YM1.52 This project contains the following underlying data: This project contains the following underlying data: • R_Code_maln.R (Rscript for analysis) • Maln_data_xlsx (Analysis dataset) Page 10 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 • Questionnaire.pdf (The questionnaire used for data collection) Data are available under the terms of the Creative Commons Zero “No rights reserved” data waiver (CC0 1.0 Public domain dedication). ull Text 26. Nitichai N, Angkatavanich J, Somlaw N, et al.: Validation of the Scored Patient-Generated Subjective Global Assessment (PG-SGA) in Thai setting and association with nutritional parameters in cancer patients. Asian Pac. J. Cancer Prev. 2019; 20(4): 1249–1255. PubMed Abstract\|Publisher Full Text 12. Leuenberger M, Kurmann S, Stanga Z: Nutritional screening tools in daily clinical practice: The focus on cancer. Support. Care Cancer. 2010; 18(SUPPL. 2): 17–27. PubMed Abstract\|Publisher Full Text 13. Sealy MJ, Ottery F, Roodenburg J, et al.: Dutch Patient-Generated Subjective Global Assessment (PG-SGA): Training Improves Scores for Comprehensibility and Difficulty. Clin. Nutr. 2015; 34: S101-S. References PubMed Abstract\|Publisher Full Text 27. Dubhashi S, Kayal A: Preoperative nutritional assessment in elderly cancer patients undergoing elective surgery: MNA or PG-SGA? Indian J. Surg. 2015; 77(2): 232–235. PubMed Abstract\|Publisher Full Text 28. Boléo-Tomé C, Monteiro-Grillo I, Camilo M, et al.: Validation of the Malnutrition Universal Screening Tool (MUST) in cancer. Br. J. Nutr. 2012; 108(2): 343–348. PubMed Abstract\|Publisher Full Text 29. 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Hematol. 2000; 34(3): 137–168. PubMed Abstract\|Publisher Full Text 9. Laky B, Janda M, Cleghorn G, et al.: Comparison of different nutritional assessments and body-composition measurements in detecting malnutrition among gynecologic cancer patients. Am. J. Clin. Nutr. 2008; 87(6): 1678–1685. PubMed Abstract\|Publisher Full Text 10. References Silva FRM, de Oliveira MGOA, Souza ASR, et al.: Factors associated with malnutrition in hospitalized cancer patients: a cross- sectional study. Nutr. J. 2015; 14(1): 123. PubMed Abstract\|Publisher Full Text 11. Klein S, Kinney J, Jeejeebhoy K, et al.: Nutrition support in clinical practice: Review of published data and recommendations for future research directions. Clin. Nutr. 1997; 16(4): 193–218. PubMed Abstract\|Publisher Full Text 12. Leuenberger M, Kurmann S, Stanga Z: Nutritional screening tools in daily clinical practice: The focus on cancer. Support. Care Cancer. 2010; 18(SUPPL. 2): 17–27. PubMed Abstract\|Publisher Full Text 13. Sealy MJ, Ottery F, Roodenburg J, et al.: Dutch Patient-Generated Subjective Global Assessment (PG-SGA): Training Improves Scores for Comprehensibility and Difficulty. Clin. Nutr. 2015; 34: S101-S. Publisher Full Text 14. Ravasco P, Monteiro-Grillo I, Vidal PM, et al.: Impact of nutrition on outcome: A prospective randomized controlled trial in patients with head and neck cancer undergoing radiotherapy. Head Neck. 2005; 27(8): 659–668. PubMed Abstract\|Publisher Full Text 15. Gavazzi C, Colatruglio S, Valoriani F, et al.: Impact of home enteral nutrition in malnourished patients with upper gastrointestinal cancer: A multicentre randomised clinical trial. Eur. J. Cancer. 2016; 64: 107–112. PubMed Abstract\|Publisher Full Text 16. Ministry of Health K: National Guideline for Integrated Management of Acute Malnutrition. 2009 (June). 17. Kaduka LU, Bukania ZN, Opanga Y, et al.: Malnutrition and cachexia among cancer out-patients in Nairobi, Kenya. J. Nutr. Sci. 2017; 6: e63. PubMed Abstract\|Publisher Full Text\|Free Full Text 18. Isenring E, Cross G, Daniels L, et al.: Validity of the malnutrition screening tool as an effective predictor of nutritional risk in oncology outpatients receiving chemotherapy. Support. Care Cancer. 2006; 14(11): 1152–1156. PubMed Abstract\|Publisher Full Text 19. Jeejeebhoy KN, Detsky AS, Baker JP: Assessment of nutritional status. J. Parenter. Enter. Nutr. 1990; 14(5 SUPPL): 193S–196S. Publisher Full Text 20. Davies M: Nutritional screening and assessment in cancer- associated malnutrition. Eur. J. Oncol. Nurs. 2005; 9(SUPPL. 2): S64–S73. Publisher Full Text 21. White JV, Guenter P, Jensen G, et al.: Consensus statement: Academy of Nutrition and Dietetics and American Society for Parenteral and Enteral Nutrition: characteristics recommended for the identification and documentation of adult malnutrition (undernutrition). JPEN J. Parenter. Enteral Nutr. 2012 May; 36(3): 275–283. PubMed Abstract\|Publisher Full Text 22. Jager-Wittenaar H, Ottery FD: Assessing nutritional status in cancer: Role of the Patient-Generated Subjective Global Assessment. Curr. Opin. Clin. Nutr. Metab. Care. 2017; 20(5): 322–329. Publisher Full Text 23. References tional Quality of reening ematic e-Based ealth Care: y Group. ng and Cachexia. isk in rt. Care s of cancer ve global bi, Kenya. weight loss 1980; 69(4): ncer 2000; 34(3): erent surements r patients. associated cross- in clinical tions for 218. ening tools t. Care Generated proves r. 2015; 34: utrition on n patients Head Neck. me enteral ointestinal Cancer. 2016; 64: 107–112. PubMed Abstract\|Publisher Full Text 16. Ministry of Health K: National Guideline for Integrated Management of Acute Malnutrition. 2009 (June). 17. Kaduka LU, Bukania ZN, Opanga Y, et al.: Malnutrition and cachexia among cancer out-patients in Nairobi, Kenya. J. Nutr. Sci. 2017; 6: e63. PubMed Abstract\|Publisher Full Text\|Free Full Text 18. Isenring E, Cross G, Daniels L, et al.: Validity of the malnutrition screening tool as an effective predictor of nutritional risk in oncology outpatients receiving chemotherapy. Support. Care Cancer. 2006; 14(11): 1152–1156. PubMed Abstract\|Publisher Full Text 19. Jeejeebhoy KN, Detsky AS, Baker JP: Assessment of nutritional status. J. Parenter. Enter. Nutr. 1990; 14(5 SUPPL): 193S–196S. Publisher Full Text 20. Davies M: Nutritional screening and assessment in cancer- associated malnutrition. Eur. J. Oncol. Nurs. 2005; 9(SUPPL. 2): S64–S73. Publisher Full Text 21. White JV, Guenter P, Jensen G, et al.: Consensus statement: Academy of Nutrition and Dietetics and American Society for Parenteral and Enteral Nutrition: characteristics recommended for the identification and documentation of adult malnutrition (undernutrition). JPEN J. Parenter. Enteral Nutr. 2012 May; 36(3): 275–283. PubMed Abstract\|Publisher Full Text 22. Jager-Wittenaar H, Ottery FD: Assessing nutritional status in cancer: Role of the Patient-Generated Subjective Global Assessment. Curr. Opin. Clin. Nutr. Metab. Care. 2017; 20(5) 322–329. Publisher Full Text 23. Elia M: ‘MUST’REPORT Nutritional screening of adults: a multidisciplinary responsibility 2003; p. 1–95. 24. Zhang Z, Wan Z, Zhu Y, et al.: Prevalence of malnutrition comparing NRS2002, MUST, and PG-SGA with the GLIM criteria in adults with cancer: A multi-center study. Nutrition. 2021 Mar 83: 111072. Epub 2020/12/29. eng. PubMed Abstract\|Publisher Full Text 25. Bauer J, Capra S, Ferguson M: Use of the scored Patient- Generated Subjective Global Assessment (PG-SGA) as a nutrition assessment tool in patients with cancer. Eur. J. Clin. Nutr. 2002; 56(8): 779–785. Publisher Full Text 26. Nitichai N, Angkatavanich J, Somlaw N, et al.: Validation of the Scored Patient-Generated Subjective Global Assessment (PG-SGA) in Thai setting and association with nutritional parameters in cancer patients. Asian Pac. J. Cancer Prev. 2019; 20(4): 1249–1255. References Nitenberg G, Raynard B: Nutritional support of the cancer patient: Issues and dilemmas. Crit. Rev. Oncol. Hematol. 2000; 34(3): 137–168. PubMed Abstract\|Publisher Full Text 23. Elia M: ‘MUST’REPORT Nutritional screening of adults: a multidisciplinary responsibility 2003; p. 1–95. 9. Laky B, Janda M, Cleghorn G, et al.: Comparison of different nutritional assessments and body-composition measurements in detecting malnutrition among gynecologic cancer patients. Am. J. Clin. Nutr. 2008; 87(6): 1678–1685. PubMed Abstract\|Publisher Full Text 24. Zhang Z, Wan Z, Zhu Y, et al.: Prevalence of malnutrition comparing NRS2002, MUST, and PG-SGA with the GLIM criteria in adults with cancer: A multi-center study. Nutrition. 2021 Mar; 83: 111072. Epub 2020/12/29. eng. PubMed Abstract\|Publisher Full Text 10. Silva FRM, de Oliveira MGOA, Souza ASR, et al.: Factors associated with malnutrition in hospitalized cancer patients: a cross- sectional study. Nutr. J. 2015; 14(1): 123. PubMed Abstract\|Publisher Full Text 25. Bauer J, Capra S, Ferguson M: Use of the scored Patient- Generated Subjective Global Assessment (PG-SGA) as a nutrition assessment tool in patients with cancer. Eur. J. Clin. Nutr. 2002; 56(8): 779–785. P bli h F ll T t 11. Klein S, Kinney J, Jeejeebhoy K, et al.: Nutrition support in clinical practice: Review of published data and recommendations for future research directions. Clin. Nutr. 1997; 16(4): 193–218. PubMed Abstract\|Publisher Full Text Publisher Full Text 53. Barbosa-Silva MCG: Subjective and objective nutritional assessment methods: what do they really assess ? Curr. Opin. Clin. Nutr. Metab. Care. 2008; 11(3): 248–254. Publisher Full Text 35. Balstad TR, Bye A, Jenssen CRS, et al.: Patient interpretation of the patient-generated subjective global assessment (PG-SGA) short form. Patient Prefer. Adherence. 2019; 13: 1391–1400. PubMed Abstract\|Publisher Full Text 54. Miyata S, Tanaka M, Ihaku D: The prognostic significance of nutritional status using malnutrition universal screening tool in patients with pulmonary tuberculosis. Nutr. J. 2013; 12(1): 1–5. 36. Marshall S, Young A, Bauer J, Isenring E: Malnourished Older Adults Admitted To Rehabilitation in Rural New South Wales Remain Malnourished Throughout Rehabilitation and Once Discharged Back To the Community: a Prospective Cohort Study. 2015; 4(4): 197–204. 55. Roulston F, McDermott R: Comparison of three validated nutritional screening tools in the oncology setting. Proc. Nutr. Soc. 2008; 67(OCE7): 2009. Publisher Full Text 56. Abe Vicente M, Barão K, Donizetti Silva T, et al.: ¿Cuáles son los métodos más eficaces de valoración del estado nutricional en pacientes ambulatorios con cáncer gástrico y colorrectal? Nutr. Hosp. 2013; 28(3): 585–591. PubMed Abstract\|Publisher Full Text 37. Todorovic V, Russell C, Stratton R: JWaME. Nutritional screening and care planning with the ‘MUST’. 2003; p. 4–9. 38. Stratton RJ, Hackston A, Longmore D, et al.: Malnutrition in hospital outpatients and inpatients: prevalence, concurrent validity and ease of use of the ‘malnutrition universal screening tool’ (‘MUST’) for adults. Br. J. Nutr. 2004; 92(5): 799–808. PubMed Abstract\|Publisher Full Text 57. Ramboer C, Verhamme M, Vermeire L: Patients’ perception of involuntary weight loss: implications of underestimation and overestimation. Br. Med. J. (Clin. Res. Ed.). 1985; 291(6502): 1091. PubMed Abstract\|Publisher Full Text\|Free Full Text 39. Cawood AL, Elia M, Sharp SKE, et al.: Malnutrition self-screening by using MUST in hospital outpatients: Validity, reliability, and ease of use. Am. J. Clin. Nutr. 2012; 96(5): 1000–1007. PubMed Abstract\|Publisher Full Text 58. Abbott J, Teleni L, McKavanagh D, et al.: Patient-Generated Subjective Global Assessment Short Form (PG-SGA SF) is a valid screening tool in chemotherapy outpatients. Support. Care Cancer. 2016; 24(9): 3883–3887. PubMed Abstract\|Publisher Full Text 40. Cawood AL, Walters ER, Sharp SKE, et al.: ‘Self-screening’ for malnutrition with an electronic version of the Malnutrition Universal Screening Tool (‘MUST’) in hospital outpatients: Concurrent validity, preference and ease of use. Br. J. Nutr. 2018; 120(5): 528–536. 59. Publisher Full Text Du H, Liu B, Xie Y, et al.: Comparison of different methods for nutrition assessment in patients with tumors. Oncol. Lett. 2017; 14(1): 165–170. PubMed Abstract\|Publisher Full Text PubMed Abstract\|Publisher Full Text 34. Persson C, Sjödén PO, Glimelius B: The Swedish version of the patient-generated subjective global assessment of nutritional status: Gastrointestinal vs urological cancers. Clin. Nutr. 1999; 18(2): 71–77. PubMed Abstract\|Publisher Full Text PubMed Abstract\|Publisher Full Text 43. Enøe C, Georgiadis MP, Johnson WO: Estimation of sensitivity and specificity of diagnostic tests and disease prevalence when the true disease state is unknown. Prev. Vet. Med. 2000; 45(1-2): 61–81. PubMed Abstract\|Publisher Full Text 63. Muscaritoli M, Lucia S, Farcomeni A, et al.: Prevalence of malnutrition in patients at first medical oncology visit: the PreMiO study. Oncotarget. 2017; 8(45): 79884–79896. eng. PubMed Abstract\|Publisher Full Text 44. Hui ASL, Walter SD: Estimating the Error Rates of Diagnostic Tests Biometrics. 1980; 36(1): 167–171. 64. Shaw C, Fleuret C, Pickard JM, et al.: Comparison of a novel, simple nutrition screening tool for adult oncology inpatients and the Malnutrition Screening Tool (MST) against the Patient- Generated Subjective Global Assessment (PG-SGA). Support. Care Cancer. 2015; 23(1): 47–54. PubMed Abstract\|Publisher Full Text 45. Skinner: Probability Proportional to Size Sampling. Introduction to Survey Sampling. 2016; p. 39–47. 46. Connor RJ: Sample Size for Testing Differences in Proportions for the Paired-Sample Design Author (s): Robert J. Connor Published by: International Biometric Society Stable. Sample Size for Testing Differences in Proporti. 1987; 43(1): 207–211. Reference Source 65. Worku E: Prevalence and Factors Associated with Undernutrition on Cancer Patients at Tikur Anbessa Specialized Hospital, Ethiopia: A cross-sectional study. 2021. 47. Ræder H, Henriksen C, Bøhn SK, et al.: Agreement between PG-SGA category and fat-free mass in colorectal cancer patients. Clinical Nutrition ESPEN. 2018 2018/10/01; 27: 24–31. PubMed Abstract\|Publisher Full Text 66. Bauer J, Capra S: Comparison of a malnutrition screening tool with subjective global assessment in hospitalised patients with cancer - Sensitivity and specificity. Asia Pac. J. Clin. Nutr. 2003; 12(3): 257–260. 48. Teixeira AC, Mariani MGC, Toniato TS, et al.: Scored Patient- Generated Subjective Global Assessment: risk identification 120(5): 528 536. PubMed Abstract\|Publisher Full Text 60. Barbosa-Silva MC, Barros AJ: Indications and limitations of the use of subjective global assessment in clinical practice: an update. Curr. Opin. Clin. Nutr. Metab. Care. 2006 May; 9(3): 263–269. PubMed Abstract\|Publisher Full Text 41. Amaral TF, Antunes A, Cabral S, et al.: An evaluation of three nutritional screening tools in a Portuguese oncology centre. J. Hum. Nutr. Diet. 2008; 21(6): 575–583. PubMed Abstract\|Publisher Full Text 61. Shike M: Nutrition therapy for the cancer patient. Hematol. Oncol. Clin. North Am. 1996; 10(1): 221–234. Publisher Full Text 42. Yang D, Zheng Z, Zhao Y, et al.: Patient-generated subjective global assessment versus nutritional risk screening 2002 for gastric cancer in Chinese patients. Future Oncol. 2019; 16(3): 4475–4483. PubMed Abstract\|Publisher Full Text 62. Planas M, Álvarez-Hernández J, León-Sanz M, et al.: Prevalence of hospital malnutrition in cancer patients: a sub-analysis of the PREDyCES® study. Support. Care Cancer. 2016; 24(1): 429–435. PubMed Abstract\|Publisher Full Text S 0 S. Publisher Full Text 14. Ravasco P, Monteiro-Grillo I, Vidal PM, et al.: Impact of nutrition on outcome: A prospective randomized controlled trial in patients with head and neck cancer undergoing radiotherapy. Head Neck. 2005; 27(8): 659–668. PubMed Abstract\|Publisher Full Text 15. Gavazzi C, Colatruglio S, Valoriani F, et al.: Impact of home enteral nutrition in malnourished patients with upper gastrointestinal cancer: A multicentre randomised clinical trial. Eur. J. Cancer. Page 11 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 30. Borges NP, Silva BDA, Cohen C, et al.: Comparison of the nutritional diagnosis, obtained through different methods and indicators, in patients with cancer. Nutr. Hosp. 2009; 24(1): 51–55. and need for nutritional intervention in cancer patients at hospital admission. Nutr. Clín. Diet. Hosp. 2018; 95–102. 49. Lunn D, Spiegelhalter D, Thomas A, et al.: The BUGS project: Evolution, critique and future directions. Stat. Med. 2009 Nov 10; 28(25): 3049–3067. PubMed Abstract\|Publisher Full Text 31. Zhang L, Lu Y, Fang Y: Nutritional status and related factors of patients with advanced gastrointestinal cancer. Br. J. Nutr. 2014; 111(7): 1239–1244. PubMed Abstract\|Publisher Full Text ( ) PubMed Abstract\|Publisher Full Text 50. Thomas A, O Hara B, Ligges U, et al. Making BUGS Open. R news. 2006; 12–17. 32. Ottery FD: Definition of standardized nutritional assessment and interventional pathways in oncology. Nutrition. 1996; 12(SUPPL.1): S15–S19. Publisher Full Text 51. Kostoulas P, Nielsen SS, Branscum AJ, et al.: STARD-BLCM: Standards for the Reporting of Diagnostic accuracy studies that use Bayesian Latent Class Models. Prev. Vet. Med. 2017 Mar 1; 138: 37–47. PubMed Abstract\|Publisher Full Text Publisher Full Text 33. Poziomyck AK, Fruchtenicht AVG, Kabke GB, et al.: Reliability of nutritional assessment in patients with gastrointestinal tumors. Revista do Colegio Brasileiro de Cirurgioes. 2016; 43(3): 189–197. PubMed Abstract\|Publisher Full Text 52. Auma C: Replication data for: Performance of Malnutrition Universal Screening Tool and Patient-Generated Global Subjective Assessment in screening for cancer-related malnutrition in Nairobi County, Kenya. [Dataset] Harvard Dataverse 2022. Publisher Full Text Version 1 © 2023 Kruger M. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ( ) PubMed Abstract Page 12 of 17 Mariana Kruger Department of Paediatrics and Child Health, Faculty of Medicine and Health Sciences, Stellenbosch University, Stellenbosch, Western Cape, South Africa Check spelling and edit. Professional grammar editing will improve the readability of the text. Avoid long sentences with high density of information. Check the following and correct: “….cute disease effect score…” under Malnutrition Universal Screening Tool. 1. Write out abbreviations when used for the first time e.g., Se and Sp. 2. Indicated clearly under the section “Test outcomes, sensitivity and specificity” what cut-off point was used for which test; either by adding respectively or linking the cut-off point to the test. 3. Please add the time needed to use both tests as it seems to be quiet time-consuming for screening tests, especially if the PGA-SGA should be done by the treating physician. 4. Is the work clearly and accurately presented and does it cite the current literature? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Yes Are all the source data underlying the results available to ensure full reproducibility? Open Peer Review Current Peer Review Status: F1000Research 2022, 11:755 Last updated: 14 OCT 2024 Open Peer Review Response to comment 3 p We have revised this by linking the cut-off to the test. As such, it now reads: The cut-off value of ≥1 for MUST and ≥4 for PG-SGA gave the best combination of the Se and Sp. Response to comment 4 p We have supplied the information on the time needed for the tests: PG-SGA and MUST in paragraph 4 and paragraph 5 of the introduction section, respectively. Competing Interests: No competing interests. Reviewer Report 15 August 2022 Reviewer Report 15 August 2022 Are sufficient details of methods and analysis provided to allow replication by others? Partly Page 13 of 17 Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Oncology, nutrition, paediatrics, ethics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Author Response 02 Nov 2023 Caroline Auma Response to comment 1 I confirm that proofreading has been done to correct the grammatical errors. Response to comment 2 We have included this in the second last line of paragraph 3 in the introduction section: Sensitivity (Se) and Specificity (Sp). Response to comment 3 We have revised this by linking the cut-off to the test. As such, it now reads: The cut-off value of ≥1 for MUST and ≥4 for PG-SGA gave the best combination of the Se and Sp. Response to comment 4 We have supplied the information on the time needed for the tests: PG-SGA and MUST in paragraph 4 and paragraph 5 of the introduction section, respectively. Competing Interests: No competing interests. F1000Research 2022, 11:755 Last updated: 14 OCT 202 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Oncology, nutrition, paediatrics, ethics I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Yes https://doi.org/10.5256/f1000research.135124.r143589 © 2022 Ngesa O. This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Oscar Ngesa Oscar Ngesa Department of Mathematics and Physical Sciences, Taita Taveta University, Voi, Kenya Below is my review of the paper titled “Performance of Malnutrition Universal Screening Tool and Patient-Generated Global Subjective Assessment in screening for cancer-related malnutrition in Nairobi, Kenya”. Nairobi, Kenya”. The study aims at evaluating the performance of the Patient Generated-Subjective Global Assessment (PG-SGA) and Malnutrition Universal Screening Tool (MUST) in screening for malnutrition among cancer patients in Kenya. The cancers of interest in this setting are head and neck, respiratory and gastrointestinal cancer. Bayesian Latent Class Model (BLCM) was employed in the analysis. It is a preferred approach in the absence of a gold standard. Generally, the paper is well written in an appropriate language and easy to follow. Below are my specific comments and queries. In the introduction, put 39% to 87% instead of 39 to 87%. ○ All citations at the end of each sentence should be placed before the period/full stop rather than after. This needs to be corrected in the entire document. ○ The noun citation styles Kaduka,Bukania,17 and Ramboer, Verhamme57 do not seem to be correct. Confirm the style. ○ Can the authors check for model stability and robustness of the estimates via sensitivity analysis? Changing the different priors. ○ The authors mentioned that they would assess convergence of the models using visual appraisal of the density plots and Gelman-Rubin Diagnostic plots. However, none of these is reported in the document. ○ In the introduction, put 39% to 87% instead of 39 to 87%. ○ All citations at the end of each sentence should be placed before the period/full stop rather than after. This needs to be corrected in the entire document. ○ The noun citation styles Kaduka,Bukania,17 and Ramboer, Verhamme57 do not seem to be correct Confirm the st le ○ All citations at the end of each sentence should be placed before the period/full stop rather than after. This needs to be corrected in the entire document. ○ Is the study design appropriate and is the work technically sound? Yes Is the study design appropriate and is the work technically sound? Yes Are sufficient details of methods and analysis provided to allow replication by others? Partly If applicable, is the statistical analysis and its interpretation appropriate? Partly Are all the source data underlying the results available to ensure full reproducibility? Yes Are the conclusions drawn adequately supported by the results? Yes Competing Interests: No competing interests were disclosed. Reviewer Expertise: Statistical Modeling; Bayesian analysis; Malnutrition Analysis I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have Oscar Ngesa Oscar Ngesa Department of Mathematics and Physical Sciences, Taita Taveta University, Voi, Kenya Below is my review of the paper titled “Performance of Malnutrition Universal Screening Tool and Patient-Generated Global Subjective Assessment in screening for cancer-related malnutrition in g epartment of Mathematics and Physical Sciences, Taita Taveta University, Voi, Kenya Page 14 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 Response to comment 5 We have included this as the last part of the results section. It reads: On the convergence of the MCMC chains, the density plots of all posterior estimates yielded unimodal distributions. Similarly, the G elman-Rubin diagnostic statistics for the parameters produced unitary ratios. These results suggest convergence of the chains. Competing Interests: No competing interests were disclosed. Author Response 09 Nov 2023 Response to comment 4 Based on the three assumptions of the latent class analysis, we only met the minimum criteria. In particular, we only had two populations thus the constancy of Se and Sp estimates could not be assessed. Secondly, the two tests (MUST and PG-SGA) target different symptoms thus we assumed conditional independence. Finally, there was no prior information on any of the parameters in particular the Se and Sp. Therefore sensitivity analysis by modifying priors wouldn't be possible. Are the conclusions drawn adequately supported by the results? Yes Page 15 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 Response to comment 3 This has been revised to align with the rest of the in-text citations. significant reservations, as outlined above. Author Response 02 Nov 2023 Response to comment 1 p Revised as recommended. Now reads 39% to 87%. Response to comment 2 We have revised this as advised. However, It looks like this is an error from the journal editors because, from the Word document that I submitted, the citations were correctly placed at the end. Caroline Auma More to Comment 2 Regarding citations coming after the full stops, I noticed the same issue after version 2 was published. I engaged the editors and they said the change couldn't be made due to F1000 journal style. More to Comment 2 Competing Interests: No competing Interests. Page 16 of 17 F1000Research 2022, 11:755 Last updated: 14 OCT 2024 The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com The benefits of publishing with F1000Research: Your article is published within days, with no editorial bias • You can publish traditional articles, null/negative results, case reports, data notes and more • The peer review process is transparent and collaborative • Your article is indexed in PubMed after passing peer review • Dedicated customer support at every stage • For pre-submission enquiries, contact research@f1000.com The benefits of publishing with F1000Research: The benefits of publishing with F1000Research: Page 17 of 17
https://openalex.org/W3124893366	https://jurnal.ugm.ac.id/v3/IJP/article/download/596/264	English	null	Synthesis and antitumor properties of some new N-(5-R-benzyl-1,3-thiazol-2-yl)-4,5-dihydro-1H-imidazole-2-carboxamides	Indonesian Journal of Pharmacy	2,020	cc-by	6,268	INTRODUCTION compound with similar structure (1) were described. The antimicrobial (Sueleyman et al., 2005; Chaudhary et al., 2011) and anticancer (Beauchard et al., 2009) activity of such compounds were reported. They are also ligands of ad renergic α2 receptor (Saczewski et al., 2006), inhibitors of cyclooxygenase (Tanaka et al., 1994), and glycogen synthase kinase-3 (Saczewski et al., 2006), which can be considered as prominent anticancer targets (Satish and Woodgett, 2008). 2-Aminothiazole and their derivatives are of great importance in the organic and medicinal chemistry field (Nevagi, 2014; Chhabriaet et al., 2016; Aejazet et al., 2015). 2-Aminothiazole core is privileged structure for the compounds with a broad spectrum of activities, such as antibacterial (Vukovic et al., 2008), antifungal (Edwardset et al., 2013), antitubercular (Al-Balas et al., 2009), anti- HIV (Venkatachalam et al., 2001), antioxidant (Chaban et al., 2019), pesticidal (Wilkes et al., 1991), anti-inflammatory (Holla et al., 2003) etc. Among 2-aminothiazole-based compounds 5- benzyl derivatives are of special interest over the last decades. Significant antimicrobal (Khalilet et al., 2015) and anticancer activities of these compounds (Krasavin et al., 2009; Pokhodylo et al., 2014; Choi et al., 2011; Schiedel et al., 2016; Finiuk et al., 2017; Ostapiuk et al., 2018) have been reported. Aminothiazole derivatives have been also used as sensitive analytical reagents (Lozynska et al., 2015; Tymoshuk et al., 2019). Indonesian Journal of Pharmacy Indonesian Journal of Pharmacy VOL 31 (3) 2020: 150–160 \| RESEARCH ARTICLE dymyr Ya Horishny1, Iryna V Drapak2, Taras I Chaban2, Yuriy V Ostapiuk3, Vasyl S Matiychuk3 1. Department of Pharmaceutical, Organic and Bioorganic Chemistry Danylo Halytsky Lviv National Medical University, Lviv, 79010, Ukraine y, , , 2. Department of General, Bioinorganic, Physical and Colloidal Chemistry, Danylo Halytsky Lviv National Medical University, 69 Pekarska, Lviv, 79010, Ukraine y 3. Department of Organic Chemistry, Ivan Franko National University of Lviv, 6 Kyryla і Mefodia, Lviv, 79005, Ukraine y 3. Department of Organic Chemistry, Ivan Franko National University of Lviv, 6 Kyryla і Mefodia, Lviv, 79005, Ukraine Info Article ABSTRACT Submitted: 02-06-2020 Revised: 30-06-2020 Accepted: 01-09-2020 Corresponding author Vasyl S Matiychuk Email: v_matiychuk@ukr.net New N-(5-R-benzyl-1,3-thiazol-2-yl)-2-morpholin-4-yl-2-oxoaceta- mides have been prepared in good yields via the reaction of N-(5-R-benzyl- 1,3-thiazol-2-yl)-2-chloroacetamides with sulfur and morfoline. These compounds react with ethendiamine to form series of novel N-[5-R-benzyl)- 1,3-thiazol-2-yl]-4,5-dihydro-1H-imidazole-2-carboxamides with excellent yields. Anticancer activity screening of synthesized compounds was carried out within the framework of Developmental Therapeutic Program of the National Cancer Institute's (DTP,NCI, Bethesda, Maryland, USA). It was showed that compounds are promising for new anticancer agents search. Keywords: organic synthesis, thiazole, imidazole, anticancer activity. MATERIALS AND METHODS Chemicals and reagents All chemicals were of analytical grade and commercially available. All reagents and solvents were used without further purification and drying. 0 Indonesian J Pharm 31(3), 2020, 150-160 \| indonesianjpharm.farmasi.ugm.ac.id Copyright© 2020 THE AUTHOR(S). This article is distributed under a Creative Commons Attribution-ShareAlike 4.0 International (CC BY-SA 4.0) The general procedure for 2-morpholin-4-yl-N- aryl-2-thioxoacetamides (2a-e) preparation. The general procedure for 2-morpholin-4-yl-N- aryl-2-thioxoacetamides (2a-e) preparation. N-{5-[2-chloro-5-(trifluoromethyl)benzyl]-1,3- thiazol-2-yl}-2-morpholin-4-yl-2- thioxoacetamide (2e) Yield 71%, mp 207-209°C. 1H NMR (400 MHz, DMSO): δ = 12.67 (s, 1H, NH), 7.89 (s, 1H, C6H3), 7.71 (d, J = 7.3 Hz, 1H, C6H3), 7.66 (d, J = 7.0 Hz, 1H, C6H3), 7.36 (s, 1H, thiazole), 4.32 (s, 2H, ArCH2), 4.08 (s, 2H, CH2), 3.73 (d, J = 2.5 Hz, 2H, CH2), 3.64 (s, 2H, CH2), 3.59 (s, 2H, CH2). Anal.calcd.for C17H15ClF3N3O2S2: C, 45.39; H, 3.36; N, 9.34. Found: C, 45.15; H, 3.23; N, 9.25. N-{5-[2-chloro-5-(trifluoromethyl)benzyl]-1,3- thiazol-2-yl}-2-morpholin-4-yl-2- thioxoacetamide (2e) Yield 71%, mp 207-209°C. 1H NMR (400 MHz, DMSO): δ = 12.67 (s, 1H, NH), 7.89 (s, 1H, C6H3), 7.71 (d, J = 7.3 Hz, 1H, C6H3), 7.66 (d, J = 7.0 Hz, 1H, C6H3), 7.36 (s, 1H, thiazole), 4.32 (s, 2H, ArCH2), 4.08 (s, 2H, CH2), 3.73 (d, J = 2.5 Hz, 2H, CH2), 3.64 (s, 2H, CH2), 3.59 (s, 2H, CH2). Anal.calcd.for C17H15ClF3N3O2S2: C, 45.39; H, 3.36; N, 9.34. Found: C, 45.15; H, 3.23; N, 9.25. N-{5-[2-chloro-5-(trifluoromethyl)benzyl]-1,3- thiazol-2-yl}-2-morpholin-4-yl-2- thioxoacetamide (2e) A suspension of 0.01mol of powdered sulfur in 10mL of morpholine was stirred for 5min. To the prepared solutions the chloroacetamides (0.05 mol) was added, and stirred for 60min room temperature. The reaction mixture was poured into 200mL water and left for 24 h. The solid precipitated was filtered off, washed with water (20mL), dried and crystallized from ethanol. ( ) Yield 71%, mp 207-209°C. 1H NMR (400 MHz, DMSO): δ = 12.67 (s, 1H, NH), 7.89 (s, 1H, C6H3), 7.71 (d, J = 7.3 Hz, 1H, C6H3), 7.66 (d, J = 7.0 Hz, 1H, C6H3), 7.36 (s, 1H, thiazole), 4.32 (s, 2H, ArCH2), 4.08 (s, 2H, CH2), 3.73 (d, J = 2.5 Hz, 2H, CH2), 3.64 (s, 2H, CH2), 3.59 (s, 2H, CH2). Anal.calcd.for C17H15ClF3N3O2S2: C, 45.39; H, 3.36; N, 9.34. Found: C, 45.15; H, 3.23; N, 9.25. The general procedure fot 4,5-dihydro-1H- imidazole-2-carboxamides (3a-e) preparation N-[5-(4-chlorobenzyl)-1,3-thiazol-2-yl]-4,5- dihydro-1H-imidazole-2-carboxamide (3b) N-[5-(4-chlorobenzyl)-1,3-thiazol-2-yl]-4,5- dihydro-1H-imidazole-2-carboxamide (3b) Yield 97%, mp 230°C (decomp.). 1H NMR (400 MHz, DMSO): δ = 7.33 (d, J = 8.3 Hz, 2H, C6H4), 7.24 (d, J = 8.3 Hz, 2H, C6H4), 7.13 (s, 1H, thiazole), 4.00 (s, 2H, ArCH2), 3.81 (s, 4H, 2CH2). Anal.calcd.for C14H13ClN4OS: C, 52.42; H, 4.08; N, 17.46. Found: C, 52.15; H, 4.10; N, 17.67. N-[5-(4-chlorobenzyl)-1,3-thiazol-2-yl]-4,5- dihydro-1H-imidazole-2-carboxamide (3b) Chemistry All the melting points were determined in an open capillary and are uncorrected. 1H- spectra were recorded on a Varian Mercury 400 (400MHz for 1H). Mass spectra were run using Agilent 1100 series LC/MSD. Agilent Technologies Inc. with an API–ES/APCI ionization mode. The elemental analysis of experimental data on contents of Carbon, Hydrogen and Nitrogen were within ±0.3 % of the theoretical values. In this work we described the synthesis and anticancer activity of N-[5-R-benzyl)-1,3-thiazol-2- yl]-4,5-dihydro-1H-imidazole-2-carboxamides. The latter are the new class of organic compounds and their biological activity is not investigated. However, the synthesis and biological properties of 150 Volodymyr Ya Horishny N-(5-benzyl-1,3-thiazol-2-yl)-4,5-dihydro-1H- imidazole-2-carboxamide (3a) Yield 85%, mp 235°C. 1H NMR (400 MHz, DMSO): δ = 7.31 - 7.17 (m, 5H, C6H5), 7.14 (s, 1H, thiazole), 4.01 (s, 2H, ArCH2), 3.83 (s, 4H, 2CH2). ESI-MS: m/z 287 [M+H]+; Anal. calcd.for C14H14N4OS: C, 58.72; H, 4.93; N, 19.57. Found: C, 58.45; H, 4.82; N, 19.43. N-[5-(2-chlorobenzyl)-1,3-thiazol-2-yl]-2- morpholin-4-yl-2-thioxoacetamide (2c) N-[5-(2-chlorobenzyl)-1,3-thiazol-2-yl]-2- morpholin-4-yl-2-thioxoacetamide (2c) Yield 99%, mp 191-193°C. 1H NMR (400 MHz, DMSO): δ = 12.67 (s, 1H, NH), 7.59 – 7.39 (m, 2H, C6H4), 7.37 – 7.17 (m, 3H, C6H4, thiazole), 4.21 (s, 2H, ArCH2), 4.14 – 3.93 (m, 2H, CH2), 3.72 (s, 2H, CH2), 3.63 (d, J = 4.1 Hz, 2H, CH2), 3.57 (s, 2H, CH2). Anal.calcd.for C16H16ClN3O2S2: C, 50.32; H, 4.22; N, 11.00. Found: C, 50.17; H, 4.11; N, 11.15. N-[5-(2-chlorobenzyl)-1,3-thiazol-2-yl]-2- morpholin-4-yl-2-thioxoacetamide (2c) N-(5-benzyl-1,3-thiazol-2-yl)-2-morpholin-4- yl-2-thioxoacetamide (2a) Yield 86%, mp 188-190°C. 1H NMR (400 MHz, DMSO): δ = 12.61 (s, 1H, NH), 7.50 – 7.16 (m, 6H, C6H4, thiazole), 4.10 (s, 2H, PhCH2), 4.08 (d, J = 4.1 Hz, 2H, CH2), 3.73 (d, J = 4.0 Hz, 2H, CH2), 3.64 (s, 2H, CH2), 3.58 (s, 2H, CH2). Anal.calcd.for C16H17N3O2S2: C, 55.31; H, 4.93; N, 12.09. Found: C, 55.12; H, 4.90; N, 12.15. Method A. 0.0015mol of the corresponding morpholin-4-yl-2-thioxoacetamide 2a-e and 4mL of ethylenediamine was stirred at 50ºС for 30min. The mixture was cooled and poured into the 30mL of water. The precipitate was filtered, washed with water, dried and recrystallized from an alcohol. Method B. 1g of sulfur was dissolved in ethylenediamine (10 mL), and stirred for 30min. To the formed solution, 0.006mol of the corresponding chloroacetamide was added with constant stirring for 10min. The mixture was continued stirred for 30min, then cooled and poured into the 100mL of water and leave for 1 day. The precipitate was filtered, washed with water, dried and recrystallized from an alcohol. N-[5-(4-chlorobenzyl)-1,3-thiazol-2-yl]-2- morpholin-4-yl-2-thioxoacetamide (2b) Yield 93%, mp 238-240°C. 1H NMR (400 MHz, DMSO): δ = 12.65 (s, 1H, NH), 7.36 (d, J = 8.2 Hz, 2H, C6H4), 7.34 – 7.26 (m, 3H, thiazole, C6H4), 4.10 (s, 2H, ArCH2), 4.07 (s, 2H, CH2), 3.73 (s, 2H, CH2), 3.64 (s, 2H, CH2), 3.58 (s, 2H, CH2). Anal.calcd.for C16H16ClN3O2S2: C, 50.32; H, 4.22; N, 11.00. Found: C, 50.49; H, 4.30; N, 10.75. Synthesis and antitumor properties 3.84 (s, 4H, 2CH2). ESI-MS: m/z 321 [M+H]+; Anal. calcd.for C14H13ClN4OS: C, 52.42; H, 4.08; N, 17.46. Found: C, 52.17; H, 3.97; N, 17.50. Three dose-response parameters were calculated for each compound. Growth inhibition of 50% (GI50) was calculated from [(Ti − Tz)/(C − Tz)] ×100−50, which is the drug concentration resulting in a 50% lower net protein increase in the treated cells (measured by SRB staining) as compared to the net protein increase seen in the control cells. The drug concentration resulting in total growth inhibition (TGI) was calculated from Ti = Tz. The LC50 (concentration of drug resulting in a 50% reduction in the measured protein at the end of the drug treatment as compared to that at the beginning) indicating a net loss of cells following treatment was calculated from [(Ti − Tz)/Tz] × 100 = -50. Values were calculated for each of these three parameters if the level of activity was reached; however, if the effect was not reached or was exceeded, the value for that parameter was expressed as more or less than the maximum or minimum concentration was tested. N-{5-[2-chloro-5-(trifluoromethyl)benzyl]-1,3- thiazol-2-yl}-4,5-dihydro-1H-imidazole-2- carboxamide (3e) Yield 95%, mp>260°C. 1H NMR (400 MHz, DMSO): δ = 7.78 – 7.56 (m, 2H, C6H4), 7.33 (s, 1H), 7.14 (s, 1H, thiazole), 4.09 (s, 2H, ArCH2), 3.81 (s, 4H, 2CH2). Anal. calcd.for C15H12ClF3N4OS: C, 46.34; H, 3.11; N, 14.41. Found: C, 46.50; H, 3.00; N, 14.62. N-{5-[3-(trifluoromethyl)benzyl]-1,3-thiazol-2- yl}-4,5-dihydro-1H-imidazole-2-carboxamide (3d) Yield 79%, mp 253-255°C. 1H NMR (400 MHz, DMSO): δ = 7.57 (s, 1H, C6H4), 7.54-7.50 (m, 3H, C6H4), 7.17 (s, 1H, thiazole), 4.13 (s, 2H, ArCH2), 3.83 (s, 4H, 2CH2). Anal.calcd.for C15H13F3N4OS: C, 50.84; H, 3.70; N, 15.81. Found: C, 50.60; H, 3.52; N, 15.58. 2-Morpholin-4-yl-2-thioxo-N-{5-[3- (trifluoromethyl)benzyl]-1,3-thiazol-2- yl}acetamide (2d) Yield 81%, mp 187-189°C. 1H NMR (400 MHz, DMSO): δ = 12.73 – 12.64 (br.s, 1H, NH), 7.67 (s, 1H, C6H4), 7.63-7.54 (br.s, 3H, C6H4), 7.38 (s, 1H, thiazole), 4.23 (s, 2H, ArCH2), 4.11 - 4.03 (m, 2H, CH2), 3.72 (s, 2H, CH2), 3.64 (s, 2H, CH2), 3.61 – 3.53 (m, 2H, CH2). Anal.calcd.for C17H16F3N3O2S2: C, 49.15; H, 3.88; N, 10.11. Found: C, 48.97; H, 3.72; N, 9.99. N-[5-(2-chlorobenzyl)-1,3-thiazol-2-yl]-4,5- dihydro-1H-imidazole-2-carboxamide (3c) Yield 95%, mp 230°C(decomp.). 1H NMR (400 MHz, DMSO): δ = 7.42 (d, J = 7.4 Hz, 1H, C6H4), 7.35 (d, J = 7.3 Hz, 1H, C6H4), 7.31 – 7.21 (m, 2H, C6H4), 7.12 (s, 1H, thiazole), 4.12 (s, 2H, ArCH2), 151 Volume 31 Issue 3 (2020) Pharmacology y Diazonium salts are important reagents in organic synthesis, which are easily obtained from readily available aromatic amines. The utilization of diazonium salts in the design and synthesis of combinatorial libraries of furane (Obushak et al., 2009; Gorak et al., 2009; Obushak et al., 2008), pyrazole (Matiichuk et al., 2008), and 1,2,3-triazole (Obushak et al., 2009) derivatives, as well as some fused heterocycles (Chaban et al., 2019; Zelisko et al., 2015; Chaban et al., 2017; Zubkov et al., 2010; Klenina et al., 2017; Chaban et al., 2018) has been shown in our previous works. In this work we developed the new method of the synthesis of N-[5-R-benzyl)-1,3-thiazol-2-yl]-4,5- dihydro-1H-imidazole-2-carboxamide 3a–e based on diazonium salt as a started reagents. At the first stage 2-chloro-N-[5-(R-benzyl)-thiazol-2-yl]- acetamides were prepared via described protocol (Scheme 1) (Obushak et al., 2004; Ostapiuk et al., 2012). Cytotoxic activity against malignant human tumor cells Cytotoxic activity against malignant human tumor cells The tested compounds were added to the culture at a single concentration (10−5M) and the cultures were incubated for 48 h. Endpoint determinations were made with a protein binding dye, sulforhodamine B (SRB). Results for each tested compound were reported as the percent growth of the treated cells when compared to the untreated control cells. The percent growth was evaluated spectrophotometrically versus not treated controls. The cytotoxic and/or growth inhibitory effects of the most active compounds were tested in vitro against the full panel of about 60 human tumor cell lines at 10-fold dilutions of five concentrations ranging from 10−4to 10−8M. The 48-h continuous drug exposure protocol was followed and an SRB protein assay was used to estimate cell viability or growth. Using the seven absorbance measurements [time zero, (Tz), control growth in the absence of drug, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percent growth was calculated at each of the drug concentrations levels. Percent growth inhibition was calculated as: It well known that chloroacetanilides react with sulfur and morfoline to form corresponding monothiooxamides (Yarovenko et al., 1999). But chloracetamide derivatives of heterocyclic amines were not investigated in this reaction. So, we study the reaction of chloracetamides 1a-e, sulfur and morfoline. The optimal conditions for the synthesis of target monothiooxamides were the next: firstly, sulfur was stirred with morfoline for 30min. (this time is needed to obtain a sufficient amount of polysulfides in the reaction mixture); − Tz) × 100 for concentrations for which Ti ≥ Tz − Tz) −Tz) ×100 for concentrations for which Ti <Tz 152 Volume 31 Issue 3 (2020) Volume 31 Issue 3 (2020) Volodymyr Ya Horishny Scheme 1 Synthesis of 2-chloro-N-[5-(R-benzyl)-thiazol-2-yl]-acetamides Scheme 1. Synthesis of 2-chloro-N-[5-(R-benzyl)-thiazol-2-yl]-acetamides. Scheme 1. Synthesis of 2-chloro-N-[5-(R-benzyl)-thiazol-2-yl]-acetamides. Scheme 2. Synthesis of N-[5-R-benzyl-1,3-thiazol-2-yl]-4,5-dihydro-1H-imidazole-2-carboxamide Scheme 3. Tautomeric transformationN-[5-R-benzyl-1,3-thiazol-2-yl]-4,5-dihydro-1H-imidazole-2- carboxamides. Scheme 3. Tautomeric transformationN-[5-R-benzyl-1,3-thiazol-2-yl]-4,5-dihydro-1H-imidazole-2 carboxamides. Scheme 3. Tautomeric transformationN-[5-R-benzyl-1,3-thiazol-2-yl]-4,5-dihydro-1H-imidazole-2- carboxamides. form N-[5-R-benzyl)-1,3-thiazol-2-yl]-4,5-dihydro- 1H-imidazole-2-carboxamides 3a-e(Scheme 2). after, the corresponding chloroacetyl derivative was added and mixture and stirred for 1 hour. This protocol affords compounds 2a–e in a very high purity and in excellent yields (Scheme 2). We exanimated the possibility of the synthesis of 4,5-dihydroimidazole-2-carboxamides 3a-e in one-pot reaction of 1, sulfur, and ethylenediamine (Scheme 2). The reaction was carried out by heating in DMF within 5–6 hours, but yields of the final products were lower and required additional crystallizations. Cytotoxic activity against malignant human tumor cells Test compounds Average growth, % Range of growth, % Most sensitive cell line (cancer line/type) GP% Positive cytostatic eﬀecta 2a 100.84 78.99-115.40 UO-31 (RenalCancer) 78.99; 0/60 2b 103.91 80.33-123.38 UO-31 (Renal Cancer) 80.33; 0/60 2c 99.72 69.30-110.27 CCRF-CEM (Leukemia) 69.30; UO-31 (RenalCancer) 72.21 1/60 2d 100.00 74.53-118.71 UACC-62 (Melanoma) 74.77; CAKI-1 (RenalCancer) 79.31; UO-31 (RenalCancer) 74.53 2/60 2e 90.13 67.14-114.39 HOP-92 (Non-Small Cell Lung Cancer)70.37; UACC-62 (Melanoma) 72.57; CAKI-1 (RenalCancer) 67.14; UO- 31 (RenalCancer) 69.52 4/60 3a 90.59 57.10-120.12 CCRF-CEM (Leukemia) 57.10;HL-60(TB) (Leukemia) 60.59; K-562 (Leukemia) 70.21; MOLT-4 (Leukemia) 71.88; RPMI-8226 (Leukemia) 59.91; SR (Leukemia) 71.04; UACC-62 (Melanoma) 71.70; A498 (RenalCancer) 61.61; 8/60 3b 80.86 40.80-121.62 CCRF-CEM (Leukemia) 54.37; HL-60(TB) (Leukemiaa) 40.80; K-562 (Leukemia) 48.68; MOLT-4 (Leukemia) 69.14; RPMI-8226 (Leukemia) 58.04;EKVX (Non- Small Cell Lung Cancer) 64.09;NCI-H23 (Non-Small Cell Lung Cancer) 68.17;HCT-15 (Colon Cancer) 67.36; SK-MEL-5 (Melanoma) 62.84;UO-31 (Renal Cancer) 59.68 15/60 3c 41.92 -53.18-95.07 CCRF-CEM (Leukemia) -13.34; HL-60(TB) (Leukemia) 8.60; K-562 (Leukemia) 10.33; MOLT-4 (Leukemia) - 17.55; SR (Leukemia) - 19.89; A549/ATCC (Non-Small Cell Lung Cancer) 23.55; NCI-H460 (Non-Small Cell Lung Cancer) 13.67; HCT-116 (Colon Cancer) 36.93; KM12 (Colon Cancer) 35.70; SF-295 (CNS Cancer) 4.89; SF-539 (CNS Cancer) 2.96; LOX IMVI (Melanoma) -53.09; M14 (Melanoma) 30.71; MDA-MB-435 (Melanoma) 30.36; UACC-62 (Melanoma) 38.95; OVCAR-4 (Ovarian Cancer) 31.04; OVCAR-8 (Ovarian Cancer) 34.53; NCI/ADR-RES (Ovarian Cancer) 36.71; 786-0 (Renal Cancer) 45.35; ACHN (Renal Cancer) 33.01; CAKI-1 (Renal Cancer) -53.18; SN12C (Renal Cancer) 38.86; UO-31 (Renal Cancer) -17.10 50/60 aRatio between number of cell lines with percent growth from 0 to75 and total number of cell lines. Table I. Overview of the preliminary anticancer assay at single dose concentration of 10μM. Table I. Overview of the preliminary anticancer assay at single dose concentration of 10μM. Cytotoxic activity against malignant human tumor cells p y y ( ) Synthesized N-(5-R-benzyl-1,3-thiazol-2- yl)-2-morpholin-4-yl-2-oxoacetamides 2a–ewere investigated in the reaction with ethendiamine. It was found. that the heating of reagents for 30 min at 50°C led to the closure of the imidazoline ring to 153 Volume 31 Issue 3 (2020) Synthesis and antitumor properties Synthesis and antitumor properties Table I. Overview of the preliminary anticancer assay at single dose concentration of 10μM. Test compounds Average growth, % Range of growth, % Most sensitive cell line (cancer line/type) GP% Positive cytostatic eﬀecta 2a 100.84 78.99-115.40 UO-31 (RenalCancer) 78.99; 0/60 2b 103.91 80.33-123.38 UO-31 (Renal Cancer) 80.33; 0/60 2c 99.72 69.30-110.27 CCRF-CEM (Leukemia) 69.30; UO-31 (RenalCancer) 72.21 1/60 2d 100.00 74.53-118.71 UACC-62 (Melanoma) 74.77; CAKI-1 (RenalCancer) 79.31; UO-31 (RenalCancer) 74.53 2/60 2e 90.13 67.14-114.39 HOP-92 (Non-Small Cell Lung Cancer)70.37; UACC-62 (Melanoma) 72.57; CAKI-1 (RenalCancer) 67.14; UO- 31 (RenalCancer) 69.52 4/60 3a 90.59 57.10-120.12 CCRF-CEM (Leukemia) 57.10;HL-60(TB) (Leukemia) 60.59; K-562 (Leukemia) 70.21; MOLT-4 (Leukemia) 71.88; RPMI-8226 (Leukemia) 59.91; SR (Leukemia) 71.04; UACC-62 (Melanoma) 71.70; A498 (RenalCancer) 61.61; 8/60 3b 80.86 40.80-121.62 CCRF-CEM (Leukemia) 54.37; HL-60(TB) (Leukemiaa) 40.80; K-562 (Leukemia) 48.68; MOLT-4 (Leukemia) 69.14; RPMI-8226 (Leukemia) 58.04;EKVX (Non- Small Cell Lung Cancer) 64.09;NCI-H23 (Non-Small Cell Lung Cancer) 68.17;HCT-15 (Colon Cancer) 67.36; SK-MEL-5 (Melanoma) 62.84;UO-31 (Renal Cancer) 59.68 15/60 CCRF CEM (L k i ) 13 34 HL 60(TB) (L k i ) 50/60 Table I. Overview of the preliminary anticancer assay at single dose concentration of 10μM. the spectrum is due to a rapid tautomeric transformation (Scheme 3). For the same reason, the NH protons of the amide group of the 4,5-dihydro-1H-imidazole ring do not appear at all. Signals of protons of the methylene group of the benzyl radical are at 4.00 - 4.13 ppm. The 4-H proton signals of the thiazole moiety are at 7.12 - 7.17 ppm. Cytotoxic activity against malignant human tumor cells Test compounds Average growth, % Range of growth, % Most sensitive cell line (cancer line/type) GP% Positive cytostatic eﬀecta 2a 100.84 78.99-115.40 UO-31 (RenalCancer) 78.99; 0/60 2b 103.91 80.33-123.38 UO-31 (Renal Cancer) 80.33; 0/60 2c 99.72 69.30-110.27 CCRF-CEM (Leukemia) 69.30; UO-31 (RenalCancer) 72.21 1/60 2d 100.00 74.53-118.71 UACC-62 (Melanoma) 74.77; CAKI-1 (RenalCancer) 79.31; UO-31 (RenalCancer) 74.53 2/60 2e 90.13 67.14-114.39 HOP-92 (Non-Small Cell Lung Cancer)70.37; UACC-62 (Melanoma) 72.57; CAKI-1 (RenalCancer) 67.14; UO- 31 (RenalCancer) 69.52 4/60 3a 90.59 57.10-120.12 CCRF-CEM (Leukemia) 57.10;HL-60(TB) (Leukemia) 60.59; K-562 (Leukemia) 70.21; MOLT-4 (Leukemia) 71.88; RPMI-8226 (Leukemia) 59.91; SR (Leukemia) 71.04; UACC-62 (Melanoma) 71.70; A498 (RenalCancer) 61.61; 8/60 3b 80.86 40.80-121.62 CCRF-CEM (Leukemia) 54.37; HL-60(TB) (Leukemiaa) 40.80; K-562 (Leukemia) 48.68; MOLT-4 (Leukemia) 69.14; RPMI-8226 (Leukemia) 58.04;EKVX (Non- Small Cell Lung Cancer) 64.09;NCI-H23 (Non-Small Cell Lung Cancer) 68.17;HCT-15 (Colon Cancer) 67.36; SK-MEL-5 (Melanoma) 62.84;UO-31 (Renal Cancer) 59.68 15/60 3c 41.92 -53.18-95.07 CCRF-CEM (Leukemia) -13.34; HL-60(TB) (Leukemia) 8.60; K-562 (Leukemia) 10.33; MOLT-4 (Leukemia) - 17.55; SR (Leukemia) - 19.89; A549/ATCC (Non-Small Cell Lung Cancer) 23.55; NCI-H460 (Non-Small Cell Lung Cancer) 13.67; HCT-116 (Colon Cancer) 36.93; KM12 (Colon Cancer) 35.70; SF-295 (CNS Cancer) 4.89; SF-539 (CNS Cancer) 2.96; LOX IMVI (Melanoma) -53.09; M14 (Melanoma) 30.71; MDA-MB-435 (Melanoma) 30.36; UACC-62 (Melanoma) 38.95; OVCAR-4 (Ovarian Cancer) 31.04; OVCAR-8 (Ovarian Cancer) 34.53; NCI/ADR-RES (Ovarian Cancer) 36.71; 786-0 (Renal Cancer) 45.35; ACHN (Renal Cancer) 33.01; CAKI-1 (Renal Cancer) -53.18; SN12C (Renal Cancer) 38.86; UO-31 (Renal Cancer) -17.10 50/60 aRatio between number of cell lines with percent growth from 0 to75 and total number of cell lines. aRatio between number of cell lines with percent growth from 0 to75 and total number of cell lines. The structures of the obtained compounds were confirmed by1H NMR, mass spectroscopy and elemental analysis. Spectroscopic data of all compounds were in accordance to the proposed structures. In 1H NMR spectra, signals of methylene group protons of N-[5-R-benzyl)-1,3-thiazol-2-yl]- 4,5-dihydro-1H-imidazole-2-carboxamides appear as a singlets at 3.81-3.84 ppm. Such a character of the spectrum is due to a rapid tautomeric transformation (Scheme 3). the spectrum is due to a rapid tautomeric transformation (Scheme 3). For the same reason, the NH protons of the amide group of the 4,5-dihydro-1H-imidazole ring do not appear at all. Signals of protons of the methylene group of the benzyl radical are at 4.00 - 4.13 ppm. The 4-H proton signals of the thiazole moiety are at 7.12 - 7.17 ppm. Cytotoxic activity against malignant human tumor cells For the same reason, the NH protons of the amide group of the 4,5-dihydro-1H-imidazole ring do not appear at all. Signals of protons of the methylene group of the benzyl radical are at 4.00 - 4.13 ppm. The 4-H proton signals of the thiazole moiety are at 7.12 - 7.17 ppm. For the same reason, the NH protons of the amide group of the 4,5-dihydro-1H-imidazole ring do not appear at all. Signals of protons of the methylene group of the benzyl radical are at 4.00 - 4.13 ppm. The 4-H proton signals of the thiazole moiety are at 7.12 - 7.17 ppm. 154 Volume 31 Issue 3 (2020) Volodymyr Ya Horishny Table 2. In vitro anticancer activity at 60 human tumor cell lines for compound 3c. Disease Celllime GI50, µM TGI µM Disease Celllime GI50, µM TGI µM Leukemia CCRF-CEM 1.56 >100 Melanoma LOX IMVI 0.15 1.12 HL-60(TB) 2.80 11.5 MALME-3M 65.9 >100 K-562 3.93 >100 M14 6.85 >100 MOLT-4 2.23 >100 MDA-MB-435 7.65 >100 RPMI-8226 5.38 >100 SK-MEL-2 11.0 55.4 SR 1.77 >100 SK-MEL-28 54.6 >100 NSC lungcancer A549/ATCC 2.23 >100 SK-MEL-5 2.81 14.6 EKVX 5.70 >100 UACC-257 5.99 >100 HOP-62 19.4 58.5 UACC-62 11.6 79.0 HOP-92 2.85 10.3 Ovarian Cancer IGROV1 3.92 >100 NCI-H226 2.57 80.9 OVCAR-3 4.99 >100 NCI-H23 6.98 >100 OVCAR-4 12.3 >100 NCI-H322M >100 >100 OVCAR-5 12.2 >100 NCI-H460 3.20 >100 OVCAR-8 4.60 >100 NCI-H522 1.97 58.1 NCI/ADR-RES 6.01 >100 Colon Cancer COLO 205 8.68 >100 SK-OV-3 40.7 >100 HCC-2998 16.2 >100 Renal Cancer 786-0 3.71 35.1 HCT-116 7.27 >100 A498 41.3 >100 HCT-15 6.13 >100 ACHN 3.93 19.6 HT29 5.81 >100 CAKI-1 3.43 28.3 KM12 2.89 23.1 RXF 393 4.27 20.3 SW-620 7.07 >100 SN12C 7.89 21.2 CNS Cancer SF-268 5.30 >100 TK-10 3.21 9.94 SF-295 2.71 9.79 UO-31 2.88 15.5 SF-539 3.95 >100 Breast Cancer MCF7 7.73 >100 SNB-19 2.99 >100 MDA-MB- SNB-75 1.57 4.43 231/ATCC 4.81 >100 U251 7.83 >100 HS 578T 8.47 5.99 Prostate Cancer PC-3 6.52 >100 BT-549 8.33 >100 DU-145 1.54 >100 T-47D 5.13 >100 MDA-MB-468 5.52 >100 Pharmacology Сpd Subpanel tumor cell lines L NSCLC ColC CNSC M OV RC PC BC MG-MID 3c 2.95 16.1 7.72 4.06 18.5 12.1 9.79 4.03 6.67 9.01 5-FU 15.1 >100 8.4 72.1 70.6 61.4 45.6 22.7 76.4 22.60 Cisplatin 6.3 9.4 21.0 4.7 8.5 6.3 10.2 5.6 13.3 9.48 Curcumin 3.7 9.2 4.7 5.8 7.1 8.9 10.2 11.2 5.9 7.41 Table III. Anticancer selectivity pattern of the most active compound 3c at the GI50 (С, μМ) and TGI (С, μМ) levels Table III. Anticancer selectivity pattern of the most active compound 3c at the GI50 (С, μМ) and TGI (С, μМ) levels Сpd Parameters Subpanel tumor cell lines L NSCLC ColC CNSC M OV RC PC BC 3c GI50 2.95 16.1 7.72 4.06 18.5 12.1 9.79 4.03 6.67 SI 3.08 0.57 1.18 2.24 0.49 0.75 0.93 2.26 1.36 TGI 85.25 67.48 89.01 69.3 >100 72.23 31.24 >100 84.33 SI** 0.91 1.13 0.87 1.12 0.78 1.08 2.49 0.78 0.92 Selectivity index at the GI50 (С, μМ) level; Selectivity index at the TGI (С, μМ) level. L – leukemia, NSCLCC – non- small cell lung cancer, ColC – colon cancer, CNSC – CNS cancer, M – melanoma, OV– ovarian cancer, RC – renal cancer, PC – prostate cancer, BC – breast cancer. Table IV. Mean growth inhibitory concentration (GI50, µM) of compound 3c in comparison with 5-FU, Cisplatin and Curcumin Selectivity index at the GI50 (С, μМ) level; *Selectivity index at the TGI (С, μМ) level. L – leukemia, NSCLCC – non- small cell lung cancer, ColC – colon cancer, CNSC – CNS cancer, M – melanoma, OV– ovarian cancer, RC – renal cancer, PC – prostate cancer, BC – breast cancer. Table IV. Mean growth inhibitory concentration (GI50, µM) of compound 3c in comparison with 5-FU, Cisplatin and Curcumin. Cisplatin and Curcumin. Сpd Subpanel tumor cell lines L NSCLC ColC CNSC M OV RC PC BC MG-MID 3c 2.95 16.1 7.72 4.06 18.5 12.1 9.79 4.03 6.67 9.01 5-FU 15.1 >100 8.4 72.1 70.6 61.4 45.6 22.7 76.4 22.60 Cisplatin 6.3 9.4 21.0 4.7 8.5 6.3 10.2 5.6 13.3 9.48 Curcumin 3.7 9.2 4.7 5.8 7.1 8.9 10.2 11.2 5.9 7.41 percent (GPmean) of 41.92%. Moreover, this derivative demonstrated cytotoxic effect on Leukemia cell lines CCRF-CEM (GP=-13.34%), MOLT-4 (GP=-17.55%) and SR (GP=- 19.89%), Melanoma cell lines LOXIMVI (GP =-53.09%), Renal Cancer cell lines CAKI-1 (GP=-53.18%), UO-31 (GP=-17.10%). Pharmacology reported as the percent of cancer cell line growth (GP%) (Table I). The range of GP% shows the lowest and the highest values founded for different cancer cell lines. gy Among newly synthesized compounds substances 2a-e and 3a-c were selected by the National Cancer Institute (NCI) Developmental Therapeutic Program for the in vitro cell line screening to investigate their anticancer activity. The human tumor cell lines were derived from nine different cancer types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers. Primary anticancer assays were performed according to the DTP protocol (NCI USA), which was described elsewhere (Boyd et al., 1995; Boyd et al., 1997; Shoemaker et al., 2006).The results of primary screening are Tested compounds 2a-e showed a low antitumor activity in the in vitro screening assay. For the compounds 3a and 3b the average levels of cell growth (GPmean) were 90.59% and 80.86 % respectively. It should be noted, that selective action of tested compounds was observed towards Leukemia cell lines (range of GP= 57.10–71.88% (compound 3a) and GP= 35.01–69.14% (3b). The most active compound 3c was found to be effective against 50 cell lines with the average cell growth 155 Volume 31 Issue 3 (2020) Synthesis and antitumor properties Table III. Anticancer selectivity pattern of the most active compound 3c at the GI50 (С, μМ) and TGI (С, μМ) levels Сpd Parameters Subpanel tumor cell lines L NSCLC ColC CNSC M OV RC PC BC 3c GI50 2.95 16.1 7.72 4.06 18.5 12.1 9.79 4.03 6.67 SI 3.08 0.57 1.18 2.24 0.49 0.75 0.93 2.26 1.36 TGI 85.25 67.48 89.01 69.3 >100 72.23 31.24 >100 84.33 SI** 0.91 1.13 0.87 1.12 0.78 1.08 2.49 0.78 0.92 Selectivity index at the GI50 (С, μМ) level; *Selectivity index at the TGI (С, μМ) level. L – leukemia, NSCLCC – non- small cell lung cancer, ColC – colon cancer, CNSC – CNS cancer, M – melanoma, OV– ovarian cancer, RC – renal cancer, PC – prostate cancer, BC – breast cancer. Table IV. Mean growth inhibitory concentration (GI50, µM) of compound 3c in comparison with 5-FU, Cisplatin and Curcumin. Pharmacology Signiﬁcant cytostatic effect was observed toward Leukemia cell lines HL-60(TB) (GP=8.60), CNS Cancer cell lines SF-295(GP=4.89) and SF-539(GP=2.96). lines for the tested compound. For the MG_MID calculation, insensitive cell lines were included with the highest concentration tested. The mentioned derivative demonstrated a high activity toward the SR Leukemia cell lines (GI50=1.77µM), NCI-H522NSC lung cancer cell lines (GI50=1.97µM), SNB-75 CNS Cancer cell lines (GI50= 1.57µM), and DU-145 Prostate Cancer cell lines (GI50=1.97µM). For some cancer cell lines the cytotoxic effect was observed: HOP-92 Non-Small Cell Lung Cancer cell lines LC5 =62.0 µM; LOX IMVI Melanoma cell lines LC50=4.48µM; and RXF Renal Cancer cell lines LC50=69.5µM. ( ) Finally, compound 3c was selected for an advanced assay against a panel of approximately sixty tumor cell lines at 10-fold dilutions of ﬁve concentrations (100µM, 10µM, 1.0µM, 0.1µM and 0.01µM) (Table II). The percentage of growth was evaluated spectrophotometrically versus controls not treated with test agents after 48h exposure and using SRB protein assay to estimate cell viability or growth. Dose–response parameters were calculated for each cell line: GI50 – molar concentration of the compound that inhibits 50% net cell growth; TGI – molar concentration of the compound leading to the total inhibition; and LC50– molar concentration of the compound leading to 50% net cell death. Furthermore, a mean graph midpoints (MG_MID) were calculated for GI50, giving an average activity parameter over all cell µ The selectivity index (SI) obtained by dividing the full panel MG-MID (μM) of the compound 3c by its individual subpanel MG-MID (μM) was consideredas a measure of compound’s selectivity. Ratios between 3 and 6 refer to moderate selectivity, ratios greater than 6 indicate high selectivity toward the corresponding cell line, while compounds not meeting ei ther of the criteria are rated non-selective (Rostom, 2006). In this context, the active compound 3с demonstrates moderate selectivity toward Leukemia cell lines at the GI50 levels (SI=3.08) and low selectivity toward Renal Cancer cell lines (SI=2.49) at the TGI levels (Table III). Volume 31 Issue 3 (2020) 156 Volodymyr Ya Horishny acp synthase Mtfabh. PLoS One, 4(5): 5617- 5621. The screening results revealed that compound 3c possesses potent in vitro antitumor activity, with MG-MID = 9.01 in comparison with standard anticancer agent 5-fluorouracil (5-FU), Cisplatin MG-MID = 22.60 and Curcumin MG-MID = 7.41 (Table IV). CONCLUSIONS Here, we have shown the development of new efficient protocol for N-[5-benzyl-1,3-thiazol- 2-yl]-4,5-dihydro-1H-imidazole-2-carboxamides synthesis. The row of N-[5-R-benzyl-1,3-thiazol-2- yl]-4,5-dihydro-1H-imidazole-2-carboxamides was synthesized with high yields. Primary anticancer assay of synthesized compounds was performed at approximately sixty human tumor cell lines panel within DTP protocol (Drug Evaluation Branch, National Cancer Institute, Bethesda, USA). The compounds with significant levels of anticancer activities have been found, that can be used for further optimization. N-[5-(2-chlorobenzyl)-1,3- thiazol-2-yl]-4,5-dihydro-1H-imidazole-2- Boyd MR., Paull KD., 1995. 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Journal of Agricultural Food Chemistry, 39(9): 1652-1657. https://doi.org/10.1021/acs.jafc.6b02032 Rostom S., 2006. Synthesis and in vitro antitumor evaluation of some indeno[1,2- c]pyrazol(in)es substituted with sulfonamide, sulfonylurea(-thiourea) pharmacophores, and some derived thiazole ring systems. Bioorg Med Chem., 14 (19): 6475-6485. https://doi.org/10.1016/j.bmc.2006.06.02 0 Available at: http://www.dtp.nci.nih.gov (Accessed 10 February 2019). https://doi.org/10.1016/j.bmc.2006.06.02 Yarovenko VN., Kosarev SA., Zavarzin IV., Krayushkin MM., 1999. Reactions of monothiooxamides with N-nucleophiles. Volume 31 Issue 3 (2020) 159 Synthesis and antitumor properties Synthesis of 4,5-dihydroiidazole-2- carboxanilides. Russ Chem Bull., 71(4): 749– 753. https://doi.org/10.1007/BF02496262 Zubkov FI., Ershova JD., Zaytsev VP., Obushak MD., Matiychuk VS., Sokolova EA., Khrustalev VN., Varlamov AV., 2010. The first example of an intramolecular Diels-Alder furan (IMDAF) reaction of iminium salts and its application in a short and simple synthesis of the isoindolo[1,2-a]isoquinoline core of the jamtine and hirsutine alkaloids. Tetrahedron Lett., 51 (52): 6822-6824. https://doi.org/10.1016/j.tetlet.2010.10.04 g Zelisko NA., Atamanyuk DA., Ostapiuk YB., Bryhas AB., Matiychuk VS., Gzella AC., Lesyk RB., 2015. Synthesis of fused thiopyrano[2,3- d][1,3]thiazoles via hetero-Diels-Alder reaction related tandem and domino processes. Tetrahedron Lett., 71(50): 9501- 9508. https://doi.org/10.1016/j.tet.2015.10.019 6 160 Volume 31 Issue 3 (2020) Volume 31 Issue 3 (2020)
https://openalex.org/W2965280594	https://hal.science/hal-02326265/document	English	null	Sulcal organization in the medial frontal cortex provides insights into primate brain evolution	Nature communications	2,019	cc-by	15,916	Céline Amiez, Jerome Sallet, William D. Hopkins, Adrien Meguerditchian, Fadila Hadj-Bouziane, Ben Suliann, Charles R E Wilson, Emmanuel Procyk, Michael Petrides To cite this version: Céline Amiez, Jerome Sallet, William D. Hopkins, Adrien Meguerditchian, Fadila Hadj-Bouziane, et al.. Sulcal organization in the medial frontal cortex provides insights into primate brain evolution. Nature Communications, 2019, 10 (1), pp.3437. 10.1038/s41467-019-11347-x. hal-02326265 Distributed under a Creative Commons Attribution 4.0 International License HAL Id: hal-02326265 https://hal.science/hal-02326265v1 Submitted on 22 Oct 2019 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution 4.0 International License ARTICLE 9-11347-x Sulcal organization in the medial frontal cortex provides insights into primate brain evolution Although the relative expansion of the frontal cortex in primate evolution is generally accepted, the nature of the human uniqueness, if any, and between-species anatomo-func- tional comparisons of the frontal areas remain controversial. To provide a novel interpretation of the evolution of primate brains, sulcal morphological variability of the medial frontal cortex was assessed in Old World monkeys (macaque/baboon) and Hominoidea (chimpanzee/ human). We show that both Hominoidea possess a paracingulate sulcus, which was pre- viously thought to be unique to the human brain and linked to higher cognitive functions, such as mentalizing. Also, we show systematic sulcal morphological organization of the medial frontal cortex that can be traced from Old World monkeys to Hominoidea species, demon- strating an evolutionarily conserved organizational principle. These data provide a new fra- mework to compare sulcal morphology, cytoarchitectonic areal distribution, connectivity, and function across the primate order, leading to clear predictions about how other primate brains might be anatomo-functionally organized. 1 Univ Lyon, Université Lyon 1, Inserm, Stem Cell and Brain Research Institute U1208, 69500 Bron, France. 2 Wellcome Integrative Neuroimaging Centre, Department of Experimental Psychology, University of Oxford, Oxford OX1 3SR, UK. 3 Department of Comparative Medicine, University of Texas MD Anderson Cancer Center, Bastrop, TX 78602, USA. 4 Laboratoire de Psychologie Cognitive, UMR7290, Université Aix-Marseille, CNRS, 13331 Marseille, France. 5 Station de Primatologie CNRS, UPS846, 13790 Rousset, France. 6 Brain & Language Research Institute, Université Aix-Marseille, CNRS, 13604 Aix- en-Provence, France. 7 Integrative Multisensory Perception Action & Cognition Team (ImpAct), INSERM U1028, CNRS UMR5292, Lyon Neuroscience Research Center (CRNL), University of Lyon 1, 69500 Lyon, France. 8 Institut des Sciences Cognitives Marc Jeannerod, UMR5229, CNRS-Université Claude Bernard Lyon I, 69675 Bron, France. 9 Montreal Neurological Institute, Department of Neurology and Neurosurgery and Department of Psychology, McGill University, Montreal, QC H3A 2B4, Canada. 10These authors contributed equally: Céline Amiez, Jérôme Sallet. Correspondence and requests for materials should be addressed to C.A. (email: celine.amiez@inserm.fr) 1 NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications TURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x H H ow is the human brain unique? Although the relative expansion of the frontal cortex in primate evolution is generally accepted, the nature of the changes that occur remains controversial. Neuroanatomy offers a window into the evolution of brain circuits and their functions. Sulcal organization in the medial frontal cortex provides insights into primate brain evolution At a macroscopic level, a large body of literature has emphasized the link between the extent of gyriﬁcation, the rapid expansion of the cerebral cortex, and the complexity of the computational processing per- formed in a given brain1. Several hypotheses have been proposed to explain the origin of sulci and gyri: genetic control1, cortical growth2, tension of white matter cortico-cortical axons3, cortico- thalamic axons4, or simply mechanical instability in a soft tissue growing non-uniformly5. Although important, previous discus- sions of cortical gyriﬁcation have not considered another major dimension of sulcal pattern organization, i.e., its variability. Inter- individual variability of traits is at the basis of many evolutionary genetic studies6,7. Thus, the primary goal of the present study was to investigate variability in sulcal phenotypes in four key primate populations to provide new evidence and understanding of the evolution of primate frontal cortex. thought to be unique to the human brain, such as mentalizing or counterfactual thinking23. Whether the MFC is anatomo- functionally comparable in the human and macaque brains remains a subject for debate24,25. Based on neuroimaging ana- tomical scans, we investigate the sulcal morphological variability in Old-world monkeys (80 rhesus monkeys [Macaca mulatta] and 88 baboons [Papio papio]) and Hominoidea (225 chimpan- zees [Pan troglodytes] and 197 humans [Homo sapiens]) to identify potential speciﬁc characteristics of the human MFC. y p p The analysis of the sulcal organization in the MFC of primates provides critical new evidence of how the frontal cortex evolved within the primate order. We here show that both Hominoidea possess a PCGS, which was previously thought to be unique to the human brain and linked to higher cognitive functions. We also conﬁrm the expansion of the most rostral part of the MFC in humans. But overall, we reveal systematic sulcal morphological organization of the MFC that can be traced from Old-world monkeys to Hominoidea, demonstrating an evolutionarily con- served organizational principle. Sulcal patterns follow a precise topographical organization8. It is important to note that sulcal organization is not random despite the presence of signiﬁcant inter-hemispheric and inter- subject variability. A straightforward example is the location of the central sulcus. Although its shape, length, and depth may vary across hemispheres and individuals, it is always present and systematically located at the same strategic antero-posterior location. Importantly, this is not a human speciﬁc feature as it is observed in all primates9. Sulcal organization in the medial frontal cortex provides insights into primate brain evolution Although the origin of gyriﬁcation is not well understood, three types of sulci can be identiﬁed in primates, based on their appearance during gestation. Primary sulci, which appear ﬁrst during gestation (e.g., central sulcus, cingulate sulcus –CGS–)10,11, are present in all hemispheres and in all individuals. By contrast, the probability of observing sec- ondary/tertiary sulci is variable. For example, the paracingulate sulcus (PCGS) is present only in about 70% of subjects at least in one hemisphere12–14. Results The paracingulate sulcus (PCGS): an innovation of the brain in Hominoidea. A major ﬁnding of the present analysis was the demonstration that a PCGS can be observed, at least in one hemisphere, in 70.1% of human brains and 33.8% of chimpanzee brains, but not in baboon and macaque brains (dependent vari- able: PCGS present (0/1), main effect species: χ2 = 242.18, df = 3, p-value = 2.2e−16, logistic regression, GLM ﬁtted using an adjusted-score approach to bias reduction) (Fig. 1). A post-hoc Tukey test demonstrated that the probability of occurrence of a PCGS is signiﬁcantly decreased from human to chimpanzee brains (estimate = 1.47562, std. error = 0.20917, z-value = 7.054, p-value < 0.001) (Fig. 1e). Interestingly, human and chimpanzee brains differed with respect to asymmetries in the PCGS. In human brains, the PCGS was present in the left hemisphere in 35% of subjects, in the right hemisphere in 12.7% of subjects, and in both hemispheres in 22.8% of subjects, a distribution that differed signiﬁcantly (dependent variable: PCGS present (0/1), main effect of left versus right hemispheres: χ2 = 19.912, df = 1, p-value = 8.109e−06, logistic regression) (Fig. 1e). In contrast, in the chimpanzee, the PCGS was present in the left hemisphere in 12.9% of subjects, in the right hemisphere in 14.2% of subjects, and in both hemispheres in 6.7% of subjects, a distribution that did not differ signiﬁcantly (dependent variable: PCGS present (0/ 1), main effect of left versus right hemispheres: χ2 = 0.12399, df = 1, p-value = 0.7247). p Importantly, several lines of evidence indicate that many pri- mary sulci are limiting sulci between cytoarchitectonic areas. For instance, the central sulcus is the limiting sulcus between the primary motor cortex (area 4) which occupies the anterior bank of this sulcus and extends for a variable distance on the precentral gyrus and the primary somatosensory cortex (area 3) that lies on its posterior bank15–17. Although there is some inter-individual variability in the relation of the precise border of these areas and macrostructural features across individual brains18, the central sulcus remains a dividing line between the motor cortical region, anteriorly, and the somatosensory cortical region, posteriorly17,19. Several studies performing single subject analysis have also demonstrated that the organization of sulci and their variability is pertinent to prediction of the location of functional areas in various brain regions, e.g., refs. 20,21. NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications Results ILS present (0/1), main effect hemispheres (left/right): χ2 = 0.68999, df = 1, p-value = 0.4062 (ns)), the baboon brain (χ2 = 3.3591, df = 1, p-value = 0.06684 (ns)), or the macaque brain (χ2 = 0.42938, df = 1, p-value = 0.5123 (ns), logistic regression). However, the ILS was present signiﬁcantly more often in the left than the right hemisphere in the chimpanzee (χ2 = 10.419, df = 1, p-value = 0.001247, logistic regression). Finally, in Hominoidea, when a PCGS was present, the ILS was almost always absent (Fig. 2b) (human: presence of ILS in hemispheres with a PCGS versus hemispheres without a PCGS: χ2 = 30.221, df = 1, p-value = 3.855e−08; chimpanzee: χ2 = 14.011, df = 1, p-value = 0.0001817, logistic regression). ILS present (0/1), main effect hemispheres (left/right): χ2 = 0.68999, df = 1, p-value = 0.4062 (ns)), the baboon brain (χ2 = 3.3591, df = 1, p-value = 0.06684 (ns)), or the macaque brain (χ2 = 0.42938, df = 1, p-value = 0.5123 (ns), logistic regression). However, the ILS was present signiﬁcantly more often in the left than the right hemisphere in the chimpanzee (χ2 = 10.419, df = 1, p-value = 0.001247, logistic regression). Finally, in Hominoidea, when a PCGS was present, the ILS was almost always absent (Fig. 2b) (human: presence of ILS in hemispheres with a PCGS versus hemispheres without a PCGS: χ2 = 30.221, df = 1, p-value = 3.855e−08; chimpanzee: χ2 = 14.011, df = 1, p-value = 0.0001817, logistic regression). When evaluating the probability of occurrence of each one of the four vertically oriented sulci along the CGS or PCGS, signiﬁcant species differences were found for PACS (dependent variable: presence of sulci (0/1), main effect species: χ2 = 43.068, df = 3, p = 2.381e−09), PRPACS (χ2 = 49.192, df = 3, p = 1.187e−10), VPCGS-P (χ2 = 108.8, df = 3, p = 2.2e−16), and VPCGS-A (χ2 = 83.311, df = 3, p = 2.2e−16) (see Fig. 3a–d). For PACS, VPCGS-P, and VPCGS-A, post-hoc analysis indicated that these sulci are present in a higher proportion of human brains compared to chimpanzee, macaque, and baboon brains. Addi- tionally, the percentage of chimpanzee brains displaying a PACS, VPCGS-A, and VPCGS-P was signiﬁcantly higher compared to macaque and baboon brains, but there was no signiﬁcant difference in the probability of occurrence of these sulci between baboon and macaque brains. Results Collectively, these ﬁndings strongly suggest that sulcal organization in primates is not ran- dom, but rather has anatomo-functional relevance that is likely associated with the evolution of increasingly sophisticated sen- sory, motor, and cognitive functions. To what extent sulcal morphology across primate brains can provide insights into the anatomo-functional organization of brains in higher primates and brain evolution in the primate order remains poorly understood. p The presence/absence of an intralimbic sulcus (ILS), which is a shallow sulcus coursing parallel and ventral to the cingulate sulcus, was also examined in the four primate samples. The probability of observing an ILS in at least one hemisphere differed between species (proportions: 27%, 18.7%, 12.5%, and 11.3% of human, chimpanzee, baboon, and macaque brains, respectively; logistic regression, dependent variable: ILS present (0/1), main effect of species: χ2 = 18.993, df = 3, p-value = 0.0002743, Fig. 2a). Post-hoc Tukey analysis showed that the presence of an ILS in human brains was signiﬁcantly higher than in all other primate species. This analysis revealed no signiﬁcant differences in the presence of the ILS between the three non-human primate species (chimpanzee versus baboon: p-value = 0.5502, chimpanzee versus macaque: p-value = 0.4176, baboon versus macaque: p-value = 0.9942). The aim of the present study is to determine how the medial frontal cortex (MFC) sulcal organization has evolved through the primate order. For instance, one study13 has shown that the cytoarchitectonic organization of the cingulate cortical region is modulated, depending on the presence/absence of the PCGS. This cingulate/paracingulate region is often neglected in comparative studies22, despite its key role in cognitive processing often We also tested for asymmetries in the presence of ILS within each species by assessing whether the ILS was more frequent in one hemisphere compared to the other. No interhemispheric differences were found in the human brain (dependent variable: 2 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x a HUMAN 734045 - LH 459453 - LH No PCGS PCGS b e CHIMPANZEE c BABOON d MACAQUE PCGS PRESENCE CGS CGS PCGS #126 - RH #8 - LH No PCGS PCGS No PCGS No PCGS % of subjects displaying a PCGS HUMAN BABOON PCGS in both H PCGS in LH PCGS in RH No PCGS 100 0 25 50 75 CHIMP * ns * *** #92 - RH #13 - LH MACAQUE Fig. 1 Presence of the paracingulate sulcus (PCGS) across primates. NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications Results The location of the cingulate sulcus (CGS) is shown in red in typical hemispheres displaying no PCGS and in those displaying a PCGS in human (a) and chimpanzee (b), as well as in typical hemispheres of baboon (c) and macaque (d) brains. The location of the PCGS is shown in yellow in typical hemispheres displaying a PCGS in the human (a) and chimpanzee brains (b). A PCGS is observed in both human and chimpanzee brains, but not in baboon and macaque brains (e). The probability of occurrence of a PCGS decreased from Human to chimpanzee. The PCGS is lateralized in the left hemisphere in human, but not in chimpanzee brains. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle e PCGS PRESENCE % of subjects displaying a PCGS HUMAN BABOON PCGS in both H PCGS in LH PCGS in RH No PCGS 100 0 25 50 75 CHIMP * ns * * MACAQUE b d Fig. 1 Presence of the paracingulate sulcus (PCGS) across primates. The location of the cingulate sulcus (CGS) is shown in red in typical hemispheres displaying no PCGS and in those displaying a PCGS in human (a) and chimpanzee (b), as well as in typical hemispheres of baboon (c) and macaque (d) brains. The location of the PCGS is shown in yellow in typical hemispheres displaying a PCGS in the human (a) and chimpanzee brains (b). A PCGS is observed in both human and chimpanzee brains, but not in baboon and macaque brains (e). The probability of occurrence of a PCGS decreased from Human to chimpanzee. The PCGS is lateralized in the left hemisphere in human, but not in chimpanzee brains. Statistics: p < 0.05, p < 0.01, *p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle Speciﬁcally, we observed that the PACS, PRPACS, VPCGS-P, and VPCGS-A sulci, spurs, or dimples, could be reliably located with respect to four anatomical landmarks: the rostral limit of the pons, the anterior commissure, the caudal limit of the genu of the corpus callosum, and the rostral limit of the corpus callosum (see “Methods” and Fig. 3). Results a The probability of occurrence of an ILS is higher in human compared with non-human primate brains. A post-hoc Tukey test indicated no signiﬁcant difference in the presence of the ILS in the three non-human primate species. Only in chimpanzee brains, the probability of observing an ILS is higher in the left than in the right hemisphere. No difference between the probability of occurrence of ILS in the left versus right hemisphere was found in human, baboon, and macaque brains. b In Hominoidea, when a PCGS is present, the ILS is almost always absent. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle (χ2 = 38.617, df = 1, p = 2.092e−08), and macaque brains (χ2 = 67.617, df = 1, p = 1.382e−14). As can be seen in Fig. 4a–d, PACS, PRPACS, and VPCGS-A are the most conserved sulci across primate species, while VPCGS-P is the least conserved sulcus. Chimpanzee brains display a sulcal organization of the dorsal MFC comparable to that of the human brain, and macaque and baboon brains display evidence of the emergence of this organization. Importantly, vertical sulci emerging from the CGS or the PCGS located in the dorsal MFC can be identiﬁed in relation to ﬁxed anatomical landmarks that are consistent across primates. sulci forming a downward facing bifurcation: the supra-rostral sulcus (SU-ROS) and the supra-orbital sulcus (SOS) (Fig. 5a). When the PCGS is absent, the fork is located at the rostral end of the CGS but, when the PCGS is present, it is more frequently located at the rostral end of the PCGS (Supplementary Fig. 2). In non-human primates, the majority of the brains displayed different sulcal patterns at the rostral end of the CGS. The form and orientation of these folds were highly variable between species. Because of this discrepancy with the observed pattern in human brains, we chose to label the sulci at the end of the CGS, the ventral (CGS-VE) and dorsal (CGS-DE) extensions of the cingulate sulcus. We viewed the CGS-VE and the CGS-DE in the nonhuman primate brains as precursors of the human SU-ROS and SOS, respectively. Ventral mPFC. The anterior cingulate cortex (ACC), the ven- tromedial prefrontal cortex (vmPFC), and the medial frontopolar cortex (mFPC) are the focus of many studies on, respectively, emotional processing29, value-based decision-making30,31, and high-order socio-cognitive processing (mentalizing)23,32,33. Results For PRPACS, post-hoc analysis indicated that its probability of occurrence was signiﬁcantly higher in human and chimpanzee brains compared to baboon and macaque brains. There were no signiﬁcant differences in the occurrence of PRPACS between human and chimpanzee brains, nor between baboon and macaque brains. Dorsal medial frontal cortex (MFC). There are several lines of evidence suggesting that the dorsal MFC is comparable in terms of cytoarchitectonic areal distribution, connectivity, and func- tional processing between the macaque and human brains25–28. The present analysis shows that the sulcal organization of this region is also well conserved. In the human brain, posterior to the genu of the corpus callosum, we identiﬁed four sulci vertical to the CGS or the PCGS when present: the paracentral sulcus (PACS), the pre-paracentral sulcus (PRPACS), the posterior (VPCGS-P), and the anterior (VPCGS-A) vertical paracingulate sulci (Fig. 3). Each one of these sulci was deﬁned as fully present, as a spur or as a dimple when only superﬁcially evident, or absent. For this analysis, we considered the presence of spurs or dimples, which were observed almost exclusively in the baboon and macaque samples, as precursors of sulci that ﬁnd full expression in the chimpanzee and human brains (see below and Supplementary Fig. 1). Our argument for considering spurs and dimples in macaque and baboon brains as precursors of each one of the four sulci examined comes from an analysis of their normalized spatial location relative to human and chimpanzee brains (see below). In summary, the four vertical sulci, or their precursors, can be found across the four-primate species examined in the current study. In human brains, the probabilities of observing the vertical sulcus PACS versus PRPACS versus VPCGS-P versus VPCGS-A are similar (dependent variable: presence of sulci (0/1), main effect vertical sulci (PACS, PRPACS, VPCGS-P, VPCGS-A): χ2 = 4.6815, df = 3, p = 0.1967). Results By contrast, these probabilities vary in chimpanzee brains (χ2 = 68.917, df = 1, p = 7.279e−15), baboons 3 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x HUMAN CHIMP BABOON MACAQUE % of subjects 100 0 25 75 a ILS PRESENCE in hemispheres with vs without a PCGS 50 % of hemispheres 100 0 25 75 50 * * ILS present No ILS ILS in both H ILS in LH ILS in RH No ILS ns * ** ns ns ns 305830 -RH #23 - LH #57 - LH HUMAN CHIMP MACAQUE #60 - LH BABOON ILS HUMAN CHIMP BABOON MACAQUE ILS PRESENCE b Fig. 2 Presence of the ILS across primates. The location of the ILS is shown in yellow in typical hemispheres of the human, chimpanzee, baboon, and macaque monkey brains (right panels). a The probability of occurrence of an ILS is higher in human compared with non-human primate brains. A post-hoc Tukey test indicated no signiﬁcant difference in the presence of the ILS in the three non-human primate species. Only in chimpanzee brains, the probability of observing an ILS is higher in the left than in the right hemisphere. No difference between the probability of occurrence of ILS in the left versus right hemisphere was found in human, baboon, and macaque brains. b In Hominoidea, when a PCGS is present, the ILS is almost always absent. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle HUMAN CHIMP BABOON MACAQUE % of subjects 100 0 25 75 a 50 ILS in both H ILS in LH ILS in RH No ILS ns * ns ns ns 305830 -RH HUMAN CHIMP ILS ILS PRESENCE HUMAN CHIMP BABOON MACAQUE % of subjects 100 0 25 75 a 50 ILS in both H ILS in LH ILS in RH No ILS ns * ns ns ns ILS PRESENCE ILS PRESENCE in hemispheres with vs without a PCGS % of hemispheres 100 0 25 75 50 * ILS present No ILS HUMAN CHIMP BABOON MACAQUE b b #57 - LH Fig. 2 Presence of the ILS across primates. The location of the ILS is shown in yellow in typical hemispheres of the human, chimpanzee, baboon, and macaque monkey brains (right panels). NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunicatio NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x b Density of the difference between the Y coordinates of PACS and of the anatomical landmark 1 in human, chimpanzee, baboon, and macaque (from top to bottom panels). This difference was normalized in relation to the brain size (total antero-posterior extent). The 0 value corresponds to the Y level of the anatomical landmark 1 in standard brains. c Density of the normalized difference between the Y coordinates of PRPACS and of the anatomical landmark 2. The 0 value corresponds to the Y level of the anatomical landmark 2 in standard brains. d Density of the normalized difference between the Y coordinates of VPCGS-P and of the anatomical landmark 3. The 0 value corresponds to the Y level of the anatomical landmark 3 in standard brains. e Density of the normalized difference between the Y coordinates of VPCGS-A and of the anatomical landmark 4. The 0 value corresponds to the Y level of the anatomical landmark 4 in standard brains. Source data are provided as a Source Data ﬁle between the Z value where this intersection is found and the Z value where the rostral limit of the genu of the corpus callosum is found as dependent variable), strongly suggesting a downward migration in the human brain. In the baboon (P. papio), the fork formed by CGS-VE and CGS-DE is also oriented downwards in the majority of hemispheres but to a lesser extent (40%) compared to the human and chimpanzee brains. In 25% of hemispheres, the fork is forward facing, incomplete in 28.75% of hemispheres, and absent in 6.25% of hemispheres (Fig. 5a). In the rhesus monkey (M. mulatta), the fork formed by CGS-VE and CGS-DE displays the same patterns as in the baboon, but in different proportions: the most frequent pattern observed is a forward-facing fork (in 45% of hemispheres), followed by a fork oriented downwards (in 23.75% of hemispheres). The fork is incomplete in 13.75% of hemispheres and is absent in 17.5% of hemispheres (Fig. 5a). Ventral to SU-ROS or CGS-VE lies the superior rostral sulcus (ROS-S). The ROS-S is a long and deep sulcus that is present in nearly 100% of hemispheres across all four species and, therefore, does not differ signiﬁcantly in its occurrence (main effect of species: χ2 = 4.5334, df = 3, p = 0.2093) (Fig. 6a). ROS-S is often connected to the accessory supra-orbital sulcus (ASOS). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x c d e cs AC cgs PRPACS 20 –30 10 0 –50 –20 –10 30 Human MNI standard Y coordinate (mm) 50 40 60 –40 AC 20 –30 10 40 0 –40 –20 –10 30 Chimpanzee standard Y coordinate (mm) HUMAN CHIMP 20 –30 10 0 –20 –10 30 Baboons standard Y coordinate (mm) AC BABOON 20 10 0 –20 –10 30 Macaque standard Y coordinate (mm) Anterior commissure LANDMARK 2 Caudal limit of the genu of the corpus callosum LANDMARK 3 Rostral limit of the genu of the corpus callosum LANDMARK 4 MACAQUE AC Normalized difference between Y coordinates of PRPACS and of anatomical landmark 2 Normalized difference between Y coordinates of VPCGS-P and of anatomical landmark 3 Normalized difference between Y coordinates of VPCGS-A and of anatomical landmark 4 Rostral limit of the pons LANDMARK 1 b Normalized difference between Y coordi- nates of PACS and of anatomical landmark 1 PACS 0 5 10 15 DENSITY HUMAN CHIMP BABOON MACAQUE 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 0.2 –0.2 0 0.2 –0.2 0 5 10 15 0 5 10 15 0 5 10 15 0 0.2 –0.2 0 0.2 –0.2 a VPCGS-A VPCGS-P PACS PRPACS VPCGS-P VPCGS-A Fig. 3 Location of vertical sulci in the dorsal MFC across primates. a Schematic representation of the location of the various sulci in the respective standard space of each primate species. The scale represents the antero-posterior level (in mm) in each brain. Fixed anatomical landmarks across primates are displayed: the rostral limit of the pons (landmark 1, in yellow), the anterior commissure (landmark 2, in red), the caudal limit of the genu of the corpus callosum (landmark 3, in light blue), and the rostral limit of the genu of the corpus callosum (landmark 4, in dark blue). b Density of the difference between the Y coordinates of PACS and of the anatomical landmark 1 in human, chimpanzee, baboon, and macaque (from top to bottom panels). This difference was normalized in relation to the brain size (total antero-posterior extent). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x The 0 value corresponds to the Y level of the anatomical landmark 1 in standard brains. c Density of the normalized difference between the Y coordinates of PRPACS and of the anatomical landmark 2. The 0 value corresponds to the Y level of the anatomical landmark 2 in standard brains. d Density of the normalized difference between the Y coordinates of VPCGS-P and of the anatomical landmark 3. The 0 value corresponds to the Y level of the anatomical landmark 3 in standard brains. e Density of the normalized difference between the Y coordinates of VPCGS-A and of the anatomical landmark 4. The 0 value corresponds to the Y level of the anatomical landmark 4 in standard brains. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x Source data are provided as a Source Data ﬁle c d e 0 e (mm) 50 40 60 40 0 mm) 0 m) 0 mm) l limit of the of the corpus um MARK 4 Normalized difference between Y coordinates of PRPACS and of anatomical landmark 2 Normalized difference between Y coordinates of VPCGS-P and of anatomical landmark 3 Normalized difference between Y coordinates of VPCGS-A and of anatomical landmark 4 b Normalized difference between Y coordi- nates of PACS and of anatomical landmark 1 0 5 10 15 DENSITY HUMAN CHIMP BABOON MACAQUE 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 5 10 15 20 0 0.2 –0.2 0 0.2 –0.2 0 5 10 15 0 5 10 15 0 5 10 15 0 0.2 –0.2 0 0.2 –0.2 PCGS-A PACS PRPACS VPCGS-P VPCGS-A cs AC cgs PRPACS 20 –30 10 0 –50 –20 –10 30 Human MNI standard Y coordinate (mm) 50 40 60 –40 AC 20 –30 10 40 0 –40 –20 –10 30 Chimpanzee standard Y coordinate (mm) HUMAN CHIMP 20 –30 10 0 –20 –10 30 Baboons standard Y coordinate (mm) AC BABOON 20 10 0 –20 –10 30 Macaque standard Y coordinate (mm) Anterior commissure LANDMARK 2 Caudal limit of the genu of the corpus callosum LANDMARK 3 Rostral limit of the genu of the corpus callosum LANDMARK 4 MACAQUE AC Rostral limit of the pons LANDMARK 1 PACS a VPCGS-A VPCGS-P a b 1 b Fig. 3 Location of vertical sulci in the dorsal MFC across primates. a Schematic representation of the location of the various sulci in the respective standard space of each primate species. The scale represents the antero-posterior level (in mm) in each brain. Fixed anatomical landmarks across primates are displayed: the rostral limit of the pons (landmark 1, in yellow), the anterior commissure (landmark 2, in red), the caudal limit of the genu of the corpus callosum (landmark 3, in light blue), and the rostral limit of the genu of the corpus callosum (landmark 4, in dark blue). Results These processes reach their epitome in human subjects, and the present analysis of sulcal organization provides insights into the evolution of the above mentioned cortical regions and thus the anatomical substrate for these higher order processes in the primate order. In chimpanzee, the fork formed by the CGS-VE and CGS-DE extensions of the cingulate sulcus was also oriented downwards in the majority of hemispheres (81.25%). However, in 17.5% of hemispheres, the fork was forward facing, and was incomplete (i.e., CGS-DE is present but CGS-VE is absent) in 1.25% of hemispheres (Fig. 5a). Interestingly, as in human brains, when the PCGS is absent, the fork is located at the rostral end of the CGS. By contrast, when the PCGS is present in chimpanzee, the fork is still located at the rostral end of the CGS—and not at the PCGS as in human—brains in the majority of hemispheres (Supplemen- tary Fig. 2). g Anterior to the genu of the corpus callosum, we identiﬁed in human brains up to 9 distinct sulci34. One of the distinct features of the vmPFC is the branching of sulci at the end of the CGS. In human brains, the rostral end of the CGS is characterized by two NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications 4 NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x The ASOS is equally present in human (88.75%) and chimpanzee (81.25%) hemispheres and is observed signiﬁcantly less often in baboon (26.25%) and macaque (21.25%) hemispheres compared to chimpanzees and humans (main effect of species: χ2 = 132.81, df = 3, p-value = 2.2e−16, logistic regression) (Fig. 6b). Ventral to ROS-S, the inferior rostral sulcus (ROS-I) is equally present in human (35%) and chimpanzee (37.5%) hemispheres, but largely absent in baboon (3%) and macaque (1.5%) (χ2 = 60.348, df = 3, p = 4.952e−13) (Fig. 6c). The assessment of the relative location of the intersection of this fork with the CGS or PCGS across primates (see “Methods”), revealed that it is located at the level of the rostral limit of the genu of the corpus callosum in humans (Fig. 5b), but is located dorsal to it in the non-human primate brains (F(3,298) = 43.506, p < 2.2e−16, GLM with species as ﬁxed effect and the difference 5 5 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x PRPACS VPCGS-P VPCGS-A PACS a b c d ns 100 0 25 50 75 % of hemispheres 100 0 25 50 75 100 0 25 50 75 ns ns * Dimple Spur Sulcus No 100 0 25 50 75 * ns % of hemispheres 100 0 25 50 75 % of hemispheres 100 0 25 50 75 100 0 25 50 75 % of hemispheres 100 0 25 50 75 HUMAN CHIMP BABOON MACAQUE 734045 - LH #70 - RH #8 - LH * * * * * ns * #4 - LH % of hemispheres % of hemispheres % of hemispheres % of hemispheres HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE Fig. 4 Presence of vertical sulci in the dorsomedial frontal cortex across primates. Probability of occurrence (±s.e.m.) of the paracentral sulcus (PA the pre-paracentral sulcus (PRPACS, b), the posterior (VPCGS-P, c) and the anterior (VPCGS-A, d) vertical paracingulate sulci in the four species. a Left panel: The probability of observing a PACS decreases from human, chimpanzee, baboon, to macaque. Posthoc analysis showed that the prese PACS was higher in Hominoidea (human and chimpanzee) than in Old-world monkeys (baboon and macaque), but that it was similar between hum chimpanzees and also between baboon and macaque. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x Posthoc analysis showed that the presence of PACS was higher in Hominoidea (human and chimpanzee) than in Old-world monkeys (baboon and macaque), but that it was similar between human and chimpanzees and also between baboon and macaque. Right panel: PACS characteristics vary across primates: it is a sulcus (blue) in 100% of hemispheres in Hominoidea, it is a spur (green) or a dimple (pink) in Old-world monkeys. It is more frequently a spur than a dimple in baboon, as opposed to macaque. b PRPACS. Left panel: the probability of observing PACS is higher in Hominoidea than in Old-world monkeys but is similar between human and chimpanzee, on one hand, and between baboon and macaque on the other hand. Right panel: PRPACS characteristics vary across primates: it is a sulcus in 100% of hemispheres in Hominoidea, but it is a spur or a dimple in Old-world monkeys. It is more frequently a spur than a dimple in baboon, as opposed to macaque. c VPCGS-P. Left panel: the probability of observing VPCGS-P decreases from human, chimpanzee, baboon, to macaque. Posthoc analysis demonstrated that the presence of the VPCGS-P was higher in human than in non-human primates. Its probability of occurrence was also higher in chimpanzee than in Old-world monkeys. It was, however, similar between macaque and baboon. Right panel: VPCGS-P characteristics vary across primates: it is a sulcus in 100% of hemispheres in Hominoidea, but it is a spur or a dimple in baboon and only a dimple in Macaque. d VPCGS-A. Left panel: The probability of occurrence of VPCGS-A decreases from human, chimpanzee, baboon, to macaque. Right panel: VPCGS-P characteristics vary across primates: it is a sulcus in 100% of hemispheres in Hominoidea, but it is more frequently a spur than a dimple in baboon, as compared with macaque. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle Fig. 4 Presence of vertical sulci in the dorsomedial frontal cortex across primates. Probability of occurrence (±s.e.m.) of the paracentral sulcus (PACS, a), the pre-paracentral sulcus (PRPACS, b), the posterior (VPCGS-P, c) and the anterior (VPCGS-A, d) vertical paracingulate sulci in the four species. a PACS. Left panel: The probability of observing a PACS decreases from human, chimpanzee, baboon, to macaque. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x Right panel: PACS characteristics vary across primates: it is a sulcus (blue) in 100% of hemis in Hominoidea, it is a spur (green) or a dimple (pink) in Old-world monkeys. It is more frequently a spur than a dimple in baboon, as opposed to ma b PRPACS. Left panel: the probability of observing PACS is higher in Hominoidea than in Old-world monkeys but is similar between human and chimpanzee, on one hand, and between baboon and macaque on the other hand. Right panel: PRPACS characteristics vary across primates: it is a su 100% of hemispheres in Hominoidea, but it is a spur or a dimple in Old-world monkeys. It is more frequently a spur than a dimple in baboon, as oppo macaque. c VPCGS-P. Left panel: the probability of observing VPCGS-P decreases from human, chimpanzee, baboon, to macaque. Posthoc analys ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11 PACS a ns 100 0 25 50 75 * % of hemispheres HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 0 25 50 75 * * HUMAN CHIMP BABOON MACAQUE HUMAN 734045 - LH PRPACS b 100 0 25 50 75 ns ns * % of hemispheres HUMAN CHIMP BABOON MACAQUE b % of hemispheres 100 0 25 50 75 * * HUMAN CHIMP BABOON MACAQUE CHIMP #8 - LH VPCGS-P c 100 0 25 50 75 * ns % of hemispheres HUMAN CHIMP BABOON MACAQUE c % of hemispheres 100 0 25 50 75 * HUMAN CHIMP BABOON MACAQUE CHIMP % of hemispheres BABOON #70 - RH VPCGS-A d 100 0 25 50 75 ns % of hemispheres HUMAN CHIMP BABOON MACAQUE d % of hemispheres 100 0 25 50 75 HUMAN CHIMP BABOON MACAQUE Dimple Spur Sulcus No MACAQUE #4 - LH % of hemispheres MACAQUE HUMAN CHIMP BABOON MACAQUE UMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE Fig. 4 Presence of vertical sulci in the dorsomedial frontal cortex across primates. Probability of occurrence (±s.e.m.) of the paracentral sulcus (PACS, a), the pre-paracentral sulcus (PRPACS, b), the posterior (VPCGS-P, c) and the anterior (VPCGS-A, d) vertical paracingulate sulci in the four species. a PACS. Left panel: The probability of observing a PACS decreases from human, chimpanzee, baboon, to macaque. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x Figure CGS-DE CGS-VE SU-ROS/SOS and CGS-VE/CGS-DE INTERSECTION PATTERNS a b c HUMAN BABOON MACAQUE Incomplete fork Only CGS-DE present No fork SU-ROS/SOS and CGS-VE/CGS-DE INTERSECTION LOCATION Rostral limit of the genu of the corpus callosum 109830 - RH CS CGS VPCGS-P PRPACS VPCGS-A PACS SOS SU-ROS #131 - LH #55 - RH #76 - RH #92 - RH #67 - LH #4 - LH #20 - LH #13 - LH #42 - RH CGS-DE CGS-VE CGS-DE CGS-VE CGS-DE CGS-VE % of hemispheres 100 0 25 50 75 734045 - LH The RP-PCGS RP-PCGS % of hemispheres 100 0 25 50 75 CHIMPANZEE Complete fork pointing downward Complete fork forward-facing CHIMP #203 - LH Intersection ΔZ * Rostral limit of the genu of the corpus callosum #60 - LH 0 10 20 30 * HUMAN CHIMP BABOON MACAQUE Normalized ΔZ value HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE SU-ROS/SOS and CGS-VE/CGS-DE INTERSECTION PATTERNS a HUMAN CHIMPANZ SU-ROS/SOS and CGS-VE/CGS-DE INTERSECTION PATTERNS a HUMAN BABOON MACAQUE Incomplete fork Only CGS-DE present No fork 109830 - RH CS CGS VPCGS-P PRPACS VPCGS-A PACS SOS SU-ROS #131 - LH #55 - RH #76 - RH #92 - RH #67 - LH #4 - LH #20 - LH #13 - LH #42 - RH CGS-DE CGS-VE CGS-DE CGS-VE CGS-DE CGS-VE % of hemispheres 100 0 25 50 75 CHIMPANZEE Complete fork pointing downward Complete fork forward-facing * #60 - LH HUMAN CHIMP BABOON MACAQUE CGS-DE CGS-VE b SU-ROS/SOS and CGS-VE/CGS-DE INTERSECTION LOCATION Rostral limit of the genu of the corpus callosum CHIMP #203 - LH Intersection ΔZ Rostral limit of the genu of the corpus callosum 0 10 20 30 * HUMAN CHIMP BABOON MACAQUE Normalized ΔZ value b b c 734045 - LH The RP-PCGS RP-PCGS % of hemispheres 100 0 25 50 75 HUMAN CHIMP BABOON MACAQUE Normalized ΔZ value hemispheres in chimpanzee. By contrast, only 2.5% of the hemispheres of baboon brains display this sulcus and no hemispheres in macaque brains (main effect of species: χ2 = 227.43, df = 3, p-value = 2.2e−16). NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x Posthoc analysis showed that the presence of PACS was higher in Hominoidea (human and chimpanzee) than in Old-world monkeys (baboon and macaque), but that it was similar between human and chimpanzees and also between baboon and macaque. Right panel: PACS characteristics vary across primates: it is a sulcus (blue) in 100% of hemispheres in Hominoidea, it is a spur (green) or a dimple (pink) in Old-world monkeys. It is more frequently a spur than a dimple in baboon, as opposed to macaque. b PRPACS. Left panel: the probability of observing PACS is higher in Hominoidea than in Old-world monkeys but is similar between human and chimpanzee, on one hand, and between baboon and macaque on the other hand. Right panel: PRPACS characteristics vary across primates: it is a sulcus in 100% of hemispheres in Hominoidea, but it is a spur or a dimple in Old-world monkeys. It is more frequently a spur than a dimple in baboon, as opposed to macaque. c VPCGS-P. Left panel: the probability of observing VPCGS-P decreases from human, chimpanzee, baboon, to macaque. Posthoc analysis demonstrated that the presence of the VPCGS-P was higher in human than in non-human primates. Its probability of occurrence was also higher in chimpanzee than in Old-world monkeys. It was, however, similar between macaque and baboon. Right panel: VPCGS-P characteristics vary across primates: it is a sulcus in 100% of hemispheres in Hominoidea, but it is a spur or a dimple in baboon and only a dimple in Macaque. d VPCGS-A. Left panel: The probability of occurrence of VPCGS-A decreases from human, chimpanzee, baboon, to macaque. Right panel: VPCGS-P characteristics vary across primates: it is a sulcus in 100% of hemispheres in Hominoidea, but it is more frequently a spur than a dimple in baboon, as compared with macaque. Statistics: p < 0.05, p < 0.01, *p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle Fig. 4 Presence of vertical sulci in the dorsomedial frontal cortex across primates. Probability of occurrence (±s.e.m NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications 6 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x Finally, we observed at the most rostral part of the frontal cortex, several sulci that are present almost exclusively in Hominoidea. Notably, we observed, exclusively in the human brain, an additional vertical sulcus joining the CGS or PCGS anterior to the rostral limit of the genu of the corpus callosum which was labeled the rostro-perpendicular paracingulate sulcus (RP-PCGS) (Fig. 5c). In addition, we observed a sulcus located ust dorsal to SOS, which joins the lateral surface of the brain but not the CGS or PCGS, called the dorsomedial polar sulcus (DMPS) in 88.75% of hemispheres in human and in 48.75% of hemispheres in chimpanzee. By contrast, only 2.5% of th hemispheres of baboon brains display this sulcus and n hemispheres in macaque brains (main effect of species: χ2 = 227.43, df = 3, p-value = 2.2e−16). A second sulcus, the ven tromedial polar sulcus (VMPS), located in the ventral MFC joining the lateral surface of the brain and sometimes the ROS- was identiﬁed more frequently in human brains (57.5% o hemispheres) than in chimpanzee brains (11.25% of hemisphere and was absent in the baboon and the macaque (main effect o species: χ2 = 126.29, df = 3, p = 2.2e−16) (Fig. 7c). NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications ARTICLE Fig. 5 Morphological characteristics of the junction between the dorsal and ventral MFC. a Rostral end of the CGS/PCGS. In human. The rostral end of CGS is characterized by two sulci forming a fork pointing downward: SU-ROS, pointing downward, and SOS, pointing upward. In chimpanzee. The fork formed by the precursors of SU-ROS and SOS, i.e., CGS-VE and CGS-DE, respectively, is also pointing as in the human brain in the majority of hemispheres. In 17.5% Fig. 5 Morphological characteristics of the junction between the dorsal and ventral MFC. a Rostral end of the CGS/PCGS. In human. The rostral end of CGS is characterized by two sulci forming a fork pointing downward: SU-ROS, pointing downward, and SOS, pointing upward. In chimpanzee. The fork formed by the precursors of SU-ROS and SOS, i.e., CGS-VE and CGS-DE, respectively, is also pointing as in the human brain in the majority of hemispheres. In 17.5% of hemispheres, the fork is forward facing (purple area), and is incomplete (green area) (i.e., SOS is present but SU-ROS is absent) in 1.25% of hemispheres. In baboon. The fork formed by CGS-VE and CGS-DE is also pointing downward in the majority of hemispheres but to a lesser extent than in Hominoidea. In 25% of hemispheres, the fork is forward facing, it is incomplete in 28.75% of hemispheres, and is absent in 6.25% of hemispheres. In macaque. The fork formed by CGS-VE and CGS-DE is displaying the same patterns as in the baboon but in different proportions: the most frequent pattern observed is a fork facing forward, followed by a fork pointing downward. The fork is incomplete in 13.75% of hemispheres and is absent in 17.5% of hemispheres. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. b Location of the SU-ROS/SOS and CGS-DE/CGS-VE intersection. The top diagram shows the location of the intersection between SU-ROS/SOS and CGS-VE/CGS-DE in a typical hemisphere of a chimpanzee. The ΔZ corresponds to the difference between the dorsoventral Z coordinates where the intersection is observed and the dorsoventral Z coordinates where the rostral limit of the genu of the corpus callosum (represented by a purple cross) is located. ΔZ was then normalized for brain size to compare across primate brains. Boxplots displaying the mean (±s.e.m.) normalized ΔZ across individuals are presented in the bottom diagram. Note that black dots represent outliers and horizontal lines the mean of each distribution. ARTICLE c Probability of occurrence (±s.e.m.) of RP-PCGS. The RP-PCGS is human-speciﬁc since it is not present in non-human primates. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle observed is a fork facing forward, followed by a fork pointing downward. The fork is incomplete in 13.75% of hemispheres and is absent in 17.5% of hemispheres. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. b Location of the SU-ROS/SOS and CGS-DE/CGS-VE intersection. The top diagram shows the location of the intersection between SU-ROS/SOS and CGS-VE/CGS-DE in a typical hemisphere of a chimpanzee. The ΔZ corresponds to the difference between the dorsoventral Z coordinates where the intersection is observed and the dorsoventral Z coordinates where the rostral limit of the genu of the corpus callosum (represented by a purple cross) is located. ΔZ was then normalized for brain size to compare across primate brains. Boxplots displaying the mean (±s.e.m.) normalized ΔZ across individuals are presented in the bottom diagram. Note that black dots represent outliers and horizontal lines the mean of each distribution. c Probability of occurrence (±s.e.m.) of RP-PCGS. The RP-PCGS is human-speciﬁc since it is not present in non-human primates. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle ROS-S ROS I ns HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 0 25 50 75 a b #92 - RH #8 - LH ROS-S ROS-I ASOS ns ns ns ns ns HUMAN CHIMP BABOON MACAQUE #30 - LH HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 0 25 50 75 HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 25 50 75 0 HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 25 50 75 0 102513 - RH ROS-I ROS-S ASOS ns ns SU-ROS SOS * a b c lci present in the ventral MFC across primates. The location of the ROS-S, ROS-I, and ASOS sulci are displayed in human, chimpanzee, baboon, aque brains in the right panels. a The probability of occurrence (±s.e.m.) of ROS-S is similar in human and non-human primates. b The probability ence (±s.e.m.) of ROS-I is comparable in human and chimpanzee and is almost absent in Old-world monkeys. c The probability of occurrence is equal in human and chimpanzee. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x A second sulcus, the ven- tromedial polar sulcus (VMPS), located in the ventral MFC, joining the lateral surface of the brain and sometimes the ROS-I, was identiﬁed more frequently in human brains (57.5% of hemispheres) than in chimpanzee brains (11.25% of hemispheres) and was absent in the baboon and the macaque (main effect of species: χ2 = 126.29, df = 3, p = 2.2e−16) (Fig. 7c). Figure 8 Finally, we observed at the most rostral part of the frontal cortex, several sulci that are present almost exclusively in Hominoidea. Notably, we observed, exclusively in the human brain, an additional vertical sulcus joining the CGS or PCGS anterior to the rostral limit of the genu of the corpus callosum which was labeled the rostro-perpendicular paracingulate sulcus (RP-PCGS) (Fig. 5c). In addition, we observed a sulcus located just dorsal to SOS, which joins the lateral surface of the brain but not the CGS or PCGS, called the dorsomedial polar sulcus (DMPS) in 88.75% of hemispheres in human and in 48.75% of 7 Discussion l f Analysis of sulcal brain morphology has proven to be a powerful method for a better understanding of the evolution of the MFC in primates. Although gyriﬁcation correlates with brain volume1,35, and therefore with the expansion of primate neocortex, the pre- sent results did not reveal random or homogeneous changes in sulcal morphology in the MFC across the four primate species examined (e.g., differences in the ACC/vmPFC but no differences in the MCC/medial premotor cortex). The observed differences were in speciﬁc regions that have expanded more during evolu- tion36. Here, we report that chimpanzees (P. troglodytes) possess a PCGS, a feature previously thought to be unique to the human brain (H. sapiens)26–28. Two differences must, however, be emphasized: (1) the probability of occurrence of a PCGS is lower in the chimpanzee than in the human brain, and (2) the presence of a PCGS is largely lateralized in the left hemisphere in the human brain, whereas it is equally present in both hemispheres in the chimpanzee (Fig. 1e). The leftward asymmetry observed in the human brain appears to be a human trait under the inﬂuence of genetic factors and the in-womb environment14. Although its functional signiﬁcance is not fully understood, it has been shown that it correlates with the involvement of the left cingulate cortex in language tasks in right-handed subjects37. Notably, only in the chimpanzee brain, the probability of occurrence of an ILS is higher in the left than in the right hemisphere (Fig. 2a). Since the ILS is observed only in hemispheres displaying no PCGS in human and chimpanzee brains (Fig. 2b), one can hypothesize that the lateralized presence of an ILS in the left hemisphere in chimpanzee brains may constitute an evolutionary reﬂection of the beginnings of the sulcal organization observed in the human brain, i.e., a PCGS lateralized in the left hemisphere and an ILS not lateralized. Furthermore, the present data demonstrate that, in Old-world monkeys, in contrast to the ACC/vmPFC region, the organization of the MFC posterior to the genu of the corpus callosum (which corresponds to the MCC, where the medial motor and premotor areas lie), has all the basic features of the human sulcal organi- zation and reaches a similar organization across Hominoidea. Speciﬁcally, Old-world monkeys display precursors—in the form of spurs or dimples—of the four vertical sulci of this region: PACS, PRPACS, VPCGS-P, and VPCGS-A. NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x Our results are in line with a recent study showing that the ACC/vmPFC is a region of high structural variability within the human brain and the macaque brain41. The present study provides an explanation of the nature of this variability: Since we observed reduced variability in the ACC/vmPFC of human and chimpanzee brains compared to Old-world monkeys, we suggest that a facing downward SU-ROS/CGS-VE morphol- ogy might be associated with a trait that is conferring an evolu- tionary advantage, and was therefore selected. in comparison with more distantly related primates. There is no evidence from cytoarchitectonic38,39 and neuroimaging24,40 stu- dies that these changes are associated with new cortical areas, suggesting instead differential expansion of (1) the rostral MFC (area 9) and mFPC (area 10), two brain regions that have been considered important for high-order socio-cognitive processing23,32,33 and (2) the vmPFC implicated in value-based decisions30,31. Our results are in line with a recent study showing that the ACC/vmPFC is a region of high structural variability within the human brain and the macaque brain41. The present study provides an explanation of the nature of this variability: Since we observed reduced variability in the ACC/vmPFC of human and chimpanzee brains compared to Old-world monkeys, we suggest that a facing downward SU-ROS/CGS-VE morphol- ogy might be associated with a trait that is conferring an evolu- tionary advantage, and was therefore selected. ARTICLE It is decreased in Old-world monkeys but equally present in baboon and macaque. Statistics: p < 0.05, 1, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle HUMAN 102513 - RH ROS-I ROS-S ASOS SU-ROS SOS ROS-I ns ns * HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 25 50 75 0 b #8 - LH CHIMP ASOS ns ns HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 25 50 75 0 ns ns *** c MACAQUE #30 - LH MACAQUE Fig. 6 Sulci present in the ventral MFC across primates. The location of the ROS-S, ROS-I, and ASOS sulci are displayed in human, chimpanzee, baboon, and macaque brains in the right panels. a The probability of occurrence (±s.e.m.) of ROS-S is similar in human and non-human primates. b The probability of occurrence (±s.e.m.) of ROS-I is comparable in human and chimpanzee and is almost absent in Old-world monkeys. c The probability of occurrence of ASOS is equal in human and chimpanzee. It is decreased in Old-world monkeys but equally present in baboon and macaque. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications 8 8 NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 0 25 50 75 DMPS VMPS * * ns * ns HUMAN CHIMP DMPS VMPS 109830 - RH 109830 - RH # 8 - LH DMPS ns a b HUMAN CHIMP BABOON MACAQUE Fig. 7 Probability of occurrence of DMPS and VMPS across primates. The location of the DMPS and VMPS sulci are displayed in human and chimpanzee brains in the right panels. The probability of occurrence of both DMPS (a) and VMPS (b) is higher in human than in chimpanzee but is very low in Old- world monkeys. Statistics: p < 0.05, p < 0.01, p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle VMPS * ns ns b HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE % of hemispheres 100 0 25 50 75 DMPS * * ns a VMPS *** ns HUMAN CHIMP DMPS VMPS 109830 - RH 109830 - RH # 8 - LH DMPS ns b HUMAN CHIMP BABOON MACAQUE s primates. The location of the DMPS and VMPS sulci are displayed in human and chimpanzee both DMPS (a) and VMPS (b) is higher in human than in chimpanzee but is very low in Old- 01, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle b a HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE HUMAN CHIMP BABOON MACAQUE CHIMP Fig. 7 Probability of occurrence of DMPS and VMPS across primates. The location of the DMPS and VMPS sulci are displayed in human and chimpanzee brains in the right panels. The probability of occurrence of both DMPS (a) and VMPS (b) is higher in human than in chimpanzee but is very low in Old- world monkeys. Statistics: p < 0.05, p < 0.01, *p < 0.001, ns non-signiﬁcant. Source data are provided as a Source Data ﬁle summarizes the major changes occurring between macaque, baboon, chimpanzee and human brains. summarizes the major changes occurring between macaque, baboon, chimpanzee and human brains. in comparison with more distantly related primates. There is no evidence from cytoarchitectonic38,39 and neuroimaging24,40 stu- dies that these changes are associated with new cortical areas, suggesting instead differential expansion of (1) the rostral MFC (area 9) and mFPC (area 10), two brain regions that have been considered important for high-order socio-cognitive processing23,32,33 and (2) the vmPFC implicated in value-based decisions30,31. NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications Discussion l f Schematic sulcal organization of the MFC in each species is represented in the respective stereotaxic space. Main changes from one species to another are indicated. The ΔZ corresponds to the difference between the dorso-ventral coordinates where the intersection is observed and the dorso-ventral Z coordinates where the rostral limit of the genu of the corpus callosum is locate The difference ΔZ is decreased in the human compared to non-human primates, showing that the intersection between the CGS with the SU-ROS/CGS-V and with the supra-orbital sulcus SOS/CGS-DE is pushed down from non-human to human primates Fig. 8 Summary of sulcal changes occurring from macaque to human. Schematic sulcal organization of the MFC in each species is represented in their respective stereotaxic space. Main changes from one species to another are indicated. The ΔZ corresponds to the difference between the dorso-ventral Z coordinates where the intersection is observed and the dorso-ventral Z coordinates where the rostral limit of the genu of the corpus callosum is located. The difference ΔZ is decreased in the human compared to non-human primates, showing that the intersection between the CGS with the SU-ROS/CGS-VE and with the supra-orbital sulcus SOS/CGS-DE is pushed down from non-human to human primates human brain34 and that the macaque default brain network is comparable to that observed in the human brain42,43. the RP-PCGS reﬂects the expansion of medial area 9, a region thought to be important for social and metacognitive processes23,32,33. p Collectively, the results presented here provide (1) strong evi- dence for a comparable organization of the MCC across primates and (2) a clear framework to test hypotheses regarding the rela- tion between the vertical sulci/spurs/dimples, cytoarchitectonic areas, and functions. For example, there is some evidence that PACS may be a sulcus occurring at the border between medial area 4 and medial area 6 in several primate species: human44–46, orangutan45,47, and macaque48. Finally, as no new areas have been identiﬁed in the human MFC in neuroimaging24,40 and cytoarchitectonic 38,39 studies, the emergence of a new sulcus in the human MFC might reﬂect the relative expansion of this region compared to other primates. For instance, we suggest that Future studies may test for relationships between the vertical sulci in the dorsal MFC and cytoarchitectonic areas. Discussion l f This result provides additional evidence that the MCC is comparable both anatomi- cally and functionally across macaque and human brains (see Procyk et al.25 for a detailed anatomo-functional comparison), contrary to what had been proposed before26–28. Importantly, we show that these sulci refer to speciﬁc and highly-reliable anato- mical features, and we provide a simple framework within which they can be identiﬁed: PACS is located at the level of the rostral limit of the pons, PRPACS at the level of the anterior commis- sure, VPCGS-P and VPCGS-A at the level of the caudal and rostral limits of the genu of the corpus callosum, respectively. These landmarks could be used to guide interpretation of func- tional neuroimaging data and, also, comparative functional stu- dies across primates. Another major difference between the primate species exam- ined was the organization of the MFC rostral to the genu of the corpus callosum. Differences were associated with (1) a sulcus only observed in the human brain, the RP-PCGS, (2) the pro- gressive emergence of the DMPS and VMPS, and (3) the SU- ROS/SOS intersection that is displaced downwards from the level of the rostral limit of the genu of the corpus callosum in humans The present results also show that the ventral MFC is highly conserved from macaque to human brains. ROS-S, which is in the center of this region, is a deep sulcus in all primate species. Our results are supported by recent studies showing that the main vmPFC node of the default mode network lies in this sulcus in the 9 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x MACAQUE HUMAN CHIMP BABOON Human MNI standard Y coordinate (mm) 20 –30 10 70 0 –50 –20 –10 30 50 40 60 –40 20 –30 10 40 0 –40 –20 –10 30 Chimpanzee standard Y coordinate (mm) 20 –30 10 0 –20 –10 30 Baboon standard Y coordinate (mm) 20 10 0 –20 –10 30 Macaque standard Y coordinate (mm) Expansion of area 9 RP-PCGS appears. SOS/SU-ROS intersection pushed down. PCGS appears PACS, PRPACS, VPCGS-A and VPCGS-P spurs become sulci. DMPS and VMPS appear. Full setup of SOS and SU-ROS precursors. PACS, PRPACS, VPCGS-P and VPCGS-A dimples become spurs. Precursors of SOS (CGS-DE) and SU-ROS (CGS-VE) appear. Expansion of area 10 ΔZ = 0 ΔZ > 0 ΔZ > 0 ΔZ > 0 Fig. 8 Summary of sulcal changes occurring from macaque to human. Discussion l f on the consistent location of the vertical sulci in the dorsal MFC, one can hypothesize that, in both the human and macaque brains, (1) the VPCGS-A, which is located at the rostral limit of the genu of the corpus callosum and is better conserved than the VPCGS-P across primates, may be a reliable landmark to distinguish the ACC from the aMCC; (2) the PRPACS, which is located at the level of the anterior commissure and highly conserved across primates, may correspond to the limit between aMCC and pMCC; (3) the PACS, which is located at the level of the rostral limit of the pons, might be the limit between pMCC and PCC. Although there is no currently available information about the four-region model in the MFC of the baboon (P. papio) and chimpanzee (P. troglodytes), one can infer, based on our demonstration that the vertical sulci examined are conserved and hold the same location as in the human and macaque brains, that these sulci may limit the same anatomo-functional regions. This hypothesis is displayed in Supplementary Fig. 3 and future studies can test directly this hypothesis. It is important to note, however, that the precise relation of sulci to cytoarchitectonic areas remains controversial49 and requires considerably more investi- gation in appropriately sectioned brains, i.e., in histological sec- tions perpendicular to the orientation of a given sulcus, as originally pointed out by Economo and Koskinas50. g y p y Besides sulci, we propose that other anatomical landmarks could help guide interpretation of the functional organization of the MFC. This is a relevant question as New-World monkeys include species with gyriﬁed brains (e.g., Capuchin and Squirrel monkeys) and species with lissencephalic brains (e.g., the mar- moset)51,52. Importantly, it has been shown that the relative position and cytoarchitecture of homologous areas in New versus Old-world monkeys are similar53,54, despite a different brain size51. On the basis of the framework provided by the present study, it would be reasonable to hypothesize that homologous cytoarchitectonic and functional regions in the MFC in New World monkeys may be identiﬁed using landmarks common to all primates: the PCC/MCC limit might be located at the level of the rostral part of the pons (where PACS is found in Old-world monkeys, apes, and human). Discussion l f Based on cytoarchitecture, Vogt has proposed a hierarchical four-region organization of the cingulate cortex28 both in human and macaque brains emphasizing the distinction between the ACC, the anterior and the posterior MCC (aMCC and pMCC), the posterior cingulate cortex (PCC), and the retrosplenial cortex. We superimposed this model on the present sulcal organization scheme in the human and macaque brains to examine whether the vertical sulci may have signiﬁcant meaning in terms of the anatomo-functional organization of the cingulate cortex. Based NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications 10 ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x expansion in motor and cognitive processes. Importantly, the brain size difference across primate species does not explain by itself the pattern of sulcal organization observed in the present study as we observed (1) different patterns of sulcal morphology within each species (controlling therefore for brain size), and (2) a consistent antero-posterior organization of the vertical sulci and their precursors in the dorsal MFC in the four species (i.e., in different brains of different sizes). Studies assessing within species inter-individual variability in sulcal morphology and behavioral performance provide evidence of the existence of relationships between structure and function. Such evidence exists for the human brain, e.g., refs. 57,58. and the chimpanzee brain, e.g., ref. 59. Indeed, Hopkins et al.59 found that male chimpanzees producing attention getting sounds showed a larger leftward asymmetry in the depth of central and ventral portions of the central sulcus compared to males that produce less attention getting sounds with females showing the opposite pattern. We also recently showed that the presence of a PCGS and an intra- limbic sulcus, particularly in the left hemisphere, was associated respectively with the production and use of attention-getting sounds by chimpanzees as well as rightward handedness (Hop- kins et al., in preparation). It is therefore reasonable to think that interindividual variability in sulcal morphology in macaque and baboon may be associated with variability in behavioral performance. Discussion l f The MCC should expand from this location to the anterior part of the genu of the corpus callosum, i.e., where the VPCGS-A is found and which corresponds to the limit between MCC and ACC. As in humans, we also predict the border between supplementary motor area and pre- supplementary motor area to be at the level of the anterior commissure in standardized stereotaxic space, i.e., at y = 0 in Talairach and Tournoux coordinates55 or y = +3 in the latest asymmetrical MNI brain56. p The present study provides critical new evidence in the context of a new comprehensive framework regarding prefrontal cortical evolution. On one hand, these results are in agreement with previous comparative connectomic studies24,40 and show a con- served organization of the MFC, suggesting the existence of a precursor architecture in the last common ancestor to human, apes, and Old-world monkeys. On the other hand, the appearance in human and ape brains of the PCGS, and the relative expansion of the rostral and dorsal part of the MFC in the human brain (emergence of the RP-PCGS and the more ventral position of the intersection between SUROS/CGS-VE, SOS/CGS-DE and CGS) (summarized in Fig. 8) might reﬂect the evolution of higher-order mentalizing processes in Hominoidea60,61 from a rudimentary form in Old-world monkeys62,63. y To conclude, the present article, by throwing light on how the MFC has evolved, paves the way to future studies assessing relationships between sulcal morphology, behavioral perfor- mance, and functional organization within and across primate species. We anticipate that this framework will be used to examine the relationships between sulcal morphology, cytoarch- itectonic areal distribution, connectivity, and function, within the studied species and also across the primate order. y Manual labeling of the sulci following visual inspection in the various species in the present study may appear as a limitation. Yet, a similar strategy has been recently employed and conﬁrmed the organization of these sulci in the human brain34. Further- more, such precise analysis of sulci in multiple species represents an important step forward towards the development of efﬁcient algorithms for automatic labeling, which are currently not available. To characterize further the evolution of sulcal patterns in primates, future studies will need to consider the assessment of more species. Discussion l f But, as no endocast exists for the MFC, it is important to note that only analysis on living species could be conducted to understand how structures on the medial surface of the brain have evolved. NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications ARTICLE NATURE COMMUNICATIONS \| https://doi.org/10.1038/s41467-019-11347-x Structure, Function, and Heritability”). All subjects provided written informed consent on forms approved by the Institutional Review Board of Washington University in St. Louis. In addition, the present study received approval (no. 15- 213) from the Ethics Committee of Inserm (IORG0003254, FWA00005831) and from the Institutional Review Board (IRB00003888) of the French Institute of Medical Research and Health. normalized on the basis of the dorso-ventral extent of the brains normalized in their respective standard space (see “Methods”) at the level of the rostral limit of the genu of the corpus callosum across primates (i.e., 100, 50, 30, and 24 mm, respectively, in human, chimpanzee, baboon, and macaque brains). In both analyses, C.A. labeled the sulci. The labeling was performed on three separate occasions, and the few remaining inconsistencies in labeling were discussed with J.S. and M.P. Regarding the human data, validation of the nomenclature used was obtained in an independent experiment in which the naming of the sulci was performed by another expert34. normalized on the basis of the dorso-ventral extent of the brains normalized in their respective standard space (see “Methods”) at the level of the rostral limit of the genu of the corpus callosum across primates (i.e., 100, 50, 30, and 24 mm, respectively, in human, chimpanzee, baboon, and macaque brains). In both analyses, C.A. labeled the sulci. The labeling was performed on three separate occasions, and the few remaining inconsistencies in labeling were discussed with J.S. and M.P. Regarding the human data, validation of the nomenclature used was obtained in an independent experiment in which the naming of the sulci was performed by another expert34. Non-human primates. High-resolution anatomical scans of chimpanzee (P. tro- glodytes) and baboon (P. papio) brains were obtained from the laboratories of Dr. William Hopkins and Dr. Adrien Meguerditchian, respectively. High-resolution anatomical scans of Macaque (M. mulatta) brains were obtained from the laboratories of Drs E. Procyk and C. Amiez, W. Hopkins, J. Sallet, F. Hadj-Bou- ziane, and S. Ben Hamed. These data are now available in the PRIMatE Data Exchange (PRIME-DE) database [http://fcon_1000.projects.nitrc.org/indi/indi- PRIME.html]65. Note that no new data were collected speciﬁcally for the purpose of the present study. Data collected initially for studies on rhesus monkeys (M. Mulatta) and baboons (P. ARTICLE papio) were conducted under local ethics agreements (licenses from the United Kingdom (UK) Home Ofﬁce; Provence and Lyon ethics committees) and in accordance with The Animals (Scientiﬁc Procedures) Act 1986 and with the European Union guidelines (EU Directive 2010/63/EU). Chimpanzee (P. troglodytes) data collection was approved by the Institutional Animal Care and Use Committees at YNPRC and UTMDACC and also followed the guidelines of the Institute of Medicine on the use of chimpanzee in research. Statistical analysis. We tested the inﬂuence of species status on the probability of a sulcus to be present with logistic regressions. In the statistical models, “species” (human, chimpanzee, baboon, macaque) was the independent variable and “Pre- sence” (0, 1) of a sulcus was the dependent variable. The lateralization of PCGS and ILS in human versus chimpanzee was assessed separately in each species using a binomial logistic regression GLMs with hemisphere (left versus right) as inde- pendent variable. To assess whether the probability of observing the four vertical sulci (i.e., PACS, PREPACS, VPCGS-P, and VPCGS-A) in the dorsal MFC was similar or different in each species, we performed binomial logistic regression GLMs in each species with these sulci (PACS, PREPACS, VPCGS-P, and VPCGS- A) as independent variable. ANOVA Chi-square tests and post-hoc Tukey tests were then applied. Note that, in the cases where only one value was observed in a speciﬁc variable (e.g., the PCGS is absent in baboon and macaque and therefore the “presence” value is 0 for all subjects), the binomial logistic regression GLM was ﬁtted using an adjusted-score approach to bias reduction (using the brglm package; https://cran.r-project.org/web/packages/brglm/brglm.pdf). Neuroimaging data analysis. Human and macaque brains were normalized in the Human (http://www.bic.mni.mcgill.ca/ServicesAtlases/HomePage) and Macaque66 MNI steretotaxic coordinate systems, respectively. Chimpanzee brains were nor- malized in the Chimpanzee standard brain developed by Dr. W. Hopkins67 [http:// www.chimpanzeebrain.org/]. Baboon brains were normalized in the Baboon standard brain developed by Dr. A. Meguerditchian68 [http://www.nitrc.org/ projects/haiko89/]. Note that normalization of all primate brains consisted in linear registrations, which has the great advantage of allowing within-species compar- isons between brains without altering relationships between sulci and gyri. It is, therefore, unlikely that such processing inﬂuences commonality and divergence of sulcal organization observed between species. Normalization of primate brains was performed with SPM12 [https://www.ﬁl.ion.ucl.ac.uk/spm/software/spm12/]. Received: 14 January 2019 Accepted: 9 July 2019 Received: 14 January 2019 Accepted: 9 July 2019 (a) To assess whether PACS, PRPACS, VPCGS-P, and VPCGS-A were located at the level of, respectively, the most rostral limit of the pons (landmark 1), the anterior commissure (landmark 2), the caudal (landmark 3), and the rostral (landmark 4) limit of the genu of the corpus callosum, we calculated the difference between the Y value of the intersection between the CGS or the PCGS (if present) and the PACS, PRPACS, VPCGS-P, and VPCGS-A and the Y value of landmarks 1, 2, 3, and 4, respectively. This difference was calculated in all four species on the normalized T1 data of these species. This difference was then normalized to take into account the different antero-posterior extent of the brains of the four species, the antero-posterior extent of the human, chimpanzee, baboon, and macaque brains being respectively 175, 110, 85, and 60 mm. The normalization performed within species was obtained by dividing the Y coordinate of the sulcus of interest, measured on brains registered linearly in the species-speciﬁc standard space, by the antero-posterior extent of the standard brain. ARTICLE In addition, we tested whether the relative distributions of Z positions of the fork formed by SU-ROS/CGS-VE, SOS/CGS-DE, and the CGS or PCGS (when present), relative to the rostral limit of the genu of the corpus callosum were similar across species using standard generalized linear models in which the independent variable was “species” (human, chimpanzee, baboon, macaque) and the dependent variable was the normalized Z relative position of this fork. This displacement was normalized across primates on the basis of the dorso-ventral extent of the brains normalized in their respective standard space at the level of the rostral limit of the genu of the corpus callosum (i.e., 100, 50, 30, and 24 mm, respectively, in human, chimpanzee, baboon, and macaque brains). The normalization performed within species was obtained by dividing the Z coordinate of the intersection between the fork and the CGS or PCGS, measured on brains registered linearly in the species- speciﬁc standard space, by the dorso-ventral extent of the standard brain. All data are presented in the Source Data ﬁle. Two levels of analysis were performed: (1) A large qualitative analysis in the whole set of data: 197 human, 225 chimpanzee, 88 baboon, and 80 rhesus monkey brains. This analysis was performed to examine whether the PCGS and the ILS were human-speciﬁc features. Towards that goal, each MRI scan was visually inspected to assess the presence or absence of these sulci in either hemisphere of each normalized brain for each species assessed. (1) A large qualitative analysis in the whole set of data: 197 human, 225 chimpanzee, 88 baboon, and 80 rhesus monkey brains. This analysis was performed to examine whether the PCGS and the ILS were human-speciﬁc features. Towards that goal, each MRI scan was visually inspected to assess the presence or absence of these sulci in either hemisphere of each normalized brain for each species assessed. All statistics were performed with R software, R Development Core Team69 under R-Studio70. Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. (2) A restricted qualitative/quantitative analysis of all sulci of the MFC in both hemispheres of a subset of 40 brains of each species. (2) A restricted qualitative/quantitative analysis of all sulci of the MFC in both hemispheres of a subset of 40 brains of each species. References 1. Zilles, K., Palomero-Gallagher, N. & Amunts, K. Development of cortical folding during evolution and ontogeny. Trends Neurosci. 36, 275–284 (2013). d h d l f h d l f l 1. Zilles, K., Palomero-Gallagher, N. & Amunts, K. Development of cortical folding during evolution and ontogeny. Trends Neurosci. 36, 275–284 (2013). 2. Toro, R. & Burnod, Y. A morphogenetic model for the development of cortical convolutions. Cereb. Cortex 15, 1900–1913 (2005). 2. Toro, R. & Burnod, Y. A morphogenetic model for the development of cortical convolutions. Cereb. Cortex 15, 1900–1913 (2005). 3. Van Essen, D. C. A tension-based theory of morphogenesis and compact wiring in the central nervous system. Nature 385, 313–318 (1997). 4. Rakic, P. Speciﬁcation of cerebral cortical areas. Science 241, 170–176 (1988). p ( ) 5. Tallinen, T., Chung, J. Y., Biggins, J. S. & Mahadevan, L. Gyriﬁcation from constrained cortical expansion. Proc. Natl. Acad. Sci. USA 111, 12667–12672 (2014). 5. Tallinen, T., Chung, J. Y., Biggins, J. S. & Mahadevan, L. Gyriﬁcation from constrained cortical expansion. Proc. Natl. Acad. Sci. USA 111, 12667–12672 (2014). (b) To assess the relative location of the intersection of the fork formed by SU- ROS/CGS-VE and SOS/CGS-DE with the CGS or PCGS across primates, we calculated the % of displacement of this intersection from the rostral limit of the genu of the corpus callosum. Towards that goal, we measured the difference between the Z value of this intersection and the Z value of the rostral limit of the genu of the corpus callosum. To compare primate brains, this difference was then (b) To assess the relative location of the intersection of the fork formed by SU- ROS/CGS-VE and SOS/CGS-DE with the CGS or PCGS across primates, we calculated the % of displacement of this intersection from the rostral limit of the genu of the corpus callosum. Towards that goal, we measured the difference between the Z value of this intersection and the Z value of the rostral limit of the genu of the corpus callosum. To compare primate brains, this difference was then 6. Harris, H. Enzyme polymorphisms in man. Proc. R. Soc. Lond. B Biol. Sci. 164, 298–310 (1966). 6. Harris, H. Enzyme polymorphisms in man. Proc. R. Soc. Lond. B Biol. Sci. 164, 298–310 (1966). 7. Przeworski, M., Hudson, R. R. & Di Rienzo, A. Adjusting the focus on human variation. Trends Genet. 16, 296–302 (2000). 7. Methods N i i Neuroimaging T1 anatomical data of 197 human (H. sapiens), 225 chimpanzee (P. troglodytes), 88 baboon (P. papio), and 80 rhesus monkeys (M. mulatta) brains were analyzed. Human subjects. High-resolution anatomical scans of the human brain were obtained from the Human Connectome Project database [http://www. humanconnectome.org/]. Only data from subjects displaying no family relation- ships were analyzed. The participants in the HCP study were recruited from the Missouri Family and Twin Registry that includes individuals born in Missouri64. Acquisition parameters of T1 anatomical scans are the following: whole head, 0.7 mm3 isotropic resolution, TR = 2.4 s, TE = 2.14 ms, ﬂip angle = 8° (more details can be found at [https://humanconnectome.org/storage/app/media/ documentation/s1200/HCP_S1200_Release_Appendix_I.pdf]). The full set of inclusion and exclusion criteria is detailed elsewhere64. Brieﬂy, the HCP subjects are healthy individuals free from major psychiatric or neurological illnesses. They are drawn from ongoing longitudinal studies64, where they had received extensive assessments, including the history of drug use, and emotional and behavioral problems. The experiments were performed in accordance with relevant guidelines and regulations and all experimental protocols were approved by the Institutional Review Board (IRB) (IRB # 201204036; Title: “Mapping the Human Connectome: Altogether comparative neuroanatomy studies show a rela- tively well conserved anatomo-functional organization of the MFC in primates24,38,40, which suggests that the precursor of the human brain was already present in the last ancestor to humans, apes, and Old-world monkeys. Differences in sulcal pattern morphology as observed in the present study could be related to the expansion of cortical areas, which in turn could be related to 11 Data availability First, this analysis consisted in assessing the characteristics of the vertical sulci in the MFC emerging from the CGS or the PCGS in all brains across species. Towards that goal, the sulci were assigned in one of 3 categories based on our qualitative observations (Supplementary Fig. 1): sulcus (a real sulcus—long and deep—can be observed), spur (a precursor sulcus, i.e., not long enough to be considered a sulcus), and dimple (locations where a slight indentation indicates a dimple of the CGS or PCGS). The source data underlying Figs. 1e, 2a, b, 3b–e, 4a–d, 5a–c, 6a–c, 7a, b, and Supplementary Fig. 2a, b are provided as a Source Data ﬁle. Human, macaque, chimpanzee, and baboon anatomical scans are available, respectively, from the Human Connectome Project database [http://www.humanconnectome.org/], the PRIMatE Data Exchange (PRIME-DE) database [https://fcon_1000.projects.nitrc.org/indi/indiPRIME. html], from Dr. W. Hopkins [http://www.chimpanzeebrain.org/], and from Dr. A. Meguerditchian [http://www.nitrc.org/projects/haiko89/]. A reporting summary for this Article is available as a Supplementary Information ﬁle. Second, we identiﬁed all sulci in the MFC and assessed any relationships that may exist between these sulci and certain ﬁxed anatomical landmarks across the species examined, such as the rostral limit of the pons, anterior commissure, caudal and rostral limits of the genu of the corpus callosum. The relationships between the location of a given sulcus and a particular anatomical landmark across species were examined as follows: ARTICLE 14. Amiez, C., Wilson, C. R. E. & Procyk, E. 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Trends Genet. 16, 296–302 (2000). 12 NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunicatio Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. Additional information 69. R Development Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2008). 69. R Development Core Team. R: A Language and Environment for Statistical Computing (R Foundation for Statistical Computing, 2008). Supplementary Information accompanies this paper at https://doi.org/10.1038/s41467- 019-11347-x. 70. RStudio Team. RStudio: Integrated Development for R (RStudio Inc., 2016). 70. RStudio Team. RStudio: Integrated Development for R (RStudio Inc., 2016). ARTICLE Regional and hemispheric variation in cortical thickness in chimpanzees (Pan troglodytes). J. Neurosci. 33, 5241–5248 (2013). 37. Toga, A. W. & Thompson, P. M. Mapping brain asymmetry. Nat. Rev. Neurosci. 4, 37–48 (2003). 38. Petrides, M. & Pandya, D. N. Comparative cytoarchitectonic analysis of the human and the macaque frontal cortex. in Handbook of Neuropsychology (eds Boller, F. & Grafman, J.) 17–58 (Amsterdam 1994). 68. Love, S. A. et al. The average baboon brain: MRI templates and tissue probability maps from 89 individuals. Neuroimage 132, 526–533 (2016). 13 NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications Acknowledgements Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ Reprints and permission information is available online at http://npg.nature.com/ reprintsandpermissions/ g This work was supported by the Human Frontier Science Program (RGP0044/2014). C. A. received funding from the French National Research Agency (ANR-18-CE32-0012- 01). J.S. was supported by a Sir Henry Dale Wellcome Trust Fellowship (105651/Z/14/Z). The Wellcome Centre for Integrative Neuroimaging is supported by core funding from the Wellcome Trust (203139/Z/16/Z). W.D.H. is supported by NIH grants NS-042867, NS-073134, and NS-092988. A.M. has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program Grant Agreement No. 716931 (716931 – GESTIMAGE – ERC-2016-STG), from the French “Agence Nationale de la Recherche” (ANR-12-PDOC-0014-01, LangPrimate Project), as well as from grants ANR-16-CONV-0002 (ILCB), ANR-11-LABX-0036 (BLRI), and ANR-11-IDEX-0001-02 (AMIDEX). F.H.-B. received funding from the French National Research Agency (ANR-15-CE37-0003). E.P. was supported by the Medical Research Foundation (FRM), Neurodis Foundation, and the Labex CORTEX ANR-11-LABX-0042 of Université de Lyon. M.P. was supported by the Canadian Institutes of Health Research (CIHR) Foundation grant FDN-143212. E.P., C.A., F.H.-B., and S.B.H. are employed by the Centre National de la Recherche Scientiﬁque. The authors thank K. Knoblauch for helpful comments on statistical analysis. The authors thank Delphine Autran-Clavagnier for data acquisition in macaques. Peer review information: Nature Communications thanks Danilo Bzdok, Christopher Petkov and Michel Thiebaut de Schotten for their contribution to the peer review of this work. Peer reviewer reports are available. Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional afﬁliations. Author contributions C.A. organized the project, coordinated the consortium to gather the dataset, and ana- lyzed the data. C.A., J.S. and M.P. interpreted data and wrote the article. W.D.H. pro- vided chimpanzee and macaque T1 data; A.M. provided baboon T1 data; F.H.-B., S.B.H., J.S., W.D.H., C.R.E.W., C.A. and E.P. provided macaque T1 data. © The Author(s) 2019 © The Author(s) 2019 NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunications 14 NATURE COMMUNICATIONS \| (2019) 10:3437 \| https://doi.org/10.1038/s41467-019-11347-x \| www.nature.com/naturecommunicatio
https://openalex.org/W4230764084	https://hal.science/hal-01606195/file/2016_Fernandez_Phytopathology_1.pdf	English	null	Abstracts of Presentations at the 2016 APS Annual Meeting	Phytopathology	2,016	cc-by-sa	263,947	To cite this version: D. Fernandez, M. Grossi de Sa, I. Mezzalira, M. Beneventi, D. Amora, et al.. Functional anal- ysis of root-knot nematode (Meloidogyne javanica) virulence genes in rice. Annual Meeting of the American-Phytopathological-Society (APS), Jul 2016, Tampa (Floride), United States. 163 p., 10.1094/PHYTO-106-12-S4.1. hal-01606195 Functional analysis of root-knot nematode (Meloidogyne javanica) virulence genes in rice D. Fernandez, M. Grossi de Sa, I. Mezzalira, M. Beneventi, D. Amora, H. Baimey, A. Petitot, Janice de Almeida, E. Saliba Albuquerque 2016 APS Annual Meeting Abstracts of Presentations Abstracts submitted for presentation at the APS Annual Meeting in Tampa, Florida, U.S.A., July 30–August 3, 2016. Recommended format for citing annual meeting abstracts, using the first abstract below as an example, is as follows: Harper, S., Cowell, S., and Dawson, W. 2016. Factors that influence the structure of Citrus tristeza virus populations. (Abstr.) Phytopathology 106:S4.1. http://dx.doi.org/10.1094/PHYTO-106-12-S4.1 The abstracts are published for citation purposes. They were not reviewed by the Phytopathology Editorial Board and were not edited by the APS editorial staff. Please send questions or comments to aps@scisoc.org. htt //d d i /10 1094/PHYTO 106 12 S4 1 The abstracts are published for citation purposes. They were not reviewed by the Phytopathology Editorial Board and were not edited by the APS editorial staff. Please send questions or comments to aps@scisoc.org. http://dx.doi.org/10.1094/PHYTO-106-12-S4.1 © 2016 h A i h h l i l S i http://dx.doi.org/10.1094/PHYTO-106-12-S4.1 © 2016 The American Phytopathological Society © 2016 The American Phytopathological Society Factors that influence the structure of Citrus tristeza virus populations S. HARPER (1), S. Cowell (2), W. Dawson (2) (1) Department of Plant Pathology, University of Florida, U.S.A.; (2) Department of Plant Path Factors that influence the structure of Citrus tristeza virus populations S. HARPER (1), S. Cowell (2), W. Dawson (2) (1) Department of Plant Pathology, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Factors that influence the structure of Citrus tristeza virus populations S. HARPER (1), S. Cowell (2), W. Dawson (2) (1) Department of Plant Pathology, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A. S. HARPER (1), S. Cowell (2), W. Dawson (2) (1) Department of Plant Pathology, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Flor S. ( ), S. Cowe ( ), W. awso ( ) (1) Department of Plant Pathology, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A. rtment of Plant Pathology, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A. A virus infection of a host is not a discrete, static entity, but a dynamic population whose members interact with one another and with their host to persist and spread. We have been using populations of Citrus tristeza virus (CTV) strains to examine this phenomenon, and have found that under constant conditions, the population structure will stabilize to an equilibrium defined by the host. HAL Id: hal-01606195 https://hal.science/hal-01606195v1 Submitted on 3 Jun 2020 L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés. HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers. Distributed under a Creative Commons Attribution - ShareAlike 4.0 International License 2016 APS Annual Meeting Abstracts of Presentations This led us to ask what determinants, other than the host, can define a population, and what factors can cause a shift in population structure. Here we examined the effect of (a) inducing stress on the host through drought, high temperature, and by inducing systemic acquired resistance through the application of salicylic acid (SA), and (b) the addition of Citrus tatter leaf virus (CTLV) and Citrus leaf blotch virus (CLBV). We found that the structure of a CTV population comprised of three strains showed no significant change between treated and non-treated plants when exposed to drought or treated with SA, nor did the addition of CTLV or CLBV. Only exposure to approximate Florida summer temperatures (36°C day/25°C night) caused a change in structure, decreasing virus titer in a strain-specific and host-specific manner. Knowledge of the factors that determine population structure will aid in developing methods to manipulate populations and prevent disease. Virus populations associated with Maize lethal necrosis (MLN) in East Africa Virus populations associated with Maize lethal necrosis (MLN) in East Africa ( ) (1) USDA-ARS Corn, Soybean and Wheat Quality Research Unit, Wooster, OH, U.S.A.; (2) Molecular and Cellular Imaging Center, The Ohio State University, Wooster, OH, U.S.A.; (3) International Institute of Tropical Agriculture (IITA), Dar es Salaam, Tanzania; (4) Venganza, Inc, Augustine, FL, U.S.A.; (5) National Agricultural Research Organization, Entebbe, Uganda; (6) Kenya Agricultural and Livestock Research Organization (KALRO), Nairobi, Kenya; (7) Plant Pathology Department, The Ohio State University, Wooster, OH, U.S.A. Maize lethal necrosis (MLN) has emerged as a devastating disease in East Africa in recent years. MLN is caused by co-infection of a maize-infecting potyvirus and maize chlorotic mottle virus (MCMV). Deep sequencing of MLN-associated samples collected in Kenya and Uganda indicate high conservation of MCMV sequences, whereas the associated potyvirus population is diverse and complex. Sugarcane mosaic virus (SCMV)-like sequences are abundant and variable across samples, and clustered into four groups with over 10% nucleotide sequence divergence. Sequence analyses indicate presence of other putative viruses in MLN-diseased tissue that require further characterization. These data are valuable to inform diagnostics, containment, and management of destructive maize viruses in East Africa. Investigating the spread of Grapevine red blotch-associated virus E. Cieniewicz (1), M. Fuchs (1), K. Perry (2) (1) Cornell University New York State Agriculture Experiment Station, U.S.A.; (2) Cornell University, U.S.A. nvestigating the spread of Grapevine red blotch-associated virus . Cieniewicz (1), M. Fuchs (1), K. Perry (2) Investigating the spread of Grapevine red blotch-associated virus E. Cieniewicz (1), M. Fuchs (1), K. Perry (2) (1) Cornell University New York State Agriculture Experiment Station, U.S.A.; (2) Cornell University, U.S.A. (1) Cornell University New York State Agriculture Experiment Station, U.S.A.; (2) Cornell University, U.S.A. Red blotch is a recently recognized viral disease of Vitis spp. The causal agent, Grapevine red blotch-associated virus (GRBaV) has a single-stranded, circular DNA genome (3,206 nt), and is a proposed member of the family Geminiviridae. GRBaV has been detected in all major grape-growing regions of the United States and Canada, likely as a result of the dissemination of infected propagation and planting material. Of major concern to the grape industry is whether GRBaV is transmitted in vineyards. While there is no indication of spread in most areas where GRBaV has been detected, within- vineyard transmission is documented in California, likely due to insect vectors. To identify insect vectors of GRBaV, a detailed census of hemipteran insects in a selected California vineyard was performed. Of more than 50 species screened in 2015, only four species of the families Cicadellidae, Membracidae, and Cixiidae consistently carried genetic elements of GRBaV, as shown by multiplex PCR. The ability of vector candidate species to S4.1 transmit GRBaV to healthy grapevines is being assessed in greenhouse transmission assays. Understanding the mechanism of transmission will inform proper management strategies to control the insect vector and minimize spread of GRBaV. transmit GRBaV to healthy grapevines is being assessed in greenhouse transmission assays. Understanding the mechanism of transmission will inform proper management strategies to control the insect vector and minimize spread of GRBaV. Epidemiological insights into an emerging virus disease: Blueberry shock virus on cranberry S. THOMAS-SHARMA (1), L. Wells-Hansen (1), R. Page (1), V. Kartanos (2), E. Saalau-Rojas (3), P. McManus (1) (1) University of Wisconsin-Madison, U.S.A.; (2) Bayer Crop Science, U.S.A.; (3) UMass Cranberry Station, U.S.A. Epidemiological insights into an emerging virus disease: Blueberry shock virus on cranberry S. THOMAS-SHARMA (1), L. Wells-Hansen (1), R. Page (1), V. Kartanos (2), E. Saalau-Rojas (3), P. McManus (1) (1) University of Wisconsin-Madison, U.S.A.; (2) Bayer Crop Science, U.S.A.; (3) UMass Cranberry Station, U.S.A. Berry scarring, where cranberry uprights (shoots) produce scarred, disfigured berries that turn prematurely red, is an emerging problem for growers in WI and MA. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. Trivapro™ fungicide was registered late in 2015, and is now commercially available for use on corn, soybeans, and wheat. This fungicide contains three broad spectrum ingredients (a strobilurin, triazole, and an SDHI) that all have different modes of action. The SDHI component, Solatenol™ is an extremely potent active ingredient that provides long residual control of many important diseases. This combination of active ingredients provides both preventive and curative disease control. Trivapro is particularly effective against many of the rust pathogens. Results from corn and wheat trials in the US in 2015 demonstrated the grower benefits of longer lasting control. Research has also shown some crop enhancement benefits. Evaluation of fungicides for management of Fusarium wilt of watermelon N. MILLER (1), M. Adams (1), L. Quesada-Ocampo (1) (1) North Carolina State University, U.S.A. Fusarium wilt of watermelon, caused by Fusarium oxysporum f. sp. niveum, is an economically important disease in the United States and worldwide. It has traditionally been managed with long crop rotations and resistant varieties. However, the development and increased prevalence of new pathogen races has reduced the number of available resistant cultivars. In light of this, new chemical control options would greatly assist growers in controlling Fusarium wilt of watermelon. This study evaluates new and existing chemical control options for their potential in limiting F. oxysporum growth in the lab and managing disease in the field. Fungicide efficacy in vitro using a fungicide-amended media assay with 10 single formulation products at four concentrations revealed that prothioconazole and ADEPIDYN™ fungicide were among the best performing fungicides. Sensitivity to these fungicides was determined for 100 isolates of Fusarium oxysporum. Field-testing of these two products at two concentrations and application methods indicated that both products significantly reduced disease incidence, but a drench treatment followed by a foliar spray treatment 14 days post transplant was superior in reducing Fusarium wilt incidence in comparison to either product used in only a drench treatment. These two products could effectively complement existing strategies in managing Fusarium wilt of watermelon. Pimaricin: A new post-harvest fungicide for selected citrus, stone, and pome fruits D. Chen (1), H. Förster (1), J. nvestigating the spread of Grapevine red blotch-associated virus . Cieniewicz (1), M. Fuchs (1), K. Perry (2) We detected Blueberry shock virus (BlShV) on symptomatic uprights using DAS-ELISA and PCR, and initiated epidemiological studies. Similar to BlShV in blueberry, symptomatic cranberry uprights ‘recover’ from symptoms in the following season. These recovered uprights continue to test positive for BlShV and had greater incidence of BlShV in seeds (71%), compared to symptomatic uprights (12%). Studies on yield in three WI marshes affected by BlShV revealed that flowering is comparable in healthy, recovered, and symptomatic uprights. By the time of harvest, symptomatic uprights produced fewer marketable fruit and had lower fruit weight when compared to healthy uprights. Yield components on recovered and healthy uprights were comparable. Phylogenetic analysis of the coat protein of cranberry isolates from WI (n=12) and MA (n=5) revealed 96-100% nucleotide similarity among isolates, and isolates did not group by geography. Analysis of the protein sequence revealed that BlShV on cranberry shares only 90% coat protein identity with BlShV on blueberry. Small RNA sequencing and assembly (sRSA) indicates genome-level single nucleotide polymorphisms in BlShV from cranberry, suggesting different strains on blueberry and cranberry. In-depth analysis of sRSA data to identify other known and unknown viruses is underway. Threat to control Canna Yellow Streak Virus; altered infectivity by associating with a Sub-viral element of novel Canna Yellow Mottle Virus D. WIJAYASEKARA (1), P. Hoyt (1), B. Dunn (1), A. Gimondo (1), J. Verchot (1) (1) Oklahoma State University U S A Threat to control Canna Yellow Streak Virus; altered infectivity by associating with a Sub-viral element of novel Canna Yellow Mottle Virus D. WIJAYASEKARA (1), P. Hoyt (1), B. Dunn (1), A. Gimondo (1), J. Verchot (1) (1) Oklahoma State University, U.S.A. Canna Yellow Mottle Virus (CaYMV) poses a significant threat to the canna industry by causing unwanted virus-like symptoms to the foliage. CaYMV was first reported in 1979 and the full genome has not been reported. In order to determine the full CaYMV genome and characterize the disease, next generation sequencing was carried out using Virion DNA extracted from infected canna plants. The full CaYMV genome consisting of 6976 bp with two associated sub-viral elements (SVE), was recovered. The CaYMV genome and two sub-viral elements were cloned and sequenced using PCR primers. With initial diagnostic experiments using multiplex PCR reported in 2015, CaYMV was found to be associated with Canna Yellow Streak Virus (CaYSV). To determine the nature of this association, analysis of naturally infected canna varieties including greenhouse-grown and field-grown canna for the presence of CaYMV, CaYSV and SVEs was conducted using specific diagnostic primers for CaYMV and SVE 0036. Among six canna varieties, one variety “Australia” tested positive for only the SVE-0036 while another variety “Striped Beauty” tested positive for all three. Sap containing SVE0036, CaYSV, or CaYMV were mixed and used to inoculate bean or lupine plants. Infectivity of CaYSV with the presence of SVE0036 altered in bean and lupine plants. These data suggest that it is important to consider the presence of SVE0036 when testing for CaYSV infectivity. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. All of the new fungicides reduced PRR incidence and soil populations significantly to very low levels as compared to the control starting with the first application. Mefenoxam was only effective when a high label rate was used in the fourth application. Most of the new fungicides also increased tree canopy size and trunk diameter compared to the control. Greenhouse studies confirmed the efficacy of these new fungicides. Once registered, these new compounds will provide highly effective treatment options and resistance management strategies in the control of PRR of citrus. spp. Thus, we evaluated the efficacy of new chemicals belonging to different modes of action (i.e., ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin) against PRR. Navel orange trees on Carrizo rootstock were inoculated with P. parasitica at planting. Applications with 12 fungicide treatments were made five weeks after planting, in spring and fall 2014, and in spring 2015. Feeder roots and adjacent soil were collected before or after application. PRR incidence and pathogen populations were determined by plating root segments and soil suspensions, respectively, on a selective medium. All of the new fungicides reduced PRR incidence and soil populations significantly to very low levels as compared to the control starting with the first application. Mefenoxam was only effective when a high label rate was used in the fourth application. Most of the new fungicides also increased tree canopy size and trunk diameter compared to the control. Greenhouse studies confirmed the efficacy of these new fungicides. Once registered, these new compounds will provide highly effective treatment options and resistance management strategies in the control of PRR of citrus. Improving genomic resources for the detection of Macrophomina phaseolina infection of strawberry A. BURKHARDT (1), K. Childs (2), M. Ramon (1), F. Martin (1) (1) USDA, U.S.A.; (2) Michigan State University, U.S.A. Macrophomina phaseolina is the causal agent of charcoal rot, which is characterized by wilting, stunting, and crown discoloration. It has had an increasingly devastating impact on strawberry production in California as treatment methods have shifted from broadcast pre-plant fumigation with methyl bromide+chloropicrin to individual bed treatment with alternative fumigants. Hundreds of M. phaseolina isolates were collected from infected hosts in multiple countries and states, including many from California. An SSR analysis revealed that, with few exceptions, the strawberry isolates were a single genotype (isolates from other hosts were not pathogenic on strawberry). Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. To quickly identify the strawberry genotype, genotype-specific primers were developed with the end goal of implementing a diagnostic assay from soil prior to planting. These primers were designed by sequencing multiple isolates and using comparative genomics to identify genotype-specific loci. However, the complexity of the differences in the genomes of strawberry and non-strawberry isolates cannot be fully resolved with this approach. Therefore, PacBio sequencing was used to assemble a strawberry isolate reference genome. The draft assembly has an N50 of 3.3 Mb with 143 contigs and ~150x genome coverage. Ongoing work to improve and annotate this assembly is being done so that it can be used to identify additional strawberry-specific loci and to understand host specificity through comparative genomics. Identifying sources of inoculum and timing of tissue infection by fungal pathogens associated with winterberry fruit rot F. PEDUTO HAND (1), S. Lin (1), N. Taylor (1) (1) The Ohio State University, U.S.A. Identifying sources of inoculum and timing of tissue infection by fungal pathogens associated with winterberry fruit rot F. PEDUTO HAND (1), S. Lin (1), N. Taylor (1) (1) The Ohio State University, U.S.A. Winterberry is a valuable deciduous ornamental plant carrying brightly colored berries during the winter that is popular as a cut green for use in holiday decorations. In the past 3-4 years, a fruit rot has been challenging winterberry growers in the midwestern and eastern U.S., in some cases leading to complete crop loss. In this study, we monitored winterberry ‘Bonfire’ and ‘Sparkleberry’ throughout the 2015 growing cycle in two Ohio nurseries to identify sources of inoculum and assess periods of tissue infection. Twigs and mummified fruit were collected during plant dormancy to determine potential sources of primary inoculum. Flowers, leaves, and berries were collected weekly from June to December to record leaf spot and fruit rot incidence and severity, as well as pathogen recovery. Spore traps were used during the growing season to monitor spore abundance in the orchards. Based on the isolation results as well as counts from the spore traps, it appears that the disease may be due to a fungal complex, including species of Alternaria, Cladosporium, Colletotrichum, Epicoccum, Fusarium, Phoma, and Phomopsis. The major source of primary inoculum appears to be the mummified fruit. Leaf spots may serve as a source of secondary inoculum. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. Li (3) (1) China Agricultural University/Postdoctoral Fellows Workstation of Haidian Science Park, China; (2) Iowa State University, U.S.A.; (3) China Agricultural University, China Worldwide distribution of the sweet potato strains of Ceratocystis fimbriata Q. LI (1), T. Harrington (2), J. Li (3) (1) China Agricultural University/Postdoctoral Fellows Workstation of Haidian Science Park, China; (2) Iowa State University, U.S.A.; (3) China Agricultural University, China ) ( ) niversity/Postdoctoral Fellows Workstation of Haidian Science Park, China; (2) Iowa State University, U.S.A.; (3) China China (1) China Agricultural University/Postdoctoral Fellows Workstation of Haidian Science Park, China; (2) Iowa State Univers Agricultural University, China Ceratocystis fimbriata is a complex of species with a wide host range and worldwide geographical distribution. However, a sweet potato strain appears to be host specialized to cause black rot of storage roots. Isolates from black rotted roots of sweet potato (Ipomoea batatas) in China, Japan, Indonesia, Papua New Guinea, New Zealand and the USA had identical ITS rDNA sequences and only minor variation in 14 microsatellite loci (only two alleles each for two loci, and the other loci monomorphic). Three Ipomoea microsatellite genotypes (C1, C2 and C3) were found among the 12 isolates from sweet potato in China, two genotypes were found in the three isolates from Japan, and the other 17 isolates had identical microsatellite markers. The DNA sequences of the mating type genes of sweet potato isolates were most similar to those of isolates of C. fimbriata from various host plants in Ecuador, a center of diversity for sweet potato. Ceratocystis fimbriata on sweet potato was first described in 1890 in eastern USA, where black rot has been known for 150 years. It is hypothesized that the fungus was introduced to Japan on sweet potato storage roots and introduced from there to China in the 1930s. Fungi associated with cankers and wounds of cultivated and wild olives in the Western Cape, South Africa C. SPIES (1), W. van Jaarsveld (2), P. Moyo (3), L. Mostert (2), F. Halleen (4) (1) ARC Infruitec-Nietvoorbij, South Africa; (2) Stellenbosch University, South Africa; (3) Stellenbosch University, South Africa; (4) ARC Infruite- Nietvoorbij, South Africa Olive (Olea europaea subsp. europaea) production in South Africa is centred in the Western Cape Province, where viticulture is the main agricultural enterprise. Wild olive trees (Olea europaea subsp. cuspidata) also occur naturally in this region. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. Adaskaveg (1) ( ) ( ) g ( ) (1) Department of Plant Pathology and Microbiology, University of California, Riverside, U.S.A. Pimaricin (natamycin) is a polyene macrolide antimycotic produced by Streptomyces natalensis. It has been demonstrated to block fungal growth by binding to ergosterol within cell membranes. In use as a food preservative for many years, no resistance to pimaricin has ever been observed in Penicillium spp. populations. Pimaricin has potential to be an effective naturally derived fungicide for postharvest management of fruit decays when used by itself (e.g., stone fruits) or in mixtures with conventional fungicides (e.g., citrus and pome fruits) for improved efficacy and as an anti-resistance strategy. We tested the in vitro toxicity of pimaricin against selected postharvest pathogens of fruit crops. Mean EC50 values for inhibition of mycelial growth of isolates of Alternaria alternata, Botrytis cinerea, Geotrichum citri-aurantii, P. digitatum, and P. expansum were 0.92 mg/L, 0.79 mg/L, 3.20 mg/L, 1.47 mg/L, and 1.14 mg/L, respectively. Treatments of inoculated citrus (G. citri-aurantii, P. digitatum), stone fruits (B. cinerea, Monilinia fructicola, Rhizopus stolonifer), and pome fruits (A. alternata, B. cinerea, P. expansum, Mucor piriformis) with 1000 mg/L pimaricin were mostly effective with a reduction in decay incidence by 65 to 95% as compared to the control. However, reduced efficacy or treatment failures were observed in some pathosystems (e.g., P. expansum on pears). The mechanism for this unique selective performance is currently being investigated. Evaluation of new fungicides for management of Phytophthora root rot of citrus W. Hao (1), M. Gray (1), H. Förster (1), J. Adaskaveg (1) (1) University of California, Riverside, U.S.A. Phytophthora root rot (PRR), caused by Phytophthora spp., is an important disease of citrus in California. For its chemical control, only mefenoxam and potassium phosphite are currently registered. Mefenoxam resistance is widespread, and overuse of phosphonates may lead to resistance in Phytophthora S4.2 spp. Thus, we evaluated the efficacy of new chemicals belonging to different modes of action (i.e., ethaboxam, fluopicolide, mandipropamid, and oxathiapiprolin) against PRR. Navel orange trees on Carrizo rootstock were inoculated with P. parasitica at planting. Applications with 12 fungicide treatments were made five weeks after planting, in spring and fall 2014, and in spring 2015. Feeder roots and adjacent soil were collected before or after application. PRR incidence and pathogen populations were determined by plating root segments and soil suspensions, respectively, on a selective medium. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. A significant increase in the number of fungi recovered at bloom indicates that flowers could serve as a point of entry for fruit infections. The ultimate goal of this study is to provide growers with appropriate management strategies. Occurrence of foliar diseases of watermelon on commercial farms in South Carolina in 2015 G. RENNBERGER (1), A. Keinath (1), P. Gerard (2) Occurrence of foliar diseases of watermelon on commercial farms in South Carolina in 2015 G. RENNBERGER (1), A. Keinath (1), P. Gerard (2) (1) Coastal Research and Education Center, Clemson University, U.S.A.; (2) Department of Mathematical Sciences, ( ), ( ) Education Center, Clemson University, U.S.A.; (2) Department of Mathematical Sciences, Clemson University, U.S.A. ( ) ( ) ( ) Coastal Research and Education Center, Clemson University, U.S.A.; (2) Department of Mathematical Sciences, Clemson Un The main foliar diseases of watermelon in the southeastern U.S. are anthracnose, downy mildew (DM), gummy stem blight (GSB) and powdery mildew (PM). A survey was conducted in South Carolina in 2015 to obtain current data on the occurrence and distribution of these diseases. A stratified two- stage cluster sampling design was used, with 6 pre-selected counties as strata, 17 randomly selected growers as the first cluster and 21 randomly selected fields as the second cluster. Diseased leaves (100 per field) were collected along 4 transects encompassing 2500 m2 with 5 equidistant sampling points along each transect. All leaves were examined microscopically, and all pathogens present were recorded. Data were analyzed with SAS survey procedures for means and proportions, logistic regression and frequency analysis. The estimated statewide probability of occurrence (and range across counties) was 0.48 (0.32-0.78) for GSB, 0.21 (0.00-1.00) for PM, 0.15 (0.00-0.49) for DM and 0.06 (0.00-0.36) for Cercospora leaf spot (CLS). No anthracnose was found. Field size significantly influenced the probability of pathogen occurrence; with increasing field size, the probabilities decreased. More than 30% of leaves had two or more pathogens. Associations between pathogen pairs were analyzed. The only significant interaction was that CLS was more likely to occur when GSB was present on the leaf. This study will help to match fungicide recommendations to the most frequent diseases. Worldwide distribution of the sweet potato strains of Ceratocystis fimbriata Q. LI (1), T. Harrington (2), J. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. Trunk pathogens of grapevines such as Eutypa lata, Neofusicoccum australe, Pleurostoma richardsiae, and species of Phaeoacremonium have been associated with olive trunk diseases in California, Italy and Spain, but the incidence of these on South African olive trees are unknown. The aim of this study was to identify fungi associated with cankers and wounds of cultivated and wild olives in the Western Cape Province of South Africa. Isolations from 125 olive and 27 wild olive trees sampled in nine S4.3 districts yielded a fungal collection dominated by an undescribed Pseudophaeomoniella species. This species has been identified from 54 olive (43%) and 15 wild olive trees (47%) in eight districts. Other fungi, including known olive trunk pathogens, occurred at incidences of 13% or less. This stands in contrast to high incidences of the Botryosphaeriaceae and Phaeoacremonium reported on olive trees in California and Italy. The high incidence and distribution of Pseudophaeomoniella sp. suggests this fungus plays an important role in olive trunk diseases in South Africa. districts yielded a fungal collection dominated by an undescribed Pseudophaeomoniella species. This species has been identified from 54 olive (43%) and 15 wild olive trees (47%) in eight districts. Other fungi, including known olive trunk pathogens, occurred at incidences of 13% or less. This stands in contrast to high incidences of the Botryosphaeriaceae and Phaeoacremonium reported on olive trees in California and Italy. The high incidence and distribution of Pseudophaeomoniella sp. suggests this fungus plays an important role in olive trunk diseases in South Africa. Multiple mechanisms of nematode control conferred by seed and root applications of microbial biocontrol agents, Trichoderma and Bacillus M. CADLE-DAVIDSON (1), G. Harman (1), R. Hendriks (2), W. Nosir (1) (1) Advanced Biological Marketing, Inc, U.S.A.; (2) Advanced Biological Marketing, Inc South Africa Division, South Africa Multiple mechanisms of nematode control conferred by seed and root applications of microbial biocontrol agents, Trichoderma and Bacillus M. CADLE-DAVIDSON (1), G. Harman (1), R. Hendriks (2), W. Nosir (1) (1) Advanced Biological Marketing, Inc, U.S.A.; (2) Advanced Biological Marketing, Inc South Africa Division, South Africa Nematode diseases of plants are estimated to contribute $150B USD in annual crop losses around the world due to Cyst (Heterodera and Globodera spp) and Root knot nematode (Meloidogyne spp) infection in species ranging from Legumes to Solanaceae to Brassicaceae among others. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. Chemical and host resistance resources are available for control however a common recommended practice is rotation to nonhost crops. Biological control of nematode diseases has been shown in the literature with the main mechanism being direct antagonism. 2015 field observations of soybeans in Minnesota USA (strip plots) and Gauteng South Africa (replicated small plots) indicated significant control of Soybean cyst and Root knot nematodes using Trichoderma and Bacillus seed treatments. Subsequently, a split root greenhouse experiment was conducted to identify strain specific activities and gain insight into control mechanisms. Tomato plant root systems were split and transplanted into two pots, both infested with M. javanica, such that individual plants would be influenced by two pots each. One of the two pots was treated with an individual biocontrol strain or strain combination as a soil drench. Twelve treatments were tested each with six replicates. The results showed excellent control of nematodes by some strains and strain combinations, synergistic effects between strains, and multiple mechanisms of control. Mechanisms and implications for population control will be discussed. Enhanced nematicidal actitivity of abamectin via nanoparticle formulation R. Guenther (1), C. Opperman (1), J. Zhu (1), D. Lindbo (2), J. Kerns (1), S. Lommel (1), T. Sit (1) (1) North Carolina State University, U.S.A.; (2) USDA-Natural Resources Conservation Service, U.S.A Abamectin (Abm) has broad nematicidal activity but its usage is limited to seed coatings due to its high affinity for soils. To overcome this limitation, Abm was infused into plant viral nanoparticles (PVNAbm) based on Red clover necrotic mosaic virus, a virus that’s normally transmitted through the soil sans vector. An added benefit of PVNAbm formulation is the increased diffusion rate of Abm through the soil. PVNAbm was found to be stable and have low affinities for soils collected from commercial fields (planted with corn, soybeans and sweet potatoes) in North Carolina even in samples with greater than 3% organic content. PVNAbm was found to increase the duration that Abm is nematicidal by 2-4 fold in comparison to commercial formulations. In sterile and live soil greenhouse testing, PVNAbm provided a 50% reduction in galling and egg quantities per root for tomatoes challenged by root knot nematode (RKN). When applied to turf, PVNAbm is capable of moving Abm below the thatch layer and a single application was found to retain appreciable turf greening when tested on turf infested with sting nematode. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. Microplot field trials are under way to determine the performance of PVNAbm under different application protocols/conditions and to quantify its effectiveness in reducing RKN/soybean cyst nematode damage in row crops and sting nematode in turf. Interaction between Fusarium and soybean cyst nematode on soybean P. OKELLO (1), A. Adhikari (1), A. Varenhorst (1), S. Osborne (2), E. Byamukama (1), F. Mathew (1) (1) South Dakota State University, U.S.A.; (2) USDA-ARS, U.S.A. The soybean cyst nematode (SCN), Heterodera glycines, and Fusarium spp. are soilborne organisms that constrain soybean [Glycine max L.] production in the United States. While Fusarium and SCN are known to coexist in soybean fields, our knowledge of how these organisms interact and the implications for management is limited. The objective of this study was to determine if any interaction occurs between Fusarium spp. and SCN on soybean under greenhouse conditions. Pre-germinated soybean seedlings of SCN-susceptible (Williams 82) and SCN-resistant (Jack) cultivars were grown in cone-tainers filled with a sand-clay soil mixture (2:1) infested with inoculum of either of the four Fusarium spp. (F. proliferatum, F virguliforme, F. sporotrichioides, and F. subglutinans). A suspension of 2,000 egg/ml suspension of SCN HG type 0 were used for inoculations at planting. The experiment was set up as factorial design with five replications for each treatment (Fusarium alone, SCN alone, and a combination of SCN and Fusarium) and repeated twice. After 39 days, disease severity as percentage of lesion length on soybean roots and SCN egg counts from soybean roots were evaluated. For disease severity, significant effects of the treatment involving Fusarium spp. was observed on the two soybean cultivars (P = 0.01). Our results from the interaction study between Fusarium spp. and SCN illustrates the need for developing targeted approaches to manage the Fusarium-SCN complex on soybean. Effect of Seed Treatments on Root-Knot Management for Soybean and Cotton W. ALJAAFRI (1), W. Aljaafri (1), G. Lawrence (1), V. Klink (1) (1) Mississippi State University, U.S.A. Biological control is being accepted as an alternative to chemicals methods. Experiments were conducted at Mississippi State University to determine the efficacy of potential biological control products for management of the root-knot nematode. Seed applied biological products were received from Albaugh, LLC. Cotton and soybean seeds were treated with nine and seventeen biological compounds, respectively. Seeds were planted in 500 cm3 of a steam sterilized sand: soil mix 10 cm Dia. Trivapro™ fungicide, a new fungicide for broad spectrum control of diseases of corn, soybeans, and wheat E. TEDFORD (1), E. Tedford (1), A. Tally (1) (1) Syngenta, U.S.A. clay pots and remained for 50 days. Each test included Abamectin and ILeVo as industry standards. Seed applied biological compounds significantly reduced root-knot nematode development on cotton and soybean. Seeds treated with Abamectin, and ALB- EXP5-1+ALB-SAR1 significantly reduced juvenile development and number of eggs produced on cotton. On soybean seeds treated with Abamectin and Bionematicide+thiabendazol+ALB-GG reduced root-knot juvenile development. Abamectin and SAR+Harpin protein+Thiabendazol reduced the number of eggs that developed on soybean roots. Seed applied biological products may have potential in nematode management. oybean cyst nematode populations to the PI 88788 source of resistance from 2000 through 2015 in Iowa and the effect Adaptation of soybean cyst nematode populations to the PI 88788 source of resistance from 2000 through 2015 in Iowa and the effects on soybean yields M. MCCARVILLE (1), C. Marett (2), M. Mullaney (2), G. Gebhart (2), G. Tylka (2) (1) Bayer CropScience, U.S.A.; (2) Iowa State University, U.S.A. Adaptation of soybean cyst nematode populations to the PI 88788 source of resistance from 2000 through 2015 in Iow soybean yields y y M. MCCARVILLE (1), C. Marett (2), M. Mullaney (2), G. Gebhart (2), G. Tylka (2) (1) Bayer CropScience, U.S.A.; (2) Iowa State University, U.S.A. Most soybean cultivars grown in Iowa with resistance to the soybean cyst nematode (SCN), Heterodera glycines, have SCN resistance genes from a breeding line named PI 88788. Each year, field experiments are conducted throughout Iowa to evaluate 60 or more SCN-resistant and three or more SCN-susceptible cultivars at up to nine locations. Data on SCN population density, SCN virulence (HG type), soybean yield, precipitation, and growing degree-days from the experiments conducted from 2000 through 2015 were compiled and analyzed to determine how these factors affected SCN reproduction and soybean yield. Reproduction of SCN was positively correlated with temperatures and negatively associated with precipitation. Reproduction of SCN on resistant soybean cultivars with resistance from either PI 88788 or another source, Peking, was positively correlated with increasing virulence on these resistance sources. Both virulence on PI 88788 and reproduction on PI 88788-derived cultivars increased from 2000 to 2015, but not so on Peking. Yields of susceptible, PI 88788-derived, and Peking-derived SCN-resistant soybean cultivars were negatively correlated with SCN reproduction. Results indicate that virulence to PI 88788 is increasing in SCN populations in Iowa, resulting in greater SCN reproduction and yield loss. This trend is problematic because almost all SCN-resistant soybean cultivars available for Iowa farmers have resistance genes from the PI 88788 breeding line. S4.4 Spatio-temporal analysis of rice blast incidence in China F. Guo (1), M. Lu (2), B. Wu (1), F. Guo (1) (1) China Agricultural University, China; (2) National Agro-t Spatio-temporal analysis of rice blast incidence in China Spatio-temporal analysis of rice blast incidence in China F. Guo (1), M. Lu (2), B. Wu (1), F. Guo (1) (1) China Agricultural University, China; (2) National Agro-tech Extension and Service Center, China Rice blast caused by Magnaporthe oryzae is a devastating disease worldwide. Knowledge on the spatial and temporal patterns of disease risk at regional scale is important to understand its epidemic. In this study, rice blast incidence was surveyed in the main rice production areas in China between June 10 and September 10 from 2009 to 2014. Rice blast often had two peaks each year, and was highest in 2012 among these years. Adaptation of soybean cyst nematode populations to the PI 88788 source of resistance from 2000 through 2015 in Iow soybean yields SILVA (1), M. Silva (1), E. Pozza (2), G. Vasco (2), P. Paula (2), E. Chaves (2), G. Dornelas (2), M. Silva (2), A. Pozza (2) (1) UFLA, Brazil; (2) Lavras Federal University, UFLA, Brazil The rust (Hemileia vastatrix) is the most important disease of coffee and the intensity depends on productivity, cropping system, plant nutrition and water availability. Thus, the objectives were to evaluate the spatio-temporal disease progress and the relation with plant nutrition and soil fertility in coffee farms irrigated by drip and center pivot, using geostatistics. The drip and pivot areas were georeferenced with 52 points in 11ha and 50 in 17ha, respectively. The disease incidence was evaluated in 60 leaves, in five plants/point at 60-days intervals, from August 2012 to May 2015, totaling 17 evaluations. It was also evaluated at each point, productivity, mineral nutrition of the plants and soil fertility annually. These data were correlated with the rust incidence. The disease progress curves were plotted and the adjustment of semivariogram models was done. Then, the data were interpolated by ordinary kriging and the maps of disease and nutrients were constructed (ArcGIS 9.2®). The disease incidence in dripping was less than 3% over the years. In the pivot, the incidence reaching in August 2013 and from June to August 2014, between 21 and 27%. There was correlation between soil fertility and plant nutrition with rust for Ca, P and K. The spatial distribution of the disease and nutrients changed over the years. The exponential model was adjusted to coffee rust over the space. The disease gradient was observed from the foci in the kriging maps. Validation and refinement of a predictive model for Sclerotinia sclerotiorum apothecial development in soybean fields J. WILLBUR (1), H. Lucas (2), B. Mueller (2), S. Chapman (2), M. Kabbage (2), D. Smith (2) (1) University of Wisconsin - Madison, U.S.A.; (2) University of Wisconsin - Madison, U.S.A. Sclerotinia stem rot (SSR), caused by Sclerotinia sclerotiorum, is one of the most important yield-limiting diseases of soybean worldwide and is a significant concern in the Great Lakes region of the USA. Phenologically-based fungicide applications are often inefficient, and fungicide application is sometimes unnecessary when weather conditions are not conducive for infection. Weather-based risk assessment tools are sometimes used to more accurately predict the timing of fungicide applications, however, such tools are not currently available for soybeans. Adaptation of soybean cyst nematode populations to the PI 88788 source of resistance from 2000 through 2015 in Iow soybean yields For the major production areas other than north eastern China, the disease incidence was highest in the Yunnan-Guizhou plateau, followed by Sichuan Basin and southern China, and lowest in central and eastern China. Spatial autocorrelation analysis revealed a significant correlation at distances ranging from 110KM to 340KM. Results of iso cluster unsupervised classification conducted in GIS on interpolated incidence on different dates could classify rice production areas into low and high disease risk regions. Although the regions with high disease risk varied among years, some regions frequently had high disease risk, such as areas along the border of Yunnan and Guangxi, the border of Sichuan and Chongqing, the border of Fujian and Guangdong, and the junction of Jiangxi, Anhui, and Zhejiang. The results of hot spot analysis were consistent with those of cluster analysis. The spatial and temporal patterns of rice blast obtained from this study provided a reference to efficiently organize resources for better management of the disease in China. Association between temperature sensitivity and virulence of Blumeria graminis f. sp. tritici Y. ZHOU (1), L. Han (1), Y. Zou (1), X. Duan (1), J. Fan (1) (1) Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China Wheat powdery mildew caused by Blumeria graminis f. sp. tritici (Bgt) is a highly destructive disease in China, which is highly sensitive to meteorological factors, especially temperature. In order to clarify the co-evolution between temperature sensitivity and virulence of Bgt, we collected a total of 90 isolates from 5 provinces (cities), including Yunnan, Sichuan, Beijing, Henan and Shaanxi, and tested the virulence of isolates according to 32 wheat varieties or lines with known powdery mildew resistant genes and sensitivities to temperature by detached leaf segment method. The results showed that the mean ET50 of tested isolates was 23.22°C, with the highest 25.15°C and lowest 19.78°C, respectively. The ET50 value of 73.34% of isolates ranged from 22.0°C to 24.0°C, and 10% of isolates below 22°C, 16.67% of isolates above 24°C. The virulence gene diversity index of the 90 Bgt isolates was 0.2370. The highest index H of isolates which from Sichuan Province is 0.2212 while the lowest index H of isolates from Henan Province is 0.1975. The results showed that there was a significant negative correlation between temperature-sensitivity and virulence diversity of B. graminis f. sp. tritici populations. Spatio-temporal aspects of rust and nutrition of coffee plants in different irrigation systems M. Adaptation of soybean cyst nematode populations to the PI 88788 source of resistance from 2000 through 2015 in Iow soybean yields In 2014, a logistic regression model using virtual weather data as inputs was developed to predict apothecial germination. The model uses 30-day averages of mean air temperature (r = - 0.59, P < 0.01) and maximum leaf wetness (r = 0.35, P < 0.01) to predict the probability of apothecial presence prior to and during the soybean flowering period. In 2015, model-based fungicide applications successfully decreased disease severity and prevented yield loss in field trials. An on-site weather station was also used to validate virtual weather data. Due to inaccuracies in virtual moisture variables, higher resolution virtual weather data was combined with data collected in Wisconsin and Michigan for model refinement. The model will be incorporated into a preliminary web-based user interface to assist growers in effectively timing fungicide applications for integrated SSR management. A forward genetic screen in Phomopsis longicolla provides unique insights into pathogenesis A forward genetic screen in Phomopsis longicolla provides unique insights into pathogenesis M. ZACCARON (1), S. Sharma (2), W. Fagundes (2), W. Hawkins (2), N. Lawson (2), J. Ridenour (2), J. Smith (2), B. Dhillon (2), J. Rupe (2), B. Bluhm (2) (1) University of Arkansas, U.S.A.; (2) University of Arkansas, U.S.A. A forward genetic screen in Phomopsis longicolla provides unique insights into pathogenesis M. ZACCARON (1), S. Sharma (2), W. Fagundes (2), W. Hawkins (2), N. Lawson (2), J. Ridenour (2), J. Smith (2), B. Dhillon (2), J. Rupe (2), B. Bluhm (2) A forward genetic screen in Phomopsis longicolla provides unique insights into pathogenesis M. ZACCARON (1), S. Sharma (2), W. Fagundes (2), W. Hawkins (2), N. Lawson (2), J. Ridenour (2), J. Smith (2), B. Dhil Bluhm (2) g p g p q g p g M. ZACCARON (1), S. Sharma (2), W. Fagundes (2), W. Hawkins (2), N. Lawson (2), J. Ridenour (2), J. Smith (2), B. Dhillon (2), J. Rupe (2), B. Bluhm (2) ma (2), W. Fagundes (2), W. Hawkins (2), N. Lawson (2), J. Ridenour (2), J. Smith (2), B. Dhillon (2), J. Rupe (2), B. ( ) (1) University of Arkansas, U.S.A.; (2) University of Arkansas, U.S.A. Phomopsis longicola (Hobbs) causes Phomopsis seed decay of soybean (Glycine max), and also causes lesions on soybean stems, pods, and petioles. P. longicola can also exist endophytically within soybean stems, which has given rise to the hypothesis that pathogenesis occurs when the pathogen transitions from endophytic or hemibiotrophic growth to necrotrophy. In this study, a forward genetic screen was performed to elucidate genetic mechanisms underlying necrotrophy, and a RAD-seq approach was adapted to characterize genomic lesions in selected mutants. A collection of 1114 tagged, insertional mutants was created via Agrobacterium-mediated transformation, and mutants were evaluated individually in soybean stems for their ability to induce necrosis and form pycnidia. Six mutants induced significantly less necrosis in soybean stems than the wild-type strain, and four formed significantly fewer pycnidia. No gain-of-function mutations (increased necrosis) were observed. A modified RAD-seq protocol developed to identify the site of insertion was applied to a mutant significantly reduced in its ability to cause stem necrosis; a single copy of the disruption cassette integrated into the presumed promoter region of a cellobiohydrolase. This study describes new tools to dissect the interaction between P. Magnaporthe oryzae zinc finger effector1 exploits rice HIRA/AS1/AS2 complex to alter plant development and increase disease susceptibility Y. Chen (1), W. Liu (2), Q. Wang (3), Y. Yang (2) (1) Penn State University U S A ; (2) Penn State University U S A ; (3) Penn State University U S A Magnaporthe oryzae zinc finger effector1 exploits rice HIRA/AS1/AS2 complex to alter plant development and increase disease susceptibility Y. Chen (1), W. Liu (2), Q. Wang (3), Y. Yang (2) (1) Penn State University, U.S.A.; (2) Penn State University, U.S.A.; (3) Penn State University, U.S.A. Rice blast, caused by Magnaporthe oryzae, is one of the most important rice diseases in the world. During the fungal infection, M. oryzae produces many effector proteins to manipulate rice cellular process and facilitate disease development. In this study, a C2H2 zinc finger effector1 (MoZFE1) protein was identified from M. oryzae and shown to enter rice cells. Overexpression of MoZFE1 in transgenic rice (MoZFE1-OX) was found to alter rice growth and development and increase disease susceptibility. RNA-seq analysis reveals suppressions of some development- and defense-related gene expressions in the MoZFE1-OX lines. Both in vitro and in vivo biochemical analyses demonstrate that MoZFE1 directly interacts with a histone chaperon HIRA and suppresses the AS2 gene expression. HIRA/AS1/AS2 is a conserved gene-silencing complex and plays a role during plant organogenesis. Lose of function mutants (rice hira, as1, and as2 lines created with CRISPR/Cas9) was shown to cause developmental and differentiation defects with some phenotypes similar to MoZFE1-OX lines. Our study suggests that MoZFE1 exploits rice HIRA/AS1/AS2 complex to alter host gene expression, thereby manipulating plant growth and development as well as defense response. Our findings will help elucidate the molecular mechanism of the rice-M. oryzae interaction and facilitate the development of new strategies to improve rice disease resistance and food security. Comparative Genomics of Two Magnaporthe oryzae Field Isolates Uncovers Novel Candidate Effector Genes J. HUANG (1), J. Huang (1), Y. Hong (1), L. Xu (1), M. Chen (1), Z. Zhong (1), X. Chen (1), H. Zheng (1), J. Bao (1) (1) Fujian-Taiwan Joint Center for Ecological Control of Crop Pests, Fujian Agriculture and Forestry University, Fuzh During the colonization process, rice blast fungus Magnaporthe oryzae secrets a repertoire of effectors into host tissue to overcome immunity and facilitate invasive growth. To better understand the biological functions of these complicated and diverse effectors, two field isolates Guy11 and FJ81278 were re-sequenced, and isolate-unique secreted proteins were identified through comparative genomics. When comparing with each other, 37 and 18 unique genes encoding secreted proteins were obtained in the genome of Guy11 and FJ81278, respectively. Weather-based risk assessment models for common leaf spot and black seed disease of strawberry caused by Mycosphaerella fragariae O. CARISSE (1) (1) Agriculture and AgriFood Canada Canada (1) Agriculture and AgriFood Canada, Canada Common leaf spot of strawberry (Mycosphaerella fragariae) is a sporadic disease. On susceptible cultivars and under favorable weather conditions, M. fragariae causes black seed disease, reduced plant vigor, and reduced yield the following year. Logistic regression analysis was used to examine the relationship between weather and outbreaks of common leaf spot and black seed. Information collected at 100 location-years was used to develop the models. The response variables were fields with an average of more than 10 lesions per leaf at the beginning of flowering (10% flowering) or with black seed symptoms on more than 5% of the fruits at harvest. Nonparametric correlation and logistic regression analysis was used to identified combinations of temperature, relative humidity, and rainfall during the spring period (bud break to 10% flowering) as predictor variables. Based on model prediction accuracy, sensitivity, and specificity, only few models correctly classified fields (> 80% correct classifications). The best predictors were temperature and rain (frequency and duration). The best models were validated with an independent data set (n = 20) and prediction accuracy was similar than the accuracy for the original data sets. Considering that management actions such as fungicide applications are not needed every year and in every field, these models could be used to identify field at risk for common leaf spot and black seed diseases. Genetics behind avirulence: Identification of novel avr-genes in Magnaporthe oryzae D. TATE (1), J. Hu (1), V. Ganeshan (1), T. Mitchell (1) (1) The Ohio State University, U.S.A. Rice blast disease, caused by Magnaporthe oryzae, accounts for 10-30% of yearly rice loss which is estimated to represent enough food to feed 60 million people. The study of interactions between M. oryzae and rice can be used to help mitigate one of the most devastating diseases plaguing a S4.5 primary food source and knowledge of this pathosystem can be used to manage related fungal diseases. The objective of this study is to find the corresponding avirulence (Avr)-gene to the Pi-2 resistance (R)-gene in hopes of better understanding the strong relationship between M. oryzae and rice. In previous studies we have identified five candidate AvrPi2 genes by Agrobacterium tumefaciens-mediated transformation; infection assays; and TAIL- PCR with the incompatible strain KJ201. These genes have been cloned into the compatible strain Chinos. Weather-based risk assessment models for common leaf spot and black seed disease of strawberry caused by Mycosphaerella fragariae O. CARISSE (1) (1) Agriculture and AgriFood Canada Canada By infecting Pi-2 R rice plants with the Chinos transformants, and assessing disease, we were able to identify and characterize the most probable cognate Avr-gene. Pathogenicity assays demonstrated that two transformed lines Chinos4842 and Chinos8832 produced a similar phenotype to KJ201, with Chinos4842 showing a nearly identical phenotype match. Further, the mutants’ ability to induce a hypersensitive response (HR) was assessed using 3,3′-diaminobenzidine (DAB) staining in leaf sheath assays. Of the transformed lines, Chinos4842 was the only line able to produce a HR. These results strongly suggest that MGG_04842 is AvrPi2. primary food source and knowledge of this pathosystem can be used to manage related fungal diseases. The objective of this study is to find the corresponding avirulence (Avr)-gene to the Pi-2 resistance (R)-gene in hopes of better understanding the strong relationship between M. oryzae and rice. In previous studies we have identified five candidate AvrPi2 genes by Agrobacterium tumefaciens-mediated transformation; infection assays; and TAIL- PCR with the incompatible strain KJ201. These genes have been cloned into the compatible strain Chinos. By infecting Pi-2 R rice plants with the Chinos transformants, and assessing disease, we were able to identify and characterize the most probable cognate Avr-gene. Pathogenicity assays demonstrated that two transformed lines Chinos4842 and Chinos8832 produced a similar phenotype to KJ201, with Chinos4842 showing a nearly identical phenotype match. Further, the mutants’ ability to induce a hypersensitive response (HR) was assessed using 3,3′-diaminobenzidine (DAB) staining in leaf sheath assays. Of the transformed lines, Chinos4842 was the only line able to produce a HR. These results strongly suggest that MGG_04842 is AvrPi2. A forward genetic screen in Phomopsis longicolla provides unique insights into pathogenesis longicola and soybean, and provides new insight into conserved mechanisms underlying stem, pod, and seed necrosis caused by P. longicola. Magnaporthe oryzae zinc finger effector1 exploits rice HIRA/AS1/AS2 complex to alter plant development and increase disease susceptibility Y. Chen (1), W. Liu (2), Q. Wang (3), Y. Yang (2) (1) Penn State University U S A ; (2) Penn State University U S A ; (3) Penn State University U S A Consistent with the known effector features, the majority of these proteins are small, cysteine-rich, specifically expressed at the early stage of infectious process, and possessed presence or absence polymorphisms among different field isolates. Meanwhile, due to the different origin of Guy11 and FJ81278, the two sets of unique genes owned the diverse distribution patterns in the strains isolated from different rice varieties (Oryza sativa ssp. indica and japonica). Furthermore, we have identified two unique proteins could suppress BAX-triggered programmed cell death in Nicotiana benthamiana so far, which indicated they are novel effectors. In conclusion, our comparative genomics approach is effective to generate a list of unique genes which could be candidate effectors and identify the novel effector genes. To whom correspondence may be addressed. E-mail: wangzh@fafu.edu.cn Molecular dissection of a complex gene locus conferring high virulence on barley cv. Bowman in the fungal cereal pathogen Cochliobolus sativus S. ZHONG (1), Y. Leng (1) Molecular dissection of a complex gene locus conferring high virulence on barley cv. Bowman in the fungal cereal pathogen Cochliobolus sativus S. ZHONG (1), Y. Leng (1) Molecular dissection of a complex gene locus conferring high virulence on barley cv. Bowman in the fungal cereal pathogen Cochliobolus sativus S. ZHONG (1), Y. Leng (1) (1) North Dakota State University, U.S.A. Molecular dissection of a complex gene locus conferring high virulence on barley cv. Bowman in the fungal cereal pathogen Cochliobolus sativus S. ZHONG (1), Y. Leng (1) (1) North Dakota State University, U.S.A. Cochliobolus sativus (Anamorph: Bipolaris sorokiniana) is a fungal pathogen causing spot blotch, common root rot, and black point in cereal crops. For spot blotch, four pathotypes (0, 1, 2 and 7) have been identified based on their differential virulence patterns on three barley genotypes (Bowman, ND5883 and NDB112), but the molecular mechanism underlying host specificity of these pathotypes is not well understood. Our previous studies indicated that isolate ND90Pr (pathotype 2) has a complex gene locus (VHv1) conferring high virulence on barley cv. Bowman. VHv1 is located in an ND90Pr-specific genomic region, which contains 43 predicted genes including two for nonribosomal peptide synthetases (NRPSs). Knocking out either of the two NRPS genes led to loss of high virulence on cv. Bowman, suggesting that both NRPSs are involved in the biosynthesis of a presumably secondary metabolite virulence factor in the pathogen. We further characterized other genes adjacent and between the two NRPS genes at the VHv1 locus and found that the two genes encoding 3-hydroxyacyl-CoA dehydrogenase and short-chain dehydrogenase/reductase (SDR), respectively, were also required for the high virulence of isolate ND90Pr on barley cv. Bowman. Oomycete species richness in cacao soil assessed by massive amplicon sequencing of the cytochrome oxidase II gene M. RATTI (1), E. Goss (1), J. Cevallos-Cevallos (2), C. Arias (2) (1) Plant Pathology Department University of Florida U S A ; (2) Centro de Investigaciones Biotecnologicas del Ecuador CIBE Bowman (2), S. Withers (3), D. Gent (4), K. Childs (5), L. Quesada-Ocampo (3) (1) University of Florida, Fort Pierce, FL, U.S.A.; (2) Van Andel Research Institute, Grand Rapids, MI, U.S.A.; (3) North Carolina State University, Raleigh, NC, U.S.A.; (4) Oregon State University, Corvallis, OR, U.S.A.; (5) Michigan State University, East Lansing, MI, U.S.A. Oomycete plant pathogens secrete effector molecules that manipulate host physiology to achieve colonization. Effector proteins have been identified in various oomycete species but little is known about the sequence, function, and evolution of effectors of downy mildew plant pathogens. Oomycete plant pathogens secrete effector molecules that manipulate host physiology to achieve colonization. Effector proteins have been identified in various oomycete species but little is known about the sequence, function, and evolution of effectors of downy mildew plant pathogens. Pseudoperonospora humuli is the causal agent of downy mildew of hop, an economically important disease that occurs worldwide and has gained importance in recent years in the United States due to an increase of hop production. The main goal of our study was to identify and make available to the oomycete community a set of candidate pathogen effectors from P. humuli using transcriptomics. Identified effector candidates can be employed in high throughput functional genomics screens, commonly referred to as effectoromics, to test hop germplasm for specific pathogen recognition by resistance R proteins. Here we report the identification of 296 sequences annotated as RXLR host-translocated effectors and the expression during P. humuli-hop interaction. Our findings highlight a set of in planta expressed RXLR candidate effectors for pathogen-assisted breeding in hop for resistance to downy mildew. What is a clone? Rethinking the ancestry of US clonal lineages of Phytophthora infestans using whole genome sequences B. KNAUS (1), J. Tabima (2), Z. Kamvar (2), C. Davis (3), H. Judelson (3), N. Grünwald (1) (1) USDA ARS, Horticultural Crops Research Laboratory, Corvallis, OR, U.S.A.; (2) Oregon State University, Botany and Plant Pathology, Corvallis, OR, U.S.A.; (3) Univ. California, Plant Pathology, Riverside, CA, U.S.A. Identifying genetic boundaries of a clone or clonal lineage is not straightforward. Identical multilocus genotypes can arise independently from unrelated clones. Similarly, lineages derived from the same ancestral clone can accumulate variants that distinguish them. Populations of the potato and tomato late blight pathogen, Phytophthora infestans, are well known for emerging as novel clonal lineages. Oomycete species richness in cacao soil assessed by massive amplicon sequencing of the cytochrome oxidase II gene M. RATTI (1), E. Goss (1), J. Cevallos-Cevallos (2), C. Arias (2) (1) Plant Pathology Department University of Florida U S A ; (2) Centro de Investigaciones Biotecnologicas del Ecuador CIBE Oomycete species richness in cacao soil assessed by massive amplicon sequencing of the cytochrome oxidase II gene M. RATTI (1), E. Goss (1), J. Cevallos-Cevallos (2), C. Arias (2) Oomycete species richness in cacao soil assessed by massive amplicon sequencing of the cytochrome oxidase II gene M. RATTI (1), E. Goss (1), J. Cevallos-Cevallos (2), C. Arias (2) (1) Plant Pathology Department, University of Florida, U.S.A.; (2) Centro de Investigaciones Biotecnologicas del Ecuador CIBE-ESPOL, Ecuador Oomycetes are ubiquitous in both natural ecosystems and crops, however diversity studies have focused in temperate regions. Emerging diseases together with climate change highlight the need to study oomycetes in other potential disease hotspots. Our aim was to assess Oomycete species richness and composition in soil samples from cacao farms of Ecuador by using high-throughput multiplexed Illumina sequencing of the cytochrome oxidase II gene (cox2). We used these nucleotide data to test the hypothesis that different oomycete species richness and composition are found between soil from cacao farms and neighboring soils, which included weeds and other cultivated crops. Eight farms were sampled covering three zones of the Coastal Region of Ecuador. Total DNA was extracted directly from collected soil samples and used for library preparation. A positive control, consisting of S4.6 gDNA from three known Phytophthora species, was included in the analysis. Amplicons of ~580bp were sequenced by Illumina MiSeq 2x300. Resulting reads were filtered before conducting megaBLAST analysis against a custom Oomycete sequence database. Most of the identified species were Phytophthora and Pythium. The majority of known pathogens, such as P. palmivora, were not restricted to cacao farms, rather they were also found in the plant communities adjacent to farms. These results suggest that wild plants and neighboring crops contribute to the ecology of Oomycete pathogens affecting cacao. gDNA from three known Phytophthora species, was included in the analysis. Amplicons of ~580bp were sequenced by Illumina MiSeq 2x300. Resulting reads were filtered before conducting megaBLAST analysis against a custom Oomycete sequence database. Most of the identified species were Phytophthora and Pythium. The majority of known pathogens, such as P. palmivora, were not restricted to cacao farms, rather they were also found in the plant communities adjacent to farms. These results suggest that wild plants and neighboring crops contribute to the ecology of Oomycete pathogens affecting cacao. Genome sequence and effector repertoire of the hop downy mildew pathogen Pseudoperonospora humuli L. CANO (1), M. Oomycete species richness in cacao soil assessed by massive amplicon sequencing of the cytochrome oxidase II gene M. RATTI (1), E. Goss (1), J. Cevallos-Cevallos (2), C. Arias (2) (1) Plant Pathology Department University of Florida U S A ; (2) Centro de Investigaciones Biotecnologicas del Ecuador CIBE Effect of red light on morphology of Peronospora belbahrii sporangia J. PATEL (1), S. Zhang (1) (1) Tropical Research and Education Center, University of Florida, U.S.A. Downy mildew of basil, caused by Peronospora belbahrii, has spread to more than 42 US states since it was first detected in Homestead, FL in 2007. Recently, our experiments showed that red light can suppress downy mildew in basil. The aim of this study was to investigate the effect of red light on biology of P. belbahrii sporangia. The red LED lights were hung 4 feet above the greenhouse bench and 5 feet between two adjacent LED lights. The LED in the spectrum of 625 nm wavelength illuminated at an average light intensity of 12 µmol.m–2.s–1. The LEDs were automatically turned on from 8 pm to 8 am each day. One set of inoculated basil plants was maintained under dark conditions and another set of inoculated basil plants were placed under the exposure of red light. Fresh sporangia were harvested from the basil plants under dark conditions and under red light. A total of fifty sporangia were examined under a microscope with 40x magnification lens. The sporangia produced under dark conditions were brown in color and had oval shaped sporangia, whereas sporangia produced under red illumination were white in color and had round shaped sporangia. The change in morphology of the sporangia is an indication of a possible direct effect of red lights on P. belbahrii sporangia production. These results warrant further investigations to determine the mechanisms underlying changes in biology of P. belbahrii sporangia affected by red light. Bean common mosaic virus isolate overcomes the bc-3 resistance allele in common bean: Virus genetic determinants defining a novel pathogenicity group mosaic virus isolate overcomes the bc-3 resistance allele in common bean: Virus genetic determinants defining a novel roup X. FENG (1), J. Myers (2), A. Karasev (1) (1) University of Idaho, U.S.A.; (2) Oregon State University, U.S.A. Bean common mosaic virus (BCMV) exists as a complex of strains that can be distinguished biologically, on a set of bean differentials, into pathogenicity groups (PG) numbered I to VII. Resistance against BCMV in Phaseolus vulgaris is governed by six recessive resistance alleles at four loci. One of these alleles, bc-3, is able to protect P. Oomycete species richness in cacao soil assessed by massive amplicon sequencing of the cytochrome oxidase II gene M. RATTI (1), E. Goss (1), J. Cevallos-Cevallos (2), C. Arias (2) (1) Plant Pathology Department University of Florida U S A ; (2) Centro de Investigaciones Biotecnologicas del Ecuador CIBE These successions of clones have historically been named within the United States as lineages US1-US24, in order of appearance. Hypothetically, these lineages can emerge through divergence from other US lineages, recombination among US lineages or as novel, independent lineages. We used 32 whole genome sequences including dominant US clonal lineages and available samples from global populations. Based on the core orthologous genes located on supercontigs 1.1 and 1.5 we observed substantial amounts of variation shared among strains within clonal lineages. We found no support for emergence of any of the US lineages from a common ancestor shared with any other US lineages. Each of the US clonal lineages fit a model where populations of new clonal lineages emerge via migration from a source population that is sexual in nature and potentially located in central Mexico or elsewhere. This work provides novel insights into patterns of emergence of clonal lineages in plant pathogen genomes and provides an evolutionary framework for definition of clones in P. infestans. Temperature and light effects on germination of Peronospora effusa R. CHOUDHURY (1), S. Koike (2), N. McRoberts (1) (1) University of California, Davis, U.S.A.; (2) UCCE Monterey County, U.S.A. Spinach downy mildew, caused by the biotrophic oomycete Peronospora effusa, is an economically important disease that is found in all spinach growing regions of the US. To effectively predict disease risk we need to understand the response of P. effusa to different environmental conditions. However, compared with several other downy mildew pathogens, relatively little research has been done on the optimal environmental conditions for growth and sporulation of P. effusa. We conducted several germination assays, exposing P. effusa sporangia to different temperature and lighting conditions. Between 5 and 25°C under constant darkness, germination of P. effusa sporangia on water agar declined following an approximately logistic decay. These results were qualitatively different from a previous study of P. effusa germination that found a bimodal response curve, with increased germination at lower and higher temperatures. Time course studies revealed that most sporangia germinated within the first eight hours of plating, regardless of incubation temperature. Sporangia exposed to green or blue light had significantly reduced germination when compared with those exposed to red, yellow, or no light. Light intensity and color significantly impacted germination, although the effect of color varied by light intensity. The use of these results in the development of a disease risk model will be discussed. Bean common mosaic virus (BCMV) exists as a complex of strains that can be distinguished biologically, on a set of bean differentials, into pathogenicity groups (PG) numbered I to VII. Resistance against BCMV in Phaseolus vulgaris is governed by six recessive resistance alleles at four loci. One of these alleles, bc-3, is able to protect P. vulgaris against all BCMV strains and against other potyviruses; bc-3 was identified as the eIF4E allele carrying mutated eukaryotic translation initiation factor gene. We characterized a novel BCMV isolate, 1755a, that was able to overcome bc-2 and bc-3 alleles in common bean. Hence, it displayed a novel pattern of interactions with resistance genes in P. vulgaris, and was assigned to a new pathogroup, PG-VIII. The IVT7214 cultivar supporting the replication of BCMV-1755a was found to have the intact homozygous bc-3 CAPS marker. The complete genome of the 1755a isolate was sequenced and found to be a recombinant between the BCMV strain NL1 (PG-I) and an unknown BCMV strain. Analysis of the recombination patterns in the genomes of NL1 and US1 (PG-I), NY15P (PG-V), US10 and RU1-OR (PG-VII), and 1755a (PG-VIII), Determination and characterization of a symptom determinant of Grapevine fanleaf virus L OSTERBAAN (1) L Osterbaan (1) M Fuchs (1) Understanding superinfection exclusion by complex populations of Citrus tristeza virus S. KANG (1), M. Bergua (1), S. Folimonova (1) (1) University of Florida, U.S.A. Superinfection exclusion (SIE) is a phenomenon in which a primary viral infection restricts a secondary infection with the same or closely related virus. For plant viruses, SIE, referred to as cross-protection, was implemented as an agricultural practice to protect crops against aggressive viruses by pre- immunizing plants with mild virus isolates. Recently we showed that SIE by Citrus tristeza virus (CTV), an economically important virus of citrus, occurs only between isolates of the same strain, but not between isolates of different strains. This work, however, was done using “pure” isolates of CTV, each containing a single virus strain, while most citrus trees in the field harbor complex populations made up of different CTV strains. The objective of this study was to assess how SIE works in plants simultaneously pre-infected with several CTV strains. Co-inoculation of citrus trees with variants of T36, T68 and/or T30 strains demonstrated that multi-strain populations equilibrate soon after establishment of infection, show uniform intra- host distribution and maintain the established equilibrium over time. SIE experiments showed that exclusion of a secondary infection by a CTV variant was triggered by the presence of another variant of the same strain in the primary population, which was in the agreement with our previous observations. The same rule appeared to be in effect when SIE by mixed virus populations was tested in a series of different citrus varieties. Potato virus Y strains co-localize and compete in single epidermal leaf cells S. MONDAL (1), S. Mondal (1), S. Gray (1), M. Ghanim (2) (1) Cornell University, U.S.A.; (2) Volcani Center, Israel In recent years, recombinant strains of Potato virus Y (PVY) have become predominant in U.S. potato crops; whereas the parental PVY strains have been disappearing. Often multiple PVY strains are found in the same field and occasionally within the same plant, but little is known about how they interact or compete within the plant. Here, we examine the spatial and temporal dynamics of PVY strain infection at the cellular and tissue levels in different hosts. Immunofluorescence and confocal microscopy was used to localize mixtures of PVY strains (PVYO and PVYNTN) in epidermal leaf cells of ‘Samsun NN’ tobacco or ‘Red Maria’ potato. Determination and characterization of a symptom determinant of Grapevine fanleaf virus L OSTERBAAN (1) L Osterbaan (1) M Fuchs (1) Determination and characterization of a symptom determinant of Grapevine fanleaf virus L. OSTERBAAN (1), L. Osterbaan (1), M. Fuchs (1) (1) Section of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, U.S.A. Determination and characterization of a symptom determinant of Grapevine fanleaf virus L. OSTERBAAN (1), L. Osterbaan (1), M. Fuchs (1) (1) Section of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, U.S.A. L. OSTERBAAN (1), L. Osterbaan (1), M. Fuchs (1) (1) Section of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, U.S.A. L. OSTERBAAN (1), L. Osterbaan (1), M. Fuchs (1) (1) Section of Plant Pathology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Sta ( ) ( ) ology and Plant-Microbe Biology, Cornell University, New York State Agricultural Experiment Station, U.S.A. Grapevine fanleaf virus (GFLV) -the causative agent of fanleaf degeneration- is one of the most devastating viral diseases of grapevines. GFLV is a bipartite (+) sense RNA nepovirus and is transmitted by the ectoparasitic nematode Xiphinema index. The five proteins encoded by RNA1 are associated with replication and protein maturation, while the three proteins encoded by RNA2 are involved in replication, cell-to-cell movement, and genome encapsidation. Infected vines exhibit a variety of symptoms, including shortened internodes, fan-like (deeply lobed) leaves, mosaic, and vein yellowing. Infected vines have decreased fruit yield and quality, and also a shortened productive lifespan. Little is known about the host-pathogen interactions leading to symptom development. Recent work with chimeras of symptomatic and asymptomatic strains of GFLV (strains GHu and F13, respectively) in the plant model species Nicotiana benthamiana has shown that symptom development in this host species is dependent on a 408-nucleotide sequence at the 3’ end of RNA1 of GFLV-GHu. A reverse genetics approach was used to characterize the symptom determinant by using agroinoculation to establish infection with a series of recombinant GFLV genomes in planta and thus determine the minimal GFLV-GHu sequence necessary for symptom development in N benthamiana A better understanding of GFLV’s biology will inform the mechanism by which the virus causes symptoms in its hosts. na. A better understanding of GFLV’s biology will inform the mechanism by which the virus causes symptoms in its hosts. Understanding superinfection exclusion by complex populations of Citrus tristeza virus S. KANG (1), M. Bergua (1), S. Folimonova (1) (1) University of Florida, U.S.A. Determination and characterization of a symptom determinant of Grapevine fanleaf virus L OSTERBAAN (1) L Osterbaan (1) M Fuchs (1) We observed that both strains systemically infect tobacco and are co-localized in cells of all leaves; however their relative prevalence changes over time. Early in infection when mosaic symptoms are observed, PVYO predominates the infection although PVYNTN is present in some cells. As infection progresses and veinal necrosis is observed, PVYNTN becomes the prevalent virus in those tissues outcompeting PVYO. Co-localization of PVYO and PVYNTN was also found in epidermal cells of potato leaves with almost all cells being infected with both viruses. Co-localization of virus strains in single cells is not a widely used approach for understanding interactions among them. The apparent non- antagonistic interaction among PVY strains could explain the multitude of emerging recombinant PVY strains discovered in potato in recent years. Ethylene inhibition can prevent aphid attraction and Potato Virus Y spread A. BAK (1), S. Chen (2), T. Antichera (2), L. Perilla (2), C. Casteel (2) (1) UCDavis, U.S.A.; (2) UCDavis, U.S.A. Vector-borne pathogens influence host characteristics relevant to host-vector contact, increasing pathogen transmission and survival. Potato Virus Y (PVY) is vector-borne and considered one of the most damaging viruses for potato worldwide. Previous studies have demonstrated aphid vectors have higher fecundity and preferentially settle on PVY-infected potatoes compared to controls. Here we demonstrate PVY infection increases ethylene production and we show that ethylene production is required for increased vector attraction to infected plants. Further, we demonstrate pathogen spread can be prevented by temporarily inhibiting ethylene signaling. Virus-induced changes in ethylene responses may mediate vector-plant interactions more broadly and thus represent a conserved mechanism for increasing transmission by insect vectors. Precision diagnostics and next-generation decision support systems N. BOONHAM (1), J. Turner (2), I. Brittain (2), D. McCluskey (3), R. Kaye (3), D. Clarke (4), N. Morant (4), D. Langton (5), F. Salinari (5), W. Charlton (6), M. Andreou (7) Precision diagnostics and next-generation decision support systems N. BOONHAM (1), J. Turner (2), I. Brittain (2), D. McCluskey (3), R. Kaye (3), D. Clarke (4), N. Morant (4), D. Langton (5), F. Salinari (5), W. Charlton (6), M. Andreou (7) (1) Fera, York, United Kingdom; (2) Fera, United Kingdom; (3) University of Hertfordshire, United Kingdom; (4) Gensys Biotech Ltd, United Kingdom; (5) Agrii, United Kingdom; (6) Bayer Crop Sciences, United Kingdom; (7) Optisense Ltd, United Kingdom Precision diagnostics and next generation decision support systems N. BOONHAM (1), J. Turner (2), I. Brittain (2), D. Oomycete species richness in cacao soil assessed by massive amplicon sequencing of the cytochrome oxidase II gene M. RATTI (1), E. Goss (1), J. Cevallos-Cevallos (2), C. Arias (2) (1) Plant Pathology Department University of Florida U S A ; (2) Centro de Investigaciones Biotecnologicas del Ecuador CIBE vulgaris against all BCMV strains and against other potyviruses; bc-3 was identified as the eIF4E allele carrying mutated eukaryotic translation initiation factor gene. We characterized a novel BCMV isolate, 1755a, that was able to overcome bc-2 and bc-3 alleles in common bean. Hence, it displayed a novel pattern of interactions with resistance genes in P. vulgaris, and was assigned to a new pathogroup, PG-VIII. The IVT7214 cultivar supporting the replication of BCMV-1755a was found to have the intact homozygous bc-3 CAPS marker. The complete genome of the 1755a isolate was sequenced and found to be a recombinant between the BCMV strain NL1 (PG-I) and an unknown BCMV strain. Analysis of the recombination patterns in the genomes of NL1 and US1 (PG-I), NY15P (PG-V), US10 and RU1-OR (PG-VII), and 1755a (PG-VIII), S4.7 indicated that P1/HC-Pro cistrons of BCMV strains may interact with most resistance genes. This is the first report of a BCMV isolate able to overcome the bc-3 resistance in common bean. An inexpensive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus huanglongbing M. KEREMANE (1), C. Ramadugu (2), D. Hall (3) ive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus huanglongbing ANE (1) C Ramadugu (2) D Hall (3) An inexpensive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus hua M. KEREMANE (1), C. Ramadugu (2), D. Hall (3) (1) USDA ARS U S A (2) U i i f C lif i Ri id U S A (3) US H i l l R h L b U S A An inexpensive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus huanglongbing M. KEREMANE (1), C. Ramadugu (2), D. Hall (3) (1) USDA ARS, U.S.A.; (2) University of California Riverside, U.S.A.; (3) US Horticultural Research Laboratory, U.S.A. Huanglongbing (HLB) is threatening citrus cultivation in California and Texas. Testing for Liberibacter (Las, the HLB pathogen) by qPCR is labor- intensive and requires expensive instrumentation. The key to HLB management is large scale testing by many interested personnel. Development of on- site testing methods will increase the chance of pathogen detection by growers, homeowners and extension agents and enables timely implementation of disease management strategies. We describe an easy method to test psyllids (HLB vectors) for the presence of the Las pathogen. The initial equipment needed includes a heating block, table top centrifuge, tubes and reaction reagents. The cost for all of the equipment needed for testing by this method will be under $300. Psyllids are captured, stored in ethanol, air-dried, and DNA extracted by heating at 80°C in extraction buffer. Pathogen DNA is amplified using a loop-mediated amplification method at ambient temperatures (65°C) for 30 minutes. Samples positive for Las will appear yellow after the reaction and negative samples will remain pink. The whole reaction can be performed within an hour in a farmhouse by non-scientific personnel with very little training. Availability of methods for large scale testing will facilitate better disease management. Comparison of two deep sequencing methods for the detection of viruses in sweet potato R. LI (1), M. Cao (2), P. Lan (3), J. Abad (4), C. Zhou (5) (1) USDA-ARS, U.S.A.; (2) Southwest University of China, China; (3) Yunnan Agricultural University, China; (4) USDA-APHIS, U.S.A.; (5) Southwest University of China, China Current methods used to detect viruses from imported accessions in plant germplasm quarantine programs face multiple challenges due to many pathogen targets, mixed infections and high genetic diversity of some pathogens. An inexpensive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus huanglongbing M. KEREMANE (1), C. Ramadugu (2), D. Hall (3) In this study, deep sequencing of small interfering RNAs (siRNAs) and RNAs (HiSeq) were compared using a sweet potato plant infected with five RNA viruses [Sweet potato chlorotic stunt virus, Sweet potato feathery mottle virus, Sweet potato virus C, Sweet potato virus G, Sweet potato virus 2] and two DNA viruses [Sweet potato leaf curl virus and Sweet potato symptomless 1]. Several bioinformatic approaches were used to resolve the viral genes and detect viruses and their quasispecies. Direct mapping of both read types against a crop-specific virus database resulted in rapid detection of the seven known viruses, whereas the blast search of assembled contigs from the HiSeq reads had low call on the DNA viruses. However, the genomic sequences of the viruses, especially the RNA viruses, were characterized to a high level of detail when analyzing the contigs from the HiSeq reads, enabling the detection of quasispecies. Deep sequencing is a powerful technique that can facilitate the detection and characterization of numerous pathogens in a single test, but both the sequencing strategy and data analysis aspects require careful consideration. Fluorogenic Detection of Plant Viruses by Helicase Dependent Amplification with Self-Quenched Primers S. MOLINA CÁRDENAS (1), A. Salazar Aguirre (1), F. Ochoa-Corona (1), J. Olson (1) (1) Oklahoma State University, U.S.A. Thermophilic helicase dependent amplification (tHDA) is an isothermal DNA amplification that does not require temperature cycling. A study of primers sharing similar thermodynamics, but different GC content in their targeted amplification products, was made to assess fluorescence using self-quenched primers (SqP) reacting in tHDA standard temperature and chemistry. Model viruses used were Rose rosette virus (RRV), High plains wheat mosaic virus (HPWMV) formerly High plains virus (HPV) and Hosta virus X (HVX). RT-PCR was used as the reference method. Temperature gradients (60 to 70°C) were performed to each set of primers. tHDA reactions with SqP were optimized assessing combinations of two primer concentrations (0.15 and 0.2 µM) with two MgSO4 concentrations (4 and 4.5 mM). The sensitivity of each primer set was determined by quantitative tHDA (qtHDA) in real time. Subsequently the products amplified by qtHDA with SqP were visualized in 2% agarose gel electrophoresis. Detection limits using a plasmid DNA carrying the target sequences for RRV and HPWMV are 0.0001 ng. There was no amplification of the HVX targeted region due to its putative high GC content. Determination and characterization of a symptom determinant of Grapevine fanleaf virus L OSTERBAAN (1) L Osterbaan (1) M Fuchs (1) Bioinformatic analyzes pipelines for analysis of millions of sequences are also made and compared with qPCR testing for multiple targets. Comparing the Next Generation Sequencing consistent qPCR for the detection of forest pathogens species and allowed to find several species related targets of Phytophthora and related Fungi. Based on the results obtained, it was possible to identify potential sources of entry, get access to contents abundancy, biodiversity and phylogeny in order and so accelerate identification process of current risk to these species for biosurveillance. Determination and characterization of a symptom determinant of Grapevine fanleaf virus L OSTERBAAN (1) L Osterbaan (1) M Fuchs (1) McCluskey (3), R. Kaye (3), D. Clarke (4), N. Morant (4), D. Langton (5), F. Salinari (5), W. Charlton (6), M. Andreou (7) (1) Fera, York, United Kingdom; (2) Fera, United Kingdom; (3) University of Hertfordshire, United Kingdom; (4) Gensys Biotech Ltd, United Kingdom; (5) Agrii, United Kingdom; (6) Bayer Crop Sciences, United Kingdom; (7) Optisense Ltd, United Kingdom ( ), ( ) (1) Fera, York, United Kingdom; (2) Fera, United Kingdom; (3) University of Hertfordshire, United Kingdom; (4) Gensys Biotech Ltd, United Kingdom; (5) Agrii, United Kingdom; (6) Bayer Crop Sciences, United Kingdom; (7) Optisense Ltd, United Kingdom The UK wheat crop is valued at £2 billion p.a., approximately 12% of which is lost to disease. Septoria leaf blotch (Zymoseptoria tritici) is the most prevalent and fungicide sprays estimated at £82million p.a. (GFK-Kynetec 2013) are used for control. Resistance is a significant problem, caused by continued overuse of chemicals with similar modes of action. Better control requires more precision in terms of spray timing as well as more careful selection of fungicides. Linking this information with risk prediction by use of disease forecasting systems is also critical. We have been developing an in-field, self-reporting, automated spore detection system, to monitor ingress of inoculum into fields. Based on cyclone air sampling technology linked to isothermal DNA amplification (LAMP) the system will sample and test air within crops, transferring data on inoculum movement wirelessly to the laboratory. In addition to inoculum, identifying resistant genotypes of a pathogen is critical to enable effective spray decisions to be made. Work has started on the development of LAMP assays to discriminate the SNPs (single nucleotide polymorphisms) responsible for azole resistance in the CYP51 gene of Septoria. The assays will be deployed on the handheld Genie III instrument, enabling decisions to be made rapidly in the field. Ultimately the two developments will converge, telling us not just when we should spray crops but also what active chemistry will be most effective. Next generation sequencing evaluation for detection of potential threats or emerging forest pest, entry point from spore and insect traps G. BILODEAU (1), E. Tremblay (2), J. Determination and characterization of a symptom determinant of Grapevine fanleaf virus L OSTERBAAN (1) L Osterbaan (1) M Fuchs (1) Béribé (3) (1) Canadian Food Inspection Agency, Canada; (2) Canadian Food Inspection Agency, Canada; (3) Natural Resources Canada, Canada dian Food Inspection Agency, Canada; (2) Canadian Food Inspection Agency, Canada; (3) Natural Resources Canada, Cana S4.8 International trade promotes the spread of exotic plant pathogens (fungi, oomycetes) that can act as entry points. Their spores that spread by wind, rain and through vectors (insects) can cause the introduction of exotic diseases responsible for the devastation of various plant species. Since it is difficult to trap and conduct investigations to these organisms, new technologies such as qPCR and metagenomics therefore can now be an option to detect without having to grow them. The DNA is extracted for metagenomics of spore trapping filters and insect traps conservation liquid collected in 2013-2014 and then sequenced with the Ion Torrent platform using fusion primers which we designed in order to three multiplex genic regions (350-400bp amplicons from ribosomal DNA region ITS1 for fungi and oomycetes and the mitochondrial DNA region ATP9-NAD9 for Phytophthora sp) and then deciphered sequences. Bioinformatic analyzes pipelines for analysis of millions of sequences are also made and compared with qPCR testing for multiple targets. Comparing the Next Generation Sequencing consistent qPCR for the detection of forest pathogens species and allowed to find several species related targets of Phytophthora and related Fungi. Based on the results obtained, it was possible to identify potential sources of entry, get access to contents abundancy, biodiversity and phylogeny in order and so accelerate identification process of current risk to these species for biosurveillance. International trade promotes the spread of exotic plant pathogens (fungi, oomycetes) that can act as entry points. Their spores that spread by wind, rain and through vectors (insects) can cause the introduction of exotic diseases responsible for the devastation of various plant species. Since it is difficult to trap and conduct investigations to these organisms, new technologies such as qPCR and metagenomics therefore can now be an option to detect without having to grow them. The DNA is extracted for metagenomics of spore trapping filters and insect traps conservation liquid collected in 2013-2014 and then sequenced with the Ion Torrent platform using fusion primers which we designed in order to three multiplex genic regions (350-400bp amplicons from ribosomal DNA region ITS1 for fungi and oomycetes and the mitochondrial DNA region ATP9-NAD9 for Phytophthora sp) and then deciphered sequences. uel Roberts Noble Foundation, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.; (3) USDA-ARS-SEFTNRL, U.S An inexpensive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus huanglongbing M. KEREMANE (1), C. Ramadugu (2), D. Hall (3) This study explored primer design criteria for tHDA with SqP for sensitive detection of plant viruses. The use of SqP reduces the cost of qPCR and qtHDA and has acceptable levels of sensitivity. tHDA with SqP bring new possibilities for field deployment primer design in biosecurity and microbial forensics. Long-term rotation history and previous crop effects on corn seedling health M. BENITEZ (1), S. Osborne (2), M. Lehman (2) (1) USDA, Northe Central Agricultural Research Laboratory, U.S.A.; (2) USDA, N Long-term rotation history and previous crop effects on corn seedling health M. BENITEZ (1), S. Osborne (2), M. Lehman (2) (1) USDA, Northe Central Agricultural Research Laboratory, U.S.A.; (2) USDA, North Central Agricultural Research Laboratory, U.S.A. M. BENITEZ (1), S. Osborne (2), M. Lehman (2) (1) USDA, Northe Central Agricultural Research Laboratory, U.S.A.; (2) USDA, North Central Agricultural Research Laboratory, U.S.A. ( ), ( ), ( ) (1) USDA, Northe Central Agricultural Research Laboratory, U.S.A.; (2) USDA, North Central Agricultural Research Laboratory, U.S.A. Diverse rotations provide benefits to agroecosystems through changes in the soil environment. A long term experiment was established to study four different four-year rotation sequences in which the crop phase prior to corn was sampled. Soils from rotations ending with soybean, sunflower, corn and pea were tested for effects on subsequent corn in the greenhouse. Corn was grown in a soil mix containing 12% of rotation soil either infested with corn root worms (CRW) or inoculated with Fusarium graminearum. Seedlings were scored for root damage and shoot vigor, rhizosphere samples collected for microbiome analyses and remaining roots scanned for root measurements. Seedlings grown in soils from rotations ending with pea and sunflower had greater vigor regardless of CRW infestation. Seedlings grown in non-inoculated soils from pea rotation had shorter roots but greater root volume and diameter. Differences in germination time and vigor were stronger between Fusarium-inoculated and non-inoculated soils originating from pea rotations, compared to other rotations. The response to the rotation ending in pea is consistent with field observations where corn grown after pea requires less fertilizer. Analyses of the rhizosphere microbiome and seedling nutrient content, together with soil physico-chemical characteristics will provide insight on the mechanisms through which corn respond positively to rotation sequences incorporating pea. Evidence for sexual reproduction in Fusicladium effusum N. CHARLTON (1), C. Mattupalli (2), B. Wood (3), C. Bock (3), C. Model for the geographic origin and spread of the globally distributed pathogen Phytophthora palmivora J. Wang (1), E. Goss (1), M. Coffey (1) (1) University of Florida, U.S.A. Model for the geographic origin and spread of the globally distributed pathogen Phytophthora palmivora J. Wang (1), E. Goss (1), M. Coffey (1) (1) University of Florida, U.S.A. The genetic structure and diversity of plant pathogens are shaped by their evolutionary history, including coevolutionary interactions with hosts, geographic spread, and long-distance migration events, often mediated by humans. In turn, diversity and dispersal of present populations impact the risk of disease epidemics and strategies for controlling disease. Phytophthora palmivora is a globally distributed oomycete that infects a broad range of cash crops and fruit trees in the tropics and subtropics, including cocoa and palms. The center of diversity of P. palmivora is in Southeast Asia, but it is an important and widespread pathogen in South America. Our multilocus sequence analysis showed that the centers of origin of the pathogen are located in the Philippines and Indonesia. We then tested alternative models for the regional and global movement of P. palmivora. We found genetic variation consistent with historical movement among Pacific Islands, likely associated with coconut. We also found evidence of a bridgehead effect in South America, in which the colonization of South America and host shift to cocoa led to further global dispersal, including gene flow back to Southeast Asia. We propose that the extensive genetic diversity in P. palmivora in Southeast Asia is the result of a complex history, including long-term co-evolution with native hosts, geographic isolation with migration, and re-introduction of genotypes from South America. The in planta regulon of the major Ralstonia solanacearum virulence regulator PhcA D. KHOKHANI (1), T. Tran Minh (1), C. Allen (1) (1) University of Wisconsin-Madison, U.S.A. PhcA, an important quorum sensing responsive virulence regulator of the bacterial wilt pathogen Ralstonia solanacearum. PhcA affects expression of virulence factors like extracellular polysaccharide, endoglucanase, and motility. However, the full extent of the regulon is unknown. We used RNAseq to test the hypothesis that the PhcA system enables Rs to transition from saprophytic to parasitic mode. An impressive 634 genes were differentially regulated at early stage of tomato wilt disease development in ΔphcA compared to the wild-type strain GMI1000, with false discovery rate of less than 0.005. Both, RNA-seq data and validation experiments indicated that multiple anion and sugar transporters are expressed at higher levels in the ΔphcA, suggesting that the pathogen uses this regulator to improve its survival at low cell densities. Model for the geographic origin and spread of the globally distributed pathogen Phytophthora palmivora J. Wang (1), E. Goss (1), M. Coffey (1) (1) University of Florida, U.S.A. Increased expression of hemagglutinins and other adhesins in ΔphcA suggest that at low population densities R. solanacearum cells may adhere more to plant surfaces and to each other. Scanning electron microscopy revealed that ΔphcA cells form thick mats in tomato xylem vessels. These were strikingly different from the smaller, looser in planta aggregates of wild-type cells. Further, ΔphcA was largely restricted near the point of infection whereas wild-type colonized tissues beyond the infection site. Taken together, the study identified new targets of PhcA regulation and shed more light on their biological roles in the disease cycle. Determination of CsrA/RsmA regulon in Xanthomonas citri subsp. citri M. ANDRADE (1) (1) Dept. Microbiology and Cell Science, CREC- University of Florida, U.S. In bacteria, it is poorly understood how post-transcriptional regulation mediated by RNA-binding proteins contributes to adaptation to environment changes during host infection. RsmA (Regulator of secondary metabolism) is a small RNA-binding protein that plays an important role in regulation of pathogenicity, motility, biofilm formation, endoglucanases and exopolysaccharide production in Xanthomonas citri subsp. citri (Xcc). In previous work, we showed that RsmA contributes to the pathogenicity by activating expression of genes encoding the type 3 secretion system (T3SS) and effectors in Xcc. However, the mechanistic understanding of Xcc RsmA in regulating motility, biofilm formation and production of exopolysaccharide and endoglucanases remains unclear. We have performed crosslinking and affinity purification followed by RNAseq analysis in order to determine the RsmA targets in Xcc cells in both rich medium and a T3SS-inducing medium. Our data revealed hundreds of putative binding mRNA targets in Xcc. Functional classification (Gene Ontology) of RsmA mRNA targets suggests Xcc RsmA is a global RNA-binding regulator that can positively and negatively affect the expression of genes in Xanthomonas. An inexpensive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus huanglongbing M. KEREMANE (1), C. Ramadugu (2), D. Hall (3) Young (2) (1) The Samuel Roberts Noble Foundation, U.S.A.; (2) The Samuel Roberts Nobl Evidence for sexual reproduction in Fusicladium effusum N. CHARLTON (1), C. Mattupalli (2), B. Wood (3), C. Bock (3), C. Young (2) (1) The Samuel Roberts Noble Foundation, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.; (3) USDA-ARS-SEFTNRL, U.S.A. Fusicladium effusum is the causal agent of pecan scab, the most prevalent and often catastrophic disease of pecan in the southeastern USA. Despite earlier efforts to determine a sexual stage, reproduction in F. effusum has been observed only by asexually produced conidia. However, the degree and distribution of genetic diversity observed within and among populations of F. effusum are typical of a sexually reproducing fungus. The mating type locus was identified in a draft genome of F. effusum and was amplified and sequenced in 14 isolates collected from different geographic locations and S4.9 cultivars, revealing that 50% of the samples contained the mtAA idiomorph and are considered mating type A (MTA). The remaining samples contained mtBA and are mating type B (MTB). A multiplex PCR screen was developed to amplify mtAA, mtBA and tubB, and used to screen 1203 isolates of F. effusum from 13 pecan populations across the southeastern USA. Analysis of this collection revealed the frequency of the mating type idiomorphs is in a 1:1 equilibrium. Select isolates were crossed in pairwise combinations on oatmeal agar and after six months, organs typical of immature sexual fruiting bodies were observed, indicating existence of a putative sexual stage. Identification of a sexual stage will provide improved understanding of the disease epidemiology and pathogen population genetics, and potentially provide insight into novel approaches to manage this damaging disease. cultivars, revealing that 50% of the samples contained the mtAA idiomorph and are considered mating type A (MTA). The remaining samples contained mtBA and are mating type B (MTB). A multiplex PCR screen was developed to amplify mtAA, mtBA and tubB, and used to screen 1203 isolates of F. effusum from 13 pecan populations across the southeastern USA. Analysis of this collection revealed the frequency of the mating type idiomorphs is in a 1:1 equilibrium. Select isolates were crossed in pairwise combinations on oatmeal agar and after six months, organs typical of immature sexual fruiting bodies were observed, indicating existence of a putative sexual stage. An inexpensive visual PCR method for field detection of Candidatus Liberibacter asiaticus associated with citrus huanglongbing M. KEREMANE (1), C. Ramadugu (2), D. Hall (3) Identification of a sexual stage will provide improved understanding of the disease epidemiology and pathogen population genetics, and potentially provide insight into novel approaches to manage this damaging disease. Ecological Impact of Signaling Chemicals on Phytophthora erythroseptica H. JIANG (1), J. Hao (1) (1) University of Maine, U.S.A. Ecological Impact of Signaling Chemicals on Phytophthora erythroseptica H. JIANG (1), J. Hao (1) (1) University of Maine, U.S.A. Phytophthora erythroseptica causes pink rot of potato, mostly with zoospores as major primary inocula. Zoosporic germination and infection of host (potato) are regulated by signaling molecules with quorum sensing mechanism. This study was to determine if the signals were species specific and if they were impacted by the environment. Exudates were obtained from high concentrations of fungi, bacteria, and oomycetes, as well as soil and roots of potato varieties. The exudates were used to treat zoospores of P. erythroseptica. Results showed that several soil microorganisms, including P. capsici, P. cactorum, Pythium irregulare, Serratia sp., Delftia sp., and Microbacterium sp. induced zoosporic germination of P. erythroseptica; Pseudomonas spp. and Bacillus amyloliquefaciens inhibited zoosporic germination; exudates of Dickeya dadantii and Pectobacterium carotovorum had no effect on zoosporic germination. Root exudate from the pink-rot-sensitive variety, ‘Dark Red Norland,’ induced zoosporic germination, while tolerant variety ‘Snowden’ did not. These results indicate that the zoosporic behavior is not only self-regulated, but also affected by other environmental organisms, such as host plants and soil microorganisms through signaling molecules. Whether same signals are shared among organisms is being investigated. This result may improve our understanding of the initiation and development of potato pink rot and contribute to breeding resistant potato cultivars. Characterization of RpfB-dependent DSF-Family Quorum Sensing Signal Turnover System in the Phytopathogen Xanthomonas L. ZHOU (1), X. Wang (1), Y. He (1) (1) Shanghai Jiao Tong University, China We demonstrate that host plant-derived reactive oxygen species (ROS) are one environmental signal that modulates the Rcs system and this stimulation is dependent on the redox sensing transcription factor, OxyR. In addition, we demonstrate a link between a novel RTX- like, cytolytic protein in lysing host plant cells, releasing ROS and stimulating the Rcs system and subsequent EPS production, thus aiding in transition between the apoplastic and xylem phases of Stewart’s wilt disease. Epidemiology and Population Biology of Grape Downy Mildew (Plasmopara viticola) in Georgia C. HONG (1), P. Brannen (1), H. Scherm (1) (1) University of Georgia, U.S.A. The epidemiology of Plasmopara viticola has been studied extensively in temperate and Mediterranean climates, but limited information is available for the southeastern U.S. where hot and humid conditions may affect survival, disease progression, and population structure of the pathogen. Downy mildew epidemic progress was monitored in 2015 in a North Georgia Vitis vinifera vineyard on cultivars Merlot and Chardonnay. Disease onset was 2 months later than predicted by a mechanistic model for oospore germination and primary infection, highlighting knowledge gaps relative to epidemiological processes in Georgia conditions. Since timing of disease onset may be related to the mode of pathogen overwintering, pairings among single-sporangium isolates were conducted to determine the potential for sexual reproduction. Oospores were observed in about half of the pairings, indicating that both mating types are present. To better understand the relative contribution of sexual and asexual reproduction and to determine the population structure of the pathogen, isolates of P. viticola were collected from 19 grape varieties in 10 vineyards throughout Georgia. Initial genotyping of 96 isolates identified only one of the previously described five cryptic species of P. viticola, clade aestivalis. This study confirmed sexual reproduction, revealed preliminary pathogen population structure, and identified knowledge gaps regarding primary infection by P. viticola in Georgia vineyards. Changes in Plasmopara obducens population structure corresponded with the emergence and rapid spread of the impatiens downy mildew epidemics C. SALGADO-SALAZAR (1), Y. Rivera (1) (1) USDA-ARS, Rutgers University, U.S.A. Plasmopara obducens is the newly emergent oomycete responsible for impatiens downy mildew disease (IDM) on cultivated Impatiens walleriana. Since IDM outbreaks began in 2004, the disease has reduced the $187 million annual production of impatiens in the U.S. by 40%. In this study, we evaluated if there was a change in P. obducens population structure associated with the U.S. epidemic. Characterization of RpfB-dependent DSF-Family Quorum Sensing Signal Turnover System in the Phytopathogen Xanthomonas L. ZHOU (1), X. Wang (1), Y. He (1) (1) Shanghai Jiao Tong University, China Characterization of RpfB-dependent DSF-Family Quorum Sensing Signal Turnover System in the Phytopathogen Xanthomonas L. ZHOU (1), X. Wang (1), Y. He (1) (1) Shanghai Jiao Tong University, China Molecules of the diffusible signal factor (DSF)-family are a class of quorum sensing (QS) signals used by the phytopathogens Xanthomonas. RpfB has recently been reported as a fatty acyl-CoA ligase (FCL) that activates a wide range of fatty acids to their CoA esters in vitro. Through genetic and biochemistry analysis, we found that RpfB represents a naturally occurring DSF-family signal turnover system in X. campestris pv. campestris (Xcc) and X. oryzae pv. oryzae (Xoo). RpfB effectively turns over DSF-family signals DSF and BDSF in vivo. RpfB FCL enzymatic activity is required for DSF and BDSF turnover in Xcc and Xoo. The expression of rpfB is growth phase-dependent and its expression is significantly enhanced when Xanthomonas cells enter the stationary phase. DSF regulates rpfB expression in a concentration-dependent manner. rpfB expression is also negatively regulated by the DSF signaling components RpfC, RpfG, and Clp. Although most of our findings have suggested that the DSF-family signal turnover systems are generally consistent in Xcc and Xoo, we also identified some discrepancies in bacterial pathogenesis and virulence factor production between the rpfB mutants of Xcc and Xoo. Therefore, our findings strongly indicated the complexity of the RpfB-dependent DSF-family signal turnover system, which is likely fine-tuned to facilitate infection process in different hosts (rice versus crucifers). Integration of plant host-derived signals to coordinate developmental processes involved in Pantoea stewartii subsp. stewartii biofilm maturation M. ROPER (1), M. Roper (1) (1) University of California, Riverside, U.S.A. Participation in complex, multicellular cooperative communities, known as biofilms, is a universal attribute of bacteria. Biofilm formation is now a major tenet of microbiology, yet we know little information about what environmental and physical cues drive biofilm formation in a natural context, such as during bacterial infection processes. Biofilms are encased in an extracellular matrix consisting primarily of exopolysaccharide (EPS), which holds these biofilms together, providing robustness to the architecture and making them difficult to clear from the host. The focus of this project is on the xylem- dwelling bacterium, Pantoea stewartii subsp. stewartii (Pss)/sweet corn pathosystem. We have placed our attention on the multi-component Rcs signal transduction system, an environmental sensing system found in Pss and other enteric bacteria. This system controls genes related to biofilm formation, namely EPS matrix production. Emergent rudbeckia downy mildew epidemics caused by Plasmopara halstedii are genetically distinct from the pathogen populations infecting sunflower Emergent rudbeckia downy mildew epidemics caused by Plasmopara halstedii are genetically distinct from the pathog sunflower Characterization of RpfB-dependent DSF-Family Quorum Sensing Signal Turnover System in the Phytopathogen Xanthomonas L. ZHOU (1), X. Wang (1), Y. He (1) (1) Shanghai Jiao Tong University, China Over 900 pre- and post-epidemic P. obducens samples were genotyped using 20 polymorphic microsatellite markers developed from a de novo genome assembly. Population structure analysis identified five populations (P1-P5). Prior to IDM epidemics, P1 dominated, existing as pure P1 or admixed with the other four genotypes (~50%) on North American native Impatiens species. The first epidemic IDM specimen collected on I. walleriana in 2004 from Tennessee showed an almost pure P1 genotype. Approximately 50% of the post-epidemic samples showed admixed genotypes, but admixture between P1 and the other four genotypes was uncommon. Genetic diversity was high for the four most abundant populations P2-P5 responsible for the IDM epidemics (0.56-0.78), but lower for P1 (0.37). These data showed that the recent emergence of IDM in the U.S. corresponded with several major shifts in P. obducens populations, with endemic P1 genotypes supplanted by novel genotypes. The high level of diversity and genetic structure in P. obducens populations may present difficulties for durable disease control. Construction of phosphodiesterase mutants in Erwinia amylovora to evaluate the role of cyclic di-GMP in virulence regulation R. KHARADI (1), R. Kharadi (1), L. Castiblanco (1), G. Sundin (1) (1) Michigan State University, U.S.A. Construction of phosphodiesterase mutants in Erwinia amylovora to evaluate the role of cyclic di-GMP in virulence regulation R. KHARADI (1), R. Kharadi (1), L. Castiblanco (1), G. Sundin (1) (1) Michigan State University, U.S.A. The second messenger molecule cyclic-di-GMP (c-di-GMP) is a key regulator of biofilm formation, motility and virulence in bacteria. Diguanylate cyclase (DGC) enzymes function in the synthesis of c-di-GMP and phosphodiesterase (PDE) enzymes are involved in the degradation of c-di-GMP. We have previously studied the role of DGCs in regulating virulence traits in the fire blight pathogen Erwinia amylovora. Here we demonstrate that all three predicted PDE genes (edp genes, for Erwinia diguanylate phosphodiesterase), edpA, edpB and edpC negatively regulate amylovoran production and biofilm formation. This was correlated with a significant increase in the volume of ooze produced and the amylovoran content of ooze in an immature- pear infection model using the single-gene deletion mutants DedpA, DedpB, and DedpC. We also observed a significant decrease in motility, virulence in immature pears, and systemic infection levels in apple shoots in the triple-gene deletion mutant DedpA/edpB/edpC. Gene expression levels of dspE and hrpL, both indicators of type III secretion in E. amylovora, were significantly reduced in DedpA/edpB/edpC compared to the wild type. Thus, c-di- GMP seems to be primarily involved in the positive regulation of amylovoran production and biofilm formation; in addition, increased levels of c-di- GMP also negatively regulate motility and type III secretion during the pathogenesis of E. amylovora. S4.10 Emergent rudbeckia downy mildew epidemics caused by Plasmopara halstedii are genetically distinct from the pathogen populations infecting sunflower Many non-commercial hosts are grown on a small scale or occur as weeds and their contributions to the yearly P. cubensis epidemic are understudied. North Carolina (NC) has diverse cucurbit production and previous studies have found that NC has relatively high genetic diversity of P. cubensis. In this study, Simple Sequence Repeats (SSRs) were identified in silico from the P. cubensis predicted transcriptome. A subset of the identified markers were screened and evaluated for reliability and polymorphism. Eleven markers were selected and applied to P. cubensis isolates collected from six commercial and three non-commercial cucurbits across four time points and three regions in NC. Population analyses revealed that the greatest genetic differentiation occurs when isolates are grouped by host species more so than when grouped by location or time. These findings show host species are playing the most significant role in P. cubensis diversification in NC. The results suggest non-commercial cucurbits could be contributing to P. cubensis inoculum for certain hosts more so than others, thus influencing the population dynamics of the pathogen. Species diversity and population structure of Pythium and Globisporangium in Long Island, NY ornamental greenhouses in 2014 M. PROANO (1), C. Ayala (2), A. Chiriboga (2), M. Daughtrey (3), C. Garzon (4), S. Marek (1), H. Melouk (1), S. Marek (4) (1) Oklahoma State University, Stillwater, OK, U.S.A.; (2) Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador, Ecuador; (3) Cornell University, Long Island Horticultural Research & Extension Center, Riverhead, NY, U.S.A.; (4) Oklahoma State University, U.S.A. Species diversity and population structure of Pythium and Globisporangium in Long Island, NY ornamental greenhouses in 2014 M. PROANO (1), C. Ayala (2), A. Chiriboga (2), M. Daughtrey (3), C. Garzon (4), S. Marek (1), H. Melouk (1), S. Marek (4) (1) Oklahoma State University, Stillwater, OK, U.S.A.; (2) Universidad de las Fuerzas Armadas ESPE, Sangolquí, Ecuador, Ecuador; (3) Cornell University, Long Island Horticultural Research & Extension Center, Riverhead, NY, U.S.A.; (4) Oklahoma State University, U.S.A. Globisporangium and Pythium are plant pathogens that cause root rots, blackleg of cuttings, and damping off of various crops. Growth of the nursery and floriculture production industries, one of the fastest growing agricultural sectors in the United States, has contributed to the increase of the economic impact of ornamental plant diseases. Emergent rudbeckia downy mildew epidemics caused by Plasmopara halstedii are genetically distinct from the pathogen populations infecting sunflower Y. RIVERA (1), C. Salgado-Salazar (1), T. Gulya (2), J. Crouch (3) (1) USDA-ARS Systematic Mycology and Microbiology Laboratory and Rutgers University, Beltsville, MD, U.S.A.; (2) USDA ARS Sunflower and Plant Biology Research Unit, Fargo, ND, U.S.A.; (3) USDA-ARS Systematic Mycology and Microbiology Laboratory, Beltsville, MD, U.S.A. (1) USDA-ARS Systematic Mycology and Microbiology Laboratory and Rutgers University, Beltsville, MD, U.S.A.; (2) USDA ARS Sunflower and Plant Biology Research Unit, Fargo, ND, U.S.A.; (3) USDA-ARS Systematic Mycology and Microbiology Laboratory, Beltsville, MD, U.S.A. The oomycete Plasmopara halstedii is regarded as the causal agent of downy mildew disease on 35 Asteraceae genera. The pathogen is also a significant problem of sunflower (Helianthus annuus) worldwide and more recently, a destructive pathogen of ornamental Rudbeckia fulgida (rudbeckia) in the U.S.A. As with other recent downy mildew epidemics on specialty crops, little is known about the pathogen causing modern outbreaks on rudbeckia. To determine if modern epidemics on rudbeckia are caused by the same P. halstedii genotypes as those historically found on native rudbeckia or to those causing disease on sunflower, we performed population genetic and phylogenetic analyses. Fifteen microsatellite markers were designed from a draft genome assembly generated from a P. halstedii isolate on sunflower. Phylogenetic and microsatellite analyses of 232 samples collected between 1883- 2014 revealed two main groups corresponding to host origin. Isolates infecting sunflower hosts were further separated into eight subgroups with no obvious pattern by year, physiological race or geographical origin. Isolates infecting rudbeckia hosts were further separated into three subgroups, one corresponding to historical pre-epidemic specimens and two modern subgroups. Our results indicate that the emergent populations of P. halstedii infecting rudbeckia are a different species from those infecting sunflower and genetically distinct from pre-epidemic populations infecting native rudbeckia. S4.11 Pseudoperonospora cubensis on commercial and non-commercial cucurbits in North Carolina: Population structure determined by simple sequence repeats E. WALLACE (1), L. Quesada-Ocampo (2) (1) NCSU, U.S.A.; (2) NCSU, U.S.A. Pseudoperonospora cubensis on commercial and non-commercial cucurbits in North Carolina: Population structure determined by simple sequence repeats E. WALLACE (1), L. Quesada-Ocampo (2) (1) NCSU, U.S.A.; (2) NCSU, U.S.A. The cucurbit downy mildew pathogen, Pseudoperonospora cubensis, is a major limiting factor in cucumber production in the United States (US). Each year, P. cubensis causes foliar destruction on cucumber, but it also infects approximately 60 host species in the Cucurbitaceae family. All major commercial cucurbits are hosts in the US. Emergent rudbeckia downy mildew epidemics caused by Plasmopara halstedii are genetically distinct from the pathogen populations infecting sunflower Seasonal assessment of the diversity of species causing Pythium diseases on three ornamental greenhouses from Long Island, NY is providing information about the temporal dynamics of Pythium and Globisporangiumspecies in these agricultural systems. DNA sequences of the ITS region were obtained to confirm the identity of 207 isolates collected seasonally from three ornamental operations in 2014. As in previous years, G. irregularesensu lato was the predominant pathogen isolated (n=126). Simple sequence repeats (SSRs) were used to characterize the structure of G. irregulare s.l. populations. PCoA of ten SSR loci defined four groups based on genetic distances. AMOVA revealed significant genetic differentiation between greenhouse populations and between genetic lineages. The main lineage included most of the isolates present in two greenhouses, which suggest a potential common source of inoculum for those facilities. ADEPIDYN™ fungicide: Cross resistance patterns in Alternaria solani G. OLAYA (1), R. Linley (2), K. Edlebeck (2), T. Harp (3) (1) Syngenta, U.S.A.; (2) Syngenta, U.S.A.; (3) Syngenta, U.S.A. ADEPIDYN™ is the first member of a new chemical group among the succinate dehydrogenase inhibitor fungicides (SDHI, FRAC Group 7), the N- methoxy-(phenyl-ethyl)-pyrazole-carboxamides. The common name for ADEPIDYN™ is pydiflumetofen. ADEPIDYN™ has high binding properties to the complex II enzymes that provide a strong fungicidal potency combined with the control of a wide range of plant pathogen species. A variety of mutations are associated with resistance to SDHI fungicides. Cross resistance patterns across SDHI fungicides are complex due to the fact that some mutations confer high levels or resistance and others do not and mutations could lead to different sensitivity shifts in different SDHIs. In Alternaria solani isolates, the in vitro sensitivity revealed incomplete cross resistance patterns between ADEPIDYN™ and boscalid, penthiopyrad, SOLATENOL™ and fluopyram. ADEPIDYN™ exhibited the highest intrinsic activity among these fungicides and across isolates with or without mutations in the sdh subunits (SdhB-H277Y, SdhB-H277R, SdhC-H134R, SdhD-D123E abd SdhD-H133R). ADEPIDYN™ was able to control early blight disease development on inoculation studies done with A. solani isolates sensitive and with different levels of resistance to boscalid and Sdh mutations. Fluopyram was also able to control the disease using higher fungicide rates and disease control with SOLATENOL™, penthiopyrad and boscalid varied according to the presence or absence of Sdh mutations. Fungicide resistance profiles of unique and clonal genotypes of gummy stem blight fungi from the southeastern United States H. LI (1), K. Stevenson (2), M. Brewer (1) (1) University of Georgia, U.S.A.; (2) University of Georgia, U.S.A. Fungicide resistance profiles of unique and clonal genotypes of gummy stem blight fungi from the southeastern United States H. LI (1), K. Stevenson (2), M. Brewer (1) (1) University of Georgia, U.S.A.; (2) University of Georgia, U.S.A. Gummy stem blight (GSB) is a devastating disease of cucurbits that is usually managed with fungicide applications. However, GSB fungi have rapidly evolved resistance to multiple classes of fungicides. To better understand the evolution and persistence of fungicide resistance in field populations, we studied resistance profiles associated with unique and clonal genotypes of GSB fungi. Nineteen Stagonosporopsis caricae isolates and 120 S. citrulli isolates collected from watermelon fields in the southeastern U.S. were genotyped with 16 SSR markers and a cytb marker for azoxystrobin (QoI) resistance, and phenotyped for sensitivity to tebuconazole (DMI), boscalid (SDHI), and fluopyram (SDHI). Cyp51 and sdhB genes of select isolates with varying sensitivity to DMI and SDHI fungicides, respectively, were sequenced. All S. caricae isolates were resistant to tebuconazole and azoxystrobin, and sensitive to boscalid and fluopyram. All S. citrulli isolates were sensitive to tebuconazole, sensitive to fluopyram with one exception, and resistant to azoxystrobin with two exceptions. Phenotypic differences in response to boscalid were detected among S. citrulli isolates, but the phenotype was not associated with clonal genotype, suggesting multiple evolution events. This study provides critical information for effectively managing both species of GSB fungi present in the southeastern U.S., while providing novel insights on the evolution of resistance in plant-pathogenic fungi. Survey for Quinone Outside Inhibitor Resistant Cercospora sojina in Virginia Soybeans T. ZHOU (1) H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A. Since the tomato and strawberry Elongator gene homologues are functional, they could be utilized to generate tomato and strawberry plants with increased disease resistance, using alternative technologies that are not considered transgenic, such as cisgenesis and intragenesis. Speed g up e d scove y o ove sou ces o e us es s ce w e A. RIAZ (1), N. Athiyannan (2), S. Periyannan (3), O. Afanasenko (4), O. Mitrofanova (5), E. Aitken (6), E. Lagudah (3), L. Hickey (2) (1) The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, St Lucia, QLD 4072, Australia, Brisbane, QC, Australia; (2) The University of Queensland, Queensland Alliance for Agriculture and Food Innovation, St Lucia, QLD 4072, Australia, Australia; (3) Commonwealth Scientific and Industrial Research Organization (CSIRO), Australia; (4) Department of Plant Resistance to Diseases of All Russian Research Institute for Plant Protection, Pushkin, 196608, Russia, Russia; (5) N. I. Vavilov Institute of Plant Genetic Resources, Bolshaya Morskaya 42- 44, Saint Petersburg, 190000, Russia, Russia; (6) The University of Queensland, School of Agriculture and Food Science, St Lucia, QLD 4072, Australia Leaf rust (LR) caused by Puccinia triticina, is among the most important diseases of wheat (Triticum aestivum L.) crops globally. Deployment of cultivars incorporating genetic resistance, particularly the combination of adult plant resistance (APR) and all stage resistance (ASR), is considered to be the most effective and sustainable method to control rust pathogen. While scoring of ASR in the glasshouse is straight forward, identification of APR in the field is often challenging due to varying micro environments. To overcome this, here we report a novel screening procedure that involves application of a rapid phenotyping method performed under accelerated growth conditions (AGC; i.e. constant light and controlled temperature) and effective elimination of known APR genes using linked DNA markers. Using this approach, screening of 300 diverse wheat accessions from the N. I. Vavilov Institute of Plant Genetic Resources (VIR) identified a set of 50 wheat accessions as potential sources for new APR to LR. In the subsequent screening of the subset under field conditions, 13 accessions consistently displayed good levels of APR. Further, in the DNA marker screening of the 300 accessions, 88 were identified to carry known APR genes (Lr34, Lr46 and Lr67). Interestingly, accessions carrying Lr67 were mostly landraces from India and Pakistan, consistent with previous reports about the likely origin of this APR gene. Survey for Quinone Outside Inhibitor Resistant Cercospora sojina in Virginia Soybeans T. ZHOU (1) H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A. ( ), y ( ), ( ), g ( ) (1) University of Tennessee-Entomology & Plant Pathology, Jackson, TN, U.S.A.; (2) University of Tennessee-Entomology & Plant Pathology, U.S.A.; (3) United States Department of Agriculture, Agricultural Research Service (USDA-ARS), U.S.A. ( ), y ( ), ( ), g ( ) (1) University of Tennessee-Entomology & Plant Pathology, Jackson, TN, U.S.A.; (2) University of Tennessee-Entomology & Plant Pathology, U.S.A.; (3) United States Department of Agriculture, Agricultural Research Service (USDA-ARS), U.S.A. Frogeye leaf spot (FLS), caused by Cercospora sojina, is a foliar disease affecting soybean, often managed by applications of Quinone outside inhibitor (QoI) fungicides. Because C. sojina isolates have developed resistance to QoIs, a better understanding of the biology and aggressiveness of isolates is important. Spore suspension inoculum prepared from QoI-resistant and -sensitive isolates were mixed in six different ratios. Greenhouse plants (cultivar Blackhawk) were treated: water (control), Quadris, or Quadris Top. Results were:1) Disease severity increased as the proportion of QoI resistance increased, indicating that QoI-resistant isolates are more aggressive; 2) Quadris inhibited FLS caused by QoI-sensitive isolates only, and when the proportion of QoI-resistance was ≤ 10%; 3) Quadris Top provided more effective control of FLS by reducing symptoms and delaying disease develop- ment. Fitness parameters measured were (i) mycelial growth and sporulation capacity, (ii) conidial germination and growth inhibition on fungicide amended media. No significant differences of the fitness parameters were observed between resistant and sensitive isolates, except for sporulation capacity and growth inhibition. Moreover, qPCR results showed resistant isolates dominated the population in all mixed inoculation tests. These results indicate there are differences in QoI resistant and sensitive C. sojina isolates in-planta that will influence effective disease management of FLS. Assessing potential virulence differences between QoI-sensitive and -resistant Cercospora sojina isolates from Mississippi soybean N. BROCHARD (1), M. Tomaso-Peterson (2), T. Allen (3), R. Melanson (3) (1) Mississippi State University, U.S.A.; (2) Mississippi State Unversity, U.S.A.; (3) Mississippi State Unversity, U.S.A. Assessing potential virulence differences between QoI-sensitive and -resistant Cercospora sojina isolates from Mississippi soybean N. BROCHARD (1), M. Tomaso-Peterson (2), T. Allen (3), R. Melanson (3) (1) Mississippi State University, U.S.A.; (2) Mississippi State Unversity, U.S.A.; (3) Mississippi State Unversity, U.S.A. Frogeye leaf spot (FLS) of soybean is caused by the fungus Cercospora sojina Hara. Quinone outside inhibitor (QoI) fungicides have historically been the standard for FLS management in MS. Survey for Quinone Outside Inhibitor Resistant Cercospora sojina in Virginia Soybeans T. ZHOU (1) H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A. However, widespread QoI resistance has been reported in the MS soybean production system. QoI resistance may be associated with a fitness cost that includes a loss of virulence. A comparison of virulence was assessed between two C. sojina sensitivity groups based on the presence or absence of the G143A substitution and termed QoI-sensitive or -resistant. Twenty four C. sojina isolates, 11 QoI-sensitive and 13 QoI-resistant, were assayed in an in planta virulence assessment. Soybean seedlings were inoculated with conidial suspensions of each C. sojina isolate. Percent FLS severity assessments were made 21 days post inoculation. QoI-sensitive and -resistant C. sojina isolates were pathogenic to soybean seedlings based on the production of typical FLS lesions. Disease severity significantly differed among isolates within sensitivity groups. No differences in disease severity were noted between sensitivity groups; however, a 4.5% increase in severity was noted for the QoI-resistant group. This suggests QoI- resistant isolates are equally virulent as QoI-sensitive isolates, indicating no fitness cost associated with QoI-resistance. Failure to control C. sojina with QoIs dictates the need for alternative management practices when high-yielding FLS-susceptible cultivars are planted in MS. Characterization of Arabidopsis Defense-related Gene Homologues in Tomato and Strawberry J. PEREIRA (1), K. Silva (2), M. Garald (3), J. Jeffrey (3), Z. Mou (3) (1) University of Florida, U.S.A.; (2) Salve Regina University, U.S.A.; (3) University of Florida, U.S.A. The United States is the largest strawberry producer, and second in tomato. Yield of these crops is threatened by a number of diseases. Durable measures for disease management have been difficult to develop. One strategy that is being pursued is utilizing defense-related genes from Arabidopsis thaliana and their homologues in other plants. The Arabidopsis Elongator complex plays an important role in plant immunity. Elongator is not specifically involved in pathogen recognition, subsequently, it may provide durable resistance. Our results show that overexpressing Arabidopsis Elongator genes in tomato and strawberry improves resistance to several bacterial and fungal pathogens. The predicted tomato and strawberry Elongator genes are highly homologous to those of Arabidopsis. Importantly, the tomato and strawberry Elongator gene homologues are able to complement the morphological and defense phenotypes in corresponding Arabidopsis Elongator mutants, indicating that they are functional. Generating transgenic plants with broad- spectrum disease resistance would be a promising alternative to conventional methods. However, recently there has been a strong anti-GMO propaganda aiming to reach the public. The durability of wheat stem rust (caused by Puccinia graminis f. sp. tritici) resistance is strengthened by the use of multiple broad-spectrum sources of resistance. Adult plant resistance (APR) is thought to provide partial effect, race non-specific resistance. APR was observed in the Ecuadorian bread wheat cv. Morocho Blanco (MB) in field tests at Njoro, Kenya. MB is susceptible to TTKSK (Ug99) at the seedling stage. A doubled haploid (DH) population was created from a cross between MB and the susceptible line LMPG-6 to identify loci associated with APR phenotypes. Eighty-eight DH lines were genotyped with approximately 90,000 SNPs using a custom Infinium assay from Illumina. Sixty-seven additional DH lines (MB x LMPG-6) and seventy-three recombinant inbred lines (MB x Faller) were used to verify SNPs associated with reduced levels of stem rust infection. Severity and infection type were assessed on adult plants in Njoro, Kenya from 2013-2015, Debre Zeit, Ethiopia in 2015, and in single race nurseries inoculated with races RCRSC, TPMKC, QTHJC, and QFCSC in Rosemount, USA from 2014-2015. Two QTL that reduce stem rust severity were identified on chromosome arms 2BS and 6AS in the three screening seasons in Kenya. However, in Ethiopia only 6AS was effective, and in the USA only 2BS was effective to race QFCSC. The current data indicates that a singular race and location should not be used to develop lines using APR as possible genotype x race interactions exist. Survey for Quinone Outside Inhibitor Resistant Cercospora sojina in Virginia Soybeans T. ZHOU (1) H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A. Survey for Quinone Outside Inhibitor Resistant Cercospora sojina in Virginia Soybeans T. ZHOU (1) H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A. Survey for Quinone Outside Inhibitor Resistant Cercospora sojina in Virginia Soybeans T. ZHOU (1) H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A. Quinone outside inhibitor (QoI) fungicides control a wide spectrum of fungal diseases, but this class of fungicides has a high risk of developing resistance. QoI fungicides are used in the management of frogeye leaf spot (FLS) caused by Cercocspora sojina. In 2014, FLS isolates from four fields in VA were tested, and QoI resistance was confirmed from two fields. In 2015, a statewide survey was conducted to estimate the prevalence of QoI resistant FLS in VA. Leaflet samples with FLS symptoms were collected from 55 soybean fields throughout the VA soybean growing region. Ten leaflets were randomly selected per sample and conidia were isolated from one lesion per leaflet. A total of 116 isolates were successfully cultured from 25 locations, and DNA was extracted from monoconidial cultures. A PCR-based assay was used to screen isolates for the G143A mutation in the cytochrome b gene that confers QoI resistance. Of the 116 isolates, 24% were positive for the G143A mutation. At the 10 locations with QoI resistant isolates, frequencies of the mutation ranged from 50 to 100%. DNA was extracted directly from leaves collected at all 55 locations, and a pyrosequencing assay was designed to quantify the frequencies of the G143A mutation within the C. sojina populations in each field. The survey confirms the presence of QoI resistant FLS in Virginia, so growers should use a different or mixed mode of action fungicides, plant resistant varieties, and rotate to non-host crops. S4.12 Fitness and competition studies of QoI resistant and sensitive Cercospora sojina isolates, the causal agent of frogeye leaf spot B. LIN (1), H. Kelly (1), H. Yu (2), A. Mengistu (3) Fitness and competition studies of QoI resistant and sensitive Cercospora sojina isolates, the causal agent of frogeye leaf spot B. LIN (1), H. Kelly (1), H. Yu (2), A. Mengistu (3) (1) University of Tennessee-Entomology & Plant Pathology, Jackson, TN, U.S.A.; (2) University of Tennessee-Entomology & Plant Pathology, U.S.A.; (3) United States Department of Agriculture, Agricultural Research Service (USDA-ARS), U.S.A. Survey for Quinone Outside Inhibitor Resistant Cercospora sojina in Virginia Soybeans T. ZHOU (1) H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A. This can accelerate discovery of new sources of resistance. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) (1) University of Minnesota, U.S.A.; (2) USDA-ARS, U.S.A.; (3) CIMMYT, Kenya; (4) Agriculture and Agri-Food Canada, Canada The durability of wheat stem rust (caused by Puccinia graminis f. sp. tritici) resistance is strengthened by the use of multiple broad-spectrum sources of resistance. Adult plant resistance (APR) is thought to provide partial effect, race non-specific resistance. APR was observed in the Ecuadorian bread wheat cv. Morocho Blanco (MB) in field tests at Njoro, Kenya. MB is susceptible to TTKSK (Ug99) at the seedling stage. A doubled haploid (DH) population was created from a cross between MB and the susceptible line LMPG-6 to identify loci associated with APR phenotypes. Eighty-eight DH lines were genotyped with approximately 90,000 SNPs using a custom Infinium assay from Illumina. Sixty-seven additional DH lines (MB x LMPG-6) and seventy-three recombinant inbred lines (MB x Faller) were used to verify SNPs associated with reduced levels of stem rust infection. Severity and infection type were assessed on adult plants in Njoro, Kenya from 2013-2015, Debre Zeit, Ethiopia in 2015, and in single race nurseries inoculated with races RCRSC, TPMKC, QTHJC, and QFCSC in Rosemount, USA from 2014-2015. Two QTL that reduce stem rust severity were identified on chromosome arms 2BS and 6AS in the three screening seasons in Kenya. However, in Ethiopia only 6AS was effective, and in the USA only 2BS was effective to race QFCSC. The current data indicates that a singular race and location should not be used to develop lines using APR as possible genotype x race interactions exist. S4.13 Utilizing mapping-by-sequencing to identify the Dominant Net Form Net Blotch Resistance Gene from Barley Line CI 5791 P. TAMANG (1), R. Brueggeman (1), J. Richards (1), T. Friesen (2) (1) North Dakota State University, U.S.A.; (2) Cereal Crops Research, USDA, U.S.A. Net form net blotch (NFNB) caused by Pyrenophora teres f. teres is a destructive foliar disease in barley growing regions worldwide. The diverse pathogen populations and complex host-parasite interactions occurring in this pathosystem has hindered breeding efforts. A dominant NFNB resistance gene identified on chromosome 6H in the line CI5791, designated RptCI5791, provides resistance to diverse isolates and is tightly linked to the dominant susceptibility locus Spt1. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) chacoense hybrid and several progeny were selected for their high yields and good tuber type. These breeding clones and molecular markers provide new tools for breeders to introduce PVY resistance into North American cultivars. Metabarcoding the soybean seed core mycobiome R. PEDROZO (1), A. Jumpponen (1), C. Little (1) (1) Kansas State University, U.S.A. The goal of this study was to explore the soybean seed core mycobiome using next-generation sequencing. Eight seed samples, representing four different locations in the state of Kansas and two soybean genotypes, were used. Seeds were surface disinfested with a 5% bleach solution (0.525% NaOCl v/v) for 1 min and dried overnight at room temperature. DNA was extracted from each ground soybean sample. After amplification of the ITS region, using ITS1F and tagged ITS4 primers, amplicons were sequenced using the Illumina MiSeq platform. Approximately 170,000 high-quality reads were produced from all samples analyzed in this study. Sequences from each sample were then taxonomically assigned using the Fungal ITS classifier from the Ribosomal Database Project (RDP). Initial analysis showed that both non-pathogenic fungi, such as Alternaria sp., Epicoccum sp. and Cladosporium sp., as well as pathogens such as Cercospora sp., Phomopsis sp. and Fusarium sp. were observed in all locations and genotypes. To better understand the soybean seed core mycobiome and its implication for global food security, additional experiments are underway to explore the presence of seedborne fungi, especially regarding the pathogenic groups, among individual soybean seeds as well as among three major soybean seed tissues including the seed coat, cotyledons, and embryo axis. Soil microbiomes associated with alternative strategies to manage Verticillium dahliae P. INDERBITZIN (1), J. Ward (2), A. Barbella (2), N. Solares (2), B. Calderon (2), D. Chellemi (2), K. Subbarao (1) (1) UC Davis, U.S.A.; (2) Driscoll’s Strawberry Associates, U.S.A. Verticillium dahliae is a fungal plant pathogen that causes Verticillium wilt, which results in significant losses in many crops. Control of V. dahliae is difficult as the pathogen can survive in soil without a host for over 10 years. Fumigation is an effective control, but the most viable option, methyl bromide, is no longer used and alternatives are expensive and subject to strict local regulations. Our goal is to develop environmentally-friendly and economically-viable alternatives to manage Verticillium wilt. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) These include organic soil amendments such as broccoli residue and a mixture of crab and feather meal, which have demonstrated disease suppressive effects in prior research, likely mediated by the soil microbiome. We performed greenhouse studies on a Verticillium sensitive host, eggplant, on which we assessed Verticillium wilt severity, and characterized soil microbial communities using 16S Illumina sequencing. The experimental setup involved a randomized complete block design with three soil amendments and 4-5 replicates for each amendment, and was performed separately for two soil types with different inoculum levels. The experiment was repeated once. We found that soil type, time of sampling, and amendment significantly impacted microbial communities; and that a sub-set of taxa was more abundant in disease suppressive treatments than in controls. Future studies will focus on the mechanistic bases of disease suppression by soilborne microbes. Root and rhizosphere microbiome responses to new tomato rootstock systems p p y R. POUDEL (1), L. Meyer (2), A. Jumpponen (3), M. Kennelly (4), C. Rivard (2), B. Cordova (5), J. Brisbane (5), K. Garrett (5) (1) Plant Pathology Department, Institute for Sustainable Food Systems, and Emerging Pathogens Institute, Gainesville, FL, U.S.A.; (2) Horticulture, Forestry, and Recreational Resources, Kansas State University, Olathe, KS, U.S.A.; (3) Division of Biology and Ecological Genomics Institute, Kansas State University, Manhattan, KS, U.S.A.; (4) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.; (5) Plant Pathology Department, Institute for Sustainable Food Systems, and Emerging Pathogens Institute, University of Florida, Gainesville, FL, U.S.A. Grafting tomato scions onto rootstocks can increase yields by providing disease resistance, stress tolerance, and increased vigor. We hypothesized that grafting affects the rhizosphere microbiome, or rhizobiome. We analyzed impacts of grafting scion BHN589 with two rootstocks BHN1028, RST-04-106 on the rhizobiome of tomato plants using rRNA gene sequences, including non-grafted and self-grafted controls. We also compared the microbial communities in roots vs. rhizosphere. In the first year, there were no observed effects of grafting or rootstock on microbial diversity in the three farm sites. Many OTUs were shared (72% in bacteria, 65% in fungi) among treatments, while some taxa were treatment-specific. Across all grafting treatments, higher diversity was found in the rhizosphere compared to the roots (p<0.001). Composition of the rhizobiome varied with root-rhizosphere compartment and farm site. Proteobacteria, Actinobacteria, Firmicutes, Bacteroidetes, and Planctomycetes were most abundant in all treatments, with the latter three being less abundant in roots. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) CI5791 lacks Spt1 as we were unable to amplify any allelic variants of Spt1. Thus, we hypothesize the high level of resistance in CI5791 is due to the dominant resistance gene RptCI5791 coupled with the absence of the dominant susceptibility locus Spt1. To further identify and characterize this broad-spectrum resistance, we used a forward genetics approach by g-irradiation and identified five putative mutants CI5791-γ3, -γ6, - γ7, -γ8, and -γ9 that are susceptible. We are utilizing the barley SEQCAP exome capture array (Nimblegen) and Illumina sequencing to identify the gene using a mapping-by-sequencing method. Mutated gene/s that map to the centromeric region of barley chromosome 6H will represent a strong candidate RptCI5791 gene. However, the possibility still exists that alleles of the CI5791 gene may not be present in the lines used in the exome capture, which would result in a failure to identify the gene. In this case we will continue pursuing the tried and true positional cloning approach. Development of molecular markers tightly linked to Potato virus Y resistance gene Rychc in a diploid potato population A. FULLADOLSA (1), S. Jansky (2), D. Halterman (3), A. Charkowski (1) (1) University of Wisconsin-Madison, U.S.A.; (2) USDA-ARS, University of Wisconsin-Madison, U.S.A.; (3) USDA-ARS, U.S.A. In the last 15 years, Potato virus Y (PVY) has been the main plant pathogen limiting seed potato certification in North America. The emergence of necrotic and recombinant PVY strains that cause mild foliar symptoms has resulted in increased PVY incidence. The use of cultivars that do not express foliar symptoms has also contributed to the problem. Several PVY resistance genes have been identified in wild relatives of potato, but few have been introduced into North American cultivars. We identified a diploid Solanum chacoense clone (CHC 39-7) in accession 275138, carrying PVY resistance gene Rychc, and used it in a cross with the susceptible, diploid Solanum tuberosum US-W4. A heterozygous resistant F1 progeny, XD3, was self- pollinated and the resulting population was phenotyped and used to develop molecular markers for genotyping. The phenotypic and genotypic results showed skewed segregation ratios, suggesting that a lethal factor is associated with susceptibility in the population. In addition, the development of markers that are more tightly linked to resistance in CHC 39-7 has aided in fine mapping Rychc and elucidating gene organization on chromosome IX. Furthermore, XD3 was crossed with a high yielding, adapted diploid S. tuberosum-S. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) Singh (2) (1) Hawkesbury Institute for the Environment, Penrith, Australia; (2) Hawkesbury Institute for the Environment, Western Sydney University, Australia; (3) School of Science and Health, Western Sydney University, Australia Yellow Canopy Syndrome (YCS) is a largely undiagnosed condition impacting sugarcane crops in Queensland, Australia, causing yield losses up to 30%. Since the first observation of YCS near Cairns in 2012, the condition has spread to all major cane growing areas and is a critical issue for the industry. Key YCS symptoms differ from leaf yellowing due to drought stress, phytotoxicity, insect attack, known diseases, nutrient deficiency or natural maturing. Despite elimination of some potential causes, the causal agent of YCS remains unknown. We aim to determine the involvement of biotic interactions and soil nutritional health in YCS development. A microbiome-based approach using Illumina MiSeq and HiSeq was applied on 598 samples to reveal the complexity of organisms (bacteria, fungi, other eukaryotes, viruses) present in YCS-affected sugarcane and rhizosphere soil. Soil nutrient status and microbial enzyme activity were also determined and did not significantly differ between affected and healthy fields. Bacterial (>25000 OTUs) and fungal (>11000 OTUs) assemblages of leaves, stalks, roots and rhizosphere soils differed between asymptomatic vs. symptomatic plants, as well as some rhizosphere microbial functions. Community shifts with YCS appeared variety-specific, and to date few consistent bacterial and fungal and no viral signals associated with YCS emerged. We envision ongoing data mining may reveal potential causal agent(s) of YCS or management practices for YCS. Illuminating the diversity of rust fungi infecting millets J. DEMERS (1), L. Castlebury (1) (1) USDA-ARS, U.S.A. Millets are a diverse assemblage of grains frequently grown in subsistence agriculture or as forage crops, and their popularity as nutritional alternative grains has recently increased. Millet pathogens are not well known, and modern collections of fungi associated with millets are lacking. Rust fungi are a significant group of pathogens that cause economically important diseases of three commonly grown millets: pearl millet (Pennisetum glaucum), foxtail millet (Setaria italica), and teff (Eragrostis tef), a millet becoming popular as a health food. 28S rDNA sequences were analyzed from over 40 herbarium specimens of rust fungi on millets, and related grasses, housed at the U.S. National Fungus Collections. Three taxa of rust fungi were observed on pearl millet, Puccinia substriata var. indica, P. substriata var. penicillariae, and P. cenchri, previously unreported on this host. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) The foxtail millet rust pathogen, Uromyces setariae-italicae, showed a high degree of intraspecific genetic diversity and was present on grasses in the Paniceae subtribes Cenchrinae and Melinidinae. The teff rust pathogen, previously known as U. eragrostidis, is actually distinct from U. eragrostidis and is identified as U. pedicellata. Knowledge of the diseases of millets is increasingly important as they become more widely grown as human food sources and pressure to increase yields in the presence of the pathogens intensifies. Genetic diversity of Colletotrichum isolates from Sorghum bicolor and S. halepense in the Southeastern United States K. XAVIER (1), C. Schardl (1), E. Buiate (1), M. Torres (2), M. Queiroz (3), S. Chopra (4), L. Vaillancourt (5) (1) Department of Plant Pathology, University of Kentucky, Lexington, KY, U.S.A.; (2) Functional Genomics Laboratory, Cornell University/Qatar Foundation, Education City, Qatar; (3) Laboratório de Genética Molecular e de Micro-organismo, Viçosa, KY, U.S.A.; (4) Department of Plant Science, Pennsylvania State University, University Park, PA, U.S.A.; (5) Department of Plant Pathology. University of Kentucky, Lexington, KY, U.S.A. Genetic diversity of Colletotrichum isolates from Sorghum bicolor and S. halepense in the Southeastern United States K. XAVIER (1), C. Schardl (1), E. Buiate (1), M. Torres (2), M. Queiroz (3), S. Chopra (4), L. Vaillancourt (5) (1) Department of Plant Pathology, University of Kentucky, Lexington, KY, U.S.A.; (2) Functional Genomics Laboratory, Cornell University/Qatar Foundation, Education City, Qatar; (3) Laboratório de Genética Molecular e de Micro-organismo, Viçosa, KY, U.S.A.; (4) Department of Plant Science, Pennsylvania State University, University Park, PA, U.S.A.; (5) Department of Plant Pathology. University of Kentucky, Lexington, KY, U.S.A. Anthracnose caused by Colletotrichum sublineola is the most important disease of grain sorghum worldwide, and it is emerging as a significant production constraint for sweet sorghum grown in the Southeastern U.S. Anthracnose is also common on the wild sorghum relative johnsongrass (S. halapense). Marker analysis using repetitive fingerprinting probes revealed that isolates from cultivated sorghum were generally distinct from isolates from S. halpense. RFLP analysis based on probes against individual sequences presumed to encode effectors and secondary metabolism enzymes showed that most isolates from johnsongrass grouped separately from most isolates from cultivated sorghum. Isolates from cultivated sorghum were generally aggressive to sweet sorghum variety Sugar Drip, while isolates from S. halapense generally caused little or no disease. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) Phylogenetic trees were inferred based on isolates from both host species using portions of the DNA lyase gene, the manganese superoxide dismutase gene, and a region between the APN1 and the MAT2 genes (MATAPN) genes. These trees were found to be congruent, and to identify two distinct species, the first one associated with the cultivated S. bicolor, and a second, new species associated with S. halepense. Strong evidence was found that cross-infection of these two species does occur, and this could be expected to complicate efforts to develop and deploy resistant sweet sorghum varieties in areas where johnsongrass is common. Phylogenetic and population analyses of the brown root-rot pathogen (Phellinus noxius) highlight the existence of two distinct populations J. STEWART (1), N. Sahashi (2), T. Hattori (2), Y. Ota (2), L. Shuey (3), R. Schlub (4), N. Atibalentia (5), F. Brooks (6), A. Tang (7), R. Lam (7), M. Leung (8), L. Chu (9), H. Kwan (9), A. Mohd Farid (10), S. Lee (10), C. Chung (11), H. Lee (11), Y. Huang (11), R. Liou (11), J. Tsai (12), P. Cannon (13), J. Hanna (14), N. Klopfenstein (14), M. Kim (15) Phylogenetic and population analyses of the brown root-rot pathogen (Phellinus noxius) highlight the existence of two distinct populations J. STEWART (1), N. Sahashi (2), T. Hattori (2), Y. Ota (2), L. Shuey (3), R. Schlub (4), N. Atibalentia (5), F. Brooks (6), A. Tang (7), R. Lam (7), M. Leung (8), L. Chu (9), H. Kwan (9), A. Mohd Farid (10), S. Lee (10), C. Chung (11), H. Lee (11), Y. Huang (11), R. Liou (11), J. Tsai (12), P. Cannon (13), J. Hanna (14), N. Klopfenstein (14), M. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) Pezizales, Cantharellales, and Xylariales were enriched in roots, whereas Pleosporales, Mortierellales, and Hypocreales were enriched in the rhizosphere. Interestingly, community modularity was higher in the roots compared to those from the rhizosphere. Ultimately, understanding rhizobiome responses to rootstocks will support vegetable production, rhizobiome-based crop breeding, and disease management. S4.14 Genetic determinants of bacterial adaptation to plants A. LEVY (1), A. Levy (1) (1) DOE Joint Genome Institute, U.S.A. Plants tightly associate with an array of diverse bacteria, with which they share complex and fascinating interactions, including parasitism, mutualism, and commensalism. These plant-associated (PA) bacteria have evolved a large set of genes that enable them to adapt to the plant environment. The functions of these genes are in many cases poorly studied and lack sufficient mechanistic understanding to facilitate microbiome engineering. Here, we sequenced 485 genomes of bacterial isolates and single cells isolated from the root environments of Arabidopsis, poplar, and maize. We then employed a large-scale comparative genomics study to characterize PA genes, and in particular root-associated (RA) genes. This was done by comparing 18 million genes from 3902 high quality and phylogenetically diverse bacterial genomes that were classified based on their isolation site. Several genomic features are common to most PA bacteria, such as the expansion of carbohydrate metabolism regulons. Many of the PA genes are encoded within putative gene operons, some of which encode for novel complex functions. Novel PA genes include a rapidly evolving virulence factor in the Acidovorax genus, a phage-like secretion system, and bacterial proteins with plant-like domains. The large PA and RA gene repository and the novel sequenced RA bacterial genomes will enable engineering of beneficial interactions for improved crop productivity and agricultural sustainability. Unravelling the sugarcane microbiome to resolve the Yellow Canopy Syndrome in Australia Unravelling the sugarcane microbiome to resolve the Yellow Canopy Syndrome in Australia K. HAMONTS (1), P. Trivedi (2), A. Garg (2), P. Holford (3), J. Grinyer (2), I. Anderson (2), B. Singh (2) (1) Hawkesbury Institute for the Environment, Penrith, Australia; (2) Hawkesbury Institute for the Environment, Western Sydney University, Australia; (3) School of Science and Health, Western Sydney University, Australia Unravelling the sugarcane microbiome to resolve the Yellow Canopy Syndrome in Australia K. HAMONTS (1), P. Trivedi (2), A. Garg (2), P. Holford (3), J. Grinyer (2), I. Anderson (2), B. (1) Colorado State University, Fort Collins, CO, U.S.A.; (2) Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan; (3) Forestry & Agricultural Research Institute, University of Pretoria, South Africa; (4) University of Guam, ANR/CES/CNAS, Mangilao, Guam, U.S.A.; (5) American Samoa Community College-CNR, Division of Community & Natural Resources, U.S.A.; (6) Department of Plant and Environmental Protection Sciences, University of Hawaii-Manoa, Honolulu, HI, U.S.A.; (7) Muni Arborist Limited, Hong Kong, China; (8) Muni Arborist Limited, China; (9) School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China; (10) Forest Research Institute, Kepong, Selango, Malaysia; (11) Department of Plant Pathology and Microbiology, National Taiwan University, Taiwan; (12) Taiwan Agricultural Research Institute, Taiwan; (13) USDA Forest Service, Forest Health Protection, Vallejo, CA, U.S.A.; (14) RMRS, USDA Forest Service, Moscow, ID, U.S.A.; (15) Department of Forestry, Environment and Systems, Kookmin University, Korea Phellinus noxius is a vastly destructive, fast-growing fungal pathogen that affects a wide range of woody hosts in pan-tropical areas, including Asia, Oceania, and Africa. This pathogen causes brown root-rot on diverse hosts, with little indication of host specificity. Pathogenic symptoms of P. noxius Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) Pearson’s correlation coefficient indicated that disease severity from the four trials was significantly correlated with average weekly temperature and total weekly rainfall in greenhouse inoculated (-0.67, p < 0.0006; 0.51, p < 0.01) and field inoculated plots (-0.72, p < 0.001; 0.58, p < 0.01) respectively. ManKocide and Actigard foliar applications were found to significantly reduce disease development over time, as indicated by the area under the disease progress curve (p < 0.05). The results of this study indicate that temperature and rainfall are key factors in determining disease development, and ManKocide and Actigard foliar applications are highly effective in controlling the disease with 3 to 4 applications early in the spring season. Whole genome sequencing of Xanthomonas perforans identifies effectors that influence breeding strategies S. TIMILSINA (1), P. Abrahamian (2), N. Potnis (3), G. Minsavage (4), F. White (4), B. Staskawicz (5), J. Jones (4), G. Vallad (4), E. Goss (4) (1) University of Florida, Gainesville, FL, U.S.A.; (2) Gulf Coast Research and Education Center, University of Florida, FL, U.S.A.; (3) United States Department of Agriculture, Charleston, SC, U.S.A.; (4) University of Florida, U.S.A.; (5) University of California-Berkeley, U.S.A. Whole genome sequencing of Xanthomonas perforans identifies effectors that influence breeding strategies S. TIMILSINA (1), P. Abrahamian (2), N. Potnis (3), G. Minsavage (4), F. White (4), B. Staskawicz (5), J. Jones (4), G. Vallad (4), E. Goss (4) (1) University of Florida, Gainesville, FL, U.S.A.; (2) Gulf Coast Research and Education Center, University of Florida, FL, U.S.A.; (3) United States Department of Agriculture, Charleston, SC, U.S.A.; (4) University of Florida, U.S.A.; (5) University of California-Berkeley, U.S.A. The management of bacterial spot caused by X. perforans on tomato is a challenge due to historical changes in the pathogen populations. The availability of genomic sequence for hosts and pathogens provides new data for effector-based breeding strategies. We examined the stability and evolution of several effectors in X. perforans, based on genome sequence from representative strains collected in Florida since 1990. Effectors AvrXv3 and XopJ4 trigger resistance in tomato cultivars carrying the corresponding R genes. However, the Florida X. perforans population has shifted from tomato race 3 to tomato race 4, such that avrXv3 is non-functional in all tomato race 4 strains with mutations specific to different phylogenetic groups. The X. perforans effector genes xopJ4 and avrBs2 remain intact in all strains. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) Donofrio (2) (1) University of Delware, U.S.A.; (2) University of Delaware, U.S.A. The heterothallic oomycete Phytophthora capsici causes disease under warm (25-28°C), wet conditions. This pathogen causes pod rot in lima bean (Phaseolus lunatus L.), an emerging disease in the mid-Atlantic region which reduces yields and limits production. In order to better understand sources of inoculum, isolates were collected from infected commercial plant samples, 13 irrigation water sources, potential weed hosts, and soil sediments in Delaware. Water filtration, baiting with fruits, or baiting with Rhododendron leaves, were used in an attempt to extract P. capsici from irrigation water. Species other than P. capsici were recovered in abundance from irrigation water, weed hosts, and soil sediments. A total of 38 isolates were recovered from symptomatic lima bean, pepper, pumpkin, and watermelon samples and positively identified as P. capsici both morphologically and molecularly. Isolates derived from single zoospores where characterized for mating type, mefenoxam sensitivity and virulence on eight commercial lima bean cultivars. The A2 mating type was prevalent among the collected isolates (78.9%) and seven isolates with resistance to mefenoxam were identified. All isolates were virulent on the eight lima bean cultivars tested. Two SSR markers were identified that co-segregate with mefenoxam sensitivity, with over 80% accuracy when used together, that could have potential use in disease management efforts. Epidemiology and management of bacterial leaf spot caused by novel Pseudomonas syringae strains on watermelon in Florida E. NEWBERRY (1), L. Ritchie (1), B. Babu (1), T. Sanchez (2), K. Beckham (2), J. Jones (2), J. Freeman (1), N. Dufault (2), M. Paret (1) (1) NFREC, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A. Major disease outbreaks occurring on watermelon and squash caused by Pseudomonas syringae were widespread in Florida and Georgia during recent years. This was recognized as a new disease in the region, and affected approximately 6,500 acres of watermelon from Florida in 2013. Field trials were conducted during the 2014 and 2015 spring seasons in Quincy and Citra, FL to examine environmental conditions which may be conducive for disease progression, and to assess the efficacy of foliar application of copper with or without Ethylene bis-dithiocarbamate (ManKocide/Kocide 3000) and foliar/drip application of plant activator (Acibenzolar S-Methyl; Actigard) in managing both transplant and field level introductions of the pathogen. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) The objective of this study was to estimate a wide-scale genetic diversity and evolutionary history of P. noxius movement. A total of 158 isolates were included from Japan, Taiwan, Australia, China (Hong Kong), Malaysia, and the Pacific islands of American Samoa, Saipan, Guam, Palau, Pohnpei, and Kosrae were sequenced at four loci including ITS, LSU-rDNA, elongation factor-1 alpha, and RNA polymerase subunit II. The results show two distinct clades of P. noxius. Currently, sequencing data are generating by Illumina sequencing of double- digest reduced representation libraries (ddRAD). Continued analyses will assess regional population structure, gene flow among populations, diversity in recently observed populations, and potential suitable climate space for specific genotypes. Microsatellite genotyping reveals high genetic diversity in Phytophthora infestans from Mexico S. SHAKYA (1), M. Larsen (2), M. Condoy (3), H. Lozoya-Saldana (3), N. Grunwald (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A.; (3) Universidad Autónoma Chapingo, Mexico Toluca valley of Mexico is considered to be the center of origin of late blight pathogen, Phytophthora infestans. This hypothesis is supported by populations being sexual, mating type ratios 1:1 and a high standing genetic diversity in HWE. However, the population structure from other areas of Mexico is understudied. We hypothesize that migration of P. infestans outside of Toluca might have resulted in admixed population structure with decreasing diversity away from Toluca. To test this hypothesis we sampled and microsatellite genotyped more than 85 isolates from Mexico in the regions of Chapingo, Juchitepec, San Geromino, Tlaxcala and Toluca. Our analysis revealed high number of multi-locus genotypes and high allelic diversity. Juchitepec samples formed two distinct clusters suggesting two differentiated populations. Tlaxcala isolates seem to be uniformly distributed possibly indicating multiple migration events and admixture. Chapingo and San Geromino samples were more similar between them. In general, all regions showed no linkage among loci suggesting presence of sexual reproduction, except for Chapingo. Contrary to our hypothesis, diversity in other populations was even higher or comparable to Toluca. These results indicate that the center of origin is larger than previously thought. Our work provides a more refined understanding of the population structure of P. infestans in areas near Toluca where the pathogen has been well studied. Characterization of isolates of Phytophthora capsici from the mid-Atlantic region, U.S. N. ABEYSEKARA (1), H. Hickman (2), S. Westhafer (2), N. Gregory (2), G. Johnson (2), T. Evans (2), N. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) Kim (15) ( ) ( ) p ( ) ( ) (1) Colorado State University, Fort Collins, CO, U.S.A.; (2) Forestry and Forest Products Research Institute, Tsukuba, Ibaraki, Japan; (3) Forestry & Agricultural Research Institute, University of Pretoria, South Africa; (4) University of Guam, ANR/CES/CNAS, Mangilao, Guam, U.S.A.; (5) American Samoa Community College-CNR, Division of Community & Natural Resources, U.S.A.; (6) Department of Plant and Environmental Protection Sciences, University of Hawaii-Manoa, Honolulu, HI, U.S.A.; (7) Muni Arborist Limited, Hong Kong, China; (8) Muni Arborist Limited, China; (9) School of Life Sciences, The Chinese University of Hong Kong, Hong Kong, China; (10) Forest Research Institute, Kepong, Selango, Malaysia; (11) Department of Plant Pathology and Microbiology, National Taiwan University, Taiwan; (12) Taiwan Agricultural Research Institute, Taiwan; (13) USDA Forest Service, Forest Health Protection, Vallejo, CA, U.S.A.; (14) RMRS, USDA Forest Service, Moscow, ID, U.S.A.; (15) Department of Forestry, Environment and Systems, Kookmin University, Korea Phellinus noxius is a vastly destructive, fast-growing fungal pathogen that affects a wide range of woody hosts in pan-tropica Oceania, and Africa. This pathogen causes brown root-rot on diverse hosts, with little indication of host specificity. Pathogen S4.15 infection can include reduced tree growth, defoliation, and branch dieback; however, P. noxius can survive as a saprophyte. Understanding the genetic diversity and evolutional history of P. noxius populations worldwide well help assess the evolutionary origins, worldwide movement, and potential ecological differences within P. noxius. The objective of this study was to estimate a wide-scale genetic diversity and evolutionary history of P. noxius movement. A total of 158 isolates were included from Japan, Taiwan, Australia, China (Hong Kong), Malaysia, and the Pacific islands of American Samoa, Saipan, Guam, Palau, Pohnpei, and Kosrae were sequenced at four loci including ITS, LSU-rDNA, elongation factor-1 alpha, and RNA polymerase subunit II. The results show two distinct clades of P. noxius. Currently, sequencing data are generating by Illumina sequencing of double- digest reduced representation libraries (ddRAD). Continued analyses will assess regional population structure, gene flow among populations, diversity in recently observed populations, and potential suitable climate space for specific genotypes. infection can include reduced tree growth, defoliation, and branch dieback; however, P. noxius can survive as a saprophyte. Understanding the genetic diversity and evolutional history of P. noxius populations worldwide well help assess the evolutionary origins, worldwide movement, and potential ecological differences within P. noxius. sustainable carbon source in application of anaerobic soil disinfestation for control of apple nursery replant disease M M l (2) Grass residues as a sustainable carbon source in application of anaerobic soil disinfestation for control of apple nursery replant disease S. Hewavitharana (1), M. Mazzola (2) (1) Washington State University, U.S.A.; (2) USDA-ARS, U.S.A. Studies were conducted to assess the efficacy of anaerobic soil disinfestation (ASD) for control of replant disease in an apple nursery setting. Treatments applied in a field trial conducted at an experimental orchard in Washington State included ASD using orchard grass residues (GR; 20 t ha–1) as the carbon source, Telone C35 (27 gal acre–1) and no treatment control (NTC). The apple rootstocks G.935, G.41, and M.9, with respectively decreasing level of tolerance to replant disease, were planted into each of six blocks per treatment with five replicates per rootstock. Post-treatment weed biomass analysis, rootstock growth analysis and rhizosphere fungal community analysis were conducted. Prior to tillage that preceded rootstock planting, ASD yielded significant weed suppression but weed biomass in fumigated plots was comparable to NTC. During the first growing season trunk diameter of G.41 and G.935 rootstocks were significantly increased by ASD compared to the NTC and it was comparable to fumigation treatment. ASD enhanced growth of M.9 relative to NTC but was lower than that for the fumigation. According to TRFLP analysis, different soil treatments possessed unique rhizosphere fungal communities. Based on this study, an increase of 5-25% gross revenue was projected using ASD based on market value of different rootstocks and sizes. ASD with grass is a potential viable soil-borne disease management practice for the apple nursery industry. Management of Fusarium wilt Tropical race 4 in bananas Management of Fusarium wilt Tropical race 4 in bananas A. DRENTH (1), J. Henderson (2), A. Drenth (2) . DRENTH (1), J. Henderson (2), A. Drenth (2) ( ) ( ) ( ) (1) The University of Queensland, Australia; (2) The University of Queensland, Australia Fusarium oxysporum f.sp. cubense Tropical race 4 (TR4) is one of the most important pathogens of banana which severely affects many banana varieties, including Cavendish. First identified in Taiwan, TR4 has spread rapidly over major banana production areas and has had a major impact on banana production. More recently it has been identified outside Southeast Asia in Mozambique, and in Jordan. The most recent incursion was detected in Australia’s main banana growing region around Tully in North Queensland in March 2015. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) Nobactra brings a new concept to control a wide range of bacterial diseases, comprised of two key elements that when combined have a strong synergistic relationship. The first component is a cocktail of 10 antagonistic bacteria and the second component is a powder formulation of essential oils. The advantages of using a cocktail of bacteria rather than a single microbe include: a) consistency in a range of soil and climatic conditions; b) significantly lower chances of resistance development due to the multiple mechanisms of activity; c) wide spectrum of activity. The innovative oil formulation overcomes the high phytotoxicity of the essential oil and allows for effective use against bacterial diseases without harming the plant. Nobactra’s solutions have shown high efficacy rates in the control of several diseases. The following are key examples: Potato scab (Streptomyces spp.) – seed treatment and soil treatment. Tomato canker (Clavibacter michiganensis subsp. Michiganensis - Cmm) Nobactra controls the distribution of Cmm in the greenhouse. Seed borne bacterial diseases (Pseudomonas, Xanthamonas, Acidovorax, Erwinia, Cmm.). Nobacta’s products are officially registered in Israel. Investigation of the anti-bacterial activity of SERENADE® ASO against bacterial diseases of crops T SMITH (1) T K bl h (2) Investigation of the anti-bacterial activity of SERENADE® ASO against bacterial diseases of crops T. SMITH (1), T. Knobloch (2) (1) Bayer, Crop Science Division - Biologics Research, U.S.A.; (2) Bayer, Crop Science Division - Small Molecule Research, France Investigation of the anti-bacterial activity of SERENADE® ASO against bacterial diseases of crops T. SMITH (1), T. Knobloch (2) T. SMITH (1), T. Knobloch (2) (1) Bayer, Crop Science Division - Biologics Research, U.S.A.; (2) Bayer, Crop Science Division - Small Molecule Research, France ( ) ( ) (1) Bayer, Crop Science Division - Biologics Research, U.S.A.; (2) Bayer, Crop Science Division - Small Molecule Rese Bacterial diseases of agricultural crops are costly with losses estimated at $10 billion per year. Current management strategies often use a multifaceted approach, including biological control. The biological-based product, SERENADE® ASO, is a known tool in integrated foliar spray programs to combat fungal diseases in fruits and vegetables, based on fungal cell membrane disrupting properties of lipopeptide chemistry produced by Bacillus subtilis QST-713. In addition, SERENADE® ASO has proved helpful to growers in managing bacterial diseases, such as Pseudomonas syringae pv. tomato and Xanthomonas campestris pv. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) The avrBsT gene is absent in race 3 strains collected in the 1990s, but present in race 3 strains collected in 2006 and the majority of race 4 strains. The conserved presence of XopJ4 and increased presence of AvrBsT among race 4 strains of X. perforans make them ideal targets for resistance breeding. Specific R-genes against these two effectors could be pyramided with Bs2 resistance to develop tomato cultivars with enhanced resistance against X. perforans for commercial production in Florida. Cybridization: A promising plant resistance for citrus canker control M. MURATA (1), A. Omar (1), C. Chase (2), J. Grosser (1), J. Graham (1) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Cybridization is a somatic hybridization system that enables the construction of novel nuclear and cytoplasmic genome combinations. In this work, cybridization was used to study the role of mitochondrial (mt) and chloroplast (cp) genes in resistance to Xanthomonas citri subsp. citri (Xcc), the cause of citrus canker. Putative cybrids were created through the fusion between nucellar callus cells of Meiwa kumquat (Fortunella crassifolia), a citrus canker resistant, and mesophyll cells of citrus canker susceptible grapefruit (Citrus paradisi). Six EST-SSR markers demonstrated that all clones have a grapefruit nuclear genome; however, four CAPS markers showed variation in the cytoplasmic genomes. In 123 cybrids, kumquat mt and cp were identified, and in 19 cybrids, kumquat mt and grapefruit cp were present. Eighty-two cybrid clones were evaluated by detached leaf assay (DLA), and clones that performed better in the DLA were evaluated by syringe inoculation of attached leaves. Q-PCR was carried out to quantify the Xcc population. S4.16 Cybrid clones showed a range of citrus canker resistance, but all clones produced a significantly lower number of lesions and a one log unit reduction of the Xcc population compared to grapefruit. Cybrid clones with grapefruit cp were more susceptible to Xcc compared to clones with kumquat cp. In addition to citrus canker resistance, these cybrids provide a valuable model to study the role of cytoplasmic genomes in plant disease resistance. Biological control of plant pathogenic bacteria G. KRITZMAN (1), G. Kritzman (2) (1) Nobactra, Israel; (2) Nobactra, Israel Bacterial diseases take a high economical toll in all areas of agriculture. Currently growers attempt to treat these outbreaks with copper compounds that have low efficacy and are detrimental to the environment. Mapping of two QTL conferring adult plant resistance to Puccinia graminis f. sp. tritici race TTKSK (Ug99) and evidence for race specificity J. BRIGGS (1), M. Rouse (2), S. Bhavani (3), C. Hiebert (4) vesicatoria, causal agent of bacterial speck and spot, on crops such as tomato and pepper. Recent research using both in vitro and in vivo tools point to two main actions contributing to the antibacterial activity of SERENADE® ASO: a direct antibacterial effect from biological chemistry produced by QST-713 during fermentation and an indirect effect on the plant. Different approaches were applied to eliminate potential activity from living cells in SERENADE® ASO to examine the role of biological chemistry. Research with Arabidopsis treated with SERENADE® ASO showed up-regulation of several pathways associated with host defense, and microscopy studies using tomato seedlings revealed transient changes in the condition of stomata openings. Additional research is ongoing to better characterize the mechanism of SERENADE® ASO against plant pathogenic bacteria. sustainable carbon source in application of anaerobic soil disinfestation for control of apple nursery replant disease M M l (2) S4.17 Efficacy of biopesticides on root diseases and pests in hydroponic production of vegetables A. POLEATEWICH (1), R. Buitenhuis (1), M. Brownbridge (1), T. Cranmer (1), A. Summerfield (1) (1) Vineland Research and Innovation Centre, Canada Soil-borne pathogens cause significant losses in hydroponic greenhouse vegetable production, even when fungicides and water treatments are employed. Several microbes and microbe-derived products protect plants from pathogens. Many new products are available but comparative efficacy data is lacking. This research evaluated biopesticide efficacy under commercial conditions to identify implications for IPM. A small scale hydroponic system was constructed to evaluate efficacy of 10 bio-pesticides on commercial greenhouse tomato and cucumber cultivars. The effect of cultivar, grafting, and substrate type on product efficacy was also determined. Products were applied as a drench at transplant to rockwool blocks and at transplant to coco or rockwool slabs. Products were compared to fungicide and water controls. Plants were challenged with Pythium spp. 3-5 days after product application and root rot, plant height and weight was evaluated after 3 weeks. Several products reduced root rot equal to or greater than the fungicide Previcur. Disease reduction varied depending on the substrate, time of year and rootstock genetics. The best performing products in the disease trials were tested for potential positive or negative effects on performance of greenhouse pests Trialeurodes vaporariorum and Tetranychus urticae. Results from this research will allow growers to make informed management decisions and derive maximum benefit from their investment in disease control products. Organic rice production has steadily increased in the U. S. over the last 20 years, with a majority of acreage being grown in the southern region. Due to warm and long growing seasons in the region, diseases are among the most important factors threatening the sustainability and productivity of organic rice. Field studies were conducted for the first time in the U. S. to evaluate the efficacy of cultivar resistance, cover crop, fertility, tillage and biocontrol agent for management of rice diseases on organically managed land in Texas in 2009 to 2014. Among 20 cultivars evaluated, Tesanai 2 had the highest level of resistance to both narrow brown leaf spot (NBLS) and brown spot followed by XL723 and XL753. Production following the incorporation of clover winter cover crop resulted in a consistent reduction in NBLS and brown spot than following winter fallow or annul ryegrass cover crop. sustainable carbon source in application of anaerobic soil disinfestation for control of apple nursery replant disease M M l (2) The continuous long distance dissemination of this fungus, as well as local spread to new plantations and fields, raises many questions concerning our ability for effective containment of this pathogen. The recent outbreak in Australia also highlighted a lack of understanding of many basic pathological and epidemiological facts of this important pathogen. Therefore, our aim was to improve detection and identification of the pathogen, destruction of infected plants, containment strategies and overall management of plantations. In the absence of high levels of resistance in economically acceptable banana varieties, containment may be a temporary but effective strategy to win valuable time to identify or develop banana varieties with high levels of resistance to Panama wilt TR4. A global program for evaluation of germplasm in multiple field sites linked with international collaborators is urgently needed. Pseudomonas sp associated with Smilax bona-nox display strong activity against Phytophthora spp G. ALI (1), A. El-Sayed (2), D. Norman (2), M. Brennan (3) (1) University of Florida, Apopka, FL, U.S.A.; (2) University of Florida/ Institute of Food and Agricultural Sciences, Apopka, FL, U.S.A.; (3) MREC/University of Florida/ Institute of Food and Agricultural Sciences, Apopka, FL, U.S.A. Smilax bona-nox is a perennial weed, which is found in natural landscapes and forests throughout the southwestern U.S. S. bona-nox displays disease tolerance and vigorous growth, which could be partially attributed to beneficial plant-associated microorganisms. In this report, we recovered 44 bacterial isolates from roots and shoots of S. bona-nox. Based on 16S rRNA gene sequencing, these isolates belonged to 14 different genera. Antimicrobial screens showed that two of these isolates, EA6 and EA14, displayed strong inhibition of Phytophthora parasitica. Phylogenetic analyses based on the rpoD gene revealed that EA6 and EA14 belonged to the genus Pseudomonas. EA6 and EA14 did not produce biofilm, but produced bio- surfactants, volatile hydrogen cyanide and sulfur compounds. Biochemical fractionation of the volatile and secreted products of these two isolates for identifying the bioactive compound(s) revealed that volatile and organic extracts did not display any anti-Phytophthora activity. In contrast, salt precipitation of the aqueous phase revealed strong anti-Phytophthora activity, which was abolished by autoclaving or proteinase K treatments. Protein fractionation analyses suggest that the antimicrobial activity of EA6 is likely due to synergistic interaction of a mixture of glucanolytic enzymes. In conclusion, these two Pseudomonas isolates can be used for organically controlling Phytophthora diseases in crops. Genetic basis for pathogenicity in Fusarium oxysporum f.sp. cepae causing basal rot in onion J. CLARKSON (1), A. Taylor (1), A. Jackson (1), A. Armitage (3), R. Harrison (3) (1) Warwick Crop Centre, University of Warwick, United Kingdom; (2) Warwick Crop Centre, University of Warwick, United Kingdom; (3) East Malling Research, United Kingdom RNA-Seq analysis of stuA mutants in Fusarium verticillioides indicates dramatic genomic wide transcriptional reprogramming M. Rath (1), N. Crenshaw (2), S. Gold (2) RNA-Seq analysis of stuA mutants in Fusarium verticillioides indicates dramatic genomic wide transcriptional reprogramming M. Rath (1), N. Crenshaw (2), S. Gold (2) (1) University of Georgia & USDA, U.S.A.; (2) USDA, U.S.A. StuA, first discovered in Aspergillus nidulans and a member of the APSES class of transcription factors, regulates several essential developmental stages in fungi such as virulence, sporulation and toxin production in phytopathogenic fungi. Fusarium verticillioides (Fv), a maize phytopathogen, produces fumonisin mycotoxins in maize. Fumonisin-contaminated corn causes fatal toxicity in livestock, is associated with neural tube birth defects and growth stunting in children and is also a potential carcinogen to humans. Our objective is to explore the impact of the stuA homolog on morphogenesis and toxin production in Fv. ?stuA mutants were generated via Agrobacterium-mediated transformation of Fv with an OSCAR deletion construct. Characterization of the mutants indicate slower vegetative growth on solid media, reduced sporulation and macroconidiation. A cracked corn assay on maize kernels and virulence assay on maize seedlings revealed reduced fumonisin accumulation and reduced virulence respectively in stuA mutant infected maize as compared to the wild-type pathogen infection. To characterize the mutants transcriptomally, RNA-sequencing of three of the mutants and the wild-type Fv strain M3125 was done via Illumina NextSeq and the Tuxedo pipeline is being utilized for differential expression analysis. Preliminary RNA-seq analysis indicates major alteration of numerous transcription factors, transporters and signaling in the stuA mutants. sustainable carbon source in application of anaerobic soil disinfestation for control of apple nursery replant disease M M l (2) An application of the soil amendment Nature Safe or Rhizogen at 90, 150 or 210 kg N/ha significantly reduced NBLS and brown spot. Use of conventional tillage practice prevented the occurrence of straighthead on all 20 cultivars evaluated in the clover winter cover crop plots. A foliar application of Serenade Max significantly reduced NBLS and sheath blight, resulting in an increase in yield. Cultivar resistance, cover crop, fertility, tillage and biocontrol can be effective tools for management of diseases in organic rice production. Elucidating the genetic basis of race differentiation in Cercospora sojina through genotype-by-sequencing approaches S. SHARMA (1), B. Dhillon (1), A. Fakhoury (2), B. Bluhm (1) (1) University of Arkansas, U.S.A.; (2) Southern Illinois University, U.S.A. Elucidating the genetic basis of race differentiation in Cercospora sojina through genotype-by-sequencing approaches S. SHARMA (1), B. Dhillon (1), A. Fakhoury (2), B. Bluhm (1) (1) University of Arkansas, U.S.A.; (2) Southern Illinois University, U.S.A. Elucidating the genetic basis of race differentiation in Cercospora sojina through genotype-by-sequencing approaches S. SHARMA (1), B. Dhillon (1), A. Fakhoury (2), B. Bluhm (1) 1) University of Arkansas, U.S.A.; (2) Southern Illinois University, U.S.A. Cercospora sojina, the causal agent of frogeye leaf spot (FLS), is a widespread pathogen of soybean in the United States. Recently, a race structure was proposed for C. sojina based on susceptibility vs resistance responses of 12 soybean differential cultivars. We hypothesize that the delineation of races in C. sojina is attributable to variation in effector gene complements among individual isolates. To test this hypothesis, we devised a genotype-by- sequencing strategy for C. sojina to facilitate association mapping of loci associated with race structure. We developed a restriction-site associated DNA sequencing (RAD-seq) approach to cost-effectively genotype large numbers of C. sojina isolates, with the ultimate goal of identifying single nucleotide polymorphisms (SNPs) for association mapping. Bulk segregant analysis was performed with two pools of C. sojina isolates (48 isolates per pool, selected based on resistant or susceptible reaction on soybean cultivar UA5612). Ion Torrent PGM sequencing identified 15,158 SNPs, and candidate pathogenicity genes were identified based on differences in SNP density and non-synonymous SNPs between the resistant and susceptible pools. Elucidating the genetic basis of race structure in C. sojina will accelerate the development of DNA-based assays to differentiate races of the pathogen, and contribute to the fundamental understanding of genetic mechanisms underlying FLS. RNA-Seq analysis of stuA mutants in Fusarium verticillioides indicates dramatic genomic wide transcriptional reprogramming M. Rath (1), N. Crenshaw (2), S. Gold (2) (1) University of Georgia & USDA, U.S.A.; (2) USDA, U.S.A. Enhancing our understanding of Fusarium maize stalk rot pathogenesis through gene association subnetwork module analyses H. ZHANG (1), H. Zhang (2), M. Kim (2), H. Yan (2), B. Yoon (2), W. Shim (2) (1) Texas A&M university, U.S.A.; (2) Texas A&M university, U.S.A. redolens, which have also been associated with basal rot. Preliminary results from a comparative analysis of genomes from pathogenic and non-pathogenic F. oxysporum isolates will also be described as well as the potential use of SIX genes as a diagnostic to enable quantification of FOC and assessment of disease risk in field or store. Quantitative pyrosequencing for rapid detection and quantification of Aspergillus flavus atoxigenic active ingredients communities Quantitative pyrosequencing for rapid detection and quantification of Aspergillus flavus atoxigenic active ingredients in complex fungal communities K SHENGE (1) B Adhik i (1) K C lli (1) R B d dh (2) P C (3) K. SHENGE (1), B. Adhikari (1), K. Callicott (1), R. Bandyopadhyay (2), P. Cotty (3) (1) USDA-ARS, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Niger K. SHENGE (1), B. Adhikari (1), K. Callicott (1), R. Bandyopadhyay (2), P. Cotty (3) (1) USDA-ARS U S A ; (2) International Institute of Tropical Agriculture (IITA) Niger ( ), ( ), ( ), y p y y ( ), y ( ) (1) USDA-ARS, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Nigeria; (3) USDA-ARS, U.S.A. ( ), ( ), ( ), y p y y ( ), y ( ) (1) USDA-ARS, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Nigeria; (3) USDA-ARS, U.S.A. Use of atoxigenic Aspergillus flavus genotypes to displace aflatoxin producers is a highly effective method for reducing aflatoxin contamination in crops. However, there is a need for improved methods for rapid assessment of treatment efficacy and residual effects. To address this need, the current study designed twenty-four quantitative pyrosequencing assays for rapid post-harvest detection and quantification of Aspergillus flavus genotypes which serve as active ingredients in the biocontrol product Aflasafe™ that is registered for use in Nigeria. The fungi must be quantified in mixed fungal populations associated with crop surfaces. Sixteen of the assays are for individual genotypes and eight are for simultaneous detection and quantification of multiple active ingredients. Assay refinement with mixtures of DNA from target and non-target genotypes indicated both accuracy and sensitivity of the assays in detecting the active ingredients in fungal populations. Quantification of target alleles by single and multiple genotype assays was highly correlated with proportions of target DNA in the mixtures. Results from tests to validate the assays with DNA extracted from treated corn samples were not consistent among assays. Proposed model for Candidatus Liberibacter asiaticus and solanacearum systemic invasion and multiplication in the psyllid host and vector J. BROWN (1), T. Rast (1), J. Cicero (2), T. Fisher (1) (1) The University of Arizona, U.S.A.; (2) University of Florida, U.S.A. Proposed model for Candidatus Liberibacter asiaticus and solanacearum systemic invasion and multiplication in the p J. BROWN (1), T. Rast (1), J. Cicero (2), T. Fisher (1) Proposed model for Candidatus Liberibacter asiaticus and solanacearum systemic invasion and multiplication in the psyllid host and vector J. BROWN (1), T. Rast (1), J. Cicero (2), T. Fisher (1) (1) The University of Arizona, U.S.A.; (2) University of Florida, U.S.A. Ca. Liberibacter asiaticus and solanacterum are the causal agents of Huanglongbing and Zebra chip disease of citrus and solanaceous crops, respectively. Bacterial effectors that interact with psyllid vector proteins during invasion of and exit from the gut, and that facilitate entry into the salivary glands were identified using functional genomics, proteomics, yeast-2 hybrid (Y2H), co-immunoprecipitation (Co-IP), and electron microscopic analyses. In silico annotation and differential expression studies of psyllid nymphs and adults, and midgut and salivary glands identified transcripts and proteins with altered expression in response to Ca. Liberibacter infection. Differentially expressed transcripts were used as bait in Y2H studies to corroborate protein- protein interactions. Those positive by Y2H were subjected to verification by bait to prey co-transformation and Co-IP. Electron micrographs of the PoP midgut revealed the presence of CLso in the ventricular lumen, apical and basal epithelial cytosol, and in the filter chamber periventricular space. CLso were also prevalent in salivary gland pericellular spaces and head epidermal cell cytosol. Collectively, the results suggest a model for invasion in which Ca. Liberibacter- and prophage-encoded effectors manipulate the host endocytic pathway into a ‘pathogen-mediated phagocytosis scenario’ leading to bacterial exit from the gut into the hemocoel, and systemic invasion of other psyllid tissues and organs. Systemic infection of split root trees by Candidatus Liberibacter asiaticus suggests rapid movement between phloem sieve tubes J. ORROCK (1), J. Orrock (1), E. Johnson (1) (1) Citrus Research and Education Center, University of Florida, U.S.A. Huanglongbing (HLB; syn. citrus greening), caused by the phloem-limited bacterium Candidatus Liberbacter asiaticus (Las), poses one of the largest threats to citrus production. In ten years it has spread to most citrus trees in Florida. Citrus production in Florida is down >70% from the pre-HLB era. Las infects a tree when an infected Diaphorina citri (Asian Citrus Psyllid) feeds on phloem sap in a leaf. After infection of the leaf, Las rapidly colonizes the root system, but remains sectored in the canopy. Enhancing our understanding of Fusarium maize stalk rot pathogenesis through gene association subnetwork module analyses H. ZHANG (1), H. Zhang (2), M. Kim (2), H. Yan (2), B. Yoon (2), W. Shim (2) (1) Texas A&M university, U.S.A.; (2) Texas A&M university, U.S.A. Enhancing our understanding of Fusarium maize stalk rot pathogenesis through gene association subnetwork module analyses H. ZHANG (1), H. Zhang (2), M. Kim (2), H. Yan (2), B. Yoon (2), W. Shim (2) (1) Texas A&M university, U.S.A.; (2) Texas A&M university, U.S.A. Fusarium verticillioides is an important stalk rot pathogen of maize. While a select number of genes associated with virulence have been characterized, our knowledge of the genetic network underpinning this mechanism is very limited. Previously, our lab identified a striatin-like protein, Fsr1, that plays a key role in stalk rot pathogenesis. To further characterize genetic networks downstream of Fsr1, we performed RNA-Seq with maize B73 stalks inoculated with F. verticillioides wild type and fsr1 mutant. Datasets were first used to infer F. verticillioides co-expression networks. Subsequently, we used a computationally efficient branch-out technique, along with an adopted probabilistic pathway activity inference method, to identify functional subnetwork modules likely involved in F. verticillioides pathogenicity. Through our analyses, potential pathogenicity-associated subnetwork modules were identified, each consisting multiple interacting genes with coordinated expression patterns, but whose collective activation level is significantly different in the wild type and the mutant. In this study we selected three putative pathogenicity genes, FvSYN1, FvEBP1 and FvCYP1, from three predicted subnetworks. We generated gene-deletion mutants and performed pathogenicity assays. Furthermore, qPCR assays were performed to determine whether these genes serve as a hub gene and impose critical regulatory control on associated genes in each subnetwork. S4.18 Basal rot of onion, caused by Fusarium oxysporum f.sp. cepae (FOC), is a soilborne fungus and part of the F. oxysporum species complex, which includes other important pathogenic formae speciales (f.spp.) adapted to particular crop hosts, as well as non-pathogenic isolates. The genetic basis for pathogenicity in F. oxysporum is poorly understood, but ‘secreted in xylem’ (SIX) genes have recently been associated with virulence in several f.spp. A set of 32 F. oxysporum isolates were tested for pathogenicity on onion and whole genome sequencing and PCR approaches used to detect putative effector genes. Seven SIX genes and two other putative effectors were identified in highly pathogenic isolates which were all absent in non-pathogenic isolates. Expression of SIX genes and other putative effectors increased in planta over 96 hours following inoculation of onion seedlings. Different SIX gene complements were also identified in other f. spp. but none were found in F. avenaceum, F. proliferatum or F. Enhancing our understanding of Fusarium maize stalk rot pathogenesis through gene association subnetwork module analyses H. ZHANG (1), H. Zhang (2), M. Kim (2), H. Yan (2), B. Yoon (2), W. Shim (2) (1) Texas A&M university, U.S.A.; (2) Texas A&M university, U.S.A. The current results demonstrate that quantitative pyrosequencing is a robust and reliable tool for rapid detection, quantification, and monitoring of multiple genotypes within complex fungal communities associated with crops. Engineering mobile RNA in Carrizo to enhance plant defense responses to control citrus greening S. Zhang (1), G. Perazzo (1), D. Gabriel (1) (1) Integrated Plant Genetics, U.S.A. Developing resistance to citrus huanglongbing C. RAMADUGU (1), M. Keremane (2), T. McCollum (3), D. Hall (3), M. Roose (1) C. RAMADUGU (1), M. Keremane (2), T. McCollum (3), D. Hall (3), M. Roose (1) (1) University of California Riverside, U.S.A.; (2) USDA ARS National Clonal Germplasm Repository for Citrus and Dates, U.S.A.; (3) US Horticultural Research Laboratory, U.S.A. ( ) ( ) ( ) ( ) ( ) (1) University of California Riverside, U.S.A.; (2) USDA ARS National Clonal Germplasm Repository for Citrus and Dates, U.S.A.; (3) US Horticultural Research Laboratory, U.S.A. Citrus huanglongbing (HLB) has caused significant damage to citrus industries in Florida and Brazil. The disease is associated with a fastidious bacterium, Candidatus Liberibacter asiaticus (Las) and transmitted by psyllids. Development of HLB resistant rootstock and scion cultivars will be beneficial to the industry. Sources of HLB resistance are not known in the genus Citrus. We have demonstrated HLB resistance in Eremocitrus and Microcitrus which are closely related genera sexually compatible with Citrus. Naturally occurring hybrids of Citrus with Eremocitrus and Microcitrus parentage were exposed to the HLB pathogen under greenhouse conditions and determined to be HLB resistant in controlled experiments. Since the resistance from these non-Citrus genera appears to be heritable, we have generated about 800 mandarin and trifoliate hybrids using Eremocitrus and Microcitrus as pollen parents. The hybrid seeds were raised in greenhouses in Florida and challenged by feeding with LAS-positive psyllids for four weeks. Preliminary results indicate the absence of Las in many hybrids. We are monitoring the putative resistant hybrid plants continuously by assaying for the presence of Las by qPCR and digital PCR, and also by observing the hybrids for expression of disease symptoms. We will confirm resistance after conducting no-choice psyllid feeding experiments. Engineering mobile RNA in Carrizo to enhance plant defense responses to control citrus greening S. Zhang (1), G. Perazzo (1), D. Gabriel (1) (1) Integrated Plant Genetics, U.S.A. Plants modulate plant defense responses against pathogens with both pro- and anti-programmed cell death (PCD) proteins. By suppressing expression of anti-PCD proteins, PCD can occur more rapidly and with stronger defense responses. There are many PCD triggers, including Reactive Oxygen Species (ROS) which activate plant defense signaling pathways over long distances. Citrus greening is caused by Ca. Liberibacter asiaticus (Las). Las produces a peroxidase that has been suggested to dampen the ROS signaling pathway. A gene silencing strategy was used to suppress an anti-PCD gene in Carrizo rootstocks. The expression levels of three citrus defense response genes (NDR1, PR1 and EDS1) were significantly higher in silenced lines than in nontransgenic (NT) controls after inoculation with Las flagellin (flg22), used as a proxy for Las. Silenced Carrizo lines were also more tolerant to heat and cold stresses than NT controls. Challenge inoculations are currently underway using approach-grafted, Las infected citrus; all sampling was taken from leaves above the approach graft union. To date, three transgenic Carrizo lines failed to become infected over a 9 month challenge period as compared with two transgenic lines that became infected within two months. Following removal of the infected source tree, two NT scions that were consistently positive have now tested negative, possibly indicating movement of the silencing signal from transgenic rootstock to NT scion. Antifungal mechanisms of a plant defensin MtDef4 are not conserved between ascomycete fungi, Neurospora crassa and Fusarium graminearum K ISLAM (1) K El-Mounadi (2) D Shah (3) Small cysteine-rich defensins are ubiquitous antifungal plant peptides which contribute to the innate immunity of plants. The plant defensin, MtDef4, inhibits the growth of the ascomycete fungi, Neurospora crassa and Fusarium graminearum, at micromolar concentrations. Previous studies have suggested that MtDef4 is transported into the cytoplasm of these fungi and exerts its antifungal activity on various intracellular targets. Here, we have investigated whether the antifungal mechanisms of MtDef4 are conserved in these fungi. We show that N. crassa and F. graminearum respond very differently to MtDef4 challenge. Membrane permeabilization is required for the antifungal activity of MtDef4 against F. graminearum but not against N. crassa. We find that MtDef4 is targeted to different subcellular compartments in each fungus. Internalization of MtDef4 in N. crassa is energy-dependent and involves endocytosis. By contrast, MtDef4 appears to translocate into F. graminearum autonomously using partially energy-dependent pathway. MtDef4 has been previously shown to bind to the phospholipid phosphatidic acid (PA). We provide evidence that the plasma membrane localized phospholipase D (PLD1), involved in the biosynthesis of PA, is needed for entry of this defensin in N. crassa, but not in F. graminearum. To our knowledge, this is the first example of a plant defensin which inhibits the growth of two ascomycete fungi via different mechanisms. Identifying genetic variation in the Sorghum PAMP response D. CHEN (1), P. Balint-Kurti (2), G. Stacey (1) (1) Divisions of Plant Science and Biochemistry, University of Missouri-Columbia, MO, 65201, U.S.A.; (2) USDA-ARS, Department of Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A. Sorghum is an important grain, forage and biofuel crop with a fully sequenced genome and a variety of available genetic resources. In comparison to studies of Arabidopsis, very little is known about the innate immunity response of crop species, including sorghum. Such studies could contribute to efforts to create crops with enhanced disease resistance. The plant innate immunity system is triggered by plant recognition of conserved pathogen molecules, so called pathogen-associated molecular patterns (PAMPs). We measured PAMP induced production of reactive oxygen species (ROS) as a quantitative measure of the immunity response in more than 50 different Sorghum genotypes. In addition, we also analyzed the production of nitric oxide (NO) as another measure of the plant response to PAMP addition. The results showed a significant variation among the phenotypes with a few responding very strongly with others showing essentially no response. Proposed model for Candidatus Liberibacter asiaticus and solanacearum systemic invasion and multiplication in the psyllid host and vector J. BROWN (1), T. Rast (1), J. Cicero (2), T. Fisher (1) (1) The University of Arizona, U.S.A.; (2) University of Florida, U.S.A. Canopy sectoring is thought to occur because of limited lateral connections of phloem sieve tubes. S4.19 To investigate if lateral movement of Las between sieve tubes is distance or tissue specific, root systems of grafted and seedling trees were split to three different heights: below crown, above crown, and above graft union (or equivalent on seedlings). The trees were then graft inoculated above one side of the split root system. Infection to both halves of the root system was monitored weekly. Contrary to our hypothesis; the graft union and crown do not play a unique role in lateral movement. Similar patterns of infection occurred in all tissue types of the grafted trees and seedling trees. Greater variability in the time between Las detection in the root systems on the inoculated side and the opposite side in all trees with the highest split suggests that vertical distance is important in lateral movement of the bacterium. Cultivar-specific wheat miRNAs during stripe rust infection in Triticum aestivum S. RAMACHANDRAN (1), N. Mueth (1), P. Zheng (1), S. Hulbert (1) (1) Washington State University, U.S.A. Wheat represents approximately 30% of the world’s production of grain crops with more than 220 million cultivated hectares. Productivity at harvest is mainly governed by genetic composition and environmental factors influencing growth and development. Understanding the genetic factors that regulate these processes will help in developing varieties with better yield potential and disease resistance. MiRNAs are regulators of gene expression in eukaryotes that control myriad processes from development to abiotic and biotic stress responses. Using 12 small RNA libraries prepared from two wheat cultivars infected with stripe rust fungus (Puccinia striiformis), we identified 43 previously-known miRNAs, 50 novel variants of known miRNAs and 93 candidate novel miRNAs. Digital gene expression revealed 20 cultivar-specific miRNAs, 6 miRNAs differentially expressed between the two cultivars and 4 miRNAs responsive to stripe rust infection. These results were validated using RT-PCR and qRT-PCR. Using different target prediction algorithms, 69 wheat miRNAs were found to target fungal genes- a majority of which coded for small, secreted, cellular proteins. These results suggest a trans-kingdom regulation of gene expression potentially involved in the cultivar-specific resistance to stripe rust. Overall, this study contributes to the current repository of wheat miRNAs and provides novel information on the as yet uncharacterized roles for miRNAs in wheat host-pathogen interactions. Proposed model for Candidatus Liberibacter asiaticus and solanacearum systemic invasion and multiplication in the psyllid host and vector J. BROWN (1), T. Rast (1), J. Cicero (2), T. Fisher (1) (1) The University of Arizona, U.S.A.; (2) University of Florida, U.S.A. Antifungal mechanisms of a plant defensin MtDef4 are not conserved between ascomycete fungi, Neurospora crassa and Fusarium graminearum K. ISLAM (1), K. El-Mounadi (2), D. Shah (3) (1) Donald Danforth Center, U.S.A.; (2) Kutztown University of Pennsylvania, U.S.A.; (3) Donald Danforth Center, Bangladesh Antifungal mechanisms of a plant defensin MtDef4 are not conserved between ascomycete fungi, Neurospora crassa and Fusarium graminearum K ISLAM (1) K El-Mounadi (2) D Shah (3) This variation provides the basis for additional experiments focused on defining specific genetic loci that control the Sorghum PAMP response. We are also conducted a transcriptomic analysis of the Sorghum PAMP response with the goal to define expression QTL (eQTL) related to pathogen resistance. The goal is to use the information obtained to aid breeding efforts to increase biotic stress resistance in Sorghum. Understanding quantitative disease resistance in rice: Defense response gene regulation and promoter patterns B. TONNESSEN (1), R. Mauleon (2), N. Alexandrov (2), J. Leach (3) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State Univers g q p g g p p B. TONNESSEN (1), R. Mauleon (2), N. Alexandrov (2), J. Leach (3) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. ( ), ( ), ( ), ( ) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. ( ) ( ) ( ) ( ) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. Sessa (2) (1) Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Israel; (2) Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Israel Xanthomonas effector kinase XopAU promotes disease symptoms by activation of host MAPKK D. TEPER (1), E. Bosis (1), G. Popov (1), A. Girija (1), G. Sessa (2) (1) Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Israel; (2) Department of Molecular Biology and Ecology of Plants, Tel Aviv University, Israel Xanthomonas euvesicatoria (Xcv) is the causal agent of bacterial spot disease of pepper. Xcv pathogenicity depends on a type III secretion system that delivers effector proteins into host cells to manipulate host signaling and promote disease. Transient expression of the Xcv effector XopAU in host and non-host plants promoted typical defense responses including phosphorylation of MAPKs, accumulation of pathogenesis-related (PR) proteins and elicitation of cell death. Genetic analysis of XopAU by insertional mutagenesis and overexpression revealed a role for this effector in the development of chlorotic disease symptoms. XopAU encode a catalytically active protein kinase and this activity is required for its biological function. Protein-protein interaction studies demonstrated that XopAU physically interacts with the plant immunity-associated MAPKK MKK2. In addition, MKK2 was shown to be directly phosphorylated by XopAU in vitro while expression of XopAU in planta promotes an increase in the phosphorylation of MKK2. Silencing of MKK2 or overexpression of catalytically inactive MKK2K99R in Nicotiana benthamiana reduced XopAU-mediated cell death and MAPK phosphorylation while silencing of MKK2 in pepper attenuated the Xcv-mediated chlorotic disease symptoms. Together, this study indicates that XopAU contributes to Xcv disease symptoms by manipulating host MAPK signaling through activation of MKK2. Dissecting the function and downstream targets of CsLOB1 in citrus S. DUAN (1), H. Jia (1), S. Gowda (1), N. Wang (1) (1) Citrus research and education center, U.S.A. Citrus canker caused by Xanthomonas citri pv. citri (Xcc) is a devastating disease affecting most citrus species. PthA4, one of Xcc Type III secretion system effectors, is responsible for citrus canker pustule formation on citrus leave. PthA4 binds to the effector binding element in the promoter of CsLOB1 to activate its gene expression, which subsequently activates genes responsible for pustule formation. CsLOB1 has not been functionally characterized and it remains unknown how CsLOB1 controls its downstream genes. CsLOB1 was highly expressed in the citrus stem phloem and cambial zone in wild-type plants. ( ) ( ) ( ) ( ) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. The results showed an increase of free SA levels in leaves, proportional to the number of COS-OGA applications. Finally, repetitive COS-OGA sprayings efficiently protected greenhouse-grown tomato against Leveillula taurica, with 90% leaf protection in terms of severity. confirmed by SA quantification in the leaves. The results showed an increase of free SA levels in leaves, proportional to the number of COS-OGA applications. Finally, repetitive COS-OGA sprayings efficiently protected greenhouse-grown tomato against Leveillula taurica, with 90% leaf protection in terms of severity. Degradation of the master regulator of plant defense NPR1 by the type III effector HopAB2 Z. FU (1), H. Chen (1), J. Chen (2), Z. Shang (1), M. Li (1), F. Liu (3) (1) University of South Carolina, Columbia, SC, U.S.A.; (2) Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China; (3) Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China g g p y yp p Z. FU (1), H. Chen (1), J. Chen (2), Z. Shang (1), M. Li (1), F. Liu (3) (1) University of South Carolina, Columbia, SC, U.S.A.; (2) Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China; (3) Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, China The plant hormone salicylic acid (SA) is required for systemic acquired resistance (SAR). NPR1, as an SA receptor, is required for SA-mediated plant defense. The expression of over 97% of SA-responsive genes is dependent on NPR1, therefore, NPR1 functions as a master regulator of plant defense. Many studies have shown that pathogen effectors suppress or disrupt MAMP-triggered immunity (MTI) and cell death. There are also isolated studies, which suggest that effectors suppress or disrupt SA-mediated plant defense. In order to provide strong and direct evidence on the targeting of SA- mediated plant defense by pathogen effectors, we tested the interactions between the SA receptor NPR1 and all type III effectors from the bacterial pathogen Pseudomonas syringae in yeast two-hybrid assays. Interestingly, we found that the type III effector HopAB2 interacts with NPR1 only in the presence of SA or its active analogs. Using Agrobacterium co-infiltration assays, we found that HopAB2 degrades GFP- tagged NPR1 protein, dependent on its E3 ligase activity and through the 26S proteasome. Using Dexamethasone (DEX)-inducible Arabidopsis transgenic plants expressing the hopAB2 gene, we found that HopAB2 degrades NPR1 native protein. ( ) ( ) ( ) ( ) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. The potential yield of rice worldwide is reduced by one third due to disease. The extensive work in breeding rice varieties that harbor resistance genes has helped to mitigate this damage. The method is effective, but the selective pressure of a single host resistance gene can be overcome after only a few generations. Enhancing the expression of basal defense response (DR) genes that are activated against a broad spectrum of pathogens could be a more sustainable approach to breeding for resilience. DR gene promoters can affect timing and intensity of expression, and rice varieties with good basal resistance have been shown to contain polymorphisms that are beneficial in the activation of the defense response. Recently, the genomes of a diverse panel of 3000 rice varieties were sequenced. This information, along with transcriptomic analyses of resistant interactions, were utilized in this study to uncover the genome-wide patterns in DR gene promoters. These patterns are potential cis-elements, and DR gene-specific combinations of these elements in multiple loci infers their functionality in fine-tuning regulation during a resistant host-pathogen interaction. These results could refine marker assisted breeding for a stable, broad spectrum resistance. COS-OGA stimulates plant innate immunity in a cumulative process that involves salicylic acid G. VAN AUBEL (1), P. Van Cutsem (2) ( ), ( ) (1) University of Namur, Belgium; (2) University of Namur, Research Unit in Plant Cellular and Molecular Biology, Belgium COS-OGA is a new patented active substance for plant protection registered in Europe against powdery mildew. COS-OGA contains chitosan oligomers (COS), plant non self-molecules combined with pectin-derived oligogalacturonides (OGA), plant self-molecules. COS-OGA mimics a plant-fungus interaction: during plant infection, fungal pathogens deacetylate their cell wall chitin into chitosan to escape plant chitin receptors. Chitosan fragmentation by plant enzymes then yields polycationic COS molecules while plant cell wall degradation by fungal polygalacturonases releases polyanionic OGA molecules. The plant defense stimulation was investigated on tomato plants after several foliar sprayings of COS-OGA. A proteomic study on leaves revealed that the elicitor treatment led mainly to an overexpression of Pathogenesis-Related proteins, among which P69 subtilisin-like proteases. Gene expression study by qRT-PCR revealed that COS-OGA significantly upregulated transcripts of salicylic acid (SA)-responsive genes while transcripts linked to ethylene and jasmonic acid pathways were not affected. The activation of SA-dependent plant defense reactions was S4.20 confirmed by SA quantification in the leaves. ( ) ( ) ( ) ( ) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. Similar to npr1-2 mutant plants, these transgenic Arabidopsis plants show sensitivity to SA-induced toxicity and are comprised in SAR. Our studies provide solid evidence that HopAB2 disrupts SA-mediated plant defense by degrading NPR1. Resistance of Carolina Gold Select rice to African strains of Xanthomonas oryzae pv. oryzicola is triggered by inactivated TAL effectors L. TRIPLETT (1), S. Cohen (2), C. Heffelfinger (3), A. Bogdanove (4), J. Leach (2) (1) The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (2) Colorado State University, U.S.A.; (3) Yale University, U.S.A.; (4) Cornell University, Ithaca, NY, U.S.A.; (5) Colorado State University, Fort Collins, CO, U.S.A. L. TRIPLETT (1), S. Cohen (2), C. Heffelfinger (3), A. Bogdanove (4), J. Leach (2) (1) The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (2) Colorado State University, U.S.A.; (3) Yale University, U.S.A.; (4) Cornell University, Ithaca, NY, U.S.A.; (5) Colorado State University, Fort Collins, CO, U.S.A. The rice pathogen Xanthomonas oryzae pv. oryzicola causes bacterial leaf streak of rice, a disease for which no single-gene resistance is available. The bacterium produces numerous transcription activator-like (TAL) effectors that activate host susceptibility genes. There are two common rice resistance mechanisms against TAL effectors: DNA polymorphisms that allow escape of TAL effector binding, and placement of TAL effector binding sites to cause specific TAL effectors to activate resistance genes. We found that the heirloom rice variety Carolina Gold Select has a third form of resistance, triggered by diverse TAL effectors. The resistance was triggered in a manner independent of the transcriptional activation domain and of the composition of repeat variable diresidues that specify host DNA target sequences. While the TAL effector central repeat region was critical to resistance, an effector with only 3.5 central repeats was sufficient for resistance activation. The resistance was mapped to a single dominant locus, Xo1, on chromosome 4. The Xo1 interval confers complete resistance to strains in the African clade of X. oryzae pv. oryzicola, representing the first dominant resistance locus against bacterial leaf streak in rice. The TAL effector triggered resistance phenotype has strong similarity to that conferred by the tomato resistance gene Bs4, suggesting that monocots and dicots have converged on the structure of TAL effectors as a pattern triggering resistance. Xanthomonas effector kinase XopAU promotes disease symptoms by activation of host MAPKK D. TEPER (1), E. Bosis (1), G. Popov (1), A. Girija (1), G. ( ) ( ) ( ) ( ) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. Further work is being conducted to explore the effects of various environmental factors on conidia production and dispersal. Detection and quantification of Bremia lactucae by spore trapping and quantitative PCR S. KUNJETI (1), A. Anchieta (2), F. Martin (3), Y. Choi (4), M. Thines (5), R. Mitchelmore (1), S. Koike (6), C. Tsuchida (1), W. Mahaffee (7), K. Subbarao (1), S. Klosterman (3) (1) University of California Davis, U.S.A.; (2) USDA Salinas, Salinas, CA, U.S.A.; (3) USDA-ARS, Salinas, CA, U.S.A.; (4) Korea University, Division of Environmental Sciences and Ecological Engineering, Seoul 136-701, Korea; (5) Biodiversity and Climate Research Center (BiK-F), Germany; (6) University of California Cooperative Extension, Monterey County, Salinas, U.S.A.; (7) USDA-ASR, U.S.A.; (8) University of California Davis, Salinas, CA, U.S.A. a (2), F. Martin (3), Y. Choi (4), M. Thines (5), R. Mitchelmore (1), S. Koike (6), C. Tsuchida (1), W. Mahaffee (7), K. (3) Subbarao (1), S. Klosterman (3) (1) University of California Davis, U.S.A.; (2) USDA Salinas, Salinas, CA, U.S.A.; (3) USDA-ARS, Salinas, CA, U.S.A.; (4) Korea University, Division of Environmental Sciences and Ecological Engineering, Seoul 136-701, Korea; (5) Biodiversity and Climate Research Center (BiK-F), Germany; (6) University of California Cooperative Extension, Monterey County, Salinas, U.S.A.; (7) USDA-ASR, U.S.A.; (8) University of California Davis, Salinas, CA, U.S.A. Bremia lactucae adversely affects marketability of lettuce. The disease has been managed with a combination of host resistance and fungicide applications with mixed success over the years. Fungicide applications are routinely applied under the assumption that inoculum is always present during favorable environmental condition. This approach could lead to fungicide resistance in B. lactucae populations. Detection and quantification of airborne B. lactucae near lettuce crops may lead to a more judicious timing of fungicide applications. For specific detection of B. lactucae, we designed a qPCR assay based on a target sequence in B. lactucae mitochondrial DNA. PCR tests using DNA from 83 geographically diverse isolates, representing 14 Bremia spp., and amplicon sequencing confirmed that the primers developed for the TaqMan assays are species-specific and only amplify template from B. lactucae. Cq values >35 were considered as nonspecific but a single sporangium could be detected at a Cq of 34. The correlation (R2) for regression between sporangial density derived from flow cytometry and Cq values derived from the qPCR was 0.86. The assay was deployed using spore traps in the Salinas Valley, where nearly half the US lettuce is produced. ( ) ( ) ( ) ( ) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. We aim to study the function of CsLOB1 and identify the putative downstream target genes of CsLOB1. To observe the physiological effect by suppressing CsLOB1 expression, a Citrus Tristeza Virus-based RNAi vector, engineered with truncated anti-sense CsLOB1, was constructed for Virus-induced Gene Silencing (VIGS). Suppression of CsLOB1 via VIGS caused decreased stem diameter and retarded plant growth. To further confirm this result, Agrobacterium-mediated transient gene expression method is being used to reveal the CsLOB1 function during pustule formation post Xcc infection on citrus leave using small hairpin (shRNA) and artificial microRNA methods. Meanwhile, Dexamethasone-inducible CsLOB1 over-expression transgenic plants were constructed to identify the putative downstream genes of CsLOB1. Trapping conidia spores of Podosphaera aphanis in high tunnels and field strawberry plots R. ONOFRE (1), D. Gadoury (2), N. Dufault (1), N. Peres (1) (1) University of Florida, U.S.A.; (2) Cornell University, U.S.A. Strawberry Powdery Mildew (SPM) caused by Podosphaera aphanis can be severe on susceptible cultivars, especially in tunnel and glasshouse production. Epidemics of SPM are largely driven by intense sporulation and conidia dispersal, which are two highly relevant factors for management strategies. The objective of this study was to evaluate SPM aerial conidia spore concentrations in plastic tunnel and field environments of Florida. A 7- day recording volumetric spore trap was installed in both the field and high tunnel plots during the 2015-16 growing season. Airborne conidial dose at each site was recorded hourly for 5 weeks, as was temperature, relative humidity, leaf wetness and rainfall. In both plots, spore counts followed a diurnal pattern, with a peak in aerial concentrations between 10 am and 2 pm. Cumulative airborne dose in the tunnel (1488 spores/m3) was notably higher than in the field (82 spores/m3). Several coincident factors likely contribute to the airborne dose in tunnel growing systems, including an inherently more favorable environment for sporulation and infection, and subsequent increased disease severity. However, factors such as residence time of conidia S4.21 within the system after becoming airborne, and effects of visible light and UV radiation on inoculum production and dispersal may also contribute to higher inoculum dose. Further work is being conducted to explore the effects of various environmental factors on conidia production and dispersal. within the system after becoming airborne, and effects of visible light and UV radiation on inoculum production and dispersal may also contribute to higher inoculum dose. Plant pathogens were collected over Florida up to 3500 m by a wing-mounted dust collection system called DART (Dust Atmospheric Recovery Technology) A SCHUERGER (1) B T h (2) T E (3) J P l i (4) A. SCHUERGER (1), B. Tench (2), T. Emmons (3), J. Palaia (4) (1) University of Florida, U.S.A.; (2) Dept. of Plant Pathology, University of Florida, U.S.A.; (3) Dept. of Engineering, Univ. of Central Florida, U.S.A.; (4) 4Frontiers Corporation, U.S.A. A. SCHUERGER (1), B. Tench (2), T. Emmons (3), J. Palaia (4) (1) University of Florida, U.S.A.; (2) Dept. of Plant Pathology, University of Florida, U.S.A.; (3) Dept. of Engineering, Univ. of Central Florida, U.S.A.; (4) 4Frontiers Corporation, U.S.A. Asian dust plumes deliver up to 64 million tonnes of dust over the NW of the USA, and African dust storms deliver over 50 million tonnes of dust over Florida each year. Recent studies have demonstrated that plant pathogens associated with Asian and African aerosols can be transported to the USA in naturally occurring dust storms. In order to initiate a long-term monitoring program of plant pathogens in Asian and African dust plumes, we have developed a dust collection system called DART (Dust Atmospheric Recovery Technology). The DART dust sampler was mounted on a F104 Starfighter jet and separately on a T6 Texan propeller driven airplane and test flown over FL in 2013-2014. The DART system utilizes a high-volume pump to pass air through 4 separate filtration units where both aerosols and microbial cells are captured. Flow rates through filters are directly correlated to increased air speed, and are inversely correlated to increased altitude. The DART dust sampler has performed nominally up to 7600 m, 0.92 Mach, and 3.5 +G’s. During initial test flights in 2013 and 2014, fungi recovered from the lower troposphere over FL (up to 3500 m) contained plant pathogens including species in the genera: Acremonium, Aspergillus, Cladosporium, Curvularia, and Fusarium. Numbers of recovered fungi, but not bacteria, increased significantly when 5 or 10 µm filters were used in the DART system compared to filter pore sizes ≤ 2 µm. Validation of the CDM ipmPIPE Forecasting System: Relating Aerial Transport of Pathogen Spores to Outbreaks of Cucurbit Downy Mildew K. NEUFELD (1), A. Keinath (2), B. Dutta (3), B. Gugino (4), D. Langston (5), M. Lewis Ivey (6), M. McGrath (7), S. Miller (8), E. Sikora (9), P. Ojiambo (1) Validation of the CDM ipmPIPE Forecasting System: Relating Aerial Transport of Pathogen Spores to Outbreaks of Cucurbit Downy Mildew K. NEUFELD (1), A. Keinath (2), B. Dutta (3), B. Gugino (4), D. Langston (5), M. Lewis Ivey (6), M. Rapid LAMP diagnostics for detection and disease alerts of Phytophthora infestans J. RISTAINO (1), A. Saville (1), R. Guenter (1), C. Lagaly (1), D. Cooper (2) (1) North Carolina State University, U.S.A.; (2) Mobile Assay, U.S.A. Timing of primary inoculum release by Monilinia vaccinii-corymbosi infecting blueberries in the US Pacific Northwest D. HARTEVELD (1), T. Peever (2), J. Pscheidt (3) (1) Washington State University U S A ; (2) Washington State University U S A ; (3) Oregon State University U S A Timing of primary inoculum release by Monilinia vaccinii-corymbosi infecting blueberries in the US Pacific Northwest D. HARTEVELD (1), T. Peever (2), J. Pscheidt (3) (1) Washington State University, U.S.A.; (2) Washington State University, U.S.A.; (3) Oregon State University, U.S.A. D. HARTEVELD (1), T. Peever (2), J. Pscheidt (3) (1) Washington State University, U.S.A.; (2) Washington State University, U.S.A.; (3) Oregon State University, U.S.A. Washington State University, U.S.A.; (2) Washington State University, U.S.A.; (3) Oregon State University, U.S.A. Mummy berry, caused by Monilinia vaccinii-corymbosi, infects highbush blueberry (Vaccinium corymbosum) and causes severe economic losses in the Pacific Northwest (PNW). Control is mainly based on calendar-based fungicide sprays. The primary inoculum source is ascospores produced from apothecia that develop on mummified berries, or pseudosclerotia, that overwinter on the soil surface. Timing of apothecial development and ascospore release varies among locations and seasons, and differences in environmental factors may be why control programs are not always effective. This study aimed to identify environmental factors involved in the timing of primary inoculum release in the PNW, and develop a predictive model for ascospore release. Apothecial development was monitored in a severely affected field from 2008-2013 in Corvallis, Oregon and in two fields in northwestern Washington during the 2015 season. Data for eleven environmental factors was obtained from in-field weather stations and Washington State University’s AgWeatherNet. Soil temperature, solar radiation and precipitation were the most significant factors influencing apothecial development demonstrated by a generalized linear model regression analysis. A forecasting model developed to predict primary inoculum release is being validated for PNW locations during the 2016 season. Outcomes of this research will improve the timing of disease management practices and overall control of mummy berry in the PNW. ( ) ( ) ( ) ( ) (1) Colorado State University, U.S.A.; (2) International Rice Research Institute, Philippines; (3) Colorado State University, U.S.A. The sensitivity of the B. lactucae-specific assay enabled detection of the pathogen within the two-week lettuce free period, and indicated that the assay can be useful for predicting inoculum load for timing of fungicide applications. Timing of primary inoculum release by Monilinia vaccinii-corymbosi infecting blueberries in the US Pacific Northwes D. HARTEVELD (1), T. Peever (2), J. Pscheidt (3) (1) Washington State University U S A ; (2) Washington State University U S A ; (3) Oregon State University U S A Plant pathogens were collected over Florida up to 3500 m by a wing-mounted dust collection system called DART (Dust Atmospheric Recovery Technology) A SCHUERGER (1) B T h (2) T E (3) J P l i (4) McGrath (7), S. Miller (8), E. Sikora (9), P. (1) Department of Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A.; (2) Clemson University, Charleston, SC, U.S.A.; (3) Department of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.; (4) Department of Plant Pathology and Environmental Microbiology, Pennsylvania State University, University Park, PA, U.S.A.; (5) Virginia Polytechnic Institute and State University, Suffolk, VA, U.S.A.; (6) Department of Plant Pathology and Crop Physiology, Louisiana State University, Baton Rouge, LA, U.S.A.; (7) Plant Pathology and Plant-Microbe Biology, SIPS, Cornell University, Riverhead, NY, U.S.A.; (8) Department of Plant Pathology, Ohio State University, Wooster, OH, U.S.A.; (9) Department of Entomology and Plant Pathology, Auburn University, Auburn University, AL, U.S.A. Cucurbit downy mildew (CDM) caused by Pseudoperonospora cubensis is considered the most damaging disease of cucurbitaceous crops worldwide. Sporangia produced by the pathogen are carried by wind currents and deposited on host plants following rain events. The CDM ipmPIPE forecasting system integrates data on current disease outbreaks and trajectories of sporangia transport from source fields to forecast the risk of new disease outbreaks in the eastern United States. The forecasting system was validated by relating the predicted risks of disease outbreak to the presence or absence of sporangia after a rain event and the onset of disease symptoms in the field. Rainwater samples from eight states, AL, GA, LA, NC, NY, OH, PA, and SC were collected from planting to the first report of disease during the 2013 to 2015 seasons. The presence of sporangia was then analyzed using primers specific to the pathogen. The pathogen was detected in 4-30% of the 203 samples collected from 2013 to 2015. Presence of sporangia in rainwater samples correctly classified disease onset in 67%, 81% and 78% of the cases, while the forecasting system correctly predicted disease onset in 75%, 85% and 60% of the cases in 2013, 2014 and 2015, respectively. Predictions from the forecasting system and presence of sporangia in rainwater samples were in agreement in 60-87% of the cases analyzed except in 2015 when presence of sporangia in rainwater samples were detected infrequently. S4.22 Phytophthora infestans, causal agent of the Irish potato famine, is a threat to food security globally and an important pathogen in the US on tomato and potato. Plant pathogens were collected over Florida up to 3500 m by a wing-mounted dust collection system called DART (Dust Atmospheric Recovery Technology) A SCHUERGER (1) B T h (2) T E (3) J P l i (4) In 2009, late blight epidemics in the eastern US were the worst in modern history due to widespread inoculum distribution from infected tomato transplants and favorable weather. That year the US-22 lineage was identified as the dominant lineage. USABlight (www.usablight.org) was launched in 2011 to improve disease detection and communication between researchers, growers, the industry, and the public about late blight. A total of 1094 late blight reports were recorded between 2011-2015. Of these, 64% were from tomato and a third primarily in the NE US. A shift in the dominant clonal lineage from mefenoxam sensitive US-22 to US-23 occurred and 82% of genotyped strains were US-23. Disease outbreaks begin each year in Florida winter tomatoes. Spread through movement of tomato fruit is suspected. We developed a loop-mediated isothermal amplification (LAMP) assay specific for detection of P. infestans. The assay can be run in the field at 65C, needs no thermocycling, and can detect 100-10 pg/ul of pathogen DNA. A prototype mobile reader developed by Mobile Assay has been used for in-field detection using a SYBR green stain and hand-held tablet. This mobile reader and LAMP assay is being tested in both the US and East Africa to improve pathogen detection and will be used to develop an Afriblight tracking database. LAMP lights the way: Early detection of airborne inoculum of Magnaporthe oryzae in turfgrass fields F. PEDUTO HAND (1), F. Peduto Hand (1), C. Villari (1), W. Mahaffee (2), T. Mitchell (1) (1) The Ohio State University, U.S.A.; (2) USDA-ARS, U.S.A. A host-specialized form of Magnaporthe oryzae (Lolium pathotype, MoL) is the causal agent of Grey Leaf Spot (GLS), a detrimental disease of perennial ryegrass. Early diagnosis of this disease is crucial for making timely management decisions. A pathogen-specific quantitative loop-mediated isothermal amplification (LAMP) assay, that is able to detect as low as 25 spores of the pathogen, was developed in our laboratory to monitor GLS airborne inoculum. To assess the suitability of the method for field use, two perennial ryegrass plots were artificially inoculated with pathogen-infested dried grass clippings at The Ohio State University Turfgrass Research Center in the summer of 2015, and five continuously running custom impaction spore traps were placed in each plot. Sampling units were replaced daily and tested with the developed qLAMP assay while plots were monitored for symptom development. Detection of the select agent Rathayibacter toxicus using recombinase polymerase amplification coupled with a lateral flow device M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Stack (5) ct agent Rathayibacter toxicus using recombinase polymerase amplification coupled with a lateral flow device ot (2), R. Mann (3), B. Rodoni (4), J. Stack (5) Detection of the select agent Rathayibacter toxicus using recombinase polymerase amplification coupled with a lateral flow device M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Stack (5) (1) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.; (2) Department of Plant Pathology, Kansas State University, Manhattan, KS, Australia; (3) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Manhattan, KS, U.S.A.; (4) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Bundoora, Australia; (5) Department of Plant Pathology, Kansas State University, Australia (1) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.; (2) Department of Plant Pathology, Kansas State University, Manhattan, KS, Australia; (3) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Manhattan, KS, U.S.A.; (4) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Bundoora, Australia; (5) Department of Plant Pathology, Kansas State University, Australia Rathayibacter toxicus is a gram-positive plant pathogen responsible for a lethal disease of livestock in Australia. An easy, sensitive in-field detection method would enhance capabilities for port of entry inspections and biosecurity surveillance surveys of annual ryegrass (Lolium rigidum) hay and seed. Recombinase polymerase amplification (RPA) is a fast and sensitive isothermal DNA amplification and detection technology. Long forward and reverse primers (32 bp) and a long probe (49 bp) labelled with FAM were designed using gene involved in toxin production. TwistDX kit was used to amplify the target DNA (at ~37°C) and a lateral flow device used to visualize the amplified products. RPA primers and probes targeting the ITS region of the host genomes were designed for use as internal controls, to enhance the reliability and accuracy of the assays. RPA assays were tested against inclusivity and exclusivity panels for high specificity and accuracy. In-field validation in South Australia confirmed the efficiency and sensitivity of the RPA assays; detected the R. toxicus from infected samples. The proposed RPA method will enhance diagnostics, surveillance, disease management, and in-field biosecurity decisions. Development of a rapid AmplifyRP Acceler8 test for grapevine red blotch-associated virus in grapevines R. LI (1), M. Fuchs (2), K. Perry (3), T. Mekuria (4), S. Detection of the select agent Rathayibacter toxicus using recombinase polymerase amplification coupled with a lateral flow device M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Stack (5) Zhang (1) (1) Agdia Inc., U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell University, U.S.A.; (4) Vintage Nurseries, U.S.A Grapevine red blotch-associated virus (GRBaV) is of unprecedented concern to the grape industry since its discovery in 2011. A rapid, reliable, sensitive, and user-friendly test is needed to quickly identify GRBaV-infected grapevines and facilitate their timely removal from vineyards. An AmplifyRP Acceler8 detection kit was developed for GRBaV for use both in laboratories and vineyards. The test is performed in a disposable detection chamber for 20 minutes using crude grapevine extracts. The test specifically detects up to 11 copies of GRBaV genomic DNA in a matrix of healthy crude extract. The test has no cross reactivity to host plant tissues and grapevine-infecting pathogens including arabis mosaic virus (ArMV), grapevine fanleaf virus (GFLV), grapevine leafroll-associated virus 1 (GLRaV-1), grapevine leafroll-associated virus 2 (GLRaV-2), grapevine leafroll-associated virus 3 (GLRaV-3), grapevine leafroll-associated virus 4 strain 5 (GLRaV-4 strain 5), grapevine fleck virus (GFkV), tomato ringspot virus (ToRSV), tobacco ringspot virus (TRSV), Xylella fastidiosa, and Botrytis cinerea. The pellets contain all the necessary reaction components and are stable for a long period of storage even at 37°C. The test has been validated using both viral DNA and crude plant extracts as templates. An Integrated Microfluidic Device for Rapid Plum Pox Virus Enrichment and Detection S. ZHENG (1), Y. Yeh (1), V. Mavrodieva (2), W. Zhang (1), Z. Liu (2) (1) The Pennsylvania State University, U.S.A.; (2) USDA, APHIS, PPQ, CPHST-Beltsville Laboratory, U.S.A. Rapid and sensitive detection is highly desirable for plant viral pathogens. However, plant samples with low virus concentrations pose significant challenges for early detection. We present a miniature disposable device, carbon nanotube size tunable enrichment microdevice (CNT-STEM), that is capable of rapidly concentrating and purifying plum pox virus (PPV) from leaf tissues. This microdevice enriches PPV in the sample by employing physical size-based exclusion. The device consists of patterned carbon nanotube (CNT) arrays and a microfluidics channel made of polydimethylsiloxane (PDMS). The microdevice captures PPV from homogenized leaf samples inside the CNT nanostructure while most of the host contaminants are removed. After elution, the PPV enriched sample is subjected to real-time RT-PCR. The procedure takes less than 2 hours, significantly faster than standard immunocapture virus enrichment procedure. Detection of the select agent Rathayibacter toxicus using recombinase polymerase amplification coupled with a lateral flow device M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Stack (5) The results showed that the microdevice increased analytical sensitivity of PPV real-time RT-PCR by up to 100 fold compared with the current USDA standard immunocapture sample preparation method. The microdevice could enable early detection of PPV by enriching virus particles directly from plant samples. The technology can also serve as a platform for enrichment of other plant viral pathogens. Plant pathogens were collected over Florida up to 3500 m by a wing-mounted dust collection system called DART (Dust Atmospheric Recovery Technology) A SCHUERGER (1) B T h (2) T E (3) J P l i (4) Results confirmed that the qLAMP assay-trap system was able to detect the pathogen’s inoculum up to two weeks before symptoms developed in the field. The implementation of this assay by practitioners has the potential to be a useful decision support tool to guide initiation and timing of fungicide applications for GLS management. Aspergillus communities contaminating red chilies with aflatoxins P. SINGH (1), P. Cotty (1) (1) University of Arizona, U.S.A. Cotty (4) (1) University of Arizona, Tucson, AZ, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Nampula, Mozambique; (3) International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria; (4) Agricultural Research Service, USDA and School of Plant Sciences, University of Arizona, Tucson, AZ, U.S.A. ty of the Aspergillus section Flavi S morphotype in Mozam Diversity of the Aspergillus section Flavi S morphotype in Mozambique L. ARONE (1), J. Augusto (2), R. Bandyopadhyay (3), P. Cotty (4) (1) University of Arizona, Tucson, AZ, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Nampula, Mozambique; (3) International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria; (4) Agricultural Research Service, USDA and School of Plant Sciences, University of Arizona, Tucson, AZ, U.S.A. Aflatoxins are potent mycotoxins that contaminate foods and feeds worldwide. These cancer causing toxins also impair development and suppress immune systems. In Mozambique rejection of groundnuts from export markets and high prevalence of liver cancer are linked to aflatoxins. To improve understanding of contamination in Mozambique, aflatoxin producers were compared with known causal agents from other regions. Since, Aspergillus section Flavi isolates with S morphology are linked to severe contamination in North America and East Africa, relationships of S morphology aflatoxin producers to previously described fungi were characterized. Partial sequences of aflatoxin transcription factor (aflR, 2.1 kb) and nitrate reductase (niaD, 2.1 kb) genes were compared among S isolates from Mozambique, Kenya, Benin, and USA. Phylogenetic analyses sorted 200 S isolates from Mozambique into four clades. Isolates in the first clade produced only B aflatoxins and were closely related to fungi previously associated with lethal aflatoxicosis in Kenya. The other three clades produce both B&G aflatoxins. One of these contained A. minisclerotigenes and another was sister to the Kenya isolates. The third B&G clade is divergent from but most closely related to both the NRRL isolate A-11612 and fungi from West Africa belonging to an unnamed taxon referred to as SBG. Fungi in all four clades produce high aflatoxin concentrations and are potentially important causes of crop contamination. RNAseq analysis reveals oxidative stress responses of Aspergillus flavus are related to stress tolerance and aflatoxin production J. FOUNTAIN (1), P. Bajaj (2), L. Yang (3), M. Pandey (2), S. Nayak (2), V. Kumar (2), A. Jayale (2), A. Chitikineni (2), S. Chen (4), R. Lee (3), B. Scully (5), R. Kemerait (3), R. Varshney (2), B. Exploration of biofilm formation and its potential link to virulence in the Goss’s wilt pathogen Clavibacter michiganensis subspecies nebraskensis M. BOTTI-MARINO (1), M. Botti-Marino (1), J. Jacobs (1), M. Chilvers (1), G. Sundin (1) (1) Michigan State University, U.S.A. Aspergillus communities contaminating red chilies with aflatoxins P. SINGH (1), P. Cotty (1) (1) University of Arizona, U.S.A. Aspergillus communities contaminating red chilies with aflatoxins P. SINGH (1), P. Cotty (1) (1) University of Arizona, U.S.A. Aflatoxins are carcinogenic, immunosuppressive metabolites that cause health concerns across the globe. Several Aspergilli have been implicated in aflatoxin contamination events with some aflatoxin-producers associated with specific crops. Red chilies (Capsicum spp.) are produced in warm regions that favor aflatoxin producers. Aflatoxin in red chilies may result in market loss and even crop destruction. In order to gain insight into causal agents of aflatoxin contamination of red chilies, naturally contaminated chilies from markets in Nigeria and the US were examined. The A. flavus L morphotype S4.23 was the most frequently isolated member of Aspergillus section Flavi (89%). Fungi with S strain morphology were also detected in chilies from both countries (10%). Multi-gene phylogenetic analyses of partial gene sequences for nitrate reductase (niaD, 2.1 kb) and aflatoxin pathway transcription factor (aflR, 1.8 kb) resolved S morphotype fungi into three distinct clades. Fungi that produced only B aflatoxins formed a single clade with reference A. flavus regardless of L or S morphology. All S morphotype fungi from chilies purchased in Nigeria produced both B and G aflatoxins and were assigned to either A. minisclerotigenes or the previously described unnamed taxon SBG on the basis of phylogenetic affinities; over 60% of these fungi were assigned to A. minisclerotigenes. The results suggest the etiology of aflatoxin contamination of chili is complex and may vary with region. was the most frequently isolated member of Aspergillus section Flavi (89%). Fungi with S strain morphology were also detected in chilies from both countries (10%). Multi-gene phylogenetic analyses of partial gene sequences for nitrate reductase (niaD, 2.1 kb) and aflatoxin pathway transcription factor (aflR, 1.8 kb) resolved S morphotype fungi into three distinct clades. Fungi that produced only B aflatoxins formed a single clade with reference A. flavus regardless of L or S morphology. All S morphotype fungi from chilies purchased in Nigeria produced both B and G aflatoxins and were assigned to either A. minisclerotigenes or the previously described unnamed taxon SBG on the basis of phylogenetic affinities; over 60% of these fungi were assigned to A. minisclerotigenes. The results suggest the etiology of aflatoxin contamination of chili is complex and may vary with region. Diversity of the Aspergillus section Flavi S morphotype in Mozambique L. ARONE (1), J. Augusto (2), R. Bandyopadhyay (3), P. Aspergillus communities contaminating red chilies with aflatoxins P. SINGH (1), P. Cotty (1) (1) University of Arizona, U.S.A. Guo (5) (1) University of Georgia, Tifton, GA, U.S.A.; (2) International Crop Research Institute for the Semi-Arid Tropics (ICRISAT), India; (3) University of Georgia, U.S.A.; (4) University of Florida, U.S.A.; (5) USDA-ARS, U.S.A. Aflatoxin contamination by Aspergillus flavus is exacerbated by drought stress in the field. Given that reactive oxygen species (ROS) both accumulate in plant tissues during drought and can stimulate aflatoxin production in vitro, we examined the responses of toxigenic isolates of A. flavus to oxidative stress focusing on secondary metabolite production using whole transcriptome sequencing. We examined two high toxin producing isolates, Tox4 and AF13, and one moderate producer, NRRL3357, in aflatoxin conducive yeast extract sucrose medium amended with various levels of H2O2. The high producers exhibited fewer differentially expressed genes (DEGs) than the moderate producer in response to increasing stress. Altered aminobenzoate degradation expression along with altered conidiation and growth also suggests reduced developmental rates in the isolates caused by oxidative stress. Aflatoxin genes were upregulated in response to increasing stress along with genes encoding aflatrem and kojic acid biosynthesis. Additional changes in primary metabolic genes also indicates adaptations to increasing stress. Given that the number and diversity of DEGs observed correlated with previously observed aflatoxin production and oxidative stress tolerance for each isolate, these data suggest that secondary metabolite production is involved in A. flavus oxidative responses. Continuing proteomic analyses using gel and iTRAQ methods are underway to confirm the transcriptome results. Hypoxia tolerance as a factor in Aspergillus flavus invasion and toxin contamination of developing maize seeds S. Chalivendra (1), C. DeRobertis (2), K. Damann (1) (1) Louisana State University, U.S.A.; (2) Louisiana State University, U.S.A. Hypoxia tolerance as a factor in Aspergillus flavus invasion and toxin contamination of developing maize seeds S. Chalivendra (1), C. DeRobertis (2), K. Damann (1) 1) Louisana State University, U.S.A.; (2) Louisiana State University, U.S.A. Aspergillus flavus predominantly colonizes the embryo and peripheral tissues (e.g., pericarp and the aleurone layer) of maize seeds but shows limited or late invasion of the endosperm. This pattern correlates with the O2 levels in seed tissues and is suggestive of a role for hypoxia tolerance in the successful invasion of the fungus in seeds. This proposal was tested by comparing A. flavus isolate responses to O2 deprivation in vitro with those in field inoculated seeds. Hypoxia (3 to 5% O2) allowed conidial germination but retarded radial growth and inhibited conidial and sclerotial development. Anoxia imposed suspended animation in most isolates. Growth differences among isolates correlated with biomass differences in infected kernels. Gene expression studies showed that canonical hypoxia genes (e.g., pdc1) were induced, while key development genes (e.g., brlA) were strongly suppressed by O2 deprivation. Aflatoxin (AF) levels were relatively high in inoculated kernels, although O2 deprivation in vitro decreased AF content in most isolates. Oleic acid addition to the growth medium enhanced toxin levels under hypoxia, consistent with high AF levels observed in seed. Isolate differences in growth, sporulation, gene expression and AF accumulation patterns correlated with their responses during seed colonization, implicating hypoxia tolerance as a key virulence factor in A. flavus. Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Vi i i T h TAREC U S A (2) Vi i i T h Tid t AREC U S A (3) U i it f D l U S A (4) U i it f Management of the disease is difficult, and little is known about the interaction of the causal bacterium Clavibacter michiganensis subsp. nebraskensis (Cmn) with its host. Bacterial cells are known to form simple to complex aggregations, termed biofilms, on biotic and abiotic surfaces, and biofilm formation contributes to the virulence of several plant pathogenic bacteria. We examined the occurrence of biofilm formation in Cmn, and the potential linkage of this phenotype to virulence. A total of 19 Cmn strains were screened for biofilm formation using two in vitro methods, quantification of biofilm formation on glass coverslips and scanning electron microscopy evaluation of biofilms produced on gold mesh grids. These strains were screened for virulence using stab and cut inoculation techniques on Goss’s wilt-susceptible corn hybrids and rated for disease symptoms 10 dpi. Results from these assays indicated variation in biofilm formation ability among strains. There did not appear to be a correlation between biofilm formation and virulence of the pathogen, suggesting that biofilm formation may play other roles in Cmn ecology. The negative regulatory function of tepR for the virulence of Burkholderia glumae in rice is exerted via the quorum-sensing master regulator gene qsmR J. HAM (1), J. Peng (1) (1) Louisiana State University Agricultural Center U S A The negative regulatory function of tepR for the virulence of Burkholderia glumae in rice is exerted via the quorum-se gene qsmR The negative regulatory function of tepR for the virulence of Burkholderia glumae in rice is exerted via the quorum-sensing master regulator gene qsmR J HAM (1) J P (1) ( ) g ( ) (1) Louisiana State University Agricultural Center, U.S.A. Burkholderia glumae causes bacterial panicle blight of rice, which is a globally emerging rice disease. The quorum-sensing system mediated by tofI and tofR plays a key role in the regulation of major virulence factors, such as toxoflavin (a phytotoxin), lipase and flagella. tepR, a newly found regulatory gene of B. glumae, negatively regulates multiple virulence factors including toxoflavin, lipase and exoprotease. In this study, a comparative RNA-seq analysis and a series of genetic research were performed to characterize the global regulatory function of tepR in B. glumae. Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Vi i i T h TAREC U S A (2) Vi i i T h Tid t AREC U S A (3) U i it f D l U S A (4) U i it f ship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic KU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Virginia Tech TAREC, U.S.A.; (2) Virginia Tech Tidewater AREC, U.S.A.; (3) University of Delaware, U.S.A., (4) University of Mary Park, MD, U.S.A. (1) Virginia Tech TAREC, U.S.A.; (2) Virginia Tech Tidewater AREC, U.S.A.; (3) University of Delaware, U.S.A., (4) University of Maryland, College Park, MD, U.S.A. Invasive brown marmorated stink bug (BMSB) feeding damages a variety of crops including field corn. In 2013, a survey of BMSB-infested corn fields in VA indicated BMSB damage is correlated with fumonisin contamination. The objective of the current study was to quantify the relationship between BMSB injury and fumonisin contamination in field corn. Field trials were conducted in DE, MD, and VA in 2015. Ears were covered with mesh bags at tasseling to prevent insect damage. BMSB treatments were 0 or 4 adults applied to bagged ears, and Fusarium treatments were water or a spore suspension of F. verticillioides applied to ears at the R3 growth stage. Treatments were arranged in a randomized complete block factorial design. At maturity, ears were harvested and stink bug damage, Fusarium infection, and fumonisin concentrations were assessed. Stink bug damage and Fusarium infection were significantly different among locations, but there was not a location by treatment interaction. Treatments with BMSB had more damaged kernels compared to those without BMSB. Both BMSB and Fusarium treatments increased Fusarium infection of kernels. However, only the BMSB treatment influenced fumonisin concentrations. Results suggest BMSB increases Fusarium infection and fumonisin contamination in field corn. Further studies are needed to understand mechanisms by which BMSB increases fumonisins, and to develop management strategies to mitigate impacts of BMSB on field corn in the region. Exploration of biofilm formation and its potential link to virulence in the Goss’s wilt path nebraskensis M. BOTTI-MARINO (1), M. Botti-Marino (1), J. Jacobs (1), M. Chilvers (1), G. Sundin (1) (1) Michigan State University, U.S.A. S4.24 Goss’s wilt is a bacterial disease of corn that has re-emerged in the Corn Belt of the United States since 2006 and can cause up to 50% yield loss in extreme cases. Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Vi i i T h TAREC U S A (2) Vi i i T h Tid t AREC U S A (3) U i it f D l U S A (4) U i it f Rodoni (2), S. Liu (1), J. Stack (1) (1) Department of Plant Pathology, Kansas State University, U.S.A.; (2) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Australia Annual ryegrass toxicity (ARGT) is an often fatal disease of livestock caused by Rathayibacter toxicus, a toxin producing, nematode-vectored (Anguina funesta) gram positive bacterium. R. toxicus is believed indigenous to Australia (mainly in Western Australia and South Australia) but is also present in South Africa. Complete genome information increases our understanding of the biology of R. toxicus. A complete genome of R. toxicus isolate WAC3373 (population RT-III from Western Australia) was obtained through de novo assembly (HGAP3 assembly pipeline: Hierarchical Genome Assembly Process) using PacBio sequences. The finished assembly revealed one chromosome of 2,346,032 bp length (no plasmid) with a mean coverage of 99X. The GC content was 61.5%. Annotation projected a total of 2277 genes, including 2221 coding sequences (CDS) of which 1601 genes were assigned a putative function and 620 designated as hypothetical proteins. A total of 1331 genes were assigned to COG function categories. The total number of RNA genes were 51 including, 45 tRNA CDS and 6 rRNA CDS (LSU rRNA, SSU rRNA and 5S rRNA were 3117-, 1486- and 121-bp in length, respectively). Genome analyses will provide a better understanding of the genetic constitution of this high consequence bacterium. Unique regions of the genomes were identified to develop effective diagnostic tools for plant biosecurity applications. Effect of biofertilizer applications on bacterial communities in the rhizosphere of citrus affected by huanglongbing J. LI (1), Y. Zhang (2), L. Li (3), U. Handique (2), N. Wang (3) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) University of Florida, U.S.A. Biofertilizer is increasingly used to maintain the health and productivity of citrus trees affected by huanglongbing (HLB) in Florida. Although the practice is of controversy regarding effectiveness, little is known about its non-target effects in the environment. We investigated the impact of soil application of bacterial biofertilizer agents (BFA) on rhizosphere microbial communities of HLB diseased citrus trees. Soil samples were collected at one week before applying BFA and 14 months after the first application respectively. Bacterial communities were profiled by high-throughput sequencing of 16S rDNA V3-V4 hypervariable region. Comparison of taxonomic communities only revealed marginal changes after application of BFA. Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Vi i i T h TAREC U S A (2) Vi i i T h Tid t AREC U S A (3) U i it f D l U S A (4) U i it f The RNA-seq analysis revealed that none of the known regulatory genes required for toxoflavin production (tofI, tofM, tofR, toxJ, and toxR) was differentially expressed between the wild type (336gr-1) and its ΔtepR derivative (LSUPB401), while the structural genes for the toxoflavin biosynthesis and transport were more highly expressed in the ΔtepR background. Similar results were also obtained from quantitative reverse transcription PCR experiments, which validated the RNA-seq analysis. Likewise, expression of tepR was not affected by the tofI/tofR-mediated quorum sensing system. Interestingly, qsmR, a key regulatory factor for flagellum biogenesis, was found to be upregulated in the ΔtepR strain and essential for both toxoflavin production and exoprotease activity. These results suggest that tepR exerts its regulatory functions on toxoflavin and exoprotease through qsmR in this pathogen. Research on Bacterial Endophytes at the Institute for Advanced Learning and Research C. MEI (1), R. Chretien (2), J. Carey (2), Y. He (2), S. Kadali (2), S. Lowman (2) (1) Institute for Advanced Learning and Research, U.S.A.; (2) Institute for Advanced Learning and Research, U.S.A. Bacterial endophytes live inside plants and represent a largely unexplored resource for enhancing sustainable agricultural production. They are also recognized as an untapped reservoir of novel natural products. We have demonstrated significant growth promotion of the bioenergy crop switchgrass by bacterial endophytes (Burkholderia phytofirmans PsJN and Panteoa agglomerans PaKM) under in vitro, growth chamber and greenhouse, as well as field conditions, particularly in low fertility soil. Currently, we have established a library of 225 bacterial endophytes isolated from various environments and characterized their abilities in N fixation, P solubilization and auxin production, as well as ACC deaminase activities. Many endophytes exhibited one or several of these plant beneficial traits. In the agricultural sector, researchers are exploring growth enhancement and disease resistance of crops important to the region such as tobacco, grapevine and tall fescue and testing both individual microbes and consortia for possible applications. In the environmental sector, scientists are using switchgrass and bacterial endophytes able to breakdown toxic organic compounds to remediate polychlorinated biphenyls (PCBs) in a contaminated waste water overflow pond and also to reduce PCBs in storm water. In the natural product sector, researchers are searching for unique natural products with wide-ranging applications as antibiotics, antivirals and anticancer agents. Genome analysis of Rathayibacter toxicus strain WAC3373 from Western Australia: Sequencing, assembly and annotation M. ARIF (1), G. Busot (1), R. Mann (2), B. Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Vi i i T h TAREC U S A (2) Vi i i T h Tid t AREC U S A (3) U i it f D l U S A (4) U i it f Thus, FT-antibody fusion protein can be expressed in citrus without deleterious effects on plant development. However, the highest expressing line exhibited precocious flowering and fruit set, presumably due to the bloom-inducing activity of the FT domain. This suggests that sufficiently high FT-scFv expression triggers flowering, which could be useful in developing rapid-flowering citrus lines to expedite breeding. The HLB resistance status of the FT-scFv grapefruit lines is now being determined. mically defined medium XF-26 for in-vitro cultivation of Xylella fastidiosa and Xylella taiwanensis isolated in Taiwan arma (1), S. Hsu (1), C. Su (2), C. Chang (3), Y. Tseng (1), F. Jan (1) Modification of chemically defined medium XF-26 for in-vitro cultivation of Xylella fastidiosa and Xylella taiwanensis isolated in Taiwan W. DENG (1), N. Sharma (1), S. Hsu (1), C. Su (2), C. Chang (3), Y. Tseng (1), F. Jan (1) (1) National Chung Hsing University, Taiwan; (2) Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taiwan; (3) University of Georgia, U.S.A. Modification of chemically defined medium XF-26 for in-vitro cultivation of Xylella fastidiosa and Xylella taiwanensis isolated in Taiwan W. DENG (1), N. Sharma (1), S. Hsu (1), C. Su (2), C. Chang (3), Y. Tseng (1), F. Jan (1) (1) National Chung Hsing University, Taiwan; (2) Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taiwan; (3) University of Georgia, U.S.A. ( ), ( ), ( ), ( ), g ( ), g ( ), ( ) (1) National Chung Hsing University, Taiwan; (2) Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taiwan; (3) University of Georgia, U.S.A. (1) National Chung Hsing University, Taiwan; (2) Taiwan Agricultural Chemicals and Toxic Substances Research Institute, Taiwan; (3) University of Georgia, U.S.A. Strains of Xylella fastidiosa (PD) and X. taiwanensis (PLS) are plant pathogenic bacteria that were respectively isolated from grapevine and pear tree in Taiwan. The bacteria are nutritionally fastidious and require a complex nutrient for ex-planta growth. The Taiwan Xylella PD and PLS strains can grow on PD2 but not on the chemically defined XF-26 medium that was developed for in-vitro cultivation of X. fastidiosa strains isolated in North America. To develop a defined medium that supports growth of the Taiwan Xylella strains, different medium formulations modified from the original XF-26 were tested. A XF basal medium containing 20 amino acids resulted in good growth of Taiwan PD and PLS strains. Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Vi i i T h TAREC U S A (2) Vi i i T h Tid t AREC U S A (3) U i it f D l U S A (4) U i it f The phyla Proteobacteria, Bacteroidetes Firmicutes, Actinobacteria, and Acidobacteria were most abundant in all samples, representing 95.0 to 97.0% of all assigned sequences. Analysis of the most prominent genera revealed that Pseudomonas, Chitinophaga, Bacillus, Pedobacter, Flavobacterium, Bradyrhizobium, and Rhizobium represented between 5% and 25% of the microbial communities in all the samples that where either treated or not treated. A general shift within the composition of the microbial communities that was independent of the application of BFA was observed after application. The results indicate that BFA application has limited effect on citrus rhizosphere bacterial communities; however, its lasting effects need to be determined. Impacts of abundance of Candidatus Liberibacter on the citrus phyto-microbiome R. BLAUSTEIN (1), J. Meyer (1), A. Conesa (2), G. Lorca (2), M. Teplitski (1) (1) Soil and Water Science Department University of Florida U S A ; (2) Microbiolog Understanding how the citrus microbiome is affected by (and impacts) Huanglongbing (HLB) may be critical to disease management. To test the hypothesis that phyto-microbiome composition and diversity are related to the prevalence of Candidatus Liberibacter, the HLB-causative agent, leaves were collected from HLB-infected Valencia trees that differed in the extent of disease symptoms and the leaf-associated microbial communities were S4.25 characterized by sequencing the V4 region of 16S rDNA. Approximately 1,900 OTUs were detected, representing 215 genera. The relative abundance of Candidatus Liberibacter, which ranged from 0.7 - 71.3%, induced PCA clustering and negatively correlated with alpha diversity, as indicated by Shannon (p<0.001) and Chao1 (p=0.001) indices. Positive interactions or co-occurence (p<0.05) of Candidatus Liberibacter with other genera were detected only with Pseudomonas and an unclassified genus of Oxylobacteraceae. These two, in turn, had negative interactions (p<0.05) with 5 genera that formed a complex network of positive interactions with 39 other genera, which likely represent the “healthy” phyto-microbiome. These interactions suggest that (1) the impacts of Candidatus Liberibacter on the microbiome are likely mediated through its key interactions with pseudomonads and oxylobacteria and/or (2) antagonistic effects of the bacteria from the core citrus microbiome on these two groups may lessen the pathogen titer and mitigate disease progression. Quick genome sequencing of “Candidatus Liberibacter” strains by use of Enrichment-Enlargement-Next generation sequencing (EEN) J. CHEN (1), Z. Zheng (2), F. Wu (3), X. Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Vi i i T h TAREC U S A (2) Vi i i T h Tid t AREC U S A (3) U i it f D l U S A (4) U i it f Deng (3) (1) USDA-ARS, U.S.A.; (2) South China Agricultural University, China; (3) South China Agricultural University, China Members of “Candidatus Liberibacter” are associated with several important plant diseases such as citrus Huanglongbing (HLB) and potato zebra chip (ZC) disease. The inability to culture and the low titers in infected hosts of the bacteria have been major obstacles for research. Whole genome sequence analysis could be an effective means to study the bacteria. Taking the advancement of Next Generation Sequencing (NGS) technology, a procedure, called EEN (enrichment-enlargement-NGS), has been developed for quick genome sequencing of liberibacters. Enrichment includes increasing the DNA ratio of liberibacter to its host using alternative hosts with higher titer capacity, removal of the highly methylated plant DNA and selection of low Ct values samples; Enlargement includes increasing the enriched DNA quantity through whole DNA amplification to meet the requirement of a sequencing platform; And the NGS step includes selection of sequencing platform and development of liberibacter-specific pipelines. With EEN, >10 G of sequence data was generated from MiSeq, followed by collection of liberibacter reads using Perl scripts, de novo or/and referenced genome assembling, and sequence annotation. Thus far, seven draft genome sequences have been generated, five HLB liberibacters (China, California, and Florida) and two ZC liberibacters (California) from plant or psyllid hosts. The EEN procedure could also benefit genome research in other fastidious prokaryotes. Phenotypic effects of anti-Candidatus Liberibacter asiaticus antibody expression in grapefruit T. MCNELLIS (1), T. Gottwald (2), J. Sinn (1), V. Orbovic (3) (1) Penn State University, U.S.A.; (2) USDA ARS, U.S.A.; (3) CREC-University of Florida, U.S.A. Citrus greening, or huanglongbing (HLB), is a globally-important, fatal citrus disease caused by the gram-negative, phloem-limited bacterium “Candidatus Liberibacter asiaticus” (CLas). HLB-resistant citrus germplasm is not available. This project aims to develop HLB-resistant grapefruit. Based on homology to Xylella fastidiosa and E. coli, the CLas NodT outer membrane transporter might export factors required for virulence. The goal of this project is to block NodT-mediated virulence factor export, potentially disrupting CLas virulence. A single-chain antibody (scFv) directed at the major predicted extracellular loop of CLas NodT was expressed in stably transformed ‘Duncan’ grapefruit. To promote protein expression, stability and mobility in the phloem, the antibody was fused to the phloem-mobile Flowering Locus T (FT) protein. Full-length, stable FT-scFv protein was expressed in multiple transgenic lines, most of which had no phenotypic abnormalities. Virulence traits and disease development by Xylella fastidiosa are impaired in a mutant on the outer membrane protein MopB H. CHEN (1), P. Kandel (1), L. Cruz (1), L. De La Fuente (1) (1) Auburn university, U.S.A. Virulence traits and disease development by Xylella fastidiosa are impaired in a mutant on the outer membrane protein MopB H. CHEN (1), P. Kandel (1), L. Cruz (1), L. De La Fuente (1) (1) Auburn university, U.S.A. Outer membrane (OM) proteins are key elements to maintain the integrity of the outer membrane, and play an important role in bacterial behavior. MopB is a major OM protein in the plant pathogen X. fastidiosa, which causes major losses on many economically important crops due to occlusion of the xylem network in plants. Based on DNA sequence analysis of the gene encoding MopB, the C-terminal part is a homologue of the OmpA family, and includes a conserved calcium binding motif. However the function of MopB protein in X. fastidiosa remains poorly understood. Here, MopB function has been studied by site-directed mutagenesis. Taking advantage of the natural competence of X. fastidiosa, mopB mutants were obtained in two different X. fastidiosa strains, the type strain ‘Temecula’ and the more aggressive ‘WM1-1’. mopB mutants in both background strains were impaired in surface attachment, biofilm formation, twitching motility and natural competence. Further analysis of the bacterial surface showed that the mopB mutants were impaired in pilus formation as observed by electron microscopy. Additionally, mopB mutants in both backgrounds showed reduced virulence as compared to WT when tested on tobacco (Nicotiana tabacum) plants as a host under greenhouse condition. These results suggest that MopB has an effect on pilus biogenesis and is important for the virulence of X. fastidiosa. Insights from comparative analyses between the genomes of two model strains of the cucurbit pathogenic bacterium Acidovorax citrulli S. BURDMAN (1), N. Eckshtain-Levi (2), D. Shkedy (3), M. Gershovits (3), G. Mateus da Silva (4), D. Tamir-Ariel (1), R. Walcott (4), T. Pupko (3) (1) The Hebrew University of Jerusalem, Rehovot, Israel; (2) The Hebrew University of Jerusalem, Rehovot, Israel; (3) Tel Aviv University, Tel Aviv, Israel; (4) University of Georgia, Athens, GA, U.S.A. Insights from comparative analyses between the genomes of two model strains of the cucurbit pathogenic bacterium Acidovorax citrulli S. BURDMAN (1), N. Eckshtain-Levi (2), D. Shkedy (3), M. Gershovits (3), G. Mateus da Silva (4), D. Tamir-Ariel (1), R. Walcott (4), T. Pupko (3) (1) The Hebrew University of Jerusalem, Rehovot, Israel; (2) The Hebrew University of Jerusalem, Rehovot, Israel; (3) Tel Aviv University, Tel Aviv, Israel; (4) University of Georgia, Athens, GA, U.S.A. Relationship between invasive brown marmorated stink bug and fumonisin contamination of field corn in the Mid-Atlantic J. OPOKU (1), H. Mehl (2), N. Kleczewski (3), K. Hamby (4) (1) Vi i i T h TAREC U S A (2) Vi i i T h Tid t AREC U S A (3) U i it f D l U S A (4) U i it f By omitting amino acids one by one or in combination from the basal medium, tyrosine was found to be an essential amino acid for cultivating Taiwan PD and PLS strains, and the bacteria can grow in the XF-26 medium supplemented with tyrosine (XF-Tyr). Moreover, replacing asparagine with aspartic acid in the XF-Tyr medium also supported bacterial growth of purified PD and PLS strains or the ones isolated from diseased tissues during primary cultivation. To simplify the preparation of the chemically defined medium, the 17 amino acids in the XF-26 medium were substituted by 0.4% (w/v) casamino acids containing tyrosine, aspartic acid, and 16 other amino acids, and the modified XF-26 (mXF-26) was demonstrated to support good growth of the Taiwan-isolated Xylella strains. Evaluation of assembling methods on determination of whole genome sequence of Xylella fastidiosa blueberry bacterial leaf scorch strain J. CHEN (1), C. Wallis (1), C. Chang (2) (1) USDA-ARS, U.S.A.; (2) University of Georgia, U.S.A. Evaluation of assembling methods on determination of whole genome sequence of Xylella fastidiosa blueberry bacterial leaf scorch strain J. CHEN (1), C. Wallis (1), C. Chang (2) (1) USDA-ARS, U.S.A.; (2) University of Georgia, U.S.A. Blueberry bacterial leaf scorch (BBLS) disease, a threat to blueberry production in the Southern USA and elsewhere, is caused by Xylella fastidiosa. Efficient control of BBLS requires knowledge of the pathogen. Research is challenging because Xylella fastidiosa is difficult to culture. This study was to study BBLS strain based on whole genome sequence analyses. A total of 13,739,924 sequence paired reads (mean = 251 bp) were generated from a BBLS strain isolated from Georgia, USA. Draft whole genome sequences (minimum contig size = 1,000 bp) were generated by both de novo assembling (DA) and referenced assembling (RA) using CLC Genomic Workbench. The DA draft genome was 2,537K (95 contigs). Sizes (bp) of RAs varied depending on reference genome sequences: 2,449K (39 contigs) with NC_010513 (M12, subsp. multiplex), 2,461K (70 contigs) with NC_010577 (M23, subsp. fastidiosa), 2,467K (96 contigs) with NC_004556 (Temecula1, subsp. fastidiosa), and 2,457K (180 contigs) with NC_002488 (9a5c, subsp. pauca). Percentage coverages were 99.0% (M12), 97.1% (M23), 97.1% (Temecula1) and 91.7% (9a5c). ANI (average nucleotide identity) values were 99.9 (M12), 97.5 (M23), 97.6 (Temecula1) and 96.2 (9a5c). These data suggest that 1) DA alone was promising yet contig number remained high; 2) For RA, reference genome sequences had significant influence on the BBLS genome size; And 3) BBLS strain is a member of subsp. multiplex based on its similarity to strain M12. pauca). Percentage coverages were 99.0% (M12), 97.1% (M23), 97.1% (Temecula1) and 91.7% (9a5c). ANI (average nucleotide identity) values were 99.9 (M12), 97.5 (M23), 97.6 (Temecula1) and 96.2 (9a5c). These data suggest that 1) DA alone was promising yet contig number remained high; 2) For RA, reference genome sequences had significant influence on the BBLS genome size; And 3) BBLS strain is a member of subsp. multiplex based on its similarity to strain M12. Bacterial Communities in Cultivated and Wild Cranberry Bogs S. SOBY (1), G. Ebadzad (1), E. Batory (1) (1) Midwestern University, U.S.A. Transitions in microbial population structure between fallow and cultivated soils may be an important early determinant of disease-suppression of the cultivated soil. Evaluation of assembling methods on determination of whole genome sequence of Xylella fastidiosa blueberry bacterial leaf scorch strain J. CHEN (1), C. Wallis (1), C. Chang (2) (1) USDA-ARS, U.S.A.; (2) University of Georgia, U.S.A. Cranberry bogs are an ideal model system for comparing native and cultivated phytobiomes because of the genetic similarity between wild and domesticated cranberry plants, and their location in the same geographic region of southeastern Massachusetts. Soil samples were collected from wild and cultivated cranberry bogs over the course of several growing seasons to determine the composition of phytobiomes in native and cultivated cranberry bogs. Population structures were determined by pyro-sequencing of the V4 region of 16S rRNA genes. Paired-end reads were assembled using PANDAseq, and analyzed using the QIIME software package to call operational taxonomic units and to calculate richness and diversity indices. Bacterial communities in both cultivated and wild bogs had different relative abundances, but were composed of five dominant phyla: Proteobacteria, Acidobacteria, Bacteriodetes, Verrucomicrobia and Actinobacteria. Principal Components Analysis based on soil bacterial populations among sampled sites indicate that sampling location or soil type are more important than cultivation effects in determining bacterial community structure. These results suggest that a descriptive population structure is associated with each bog type, independent of cultivation status, season, or sampling year. Host range potential of bacterial soft rot pathogens isolated from Phalaenopsis and Oncidium orchids R. CATING (1), B. Robinson (1), A. Palmateer (2), K. Frost (3) (1) Oregon State University, U.S.A.; (2) University of Florida, U.S.A.; (3) Oregon State University, U.S.A. Bacteria from the genera Pectobacterium and Dickeya (formerly Erwinia chrysanthemi) are known to cause soft rot disease in many plant species. Since 2008, bacterial soft rot caused by a Dickeya sp. has been reported on several orchid genera in South Florida. Ten isolates (5 from Phalaenopsis, 5 from Oncidium) were identified as belonging to the Dickeya or Pectobacterium genera based on phenotypic and 16S sequence data and were cultured on nutrient agar for 24 h at 28°C. Cultures were suspended in sterile water and adjusted to a concentration of 1 × 108. Carrots, onions, potatoes (R. Burbank), and Phalaenopsis orchids were inoculated by placing a 10 µL drop of bacterial suspension directly over a wound made with a sterile pipette tip. Inoculated plant material was incubated at 28°C and evaluated for symptoms at 24, 48, 72, and 96 h. Bacteria were re-isolated from symptomatic tissue and identified based on the 16S and Pel genes. Orchid isolates caused soft rot symptoms in onion, potato, carrot, and Phalaenopsis orchids. Recently, Dickeya spp. and a newly proposed species, D. Virulence traits and disease development by Xylella fastidiosa are impaired in a mutant on the outer membrane protein MopB H. CHEN (1), P. Kandel (1), L. Cruz (1), L. De La Fuente (1) (1) Auburn university, U.S.A. Bacterial fruit blotch (BFB) of cucurbits, caused by Acidovorax citrulli (Ac), has been responsible for serious economic losses in watermelon and melon production worldwide. Ac strains are divided into two main groups based on genetic traits. While group I strains have been mainly isolated from melon, most group II strains were isolated from watermelon. The genome sequence of the group II model strain AAC00-1 is available since 2007. We recently sequenced the genome of the group I model strain M6. The M6 genome is 4.85 Mb in length, which is approximately 500 Kb shorter than that of S4.26 AAC00-1. Comparative analysis revealed this size difference is mainly explained by eight AAC00-1 genomic regions (FAs; for fragments of AAC00-1), ranging from ~35 to 120 Kb and distributed throughout the AAC00-1 genome. The FAs are absent in the M6 genome. While AAC00-1 possesses over 500 open reading frames (ORFs) that are absent in M6, only about 120 M6 ORFs are absent in AAC00-1. Coverage analyses with draft genomes of other strains support the notion that significant portions of the FAs are discriminatory between groups I and II. Moreover, GC content, effective number of codon values’ and COG analyses support these FAs were introduced into group II strains by horizontal gene transfer. This study provides the first comprehensive genome comparison between the two major groups of Ac. Further investigation is needed to sharpen the distinctive genetic markers of the two groups. Bacteriophages: The over-looked component of the phyllosphere of Xanthomonas-diseased plants D. RITCHIE (1), M. Munster (2), S. Butler (2) (1) North Carolina State University, U.S.A.; (2) North Carolina State University and Plant Disease & Ins Bacteriophages: The over-looked component of the phyllosphere of Xanthomonas-diseased plants D. RITCHIE (1), M. Munster (2), S. Butler (2) (1) North Carolina State University, U.S.A.; (2) North Carolina State University and Plant Disease & In Bacteriophages: The over-looked component of the phyllosphere of Xanthomonas-diseased plants D. RITCHIE (1), M. Munster (2), S. Butler (2) (1) North Carolina State University, U.S.A.; (2) North Carolina State University and Plant Disease & Insect Clinic, U.S.A. ( ), ( ), ( ) 1) North Carolina State University, U.S.A.; (2) North Carolina State University and Plant Disease & Insect Clinic, U.S.A. Bacteriophages specifically infect and replicate within bacteria. Phages are the most numerous, diverse, and ubiquitous entities on earth, but little is known about their existence in the phytobiome, particularly the phyllosphere. Bacteria in the genus Xanthomonas cause foliar diseases in many plants. Our objective was to determine occurrence of phages on plants infected with Xanthomonas. Foliar samples from diseased Brassica spp., crape myrtle, Corylus sp., Nandina, pepper, poinsettia, Prunus spp., and Verbena were assayed. Diseased tissue was chopped/soaked in 1-3 ml sterile deionized water (SDW). Non-enrichment phage extraction used either filtering (0.22 um) and/or treating with 10% chloroform. Indicator/host bacteria were isolated from the same disease sample or bacteria that were isolated previously from similar samples. Bacteria isolated from samples were identified using Biolog, ITS region, and elicitation of HR in a non-host plant. Bacteria lysed by phages represented 4 Xanthomonas spp. and 5 pathovars (pv). Phage titers greater than 103 pfu/ml extraction SDW were detected representing all 8 plant genera. Plaque morphologies were hazy Xanthomonas-disease microbiota. Comparative genomic analyses of 15 Acidovorax pathogens provide insights into the emergence of a new turfgrass dis ifi it Comparative genomic analyses of 15 Acidovorax pathogens provide insights into the emergence of a new turfgrass disease and the host specificit Comparative genomic analyses of 15 Acidovorax pathogens provide insights into the emergence of a new turfgrass disease and the host specificity Comparative genomic analyses of 15 Acidovorax pathogens provide insights into the emergence of a new turfgrass disease and the host specificity Evaluation of assembling methods on determination of whole genome sequence of Xylella fastidiosa blueberry bacterial leaf scorch strain J. CHEN (1), C. Wallis (1), C. Chang (2) (1) USDA-ARS, U.S.A.; (2) University of Georgia, U.S.A. solani, have been reported to cause soft rot disease of potato tubers in other parts of the world, and there is concern that new species will end up in the US. We are continuing work on the orchid strains to determine their appropriate species designation. Further evidence for host preference among Acidovorax citrulli strains based on a detached melon fruit assay L. YAN (1), R. Walcott (2), B. Hu (1) Further evidence for host preference among Acidovorax citrulli strains based on a detached melon fruit assay L. YAN (1), R. Walcott (2), B. Hu (1) Further evidence for host preference among Acidovorax citrulli strains based on a detached melon fruit assay L. YAN (1), R. Walcott (2), B. Hu (1) ( ) ( ) ( ) (1) Nanjing Agricultural University, China; (2) University of Georgia, U.S.A. Bacterial fruit blotch (BFB), caused by Acidovorax citrulli, is a serious threat to cucurbit crop production. A. citrulli strains can be divided into two genetically distinct groups with group I strains being associated with a range of cucurbits and group II strains being associated with watermelon. Despite this, seedling pathogenicity assays for A. citrulli lack the sensitivity to distinguish the groups. In this study, we developed an immature, detached melon (cv. Joaquin Gold) fruit assay that clearly distinguished the two A. citrulli groups. We observed that four group I strains induced typical water-soaked lesions on melon fruit rind tissue 7 to 10 days after pin-prick inoculation. In contrast, four group II strains did not induce symptoms on detached melon fruits. Additionally, a type III secretion (T3S) system mutant of the group I strain, M6 (M6?hrcV), failed to induce watersoaking in the same assay. Based on these observations, we conclude that group I A. citrulli strains have the capacity to infect immature melon cv. Joaquin Gold fruit, while group II strains do not. The difference in the ability of group I and group II A. citrulli strains to induce watersoaking on immature melon fruit is determined by T3S effectors. Further investigation using this detached melon fruit assay will help to identify the A. citrulli T3S effectors responsible for this differential pathogenicity phenotype. Characterization and Management of Ralstonia solanacearum Causing Bacterial Wilt of Tomato in Louisiana A. JIMENEZ MADRID (1), A. Jimenez (1), M. Lewis Ivey (1) (1) Louisiana State University, U.S.A. Ralstonia solanacearum (Rs), the causal agent of bacterial wilt of tomato can cause severe economic losses to tomato growers in Louisiana. Traditional management tactics such as soil fumigation and crop rotation are ineffective. Resistant cultivars lack durability and have undesirable horticultural traits. For these reasons, many producers in Louisiana have abandoned their fields for tomato production. Although R. solanacearum is endemic in Louisiana, pathogen populations have not been characterized. Tomato samples with bacterial wilt symptoms were collected from three parishes in 2015. Comparative genomic analyses of 15 Acidovorax pathogens provide insights into the emergence of a new turfgrass disease and the host p y Q. ZENG (1), J. Wang (2), P. Giordano (3), F. Bertels (4), M. Chilvers (5), J. Vargas (5), G. Sundin (5), N. Mitkowski (6) (1) The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (2) Michigan State Univ, U.S.A.; (3) Bayer Corporation, Canada; (4) University of Basel, Switzerland; (5) Michigan State University, U.S.A.; (6) URI, U.S.A. Q. ZENG (1), J. Wang (2), P. Giordano (3), F. Bertels (4), M. Chilvers (5), J. Vargas (5), G. Sundin (5), N. Mitkowski (6) (1) The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (2) Michigan State Univ, U.S.A.; (3) Bayer Corporation, Canada; (4) University of Basel, Switzerland; (5) Michigan State University, U.S.A.; (6) URI, U.S.A. Bacterial etiolation and decline (BED), caused by Acidovorax avenae subsp. avenae (Aaa), is an emerging disease of creeping bentgrass on golf course putting greens. The causal agent of BED belongs to the genus Acidovorax, members of which can infect various hosts with strong host specificity. In this research, we aim to understand the emergence of BED, and identify genes in turf Aaa that define its host specificity. Eighteen Acidovorax draft genomes (14 turf, 3 maize, and 1 orchid pathogenic strains) were generated. Using whole genome SNP analysis, the 14 turf-pathogenic Aaa isolates were grouped into three clades. High levels of single nucleotide polymorphisms, gene deletions, insertions, and inversions were detected within the turf-pathogenic Aaa isolates, suggesting that the 2009-2011 BED outbreak was caused by a genetically diverse population. Population genomic analyses identified genes S4.27 whose sequences are conserved in turfgrass pathogenic Aaa in comparison to the closely related maize pathogenic Aaa isolates. Interestingly, many of these genes identified are located within the type III secretion system pathogenicity island, an important virulence factor of phytopathogenic bacteria. We identified homologous recombination as the potential cause of the gene conservation observed in the T3SS pathogenicity island. Our results suggested a strong host selection during the evolution of the turf-pathogenic Aaa. Epiphytic survival of Pantoea ananatis on Florida Pusley (Richardia scabra L.) B. DUTTA (1), R. Gitaitis (1), F. Anderson (1) (1) The University of Georgia, U.S.A. Pantoea ananatis, the causal organism of center rot of onion (Allium cepa L.), can survive on different weeds, but in a previous survey it was most commonly found on Florida pusley (Richardia scabra L.). The epiphytic survival of P. ananatis on Florida pusley under different temperature and moisture regimes was investigated. Further evidence for host preference among Acidovorax citrulli strains based on a detached melon fruit assay L. YAN (1), R. Walcott (2), B. Hu (1) Bacterial wilt was confirmed using commercial Rs-specific immunostrips and the bacterial streaming test. Fifteen isolates were recovered from four fields and a greenhouse and all belonged to phylotype II. Thirty-three percent of the strains were characterized as biovar 1 and 20% as biovar 3. The remaining strains did not conform to a published biovar pattern. None of the isolates were Race 3 biovar 2 based on results from R3bv2-specific PCR. Grafting with resistant rootstock varieties is being explored as a sustainable management strategy. Louisiana tomato growers were surveyed for their willingness to use grafted plants with Rs resistance. Sixty-six percent of the growers indicated that they would be willing to use Rs-resistant grafted plants but ranked not knowing how to graft and the cost of grafted plants as the top two factors that would hinder their adoption of this strategy. Development of green fluorescent protein-expressing Xanthomonas gardneri strains to study tomato infection processes F. ROTONDO (1) (1) The Ohio State University U S A (1) The Ohio State University, U.S.A. Bacterial leaf spot (BLS) is a serious disease of tomato, causing millions of dollars in losses worldwide. While BLS is associated with several species of Xanthomonas, X. gardneri is the predominant species in Ohio where it causes severe damage to processing and fresh market tomatoes. To improve our understanding of BLS disease development, two green fluorescent protein (GFP)-labeled X. gardneri strains were developed to study colonization in planta. Wild-type X. gardneri strains SM605-2011 and SM775-2012 were isolated from tomatoes produced in Ohio. Each strain was transformed by electroporation utilizing a GFP-expressing kanamycin/tetracycline-resistant plasmid, CGT46339. Overall, transformation efficiency for this system was low, approaching 102 CFU/ug DNA. Two GFP-expressing transformants, SM605-2011gfp and SM775-2012gfp, were evaluated against their wild-type counterparts for growth in vitro. No significant differences were observed based on optical density measurements (Permutation test, p>0.05), and both reached the stationary phase at 1.8 OD600 and 109 CFU/ml. Stability of the GFP marker and virulence of the transformed strains will be determined. These strains will be used to study leaf, fruit and seed infection using confocal microscopy (Leica TCS SP5). The transformed strains will be a powerful tool to visualize infection processes and provide critical information for disease management. Development of green fluorescent protein-expressing Xanthomonas gardneri strains to study tomato infection processes F. ROTONDO (1) Development of green fluorescent protein-expressing Xanthomonas gardneri strains to study tomato infection process F. ROTONDO (1) Development of green fluorescent protein-expressing Xanthomonas gardneri strains to study tomato infection processes F ROTONDO (1) elopment of green fluorescent protein-expressing Xanthomonas gardneri strains to study tomato infection processes OTONDO (1) Comparative genomic analyses of 15 Acidovorax pathogens provide insights into the emergence of a new turfgrass disease and the host Weed seedlings were spray-inoculated with 103 colony forming units (CFU)/ml of rifampicin-resistant strain Pan 97- 1rif and incubated in a growth chamber at 15.5° or 21.1°C at 65% RH for 96 h, which represented mean environmental conditions during mid-March to mid-May in Vidalia, GA. P. ananatis survived for 96 h when incubated at 21.1°C with a mean population of 3.2 × 101 CFU/g of leaf tissue. In contrast, no viable bacteria were detected after 96 h at 15.5°C. Survival of P. ananatis on Florida pusley was also monitored during alternating 12-h wet and dry periods, or continuous wet or dry periods for 96 h at 15.5° or 21.1°C. Compared to initial or continuous dry periods, P. ananatis survived significantly better with a 12/12-h wet/dry cycle or a continuous 24-h wet period at both 15.5° and 21.1°C. Unlike at 15.5 ?C, P. ananatis populations (1.8 × 101 CFU/g) survived for 96 hours post inoculation at 21.1 ?C under a 12-h of dry/12-h wet cycle. These results demonstrate that P. ananatis can survive on Florida pusley leaves under conditions of 21.1°C and prolonged leaf wetness and may potentially serve as a source of inoculum to onion. Genetic mapping of pathogenesis in the northern corn leaf blight fungus However, the importance of Fusarium associated with root disease of corn in South Dakota, is still unknown. In 2015, a survey of 50 corn fields from 23 counties in South Dakota, was conducted to characterize the Fusarium spp. associated with root diseases of corn. A total of 250 samples (5 plants per field) were collected between V1 (first leaf) to V3 (third leaf) growth stages. Root pieces from each sample were surface sterilized and placed on potato dextrose agar (PDA) for 7 to 10 days at 25°C under 12 hour fluorescent light/dark conditions. Isolates of Fusarium were hyphal-tipped, cultured, and then identified microscopically based on colony growth and spores produced on PDA. Representative Fusarium isolates were confirmed by sequencing of the translation elongation factor (EF1-α) gene region. At least seven Fusarium spp. were identified. Fusarium graminearum and F. oxysporum were the most frequently recovered (≥20%) Fusarium spp. from the corn roots. In the aggressiveness study to compare Fusarium isolates in the greenhouse, significant differences in aggressiveness (P ≤ 0.05) was observed among the Fusarium spp. with the most aggressive isolates being F. graminearum (FG13) and F. acuminatum (FA8). Results from this research will help us develop disease management strategies for corn farmers in South Dakota to maximize their yield. Understanding sexual fertility in Aspergillus flavus through analysis of F1 progeny R. GELL (1), I. Carbone (1) (1) C f d l h h C li S i i S A ( ) ( ) (1) Center for Integrated Fungal Research, North Carolina State University, U.S.A. Aspergillus flavus produces aflatoxin, which is a constant threat and economic burden to corn and oil seed crops worldwide. In order to manage this problem more cost effectively, a greater understanding of A. flavus biology and genetics is required. One important question is how genetic information moves between strains. A. flavus, previously thought of as only asexual, has recently been found to undergo sexual reproduction both in laboratory crosses and in the field. During the mating process, the sclerotium, a survival structure, of one strain acts as the female parent providing both the mitochondria and a matrix for the ascocarps and progeny to grow, while a spore or propagule from a second strain fertilizes as the male. The fertility of mating pairs is highly variable and influenced by the directionality of the cross. Genetic mapping of pathogenesis in the northern corn leaf blight fungus Genetic mapping of pathogenesis in the northern corn leaf blight fungus S. MIDEROS (1), C. Chung (2), J. Poland (3), G. Turgeon (4), R. Nelson (4) (1) University of Illinois at Urbana Champaign, U.S.A.; (2) National Taiwan University, Taiwan; (3) Kansas State University, U.S.A.; (4) Cornell University, U.S.A. Genetic mapping of pathogenesis in the northern corn leaf blight fungus S. MIDEROS (1), C. Chung (2), J. Poland (3), G. Turgeon (4), R. Nelson (4) (1) University of Illinois at Urbana Champaign, U.S.A.; (2) National Taiwan University, Taiwan; (3) Kansas State University, U.S.A.; (4) Cornell University, U.S.A. ( ), g ( ), ( ), g ( ), ( ) (1) University of Illinois at Urbana Champaign, U.S.A.; (2) National Taiwan University, Taiwan; (3) Kansas State University, U.S.A.; (4) Cornell University, U.S.A. Setosphaeria turcica (=Exserohilum turcicum) causes northern leaf blight (NLB) of maize (Zea mays). Identification of genes associated with pathogenesis/virulence suggests possible targets for control, generates molecular tools to characterize populations of the pathogen, and provides valuable information to breeders and maize geneticists. In this study we used a forward genetics approach to i) identify fungal genes that determine race-specific reactions with two maize resistance genes, and ii) determine the genetic regions for other virulence mechanisms in the pathogen. We generated a cross between a race 1 and a race 23N strain and collected 220 single-ascosporic progeny. The ability of each progeny to cause disease on maize lines with or without the Ht1 and Ht2 genes was evaluated in greenhouse experiments. Mycelial abundance, radial growth, sporulation and melanin content were visually evaluated on artificial media. Genotyping-by-sequencing generated 2,078 single nucleotide polymorphic markers (SNPs). Using 310 high- quality SNPs we calculated a genetic map composed of 22 linkage groups spanning 1981.1 cM. Genetic mapping pinpointed regions of interest for the avrHt1, avrHt2, and the melanin biosynthetic locus, as well as quantitative trait loci for mycelial abundance, speed of growth, and sporulation. Molecular markers for the identification of fungal strains capable of causing disease in maize plants with Ht1 and Ht2 genes are being validated. Characterizing Fusarium species infecting corn roots in South Dakota A. ADHIKARI (1), P. Okello (1), B. Kontz (1), M. Dunbar (1), A. Varenhorst (2), F. Mathew (2) (1) South Dakota State University, U.S.A.; (2) South Dakota State University, U.S.A. There are multiple Fusarium spp. that are associated with root diseases on corn, Zea mays, in the United States. Improvement of the environmental DNA extraction method for profiling the culturable microbiome of bacterial wilt-conducive and non- conducive soils R. GICANA (1), W. Deng (1), J. Wu (1) R. GICANA (1), W. Deng (1), J. Wu (1) (1) National Chung Hsing University, Taiwan The culturable microbiome profile of the bacterial wilt-conducive and nonconducive soils were compared using the PCR-DGGE profile of the V4-V5 hypervariable region of the 16S rRNA, and using the high throughput sequencing (HTS) analysis of the V1-V3 region of the 16S rRNA. PCR-DGGE results showed DNA fingerprints unique only to either the conducive or the nonconducive soils, which was confirmed by HTS analysis with 63 culturable taxa unique only to the nonconducive soil compared to 94 in the conducive soil. Exclusively cultured taxa in the nonconducive soil belong to the Phylum Planctomycete, Nitrospirae and Gemmatimonadetes while those enriched in the conducive soil belong to the Phylum Verrucomicrobia, Alphaproteobacteria and Gammaproteobacteria. While studying the culturable microbiome of the two soil types, we have improved a previous DNA extraction method to generate high quality DNA used in this study. The modified method needs no further purification steps, yields more DNA which are unsheared and with high molecular weight, with low humic acid content, and can extract DNA from bacteria of different cell wall compositions. The microbiome was cultured using low nutrient medium and with an improved DNA isolation technique we were able to capture some taxa previously S4.28 reported uncultured or were recalcitrant to culturing under traditional laboratory methods such as Nitrospira calida, Planctomyces sp., and several genera belonging to the classes Gemm-3 and Gemm-5. reported uncultured or were recalcitrant to culturing under traditional laboratory methods such as Nitrospira calida, Planctomyces sp., and several genera belonging to the classes Gemm-3 and Gemm-5. Introduction and seed transmission of Fusarium proliferatum in the field A. REYES GAIGE (1), W. Yue (2), C. Toomajian (2), J. Stack (2) (1) Kansas State University, U.S.A.; (2) Kansas State University, U.S.A. Fusarium proliferatum (Fp) is a seed-borne fungus in maize seed. It has a broad host range with worldwide distribution, and it is a prolific producer of mycotoxins. The introduction of this seed-borne organism into new environments can result in geographic range expansion of undesirable populations. The potential introduction of Fp via infected seed planted into a new environment and subsequent dispersal from that site via seed transmission was studied. Genotyping-by-sequencing (GBS) was used to identify unique polymorphisms in Fusarium species isolated from two maize genotypes. Improvement of the environmental DNA extraction method for profiling the culturable microbiome of bacterial wilt-conducive and non- conducive soils R. GICANA (1), W. Deng (1), J. Wu (1) The polymorphisms were used to design primer sets to accurately detect specific isolates of Fp by end-point PCR. Extensive exclusion panels were performed to validate the accuracy of the primer sets and two Fp isolates with unique GBS loci from two maize hybrids were identified. In field experiments, 2 plots each containing 6 subplots (3 of each hybrid) were established (completely randomized design). At harvest, 20 random maize ears were collected from each subplot, and 6 ears/subplot were used. 50 kernels/ear were sampled, 1855 isolates collected, and DNA extracted; 817 isolates were identified as Fp and 751 as F. verticillioides. 23 isolates were idenfitied as the unique GBS locus isolates thus indicating that transmission from the planted seed to the next generation of seed occurred. AFLP fingerprinting wil confirm their identify as the original isolates that were characterized with GBS. Genetic mapping of pathogenesis in the northern corn leaf blight fungus We are examining crosses that exhibit high fertility with one direction, but low fertility when male and female parents are reversed. Genome wide polymorphism screening using double digest Restriction Associated DNA sequencing (ddRADseq) was performed for 36 progeny from each direction of two biased crosses. These data are being used to provide an understanding of genetic inheritance and recombination in A. flavus and serve as genetic markers for mapping genomic regions that may bias fertility. By understanding these aspects of A. flavus genetics, we create opportunities to utilize strain fertility in the selection of biological control agents. Progress on genomic analysis and taxonomy of the rice blast fungus (Pyricularia oryzae) and other Magnaporthales specie N. ZHANG (1), G. Cai (2), D. Bhattacharya (3), J. Luo (3), H. Qiu (3) (1) Rutgers University, U.S.A.; (2) USDA, ARS, U.S.A.; (3) Rutgers University, U.S.A. Progress on genomic analysis and taxonomy of the rice blast fungus (Pyricularia oryzae) and other Magnaporthales specie N. ZHANG (1), G. Cai (2), D. Bhattacharya (3), J. Luo (3), H. Qiu (3) (1) Rutgers University, U.S.A.; (2) USDA, ARS, U.S.A.; (3) Rutgers University, U.S.A. The order Magnaporthales includes the rice blast fungus Pyricularia oryzae (=Magnaporthe oryzae), the take-all pathogen Gaeumannomyces graminis, and saprotrophs. Recent phylogenomic analyses strongly support the idea that the sexual genus Magnaporthe is polyphyletic and that the placement of the rice blast fungus in this genus was based on an incorrect morphological identification in the 1970’s. The asexual genus Pyricularia is the older (1880s) and correct name for the blast fungus, which corresponds well with pathogenicity, and ecological and evolutionary features. Following the new “One Fungus One Name” nomenclature, we propose to use Pyricularia oryzae for the rice blast fungus, and treat Magnaporthe oryzae as a synonym that can continue to be included in publications as Pyricularia oryzae (=Magnaporthe oryzae). This will help to bridge the literature and knowledge gap for this important species. In January 2016, a workshop entitled “Comparative genomic approaches to understanding the evolution of Magnaporthales” was held at Rutgers University. About 30 working group members are currently analyzing genome data from selected Magnaporthales lineages to understand the evolution of pathogenicity and life-style adaptation. Specifically, the following research topics are being addressed: OrthoMCL analysis of gene families, the secretome, positive selection acting on orthologous genes, MAT, secondary metabolism, and the molecular clock for Magnaporthales. S4.29 Anything But Barren: Fungal Diversity and Functions in the Pine Barrens N. ZHANG (1), J. Luo (2), S. Miller (2), E. Walsh (2), S. Bonos (2), Z. Helsel (2) (1) Rutgers University, U.S.A.; (2) Rutgers Univ, U.S.A. Pine barrens ecosystem has acidic and nutrient-poor soils, where switchgrass and other stress tolerant species are dominant understory plants. Our results indicate that grass (Poaceae) roots in the pine barrens are one of the major reservoirs of novel fungi with 47% being undescribed species. Importantly, we observed that Acidomelania panicicola (Leotiomycetes), a new genus and species we described from switchgrass in the New Jersey Pine Barrens increases root hair growth of switchgrass and rice in low nutrient conditions. We also described another new genus Pseudophialophora (Magnaporthales, Sordariomycetes) that contains several pathogenic species. Naming and description of a number of other new fungal taxa are in progress. We compared the root fungal community between the wild switchgrass from the Pine Barrens and cultivated switchgrass in managed farms using both culture and metagenomic methods. Ascospore biology of Parastagonospora nodorum under Norwegian field conditions A. FICKE (1), B. Asalf (2), A. Ruud (3) (1) Norwegian Institute for Bioeconomic Research, Norway; (2) Norwegian Institute of Bioeconomy Research, Norway; (3) Norwegian University of Life Sciences, Norway Identity, characteristics, and fungicide sensitivity of isolates in the Diaporthe species complex associated with soybean stem disease in Minnesota D. MALVICK (1), C. Floyd (2) (1) University of Minnesota U S A ; (2) University of Minnesota U S A Identity, characteristics, and fungicide sensitivity of isolates in the Diaporthe species complex associated with soybean stem disease in Minnesota D. MALVICK (1), C. Floyd (2) (1) University of Minnesota U S A ; (2) University of Minnesota U S A Identity, characteristics, and fungicide sensitivity of isolates in the Diaporthe species complex associated with soybean stem disease in Minnesota D. MALVICK (1), C. Floyd (2) (1) University of Minnesota, U.S.A.; (2) University of Minnesota, U.S.A. Late season stem diseases of soybean with symptoms of browning, canker, and top dieback are common in Minnesota. Characteristics of causal agents and factors that influence disease development are poorly understood. The objectives of this study were to: (i) assess the identity and relatedness among isolates in the Diaporthe spp. complex associated with both symptoms and asymptomatic infection, (ii) compare virulence among the isolates, and (iii) assess fungicide sensitivity. Isolates of D. longicolla (n=13) and D. phaseolorum var. caulivora (n=11) were obtained from symptomatic and asymptomatic plants across MN. The ITS and beta-tubulin genes were sequenced for all isolates and a phylogenetic tree with four branches was formed. Seedling stems of three soybean cultivars were inoculated with the isolates, and lesion length was measured to reveal significant differences in virulence among isolates. The groups in the consensus tree were associated with isolate species and virulence. No significant differences in sensitivity to the fungicides Folicure® 3.6F and Headline® were detected among isolates in vitro, and the mean EC50 for all isolates was 0.37 to 0.38 ug/ml. These studies reveal genotypic and virulence differences, but not differences in fungicide sensitivity, among isolates in the Diaporthe spp. complex from symptomatic and asymptomatic soybean stems, and suggest explanations for inconsistent disease development and avenues for disease management. Progress on genomic analysis and taxonomy of the rice blast fungus (Pyricularia oryzae) and other Magnaporthales specie N. ZHANG (1), G. Cai (2), D. Bhattacharya (3), J. Luo (3), H. Qiu (3) (1) Rutgers University, U.S.A.; (2) USDA, ARS, U.S.A.; (3) Rutgers University, U.S.A. Virulence of Rhizoctonia solani anastomosis groups on cotton, corn, rice and soybean K. URREA (1), C. Rothrock (1), S. Winters (1) (1) University of Arkansas, U.S.A. Rhizoctonia solani is an important pathogen on numerous crops. Isolates of R. solani were recovered from major row crops in the southern United States as part of a Rhizoctonia survey, and represented anastomosis groups (AG) 2, 4, 7, and 11. Virulence was evaluated for two isolates of each AG using stand and hypocotyl/coleoptile ratings on soybean, corn, rice and cotton using colonized millet seed inoculum placed 1-cm from seed at the time of planting (seed rot assay) or seedlings 7 days after planting (hypocotyl/coleoptile assay) in controlled environmental experiments at 25°C. Plant stands and discoloration ratings were recorded 7 days after planting or hypocotyl/coleoptile inoculation. In the seed rot assay, isolates of AG2 caused significant stand loss and brown-reddish discoloration of coleoptiles in corn. In cotton, the AG4 isolates were highly virulent causing significant stand loss compared to other AGs. In soybean, there were not significant differences in stands among isolates. In the hypocotyl assay on cotton, AG4 isolates caused significant damping-off. There were no significant differences in number of seedlings for corn, rice or soybeans. In all four crops, AG4 and AG11 isolates caused reddish brown lesions on hypocotyls or coleoptiles. This research supports the results of the soil and crop survey that crop increases the recovery of AGs of R. solani that are virulent on that crop. Standardizing water potential of salt-amended growth media at different temperatures for microbiological studies I. AUJLA (1), T. Paulitz (2) (1) Department of Plant Pathology, Washington State University, U.S.A.; (2) USDA-ARS, Washington State University, U.S.A. Standardizing water potential of salt-amended growth media at different temperatures for microbiological studies I. AUJLA (1), T. Paulitz (2) (1) Department of Plant Pathology, Washington State University, U.S.A.; (2) USDA-ARS, Washington State University, U.S.A Progress on genomic analysis and taxonomy of the rice blast fungus (Pyricularia oryzae) and other Magnaporthales specie N. ZHANG (1), G. Cai (2), D. Bhattacharya (3), J. Luo (3), H. Qiu (3) (1) Rutgers University, U.S.A.; (2) USDA, ARS, U.S.A.; (3) Rutgers University, U.S.A. Both methods suggest that Leotiomycetes are dominant fungi in the switchgrass roots from pine barrens soils, while Sordariomycetes are dominant in the roots growing in the rich farm soils. More experiments are being conducted to test our hypothesis that Acidomelania and other Leotiomycetes play a role in increasing plant tolerance to abiotic stresses (e.g., low pH, low nutrients, drought) and contribute to improved establishment in poor soils. Results from this work will facilitate ecological and evolutionary studies on root-associated fungi. Improved plant-based culture media for growth and sporulation of Cercospora janseana S. UPPALA (1), B. Liu (2), M. Wu (2), L. Zhou (3), X. Zhou (4) (1) Texas A & M AgriLife Research Center, U.S.A.; (2) Hunan Agricultural University, China; (3) Texas A&M AgriLife Research, U.S.A.; (4) Texas A&M AgriLife Research Center, U.S.A. Cercospora janseana is the fungus causing narrow brown leaf spot, a disease that has increased its importance in recent years in the U. S. In vitro studies are essential to better understand the biology of this pathogen and to develop effective strategies for management of this disease. However, the fungus grows very slowly and produces sparse conidia in common agar media. An In vitro study was conducted to evaluate various plant-based agar media in comparison with common agar media for their effects on radial growth and sporulation of C. janseana. Included in this study were extracts of dried rice leaves, stems, straw, roots and hulls; fresh rice leaf extract, dried extracts of barnyard grass leaves and sorghum leaves, clarified V8, and potato dextrose agar. All plant-based agar media were tested at four concentrations (25, 50, 100 and 200 g/L). Petri plates with various media were plated with a disc of 1-week-old actively growing fungus at the center and incubated at 28 ºC with the 12:12 h (light:dark) cycle. Radial growth was measured at weekly intervals for 2 weeks and sporulation was measured at the end of 2 weeks. Radial growth and sporulation differed among various media and their concentrations. Among the various media tested, dried rice leaf extract, dried barnyard grass, fresh rice leaf extract and V8 media consistently supported highest radial growth of the fungus. V8 medium also supported the maximum production of conidia of this fungus. Virulence of Rhizoctonia solani anastomosis groups on cotton, corn, rice and soybean K. URREA (1), C. Rothrock (1), S. Winters (1) (1) University of Arkansas, U.S.A. Evaluation of chemotype, pathogenicity, and aggressiveness of Fusarium graminearum isolates of wheat and soybean B. MUELLER (1), B. Mueller (1), C. Groves (1), D. Mueller (1) (1) University of Wisconsin-Madison, U.S.A. Evaluation of chemotype, pathogenicity, and aggressiveness of Fusarium graminearum isolates of wheat and soybean B. MUELLER (1), B. Mueller (1), C. Groves (1), D. Mueller (1) (1) University of Wisconsin-Madison, U.S.A. Evaluation of chemotype, pathogenicity, and aggressiveness of Fusarium graminearum isolates of wheat and soybean B. MUELLER (1), B. Mueller (1), C. Groves (1), D. Mueller (1) (1) University of Wisconsin-Madison, U.S.A. Fusarium graminearum is the causal agent of head blight (FHB) of wheat and damping off disease of soybean. F. graminearum can be classified as one of three chemotypes: 3-acetyldeoxynivalenol (3-ADON), 15-acetyldeoxynivalenol (15-ADON), and nivalenol (NIV). Besides causing yield loss, F. graminearum in wheat, reduces seed quality by contaminating grain with deoxynivalenol (DON) and NIV and can threaten animal and human health. Previously, 15-ADON was found to be the predominant F. graminearum chemotype in North America. Recently, however, the more aggressive 3- ADON chemotype is increasing in prevalence in Canada and the United States. Twelve total isolates of F. graminearum were collected in Wisconsin, Kansas, Iowa and Indiana from soybean and wheat between 2007 and 2015. Characterization of F. graminearum isolates were done using polymerase chain reaction (PCR) chemotyping. Thus far, all 12 isolates are the 15-ADON chemotype. Additional isolates will be collected in Wisconsin and further research will evaluate pathogenicity and aggressiveness on soybean and soft red winter wheat in Wisconsin. Results of this study will pave the way for the development of improved management options for Wisconsin farmers. Pathogenicity and Virulence of Alternaria alternata on ‘Alamo’ Switchgrass S. COLLINS (1), B. Ownley (1) (1) University of Tennessee, U.S.A. Alternaria alternata causes foliar disease in switchgrass (Panicum virgatum), and was the most frequently isolated fungus from a survey of 30 commercial switchgrass seed lots. The aims of this study were to determine 1) pathogenicity and virulence of A. alternata isolates recovered from commercial switchgrass seed, and 2) the impact of isolates on seed germination, plant health, and biomass. Tests were arranged in a completely randomized design and data were analyzed with mixed model ANOVA and F-LSD (SAS 9.4). Six isolates were screened for pathogenicity with a detached leaf assay using ‘Alamo’ switchgrass. All A. alternata isolates were pathogenic on detached leaves (P<0.05), with lesion areas ranging from 8.8% to 23.3%. Isolates of A. alternata were inoculated onto surface-sterilized ‘Alamo’ seed at 105 conidia/ml. Plant stand and health, and biomass yield were measured. Standardizing water potential of salt amended growth media at different temperatures for microbiological studies I. AUJLA (1), T. Paulitz (2) (1) Department of Plant Pathology, Washington State University, U.S.A.; (2) USDA-ARS, Washington State University, U.S.A. ( ), ( ) (1) Department of Plant Pathology, Washington State University, U.S.A.; (2) USDA-ARS, Washington State University, U.S Temperature and moisture profoundly influence the activity of fungi. Water potential (a measure of available water) for optimal growth of a fungi may differ with temperature. Depending on the habitat and life stage of the fungus, different components of the water potential (WP) become important. The matric component of WP dominates in soil but following plant infection, osmotic effects are important. Nutrient media amended with NaCl or KCl and polyethylene glycol (PEG) are used to study the response of fungi to osmotic and matric potentials, respectively. The advent of more precise WP measuring devices such as WP4C (Decagon Devices, Pullman, WA), using the chilled-mirror dew point technique, allowed us to standardize salt- amended potato dextrose broth with or without agar for a range of basal nutrient media from -0.2 to -9 MPa at 20°C, using NaCl and KCl for osmotic potential and PEG-8000 for matric potential. The same salt and nutrient media combinations standardized at 20°C were also measured for WP at 15, 25, 30 and 35°C. WP was extrapolated for 4 and 10°C with linear/curvilinear regression due to the temperature range limits of the instrument between 15 and 40°C. These standardized tables are more accurate than previous literature due to technological advancements and will be useful for studying the response of different life stages of fungi to osmotic and matric potential. ), ( ) or Bioeconomic Research, Norway; (2) Norwegian Institute of Bioeconomy Research, Norway; (3) Norwegian University o ) ( ) ( ) n Institute for Bioeconomic Research, Norway; (2) Norwegian Institute of Bioeconomy Research, Norway; (3) Norwegian s, Norway S4.30 P. nodorum (syn. Stagonospora nodorum) is the dominant pathogen of the Norwegian leaf blotch disease complex on wheat and can cause substantial yield losses. To improve the forecasting model for leaf blotch diseases, available at our web-based pest- and disease forecasting platform (vips- landbruk.no), we studied ascospore biology of P. nodorum. Airborne ascospores were monitored with Burkard volumetric spore traps for three years in southeastern Norway. In the laboratory, we conducted wind tunnel studies to examine the effect of simulated rain and light conditions on ascospore discharge. We determined the mating types for 117 P. nodorum isolates (MAT1 and MAT2) using MAT-specific primers and inoculated wheat plants with opposite mating types to follow ascocarp maturation under field conditions. Genetic characterization of the Colletotrichum gloeosporioides complex in apple orchards of North Carolina B. HOGE (1), M. Cubeta (1), D. Ritchie (1) (1) North Carolina State University, U.S.A. Genetic characterization of the Colletotrichum gloeosporioides complex in apple orchards of North Carolina B. HOGE (1), M. Cubeta (1), D. Ritchie (1) (1) North Carolina State University, U.S.A. Glomerella leaf spot caused by fungi in the Colletotrichum gloeosporioides (Cg) species complex is an important disease of apple (Malus domestica). It is associated with leaf and fruit spots, resulting in yield loss and premature defoliation of trees. While several studies have characterized the genotypic diversity of species complexes within Colletotrichum, little information is available on pathogen diversity on apple cultivars in North Carolina (NC). Development of sustainable management strategies requires consideration of both host and pathogen factors and information on their association with disease incidence and severity. The primary objective of this study was to examine the genetic diversity of field populations of Cg sampled from infected fruits and leaves of cultivars Gala, Golden Delicious, Pink Lady, Granny Smith, and Yellow Transparent in 15 orchards in NC. Genomic DNA was extracted from a phenotypically diverse subsample (n=50) of 150 isolates and sequence analysis of four independent genetic loci was performed to identify multi-locus haplotypes (MLHs). Eight distinct MLHs were detected, and three well-supported lineages corresponding to species in the Cg complex were identified. Isolates identified as MLH1 belonged to the Cg clade and were sampled from three of five cultivars. MLH2 isolates were identified as C. fructicola, and sampled from four of five cultivars with some overlap. The remaining MLHs were restricted to a single cultivar or isolate. Fitness ability and fungicide sensitivity of Lambertella corni-maris isolates from apple A. AMIRI (1), A. Hawkins (2), K. Mulvaney (2) (1) Washington State University, U.S.A.; (2) Washington State University, U.S.A. Lambertella corni-maris is a newly reported postharvest decay of apple. Additional knowledge is needed to understand disease ecophysiology and develop adequate control strategies. In this study, we investigated fitness ability of three isolates in vitro i.e. growth on different media, sensitivity to pH and osmotic stress and in vivo pathogenicity on nine apple cultivars. All three isolates grow significantly faster on apple juice agar compared to potato dextrose agar whereas growth on V8 agar and malt extract agar was intermediate. Isolates grew equally at pH ranging from 3 to 7 and growth was inhibited at pH ≥8. Significant sensitivity to osmotic stress was only observed at higher KCl concentrations (1M). L. Evaluation of chemotype, pathogenicity, and aggressiveness of Fusarium graminearum isolates of wheat and soybean B. MUELLER (1), B. Mueller (1), C. Groves (1), D. Mueller (1) (1) University of Wisconsin-Madison, U.S.A. Foliar disease ratings of plants from seed inoculated with all isolates was significantly greater than in untreated controls (P<0.0001). Isolates were inoculated (105 conidia/ml) on foliage of whole plants. All isolates caused greater foliar disease severity than untreated controls (P=0.10), and two isolates significantly reduced shoot fresh weight (P=0.05). Although A. alternata can cause foliar disease, isolates did not reduce plant stand, suggesting that this fungus may cause less economic losses than fungi that affect stand establishment, such as Bipolaris and Fusarium spp. Standardizing water potential of salt amended growth media at different temperatures for microbiological studies I. AUJLA (1), T. Paulitz (2) (1) Department of Plant Pathology, Washington State University, U.S.A.; (2) USDA-ARS, Washington State University, U.S.A. In the field, ascospore dispersal was not associated with rain events nor any specific day/night periodicity, but with daily relative humidity above 70%. In the laboratory, over 95% of ascospores were released during simulated rain events at 100% relative humidity, independent of light conditions. Mating type ratios of P. nodorum varied with location between 1:1 and 1:2 (M1:M2). Mature ascocarps formed 12 months after inoculation, corresponding to 551 degree-days (base 10°C) for the 2014/2015 season. We conclude that ascocarp formation can take an extended period of time and that daily humidity above 70% is sufficient for ascospore release in the field. Genetic characterization of the Colletotrichum gloeosporioides complex in apple orchards of North Carolina B. HOGE (1), M. Cubeta (1), D. Ritchie (1) (1) North Carolina State University, U.S.A. corni-maris was pathogenic to all nine test apple cultivars, however, Honey Crisp, Fuji, Pixata, and Gala were significantly more susceptible compared to Cameo, Braeburn and Red Delicious. Furthermore, the sensitivity of 100 L. corni-maris isolates to fludioxonil, boscalid, pyraclostrobin, pyrimethanil and thiabendazole was evaluated using a mycelial growth inhibition assay. Mean EC50 values were 0.07, 0.84, 1.32, 2.45 and 3.68 µg/ml, respectively. Fludioxonil and pyrimethanil were highly effective in controlling the disease on detached fruit whereas thiabendazole was the least effective. Our results indicate that L. corni-maris may be able to survive extreme conditions and is better controlled by postharvest rather than preharvest fungicides. Host colonization and substrate utilization by wood-colonizing Ascomycete fungi in the grapevine trunk disease complex E. GALARNEAU (1), C. Wallis (2), K. Baumgartner (2) (1) University of California-Davis, U.S.A.; (2) USDA-Agricultural Research Service, U.S.A. Grapevine trunk diseases cause chronic wood infections (cankers) in mixed infections within the same vine. To determine the synergistic interactions of trunk-pathogen communities and their impact on the host we are characterizing, on a pathogen-by-pathogen basis, fungal damage to woody cells and tissues. Our focus is on two taxonomically unrelated Ascomycetes: Botryosphaeria dieback pathogen Neofusicoccum parvum and Eutypa dieback pathogen Eutypa lata. After inoculating plants and identifying the host-defense compounds that accumulated in wood cankers, we tested their effects on fungal growth in vitro. Both fungi significantly degraded and utilized rutin, epicatechin, and piceid, suggesting that these fungi tolerate and possibly subvert the biochemical host-defense response. In contrast, fungal growth was significantly inhibited by lignin and gallic acid. Only E. lata discolored solid media amended with gallic acid, signifying phenoloxidase activity, which is consistent with its status as a soft-rot fungus. Previous work showed E. lata colonizes the ray parenchyma, xylem vessels and fibers, which we verified are also proliferated by N. parvum. The ability of these fungi to degrade wood was evaluated in planta by imaging the microscopic features of soft rot. As both fungi have similar gene families associated with cell-wall degradation and secondary metabolism, it is not surprising that they may share similar strategies for colonization and substrate utilization. Virulence of Botrytis prunorum associated with blossom blight on Japanese plum in Chile E. FERRADA (1), B. Latorre (1), J. Zoffoli (1), G. Diaz (2) (1) Pontificia Universidad Católica de Chile, Chile; (2) Universidad de Talca, Chile Blossom Blight (BB) is an important disease that affects the productivity of stone fruits. Recently, B. prunorum was identified causing BB on Japanese plum (Prunus salicina) in Chile. Symptoms consisted on a light brown necrosis of flowers. The objective of this study was to compare the virulence exhibited by isolates B. prunorum and isolates of B. cinerea affecting plums. Virulence tests were conducted with seven isolates of B. prunorum on detached plum flowers and mature fruits ‘Larry Ann’ inoculated with a conidial suspension (106 conidia/mL) and mycelial plugs (4 mm in diameter). Mature fruits of ‘Granny Smith’ apples and ‘Hayward’ kiwifruits were similarly inoculated. Flowers and fruits were incubated for at least 48 h at 20°C in humid chambers. Seven B. cinerea isolates were included in all virulence tests. In addition, the virulence of B. prunorum and B. Host colonization and substrate utilization by wood-colonizing Ascomycete fungi in the grapevine trunk disease complex E. GALARNEAU (1), C. Wallis (2), K. Baumgartner (2) (1) University of California-Davis, U.S.A.; (2) USDA-Agricultural Research Service, U.S.A. cinerea was studied on detached bean leaves that were inoculated with a mycelial plug (4 mm) incubated for 4 d at 20°C in humid chambers. All isolates of B. prunorum and B. cinerea were pathogenic on flowers and fruits of plums as well as on apple and kiwifruit and bean leaf. All isolates of B. prunorum were significantly (P<0.001) less virulent than B. cinerea isolates. Differences in virulence between B. cinerea and B. prunorum can be attributed to differences in the genetic expression of virulence factors. These results suggest that B. prunorum plays a secondary role in the epidemiology of BB of plums. Modelling potential inoculum availability of Mycosphaerella nawae in persimmon leaf litter using Bayesian growth curves A. VICENT (1), J. Martínez-Minaya (2), A. López-Quílez (3), D. Conesa (3) (1) Instituto Valenciano de Investigaciones Agrarias (IVIA). Moncada 46113, Valencia, Spain., Valencia, Spain; (2) Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada 46113, Valencia, Spain., Valencia, Spain; (3) Departament d’Estadística i Investigació Operativa, Universitat de València. Burjassot 46100, Valencia, Spain., Valencia, Spain (1) Instituto Valenciano de Investigaciones Agrarias (IVIA). Moncada 46113, Valencia, Spain., Valencia, Spain; (2) Instituto Valenciano de Investigaciones Agrarias (IVIA), Moncada 46113, Valencia, Spain., Valencia, Spain; (3) Departament d’Estadística i Investigació Operativa, Universitat de València. Burjassot 46100, Valencia, Spain., Valencia, Spain Circular leaf spot, caused by Mycosphaerella nawae, is a serious disease of persimmon (Diospyros kaki) inducing necrotic lesions on leaves, defoliation and fruit drop. The disease was initially restricted to humid regions in Japan and South Korea and in 2008 it was detected in arid areas in Spain. The fungus forms pseudothecia in leaf litter during winter and ascospores are released in spring infecting susceptible leaves. Persimmon growers are advised to spray with fungicides during the period of inoculum availability, which was defined based on ascospore counts. In order to develop a model of potential inoculum availability, samples of leaf litter were collected weekly in six affected orchards from 2010 to 2015. Leaves were soaked, placed in a wind tunnel, and released ascospores were counted under the microscope. Hierarchical Bayesian methods were used to fit the dynamics of ascospore production in the leaf litter to Gompertz and logistic growth curves, including temperature and rainfall as nonlinear effects. MCMC methods were used to approximate the posterior distribution of the parameters. Results showed that accumulated degree days best described ascospore dynamics with both Gompertz and logistic curves. Nutritional and environmental effects on conidial germination and appressorium formation of Phyllosticta citricarpa, the citrus black spot pathogen These results provide a better understanding of fungal biology and a robust and convenient system for further applications such as screening for efficacious fungicides. Host colonization and substrate utilization by wood-colonizing Ascomycete fungi in the grapevine trunk disease complex E. GALARNEAU (1), C. Wallis (2), K. Baumgartner (2) (1) University of California-Davis, U.S.A.; (2) USDA-Agricultural Research Service, U.S.A. Host colonization and substrate utilization by wood-colonizing Ascomycete fungi in the grapevine trunk disease complex E. GALARNEAU (1), C. Wallis (2), K. Baumgartner (2) (1) University of California-Davis, U.S.A.; (2) USDA-Agricultural Research Service, U.S.A. The predictive ability of the models was evaluated using ascospore data from two orchards not used in model development. The resulting best model was implemented in a disease warning system to schedule fungicide sprays for the control of circular leaf spot. Factors affecting germination of pseudosclerotia of Monilinia vaccinii-corymbosi on lowbush blueberry T CASE (1) S Annis (1) In the lowbush blueberry (Vaccinium angustifolium) industry of Maine, substantial yield loss can occur annually from the disease, mummy berry, caused by Monilinia vaccinii-corymbosi, if left unmanaged. The primary objective of this study is to better understand the environmental conditions that favor the germination of pseudosclerotia. We have conducted in vitro incubation experiments for the past two years, in which the germination of apothecia from pseudosclerotia has been observed in response to chill-hours (time between 0-7°C). To further identify conditions affecting the germination of pseudosclerotia, we have incubated pseudosclerotia over a wide range of chill-hours (800-2380 chill-hours), under three different soil saturations (50, 60 and 75%), as well as three different post-chill temperature regimes (10, 13 and 16°C). Germination of pseudosclerotia was greater under the highest temperature we tested (16°C), and moderate soil moisture conditions (60%). The development of apothecia from pseudosclerotia was affected by chill- hours, but varied substantially between treatments from different fields. Identifying the environmental conditions favoring the development of apothecia in the lab assists in managing this pathogen in the field through the development of a predictive model for germination. We will use this model in continuing our program for forecasting mummy berry, which gives growers an opportunity to effectively manage this disease. Nutritional and environmental effects on conidial germination and appressorium formation of Phyllosticta citricarpa, the citrus black spot pathogen N. WANG (1), M. Dewdney (2) N. WANG (1), M. Dewdney (2) (1) Department of Plant Patholog ( ) y ( ) (1) Department of Plant Pathology, Citrus Research and Education Center, University of Florida, U.S.A.; (2) mmdewdney@ufl.edu Department of Plant Pathology, Citrus Research and Educatio Citrus black spot caused by Phyllosticta citricarpa has been present in Florida since 2010 and can reduce fruit yield and marketability. Conidial germination and appressorium formation (AF) are essential for successful infection by P. citricarpa, although the conditions required for this process are poorly understood. In this study, the effects of citrus juices, concentration, pH, various carbon and nitrogen sources, and environmental conditions were evaluated in vitro. All tested juices, especially Valencia (> 85%, P < 0.05), favored germination and AF, whereas sterile water rarely stimulated S4.31 germination (< 1%). Juice quality analysis using Pearson’s correlation test revealed a moderate relationship between the Brix/Acid ratio and germination (r = 0.783, P < 0.05). The Valencia juice effect was concentration- and pH-dependent, and the maximum rate was reached in 1.5% juice with pH of 3.4. Most carbon, nitrogen, or complex sources did not favor germination or AF with exception of PDB, ammonium nitrate, and yeast extract. An incubation period of 18 to 24 h at 24°C were required for peak germination and AF. Further analysis of critical juice components using synthetic juice revealed sugars, salts, citric acid, and thiamine combined were most important for germination and AF (> 80%, P > 0.05). These results provide a better understanding of fungal biology and a robust and convenient system for further applications such as screening for efficacious fungicides. germination (< 1%). Juice quality analysis using Pearson’s correlation test revealed a moderate relationship between the Brix/Acid ratio and germination (r = 0.783, P < 0.05). The Valencia juice effect was concentration- and pH-dependent, and the maximum rate was reached in 1.5% juice with pH of 3.4. Most carbon, nitrogen, or complex sources did not favor germination or AF with exception of PDB, ammonium nitrate, and yeast extract. An incubation period of 18 to 24 h at 24°C were required for peak germination and AF. Further analysis of critical juice components using synthetic juice revealed sugars, salts, citric acid, and thiamine combined were most important for germination and AF (> 80%, P > 0.05). A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. Polymyxa betae is an obligate pathogen capable of vectoring several viruses of sugar beet that can cause large losses in production. In the past, quantifying P. betae infection levels required time-consuming staining and visual examination using light microscopy. A new species-specific quantitative real-time PCR assay that allows for rapid quantification of P. betae infection levels in sugar beet roots has been developed. The assay has been designed to target the P. betae internal transcribed spacer (ITS) region of the genomic rDNA using sequence data from forty isolates. To achieve species-specificity, the assay primers and probe were designed against nineteen Polymyxa graminis ITS sequences from GenBank. In practice, the assay has demonstrated species-specificity in all fifteen P. betae isolates tested and has not amplified P. graminis isolates. Three internal plant controls, two targeting single copy nuclear genes and one targeting the ITS region of the rDNA, have been multiplexed with the pathogen assay to standardize results across samples. Further, the assay has also been optimized for digital droplet PCR, which allows for more precise quantification of minor differences in infection levels than conventional real time PCR. These tools will allow for more efficient quantification of P. betae infection levels in experimentation evaluating host resistance, variation in aggressiveness among isolates and virus transmission studies. Ray blight disease (Stagonosporopsis tanaceti) development in pyrethrum M. BHUIYAN (1), T. Groom (2), M. Nicolas (3), P. Taylor (3) (1) The University of Melbourne, Australia; (2) Botanical Resources Australia, A Ray blight disease (Stagonosporopsis tanaceti) development in pyrethrum M. BHUIYAN (1), T. Groom (2), M. Nicolas (3), P. Taylor (3) (1) The University of Melbourne, Australia; (2) Botanical Resources Australia, Australia; (3) The University of Melbour ersity of Melbourne, Australia; (2) Botanical Resources Australia, Australia; (3) The University of Melbourne, Australia Pyrethrum (Tanacetum cinerariifolium) is commercially cultivated in Australia for pyrethrins which are used as natural pesticides. Ray blight caused by S. tanaceti is considered as the most devastating fungal disease and is recognized as one of the limiting factors of pyrethrum production. The pathogen is known to infect the flowers and leaves however, little is known about the mechanism of infection and process of tissue colonisation. Relationship between genetic and morphological characters of Botrytis cinerea isolates obtained of blueberry fields in southern Chile E. BRICEÑO (1), E. Briceño (1), S. Aguirre (2), A. Behn (2) (1) Universidad Austral de Chile, Chile; (2) Univ austral Chile, Chile Relationship between genetic and morphological characters of Botrytis cinerea isolates obtained of blueberry fields in southern Chile E. BRICEÑO (1), E. Briceño (1), S. Aguirre (2), A. Behn (2) (1) Universidad Austral de Chile, Chile; (2) Univ austral Chile, Chile Botrytis cinerea is the most important pathogen in blueberry crops (Vaccinium spp.) in southern Chile. The climate conditions of the zone and the presence of some populations fungicide resistant, increase its incidence. Due to the high morphological variability observed on Botrytis populations, the aim of this work was to characterize isolates from eight locations (Lat. 39º06’S to 41º28’S), in order to establish some relationship between morphological and genetic variability. For this, 39 monosporic cultures of B. cinerea obtained from mature blueberry fruits were morphologically characterized about mycelia color, sporulation level, growth rate, conidia size and sclerotia production. On the other hand, genetic characterization was done by PCR detection of transposons Boty and Flipper, to determinate their genotype. In addition, coding fragments for the MS457 gene were sequenced and phylogenetic tree was built. The genetic studies revealed the presence of four genotypes, predominating Vacuma in the geographic region. S4.32 The results indicate that the presence of transposable elements does not affect the growth rate of the isolates. However, different sizes of conidia are distinguished by genotype, possessing at least one of the transposons conidia size increases, then Transposa presented conidia larger while Vacuma the smaller. On the other hand Boty produced the bigger sclerotia. Mining of abiotic and biotic factors for prediction of soybean sudden death syndrome (SDS) symptoms Z. NOEL (1), M. Roth (1), J. Wang (1), E. Papenfuss (1), D. Kramer (1), M. Chilvers (1) (1) Michigan State University, U.S.A. Fusarium virguliforme is the causal agent of soybean sudden death syndrome (SDS) in the U.S. The pathogen infects roots initially, but foliar chlorosis and necrosis caused by a phytotoxin, do not usually appear until later in the growing season when it is too late to consider replanting. In this study, parameters measured before planting were used as indicators of SDS development later in the season. Prior to planting, soil nutrients (pH, K, Ca, Mg, Zn, Mn, Cu, Fe, and organic matter) soybean cyst nematode counts (eggs, juveniles, and adults), and F. virguliforme inoculum density were used to determine which variables or combination of variables helped explain SDS development throughout the growing season. Identifying Diversity Within Corynespora cassiicola, Cause of Target Spot of Cotton, Tomato, and Soybean in the Southeastern U.S. L. SUMABAT (1), M. Brewer (1), R. Kemerait (2) (1) University of Georgia, U.S.A.; (2) University of Georgia, U.S.A. Preliminary results from Multilocus Sequence Typing of the soilborne fungal pathogen Sclerotium rolfsii P. SORIA (1), M. Smith (1), N. Dufault (1) (1) University of Florida, U.S.A. Preliminary results from Multilocus Sequence Typing of the soilborne fungal pathogen Sclerotium rolfsii P. SORIA (1), M. Smith (1), N. Dufault (1) (1) University of Florida, U.S.A. Sclerotium rolfsii is an important soilborne fungal pathogen of hundreds of plant species worldwide, including economically important crops such as tomato and peanut in the southeastern US. Currently, molecular studies are scarce for this pathogen, leading to a lack of an experimental framework to study the biology S. rolfsii using genetic methods. There is, however, evidence for significant phenotypic variation among S. rolfsii isolates in terms of virulence, growth rate, sclerotial size, and temperature tolerance. Methods of measuring genetic variation are currently limited to ambiguous mycelial compatibility groups, and few sequence analyses using the ITS (internal transcribed spacer) rDNA. To accurately characterize genetic diversity among phenotypically variable S. rolfsii populations, a more elaborate and sequence-based experimental framework is required. Multilocus Sequence Typing (MLST) was chosen as a sequence-based method for cryptic species recognition and for identifying genetic diversity of S. rolfsii in the Southeastern US. Several housekeeping genes (LSU, RPB1, RPB2, TEF, and MCM7) that are commonly used in MLST analyses were evaluated to choose the most informative loci to characterize the population structure of S. rolfsii. The resulting MLST scheme will contribute to establishing a molecular framework for this important soilborne pathogen and will increase the current understanding of the biology and epidemiology of S. rolfsii. Relationship between genetic and morphological characters of Botrytis cinerea isolates obtained of blueberry fields in southern Chile E. BRICEÑO (1), E. Briceño (1), S. Aguirre (2), A. Behn (2) (1) Universidad Austral de Chile, Chile; (2) Univ austral Chile, Chile Principle components analysis allowed for dimensional reduction and indicated which variables were most important for disease prediction. Disease symptoms at R5 and R6 were significantly correlated with soybean cyst nematode data and F. virguliforme inoculum density. Other multivariate techniques are currently being performed to allow for a better understanding of the factors that contribute to the disease development of SDS. Effects of inoculum substrates on root rot of soybean caused by Fusarium oxysporum and Fusarium graminearum D. CRUZ (1), D. Mayfield (2), Y. Meng (1), G. Munkvold (1), L. Leandro (1) (1) Iowa State University, U.S.A.; (2) Iowas State University, U.S.A. Effects of inoculum substrates on root rot of soybean caused by Fusarium oxysporum and Fusarium graminearum D. CRUZ (1), D. Mayfield (2), Y. Meng (1), G. Munkvold (1), L. Leandro (1) 1) Iowa State University, U.S.A.; (2) Iowas State University, U.S.A. Fusarium oxysporum (Fo) and F. graminearum (Fg) are associated with the Fusarium seedling disease and root rot complex in soybeans. Published soil infestation methods have limitations in the expression of disease. Our objective was to compare the efficacy of infested substrates for generating root rot caused by Fo and Fg and evaluate adverse effects of non-infested substrates on roots. Autoclaved millet and rice hulls infested with Fo and Fg were added to soil at a rate of 5% (by vol.). Susceptible cultivars Jack and MN1805 were evaluated 2 weeks after planting for root length and root rot using the WinRhizo image analysis software; root rot was also estimated visually on a percent scale. Cultivars did not differ in root length and root rot assessed by image analysis, but visually-assessed root rot was greater in Jack (P=0.008). There was a significant interaction between substrate and Fusarium species for root rot assessed by both methods (P<0.0001). Millet infested with Fg produced the greatest root rot severity (37%), followed by millet infested by Fo (24%), whereas infested rice hulls resulted in low root rot (15 and 10% for Fo and Fg, respectively). Root rot for non-inoculated substrates was less than 5%. Infested and non-infested millet reduced root length compared to rice hulls. Pathogen colonization was assessed using qPCR. Our study suggests that millet is more effective for producing seedling root rot but can cause some adverse effects on root growth. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. The first detection of potato wart in North America occurred in 1909 in Newfoundland. More recently in 2000, it was found in a field of Prince Edward Island. We sequenced and assembled the genomes of two different genotypes: S. endobioticum MB42 from the Netherlands and S. endobioticum DAOM 229326 from Canada. The genome size ranged from 20.6 to 23.5 Mb and between 8000 and 8800 genes were predicted for the respective strains. Carbohydrate degrading enzymes and effectors are being analyzed to help the development of more robust genotyping and pathotyping assays and to understand better the evolution of this early lineage of plant pathogens. Origin and consequence of myrtle rust (Puccinia psidii) in the New Caledonian biodiversity hotspot S. JULIA (1), M. Laurent (2), C. Fabian (1) Origin and consequence of myrtle rust (Puccinia psidii) in the New Caledonian biodiversity hotspot S. JULIA (1), M. Laurent (2), C. Fabian (1) (1) Institut Agronomique Néo-Calédonien, New Caledonia; (2) CIRAD (Centre de coopération internationale en recherche agronomique pour le développement), New Caledonia S. JULIA (1), M. Laurent (2), C. Fabian (1) (1) Institut Agronomique Néo-Calédonien, New Caledonia; (2) CIRAD (Centre de coopération internationale en recherche agronomique pour le développement), New Caledonia S. JULIA (1), M. Laurent (2), C. Fabian (1) (1) Institut Agronomique Néo-Calédonien, New Caledonia; (2) CIRAD (Centre de coopération internationale en recherche agronomique pour le développement), New Caledonia S. JULIA (1), M. Laurent (2), C. Fabian (1) (1) Institut Agronomique Néo-Calédonien, New Caledonia; (2) CIRAD (Centre de coopération internationale en recherche agronomique pour le dé l ) N C l d i The myrtle rust disease, caused by the fungus Puccinia psidii, is considered as one of the main environmental threat to plants belonging to Myrtaceae family worldwide. In New Caledonia, a biodiversity hotspot renowned for its extraordinary botanical richness and endemicity, the fungus was first reported in 2013. The Myrtaceae, the principal target of P. psidii, holds a prominent position in the New Caledonian flora by being the most species-rich family. Taking into account the ecological importance of Myrtaceae in many ecosystems, it was agreed to establish a disease management approach to mitigate the spread and the potential long term impacts of the pathogen. In this perspective, it is essential to refine our understanding of the dynamics of the host-pathogen-environment interactions. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. Hadziabdic (1) (1) University of Tennessee, Knoxville, TN, U.S.A.; (2) United States Department of Agriculture, Agricultural Research Service, Charleston, SC, U.S.A.; (3) University of Tennessee, U.S.A.; (4) University of Tennessee, Knoxville, U.S.A.; (5) Universtiy of Tennessee, Knoxville, TN, U.S.A.; (6) Tennessee Valley Authority, Knoxville, TN, U.S.A.; (7) United States Department of Agriculture, Agricultural Research Service, Poplarville, MS, U.S.A.; (8) United States Department of Agriculture, Forest Service, Cleveland, TN, U.S.A.; (9) United States Fish and Wildlife Service, Cookeville, TN, U.S.A. Pityopsis ruthii is an endangered species endemic to the Hiwassee and Ocoee rivers in Tennessee. Due to limited information regarding biology and genetics of P. ruthii, our research over the past six years have focused on conservation and recovery efforts. Using applied and molecular techniques, a basic understanding of the biology, propagation, population genetics, and diseases of P. ruthii has been gained. Micropropagation protocols using leaf and receptacle tissues have been developed. Clones from these experiments had a 73% survival rate one year after introduction to native habitats. Vegetative propagation of stems is highly successful. Seed germination for long-term stored seeds was low (<1%) and highly variable for freshly collected seeds across different locations. A total of 814 individuals from both rivers were analyzed using chloroplast and nuclear microsatellites and indicated higher genetic diversity than initially hypothesized. Analyses of chloroplast and nuclear microsatellites showed differences between the respective data sets, indicating presence of gene flow and population structure. Two diseases were identified from greenhouse grown plants: aerial blight, caused by Rhizoctonia solani, and powdery mildew, caused Golovinomyces cichorecearum. Understanding the biology and genetics of P. ruthii will facilitate long-term conservation efforts and provide information on reintroduction and preservation of biodiversity for this endangered species. Genome sequencing of Synchytrium endobioticum offers insight into its carbohydrate degrading enzymes and effectors H. NGUYEN (1), B. van de Vossenberg (2), T. van der Lee (2), D. Joly (3), D. Smith (4), M. van Gent-Pelzer (2), P. Bonants (2), C. Levesque (1) (1) Agriculture and Agri-Food Canada, Ottawa, ON, Canada; (2) Wageningen University and Research Centre, Netherlands; (3) University of Moncton, Moncton, NB, Canada; (4) Canadian Food Inspection Agency, Canada Synchytrium endobioticum is an obligate fungal parasite causing severe and persistent potato wart disease that render potato tubers unmarketable. Therefore, quarantine measures are enforced worldwide to prevent the spread of this economically important plant disease. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. Oxygen and an aerial state are independently required for asexual development of Aspergillus species M CHI (1) K Craven (2) Oxygen and an aerial state are independently required for asexual development of Aspergillus species M. CHI (1), K. Craven (2) (1) The Samuel Roberts Noble Foundation, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A. M. CHI (1), K. Craven (2) (1) The Samuel Roberts Noble Foundation, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A. Asexual sporulation is one of the primary means for aerial dissemination of many plant pathogenic fungi. The generation of asexual spores usually involves dynamic changes in cellular polarity and morphogenesis (e.g. from filamentous hyphae to oval spores). While sporulation appears to be suppressed in planta, it is seemingly de-repressed outside of plant tissues, where it encounters an aerial environment. In this study, we identify key components that allow fungi to distinguish between in planta and aerial environments. We observed conidiophore development of several Aspergillus species in an artificial environment that mimicked the in planta condition, but where we could enforce either high or low oxygen conditions. This experimental design enabled us to reveal that not only oxygen but also the physical, aerial state are positive environmental factors inducing conidiogenesis in most of the aspergilli tested in this study. Transcriptional analysis combined with direct observation of conidiation-defective mutants revealed that expression of key regulatory genes for conidiophore development is indeed regulated by these environmental cues and that the production of reactive oxygen species is required for polarized cell differentiation. Our findings provide not only novel insights into how fungi respond to an aerial environment but also inform an improved model for the regulatory pathway controlling conidiophore development. Current Status and Ongoing Conservation Efforts for the Federally Endangered Species Pityopsis ruthii T. EDWARDS (1), P. Wadl (2), R. Trigiano (3), E. Hatmaker (3), S. Boggess (3), P. Moore (1), M. Staton (5), W. Klingeman (1), E. Bernard (1), A. Dattilo (6), B. Ownley (1), T. Rinehart (7), M. Pistrang (8), J. Skinner (1), G. Call (9), A. Windham (1), D. Development of a multiplex PCR microsatellite marker set for Raffaelea lauricola, and its potential applications T. DREADEN (1), M. Hughes (2), J. Smith (2) (1) USDA FS, U.S.A.; (2) University of Florida, U.S.A. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. Therefore, this study was undertaken to identify disease development within the whole plant using traditional culturing and histopathology, and elucidate the life cycle of S. tanaceti. Ray blight disease development in pyrethrum plants was the result of colonisation of the parenchyma cells in the hypodermis and cortex with no infection of the vascular tissues. Pycnidia developed in severely infected tissues which developed a deep brown discolouration due to necrosis caused by intra- or inter cellular colonization. Secondary infection of upper flower stems, flower buds and flower tissues most likely occurred from pycniospores released from the pycnidia in the infected leaves and petioles and being disseminated through wind, rain or water-splash. The pathogen did not infect roots of plants with infected crown tissue. This process of infection and colonisation of pyrethrum plants provides a better understanding of the complete life/disease cycle which will lead to improved control measures through the strategic use of fungicides. S4.33 Target spot, caused by the fungus Corynespora cassiicola, is a disease that is emerging on crops in the southeastern U.S., including cotton. Previous studies suggest that populations from cotton, tomato, and soybean in the U.S. are host specific and genetically differentiated based on host of origin. Although variation in aggressiveness to cotton was observed, no genetic variation was detected within populations sampled from cotton. The aim of this study was to develop markers to detect genetic variation in C. cassiicola. Variable microsatellite motifs with conserved flanking regions were identified in draft genomes of two isolates from cotton, an isolate from tomato, and an isolate from soybean. Thirty-one sets of primers were designed, and then tested for amplification of these four isolates. The nineteen loci that amplified well were evaluated on a panel of 15 isolates from diverse hosts of origin and geographic regions. Fourteen of the markers were robust and informative. Preliminary results showed diverse genotypes among C. cassiicola populations from different hosts, yet there was no genotypic variation detected within tomato or cotton isolates sampled from different cultivars, states, and years, suggesting that populations from these hosts represent distinct clonal genotypes. Determining levels of variation among isolates of C. cassiicola from cotton is critical for screening for cultivar resistance and identifying fungicides for management of target spot. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. applantum complex and related species, G. curtisii, G. meredithae, G. sessile, G. tsugae, G. tuberculosum, G. weberianum-subamboinens species complex, G. zonatum, and Tomophagus colossus (syn. G. colossus). Wood block decay assays are underway to quantify decay ability of select species. Future studies will investigate differences in decay rates and pathogenicity, host specialization and phylogeography of the most commonly observed Ganoderma taxa collected in the Southeastern United States. Progress of severity of brown rust and orange rust during two sugarcane crop seasons in Florida S. SANJEL (1), M. Hincapie (1), B. Chaulagain (1), J. Comstock (2), R. Raid (1), P. Rott (1) (1) University of Florida, U.S.A.; (2) USDA-ARS, U.S.A. Brown rust (caused by Puccinia melanocephala) and orange rust (caused by Puccinia kuehnii) are currently the two most damaging fungal diseases of sugarcane in Florida. To determine progress of these two diseases during the crop season, brown rust susceptible cultivar CL90-4725 and orange rust susceptible cultivar CL85-1040 were planted in field experiments at Belle Glade in November 2013 and in November 2014. Intensity of pathogen sporulation and percent rust affected area were assessed on the top visible dewlap leaf (TVD) and the fourth leaf below TVD every two weeks. Brown rust symptoms were mainly observed from mid-March to mid-July in 2014 and from mid-April to mid-August in 2015. Sporulation intensity and leaf damage was highest in May-June 2014 but this period shifted to June-July in 2015. In contrast, orange rust symptoms were observed all year round, but sporulation and percent diseased leaf area peaked from mid-May to early August. Later on, disease severity decreased until October when another significant increase of orange rust occurred until the end of the year. This pattern of orange rust progress was similar during the 2015 and 2016 crop seasons. Overall, disease severity was higher for orange rust than for brown rust and orange rust impacted sugarcane growth for a longer period of time during the crop season. Conducive environmental conditions for disease progress remain to be determined but appear to be broader for orange rust than for brown rust. Host specificity of Colletotrichum sp. on tree tomato (Solanum betaceum) and mango (Mangifera indica) crops S. RESTREPO (1), L. Cabrera-Villamizar (1), C. pardo (1), S. Rojas (1), P. Rojas (1), G. Danies (1), P. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. In order to reach this goal, we have conducted field-based assessments of the host range and disease monitoring on several species in a semi-controlled environment during two years. A genotyping survey of the rust showed that a unique genotype has been established throughout the whole territory. To date, P. psidii was reported on at least 40 host species. The results showed a wide variation in responses to the disease within Myrtaceae and across different habitats, as previously showed in other locations where the rust occurs. Intraspecific variability was also observed among a range of susceptible species. S4.34 Laurel wilt is an emerging disease of trees in the family Lauraceae characterized by systemic wilt and rapid mortality. It is caused by the fungus Raffaelea lauricola and its vector, the redbay ambrosia beetle (Xyleborus glabratus) that was likely introduced to the southeastern United States from Asia. The origin of the US introduction and the population genetics of this devastating pathogen have not yet been characterized. To help facilitate this we developed a panel of microsatellite markers that can be multiplexed, amplify two or more loci in a single reaction, into eight PCRs. The marker set can be used to screen 11 R. lauricola isolates per 96 well PCR plate, eliminating the need to combine samples post amplification. The marker set reduces cost by using conventional hot start Taq, 15ul reaction volumes, multiplexing PCRs and fluorescein-12-dUTP to label PCR amplicons instead of fluorescently labeled primers. Future work will utilize the microsatellite marker set to investigate the genetic structure of R. lauricola within both the US and Asian populations and generate information regarding the number of introductions, possible origins of the introductions into the US and potential for development of new strains. Genome and transcriptome analysis of Phellinus noxius, a wood decay fungus H. LEE (1), C. Chen (2), I. Tsai (3), R. Liou (2), C. Chung (2) (1) National Taiwan University, Taiwan; (2) National Taiwan University, Taiwan; (3) Genome and transcriptome analysis of Phellinus noxius, a wood decay fungus H. LEE (1), C. Chen (2), I. Tsai (3), R. Liou (2), C. Chung (2) (1) National Taiwan University, Taiwan; (2) National Taiwan University, Taiwan; (3) Academia Sinica, Taiwan Phellinus noxius, which causes brown root rot disease in more than 200 tree species, has recently gained much attention in East Asia. However, little is known about the genome of P. noxius. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. In this study, four mate-pair libraries of a P. noxius basidiospore isolate A42 were sequenced by using Illumina platform. Genome assembly and ab intio gene prediction resulted in a draft genome of ~32 Mb (N50 = 1.55 Mb) which includes 11,025 coding sequences. Both A and B mating type loci were identified. Sequence alignment of A42 and a Japanese isolate showed high similarity in B locus and one of the two HD1-HD2 pairs in A locus. Sequence analysis of the B locus of 12 basidiospore isolates from a single basidiocarp showed no polymorphism, which suggests a bipolar mating system. To facilitate gene annotation and to better understand the expression profiles of genes involved in stress responses, transcriptomes under various stress conditions were examined, encompassing temperature shift and nutrient deficiency (minimal medium, carbon deficiency, nitrogen deficiency). Genes involved in nutrient metabolism, secondary metabolites biosynthesis, hydrophobin production, and stress responses were induced. A particularly up-regulated pattern was found in the glyoxylate metabolism pathway which produces oxalate, an organic acid known to facilitate the activity of cell-wall degrading enzymes, as a byproduct. Investigations of the stress-related genes in disease development are underway. Ganoderma Species Associated with Declining or Dead Trees in the Southeastern United States A. LOYD (1), J. Smith (2), R. Blanchette (3), B. Held (3), C. Barnes (4), M. Schink (5) (1) Bartlett Tree Experts/University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) University of Minnesota, U.S.A.; (4) Departamento Nacional de Proteccion Vegetal, INAP, Ecuador; (5) Self, Port Crane, NY, U.S.A. Ganoderma is a large and diverse genus of wood decay fungi that can cause a white rot of the lower bole or roots of hardwood, conifer and palm trees. The pathogenicity and decay ability within the genus is understudied. Some, such as G. zonatum are aggressive pathogens, and have been associated with tree failure and mortality of mature palms, while others, such as G. applanatum s.l., are often saprophytic causing decay in old, weakened tress, but can also be facultative pathogens. Ganoderma species identification tools are needed to assist arborists with predicting tree failure when fruiting bodies of Ganoderma are present. Based on a preliminary survey of Ganoderma species found in Southeastern United States, over 100 isolates representing different taxa were collected from living tree species and dead wood across 4 states: Florida, Georgia, South Carolina and North Carolina. Taxa investigated in the survey were the G. A real-time PCR and digital droplet PCR assay for quantification of Polymyxa betae in sugar beet roots B. SMITH (1), F. Martin (1) (1) USDA-ARS, U.S.A. Jiménez (2) (1) Universidad de los Andes, Colombia; (2) Universidad Militar Nueva Granada, Colombia Colletotrichum species are one of the main problems in crop production in Colombia. Several research efforts have been devoted to characterize the populations of this fungus. However, the phylogenetic relationships among species within this genus are still uncertain. Colletotrichum species have been traditionally classified using morphological traits and based on their host, despite the fact that the host range of these species is still unclear. In Colombia, the phylogenetic relationships of Colletotrichum species have been determined using molecular markers as well as morphological and physiological characteristics. Preliminary results have suggested host specificity among the species assessed. However, this assays were conducted by directly damaging the plant tissue. The aim of this study was to investigate host-pathogen interactions among isolates collected from tree tomato and mango from the pre-penetrating up to the colonization stages of the pathogen. Isolates were thoroughly characterized molecularly and microscopically and were subsequently inoculated onto both hosts. Strains of C. gloeosporioides, C. asianum, C. theobromicola, and C. tamarilloi were inoculated on whole-plants and detached-leaves. Pathogenicity, latent period, aggressiveness, and sporulation were recorded to evaluate the fitness of each species on the different hosts. Preliminary results indicate the presence of host preferences among the different species of Colletotrichum assessed. Arbuscular mycorrhizal fungal communities associated with organic and conventional onion crops in the Columbia Basin of Washington L. DU TOIT (1), A. Knerr (2), D. Wheeler (2), D. Schlatter (3) (1) W hi S U i i U S A (2) W hi S U i i U S A (3) USDA ARS U S A Evaluating isolate aggressiveness of Fusarium oxysporum f.sp. niveum in Florida T. SANCHEZ (1) (1) University of Florida, U.S.A. Fusarium wilt, caused by Fusarium oxysporum f. sp. niveum, is a major soil borne disease of watermelon with limited host resistance available. Understanding isolate aggressiveness is important for variety selection and other management strategies. Pathogenicity tests of 72 isolates collected from 10 Florida counties were conducted to examine their aggressiveness on the variety Black Diamond under greenhouse settings. Seedlings were inoculated with a conidial suspension using a root dip method. Aggressiveness was measured by the percentage of wilted plants and AUDPC for incidence, over a 4 week period after inoculation. Significant (P<0.05) differences between isolates were found using ANOVA. Based on wilted plants, isolates were characterized into 4 aggressiveness levels; not aggressive, weak, medium and severe (0%, 1-32%, 33-67% and 68-100% wilt respectively). It was observed that 33.3% of the isolates were not aggressive, 15.3% weak, 25.0% medium, and 26.4% severe. Quantitative diversity indices (Simpson’s = 0.27 and Shannon entropy H’ = 1.35) indicate that these aggressiveness levels are highly diverse in Florida, and that no single level was dominant. Characterizing the aggressiveness is a necessary step for race determination, as well as the assessment of various integrated management strategies. Further research is presently being conducted that evaluates the efficacy of current integrated management strategies on isolates with a severe aggressiveness rating. Characterization of fungi associated with tomato leaf mold in the United States L. GARBER (1), N. LeBlanc (2), A. Orshinsky (1), C. Smart (3) (1) University of Minnesota, U.S.A.; (2) University of Minnesota, U.S.A.; (3) Cornell Tomato leaf mold is one of the most prevalent diseases affecting high tunnel-grown tomatoes in the USA. Disease symptoms include chlorotic to necrotic lesions, defoliation and decreased yields. Previous studies focused on the fungus, Passalora fulva (syn: Cladosporium fulvum, Fulvia fulva) as the causal pathogen of leaf mold. Our initial preliminary data suggests that sporulation on lesions is associated with a number of Cladosporium spp., primarily C. cladosporioides and C. pseudocladosporioides. Leaf mold-associated fungi were collected from MN and NY by surface sterilization of symptomatic tissue and by direct plating of spores from lesions. Single spore isolates were generated and PCR amplification of internal transcribed spacer regions and alpha-actin were performed. Amplicons were sequenced and aligned with available reference species sequences obtained from GenBank using Seaview. A majority of leaf mold isolates collected fell within the C. cladosporioides complex. Representative isolates from each Cladosporium spp. did not appear to be pathogenic. Fungicide resistance and host susceptibility of Colletotrichum orbiculare infecting cucurbit crops in North Carolina N NOËL (1) L Quesada-Ocampo (1) Fungicide resistance and host susceptibility of Colletotrichum orbiculare infecting cucurbit crops in North Carolina N. NOËL (1), L. Quesada-Ocampo (1) (1) NCSU, U.S.A. Fungicide resistance and host susceptibility of Colletotrichum orbiculare infecting cucurbit crops in North Carolina Ë Cucurbit anthracnose, caused by the ascomycete Colletotrichum orbiculare syn. lagenarium, is a widespread fruit and foliar disease that results in yield losses across the United States. In North Carolina, favorable environmental conditions, host availability, and long growing seasons offer ample opportunity for disease development, resulting in significant economic losses. Application of effective fungicides and planting of resistant cultivars, along with appropriate cultural practices, have proven effective in reducing anthracnose. However, the appearance of new races of the pathogen and development of fungicide resistance has limited the efficacy of these control strategies. The objective of this study is to characterize C. orbiculare populations infecting cucurbit crops in North Carolina, determine their sensitivity to fungicides, and their virulence in watermelon cultivars. Isolates obtained from cucurbit fields in North Carolina and elsewhere for comparison, where evaluated for fungicide sensitivity in vitro with several fungicides and various concentrations. Local isolates of different races were used to evaluate host susceptibility of 24 popular cultivars of watermelon (Citrullus lanatus) in field and greenhouse trials. Results indicate that an integrated disease management strategy that combines effective fungicides and host tolerance will be required to control cucurbit anthracnose in North Carolina. Arbuscular mycorrhizal fungal communities associated with organic and conventional onion crops in the Columbia Basin of Washington L. DU TOIT (1), A. Knerr (2), D. Wheeler (2), D. Schlatter (3) (1) W hi S U i i U S A (2) W hi S U i i U S A (3) USDA ARS U S A Arbuscular mycorrhizal fungal communities associated with organic and conventional onion crops in the Columbia Basin of Washington L. DU TOIT (1), A. Knerr (2), D. Wheeler (2), D. Schlatter (3) (1) Washington State University, U.S.A.; (2) Washington State University, U.S.A.; (3) USDA ARS, U.S.A. Arbuscular mycorrhizal fungal communities associated with organic and conventional onion crops in the Columbia B L. DU TOIT (1), A. Knerr (2), D. Wheeler (2), D. Schlatter (3) ( ) ( ) ( ) ( ) (1) Washington State University, U.S.A.; (2) Washington State University, U.S.A.; (3) USDA ARS, U.S.A. Onion mycorrhizal associations were assessed in conventional vs. organic fields in the Columbia Basin of the Pacific Northwest USA in 2014. Soil was sampled from five sites in each of four conventional and five organic fields prior to planting in March. Arbuscular mycorrhizal fungi (AMF) were baited from each soil by planting seed of the onion cv. Tamara. Plants also were sampled from each field at bulb initiation (mid-June). Onion roots were stained S4.35 to quantify mycorrhizal colonization, and DNA was extracted for 454 pyrosequencing of AMF communities using 18S rDNA and Glomerales-specific primers. AMF were prevalent in all fields, with no significant differences in colonization of baited roots in soil samples from organic vs. conventional fields (mean ± SE of 77 ± 4 vs. 75 ± 4%, respectively). Baited AMF communities from spring soil samples differed in community structure in conventional vs. organic fields (PERMANOVA Pseudo-F = 4.08, P = 0.02), though AMF richness and evenness did not differ significantly (t-tests and Welch-Satterthwaite t-test P ≥ 0.2). AMF communities in mid-summer root samples also differed in community structure (PERMANOVA Pseudo-F = 7.92, P = 0.0005), with greater richness (Welch-Satterthwaite t-test P = 0.02) but similar evenness in organic vs. conventional fields. Differences in AMF communities between organic and conventional fields were attributed mostly to differences in relative abundance of Glomus and Claroideoglomus spp. to quantify mycorrhizal colonization, and DNA was extracted for 454 pyrosequencing of AMF communities using 18S rDNA and Glomerales-specific primers. AMF were prevalent in all fields, with no significant differences in colonization of baited roots in soil samples from organic vs. conventional fields (mean ± SE of 77 ± 4 vs. 75 ± 4%, respectively). Baited AMF communities from spring soil samples differed in community structure in conventional vs. Arbuscular mycorrhizal fungal communities associated with organic and conventional onion crops in the Columbia Basin of Washington L. DU TOIT (1), A. Knerr (2), D. Wheeler (2), D. Schlatter (3) (1) W hi S U i i U S A (2) W hi S U i i U S A (3) USDA ARS U S A organic fields (PERMANOVA Pseudo-F = 4.08, P = 0.02), though AMF richness and evenness did not differ significantly (t-tests and Welch-Satterthwaite t-test P ≥ 0.2). AMF communities in mid-summer root samples also differed in community structure (PERMANOVA Pseudo-F = 7.92, P = 0.0005), with greater richness (Welch-Satterthwaite t-test P = 0.02) but similar evenness in organic vs. conventional fields. Differences in AMF communities between organic and conventional fields were attributed mostly to differences in relative abundance of Glomus and Claroideoglomus spp. Determining infection courts for Fusarium oxysporum f. sp. niveum leading to watermelon seed infestation A. PETKAR (1), P. Ji (1) (1) University of Georgia, U.S.A. Determining infection courts for Fusarium oxysporum f. sp. niveum leading to watermelon seed infestation A PETKAR (1) P Ji (1) Determining infection courts for Fusarium oxysporum f. sp. niveum leading to watermelon seed infestation A. PETKAR (1), P. Ji (1) Determining infection courts for Fusarium oxysporum f. sp. niveum leading to watermelon seed infestation A. PETKAR (1), P. Ji (1) (1) University of Georgia, U.S.A. Fusarium wilt of watermelon caused by Fusarium oxysporum f. sp. niveum (FON) is a seedborne and seed transmitted disease. Seeds latently infected by the pathogen serve as a significant inoculum source and may result in serious disease in the field. It is unknown how watermelon seeds can get infected by the pathogen. To determine potential infection courts leading to watermelon seed infestation, experiments were conducted in 2013 to 2015 using different methods to inoculate watermelon plants with FON. Watermelon seeds were harvested when mature and seed infestation by FON was determined by isolation on peptone PCNB agar and real-time polymerase chain reaction (PCR) analysis. Swab-inoculation of immature fruit resulted in infestation of 18 to 54% of the seedlots under field conditions and 0.5 to 13% under greenhouse conditions when seeds were not surface sterilized before isolation. Seed infestation was detected on 0 to 15% of the seedlots when seeds were surface sterilized before isolation. Inoculation of stigma of female flowers resulted in infestation of 0 to 14% of the seedlots. Inoculation of peduncle also resulted in infestation of 0 to 10% of the seedlots in the field experiments when seeds were not surface sterilized before isolation. The results suggest that watermelon tissues including immature fruit, flowers and peduncle could be potential infection courts of FON leading to seed infestation by the pathogen. Evaluating isolate aggressiveness of Fusarium oxysporum f.sp. niveum in Florida T. SANCHEZ (1) 1) University of Florida U S A Evaluating isolate aggressiveness of Fusarium oxysporum f.sp. niveum in Florida T. SANCHEZ (1) (1) University of Florida, U.S.A. Willis (2) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Greenhouse Evaluation of Dosage Responses of the Tomato I3 Gene to Fusarium oxysporum f. sp. lycopersici race 3 C. LAND (1), C. Land (2), G. Vallad (2), S. Hutton (2), R. Willis (2) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Fusarium oxysporum f. sp. lycopersici (FOL) causes an aggressive vascular wilt disease on tomato. The goal of this study was to evaluate increasing soil levels of FOL on tomato plants with and without the I3 gene conferring resistance to race 3 FOL isolates. Soil inoculum of a race 3 FOL isolate was produced in a corn meal:sand medium and diluted with a pasteurized field soil to levels of 104 to 101 cfu/g soil. Tomato seedlings with and without the I3 gene were transplanted to pots filled with 6 levels of infested soil, including a non-infested soil, and periodically evaluated for plant height and disease severity over 2 months. Afterwards, plants were uprooted, shoot fresh weights were measured, and roots and hypocotyls were plated onto Komada’s medium. A significant difference in total biomass was observed between I3 homozygous and heterozygous plants, with I3 homozygous plants exhibiting 20% greater growth compared to susceptible plants regardless of the initial level of soil inoculum. Plants grown in non-infested soils were symptomless, exhibiting either undetectable or very low levels of FOL recovery from the hypocotyls. Heterozygous I3 plants grown in 10 cfu/g of FOL infested soil exhibited 70% hypocotyl infection, which was the highest rate of vascular infection of all treatments. Fusarium oxysporum f.sp. lycopersici: Small Chromosomes Define Friend vs Foe K. FENSTERMACHER (1) (1) Penn State University, U.S.A. Fusarium oxysporum f.sp. lycopersici: Small Chromosomes Define Friend vs Foe K. FENSTERMACHER (1) Fusarium is a genus of filamentous fungi well-known for causing persistent wilt diseases on almost all cultivated crops. The Fusarium oxysporum Species Complex has no known sexual stage, yet is diverse and contains over 120 formae speciales that cause disease on different plant hosts. In 2010, Ma et al. sequenced F. oxysporum f.sp. lycopersici (Fol) to find virulence loci of differing GC content throughout the genome, including small, conditionally dispensable chromosomes (CDCs) that do not match known Fusarium sequences, contain genes with no known homologs, can convey pathogenicity when transferred from a pathogenic strain to a non-pathogenic relative—making them functionally similar to virulence plasmids in bacteria. Evaluating isolate aggressiveness of Fusarium oxysporum f.sp. niveum in Florida T. SANCHEZ (1) (1) University of Florida, U.S.A. CDCs could explain the high level of diversity observed in a microbe with no sexual recombination, and the inconsistent number and sizes of chromosomes consistently found in these plant pathogens. This project sampled over 40 sites, with and without known cases of Fol, to examine 1) the prevalence of CDCs where Fol is found, 2) the variation among CDCs and pathogen phenotypes, and 3) the variation within genetic backgrounds harboring different CDCs. This data will help determine the roles of CDCs in the context of the populations and growing systems in which they are found, to analyze the potential of previous or future horizontal chromosome transfer events under various conditions, and analyze the risk factors and effects on agricultural systems. Root knot nematode effects on metabolic profiles of susceptible and resistant grapevine rootstocks C. Wallis (1) (1) USDA-ARS Root knot nematodes (Meloidogyne spp.) can negatively impact newly planted and stressed vineyards. Nematode infestations also may increase grapevine susceptibility to other stresses such as water deficit or various diseases. However, little is known about direct or indirect effects of nematode feeding on grapevine physiology, or potential underlying mechanisms that impart resistance to certain rootstock cultivars. Therefore, this study assessed metabolite profiles in Cabernet Sauvignon grapevines grafted to either susceptible (O39-16) or resistant (Freedom) rootstocks established in root knot nematode-infested soil or steam-sterilized soil. Grapevines of both cultivars grown in soil infested with nematodes had reduced fructose and glucose levels compared to control grapevines grown in non-infested soil. Phenolic profiles in resistant grapevines did not differ due to exposure to nematodes, and only a few phenolic compounds increased in susceptible grapevines due to nematode feeding. Regardless of the presence of nematodes, root phenolic profiles between resistant and susceptible rootstocks were dissimilar, with different individual compounds more prevalent in one cultivar than the other. Taken together, these results demonstrated that nematode infestations altered root physiology, and that resistant grapevines expressed phenolic profiles different from susceptible grapevines. Evaluating isolate aggressiveness of Fusarium oxysporum f.sp. niveum in Florida T. SANCHEZ (1) (1) University of Florida, U.S.A. Saparrat (3) (1) Facultad de Ciencias Agrarias y Forestales, La Plata, Argentina; (2) Centro de Investigaciones de Fitopatopatologia Facultad de Ciencias Agrarias y Forestales UNLP, La Plata, Argentina; (3) Catedra de Microbiologia -INFIVE Facultad de Ciencias Agrarias y Forestales UNLP, La Plata, Argentina In silico screening of genes coding for secondary metabolites in the phytopathogenic fungus Stemphylium lycopersici P. BALATTI (1), E. Franco (2), S. Lpez (2), M. Saparrat (3) (1) Facultad de Ciencias Agrarias y Forestales, La Plata, Argentina; (2) Centro de Investigaciones de Fitopatopatologia Facultad de Ciencias Agrarias y Forestales UNLP, La Plata, Argentina; (3) Catedra de Microbiologia -INFIVE Facultad de Ciencias Agrarias y Forestales UNLP, La Plata, Argentina ( ), ( ), p ( ), p ( ) (1) Facultad de Ciencias Agrarias y Forestales, La Plata, Argentina; (2) Centro de Investigaciones de Fitopatopatologia Facultad de Ciencias Agrarias y Forestales UNLP, La Plata, Argentina; (3) Catedra de Microbiologia -INFIVE Facultad de Ciencias Agrarias y Forestales UNLP, La Plata, Argentina Secondary metabolites (SMs) are a highly diverse set of low molecular weight bioactive compounds that play different biological roles. They are dispensable when microorganism are cultivated in vitro, but usually play a key role in nature, providing organisms with adaptive advantages in different niches. Among them, toxins produced by phytopathogenic fungi play crucial roles in virulence and host nutrition. We hypothesized that the genome of Stemphylium lycopersici strain CIDEFI-216, an etiological agent of tomato gray leaf spot, contains several gene clusters that code for the synthesis of SMs, such as those involved in the biosynthesis of virulence factors. We searched by means of the bioinformatic tools Secondary Metabolite Unknown Region Finder (SMURF) and Antibiotics and Secondary Metabolite Analysis Shell (antiSMASH) enabling the ClusterFinder algorithm for potential SMs gene clusters within the draft genome sequence of the fungus. This lead to the prediction of 32, 33 and 64 additional SMs gene clusters by SMURF, antiSMASH and ClusterFinder, respectively. They included polyketides synthase (PKS), non-ribosomal peptide synthase (NRPS), terpene synthase, dimethylallyl tryptophan synthase, hybrid PKS-NRPS, lanthipeptide synthase, fatty acid synthase and others gene clusters of unknown nature. This knowledge provides critical information to understand the biological bases of the tomato-S. lycopersici interaction. Greenhouse Evaluation of Dosage Responses of the Tomato I3 Gene to Fusarium oxysporum f. sp. lycopersici race 3 C. LAND (1), C. Land (2), G. Vallad (2), S. Hutton (2), R. Evaluating isolate aggressiveness of Fusarium oxysporum f.sp. niveum in Florida T. SANCHEZ (1) (1) University of Florida, U.S.A. The frequency with which C. cladosporioides and C. pseudocladosporioides are associated with leaf mold lesions suggests that they may be important in the epidemiology and etiology of the disease. Studies regarding the association between C. cladosporioides, C. pseudocladosporiodes and leaf mold of tomatoes are ongoing and may lead to the development of unique disease management strategies in high-tunnel tomatoes. Mitochondrial and nuclear gene sequences to infer the phylogeny of Pezizomycotina (Ascomycota) E. Franco (1), S. Lopez (1), M. Saparrat (2), P. BALATTI (1) (1) Facultad de Ciencias Agrarias y Forestales, Argentina; (2) Faculad de Ciencias Agrarias y Forestales, Argentina Mitochondrial and nuclear gene sequences to infer the phylogeny of Pezizomycotina (Ascomycota) E. Franco (1), S. Lopez (1), M. Saparrat (2), P. BALATTI (1) Pezizomycotina is the largest subphylum of Ascomycota. It includes filamentous species widespread in nature, where they live as saprotrophs, mutualists and/or parasites of animal, plants and fungi. We evaluated the phylogeny of such an important subphylum not only by analyzing the sequence of nuclear genes but also of the core mitochondrial ones, since this organelle may provide additional information. Hence, we compared the phylogenetic reconstitution provided by the concatenated nucleotide sequences of 12 core mitochondrial genes (atp6, cob, cox1-3, nad1-6 and nad4L) and the individual and concatenated nucleotide sequences of 5 widely used nuclear genes (β-tub, tef-1α, gpd, rpb1 and rpb2) of 28 Pezizomycotina species. Alignments were generated with ClustalW and automatically curated with Gblocks 0.91b. Best-fit model of evolution was selected with jModelTest S4.36 2.1.7 and data matrices were analyzed under maximum likelihood and bayesian criteria in PhyML 3.0 and MrBayes 3.2, respectively. We found that only rpb2 and the concatenated nuclear and mitochondrial datasets clustered species in accordance with the current systematic of this subphylum, although incongruences were observed in some internal nodes. Discrepancies between the remaining datasets are indicative of the complex evolutionary histories and confirmed that the genes for an accurate phylogenetic estimation should be chosen with special care. In silico screening of genes coding for secondary metabolites in the phytopathogenic fungus Stemphylium lycopersici P. BALATTI (1), E. Franco (2), S. Lpez (2), M. Influence of root exudates and soil on attachment of Pasteuria penetrans to Meloidogyne arenaria C. LIU (1), P. Timper (2) Chen (2) (1) Department of Plant Biology, University of Minnesota, U.S.A.; (2) Southern Research and Outreach Center, University of M Comparison of ITS1 versus ITS2 regions for characterizing fungal communities colonizing soybean cyst nematode W. HU (1), K. Bushley (1), S. Chen (2) Comparison of ITS1 versus ITS2 regions for characterizing fungal communities colonizing soybean cyst nematode W. HU (1), K. Bushley (1), S. Chen (2) (1) Department of Plant Biology, University of Minnesota, U.S.A.; (2) Southern Research and Outreach Center, University of Minnesota, U.S.A. W. HU (1), K. Bushley (1), S. Chen (2) (1) Department of Plant Biology, University of Minnesota, U.S.A.; (2) Southern Research and Outreach Center, University of Minnesota, U.S.A. . Chen (2) ogy, University of Minnesota, U.S.A.; (2) Southern Research and Outreach Center, University of Minnesota, U.S.A. y ( ), ( ) nt of Plant Biology, University of Minnesota, U.S.A.; (2) Southern Research and Outreach Center, University of Minnesota ( ), y ( ), ( ) 1) Department of Plant Biology, University of Minnesota, U.S.A.; (2) Southern Research and Outreach Center, University o The development of next generation sequencing has enabled investigation of microbial communities from a wide range of environmental samples. We examined fungal communities associated with cysts of soybean cyst nematode (SCN) to understand the effects of different crop rotation practices. The primers most frequently used for fungal communities on the Illumina Miseq platform target either the rRNA internal transcribed spacer 1 region (ITS1) or the ITS2 region. In order to assess which primers are better for amplifying fungal communities in SCN cysts, we performed 300 bp paired-end sequencing on two Miseq runs using fungal ITS1f/ITS2r and 5.8SR/ITS4 primers. Cysts of the SCN were collected from a soybean-corn rotation field in Waseca, MN, and washed with 0.2% Tween™ 20. The DNA was extracted from 50 cysts using the CTAB method, and normalized for PCR reactions. After quality control, 361224 (ITS1) and 230404 (ITS2) reads were used for OTU picking in QIIME. OTUs were blasted against the UNITE database with the default setting in QIIME. About 99% of the ITS1 region sequences were classified as fungal sequences, however, 99% of the ITS2 region sequences were not fungal sequences. In addition, significant changes in the relative abundance of some taxa was observed between rotation treatments for only the ITS1 sequences. Influence of root exudates and soil on attachment of Pasteuria penetrans to Meloidogyne arenaria C. LIU (1), P. Timper (2) Pasteuria penetrans is a widely distributed endospore-forming bacterium which is a hyperparasite of Meloidogyne spp. Previous research showed that root exudates can reduce attachment of P. penetrans to Meloidogyne arenaria. The objective of this study was to determine if soil microorganisms modify the effect of root exudates on attachment of P. penetrans spores to M. arenaria. The following treatments were set up in both sand and clay soil: natural soil with a plant, sterilized soil with a plant, and natural soil without plant. For treatments with a plant, 1-month-old eggplant seedlings were used. All treatments were incubated in greenhouse for 3 days, and soil solutions were obtained by vacuum filtering saturated soil. Controls included phosphate- buffered saline (PBS) as well as root exudates in water. Second stage juveniles (J2) were incubated in 5 ml of different solutions for 6 h, rinsed and incubated in PBS for another 6 h with 105 P. penetrans spores. Compared to J2 incubated in PBS, attachment was significantly reduced when root exudates were present, with root exudates having a greater effect on attachment in sand than in clay. Spore attachment was always higher in clay than in S4.37 sand solutions. Root exudates reduced spore attachment more in sterilized soil than in natural soil. The presence of microorganism and chemical composition of different soils may interact with root exudates to affect endospore attachment. sand solutions. Root exudates reduced spore attachment more in sterilized soil than in natural soil. The presence of microorganism and chemical composition of different soils may interact with root exudates to affect endospore attachment. Soybean Cyst Nematodes of Ohio: Deciphering mechanisms of virulence E. WALSH (1), T. Miller (2), A. Grenell (3), B. Cassone (4), C. Taylor (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A.; (3) The College of Wooster, U.S.A.; (4) Brandon University, Canada rsity, U.S.A.; (2) The Ohio State University, U.S.A.; (3) The College of Wooster, U.S.A.; (4) Brandon University, Canada Soybean cyst nematodes (SCN; Heterodera glycines) are the most yield-reducing pest of soybean in North America. Resistant soybean cultivars are widely utilized for management purposes, however SCN populations are quickly adapting to overused sources of resistance. Counties across Ohio were surveyed for SCN populations and their virulence against the available sources of resistance was tested with HG Type assays. Influence of root exudates and soil on attachment of Pasteuria penetrans to Meloidogyne arenaria C. LIU (1), P. Timper (2) We conclude that primers targeting the fungal ITS1 region rather than ITS2 region are more suitable for characterizing fungal communities in SCN cysts. Gossypium arboreum accessions resistant to Rotylenchulus reniformis identified S. STETINA (1), J. Erpelding (1) (1) USDA, Agricultural Research Service, U.S.A. Reniform nematode (Rotylenchulus reniformis) is a serious pest of upland cotton (Gossypium hirsutum) in the southeastern United States. Resistance does not exist naturally in G. hirsutum and no resistant cultivars are available. To identify sources of reniform nematode resistance, 225 G. arboreum accessions were evaluated in repeated growth chamber experiments. Root infection was measured on 3 plants 4 weeks after inoculation with 1,000 nematodes. The 16 accessions supporting the fewest infections were evaluated again in confirmation tests lasting 8 weeks. The numbers of nematodes extracted from soil and eggs extracted from roots of 5 plants were combined and totals were analyzed (ANOVA and differences of least squares means, P ≤ 0.05). Susceptible controls G. hirsutum cv Deltapine 16 and G. arboreum A2-101 had 85,999 and 46,902 reniform nematodes, respectively; the resistant control G. arboreum A2-190 had 1,665 reniform nematodes, and only 136 reniform nematodes remained in fallow pots. All 16 accessions tested supported smaller reniform nematode populations than the susceptible controls, ranging from 10,883 to 707 individuals. Nine accessions (A2-254, A2- 272B, A2-294, A2-362, A2-456, A2-497, A2-514, A2-558, and A2-665) supported reniform nematode populations comparable to the resistant control, and accession A2-354 had fewer reniform nematodes than the resistant control. Movement of Fluopyram in Sandy Soil to Affect Meloidogyne incognita Motility T. FASKE (1), K. Hurd (2) (1) University of Arkansas, Cooperative Extension Service, U.S.A.; (2) University of Arkansas, Cooperative Extension Service, U.S.A. Influence of root exudates and soil on attachment of Pasteuria penetrans to Meloidogyne arenaria C. LIU (1), P. Timper (2) Of the populations tested to date, 96% are infective on PI88788, the most prevalent source of resistance in commercial varieties. The objective of this study was to determine how SCN populations have overcome this source of resistance. Nine SCN populations from across Ohio were selected, which vary in their HG Type and/or level of virulence. These populations were grown on both PI88788 and a susceptible cultivar, Lee 74. Female SCN were harvested 15 days after inoculation with juvenile worms, total RNA was extracted, and submitted for sequencing on the Illumina Hi-Seq 2500. Interesting expression patterns were found common among virulent populations such as putative effectors and genes related to digestion, the immune response, and detoxification. Also, populations avirulent on PI88788 share the expression of an effector not present in virulent populations, indicating that the loss of this effector may play a role in virulence to this resistance source. Further work includes cloning identified candidate genes to determine their role in this interaction. Soybean cyst nematode (SCN; Heterodera glycines) has a significant impact on the soybean industry, costing $90 million dollars annually. SCN has been managed mostly through the use of resistant cultivars and non-host crop rotations. Recently, resistance-breaking populations of SCN have made the use of SCN-resistant cultivars far less effective in controlling this nematode. Reliance on crop rotation has now become significantly more important for SCN control. However, little is known about how widely used non-host crops can impact SCN populations. Previous reports suggest that SCN numbers can be reduced during rotations with maize but the mode of action in this interaction is unknown. This study aimed to identify parental lines from the maize Nested Association Mapping population that are most effective at decreasing SCN populations through the production of allelopathic chemicals in root exudates. Field experiments were established using a simulated crop rotation scheme with maize and soybean. When exposed to developing maize seedlings, nematodes showed altered infection rates on soybean. A controlled experiment to complement field data collected showed significant differences in cyst production after pretreatment with maize lines. Soybean growers will ultimately directly benefit from this research by gaining knowledge on biochemical characteristics that can improve non-host selection and aid in further reducing SCN nematode populations in infested fields. Comparison of ITS1 versus ITS2 regions for characterizing fungal communities colonizing soybean cyst nematode W. HU (1), K. Bushley (1), S. Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by viruses C RUARK (1) S Koenning (2) M Mitchum (3) E Davis (2) C Opperman (2) S Lommel (2) T Sit (2) Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by viruses Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by viruses C. RUARK (1), S. Koenning (2), M. Mitchum (3), E. Davis (2), C. Opperman (2), S. Lommel (2), T. Sit (2) (1) North Carolina State University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) University of Missouri, U.S.A. C. RUARK (1), S. Koenning (2), M. Mitchum (3), E. Davis (2), C. Opperman (2), S. Lommel (2), T. Sit (2) (1) North Carolina State University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) University of Missou C. RUARK (1), S. Koenning (2), M. Mitchum (3), E. Davis (2), C. Opperman (2), S. Lommel (2), T. Sit (2) (1) North Carolina State University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) University of Mis Five viruses were previously discovered within Illinois greenhouse cultures of soybean cyst nematode (SCN; Heterodera glycines). In this study, the five viruses have been identified in SCN greenhouse and field populations from North Carolina (NC) and Missouri (MO). The prevalence and titers of viruses in SCN were examined using qRT-PCR in 47 greenhouse cultures and 25 field populations [20 from NC and 5 from MO]. Viral titers within SCN greenhouse cultures were similar throughout juvenile development, and the presence of viral anti-genomic RNAs within egg, second-stage juvenile (J2), and pooled J3 and J4 stages suggests active viral replication within the nematode. Overall, viruses were detected more frequently and at higher titers within greenhouse cultures versus field populations. Five greenhouse cultures harbored all five known viruses whereas in most populations a mix of viruses was detected. In contrast, three greenhouse cultures of similar descent to one another did not possess any detectable viruses and primarily differed in location of the cultures (NC versus MO). Furthermore, several of these SCN viruses were also present in the related nematodes species Heterodera trifolii (clover cyst) and Heterodera schachtii (beet cyst). Movement of Fluopyram in Sandy Soil to Affect Meloidogyne incognita Motility T FASKE (1) K Hurd (2) ), K. Hurd (2) y of Arkansas, Cooperative Extension Service, U.S.A.; (2) University of Arkansas, Cooperative Extension Service, U.S.A. Fluopyram is nematicidal to Meloidogyne incognita. It has limited xylem movement, thus direct contact is important for nematode suppression; however, the effective movement of fluopyram in sandy soil is unknown. The objective of this study was to determine the movement of fluopyram from treated seed and a water dilution in sandy soil. Treatments consisted of 0.15 mg abamectin, 0.25 mg fluopyram, and 0.75 mg thiodicarb + imidacloprid treated cotton seed, while water dilutions of 25 µg abamectin and 25 µg fluopyram were used in the second experiment. Each treatment was placed on the surface of the soil column and flushed with 500 µl of water. The soil was removed after 24 h from three, 5-cm long segments and mixed with an equal volume of water. A portion of the supernatant was placed into a well, which contained 30-40 J2 in 500 µl of water. In the first experiment, a higher percentage of immotile J2 were observed from fluopyram collected in the 0-5 and 5-10 cm segments than abamectin. No nematode immotility was observed with thiodicarb or water control. In the second experiment, a higher percentage of immotile J2 were observed in the 5-10 cm segment from the water dilution of abamectin and fluopyram compared to the treated seed. No nematode immotility was observed past the 10-cm depth for either nematicide. The effective movement of fluopyram in sandy soil within 24 h was limited to the upper 10-cm of soil, which was similar to that of abamectin. S4.38 Root Knot Nematode (Meloidogyne incognita) populations on eggplant could be reduced using Marigold extracts A. HAMEED (1), N. Liaqat (2), K. Riaz (2), M. Alam (2), S. Sarfraz (2), S. Jameel (2) (1) University of Agriculture, Faisalabad, Pakistan; (2) University of Agriculture Faisalabad, 38040-Pakistan, Pakistan Root knot nematode diseases are gaining importance in vegetables in Pakistan. Chemical pesticides are often hazardous and are discouraged due to their non-targeted effects. Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by Baas (2), B. Werling (3), H. Melakeberhan (1) (1) Michigan State University Department of Horticulture, East Lansing, MI, U.S.A.; (2) Michigan State University Extension-St. Joseph County, Centreville, MI, U.S.A.; (3) Michigan State University Extension-Oceana County, Hart, MI, U.S.A. Cover crops are often used for reducing soil erosion, retaining soil nutrients, and building organic matter. Free-living nematodes (nematodes that do not feed on plants) are well-established bioindicators and may respond to changes in soil ecology due to cover cropping. Some cover crop cultivars are also marketed for managing plant-parasitic nematodes. This study assessed the impact of common Michigan cover crops on plant-parasitic and free-living nematodes in carrot production systems. Research was conducted at 4 sites in Michigan where cover crops were grown in the fall preceding summer carrot production. Oats, ‘Defender’ radish, dwarf essex rape, a mixture of oats and ‘Defender’ radish, and a fallow control were treatments at each site. Black oats and ‘Graza’ radish were also grown at sites 3 and 4 while ‘Image’ radish was grown only at site 4. Lesion nematode (Pratylenchus sp.) was present at all four sites. At sites 1, 3, and 4, lesion nematode densities were relatively small (less than 20 nematodes/100 cm3 soil) and not significantly (P > 0.05) affected by cover crops. At site 2, during carrot production, lesion nematode density was significantly (P < 0.05) greater following ‘Defender’ radish than fallow or other cover crop treatments. Similarly, at site 2 during carrot production, free-living nematode densities were greater following ‘Defender’ radish or oats than fallow, but were not significantly affected by cover crops at the other sites. Mode of action studies on the nematicide fluensulfone P. NAVIA GINE (1) (1) 1973, Wellington, FL, U.S.A. Fluensulfone has proven efficacy in the field against a range of nematode species. The mode of action of fluensulfone is however currently unknown. Fluensulfone was found to have nematicidal activity against C. elegans but at concentrations ≥100-fold greater than those reported to be effective against the PPN Meloidogyne javanica. Fluensulfone affected a number of C. elegans behaviours, including locomotion, pharyngeal pumping, egg laying and development. The pharyngeal effects suggested that fluensulfone might influence feeding behaviour in PPNs. In PPNs, the behaviour of the stylet mouth spear is critical in feeding and host invasion. The action of fluensulfone on the stylet of the PPN Globodera pallida was therefore investigated, to validate observations made with C. elegans. Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by The pharmacology of the stylet was also investigated, using knowledge of the C. elegans pharynx as a guide. Fluensulfone stimulated stylet activity and blocked 5-HT-stimulated activity. If metabolic impairment is identified as the causative agent of fluensulfone nematicidal activity this must be further studied through metabolomics and other techniques. Movement of Fluopyram in Sandy Soil to Affect Meloidogyne incognita Motility T FASKE (1) K Hurd (2) In this study, amongst the several powder plant extracts (5 gram equivalent) of selected indigenous plants such as Marigold (Tagetes erecta), Akk (Calotropis procera), Kanair (Nerium oleander) and Neem (Azadirachta indica), the marigold extracts not only decreased the root galling index (55.1%) and number of knots per plant on root system (47.4%) in the susceptible eggplant variety (cluster king) but also increased the germination (28%) and plant vigor (32%) as compared to control plants harvested. Kanair (42.09 & 32.8%), Neem (37.56 & 28.5%) and Akk (31.51 & 21.69%) were also effective. These extracts were applied as seed treatment followed by drenching at an interval of ten days post emergence upto one month. Data was collected after two months and correlated scientifically. Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high in Soybean cyst nematode culture collections and field populations from North Carolina and Missouri reveal high incidences of infection by If nematode infection with viruses is truly more common than first considered, the potential influence on nematode biology, pathogenicity, ecology, and control warrants continued investigation. Potential of trap cropping for managing root-knot nematode B WESTERDAHL (1) Potential of trap cropping for managing root-knot nematode B. WESTERDAHL (1) Potential of trap cropping for managing root-knot nematode B. WESTERDAHL (1) (1) University of California Entomology & Nematology Department, U.S.A. ( ) (1) University of California Entomology & Nematology Department, U.S.A. ( ) (1) University of California Entomology & Nematology Department, U.S.A. Trap cropping is a nematode management technique that has been tested periodically since the late 1800’s. A susceptible host is planted and larvae of a sedentary parasitic nematode such as root-knot are induced to enter and establish a feeding site. Once this has occurred, and the female begins to mature, she is unable to leave the root. The plants are then destroyed before the life cycle of the nematode can be completed, trapping nematodes within the root. By itself, trap cropping is not likely to provide the same level of control as a fumigant nematicide, because not all nematodes are induced to enter the roots. However, the potential for loss of registration of fumigants for various environmental reasons is great enough that an IPM approach using two or more techniques in combination that will each provide partial control of the nematode population is warranted. Several years of field testing of trap crops with and without a biological nematicide for management of root-knot nematode on carrots demonstrated that trap cropping resulted in statistically significant increases in weight and number of marketable carrots. A wet fallow treatment, in which a trap crop was not planted, but the weeds that germinated following irrigation served to trap nematodes within the roots, followed by tillage two weeks later, also produced a statistically greater yield of marketable carrots than the untreated. Cover cropping affects plant-parasitic and free-living nematodes in Michigan carrot production Z. GRABAU (1), Z. Maung (1), D. Noyes (1), D. Brainard (1), D. Baas (2), B. Werling (3), H. Melakeberhan (1) (1) Michigan State University Department of Horticulture, East Lansing, MI, U.S.A.; (2) Michigan State University Extension-St. Joseph County, Centreville, MI, U.S.A.; (3) Michigan State University Extension-Oceana County, Hart, MI, U.S.A. Cover cropping affects plant-parasitic and free-living nematodes in Michigan carrot production Z. GRABAU (1), Z. Maung (1), D. Noyes (1), D. Brainard (1), D. Breadth of resistance of Phytophthora fruit rot resistant watermelon germplasm to Phytophthora capsici isolates from across United States of America Breadth of resistance of Phytophthora fruit rot resistant watermelon germplasm to Phytophthora capsici isolates from America C. KOUSIK (1), J. Ikerd (1), M. Mandal (2) (1) U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, U.S.A.; (2) ORISE Participant, U.S. Vegetable Laboratory, USDA, ARS., U.S.A. Phytophthora fruit rot of watermelon caused by Phytophthora capsici is particularly severe in southeastern U.S where optimal conditions for disease development are prevalent. We have developed Phytophthora fruit rot resistant (PFR) germplasm lines (USVL020-PFR, USVL203-PFR, USVL489-PFR and USVL782-PFR) for use in breeding programs. The breadth of resistance of these lines to 20 P. capsici isolates collected from various states and different crops was evaluated. Mature fruit of PFR lines or susceptible checks ‘Mickylee’ and ‘Sugar Baby’ were collected from plants grown in a field. Fruit were inoculated by placing a 7-mm agar plug from a 4-day-old colony of a given P. capsici isolate. Inoculated fruit were kept in a walk-in-humid chamber (>95% RH, temperature 26±2 ºC) for 5 days. Ratings on lesion diameter and visible pathogen growth were then recorded. Variability in aggressiveness among the 20 isolates on checks and some PFR lines was observed. Check cultivars were susceptible to all isolates whereas PFR lines were significantly more resistant. However, PFR lines were not immune to all isolates. Significant differences in lesion size on PFR lines for some isolates was observed. In most cases very small lesions (0.8mm) formed on PFR lines under the agar plug and on the fruit surface. Our results suggest that watermelon cultivars developed using these PFR lines as resistant sources will be able to provide resistance against Phytophthora fruit rot across many states. The Effect of Increased Soil Fertility on Seedling Disease Development of Soybean in Ohio M. EYRE (1), S. Culman (1), A. Dorrance (1) (1) The Ohio State University, U.S.A. Low or high soil fertility levels have been shown to have potential secondary impacts on root pathogens, some of which decrease yield. Fertility recommendations for soybean in Ohio are over 20 years old and one proposed scenario is that more phosphorus and potassium may be required to support the increase in yields over the past 2 decades. Therefore, the objective of this study was to evaluate the impact of higher rates of P, K, and P + K on seedling diseases caused by oomycetes in both field and greenhouse trials. Temporal analysis of oomycete communities associated with soybean and corn M. CHILVERS (1), J. Rojas (1) (1) Michigan State University, U.S.A. Corn and soybean rotation is typically practiced in field crop production, with the purported benefit of reducing pathogen inoculum density. This management practice has been studied before with concentration on a single pathogen species. There is limited information on how the associated oomycete community is modulated by using different crops in the rotation. We hypothesized that the genetic background of the hosts and their different growth stages could have an effect on the associated oomycete community. In 2013-14, field trials including two soybean cultivars (cv. Sloan and S4.39 Archer) and two corn cultivars (cv. DK5261 and P0255AMXT) were planted in in a randomized block design with non- and inoculated treatments. The inoculated treatment consisted of a mixed inoculum of equal parts of Pythium irregulare, Py. sylvaticum, Py. ultimum, Py. heterothallicum, Py. oopapillum and Phytophthora sojae colonized rice. Soil and root samples were collected at different growth stages and subjected to DNA extraction. Samples were sequenced for ITS 1 amplicons. The results showed a variability in community composition between the two crops, however cultivars did not significantly vary in community composition despite different genetic backgrounds. Host could play an important role in filtering the associated oomycete community, however it did not effectively change the composition, suggesting this specific rotation system might not be conducive to pathogen inoculum reduction. Survey data reveals potential interactions between soil-borne fungi and oomycetes isolated in the midwest E. LERCH (1), A. Fakhoury (2), M Chilvers (3), A Robertson (1) (1) Iowa State University, IA, U.S.A., (2) Sourthern Illinois University, IL, U.S.A., (3) Michigan State University, Survey data reveals potential interactions between soil-borne fungi and oomycetes isolated in the midwest E. LERCH (1), A. Fakhoury (2), M Chilvers (3), A Robertson (1) (1) Iowa State University, IA, U.S.A., (2) Sourthern Illinois University, IL, U.S.A., (3) Michigan State University, Root rot pathogens, such as Fusarium and Pythium species are frequently isolated together from damped-off soybean seedlings. Consequently, they are hypothesized to form a multi-species complex that infects soybean, however the interaction has not been extensively studied and is not well understood. The objective of this research was to identify potential interactions between fungi and oomycetes. Data from 2012 Midwest survey funded by the USDA- NIFA and USB/NCSRP was used. Data comprised species recovered from seedlings collected by soybean pathologists from fields with seedling disease or a history of disease. Breadth of resistance of Phytophthora fruit rot resistant watermelon germplasm to Phytophthora capsici isolates from across United States of America Field trials were carried out at two locations in 2014 and three locations in 2015 at fields each with a known history of seedling disease. Stand counts, percent emergence, and yield were not significantly different between treatments at any of the locations. However, a greater number of Pythium spp. were recovered from plants with the addition of K. In greenhouse assays using soil collected from the same infested fields, final stand and root weights were significantly (P<0.05) higher in pots without fertility treatments for one of the two locations. Further monitoring of the influence of higher rates of K at the time of planting on disease development of soil borne diseases caused by oomycetes is warranted. Cold stress at planting increase susceptibility to damping-off caused by Pythium sylvaticum M. SERRANO (1), A. Robertson (1) (1) Iowa State University, U.S.A. Pythium sylvaticum is an Oomycete that causes damping-off of soybeans in Iowa, particularly when cool weather occurs at planting. Seed treatments are applied to over 90 percent of soybean seed in Iowa to protect germinating seedlings and consequently plant stand. The objectives of this experiment were to (i) evaluate the effect cold stress at different times after planting, and (ii) the effect of seed treatments on damping off. Soybean seed was treated with Intego Suite, Intego Suite + mandestrobin or left untreated and planted into 8 oz cups inoculated with P. sylvaticum or non-inoculated. The cups were placed in growth chamber and subjected to 96h of cold stress (4°C) at 0h or 24h after planting. A significant three-way interaction was detected between cold stress, inoculation and seed treatment (P<0.0001). In general, emergence of untreated seeds was reduced more than 70% when subjected to periods of cold stress (P<0.05). Emergence was improved with seed treatments (P<0.0001). These data indicate that periods of cold stress at planting increases the susceptibility of soybean to damping-off caused by P. sylvaticum and that seed treatments reduce damping-off under these adverse conditions. Pathogenicity and Virulence of Oomycete Species on Common Bean D. ROSSMAN (1), M. Chilvers (1), A. Rojas (2), J. Jacobs (2) (1) Michigan State University, U.S.A.; (2) Michigan State University, U.S.A. Common bean is often affected by soilborne oomycetes that cause seed and seedling rot. A study was conducted to determine the pathogenicity and virulence of 24 Pythium spp. and Phytopythium aff. Temporal analysis of oomycete communities associated with soybean and corn M. CHILVERS (1), J. Rojas (1) (1) Michigan State University, U.S.A. Hierarchical clustering was used to identify groups of isolates frequently recovered together from the same field. When data were analyzed including all fungi and oomycetes, regardless of pathogenicity, six distinct clusters were identified. Within each cluster were fungal and oomycete pathogens and non-pathogens. When data were reanalyzed with only pathogenic species, four distinct clusters were present. Cluster 3 contained only Fusarium species, while cluster 2 contained several Pythium species as well as Phytophthora sojae, Phytophthora sansomeanea and F. verticillioides. The remaining two clusters contained Fusarium and Pythium species. These data should allow soybean pathologists to identify potential interactions between specific species of soil-borne fungi and oomycetes that can be explored and used to enhance our understanding of root rot on soybean. Identification and characterization of potential oomycete pathogens from the University Agricultural Laboratory in Fresno, California M. ELLIS (1), H. Deniston-Sheets (1), D. Sieperda (2), J. Bushoven (1) (1) Plant Science Department, California State University, Fresno, U.S.A.; (2) California State University, Fresno, U.S.A. Identification and characterization of potential oomycete pathogens from the University Agricultural Laboratory in Fresno, California M. ELLIS (1), H. Deniston-Sheets (1), D. Sieperda (2), J. Bushoven (1) (1) Plant Science Department, California State University, Fresno, U.S.A.; (2) California State University, Fresno, U.S.A. The University Agricultural Laboratory (UAL) at California State University, Fresno is a 1000-acre farm that grows a wide range of fruit, nut, vegetable, and field crops. Many of the crops grown on the UAL are negatively impacted by oomycetes; however, identification of pathogenic species has not been determined. Therefore, the goal of this research was to identify and characterize potential sources of oomycete pathogens from soil and irrigation reservoirs on the UAL. Oomycetes were baited over a 5-day period using pears or cucumbers as bait. Symptomatic fruit tissue was excised and plated on the oomycete selective medium PARP. Isolates were then characterized using morphology and DNA sequencing of the internal transcribed spacer region. Forty-four isolates of Pythium spp., 6 isolates of Phytophthora spp., 4 isolates of Phytopythium spp., and 1 isolate of a Pythiogeton sp. were identified. Pythium spp. (86%), Phytophthora spp. (11%), and Phytopythium spp. (3%) were identified from the irrigation reservoir. The most common species isolated from irrigation water were Pythium oopapillum (54%) and Pythium dissotocum (25%). Of the isolates baited from soil 75% were Pythium spp., while 13%, 8%, and 4% were Phytopythium spp., Phytophthora spp., and a Pythiogeton sp. respectively. These results suggest that the UAL has a diverse number of potential oomycete pathogens and that irrigation reservoirs may act as a source for potential inoculum in farming operations. Development of a split-root technique for canola (Brassica napus L.) G. BRUCE (1), J. Robson (2), M. McDonald (2) (1) Agriculture and Agri-Food Canada, Canada; (2) University of Guelph, Canada Canola (Brassica napus) is an important oilseed crop in Canada. A split-root system was developed for studies of host-pathogen interaction with root pathogens. Canola seed was germinated on moistened filter paper within pouches. After 7 days, the root tip of each seedling was clipped back to stimulate secondary branching. Each seedling was then carefully planted into soilless mix and allowed to grow for 18 days. When uprooted, each seedling had a pair of secondary roots. One root of each pair was then potted into each side of a specially modified plant pot for subsequent growth and assessment. Estimates of viable resting spores of Plasmodiophora brassicae using propidium monoazide and qPCR B. GOSSEN (1), F. Al-Daoud (2), M. McDonald (2) (1) Agriculture and Agri-Food Canada Canada; (2) University of Guelph Canada Estimates of viable resting spores of Plasmodiophora brassicae using propidium monoazide and qPCR B. GOSSEN (1), F. Al-Daoud (2), M. McDonald (2) (1) Agriculture and Agri-Food Canada, Canada; (2) University of Guelph, Canada Estimates of viable resting spores of Plasmodiophora brassicae using propidium monoazide and qPCR B. GOSSEN (1), F. Al-Daoud (2), M. McDonald (2) (1) Agriculture and Agri-Food Canada, Canada; (2) University of Guelph, Canada Plasmodiophora brassicae [clubroot of Brassica spp.] persists in soil as long-lived resting spores. Quantitative polymerase chain reaction (qPCR) estimates of spore concentration do not distinguish between viable and non-viable spores. Propidium monoazide (PMA) penetrates non-viable cells and binds to DNA when exposed to high-intensity light, and so inhibits amplification with qPCR. The impact of pre-treating resting spores with PMA and light prior to qPCR (PMA-PCR) was assessed. Spore suspensions from 6-wk-old (immature) and 9-wk-old (mature) clubs were heat-treated, and concentrations of spores were estimated using qPCR and PMA-PCR. Spore viability was assessed using bioassays on canola seedlings. Prior to heat treatment, comparison of estimates from qPCR and PMA-PCR indicated that all mature resting spores were viable, but ≥ 74% of immature spores were not viable. This was consistent with substantially higher clubroot severity in plants inoculated with mature resting spores than immature spores. Heat treatment produced little or no change in estimates of mature spores with qPCR, but substantially reduced both estimates of spore concentration from PMA-PCR and clubroot severity in bioassays. For immature spores, PMA-PCR did not consistently demonstrate a heat treatment effect. We conclude that PMA allows for estimation of viable resting spores using qPCR. Phenotypic variation between two clades of Phytophthora cinnamomi isolated from avocado fields in California R. BELISLE (1), B. McKee (2), W. Hao (2), P. Manosalva (2) (1) University of California, Riverside, U.S.A.; (2) University of California Riverside, U.S.A. Identification and characterization of potential oomycete pathogens from the University Agricultural Laboratory in Fresno, California M. ELLIS (1), H. Deniston-Sheets (1), D. Sieperda (2), J. Bushoven (1) (1) Plant Science Department, California State University, Fresno, U.S.A.; (2) California State University, Fresno, U.S.A. The modified pots were created by placing two small plastic bags filled with potting mix into a square pot to create two equal but independent sections of soil per pot. A bamboo support was inserted between the bags. The roots were placed into each bag and covered with soil. The young seedling was loosely tied to the support, then sand was added to cover the soil and support the seedling. This resulted in almost 100% survival of the treated seedlings. As an initial proof of concept, seedling roots on one side of each pot were inoculated with resting spores of Plasmodiophora brassicae 7 days after potting and visually assessed for clubroot symptoms at 42 days after inoculation. Clubroot symptoms developed on the inoculated side but not on the control. Breadth of resistance of Phytophthora fruit rot resistant watermelon germplasm to Phytophthora capsici isolates from across United States of America vexans on two common bean (Phaseolus vulgaris) cultivars, ‘Red Hawk’ kidney bean and ‘Zorro’ black bean from Andean and Meso-American gene pools, respectively. A seedling assay was conducted at 20°C in a growth chamber. Oomycete inocula were added to the root zone at the time of planting, and seedlings were harvested after 12 days. Root length, root area, root dry weight, and emergence were quantified. Seedling assay results indicated that 12 species caused significant root length and root area reductions from the control. Similarly, 11 of the same oomycete species caused significant reductions in root dry weight. Four Pythium spp. and Phytopythium aff. vexans caused significantly lower emergence than the control and were considered to be seed rot pathogens. An in vitro seed assay was conducted at 20°C and 26°C, in which disease S4.40 ratings were assessed. In this assay, 13 species caused disease severity that was significantly higher than the control. Moreover, it was observed that the virulence of certain oomycete species varied depending temperature and host gene pool. Findings help elucidate the oomycete species that are most aggressive and identify factors that may influence oomycete species virulence on common bean. ratings were assessed. In this assay, 13 species caused disease severity that was significantly higher than the control. Moreover, it was observed that the virulence of certain oomycete species varied depending temperature and host gene pool. Findings help elucidate the oomycete species that are most aggressive and identify factors that may influence oomycete species virulence on common bean. Putative sucrose esters from Petunia x hybrida may contribute to this host’s reduced susceptibility to Phytophthora infestans M. BECKTELL (1) (1) Colorado Mesa University U S A Putative sucrose esters from Petunia x hybrida may contribute to this host’s reduced susceptibility to Phytophthora infestans M. BECKTELL (1) (1) Colorado Mesa University, U.S.A. Petunias have been established as a less susceptible host of P. infestans, but the reasons for this have not been elucidated. We have reported that petunia leaf extracts contain a potent zoosporicide. Previous biochemical analyses of crude leaf extracts suggested the presence of sucrose esters, a class of compounds known to be produced by the trichomes of Petunia and Nicotiana species. Subsequent experiments with commercially available sucrose esters have revealed that sucrose monolaurate (SM), an amphiphilic sucrose monoester known to interact with cell membranes, exhibits zoospore lysing activity. The goal of our current work is to confirm the presence of and identify the chemical structure of the putative sucrose esters present in petunia leaf extracts. Samples were purified using hydrophilic-lipophilic balanced cartridges and an established protocol for the purification of sucrose esters from Nicotiana tabacum. The purified samples were analyzed, along with SM as a control, via LCMS-ELSD, 1H and 13C NMR, and TLC. The data shows a lack of monoesters, excluding SM as the bioactive component, but supports the presence of several known sucrose di- and polyesters. Analyses are ongoing to deduce the specific chemical structures of these sucrose esters. Results from this work will bring us one step closer to understanding an aspect of the petunia-late blight interaction that has received little attention. An optimized multiplex detection protocol captures divergent isolates of yellow dwarf virus species in Kansas wheat A. LANEY (1), R. Acosta-Leal (1), A. Whitfield (1), D. Rotenberg (1) (1) Kansas State University, U.S.A. We assessed WCM immigration onto trap plants and the influence of alternative hosts such as downy brome (Bromus tectorum L), and volunteer wheat on WCM infestation and WSMV infection of wheat. Wheat trap plants were monitored weekly in the fall over three years to assess the relationship among patterns of WCM infestation and WSMV infection with cumulative growing degrees days (GDD) of spring wheat, daily mean temperature with respect to alternative hosts. The relationship among GDD on WCM infestation and WSMV infection across alternative hosts was non-linear. Mite infestation and virus infection was positively correlated with daily mean temperature (P < 0.001) in the fall. The risk of WCM infestation and WSMV infection on trap plants from downy brome and volunteer wheat plots was higher during the late fall when compared to the bare ground control plots (P < 0.05). Overall, daily mean temperature is a better predictor of WSMV risk and the Wheat streak mosaic infection on emerging crop increases with the presence of downy brome and volunteer wheat species during the fall. A full genome cDNA clone of Cherry rusty mottle associated virus induces disease in sweet cherry D. VILLAMOR (1), S. Pillai (1), K. Eastwell (1) (1) Washington State University, U.S.A. Cherry rusty mottle-associated virus (CRMaV), a member of the family Betaflexiviridae, is associated with cherry rusty mottle disease (CRMD). To date, fulfillment of Koch’s postulates for this virus has yet to be demonstrated. A full genome of CRMaV isolate 98CI194 was cloned into a modified pORE-E4 Agrobacterium binary vector between the ENTCUP2 promoter and the Nos-t terminator. Leaf agroinfiltration with the binary vector carrying ENTCUP2-CRMaV failed to infect Nicotiana benthamiana, N. occidentalis 37B, and N. glutinosa whereas agro-inoculation using either the bark flap technique or root dipping resulted in the induction of CRMD symptoms in Krymsk 6 virus-tested cherry rootstock. Further confirmation of infection was verified by RT-PCR detection of CRMaV amplicon and by positive reaction of symptomatic leaves in ELISA and Western blotting using an antibody against the coat protein of CRMaV 98CI194. Finally, bark patch inoculation using lignified wood from one of the CRMaV positive, agroinoculated, Krymsk 6 trees resulted in CRMD symptoms on virus-tested sweet cherry seedling rootstock Prunus avium ‘Mazzard’. These results provide conclusive evidence of the etiology of CRMD to be caused by CRMaV. This infectious clone provides a tool for future studies of genome expression and pathogenesis of CRMaV. An optimized multiplex detection protocol captures divergent isolates of yellow dwarf virus species in Kansas wheat A. LANEY (1), R. Acosta-Leal (1), A. Whitfield (1), D. Rotenberg (1) (1) Kansas State University, U.S.A. An optimized multiplex detection protocol captures divergent isolates of yellow dwarf virus species in Kansas wheat A. LANEY (1), R. Acosta-Leal (1), A. Whitfield (1), D. Rotenberg (1) (1) Kansas State University, U.S.A. Barley yellow dwarf (BYD) is a complex aphid-transmitted virus disease that can be caused by multiple yellow dwarf virus (YDV) species classified in the family Luteoviridae. Previous research revealed that Barley yellow dwarf virus-PAS (PAS) is prevalent in KS. However, currently available multiplex RT-PCR detection assays designed for a subset of YDVs do not include PAS, and primers we designed to the reference sequence of PAS (NC_002160) did not detect all KS isolates. The objectives of this study were to i) sequence two KS field isolates of PAS and one of PAV to enable primer design, and ii) to modify and optimize a multiplex RT-PCR assay to detect six YDV species, including divergent isolates of KS PAS. To test the new assay, 101 archived single-plant leaf samples obtained from a wheat virus survey were tested to determine YDV species composition. We found that PAS was the predominant isolate detected with 59% incidence followed by PAV with 57%; mixed infections of PAS and PAV (30%) were common. Single infections for PAS and PAV (23% each) were also found. The other YDVs were only detected in mixed infections of PAS and/or PAV in 12%, or less, of the samples. These include Cereal yellow dwarf virus-RPV, BYDV-SGV, BYDV-MAV, and a variant of Maize yellow dwarf virus-RMV first identified in MT. This new protocol will provide a tool for examining the association between BYD disease development and YDV species composition in Kansas wheat fields. Impact of downy brome (Bromus tectorum) and volunteer wheat on risk of winter wheat infection by Wheat streak mosaic virus N. RANABHAT (1), Z. Miller (1), E. Lehnhoff (2), F. Menalled (1), T. Seipel (1), M. Burrows (1) (1) Montana State University, U.S.A.; (2) New Mexico State University, U.S.A. Wheat streak mosaic is caused by Wheat streak mosaic virus (WSMV) which is vectored by the wheat curl mite (WCM, Aceria tosichella Keifer). The mite and virus can survive on multiple grass species that act as reservoirs for WSMV. Understanding the effect of alternative hosts on risk of WCM infestation and WSMV infection in newly emerged winter wheat crops is crucial for disease management. Phenotypic variation between two clades of Phytophthora cinnamomi isolated from avocado fields in California R. BELISLE (1), B. McKee (2), W. Hao (2), P. Manosalva (2) (1) University of California, Riverside, U.S.A.; (2) University of California Riverside, U.S.A. Phytophthora cinnamomi (Pc) is the causal agent of Phytophthora root rot (PRR), the most serious disease of avocado worldwide. Pagliaccia et al. (2013) first assessed the genetic diversity of this pathogen in California and identified 16 genotypes corresponding to two distinct clades of A2 mating type isolates. Additionally, differences in virulence were observed between these two clades after infecting avocado rootstocks with different levels of tolerance to Pc. To reveal additional phenotypic differences in these specific genotypes, three representative isolates from clade I and clade II were characterized for: i) mycelial growth rate at different temperatures, ii) zoospore production, iii) zoospore germination, and iv) fungicide sensitivity. More virulent Pc isolates of clade II showed slower growth but an earlier release of zoospores. The molecular interaction between these Pc isolates and their hosts are being studied using lupin and avocado plants. Pc nuclear and mitochondrial genes will be used to study the genetic relationship among isolates from these two clades. Our data suggests that the isolates representing clade II differ in most of the phenotypic traits assessed. Fungicide efficacy among these isolates is being tested under greenhouse conditions using avocado seedlings. This work represents an integrated approach to study the molecular interaction of P. cinnamomi with its host and provide practical solutions to manage this pathogen in the field. S4.41 Phylogenetic relationships among Philippine isolates of Peronosclerospora based on sequence analysis of multiple mitochondrial loci F. DELA CUEVA (1), M. Samaco (1), M. Ramon (2), F. Martin (2) (1) Institute of Plant Breeding, University of the Philippines Los Baños, Philippines; (2) USDA ARS, U.S.A. Phylogenetic relationships among Philippine isolates of Peronosclerospora based on sequence analysis of multiple mitochondrial loci F. DELA CUEVA (1), M. Samaco (1), M. Ramon (2), F. Martin (2) (1) Institute of Plant Breeding University of the Philippines Los Baños Philippines; (2) USDA ARS U S A Downy mildews cause substantial yield losses of sugarcane and corn in the Philippines with several species of Peronosclerospora (P. sacchari, P. spontanea, P. miscanthi, and P. philippinensis) commonly reported. Due to the obligate nature of the pathogen only spore morphology and host genus have been used as the bases of species identification. However, these parameters are highly effected by environmental conditions. Therefore, molecular tools are needed for investigating pathogen diversity and taxonomy. Comparative analyses of world-wide cucumber green mottle mosaic virus (CGMMV) isolates T. PITMAN (1), B. Falk (1) (1) UC Davis, U.S.A. Phenotypic variation between two clades of Phytophthora cinnamomi isolated from avocado fields in California R. BELISLE (1), B. McKee (2), W. Hao (2), P. Manosalva (2) (1) University of California, Riverside, U.S.A.; (2) University of California Riverside, U.S.A. Primers have been developed for amplification of three mitochondrial loci from fifty-four isolates of Peronosclerospora recovered primarily from sugarcane representing geographically diverse regions in the Philippines. Limited sequence divergence of the mitochondrial loci was observed among these isolates. Additional isolates recovered from corn are being sequenced and added to the analysis to evaluate the taxonomy and phylogenetic relationships among corn and sugarcane downy mildews. Studies involving a broad sampling of the pathogen from other grasses in the Philippines and neighboring Asian countries are being conducted to assess the diversity and identify species of tropical graminicolous downy mildews. To facilitate rapid pathogen identification, species-specific molecular diagnostic assays targeting mitochondrial loci are under development. This is especially important for detection of the USDA-APHIS Select Agent P. philippinensis. Identification of conserved transcriptome responses to propagative plant viruses in insect vectors K. MARTIN (1), K. Barandoc-Alviar (1), D. Rotenberg (1), A. Whitfield (1) (1) Kansas State University, U.S.A. The plant Nucleorhabdovirus, Maize mosaic virus (MMV), is vectored by the corn planthopper, Peregrinus maidis. Previously, we conducted an RNA- Seq project to identify differentially expressed (DE) transcripts in adults during infection with MMV. We identified a total of 144 transcripts, with 77 genes upregulated and 67 genes downregulated. To identify conserved transcriptome responses in vectors to propagative viruses, we compared the DE transcripts in our system to two other systems: white backed planthoppers, Sogatella furcifera, exposed to Southern rice black-streaked dwarf virus (SRBSDV, Fijivirus), and the black-faced leafhopper, Graminella nigrifrons, infected with Maize fine streak virus (MFSV, Nucleorhabdovirus). In a tblastx analysis with a cut-off value of 10-3, we identified DE transcripts that are shared between systems. Twenty three transcripts were shared between all three systems. Two transcripts were unique to P. maidis and G. nigrifrons and 38 transcripts were unique to P. maidis and S. furcifera. Protein binding was the largest category of the 144 P. maidis transcripts (19), 10 were shared in all three systems. Functional characterization is underway on transcripts potentially involved in viral replication, intercellular movement and defense. The responsive genes identified in these vectors of propagative viruses may represent a conserved anti-viral strategy and could be targets for resistance against a diverse range of viruses. An optimized multiplex detection protocol captures divergent isolates of yellow dwarf virus species in Kansas wheat A. LANEY (1), R. Acosta-Leal (1), A. Whitfield (1), D. Rotenberg (1) (1) Kansas State University, U.S.A. An optimized multiplex detection protocol captures divergent isolates of yellow dwarf virus species in Kansas wheat A. LANEY (1), R. Acosta-Leal (1), A. Whitfield (1), D. Rotenberg (1) (1) Kansas State University, U.S.A. Our results are consistent with previous research indicating that mycoviruses are commonly found in isolates of R. solani. More than 20 new viruses, belonging to at least three different virus families have been discovered. Future experiments will determine the effects of mycovirus presence on growth and virulence of the fungus, and to determine rates of horizontal transmission. Spatial analysis will determine whether interactions exist between Rhizoctonia spp., AG, and mycovirus distribution in fields, as well as crop and soil properties. Studies on Groundnut rosette disease and implications of the newly reported Groundnut ringspot virus for groundnut production in Ghana A. APPIAH (1), R. Tegg (2), S. Offei (3), C. Wilson (2) (1) Tasmanian Institute of Agriculture, School of Land and Food, University of Tasmania, Australia, Hobart, Australia; (2) Tasmanian Institute of Agriculture, School of Land and Food, University of Tasmania, Australia, New Town, Australia; (3) School of Agriculture and Consumer Sciences, University of Ghana, Legon, Accra, Ghana, Accra, Ghana Studies on Groundnut rosette disease and implications of the newly reported Groundnut ringspot virus for groundnut production in Ghana A. APPIAH (1), R. Tegg (2), S. Offei (3), C. Wilson (2) (1) Tasmanian Institute of Agriculture, School of Land and Food, University of Tasmania, Australia, Hobart, Australia; (2) Tasmanian Institute of Agriculture, School of Land and Food, University of Tasmania, Australia, New Town, Australia; (3) School of Agriculture and Consumer Sciences, University of Ghana, Legon, Accra, Ghana, Accra, Ghana Groundnut rosette is a devastating disease of groundnut (peanut) and presents a major constraint to crop production. Twelve cultivars of groundnut were screened in field trials for resistance to Groundnut rosette disease (GRD), caused by infection with Groundnut rosette assistor virus (GRAV), Groundnut rosette virus (GRV) and its satellite RNA. GRAV infection rates were higher in the wet (13.9–100%) than the dry (11.8–61.8%) season. Four local varieties were rated as resistant along with three of six improved varieties previously regarded as possessing resistance to GRD. These resistant cultivars could be used in the interim management of the disease in the field and further exploited in breeding programmes for the development of virus- resistance. Groundnut ringspot virus (GRSV)-like symptoms were observed within these trials. The identity of the virus was confirmed by ELISA, RT- PCR and amplicon sequencing, and represents a first record of GRSV for Ghana. GRSV was found in most cases in mixed infection with the GRD agents. An optimized multiplex detection protocol captures divergent isolates of yellow dwarf virus species in Kansas wheat A. LANEY (1), R. Acosta-Leal (1), A. Whitfield (1), D. Rotenberg (1) (1) Kansas State University, U.S.A. Moreover, the construction of this infectious clone opens a path forward to pursue etiological studies of other stone fruit virus-like diseases. S4.42 CGMMV is a +ssRNA Tobamovirus that infects curcurbit crops. It is capable of contaminating seeds inside the seed coat, making it a problem with the increased globalization of seed trade. 2013 was the first report of this virus in North America, which prompted federal and state responses and quarantine procedures where it was detected. Subsequent finds in 2014 and 2015 occurred in California, suggesting this is a recurring problem with global seed movement. Our efforts focus on better detection methods in seed. Currently, industry standard for detection is ELISA, which relies on antibody recognition of coat protein. Industry participants have described isolates that are not detected with available ELISA kits, but are identified with molecular assays. We are working to improve detection methods for CGMMV by serological and molecular methods. To support those efforts we need a better understanding of the diversity of CGMMV isolates. We previously sequenced two California isolates and found them to share 90% sequence homology. GenBank has 197 accessions for CGMMV, of which 42 are full genome sequences. Of these, the majority are Asian in origin. Given that CGMMV was originally described in England and is endemic to Europe and the Middle East, we worked with collaborators to amass a collection of 35 isolates from regions where CGMMV is endemic. We used enriched virion samples for next generation sequencing, and bioinformatics to analyze the sequence information. The virosome of Rhizoctonia solani and its effect in fungal pathogenicity T. STETINA (1), C. Rothrock (1), T. Spurlock (2), I. Tzanetakis (1) (1) University of Arkansas, U.S.A.; (2) University of Arkansas, U.S.A. Rhizoctonia solani consists of at least 14 genetically distinct anastomosis groups (AG). Mycoviruses have been shown to play an important in fungal epidemiology and pathogenicity. The goals of this research are to understand mycovirus epidemiology in Rhizoctonia solani on important row crops in Arkansas and their potential to influence pathogenicity. Mycoviruses have been extracted from 190 geotagged isolates from five different fields using cellulose affinity chromatography. The cDNA from double-stranded RNA enriched material was sequenced and sequences analyzed to determine whether they belong to known or undescribed viruses. Detection primers were designed for each virus and then used in all geotagged isolates. Discovery of various dsRNA mycoviruses in Trichoderma spp. causing green mold disease of shiitake Lentinula edides D KIM (1) ( ) (1) Chonbuk National University, South Korea A total of 315 fungal isolates causing green mold disease were collected from contaminated artificial logs and sawdust bags used for cultivating shiitake mushroom Lentinula edodes in Korea and were analyzed for the presence of dsRNA. dsRNA, which was purified using dsRNA-specific chromatography and verified by dsRNA-specific RNaseIII digestion, was detected in 32 isolates. The molecular taxonomy of dsRNA-infected isolates based on the ITS region of the rDNA indicated that all isolates belonged to the Trichoderma spp. The number and size of dsRNAs varied among isolates and the band patterns could be categorized into 15 groups. The most common dsRNA group, groupVI, occurred in 10 isolates encompassing three species of Trichoderma. Partial cDNA clones were obtained from two selected dsRNA groups and the sequence comparison of the cloned fragments of these dsRNAs revealed a high degree of similarity to sequences of a hypothetical protein and polyprotein genes of other mycoviruses, indicating the existence of mycoviruses in Trichoderma spp. Northern blot analysis using cloned cDNA showed specific hybridizing patterns in the dsRNA bands for isolates from which the clones were obtained, suggesting that many different mycoviruses, which have not been identified yet, exist in Trichoderma spp. An optimized multiplex detection protocol captures divergent isolates of yellow dwarf virus species in Kansas wheat A. LANEY (1), R. Acosta-Leal (1), A. Whitfield (1), D. Rotenberg (1) (1) Kansas State University, U.S.A. The identification of GRSV in groundnut fields in Ghana highlights the need for its inclusion in future groundnut-breeding programs. Genetic diversity analysis of the coat protein gene of GRAV revealed more than 97% nucleotide and amino acid identity among the Ghanaian, Nigerian and Malawian isolates, providing a good opportunity for the development of pathogen-derived resistance to combat the disease. Detection, Distribution and Effect of Hop stunt viroid on different hop cultivars M. KAPPAGANTU (1), J. Bullock (1), S. Kenny (2), K. Eastwell (2) (1) Washington State University, U.S.A.; (2) IAREC-Washington State University, U.S.A Hop stunt disease caused by Hop stunt viroid (HSVd) is a growing threat to hop cultivation globally. It severely impacts growth and yield of certain hop cultivars. In 2004, HSVd was first detected in hop yards of Washington State, the major hop growing area in the United States. RT-PCR has been the major technique used for HSVd diagnosis; however, it is costly and sample handling is technically challenging. A more robust Reverse Transcription- Recombinant Polymerase Assay (RT-RPA) was developed to facilitate larger sample numbers. To understand the current distribution of HSVd in Washington, a survey was conducted and it revealed that 17% of hops grown in this region are infected with HSVd. Symptom expression is highly variable among different hop cultivars infected with HSVd. To understand the effect of HSVd, a 5 year study was conducted of six cultivars of hop to determine the impact on yield. Over the study period, the average cone yield of infected cultivars Glacier, Cascade and Willamette was reduced by 62%, 14% and 34%, respectively, compared to non-inoculated healthy plants. No significant yield reduction was observed in cultivars Nugget, Columbus and Galena cultivars. In the final year 2013, horticultural parameters of Willamette and Nugget were also measured. Complete genome sequence of a Watermelon mosaic virus isolate from watermelon after 50 years of its detection in the United States of America A. ALI (1), N. Rajbanshi (1) (1) The University of Tulsa, Tulsa, OK, U.S.A. Complete genome sequence of a Watermelon mosaic virus isolate from watermelon after 50 years of its detection in the United States of America A. ALI (1), N. Rajbanshi (1) (1) The University of Tulsa, Tulsa, OK, U.S.A. Watermelon mosaic virus (WMV) belong to one of the important plant virus genus Potyvirus. This virus has been detected for the first time in 1965 in the Rio Grande Valley of Texas but so far no complete genome of WMV is available from cucurbits in the United States (US). WMV isolate (TX29) was collected from Rio Grande Valley of Texas during a survey in 2010 which showed severe mosaic in a watermelon plant. The complete genome of WMV-TX29 isolate was found to be 10,049 nucleotides excluding the poly-A tail. Nucleotide sequence comparison of TX29 isolate showed more sequence similarities with a WMV-French isolate. This is the first report of the complete genomic sequence of a WMV isolated from cucurbits in the US. Interactions between Bell pepper endornavirus and acute viruses C. ESCALANTE (1), R. Valverde (1) (1) Louisiana State University Agricultural Center, U.S.A. ( ) ( ) (1) Louisiana State University Agricultural Center, U.S.A. Persistent plant viruses do not cause detectable symptoms in their plant hosts. In contrast, acute viruses cause symptoms, and in most cases, disease. Most bell pepper (Capsicum annuum) cultivars are infected with the persistent virus Bell pepper endornavirus (BPEV). To study possible interactions between endornaviruses and acute viruses, we developed two near-isogenic lines of the bell pepper cultivar Marengo; one BPEV-infected and the other BPEV-free. The two lines were mechanically inoculated individually, with a mild (M) and a severe (S) isolate of the acute virus Pepper mild mottle virus (PMMoV). Symptoms were recorded and relative amounts of PMMoV determined by ELISA. When the two lines were inoculated with PMMoV-M, all plants reacted with mosaic symptoms. In contrast, when PMMoV-S was inoculated, BPEV-free plants reacted with systemic necrosis and mosaic while BPEV-infected plants reacted only with mosaic. Results of the ELISA test showed that plants of the BPEV-infected line yielded consistently less PMMoV than plants of the BPEV-free line; however, the differences were not statistically significant. The two bell pepper lines were also inoculated with Tomato spotted wilt virus, Potato virus Y, Cucumber mosaic virus, and Tobacco mosaic virus, and in all cases, plants reacted with similar symptoms. Complete genome sequence of a Watermelon mosaic virus isolate from watermelon after 50 years of its detection in the United States of America A. ALI (1), N. Rajbanshi (1) (1) The University of Tulsa, Tulsa, OK, U.S.A. Results obtained in this investigation suggest that BPEV inhibit the systemic necrotic reaction of Marengo bell pepper caused by PMMoV-S. Characterization of recombinant Potato virus YNTN (PVYNTN) isolates from Sulawesi, Indonesia A. KARASEV (1), M. Chikh-Ali (1), N. Bosqui-Perez (1), D. Vander Pol (1), D. Sembel (2) (1) University of Idaho, U.S.A., (2) Universitas Sam Ratulangi, Manado In the last three decades, Granola, a potato cultivar originally from Germany, has become the most common cultivar for fresh consumption in Indonesia. In August 2014, a survey was conducted in Sulawesi, where potato fields cultivated with Granola and its selection, cultivar Super John were surveyed for Potato virus Y (PVY) presence using visual symptoms and serology. PVY was found in Sulawesi for the first time. Samples determined to be positive for PVY were subsequently typed to strain using RT-PCR assays. All PVY isolates sampled were identified as PVYNTN recombinants, with three recombination junctions in P3, VPg, and CP regions of the genome. Three local PVY isolates were subjected to whole genome sequencing and subsequent sequence analysis. The whole genomes of the Indonesian PVYNTN isolates I-6, I-16 and I-17, were found to be closely related to the European PVYNTNa. This recombinant type was shown previously to cause potato tuber necrotic ringspot disease (PTNRD) in susceptible potato cultivars. The dependence of potato farmers on mostly a single cultivar, Granola, may have given a competitive advantage to PVYNTN over other PVY strains resulting in the predominance of the PVYNTN recombinant. The dominance of PVYNTN in Sulawesi, and possibly in Indonesia as a whole, represents a potential risk to any newly introduced potato cultivar to the country, especially cultivars susceptible to PTNRD. Variation in aphid abundance and Potato Virus Y incidence in Oregon potato S. BAG (1), K. Frost (1), S. Rondon (1), B. Charlton (2), D. Walenta (3) (1) HAREC, OSU, U.S.A.; (2) Klamath Basin Research and Extension Center, OSU Variation in aphid abundance and Potato Virus Y incidence in Oregon potato S. BAG (1), K. Frost (1), S. Rondon (1), B. Charlton (2), D. Walenta (3) (1) HAREC, OSU, U.S.A.; (2) Klamath Basin Research and Extension Center, OSU, U.S.A.; (3) OSU Extension Servic U, U.S.A.; (2) Klamath Basin Research and Extension Center, OSU, U.S.A.; (3) OSU Extension Service, U.S.A. Potato virus Y (PVY) is an economically important potato pathogen that can induce potato tuber necrotic ringspot disease. Complete genome sequence of a Watermelon mosaic virus isolate from watermelon after 50 years of its detection in the United States of America A. ALI (1), N. Rajbanshi (1) (1) The University of Tulsa, Tulsa, OK, U.S.A. More than 25 aphids species are reported to vector PVY in a non-persistent manner. Green peach aphid (GPA, Myzus persicae) and bird cherry-oat aphid (Rhopalosiphum padi L.) are considered to be the primary PVY vectors. In 2015, aphid abundance and PVY incidence were measured in nine commercial potato fields in Oregon. Aphids were collected weekly, counted and identified based on morphological characteristics. Potato leaf tissue was sampled from each location biweekly and assayed for PVY using ELISA. PVY strain composition of ELISA positive samples was determined using an RT-PCR-based strain-typing methodology. Potato aphid (Macrosiphum euphorbiae) and GPA were less abundant than all the other unidentified aphids combined. PVY incidence was highest in Klamath (8.4%), followed by Umatilla (3.3%), Union (1.75%), and Morrow (0%) counties. Strain typing of the ELISA positive samples revealed the PVYN:O strain to be most prevalent (56%) followed by PVYO (10.34%), PVYNTN (5.74%) and a mixed infection of PVYNTN and PVYNO (1.14%). Potato fields with border crops as sudan grass had lower PVY incidence and fewer aphids captured when compared to fields without border crops. Also, traps placed in fields adjacent to hay crops captured more aphids and had higher PVY incidence when compared to traps placed in fields adjacent to maize, mint, sunflower or wheat. Isolation and characterization of FRs551, a bacteriophage associated with the phytobacterium Ralstonia solanacearum strain UW551 M. STULBERG (1), J. Mershon (2), A. Ahmad (3), D. Mollov (4), Q. Huang (2) (1) FNPRU, USDA/ARS & ORISE, Beltsville, MD, U.S.A.; (2) FNPRU, USDA/ARS, U.S.A.; (3) FNPRU, USDA/ARS & Department of Plant Pathology, Faculty of Agriculture, Minia University, El-minia 61519, Egypt, U.S.A.; (4) NGRL, USDA/ARS, Beltsville, MD, U.S.A.; (5) FNPRU, USDA/ARS, Beltsville, MD, U.S.A. Ralstonia solanacearum species complex members are soil-borne, plant pathogenic bacteria that cause devastating diseases in a wide range of economically important crops around the world, with global losses in potato alone exceeding $950 million annually. The race 3 biovar 2 strains of R. solanacearum causing potato brown rot are listed as select agent pathogens due to their potential threat to U.S. agriculture. Genome analysis of the select agent strain UW551 revealed the presence of a putative intact prophage sequence with high homology to the family Inoviridae. Active virions of this phage, designated ΦRs551, were isolated from the supernatant of UW551 cells grown under normal culture conditions and were found to infect other R. solanacearum strains. Mixed Infection of Two Economically Important Tospoviruses in Nicotiana benthamiana K. ZHAO (1), C. Rosa (1) Mixed Infection of Two Economically Important Tospoviruses in Nicotiana benthamiana K. ZHAO (1), C. Rosa (1) (1) The Pennsylvania State University, U.S.A. Mixed Infection of Two Economically Important Tospoviruses in Nicotiana benthamiana K. ZHAO (1), C. Rosa (1) (1) The Pennsylvania State University, U.S.A. ( ) ( ) (1) The Pennsylvania State University, U.S.A. ( ) ( ) (1) The Pennsylvania State University, U.S.A. Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) are two economically important tospoviruses. Mixed infection of TSWV and INSV was first reported on tomato plants from Italy in 2000; and mixed infection has also been found in tobacco in many states in the US for more than two years. Sialer and Gallitelli suggested that mixed infection in tomato could represent progressive adaption of INSV to vegetable crops. To test this hypothesis, healthy Nicotiana benthamiana plants were mechanically inoculated with different ratios of TSWV/INSV and examined for how mixed S4.43 infections influence disease development. After two weeks, systemically infected leaves were collected to measure the viral titer by ELISA. Results show that TSWV is a poor competitor compared to INSV, since INSV could establish infection and reach high titer even when inoculated with equal amount of TSWV inoculum, but TSWV was not able to infect N. benthamiana efficiently in the presence of INSV. Mixed infections of tospoviruses are commonly found in nature, but the interaction between these two economically important tospoviruses and how mixed infections affect viral host range have not been studied yet. Our results provide insights into the interaction between TSWV and INSV in N. benthamiana and basic knowledge for future tospovirus mixed infection studies. infections influence disease development. After two weeks, systemically infected leaves were collected to measure the viral titer by ELISA. Results show that TSWV is a poor competitor compared to INSV, since INSV could establish infection and reach high titer even when inoculated with equal amount of TSWV inoculum, but TSWV was not able to infect N. benthamiana efficiently in the presence of INSV. Mixed infections of tospoviruses are commonly found in nature, but the interaction between these two economically important tospoviruses and how mixed infections affect viral host range have not been studied yet. Our results provide insights into the interaction between TSWV and INSV in N. benthamiana and basic knowledge for future tospovirus mixed infection studies. Genetic diversity, host range and disease resistance to the emerging Tomato mottle mosaic virus on tomato X. SUI (1), R. Li (1), C. Padmanabhan (1), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. Genetic diversity, host range and disease resistance to the emerging Tomato mottle mosaic virus on tomato X. SUI (1), R. Li (1), C. Padmanabhan (1), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. Since its first discovery in 2013 in Mexico, Tomato mottle mosaic virus (ToMMV), a new tomato-infecting tobamovirus is now present in a number of countries (i.e., Brazil, China, and Israel) and several states in the U.S. There is little information available on the molecular and biological properties of this emerging virus. With approximately 80-85% nucleotide sequence identity to two other common tobamoviruses, Tomato mosaic virus (ToMV) and Tobacco mosaic virus (TMV), there is a need to understand the genetic diversity and serological and biological properties of this emerging virus in order to develop a species-specific detection method, and to assess disease resistance durability in tomato cultivars. Comparative host range evaluation showed similarity in host species infected by three tobamoviruses. However, ToMMV was more aggressive on host plants than that of TMV and ToMV. To evaluate whether the emerging ToMMV could break the disease resistance, three commercial tomato cultivars were screened along with control Moneymaker tomato through mechanical inoculation. We observed different responses in tomato cultivars to the three tomato-infecting tobamoviruses. ToMMV exhibited aggressiveness and broke the resistance in a tomato cultivar that had demonstrated full resistance to both TMV and ToMV. This resistance breaking in tomato by the emerging ToMMV presents a serious concern for tomato growers. The mechanism of such resistance breaking will be discussed. Diversity of aflatoxin-producing fungi in non-agriculture soils of Zambia P. KACHAPULULULA (1), J. Akello (2), R. Bandyopadhyay (3), P. Cotty (4) (1) University of Arizona, Tucson, AZ, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Lusaka, Zambia; (3) International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria; (4) USDA-ARS/University of Arizona, Tucson, AZ, U.S.A. Diversity of aflatoxin-producing fungi in non-agriculture soils of Zambia ( ) k ll ( ) d dh ( ) ( ) Aflatoxins are cancer-causing, immunosuppressive toxins that contaminate maize and groundnut, important staples in Zambia. Aspergillus flavus, A. parasiticus and two unnamed taxa are frequently implicated as causal agents of contamination in Africa. Most of Zambia’s arable land is uncultivated. Aspergillus communities in crops may have originated in uncultivated regions and soils in these areas may serve as reservoirs of toxin-producers. Understanding fungal communities in cultivated and uncultivated areas may facilitate aflatoxin management. Identification of conserved sequences within tospovirus genomes for broad-spectrum resistance using RNA interference (RNAi) J. OLIVER (1), D. Rotenberg (1), A. Whitfield (1) (1) Kansas State University, U.S.A. Identification of conserved sequences within tospovirus genomes for broad-spectrum resistance using RNA interference (RNAi) J. OLIVER (1), D. Rotenberg (1), A. Whitfield (1) (1) Kansas State University, U.S.A. Tomato spotted wilt virus (TSWV) and other tospoviruses cause significant yield losses in numerous crop species worldwide. Genetic resistance is the most effective management tool for tospovirus diseases in tomato; however, resistance breaking isolates of TSWV have emerged which are capable of overcoming the currently deployed resistance genes. An alternative to traditional resistance genes is transgenic resistance through the process of RNA interference (RNAi). Though effective against tospoviruses, the sequence-specific nature of RNAi means that resistance can break down if host plants are challenged with genetically diverse populations of tospoviruses. To examine viral sequence diversity, we used publically available viral sequences of TSWV and other tospoviruses. Our approach focused on identifying short, highly conserved regions among 2679 TSWV sequences and across 589 additional sequences from seven tospovirus species. This analysis allowed us to locate sequence conservation within each of the five tospovirus genes. The identified sequences corresponded to motifs of known biological importance. Based upon their high conservation at the nucleotide level, correspondence with known biological functions, and low potential for off-target effects in tomato, these sequences have been incorporated into anti- viral RNAi constructs for tomato transformation. functions, and low potential for off-target effects in tomato, these sequences have been incorporated into anti- formation. Correlation of endogenous pararetrovirus with symptoms of citrus blight disease A. ROY (1), J. Hartung (2), A. Stone (1), J. Sharo (2), R. Brlansky (3), W. Schneider (1) (1) USDA-ARS-FDWSRU, U.S.A.; (2) USDA-ARS-MPPL, U.S.A.; (3) University of Florida, CREC, U.S.A. Citrus blight (CB) is a major economic problem in citrus groves in Florida. Even though the disease was first described in Florida in 1894 the causal agent of CB is still unknown. A number of biotic and abiotic agents have been postulated as potential causes of CB. CB occurs in a wide variety of locations with diverse environments and is root graft transmissible, suggesting that the disease is caused by a systemic infectious biotic agent. Recently, we documented a population of endogenous pararetrovirus (EPRV) sequences in a root stock susceptible to CB, carrizo citrange. Genetic diversity, host range and disease resistance to the emerging Tomato mottle mosaic virus on tomato X. SUI (1), R. Li (1), C. Padmanabhan (1), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. Soils from fields cropped to maize and groundnut (161) and from uncultivated land (53) were assayed for Aspergillus section Flavi from 2012 to 2016. Communities in both wild and cultivated soils were dominated by A. parasiticus (90% and 50%, respectively) and the A. flavus L morphotype (5% and 31%, respectively). Phylogenetic analyses with partial gene sequences for nitrate reductase (niaD, 1.8 kb) and the aflatoxin pathway transcription factor (aflR, 2.1 kb) resolved 20 A. parasiticus isolates from cultivated soils into 4 well supported lineages. Relationships among the 4 lineages and A. parasiticus from other regions were evaluated. Impacts of fungi present prior to introduction of crops may cause differences in the etiology of aflatoxin contamination among regions. Characterization of fungi from non-cultivated soils may provide insight on which fungi are best adapted to the region and allow for improved aflatoxin management. Differential effects of DON and 15-ADON on maize seedling growth Y. MENG (1), J. Li (2), G. Munkvold (3), C. Liang (2), L. Luo (2) (1) China Agricultural University; Iowa State University; Hexi University, China; (2) China Agricultural University, China; (3) Iowa State University, U.S.A. Differential effects of DON and 15-ADON on maize seedling growth Y. MENG (1), J. Li (2), G. Munkvold (3), C. Liang (2), L. Luo (2) (1) China Agricultural University; Iowa State University; Hexi University, China; (2) China Agricultural University, China; (3) Iowa State University, U.S.A. niversity; Iowa State University; Hexi University, China; (2) China Agricultural University, China; (3) Iowa State University Fusarium graminearum (Fg) is a common pathogen causing maize seedling blight in China. Trichothecene mycotoxins produced by Fg have been implicated as pathogenicity factors in seedling blight, but little is known about the effects of specific toxins, such as DON and 15-ADON. In order to study the effect of both mycotoxins on seed germination, maize seeds were soaked in 20 μg ml–1 of DON or 15-ADON for 12h and germinated by rolled paper method; seed germination was counted daily and root length and seedling weight were measured after 7 days. The results showed both mycotoxins decreased seed germination significantly compared to the control, but there was no significant difference between DON and 15A-DON treatments. However, 15 A-DON displayed significantly reduced root length and lateral root numbers compared to DON. We speculated that 15-ADON production by F. Diversity of aflatoxin producing fungi in Malawi C. CHING’ANDA (1), J. Atehnkeng (2), R. Bandyopadhyay (3), P. Cotty (4) (1) University of Arizona, Tucson, AZ, U.S.A.; (2) International Institute of Tropical Agriculture (IITA), Lilongwe, Malawi; (3) International Institute of Tropical Agriculture (IITA), Ibadan, Nigeria; (4) USDA-ARS, The University of Arizona, School of Plant Sciences, Tucson, U.S.A. Genetic diversity, host range and disease resistance to the emerging Tomato mottle mosaic virus on tomato X. SUI (1), R. Li (1), C. Padmanabhan (1), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. graminearum in early stages of infection plays an important role in the poor development of roots, which is a typical symptom of maize seedling blight in the field. Complete genome sequence of a Watermelon mosaic virus isolate from watermelon after 50 years of its detection in the United States of America A. ALI (1), N. Rajbanshi (1) (1) The University of Tulsa, Tulsa, OK, U.S.A. Electron microscopy showed that ΦRs551 is a filamentous-type phage approximately 1200 nm long and 7 nm wide. ΦRs551 has a circular DNA genome that is 7929 nt in length, with 100% identity to the putative prophage sequence in the UW551 genome. It is most closely related to ΦRS603, another filamentous phage of Ralstonia isolated from soil. Our draft ΦRs551 genome contains open reading frames (ORFs) similar to those found in other Inoviridae members, but has a unique overall ORF makeup not found in any other phage relative. S4.44 Identification of conserved sequences within tospovirus genomes for broad-spectrum resistance using RNA interference (RNAi) J. OLIVER (1), D. Rotenberg (1), A. Whitfield (1) (1) Kansas State University, U.S.A. Five EPRV sequences were obtained from healthy, huanglongbing (HLB)-infected and blight-affected carrizo citrange, but only one pararetrovirus sequence, termed Citrus blight associated pararetrovirus (CBaPRV), was found as RNA in all CB affected plants but not in any of the HLB diseased or healthy plants. To assess the correlation between active CBaPRV RNA and CB, samples from 8 healthy trees and 42 citrus trees diagnosed as blighted using the syringe injection technique were collected from four locations in Florida. Reverse transcription-PCR with primers specific to CBaPRV amplified the active RNA from CB affected roots, leaves, bark and flowers, but not from any healthy or HLB infected tissues. While there is no direct evidence that CBaPRV is the causal agent of blight, the presence of viral RNA is tightly correlated with CB symptoms. Genetic diversity, host range and disease resistance to the emerging Tomato mottle mosaic virus on tomato X. SUI (1), R. Li (1), C. Padmanabhan (1), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. in fields under continuous maize and groundnut cultivation and aflatoxin contamination along the crop value chains J. AUGUSTO (1), J. Atehnkeng (2), J. Akello (3), P. Cotty (4), R. Bandyopadhyay (5) (1) Intl Inst of Tropical Agriculture, Nampula, Mozambique; (2) International Institute of Tropical Agriculture, Lilongwe, Malawi; (3) International Institute of Tropical Agriculture, Lusaka, Zambia; (4) University of Arizona, Tucson, AZ, U.S.A.; (5) International Institute of Tropical Agriculture, Ibadan, Nigeria (1) Intl Inst of Tropical Agriculture, Nampula, Mozambique; (2) International Institute of Tropical Agriculture, Lilongwe, Malawi; (3) International Institute of Tropical Agriculture, Lusaka, Zambia; (4) University of Arizona, Tucson, AZ, U.S.A.; (5) International Institute of Tropical Agriculture, Ibadan, Nigeria Aflatoxins are routinely reported in maize and groundnut in Mozambique, but the profile of aflatoxigenic fungal isolates in the fields continuously cultivated with maize or groundnut and the extent of aflatoxin accumulation along the maize and groundnut value chains are unknown. Georeferenced soil samples were taken from 250 farmers’ fields at harvest in 2013 and 2014 in different agro-ecological zones (AEZ) to determine Aspergilli profile. The fields had been under continuous maize or groundnut cultivation from two to five years. Also, aflatoxin content was measured in 300 maize and groundnut samples collected along the crops’ value chains. Inoculum density of Aspergilli was high (>2,000 CFU/g) in fields under extended (³3 years) continuous cultivation, and low-medium (0-2,000 CFU/g) either in fields with <3 years of consecutive cultivation, or at high altitude AEZ (>1,000m), irrespective of crop. Aspergillus flavus S (34%) and L (31%) morphotypes were most frequently isolated from fields under intensive cultivation (³3 years), but L morphotype was predominant (>50%) in fields with <3 years of continuous cultivation, irrespective of crop or AEZ. Aflatoxin content was high in groundnut leftovers used for human consumption after sorting (max. 3,874 ppb), groundnut from storage (max. 1,777 ppb) and groundnut sold at marketplaces (max. 720 ppb). These results suggest that targeted aflatoxin management practices may be required along the crop value chains in Mozambique. Influence of corn GMO traits for insect control on aflatoxin and fumonisin contamination in Texas T. ISAKEIT (1), T. Isakeit (1), S. Murray (2), J. Pekar (2) (1) Texas A&M University, U.S.A.; (2) Texas A&M University, U.S.A. Contamination of corn with aflatoxin and fumonisin is a chronic problem throughout most of Texas. Insect damage to ears can be a predisposition factor, so controlling insects using GMO traits could be a management tool. Diversity of aflatoxin producing fungi in Malawi S4.45 Aflatoxins are toxic fungal metabolites that often contaminate Malawi’s food and feed. These toxins cause immune suppression, stunting, and cancer. Aspergillus flavus and A. parasiticus are the most frequently reported causes of contamination. Aflatoxin producers vary widely in genetic diversity, aflatoxin production, and agro-ecological preference. Little is known about the diversity of fungi causing aflatoxin contamination in Malawi. The current study examined DNA variability among aflatoxin producers in Malawi. Maize and groundnuts were sampled across the five agro-ecological zones of Malawi and fungi infecting crops and belonging to Aspergillus section Flavi were identified. The A. flavus L morphotype comprised 44% of the infecting fungi. Fungi with S morphology composed 40% and A. parasiticus made up 15%. Use of molecular phylogenetics to assign S morphology fungi to species is underway. Fungi that did not produce aflatoxins (793) and belonged to the A. flavus L morphotype were characterized at 17 microsatellite loci in order to identify fungi of potential value as biocontrol agents for the aflatoxin prevention. 350 microsatellite haplotypes were detected. Knowledge of variability among the aflatoxin producers may also provide opportunity to improve aflatoxin management. Effects of nutrients and microorganisms on aflatoxin production by Aspergillus flavus during successive generations of subculturing O. OMOLEHIN (1), Y. Ruarang (2), R. Brown (3), Z. Chen (2) (1) Louisiana State University, U.S.A.; (2) Department of Plant Pathology and Crop Physiology, Louisiana State University, U.S.A.; (3) Southern Regional Research Center, USDA-ARS., U.S.A. Aspergillus flavus infects major agricultural crops, and produces aflatoxin which has been linked to increased liver cancer in humans. Factors such as nutrition, pH and temperature are known to influence A. flavus growth and subsequent aflatoxin production. However, the long-term effects of various media and the presence of other microorganisms on aflatoxin production have not been well studied. In the present study, A. flavus AF13 strain was grown on media containing different levels of nutrients, such as V8, Potato Dextrose Agar (PDA) and Luria Broth (LB), and subcultured for 56 generations. The highest aflatoxin production was observed with A. flavus grown on PDA, which was about 100 times higher than on LB or V8. Aflatoxin production appears to increase with successive transfers of A. flavus on the PDA medium. On the other hand, aflatoxin production seems to decrease slowly with subsequent transfer when A. flavus is grown on V8 or LB medium. Diversity of aflatoxin producing fungi in Malawi At the 39th generation on LB medium, aflatoxin production decreased to a level that was undetectable with HPLC. A. flavus that produced no detectable aflatoxins on LB medium regained the ability to produce high levels of aflatoxin when transferred from LB to PDA medium. These results confirmed earlier studies which have demonstrated that nutrients, especially the carbohydrates in PDA, play a key role in aflatoxin production over time. The impact of microorganisms on aflatoxin production in LB and V8 media will be discussed. Mycotoxins contamination on hybrids and landraces of stored maize grain in México L. VASQUEZ-SILLER (1), K. Ordóñez-Morales (2), J. Soria-Ruíz (3) (1) Universidad Autónoma Agraria Antonio Narro, Mexico; (2) Semillas Berestain, Mexico; (3) Instituto nacional de Investigaciones Forestales Agricolas y Pecuarias (INIFAP), Mexico Mycotoxins contamination is a human and animal health problem because the exposure to such substances present on grains infected by several fungi can cause diseases; thus this work explored mycotoxins contamination levels on maize grain in México. A survey was conducted in six locations forming three sets of samples including hybrids and landraces collected by UAAAN, a seed enterprise and cooperating farmers. 31 samples were tested for moisture content (MC) by constant temperature oven method; phytopathogenic fungi detected and counted by freeze blotter test, and aflatoxins B1, total fumonisins and deoxynivalenol contamination levels determined with inverted ELISA test (Enzyme-Linked-Immune-Sorbent-Assay) (Romer Labs®). General mean of MC was 11.63%. Potentially toxigenic phytopathogenic fungi detected were Fusarium verticillioides 7.6%, Fusarium graminearum 2%, Aspergillus spp. 9.2% and Penicillium spp. 1.1%. Mycotoxins levels were 0.106-2.665 ppm of total fumonisins, 0.64-2.67 ppm of deoxynivalenol and 0 ppb of aflatoxins B1. Mycotoxins levels detected on maize grain were safe for consumption according to Food and Drug Administration (FDA) recommendations, which are: 2-4 ppm of total fumonisins, 5-10 ppm of deoxynivalenol and 20 ppb of aflatoxins B1. Some mycotoxins detection levels were near the FDA risk values, indicating the need to regulate also other mycotoxins than total aflatoxins, which currently is the only one regulated by the Government of México. Profile of Aspergilli in fields under continuous maize and groundnut cultivation and aflatoxin contamination along th Mozambique in fields under continuous maize and groundnut cultivation and aflatoxin contamination along the crop value chains The objective of our field experiments was to evaluate the impact of three different GMO traits in one parent hybrid (N78S) on mycotoxin contamination. Hybrids were planted in two-row, 22 ft-long plots in a randomized, complete block design near College Station in 2014 and 2015 and were inoculated with either Aspergillus flavus or Fusarium verticillioides or both. Overall, in 2014, the Viptera-trait hybrid expressing Cry1Ab and Vip3Aa20 Bt proteins accumulated less aflatoxin (28 parts per billion (ppb) average, range 1-84 ppb) than hybrids that expressed only Cry1Ab (161 ppb, range 38-340 ppb), or no Bt proteins (175 ppb, range 54-410 ppb). Fumonisin was significantly (P=0.05) reduced in the Viptera-trait hybrid (0.3 parts per million, ppm) compared with the other hybrids (5.5 ppm and 7.5 ppm). In 2015, there was greater drought stress at flowering, which increased overall aflatoxin levels. The Viptera hybrid had the lowest average aflatoxin, 147 ppb, compared with the other hybrids (346 ppb and 274 ppb). The average fumonisin was 3 ppm with the Viptera hybrid, compared with the other hybrids (9.5 ppm and 12 ppm). These results show that in some years, the insect-control traits in some GMO hybrids will not be sufficient to reduce aflatoxin. S4.46 Conidial versus sclerotial production: Implications for corn and soil niche specialization in Aspergillus flavus R. SWEANY (1), C. DeRobertis (1), K. Damann (1) Conidial versus sclerotial production: Implications for corn and soil niche specialization in Aspergillus flavus R. SWEANY (1), C. DeRobertis (1), K. Damann (1) (1) Louisiana State University Agricultural Center, U.S.A. Aspergillus flavus contaminates corn and other oil-seed crops with carcinogenic aflatoxin. The populations of A. flavus differ between soils and crops. In Louisiana, native A. flavus isolated from soil and corn in 11 fields revealed only 6 of 16 vegetative compatibility groups (VCG) infected corn. During inoculum production for an infectivity titration experiment to verify niche specialization, two predominant VCGs in corn produced approximately 15 times more conidia than the most widespread soil VCG. Instead of producing conidia, the widespread soil VCG produced copious sclerotia. To test the hypotheses that corn infecting strains produce more conidia and soil inhabiting strains more sclerotia, three isolates from each of 16 VCGs were grown on Czapek Dox and Wickerham solid media and assessed for conidial or sclerotial production respectively. cing Aspergillus flavus community structures in fields treated with the atoxigenic biocontrol A. flavus AF36 Cott (2) ( ), y ( ) 1) University of Arizona, U.S.A.; (2) USDA-ARS, University of Arizona, U.S.A. ( ), y ( ) 1) University of Arizona, U.S.A.; (2) USDA-ARS, University of Arizona, U.S.A. Aflatoxins, toxic and carcinogenic metabolites produced by several fungi in Aspergillus section Flavi, are perennial contaminants of cottonseed in Arizona and impact crop value. Atoxigenic strains of A. flavus have been used to displace aflatoxin producers and manage aflatoxins in cottonseed in Arizona for over two decades. Previous work indicated applications benefit both treated and subsequent crops and that atoxigenic strains in treated soils decrease over time to reach equilibrium with the native A. flavus community. Crop rotation has been implicated as a factor influencing atoxigenic strain retention. The present study sought to determine factors influencing both retention of the biocontrol AF36 and reestablishment of the S strain. Weather, soil type and geography were all found to influence the community structure of A. flavus with hot temperatures, heavy soils, and low elevations favoring the S strain. Regression analyses indicated significant differences among regions in rate of change of the populations of both AF36 and the S strain as a function of time after application. Populations of A. flavus in soils are highest immediately after harvest. Steep declines occur during the few months following harvest. Both the S strain and AF36 followed similar patterns, fitting an exponential decay model. However, quantities of S strain propagules decline slower than AF36, suggesting the S strain may be adapted to resist soil factors that reduce A. flavus viability. Correlation between the disease incidence of Fusarium head blight and the mycotoxin content of winter cereal crops in Korea H. SHIM (1), H. Shim (2), I. Kang (2), D. Shin (2), J. Roh (2), S. Heu (2) (1) National Institute of Crop Science, South Korea; (2) National Insitute of Crop Science, South Korea In 2015, diseased grain rates of Fusarium Head Blight of winter cereal crops in southern region of Korea were 9.5% at an average and the incidence is slightly higher than compared to the previous year’s. Diseased grain rates of Fusarium Head Blight on barley were 18.8% in Jeollanamdo province and that was the highest in Boseonggun with 26.4%. Diseased grain rates of Fusarium Head Blight on wheat was 19.2% in Jeollanamdo province and 12.9% in Jeollabukdo province. It showed the higher incidence in Haenamgun, Goseonggun especially with 35%. cing Aspergillus flavus community structures in fields treated with the atoxigenic biocontrol A. flavus AF36 Cott (2) Mycotoxin content was investigated from the seed samples which have been collected by each region. Deoxynivalenol was 13.3 mg/kg in Sacheon city and 16.3 mg/kg in Goseonggun which was the higher disease incidence than the other regions. Zearalenone contents of the seed samples which have been collected in Sacheon city, Buangun, and Gangjingun as the highest incidence region were 1.15 mg/kg, 1.11 mg/kg, 1.62 mg/kg respectively. Fumonisin contents of the seed samples which have been collected in Sacheon city, Boseonggun, and Haenamgun were 2.54 mg/kg, 2.19 mg/kg, 2.64 mg/kg respectively. Correlation coefficient between the diseased grain rate of Fusarium Head Blight and the Zearalenone content was 0.81. in fields under continuous maize and groundnut cultivation and aflatoxin contamination along the crop value chains Generally corn VCGs produced more conidia; two VCGs comprising 91% of corn isolates produced significantly more conidia (3-60X) than 9 of 10 strictly soil VCGs. Conversely, those two corn VCGs produced very limited sclerotia, whereas all strictly soil VCGs produced significantly more sclerotia (10-100X). High conidial production appears to correlate with corn infection, whereas production of resting bodies appears to correlate with survival in the soil. Suppression of conidial production toward sclerotial production may reduce corn infection and limit outbreaks of aflatoxin contamination in corn. Seasonal population dynamics of mycotoxigenic Aspergillus section Nigri species on grapes and in vineyard soil using ddPCR J. PALUMBO (1), T. O’Keeffe (1), M. Fidelibus (2) (1) USDA ARS, U.S.A.; (2) University of California, U.S.A. Several species of Aspergillus section Nigri (black Aspergilli), including mycotoxin producers, are common residents of grape vineyards, but measuring relative population sizes of individual species using traditional isolation and identification methods is impractical. We developed species-specific primers and probes for quantitative droplet digital PCR (ddPCR) to simultaneously detect and quantify A. niger, A. carbonarius, A. welwitschiae and A. tubingensis populations from grape and soil samples. Samples were collected at berry set, veraison, harvest and raisin stages from two vineyards over two consecutive years. Plate counts showed that total fungal counts were 102-105 cfu/g on berries and 104-105 cfu/g in soil. Plate counts of black Aspergilli in soil were between 102 and 104 cfu/g, independent of sampling time, while counts of black Aspergilli on berries increased from <101 cfu/g at berry set to >103 cfu/g on raisins. Distribution of Aspergillus species using ddPCR showed that in soil, relative abundance of each species varied widely among samples, but A. niger was the most abundant species in 52 of 60 samples. On grapes, relative abundance of A. carbonarius was higher on mature grapes and raisins, and correlated with ochratoxin levels in those samples. This ddPCR method is a useful tool to describe fungal population dynamics in the environment and to monitor mycotoxigenic Aspergillus species during grape and raisin production. Factors influencing Aspergillus flavus community structures in fields treated with the atoxigenic biocontrol A. flavus AF36 R. JAIME (1), P. Cotty (2) Mycotoxins are a chemically diverse group of toxic secondary metabolites produced by fungi. In order to cope with these mycotoxins, plants have the potential to modify the chemical structures as part of their defense mechanism. The resulting metabolic products are neither routinely screened at grain Evaluation of sorghum germplasm for resistance to grain mold and mycotoxin contamination in the Mid-Atlantic B. ACHARYA (1), N. McMaster (2), M. Balota (1), D. Schmale (2), H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A.; (2) Virginia Tech, U.S.A. Evaluation of sorghum germplasm for resistance to grain mold and mycotoxin contamination in the Mid-Atlantic B. ACHARYA (1), N. McMaster (2), M. Balota (1), D. Schmale (2), H. Mehl (1) (1) Virginia Tech Tidewater AREC, U.S.A.; (2) Virginia Tech, U.S.A. Grain sorghum production is increasing in the Mid-Atlantic region due to demand for locally sourced animal feed. Grain mold, caused by a complex of fungal species, infects grains, deteriorates quality, and reduces yield 30-100%. Some grain mold fungi produce mycotoxins that are toxic to both humans and animals, but little is known about the fungi and mycotoxins associated with sorghum grain in the Mid-Atlantic. The objective of this study was to evaluate sorghum germplasm for susceptibility to grain mold and mycotoxin contamination. Under natural field conditions, 375 genetically diverse sorghum accessions from more than 25 countries were evaluated for agronomic traits (panicle type and grain color), grain mold severity, and deoxynivalenol (DON) contamination. Mold severity ranged from 0-90%, and DON ranged from undetectable to10 ppm. Grain mold severity and DON concentrations varied among panicle types and grain colors (P<0.001). Overall, open panicles with dark colored grains had lower mold severity and DON concentrations than compact panicles with light colored grains. Given the potential for grain mold and DON contamination in sorghum grown in the Mid-Atlantic, future work will focus on characterization of the causal agents of grain mold and their associated mycotoxins and identification of sorghum germplasm and agronomic traits associated with reduced susceptibility to mold and mycotoxin contamination. Seasonal population dynamics of mycotoxigenic Aspergillus section Nigri species on grapes and in vineyard soil using ddPCR J. PALUMBO (1), T. O’Keeffe (1), M. Fidelibus (2) (1) USDA ARS, U.S.A.; (2) University of California, U.S.A. Factors influencing Aspergillus flavus community structures in fields treated with the atoxigenic biocontrol A. flavus AF36 R. JAIME (1), P. Cotty (2) Factors influencing Aspergillus flavus community structures in fields treated with the atoxigenic biocontr R. JAIME (1), P. Cotty (2) (1) University of Arizona, U.S.A.; (2) USDA-ARS, University of Arizona, U.S.A. Analysis of deoxynivalenol and deoxynivalenol-3-glucoside in Canadian spring wheat varieties inoculated with Fusarium graminearum D. FERNANDO (1), C. Amarasinghe (1), S. Simsek (2) Analysis of deoxynivalenol and deoxynivalenol-3-glucoside in Canadian spring wheat varieties inoculated with Fusarium graminearum D. FERNANDO (1), C. Amarasinghe (1), S. Simsek (2) (1) University of Manitoba, Winnipeg, MB, Canada; (2) North Dakota State University, Fargo, ND, U.S.A. Analysis of deoxynivalenol and deoxynivalenol-3-glucoside in Canadian spring wheat varieties inoculated with Fusar D. FERNANDO (1), C. Amarasinghe (1), S. Simsek (2) Mycotoxins are a chemically diverse group of toxic secondary metabolites produced by fungi. In order to cope with these mycotoxins, plants have the potential to modify the chemical structures as part of their defense mechanism. The resulting metabolic products are neither routinely screened at grai S4.47 elevators nor regulated by proper legislations. The objective of this study is to analyse the deoxynivalenol (DON) and deoxynivalenol-3-glucoside (D3G) content in Canadian spring wheat varieties grown in two locations, inoculated with different chemotypes of Fusarium graminearum. According to the analysis of variance, significant differences were observed among the varieties for fusarium head blight (FHB) disease index, fusarium damaged kernel percentage (%FDK), DON and D3G content. When considered the effect of chemotype, significant differences were observed for FHB disease index, %FDK and DON content. The D3G content and D3G/DON ratio were not significantly different between the chemotypes. Correlation analysis showed a strong positive correlation between DON and D3G. The highest D3G/DON ratio was observed in moderately resistant varieties. The susceptible varieties showed lower D3G/DON ratio compared to the moderately resistant varieties. The current study indicated that Canadian spring varieties produce D3G upon Fusarium infection and testing for D3G in food and feed safety assessments in Canada should be considered. Seed Quality of Maize Cultivars Infected With Pantoea agglomerans H. SILVA-ROJAS (1), G. Morales-Valenzuela (1), L. Cordova-Tellez (1), D. Ochoa Martínez (1), A. Carballo-Carballo (1) (1) Colegio de Postgraduados, Mexico Seed Quality of Maize Cultivars Infected With Pantoea agglomerans H. SILVA-ROJAS (1), G. Morales-Valenzuela (1), L. Cordova-Tellez (1), D. Ochoa Martínez (1), A. Carballo-Carballo (1) (1) Colegio de Postgraduados, Mexico Pantoea agglomerans is a important seed borne pathogen of maize. There are reports about transmission rates, however, information concerning the quality seed is weak. The aim of this study was to evaluate the quality of seed produced in infected plants with Pantoea agglomerans. Dent corn hybrids Triunfo (comercial) and 9Bx52 (experimental) were sown in Experimental Station of Colegio de Postgraduados, Montecillo, Mexico. Disease assessments were made once a week during crops cycle. Analysis of deoxynivalenol and deoxynivalenol-3-glucoside in Canadian spring wheat varieties inoculated with Fusarium graminearum D. FERNANDO (1), C. Amarasinghe (1), S. Simsek (2) Seeds were harvested and separed acording severity of mother plant (healthy, non-systemic and systemic). Seed quality was evaluated with standard test germination in laboratory and vigour in greenhouse. Disease incidences of sistemic infection were 3.6% for Triunfo and 2.8% for 9Bx52. The results show that 1000-seed weight was lower in systemically infected plants. All variables in laboratory and greenhouse were significantly lower in seed plants with systemic infection. These results indicate that the severity of infection from the mother plant affects the quality of the seed. Rapid detection of preexisting internal Leuconostoc spp. spoilage populations in fresh-cut carrots during storage K. BRITT (1), T. Suslow (1) 1) UC Davis, U.S.A. Rapid detection of preexisting internal Leuconostoc spp. spoilage populations in fresh-cut carrots during storage K. BRITT (1), T. Suslow (1) (1) UC Davis, U.S.A. During an extended period of abnormally short quality retention in mixed component packaged salads, due primarily to rapid decay, a root-cause investigation was undertaken. From an initial investigative assessment, this study focused on identification of the primary underlying microbiological cause and suggested the need for a rapid detection screening of raw material. Analysis of several lots of unprocessed product, approx. 6 cm abrasively peeled carrot plugs, revealed the accumulation of an aqueous slime in the shipping bag void space and around the extremely softened plug surfaces. This premature diagnostic sign of lactic acid bacteria (LAB) spoilage, specifically Leuconostoc spp., developed in cold storage (2.5°C) after two weeks. Efforts were undertaken to determine whether the Leuconostoc was internalized in raw material or primarily environmental contamination with a proliferating reservoir of LAB in the primary processing and packaging environment. Polymerase chain reaction (PCR) primers specific for the amplification of a sequenced region of the Leuconostoc 16S ribosomal RNA gene confirmed the taxonomic identity. Total initial LAB and Leuconostoc bacterial populations isolated from symptomatic carrots ranged from log?? 7.5–8.5 and log?? 3.5–4.0 CFU/g carrot tissue weight respectively, and increased log?? 2 CFU/g log?? and 3.5 CFU/g respectively in population density on asymptomatic and symptomatic raw carrot material during two week refrigerated storage. Controlling bacterial soft rot in tomato fruit that have been inoculated through lenticels located around the stem attachment J. BARTZ (1), D. Spiceland (1), M. Elkahky (3), J. Brecht (4) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) Faculty of Agriculture, Mansoura University, Egypt; (4) Horticultural Sci.; University of Florida, U.S.A. Assessment of hermetic storage of maize under different environmental conditions B. LANE (1), C. Woloshuk (1) (1) Purdue University, U.S.A. Assessment of hermetic storage of maize under different environmental conditions B. LANE (1), C. Woloshuk (1) (1) Purdue University, U.S.A. Storage of maize is a significant challenge for farmers in developing countries. Fungal growth and mycotoxin accumulation are some of the leading causes of postharvest losses, which are estimated to be $4 billion annually in the Sub-Sahara. One of the largest postharvest threats is the growth of Aspergillus flavus and the accumulation of aflatoxin. As maize production in Africa continues to grow, so does the need for storage methods that will help mitigate postharvest losses. This study investigated the use of Purdue Improved Crop Storage (PICS) bags, a three-layer hermetic-storage system, for the prevention of grain rewetting in humid environments in order to mitigate fungal growth and aflatoxin accumulation. Maize (14% grain moisture) was stored in PICS and woven polypropylene bags in Indiana and Arkansas, two environments contrasting in temperature and relative humidity. After 3 months of summer storage, moisture in the woven bags increased significantly in both environments (as high as 15.9%). Populations of storage insects and fungi also increased in the woven bags, with the greatest change under the Arkansas environment. In contrast, grain in the PICS bags was not affected by the two environments; grain moisture remained unchanged, there were no storage insects, and fungal populations increased only slightly. These results provide evidence that PICS bags can be used for the safe storage of maize in the highly-variable environment of Sub-Saharan Africa. Bitter rot on apple: Comparative epidemiology of Colletotrichum species and growth temperature R. MOREIRA (1), E. Zielinski (2), A. Filho (3), L. May De Mio (2) (1) Federal University of Parana, Brazil; (2) Federal University of Parana, Brazil; (3) University of Sao P Bitter rot on apple: Comparative epidemiology of Colletotrichum species and growth temperature R. MOREIRA (1), E. Zielinski (2), A. Filho (3), L. May De Mio (2) (1) Federal University of Parana, Brazil; (2) Federal University of Parana, Brazil; (3) University of Sao Paulo, Brazil pp p p gy p g p R. MOREIRA (1), E. Zielinski (2), A. Filho (3), L. May De Mio (2) (1) Federal University of Parana, Brazil; (2) Federal University of Parana, Brazil; (3) University of Sao Paulo, Brazil The Colletotrichum genus was reclassified, and based on this new classification were described five species causing Bitter Rot on apple belonging to two complex, C. acutatum complex (CaC: C. Analysis of deoxynivalenol and deoxynivalenol-3-glucoside in Canadian spring wheat varieties inoculated with Fusarium graminearum D. FERNANDO (1), C. Amarasinghe (1), S. Simsek (2) Tomato fruit harvested from wet plants or exposed to rainfall prior to packing have a high potential for postharvest decays. In laboratory tests, bacterial soft rot (Pectobacterium carotovorum) development during storage of fruit inoculated in the stem depression was delayed by air-drying, wiping dry, or chlorine washes with efficacy linked with the interval between inoculation and treatment as well as type of treatment. Fruit that were exposed to water alone did not develop decay during storage. Incidence of bacterial soft rot was rare (0 to 13%) among inoculated fruit that were washed with chlorinated water, or wiped dry within 10 sec after dip inoculation, whereas lesions developed among > 50% of fruit washed or wiped dry at 60 min post inoculation. Incidence among non-treated controls was 100% within the 5-day storage. In three separate experiments, bacterial soft rot was reduced by an average of 52, 60, and 20% when fruit were air-dried (30 min) at 1, 4 or 18 h after inoculation, respectively. Corresponding values for washing in chlorinated water (200 ppm, pH 6.5) for 1 min averaged 48, 40 and 2%, respectively. Although evidence does not indicate that the pathogen is introduced into stem scar infection courts by contact such as occurs with wound inoculation, interventions must be initiated within an hour or less after stem scar exposure to inoculum in order to minimize the decay potential. Use of induced resistance for the management of postharvest decay of fruit G. ROMANAZZI (1) (1) Marche Polytechnic University, Italy Up to 25-30% of fruit harvested nowadays is lost, and decay causing fungi are among the main causes of such losses. Harvested produce has high economic impact, covering the costs of production, harvesting, transport, eventual cold storage, exposure on the shelf of stores (so called shelf life) and final life at the home of consumer. During this period, the fruit can be exposed to a list of fungi able to take profit of storage conditions, with high humidity, low temperatures, and senescence, that make weaker the fruit reaction to the infection. The main postharvest pathogen is Botrytis cinerea, but considerable losses of fruit are caused, according to the species, by Penicillia, Colletotrichum spp., Moniliniae, Rhizpous spp., Mucor spp., Alternaria spp., Cladosporium spp. and some other decay causing fungi. Nowadays the most frequent way to prevent postharvest decay relies in the use of synthetic fungicides, but considering their limitations, alternatives based on the use of biocontrol agents, physical means, decontaminating agents, and natural compounds are deeply studies and often used in the field and/or in packinghouses. Most of these alternative treatments have direct antimicrobial activity and at the same time they elicit the host defenses. The different ways to apply the induced resistance, with negative and positive aspects, mechanisms involved and tools useful to track the physiological changes occurring in the host tissues will be discussed. Susceptibility of sweetpotato (Ipomoea batatas) cultivars to fungal and bacterial diseases E. PALENCIA (1), L. Quesada-Ocampo (1) (1) North Carolina State University, U.S.A. Sweetpotato [Ipomoea batatas], is the number one vegetable crop in the state of North Carolina. This crop is a high-nutrient content food and a great source of pro-vitamin A, vitamin C, calcium, and iron. Field and post-harvest diseases cause yield and economical losses during sweetpotato production. Rhizopus soft rot, caused by the ubiquitous R. stolonifer, Fusarium root rot, associated with F. solani and F. oxysporum f. sp. batatas infections, and Streptomyces soil rot, caused by Streptomyces ipomoeae, are among the most detrimental diseases of sweetpotato in North Carolina. Management of sweetpotato diseases through application of antimicrobial chemical compounds is limited since few products are labeled for use in sweetpotato and due to residue restrictions for export markets. Development of cultivars with genetic resistance is the most desirable and economical means of managing plant diseases. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain GRAS (generally regarded as safe) salts were preliminary selected in in vitro tests against Botrytis cinerea, the causal agent of tomato postharvest gray mold, and added at 2% wet basis (wb) to emulsion matrixes prepared with hydroxypropyl methylcellulose (HPMC) and glycerol (ratio 3:1 dry basis, db), beeswax (BW) and oleic acid (ratio 5:1 db), and tween 80 (1.5% wb). The final emulsion solid concentration was modified to 7-10% wb in order to obtain formulations with a viscosity range of 100-150 cp. Selected stable coatings were tested in vivo against gray mold on cherry tomatoes cv. Josefina artificially inoculated with B. cinerea 24 h before coating (curative activity) and incubated at 20ºC for up to 15 days. Among these edible coatings, the most effective were those containing sodium propionate (SP), potassium carbonate (PC), ammonium phosphate (APh), and ammonium carbonate (AC), and they were further tested in vivo with fruit subjected to cold storage. Gray mold incidence and severity were determined on cherry tomatoes inoculated with B. cinerea, coated 24 h later, and stored at 5ºC for 7 and 14 days followed by a shelf life period of 7 days at 20ºC. The antifungal activity of the coatings was fungistatic rather than fungicidal. Although the HPMC-BW coatings formulated with SP were the most effective in reducing gray mold on cold-stored cherry tomatoes, coatings containing AC were selected due to their better performance for overall fruit quality preservation. Analysis of IAA and ABA in HLB diseased citrus trees Z. PANG (1), H. Wang (1), N. Wang (1) (1) Citrus Research and Educational Center, U.S.A. ( ) g ( ) g ( ) (1) Citrus Research and Educational Center, U.S.A. HLB, caused by Candidatus Liberibacter asiaticus (Las), is a disastrous disease resulting in defoliation, twig dieback, root decline and fruit drop. Among these symptoms, preharvest fruit drop directly poses a disastrous threat to the citrus industry by decreasing yield. At early developmental stage of fruit, dropping always occur at AZ-A zone which is between branch and peduncle. Assessment of hermetic storage of maize under different environmental conditions B. LANE (1), C. Woloshuk (1) (1) Purdue University, U.S.A. nymphae, C. paranaense, C. melonis) and C. gloeosporioides complex (CgC: C. fructicola, C. siamense). However, are unknown the optimum conditions for the development and the impact of each species in the disease progress. Therefore, the aim of this study was to determine, for each species, the optimum temperature and evaluate the infection and colonization in fruits of the cultivar Gala. The optimum temperature for the development of the colonies was 22-24° C for CaC, and 25-26°C for CgC. The progress of the disease was significantly higher in fruit inoculated with CaC than in fruit inoculated with CgC. The incubation period was 2 to 3 days for all isolates in wounded fruit, while in unwounded S4.48 the period was 18-25 days. The latency period was 7 days to C. paranaense and 6 days to C. nymphae with fruit wounded but in unwounded fruit, only C. paranaense spolulates with higher latency period (33 days). the period was 18-25 days. The latency period was 7 days to C. paranaense and 6 days to C. nymphae with fruit wounded but in unwounded fruit, only C. paranaense spolulates with higher latency period (33 days). Bioactive effect of triterpene extracts in mix with Pseudomonas to control Gaeumannomyces graminis var. tritici on wheat plants E. MOYA ELIZONDO (1), J. González (2), T. Quezada (2), G. Silva (2) (1) Departamento de Producción Vegetal, Universidad de Concepción, Chile; (2) Universidad de Concepción, Chile Use of induced resistance for the management of postharvest decay of fruit G. ROMANAZZI (1) (1) Marche Polytechnic University, Italy The present work was conducted to evaluate sweetpotato genotypes for resistance to economically relevant post-harvest diseases in sweetpotato production. For this purpose, replicated greenhouse and growth chamber experiments were performed to screen for resistance and susceptibility of 16 sweetpotato varieties to 8 microbial isolates. The results suggested that sweetpotato genotypes differed in resistance to the tested phytopathogenic isolates. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Here, different hormones level at peduncle area were dynamically monitored both in healthy and HLB infected trees during the fruit maturation to find if hormone imbalance in HLB infected peduncles caused preharvest fruit drop. Indole-3-acetic acid (IAA) level was lower in HLB infected trees at the early stage of fruit ripening compared to healthy trees. Level of abscisic acid (ABA), which promote fruit mature and abscission, was higher in Las infected trees. Expression analysis of hormone related genes also matched the hormone detection. We will also report our results in preventing preharvest fruit drop using different hormones. Seasonal variation in the antibacterial activity of latex-like resin from Sciadopitys verticillata K. GWINN (1), D. Yates (1), B. Ownley (1), N. Labbe’ (1) (1) University of Tennessee, U.S.A. Japanese umbrella pine, Sciadopitys verticillata, produces a latex-like resin with antibacterial activity. To determine seasonal antibacterial activity, resin was collected in summer and winter of 2015, stored at -20°C, autoclaved twice, and suspended in sterile deionized water. Resin was tested within two months of collection. Bacterial suspensions (100 µl) from nutrient broth (NB) overnight cultures were mixed with NB (100 µl), treated with resin suspension, and incubated overnight at 26°C with shaking. Treatments were: 0 (control), 25, 50, and 100 µl resin suspension diluted in water (total volume 100 µl). Colony-forming units were determined with dilution plating. Separate tests were conducted with winter and summer-collected resins, and bacterial species were evaluated individually. Data (number of treatment colonies relative to control colonies) were analyzed with mixed models ANOVA. Differences were significant at P=0.05. Exposure to resin collected in winter reduced numbers of colonies of Bacillus cereus, Erwinia amylovora, Agrobacterium tumefaciens, and Escherichia coli, and increased numbers of Xanthomonas campestris, Pseudomonas fluorescens (Pf), and Pseudomonas syringae. Exposure to resin collected in summer affected population growth of two bacterial species; colony counts of E. amylovora decreased and those of P. fluorescens increased. Future research will address the role of chemical composition and resin concentration on antibacterial activity. Bioactive effect of triterpene extracts in mix with Pseudomonas to control Gaeumannomyces graminis var. tritici on wheat plants E. MOYA ELIZONDO (1), J. González (2), T. Quezada (2), G. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Silva (2) (1) Departamento de Producción Vegetal, Universidad de Concepción, Chile; (2) Universidad de Concepción, Chile S4.49 Extracts rich in saponins from quillay (Quillaja saponaria Mol.), an endemic Chilean native tree, and rhizobacteria of the genus Pseudomonas, which produce antimicrobial compounds such as 2,4-Diacetylphloroglucinol (2,4-DAPG), have been associated with reduction of the fungus Gaeumannomyces graminis var. tritici (Ggt), causal agent of take-all disease of wheat. However, reports about bioactivity between these bacteria and this natural triterpenoids are not available. Through in vitro and in vivo tests the interaction between both new sources of take-all disease control were assessed. Results showed that quillay extracts Vet SAP® (>90% of saponins) and QL1000® (~8% of saponins) controlled Ggt in vitro and had a differential effect according to their purity on populations of 2,4-DAPG-producing P. protegens, with Vet SAP® not affecting the bacterial growth at 800 ppm of quillay saponins. On wheat seedlings, Vet SAP® did not affect the bacterial populations of the three bacterial strains assessed, although, quillay saponins at concentration of 7,360 ppm reduced bacterial biofilm formation on the rhizosphere. Strains of P. protegens had a variable antagonistic activity on wheat seedling roots and kept their disease control ability when were combined with different concentrations of Vet SAP®. These results suggest that mixtures of the antagonistic bacteria and plant extract to control G. graminis var. tritici could be useful in developing a biopesticide. Protection of wheat against Zymoseptoria tritici using sugar beet extract-based resistance inducers g y p g g A. SIAH (1), S. Mejri (2), A. Ghinet (3), B. Rigo (3), C. Abuhaie (3), M. Magnin-Robert (4), B. Randoux (4), P. Reignault (4), P. Halama (2) (1) Institut Charles Viollette (EA 7394), Institut Supérieur d’Agriculture, Lille, France; (2) Institut Charles Viollette (EA 7394), Institut Supérieur d’Agriculture, France; (3) Laboratoire de Pharmacochimie (INSERM U995-LIRIC unit), Hautes études d’ingénieur, France; (4) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d’Opale, France In the current context of sustainable agriculture, the use of resistance inducers (RIs) is a promising approach to control crop diseases. A total of 73 putative RIs, including sugar beet (SB) extract-based products, phytohormones, vitamins, and amino acid-based RIs, such as GABA and BABA, were assessed for their efficacy on wheat against Zymoseptoria tritici, a major pathogen on this crop. The experiments were performed in the greenhouse using third-leaf stage plants of the susceptible cv. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Quantitative assessment of the efficacy of ZnO nanoparticles against selected fruit fungal contaminants D. SARDELLA (1), R. Gatt (2), S. Decelis (3), V. Valdramidis (4) (1) University of Malta, Malta; (2) University of Malta, Metamaterials Unit, Malta; (3) Ministry for Health, Malta; (4) University of Malta, Faculty of Health Sciences, Malta Quantitative assessment of the efficacy of ZnO nanoparticles against selected fruit fungal contaminants D. SARDELLA (1), R. Gatt (2), S. Decelis (3), V. Valdramidis (4) (1) University of Malta, Malta; (2) University of Malta, Metamaterials Unit, Malta; (3) Ministry for Health, Malta; (4) University of Malta, Faculty of Health Sciences, Malta The use of traditional antifungals to control postharvest diseases has been reviewed or banned during the past years. Nanoparticles represent an alternative for postharvest disease management, either as coating agents or embedded into warehouses’ filters. The efficacy of zinc oxide (ZnO) was assessed following the identification of the optimal growth temperature of Penicillium expansum, Alternaria alternata, Botrytis cinerea and Rhizopus stolonifer. Fungi were inoculated (105-106 spores/mL) onto SDA plates and incubated at temperatures from 5°C to 35°C under constant aw. Mycelium diameters were measured and then fitted to a linear model to estimate the growth rate (μ) and the apparent lag time (λ). The properties of ZnO (3 to 15 mM) were evaluated for the previously identified optimal growth temperature by mycelium diameter and optical density (OD) measurements recorded every 20 min for up to 6 days with a 96-well microtitre reader at 600 nm. P. expansum and B. cinerea could grow even at 5°C while R. stolonifer had the highest μ = 0.383 ± 0.002 (cm/h) at 25°C and the shortest λ = 17.703 ± 0.228 (h) at 18°C. The antifungal tests showed R. stolonifer and P. expansum as the most sensitive to ZnO being completely inhibited even at 3 mM. A. alternata and B. cinerea were inhibited at 12-15 mM. These results were confirmed by the OD assay. ZnO nanoparticles thus appear as an effective antifungal and their efficacy can be assessed by quantitative mycological studies. Recommendation of Clove oil to minimize Penicillium based postharvest losses in pomegranates (Punica granatum L) M. ALAM (1), M. Alam (2), A. Rehman (3), M. Amin (4), K. Riaz (3) Recommendation of Clove oil to minimize Penicillium based postharvest losses in pomegranates (Punica granatum L) M. ALAM (1), M. Alam (2), A. Rehman (3), M. Amin (4), K. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Alixan treated with each putative RI using foliar application two days before inoculation with the pathogenic strain T02596. Disease symptom scoring three weeks post-inoculation revealed significant reductions of leaf chlorosis and necrosis as well as sporulation (pycnidial density) in plants treated with saccharine, salicylic acid, abscisic acid, GABA, BABA, vitamins C and BX, probenazole and 33 SB extract- based RIs, such as RI2474, which was the most efficient molecule (up to 71% disease reduction). Among the 73 tested molecules, 25 displayed an in vitro inhibiting effect on Z. tritici growth. The three most efficient SB extract-based RIs without biocide effect, including RI2474, were selected in order to improve their efficacy by optimizing the dose, the formulation and application conditions. In addition, cytological, biochemical and molecular approaches will be set up to identify the plant defense mechanisms involved in the protection conferred by the three selected RIs. Switchgrass Extractives Inhibit Plant Pathogenic Fungi A. BRUCE (1), B. Ownley (1), J. Tao (1), N. Labbe (1), K. Gwinn (1), D. D’Souza (1), N. Moustaid-Moussa (2) (1) University of Tennessee, U.S.A.; (2) Texas Tech University, U.S.A. Switchgrass is a leading contender for biofuel production. Cellulosic ethanol yield is reduced by a phenolic-rich, nonstructural portion of switchgrass, called extractives. Switchgrass extractives hinder enzyme activity and microorganism growth utilized during the biofuel conversion process. Switchgrass would be a more cost-effective feedstock if the extractives portion was developed for use as a biopesticide. The aim of this study was to assess different concentrations of switchgrass extracts for inhibition of Fusarium oxysporum, Fusarium graminearum, Alternaria alternata, Botrytis cinerea, and Bipolaris oryzae. Crude ethanol extracts were evaluated for inhibition of fungal plant pathogens with disk diffusion and spore germination assays. Colony growth was measured in disk diffusion assays on water agar; treatments were disks treated with 60X and 75X concentrations of extracts, and a ‘no extracts’ control. Inhibition of mycelial growth was greatest in disk diffusion assays with A. alternata and F. graminearum at 75X, and F. oxysporum at 60X. Spore germination was tested by incubating spores in a water control, or in extracts concentrated at 10X and 15X for 24 h, followed by repeated sampling over a 4-hour period. Germ tube growth was observed microscopically. Spore germination of all fungi was significantly inhibited by both extracts concentrations at all time points. Results indicate that switchgrass extracts have biopesticide potential. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Results show that in average seed treatments with Heads Up alone or Heads Up plus other compound improved soybean yields 2.08 bushels per acre over untreated control. Seed treatments effectively reduced or suppressed the occurrence of SDS and white mold in soybean. Compared with control, the probabilities of yield response to a treatment were 63.5%, 7%, 29.5% for increase, no change, and decrease, respectively. Compared with synthetic chemicals, there is a slightly larger number of low responsive trials for Heads Up, which is often seen in field trials with biological control products. However, the low cost (<$2 per acre) and its biological nature makes Heads Up a good candidate for soybean disease management. To improve consistency, factors leading to low responsiveness are investigated and addressed. sudden death syndrome and white mold. Eleven-year Iowa data together with some data from other mid-west soybean productions states were pooled and analyzed. Results show that in average seed treatments with Heads Up alone or Heads Up plus other compound improved soybean yields 2.08 bushels per acre over untreated control. Seed treatments effectively reduced or suppressed the occurrence of SDS and white mold in soybean. Compared with control, the probabilities of yield response to a treatment were 63.5%, 7%, 29.5% for increase, no change, and decrease, respectively. Compared with synthetic chemicals, there is a slightly larger number of low responsive trials for Heads Up, which is often seen in field trials with biological control products. However, the low cost (<$2 per acre) and its biological nature makes Heads Up a good candidate for soybean disease management. To improve consistency, factors leading to low responsiveness are investigated and addressed. Efficacy of synthetic and biorational products against powdery mildew of flowering dogwood P. LIYANAPATHIRANAGE (1), T. Simmons (1), M. Kabir (1), K. Addesso (1), F. Baysal-Gurel (1) (1) Tennessee State University, U.S.A. Flowering dogwood (Cornus florida L.) is a popular ornamental plant in home gardens and commercial settings. Powdery mildew, caused by Erysiphe pulchra, may cause cosmetic damage on flowering dogwood by attacking tender shoots and leaf surfaces that cause for dead patches and premature defoliation or kill seedlings. Various products are available or in development that have the potential to contribute to the management of powdery mildew of dogwood. In this study the effect of biorational products (Triact 70-neem oil, thymol and cedar oil) and fungicide (Cleary’s 3336F) on dogwood powdery mildew severity was evaluated. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Treatments were applied using hand-held sprayer every 7 days for a total of five applications. The experiments were done twice, each with three replicates per treatment. The severity of powdery mildew on foliage and phytotoxicity were evaluated using a scale of 0-100% foliage affected. In both experiments, the severity of powdery mildew was low to moderate (~15 and 30% foliage affected-non- treated control). Triact 70, thymol and Cleary’s 3336F provided significant control of powdery mildew compared to the non-treated control in both experiments. Powdery mildew disease progress was lower in dogwood plants treated with Triact 70, thymol, Cleary’s 3336F and the high rate of cedar oil than in those non-treated control and the low rate of cedar oil. However, thymol and the high rate of cedar oil were slightly phytotoxic on dogwood foliage. Antifungal effect of nano sized zero valent iron (nZVI) to special fungal pathogens in paprika and tomato crop M. PARK (1), J. Kim (1), Y. Lee (2), S. Kim (2) (1) Cheorwon Plasma Research Institute, South Korea; (2) Department of Applied Plant Sciences College of Agriculture and Life Sciences/Gangwon National Univ., South Korea Zero-valent iron (ZVI) has been utilized in environmental remediation including the removal of pollution or contaminants such as heavy metals, phosphate as an ingredient of fertilizers and pesticides in groundwater, soil and/or sediment. Recently, many studies have shown that the nano-sized ZVI (nZVI) have antibacterial and antifungal activity by oxidative reaction of the nZVI. Here, we are trying to apply the ZVI or nZVI as antifungal agent instead biocidal chemical compound (fungicide) for Collectorichum acutatum, Fusarium oxysporum f. sp. Lycopersici, Sclerotinia minor, S. sclerotiorum, and Rhizoctonia solina, the causal agent of major fungal disease of paprika and tomato crop in Korea. The result of antifungal activity tests showed that ZVI and nZVI were the most efficient antifungal agent to all five fungal pathogens. Especially, nZVI was more efficient to inhibit the hyphal growth than ZVI in five fungal pathogens. Natural products reduce grey mold of tomato caused by Botrytis cinerea F. AHMED (1), A. Alvarez (1), B. Sipes (1) (1) University of Hawaii at Manoa, U.S.A. Grey mold is the most important postharvest disease of tomatoes in Hawaii. Treatments with edible, natural products are needed to reduce losses and contribute to food sustainability. Identification of volatiles with nematicidal activities from Sphingobacterium nematocida ZY-71-1 and control efficiency to Meloidogyne incognita J. CHANG (1), W. Mu (2), J. Xi (2), J. Song (2) (1) Sanmenxia Tobacco Company Lushixian Branch, China; (2) Zhengzhou Tobacco Research Institute of CNTC, China Identification of volatiles with nematicidal activities from Sphingobacterium nematocida ZY-71-1 and control efficiency to Meloidogyne incognita J. CHANG (1), W. Mu (2), J. Xi (2), J. Song (2) (1) Sanmenxia Tobacco Company Lushixian Branch, China; (2) Zhengzhou Tobacco Research Institute of CNTC, China Chemical pesticide is an effective way in controlling tobacco disease, but it caused some problems such as environment pollution, ecological balance destruction and chemical residues. So biology control of tobacco disease attracts more and more attention. The usage of micro-organisms or microbial metabolites is an important approach in controlling nematodes. Sphingobacterium nematocida ZY-71-1?a bacterial endophyte, was isolated from tobacco leaves, which producing volatiles with nematicidal activities. The biocontrol effect of the bacterium towards Meloidogyne incognita was evaluated under Petri dish test and greenhouse assay. The mortality rate to the nematode caused by S. nematocida ZY-71-1 was 100% and the inhibition rate to the hatch of the eggs of the nematode was also 100%. Additionally, nematicidal bioactivity materials produced by S. nematocida ZY-71-1 were determined. By GC/MS analysis and pure chemicals confirmation, 5 volatiles with nematicidal activities against juveniles and eggs of M. incognita were identified from S. nematocida ZY-71-1, including benzeneacetaldehyde, 2-nonanone, decanal, 2-undecanone and dimethyl disulfide. This study provided an effective way for biological control of nematode and established foundation on controlling the nematode. The Role of the Citrus Microbiome in Tree Health and Tolerance to Pathogens N. GINNAN (1), T. Dang (2), P. Ruegger (2), J. Borneman (2), P. Rolshausen (2), G. Vidalakis (2), S. Bodaghi (2), M. Roper (2) (1) University of California Riverside - Department of Plant Pathology and Microbiology, Riverside, CA, U.S.A.; (2) University of California Riverside, U.S.A. N. GINNAN (1), T. Dang (2), P. Ruegger (2), J. Borneman (2), P. Rolshausen (2), G. Vidalakis (2), S. Bodaghi (2), M. Roper (2) (1) University of California Riverside - Department of Plant Pathology and Microbiology, Riverside, CA, U.S.A.; (2) University of California Riverside, U.S.A. There is compelling evidence that the health of an animal or plant is linked to the structure and activities of its associated microbiome. The goal of this project is to identify naturally occurring microbes or consortia of microbes that either directly or indirectly protect citrus trees from plant pathogens. We have, thus far, isolated and identified the culturable microbes of the roots, leaves, and budwood of citrus trees collected from California and Florida. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Riaz (3) (1) Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan, Faisalabad, Pakistan; (2) Department of Plant Pathology, University of Agriculture Faisalabad, 38040-Pakistan, Faisalabad, Pakistan; (3) Department of Plant Pathology, University of Agriculture Faisalabad, 38040- Pakistan, Faisalabad, Pakistan; (4) Institute of Horticultural Sciences, University of Agriculture Faisalabad, 38040-Pakistan, Faisalabad, Pakistan ( ) ( ) ( ) ( ) ( ) (1) Department of Plant Pathology, University of Agriculture, Faisalabad, Pakistan, Faisalabad, Pakistan; (2) Department of Plant Pathology, University of Agriculture Faisalabad, 38040-Pakistan, Faisalabad, Pakistan; (3) Department of Plant Pathology, University of Agriculture Faisalabad, 38040- Pakistan, Faisalabad, Pakistan; (4) Institute of Horticultural Sciences, University of Agriculture Faisalabad, 38040-Pakistan, Faisalabad, Pakistan Plant essential oils are important alternate to synthetic fungicides for the management of postharvest diseases in fruits. In the present study, among the tested clove (Syzygium aromaticum), coriander (Coriandrum sativum) and oregano (Oreganum vulgare) essential oils, clove oil not only inhibited the mycelial growth of Penicillium expansum and Penicillium implicatum at the tested concentrations (25, 50, 100 and 250 µl/L, performing maximum at 250 µl/L giving 100% fungal growth inhibition), but also showed 64 and 45% disease reduction against P. expansum and P. implicatum respectively, when applied as curative treatment to the artificially inoculated fruits (1 × 106 spores/mL). In addition, Clove oil treatment also reduced weight loss and maintained soluble solid contents, titratable acidity and ascorbic acid contents of pomegranate fruits for about 4 weeks at ambient temperature (25°C). Hence, it is concluded that clove oil can be used for postharvest disease management, preservation and extension of shelf life of pomegranate fruits. Summary of 11-year field trials of using Heads Up® - a Saponin plant protectant to manage soybean diseases S. Navi (1), X. Yang (1) (1) Iowa State University, U.S.A. Summary of 11-year field trials of using Heads Up® - a Saponin plant protectant to manage soybean diseases S. Navi (1), X. Yang (1) (1) Iowa State University, U.S.A. Heads Up® is a plant protectant that induces systemic acquired resistance of plants when applied as seed treatments or foliar sprays. Since 2005, field trials have been established in multi-locations of Iowa State University Research Farms to determine effectiveness of seed treatment to manage soybean S4.50 sudden death syndrome and white mold. Eleven-year Iowa data together with some data from other mid-west soybean productions states were pooled and analyzed. Development of edible composite coatings with antifungal GRAS salts for reduction of postharvest gray mold of cherry tomato fruit L. PALOU (1), C. Fagundes (2), A. Monteiro (2), M. Pérez-Gago (3) (1) Institut Valencià d’Investigacions Agràries (IVIA), Spain; (2) Universidade Federal de Santa Catarina, Brazil; (3) IVIA, Spain Based on the hypothesis that inhibition of Botrytis spore germination will significantly reduce postharvest losses, botanicals were tested for their effects on conidia. Ten Botrytis isolates from rotting tomato fruit were identified morphologically and confirmed by DNA sequence analysis. Leaves of candidate plants were frozen at -20C° and plant fluids were sterilized by passing through a 0.22 µm millipore. Extracts were evaluated by adding Botrytis spores (106/ ml) with sterilized malt extract broth to multi-well microplates. Changes in optical density measured at 6, 12, and 24 hours after inoculation were analyzed with Gen5 software. Capsicum chinense cultivars Datil and C. annuum Carnival completely inhibited fungal germination in preparations containing 30 and 40% leaf extract at all evaluation times. These extracts were superior to all other extracts tested. Extracts of Waltheria indica and C. frutescens had intermediate antifungal activity. Aloe vera, Tagetes patula and Capsicum annuum cultivars Red pepper, Criolla de cocina, New Mexico, showed no measurable antifungal activity. Extracts from the two most promising pepper cultivars, Datil and Carnival, are being evaluated as pre-harvest sprays in the greenhouse and as edible coatings on tomato fruit postharvest to reduce grey mold. Identification of volatiles with nematicidal activities from Sphingobacterium nematocida ZY-71-1 and control efficiency to Meloidogyne incognita J. CHANG (1), W. Mu (2), J. Xi (2), J. Song (2) (1) Sanmenxia Tobacco Company Lushixian Branch, China; (2) Zhengzhou Tobacco Research Institute of CNTC, China We isolated both fungi and bacteria, and sequenced the amplified 16S rRNA gene from bacteria and the ITS region of the ribosomal operon from fungi for microbial identification. A culture-independent analysis of the citrus microbiome using an Illumina MiSeq platform has also allowed us to reveal a more comprehensive view of the microbial communities that inhabit citrus. We hypothesize that the citrus microbiome and their associated metabolomes can be used to reduce or prevent colonization of citrus pathogens, such as Candidatus Liberibacter asiaticus (CLas). We are currently screening compounds produced by these isolated citrus microbes for inhibitory properties against CLas. We envision the discovery of bactericide producing microbes that S4.51 could be directly transferred and established on trees, or the naturally produced bactericide could be purified and applied to combat Clas and/or other pathogens. could be directly transferred and established on trees, or the naturally produced bactericide could be purified and applied to combat Clas and/or other pathogens. Fungal antagonism of bacteria isolated from Cultivated and Wild Cranberry Bogs S. SOBY (1), G. Ebadzad (1), E. Batory (1) (1) Midwestern University, U.S.A. Natural environments are characterized by a relative equilibrium that can be brought into disequilibrium by the introduction of agricultural plants. Both natural and agronomic systems should thus contain microbes that contribute to system equilibria by suppressing parasitic organisms. However, few biological niches have been systematically explored for biological control agents. Cranberry bogs in southeastern Massachusetts are a good model system for these studies because of the genetic similarity between wild and domesticated cranberry plants, and their location in the same geographic region. Soil and plant samples were collected from wild and cultivated bogs over several growing seasons to determine the microbial composition of cranberry bogs. 68 bacterial isolates were assessed for in vitro inhibition of the pathogenic fungi, Phomopsis sp., Colletotrichum sp., and Coleophoma sp. isolated from cranberry, as well as Trichoderma sp. Individual isolates varied in antifungal ability within and between fungi. Bacillus spp. were most antagonistic to these fungi, but several other bacterial genera were active against one or more of the fungi as well. In a preliminary assessment, 82% of the chosen bacterial isolates had activity against Phomopsis, 44% against Colletotrichum, 63% against Coleophoma, and 19% against Trichoderma. These results suggest that bog soils and plants harbor a large number of candidates for development as biological control agents in agriculture. Integration between 2,4-DAPG-producing Pseudomonas and fluquinconazole to control take-all disease of wheat in Chile E. MOYA ELIZONDO (1), C. Vera (1), R. Madariaga Burrows (2) (1) Universidad de Concepción, Chile; (2) Instituto de Investigaciones Agropecuarias, INIA Quilamapu, Chile Take-all disease of wheat, caused by the fungus Gaeumannomyces graminis var. tritici (Ggt) could be controlled by integrating fungicide seed treatments with 2,4-Diacetylphloroglucinol-producing Pseudomonas spp. (2,4-DAPG). This research assessed the effect on yield parameters and disease damage associated with the integrated use of seed treatments with two Chilean 2,4-DAPG-producing Pseudomonas spp. (106 ufc/g grain seeds) and the triazole fungicide fluquinconazole (1.8 mL/g grain seeds). A field experiment was established in Chillán, Chile, during crop season 2015. Experimental field plots of 2.4 m2 with winter wheat cv. Maxwell were seeded to assess combinations with or without the mentioned seed treatments, plus two untreated control (infested or non-infested with Ggt) on a randomized block design. Seed treatments where both strains of Pseudomonas were mixed with the fungicide reduced plant and root rot disease severity damage (50 and 49%, respectively) and white heads (32%), while increased yield (34%) and grain quality (2.1%) with respect to the Ggt infested control (P≤0.05). Single use of bacterial seed treatments without the fungicide was not different from this control (P≥0.05). Fluquinconazole alone had a lower grain yield (13%) and wheat test weight (1.4%) than when it was mixed with the two bacterial strains. Results suggest that integration of 2,4-DAPG-producing Pseudomonas with fungicides could reduce take-all disease and increase yield in wheat crops. Identification of volatiles with nematicidal activities from Sphingobacterium nematocida ZY-71-1 and control efficiency to Meloidogyne incognita J. CHANG (1), W. Mu (2), J. Xi (2), J. Song (2) (1) Sanmenxia Tobacco Company Lushixian Branch, China; (2) Zhengzhou Tobacco Research Institute of CNTC, China Heu (2) (1) National Institute of Crop Science, South Korea; (2) National Institue of Crop Science, South Korea Incidence of soybean frogeye leaf spot in Korea and selection for antagonistic bacteria controlling Cercospora sojina Hara I. KANG (1), I. Kang (1), H. Shim (2), D. Shin (2), J. Roh (2), S. Heu (2) (1) National Institute of Crop Science South Korea; (2) National Institue of Crop Science South Korea Soybean frogeye leaf spot caused by the fungus Cercospora sojina Hara, has been seen a steady increase in the incidence and prevalence on soybean in Korea. The nationwide survey of the occurrence of frogeye leaf spot showed that 11 out of 28 regions in 2015 had been infected with Cercospora sojina Hara. To control the disease, an isolate which showed strong inhibitory effect on the mycelial growth and conidial germination of Cercospora sojina Hara was selected among the antagonistic bacterial isolates collected from soybean grown soil and the bacterial isolate was identified as Paenibacillus polymyxa using 16S rRNA sequence analysis. Bacillus subtilis QST713(Serenade) was also inhibitory effect on the mycelial growth and conidial germination. The inhibition rates of these antagonistic bacteria were 53%, 75%, respectively. They could be effectively used for controlling soybean frogeye leaf spot. Integration between 2,4-DAPG-producing Pseudomonas and fluquinconazole to control take-all disease of wheat in Chile E. MOYA ELIZONDO (1), C. Vera (1), R. Madariaga Burrows (2) (1) Universidad de Concepción, Chile; (2) Instituto de Investigaciones Agropecuarias, INIA Quilamapu, Chile Identification of volatiles with nematicidal activities from Sphingobacterium nematocida ZY-71-1 and control efficiency to Meloidogyne incognita J. CHANG (1), W. Mu (2), J. Xi (2), J. Song (2) (1) Sanmenxia Tobacco Company Lushixian Branch, China; (2) Zhengzhou Tobacco Research Institute of CNTC, China Relative efficacy of Zambian atoxigenic Aspergillus flavus strains against aflatoxin-producing Aspergillus species J. AKELLO (1), T. Dubois (2), J. Atehnkeng (3), M. Mukanga (4), H. Njapau (5), J. Augusto (6), P. Cotty (7), R. Bandyopadhyay (3) (1) International Institute of Tropical Agriculture (IITA-Zambia), Zambia; (2) The World Vegetable Center (AVRDC) Eastern and Southern Africa, Arusha, Tanzania; (3) International Institute of Tropical Agriculture (IITA-Nigeria), Ibadan, Nigeria; (4) Zambia Agricultural Research Institute, Mount Makulu Central Research Station, Lusaka, Zambia; (5) National Institute for Scientific and Industrial Research (NISIR), Lusaka, Zambia; (6) International Institute of Tropical Agriculture (IITA-Mozambique), Nampula, Mozambique; (7) USDA-ARS, School of Plant Sciences, University of Arizona, Tucson, U.S.A. In Zambia, fields cropped to maize and groundnuts are predominantly colonized by aflatoxin producing Aspergillus flavus S-morphotypes and A. parasiticus. Consequently, Zambian staple foods are often contaminated with high aflatoxin levels (>20 ppb) while in the field and during storage. This study aimed at identifying the best atoxigenic A. flavus L-strains for use as biocontrol agents. In vitro, the most toxigenic L (25) and SBG (10) strains were selected out of the entire isolate library based on qualitative and quantitative TLC assessment. Competition assays were performed by co- inoculating 14 atoxigenic candidate strains and the 35 selected toxigenic ones on sterile maize grains in sterile vials. All the 14 atoxigenic strains outcompeted the toxigenic ones and reduced aflatoxin production by >96% in vitro. Eight of the 14 atoxigenic strains were selected and formulated into two mixes, with each mix comprising of 4 strains. Each mix was applied to maize and groundnut fields between 2012 and 2015 at a rate of 10 kg/ ha, 2-3 weeks before flowering. Treating fields with the two mixes altered Aspergillus soil community structure and significantly reduced aflatoxin levels in maize and groundnuts by 89.6% compared to untreated ones. Thus, the 8 atoxigenic strains hold promise for fighting aflatoxin burden in maize and groundnut. Adopting such strains to address aflatoxin could improve grain nutritional quality and safety for better health of the Zambian populace. Incidence of soybean frogeye leaf spot in Korea and selection for antagonistic bacteria controlling Cercospora sojina Hara I. KANG (1), I. Kang (1), H. Shim (2), D. Shin (2), J. Roh (2), S. Stopping the cereal killer: Exploring biological control to mitigate Fusarium head blight of wheat A. BEHARI (1), G. Kuldau (1) (1) Pennsylvania State University, U.S.A. Fusarium head blight (FHB) is a devastating fungal disease impacting wheat and other cereal production globally. Exacerbating the impact of disease is the in planta production of the toxic fungal secondary metabolite and virulence factor, deoxynivalenol (DON). DON is an inhibitor of the eukaryotic ribosome thereby blocking protein translation, and in toxicological studies has been found to affect the gastrointestinal, immune, and endocrine systems of humans and animals. Preventing DON accumulation in grain ultimately begins with preventing infection by FHB causing Fusarium species. Current mitigation strategies to combat FHB include the use of moderately resistant varieties of wheat, crop rotations with non-hosts, and fungicides, but even in conjunction these strategies provide only minimal to moderate control when environmental conditions are conducive to disease. This research aims to identify and characterize biological control agents to mitigate FHB through media based screening of samples from different production systems and geographic locations for Fusarium graminearum inhibiting and DON degrading microbes. Exploring multiple sources including various soils, plant material, silage, and haylage will allow for a diverse range of microbes exhibiting the aforementioned capabilities to be studied for their specific mechanisms, potential common genetic basis, and potential use in microbial consortia for disease control. S4.52 Characterization and distribution of natural populations of Cryptococus flavescens across the United States F. ROTONDO (1), B. McSpadden Gardner (2), P. Paul (1) (1) The Ohio State University, Wooster, OH, U.S.A.; (2) The Ohio State University, Wooster, U.S.A. Fusarium head blight (FHB) is an important disease of small grains caused primarily by Fusarium graminearum. This disease causes grain yield and quality losses under conducive conditions. The yeast Cryptococcus flavescens is an effective biological control agent of FHB on wheat. In this study, the biogeography and genetic diversity of natural populations of C. flavescens across the United States were characterized. Wheat spikes samples were collected at flowering and at harvest and assayed for C. flavescens using specific PCR primers designed on the heat shock protein hps70KDa. This assay is 1,000 times more selective for C. flavescens than other Cryptococcus spp. Samples with > 106 cells/g of wheat tissue were subjected to microbial isolation, and colonies with C. flavescens-like morphology were selected and characterized using amplified ribosomal DNA restriction analysis (ARDRA) profiling and multilocus genotyping. The results showed that C. flavescens was widespread across the country, albeit at low incidence (approximately 10%, n = 320). Inhibition of Penicillium expansum growth on Golden Delicious apples by wild yeasts from various plant materials V. TOURNAS (1), E. Katsoudas (2) Inhibition of Penicillium expansum growth on Golden Delicious apples by wild yeasts from various plant materials V. TOURNAS (1), E. Katsoudas (2) Inhibition of Penicillium expansum growth on Golden Delicious apples by wild yeasts from various plant ma V. TOURNAS (1), E. Katsoudas (2) (1) Center for Food Safety and Applied Nutrition/FDA, U.S.A.; (2) Northeast Regional Lab/ORA/FDA, U.S.A. ( ), ( ) (1) Center for Food Safety and Applied Nutrition/FDA, U.S.A.; (2) Northeast Regional Lab/ORA/FDA, U.S.A. ( ), ( ) (1) Center for Food Safety and Applied Nutrition/FDA, U.S.A.; (2) Northeast Regional Lab/ORA/FDA, U.S.A. Wild yeast strains previously isolated from various fruits were tested for their ability to inhibit Penicillium expansum in vivo (on Golden Delicious apples) in an effort to determine their potential as biocontrol agents (BCAs). Tests were performed at room temperature (~21°C) for up to 2 weeks and at refrigeration (3°C) for up to 2 months. Candida oleophila was used as positive BCA control. All strains tested showed some degree of biocontrol activity against P. expansum. At room temperature, activity (lesion size reduction) ranged between 37 and 50% during the first week. After 14 days of incubation, decays on yeast-treated apples were slightly smaller (16-19%) than those on apples treated with P. expansum alone. At refrigeration, lesion reduction ranged between 54 and 76% in the first month of storage and between 60 and 76% after 2 months of incubation. Decay incidence was 100% in apples stored at room temperature and 25-62% in those kept under refrigeration. The inhibitory activities of the wild yeasts were similar to those exhibited by C. oleophila. These strains showed potential as BCAs against P. expansum on stored apples. Evaluation of potential biocontrol agents against fungal plant pathogens affecting strawberry R. BITTER (1), G. Holmes (2) Stopping the cereal killer: Exploring biological control to mitigate Fusarium head blight of wheat A. BEHARI (1), G. Kuldau (1) (1) Pennsylvania State University, U.S.A. Kwak (2) Selection and characterization of probiotics for rye silage C JEON (1) H Kim (1) J Kim (1) Y Kwak (2) p y g C. JEON (1), H. Kim (1), J. Kim (1), Y. Kwak (2) (1) Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea; (2) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea ( ), ( ), ( ), ( ) (1) Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea; (2) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea Silage is feed of livestock. Forage crops are at risk from infection by mycotoxins producing fungi. Rye has considerate as a highly quality of forage. After heading stage, the biomass of rye increased up to 30%. However, lignification caused low fermentation efficiency and coefficient digestibility. This study was performed to develop probiotics which improve quality of silage in nutrient aspect. During the fermentation period of the rye silage, dominant microorganisms were selected. Those were confirmed antimicrobial activities against fungi and were tested acidification ability. The isolates confirmed ability of fibrinolysis through the four enzyme (cellulase, xylanase, esterase, chitinase). The final selected strains were added to the rye silage and then confirmed the effect by real-time PCR in antifungal activity. Total 180 isolates were isolated from the rye silages during various fermentation period. Among 180, 8 isolates showed antimicrobial activities against Fusarium moniliforme. two isolates among antimicrobial activities isolates showed strong acidification ability. Also a single isolate was selected that was effective all four enzymes. In real-time PCR results, the mycotoxin gene was detected significantly low in the rye silage, which added antimicrobial activity isolate. In case developed microorganism product was applied at other stock feed as well as rye silage, production of high quality forage will expand. Inhibition of Penicillium expansum growth on Golden Delicious apples by wild yeasts from various plant materials V. TOURNAS (1), E. Katsoudas (2) Stopping the cereal killer: Exploring biological control to mitigate Fusarium head blight of wheat A. BEHARI (1), G. Kuldau (1) (1) Pennsylvania State University, U.S.A. Spikes were the preferred tissue of colonization. When present, populations of C. flavescens ranged from 105-107 cells/g of tissue. Plant growth stage influenced C. flavescens colonization. This work confirms that C. flavescens is found across a broad geographical area in close association with wheat. Further, it demonstrates that C. flavescens is a non-invasive biological control option for FHB in the regions sampled. Biocontrol of Fusarium head blight with Bacillus spp. K. BOWEN (1), J. Anderson (1), K. Liu (1) (1) Auburn University, U.S.A. Fusarium head blight, caused by Fusarium graminearium, has become a cause of concern in many portions of Alabama. This disease was severe in Southern AL, due to frequent rainfall during flowering in 2015. Infection of F. graminearium reduces quantity and quality of yield by infecting the kernels of many grain crops; it also produces the mycotoxin, deoxynivalenol (DON). Wheat grain was collected from fungicide treated plots and found to be infested with Fusarium spp. Cultures were established from single spores and species were determined from morphology of spores and color of the fungus when mature. Biocontrol potential of selected Bacillus strains against F. graminearum was assayed using a dual culture method. Based on inhibition of fungal growth and spore production, strains were selected that were most suppressive against F. graminearum. In order to test the effectiveness of these strains in the field, five Bacillus strains will be applied to wheat heads during flower, and disease intensity will be rated 3 weeks later. Results of the field study will be reported. Any reduction in Fusarium head blight by a biological agent might contribute to overall management of this disease. Selection and characterization of probiotics for rye silage C. JEON (1), H. Kim (1), J. Kim (1), Y. Kwak (2) (1) Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea; (2) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea Selection and characterization of probiotics for rye silage C. JEON (1), H. Kim (1), J. Kim (1), Y. Kwak (2) (1) Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea; (2) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea Selection and characterization of probiotics for rye silage C. JEON (1), H. Kim (1), J. Kim (1), Y. Evaluation of potential biocontrol agents against fungal plant pathogens affecting strawberry R. BITTER (1), G. Holmes (2) (1) California Polytechnic State University, U.S.A.; (2) Cal Poly Strawberry Center, U.S.A. Evaluation of potential biocontrol agents against fungal plant pathogens affecting strawberry R. BITTER (1), G. Holmes (2) (1) California Polytechnic State University, U.S.A.; (2) Cal Poly Strawberry Center, U.S.A. ( ), ( ) (1) California Polytechnic State University, U.S.A.; (2) Cal Poly Strawberry Center, U.S.A. Botrytis cinerea, Fusarium oxysporum f. sp. fragariae and Macrophomina phaseolina are important pathogens that limit commercial strawberry production in California. The objective of this study was to evaluate eight species of bacteria (19 strains) for their ability to inhibit mycelial growth of all three pathogens in vitro. Percent inhibition of fungal growth due to the presence of each bacterium was determined relative to growth in the absence of each bacterium. Three strains of Bacillus amyloliquefaciens provided the greatest inhibition of B. cinerea, F. oxysporum f. sp. fragariae and M. phaseolina by an average of 43.7%, 47.8%, 42.7%, respectively, between replications. Significant differences in fungal inhibition were observed between strains of the same bacterial species, suggesting inhibition is strain-dependent. Growth inhibition caused by five B. subtilis strains varied from 39.5% to 0.6% for B. cinerea, 43.1% to 0.6% for F. oxysporum f. sp. fragariae, and 39.8% to 5.8% for M. phaseolina. Four B. licheniformis strains also displayed similar levels of strain-dependent inhibition. Bacterial antagonists that limit the growth of these soilborne and foliar pathogens could provide an alternative to the use of chemical fumigants and fungicides, or could be utilized to enhance the effectiveness of conventional or organic disease control treatments such as anaerobic soil disinfestation. Effects of Clonostachys species on charcoal rot disease caused by Macrophomina phaseolina in soybean A. ADESEMOYE (1), H. Wei (1) (1) University of Nebraska Lincoln, U.S.A. Curvularia prsadii as a biological control agent for barnyard grass (Echinochloa crus-galli) in rice fields Y. SHABANA (1), A. Abu Tabl (1) (1) Mansoura University, Egypt Curvularia prsadii as a biological control agent for barnyard grass (Echinochloa crus-galli) in rice fields Y. SHABANA (1), A. Abu Tabl (1) (1) Mansoura University, Egypt Echinochloa crus-galli is one of the most important damaging weeds of rice paddies. Three isolates of Curvularia prasadii (CP01, CP02, and CP0 3) were isolated from the rice weed, E. crus-galli in three governorates (Dakahlia, Kafr El-Sheik, and El-Garbeia) in Egypt in three consecutive growing seasons (from 2012 to 2014). The biocontrol activity of these fungal isolates was determined on the weed seedlings in the greenhouse using spore suspension of 106 conidia/ml. All of the three isolates tested produced 40-60% disease severity on the weed seedlings with no significant differences among them. Host range studies were conducted to determine the host specificity and safety of the three isolates of the bio agent, C. prsadii against 23 non-target plant species and cultivars of economic importance grown in Egypt. No disease symptoms appeared on any of the non-target plants tested. The optimum nutritional and physical conditions for the production of the biocontol agents were determined and will be discussed. Biocontrol of Tubakia leaf blight by Paenibacillus peoriae(HB774) H. YUN (1), Y. Kim (1) (1) Seoul National University, South Korea South Korea has shown decreasing proportion of coniferous plants and increasing proportion of deciduous plants due to the effect of global warming. With the growing significance of deciduous forests, the national forest damage by ongoing Tubakia leaf blight of the oak is more severe than before. In response to the current critical forest situation of Korea, this study aims to develop the efficient biocontrol agent of the Tubakia leaf blight. Among 30 bacteria isolated from soil and plants, HB774 showed the highest inhibition rate (85.8%) of the fungal mycelial growth, which was selected as a potentially effective biocontrol agent. As a result of the 16S ribosomal DNA sequence analysis, a gram-positive bacterial isolate was identified as Paenibacillus peoriae and deposited as a patent strain in Rural Development Administration of Korea. This isolate inhibited the mycelial growth by 69.7% and 75.6% at the bacterial concentrations of 1 × 106 CFU/ml and 1 × 108 CFU/ml, respectively, of which the inhibitory rates were identical to or significantly higher than those of the fungicides, propiconazole and iminonctadine-triacetate. Effects of Clonostachys species on charcoal rot disease caused by Macrophomina phaseolina in soybean A. ADESEMOYE (1), H. Wei (1) (1) University of Nebraska Lincoln, U.S.A. ( ), g ( ) (1) Hampton Roads Agricultural Research and Extension Center, Virginia Tech, U.S.A.; (2) Hampton Roads Agricultural Research and Extension Center, Virginia Tech, U.S.A. Calonectria pseudonaviculata (Cps) is the causal agent of boxwood blight. This disease first observed in USA in 2011 has significantly impacted the nursery and landscape industries. The objective of this study was to develop biologically-based, long-term solutions to managing this emerging disease at production facilities and in the landscapes. Over 1000 bacterial strains recovered from irrigation water in Virginia, USA were screened for their potential as biological control agents against microsclerotia of Cps. Microsclerotia were harvested from potato dextrose agar cultures and plated on nutrient agar in 48-well plates. Two days later, fresh bacterial cultures grown in nutrient broth were added to the culture of Cps. Three replicate wells per bacterial strain were used. Sterile nutrient broth was added to three wells per plate as controls. Microsclerotia germination and resultant mycelial growth was measured based on a 0-to-5 rating scale at five and ten days after adding bacterial cultures. It was found that approximately 30% of screened bacterial strains greatly suppressed microsclerotial germination and mycelial growth with average ratings of one or less. These bacteria are promising biological control agents (BCAs) for boxwood blight and warrant further evaluation using detached leaf assay, greenhouse and field in-vivo tests. Characterization and distribution of natural populations of Cryptococus flavescens across the United States K. PARK (1), J. Shin (1), K. Myeong (1), S. Lee (1), S. Lee (1), M. Kim (1), K. Kim (1) (1) National Institute of Horticultural and Herbal Science, South Korea Fusarium head blight (FHB) is an important disease of small grains caused primarily by Fusarium graminearum. This disease causes grain yield and quality losses under conducive conditions. The yeast Cryptococcus flavescens is an effective biological control agent of FHB on wheat. In this study, the biogeography and genetic diversity of natural populations of C. flavescens across the United States were characterized. Wheat spikes samples were collected at flowering and at harvest and assayed for C. flavescens using specific PCR primers designed on the heat shock protein hps70KDa. This assay is 1,000 times more selective for C. flavescens than other Cryptococcus spp. Samples with > 106 cells/g of wheat tissue were subjected to microbial isolation, and colonies with C. Effects of Clonostachys species on charcoal rot disease caused by Macrophomina phaseolina in soybean A. ADESEMOYE (1), H. Wei (1) (1) University of Nebraska Lincoln, U.S.A. Charcoal rot disease caused by Macrophomina phaseolina in soybean greatly disrupt stand establishment and cause high yield losses. The pathogen was estimated to cause yield losses of over 270,000 bushels in Nebraska (NE) in 2011. Chemicals are used against many diseases but not consistently effective in the management of soilborne diseases such as charcoal rot. In 2014 and 2015, surveys were conducted in soybean fields in NE. Infected S4.53 samples were collected, plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated in the dark for 4 days at 25°C. Morphology was examined and genomic DNA was extracted from isolates using MoBio microbial isolation kits. Two genes of interest, ITS and β- tubulin, were PCR-amplified. Amplicons were sequenced at the University of California Riverside Gencore. The pathogen M. phaseolina was identified but different species of Clonostachys (anamorph: Bionectria spp.), including isolates identical to C. rhizophaga, C. byssicola, and C. rosea were also recovered from the samples. It appeared from the field samples that Clonostachys spp. had negative impacts on the population of M. phaseolina. In repeated in vitro assay as well as in vivo co-inoculation through Rolled Towel Assays indicated suppression of M. phaseolina by Clonostachys spp. There is need for further studies to better understand the biocontrol efficacy of each Clonostachys sp. as well as the mechanisms involved. samples were collected, plated onto potato dextrose agar amended with 0.01% tetracycline (PDA-tet) and incubated in the dark for 4 days at 25°C. Morphology was examined and genomic DNA was extracted from isolates using MoBio microbial isolation kits. Two genes of interest, ITS and β- tubulin, were PCR-amplified. Amplicons were sequenced at the University of California Riverside Gencore. The pathogen M. phaseolina was identified but different species of Clonostachys (anamorph: Bionectria spp.), including isolates identical to C. rhizophaga, C. byssicola, and C. rosea were also recovered from the samples. It appeared from the field samples that Clonostachys spp. had negative impacts on the population of M. phaseolina. In repeated in vitro assay as well as in vivo co-inoculation through Rolled Towel Assays indicated suppression of M. phaseolina by Clonostachys spp. There is need for further studies to better understand the biocontrol efficacy of each Clonostachys sp. as well as the mechanisms involved. Trichoderma asperellum survival and parasitism of Sclerotinia sclerotiorum sclerotia in different soil types M. LOBO (1), A. Geraldine (2), F. Yoshida (3), E. Civardi (3), E. Effects of Clonostachys species on charcoal rot disease caused by Macrophomina phaseolina in soybean A. ADESEMOYE (1), H. Wei (1) (1) University of Nebraska Lincoln, U.S.A. Barbosa (1) (1) Embrapa (Brazilian Agricultural Research Corporation), National Center for Rice and Beans Research, Santo Antônio de Goiás, Brazil; (2) Instituto Federal Goiano, Rio Verde, Brazil; (3) Universidade Federal de Goiás, Goiânia, Brazil Trichoderma asperellum survival and parasitism of Sclerotinia sclerotiorum sclerotia in different soil types M. LOBO (1), A. Geraldine (2), F. Yoshida (3), E. Civardi (3), E. Barbosa (1) (1) Embrapa (Brazilian Agricultural Research Corporation), National Center for Rice and Beans Research, Santo A Federal Goiano, Rio Verde, Brazil; (3) Universidade Federal de Goiás, Goiânia, Brazil Effectiveness of bioagent Trichoderma asperellum is influenced by physical, chemical, biological and environmental factors, but there is no clear idea of how such variables affect antagonist endurance. This study assessed the survival of T. asperellum and the parasitism of Sclerotinia sclerotiorum sclerotia in different Brazilian soils. The test was carried out in a completely randomized design, arranged in a 4 × 6 factorial schedule with five repetitions. Experimental factors comprised six soils of distinct texture and fertility profiles, and four biocontrol treatments with three T. asperellum isolates and a water control. Plots consisted of 12cm x 22cm x 3cm transparent plastic containers, with sclerotia distributed over 300g of soil. After spraying with 2.0 × 1012 T. asperellum conidia ha–1 and soil moisture adjusted to 100% field capacity, treatments were incubated at 20ºC with 12h photoperiod for 28 days. Weakly assessments considered sclerotia parasitism, area under antagonist survival curve (AUASC), culturable fungi and bacteria, and bacterial diversity estimated with Biolog Ecoplates. Principal component analysis showed that parasitism of S. sclerotiorum sclerotia was proportional to T. asperellum AUASC. In contrast, antagonist survival was negatively affected by soil pH, bacterial density and diversity, organic matter, Ca, Mg and K contents. Biocontrol efficiency varied according to soil source. In-vitro control of Calonectria pseudonaviculata by bacteria recovered from irrigation water X. YANG (1), C. Hong (2) In-vitro control of Calonectria pseudonaviculata by bacteria recovered from irrigation water X. YANG (1), C. Hong (2) (1) Hampton Roads Agricultural Research and Extension Center, Virginia Tech, U.S.A.; (2) Hampton Roads Agricultural Research and Extension Center, Virginia Tech, U.S.A. X. YANG (1), C. Hong (2) (1) Hampton Roads Agricultural Research and Extension Center, Virginia Tech, U.S.A.; (2) Hampton Roads Agricultural Research and Extension Center, Virginia Tech, U.S.A. Effects of Clonostachys species on charcoal rot disease caused by Macrophomina phaseolina in soybean A. ADESEMOYE (1), H. Wei (1) (1) University of Nebraska Lincoln, U.S.A. flavescens-like morphology were selected and characterized using amplified ribosomal DNA restriction analysis (ARDRA) profiling and multilocus genotyping. The results showed that C. flavescens was widespread across the country, albeit at low incidence (approximately 10%, n = 320). Spikes were the preferred tissue of colonization. When present, populations of C. flavescens ranged from 105-107 cells/g of tissue. Plant growth stage influenced C. flavescens colonization. This work confirms that C. flavescens is found across a broad geographical area in close association with wheat. Further, it demonstrates that C. flavescens is a non-invasive biological control option for FHB in the regions sampled. The potential use of a Cladosporium sp. as a biological control agent for white mold caused by Sclerotinia sclerotiorum B. ROBINSON (1), R. Cating (2), K. Frost (2) (1) Oregon State University; Hermiston High School, U.S.A.; (2) Oregon State University, U.S.A. The potential use of a Cladosporium sp. as a biological control agent for white mold caused by Scler B. ROBINSON (1), R. Cating (2), K. Frost (2) (1) Oregon State University; Hermiston High School, U.S.A.; (2) Oregon State University, U.S.A. The potential use of a Cladosporium sp. as a biological control agent for white mold caused by Sclerotinia sclerotiorum B. ROBINSON (1), R. Cating (2), K. Frost (2) 1) O S U i i H i Hi h S h l U S A (2) O S U i i U S A Sclerotinia scelortiorum (Lib.) de Bary is a pathogenic ascomycete known to cause disease in more than 400 plant species, including white mold in potato. In this study, a fungal contaminant found associated with S. sclerotiorum that appeared to inhibit S. scelortiorum growth was isolated, sub- cultured, and identified. Morphological characteristics and sequence data from internal transcribed spacers 1 and 2, 5.8s rDNA, and Actin and Calmodulin genes were consistent with the unknown fungus belonging to the Cladisporium genus. A bioassay was used to examine inhibition of S. sclerotiorum growth by the Cladisporium isolate. A 5mm plug of the Cladisporium isolate was plated on potato dextrose agar and left to grow for 72 hours before a 5mm plug of S. sclerotiorum was plated 50mm from the Cladisporium plug. Isolates of S. sclerotiorum and Cladisporium were plated alone as controls. The plates (n=10 per treatment) were incubated for 4 days at room temperature with a 12:12 photoperiod. Radial growth of S. sclerotiorum mycelia was measured and the experiment was repeated in entirety one time. Averaging over both experiments, the radii of S. sclerotiorum plated with the unknown isolate was reduced by 34.1 mm (Paired t-test: t = 27.4, df = 19, p < 0.001) when compared to the radii of the S. sclerotiorum plated alone. These data indicate that the Cladisporium isolate has potential use as a bio-control agent against S. sclerotiorum and should be further evaluated in vivo. Infection duration of entomopathogenic fungi Aspergillus spp. and Fusarium spp. against Bemisia tabaci W. Anwar (1), H. Mushtaq (1), M. Haider (1), A. Shahid (1), U. Hameed (1), M. Ur-Rehman (1) (1) Institute of Agricultural Sciences, University of the Punjab, Lahore, Pakistan, 54590, Pakistan Entomopathogenic fungi are emerging as biocontrol of insects and new fungi are isolated from different insects with passage of time. Curvularia prsadii as a biological control agent for barnyard grass (Echinochloa crus-galli) in rice fields Y. SHABANA (1), A. Abu Tabl (1) (1) Mansoura University, Egypt In the simultaneous treatment and pre-treatment of HB774, its control efficacies were high with 80-90%, showing no significant differences from those of propiconazole and iminonctadine-triacetate. This suggests P. peoriae HB774 has a high potential to be developed as an efficient biocontrol agent against the Tubakia leaf blight on oaks. S4.54 Acaromyces ingoldii inhibits the laurel wilt pathogen, Raffaelea lauricola in vitro R. OLATINWO (1), S. Fraedrich (2) (1) USDA Forest Service, Southern Research Station, U.S.A.; (2) USDA Forest Service Acaromyces ingoldii inhibits the laurel wilt pathogen, Raffaelea lauricola in vitro R. OLATINWO (1), S. Fraedrich (2) (1) USDA Forest Service, Southern Research Station, U.S.A.; (2) USDA Forest Service, Southern Research Station, U.S Laurel Wilt is a destructive disease of redbay (Persea borbonia and other species in the laurel family (Lauraceae) in the southeastern United States (US)). The disease is caused by Raffaelea lauricola, a fungal symbiont of the redbay ambrosia beetle (Xyleborus glabratus. The beetle and the fungus were introduced into the US probably with wood packing materials. This investigation was prompted by antifungal activities demonstrated by an Acaromyces species isolated from a loblolly pine bolt in Louisiana during routine laboratory microbial isolations Our objective was to evaluate the inhibitory property of the Acaromyces species against R. lauricola. We obtained isolates of R. lauricola from fresh laurel wilt infected tissues collected from sassafras trees in Louisiana and Texas, and evaluated the competitive ability of the Acaromyces and the fungistatic activities of secondary metabolites produced by the fungus against R. lauricola in vitro. Results showed a strong inhibition of R. lauricola mycelia growth by the Acaromyces species. Growth inhibition was significant as pigmented secondary metabolites produced by Acaromyces diffuses through the media. Only minimal direct competition was observed between the two fungi. R. lauricola isolates inoculated 7-day after Acaromyces, showed no growth and no spore germination. Future experiments will examine the identity, efficacy, and potential benefits of secondary metabolite(s) produced by the Acaromyces species. Applications of Bacillus amyloliquefaciens PMB01 for managing Fusarium wilt of cucumber Applications of Bacillus amyloliquefaciens PMB01 for managing Fusarium wilt of cucumber W. DENG (1), H. Chou (2), I. Yang (3), Y. Lin (4), H. Wang (2), S. Chuang (2), T. The potential use of a Cladosporium sp. as a biological control agent for white mold caused by Sclerotinia sclerotiorum B. ROBINSON (1), R. Cating (2), K. Frost (2) (1) Oregon State University; Hermiston High School, U.S.A.; (2) Oregon State University, U.S.A. The aim of present study was to observe the steps of infection of different Aspergillus and Fusarium species and their duration of infection against Bemisia tabaci. To observe the infection duration, spore suspension of different species of Aspergillus and Fusarium already isolated from insects were used on nymphal stage of whitefly and data regarding duration of spore germination, penetration, colonization and infection was determined under control conditions. It was observed that spores of Aspergillus species germinate quickly as compare to Fusarium species. There was no significant difference in spore germination on species level. Penetration time of Aspergillus and Fusarium species vary and colonization and infection duration of Aspergillus species was less as compared to Fusarium species against nymphal stage of whitefly. In this study, it was observed that Aspergillus is more aggressive as compare to Fusarium species and also dependent on isolates of insect associated fungi. Further studies required on genetic level that makes entomopathogenic fungi more aggressive on different stages of infection process. Curvularia prsadii as a biological control agent for barnyard grass (Echinochloa crus-galli) in rice fields Y. SHABANA (1), A. Abu Tabl (1) (1) Mansoura University, Egypt Huang (2) (1) National Chung Hsing University, Taichung, Taiwan; (2) Kaohsiung District Agricultural Research and Extension Station, Pingtung, Taiwan; (3) Taiwan Agricultural Mechanization Research and Development Center, Taipei, Taiwan; (4) National Pingtung University of Science and Technology, Pingtung, Taiwan ( ), ( ), g ( ), ( ), g ( ), g ( ), g ( ) (1) National Chung Hsing University, Taichung, Taiwan; (2) Kaohsiung District Agricultural Research and Extension Station, Pingtung, Taiwan; (3) Taiwan Agricultural Mechanization Research and Development Center, Taipei, Taiwan; (4) National Pingtung University of Science and Technology, Pingtung, Taiwan Cucumber Fusarium wilt, caused by Fusarium oxysporum f. sp. cucumerinum (Foc), is the most devastating disease on cucumber. Currently, no effective chemicals are available for disease control; however, prior studies showed that antagonistic rhizobacteria can reduce the disease severity, suggesting that biocontrol agents might be potential solutions for the management of Fusarium wilt disease. In this study, soil bacteria with inhibitory activity on in-vitro Foc growth were isolated. Among them, Bacillus amyloliquefaciens PMB01 showed the best antagonistic activity against Foc in confrontation assays. The efficacy of PMB01 on controlling Fusarium wilt disease of cucumber (FWC) was evaluated in field experiments, and the results showed that applications of the formulated PMB01 reduced the disease severity of FWC down to less than 15%. Prior research also revealed that the formulated PMB01 effectively controlled bacterial wilt disease of tomato (BWT), caused by Ralstonia solanacearum, in field trials. Whole genome shotgun sequencing showed that PMB01 harbors genes involved in the biosynthesis of cyclic lipopeptides, bacillibactin, and bacilysin, which might collectively contribute to its biocontrol activity. Toxicology testing demonstrated that PMB01 was non-toxic to mammals. In 2016, the biopesticide Saviour® containing B. amyloliquefaciens PMB01 as the active ingredient was developed and commercialized in Taiwan for integrated management of BWT and FWC. Field Performance of Endophytic Actinomycetes in Relation to Plant Growth Promotion and Biological Control of Fusarium oxysporum a Pathogen of Tomato K EL TARABILY (1) A Alkh j h (1) Field Performance of Endophytic Actinomycetes in Relation to Plant Growth Promotion and Biological Control of Fusarium oxysporum a Pathogen of Tomato K. EL-TARABILY (1), A. Alkhajeh (1) (1) United Arab Emirates University, Uae Evaluation of the biocontrol potential of different Pseudomonas species in suppressing Pythium root rot in petunia plugs D. MARTIN (1), M. Jones (2), C. Taylor (2), F. Peduto Hand (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A. SDS frequently appears in circular to oblong patches in a soybean field and are often found unevenly distributed across the same field, while conditions in the rest of the field may appear to be naturally suppressive to the disease. In the SDS-suppressive areas, soybean plants may be less affected by the pathogen because of the activity of other soil microbes. There is a lack of comprehensive studies that investigate the role of soil inhabiting bacteria and fungi in affecting the development of the SDS. By coupling metabarcoding with paired-end Illumina MiSeq sequencing, we identified key microbial taxa involved in the suppression or promotion of the SDS pathogen. Statistical analyses revealed significant differences in fungal and bacterial community composition between suppressive and conducive soils. A microbial consortia involving members of Actinobacteria, Fusarium oxysporum, Trichoderma, Penicillium, Metacordycpes, and Purpureocillium were found consistently associated with SDS suppression. In contrast, members of the Fusarium solani sp. complex were frequently present in conducive soils. Isolation of endophytic bacteria associated with roots from citrus infected with Phytophthora nicotianae A. OLIVARES (1), J. Hernandez-Mendoza (1), S. Nelson (2), V. Ancona (1) (1) Texas A&M Kingsville Citrus Center, U.S.A.; (2) Texas A&M Kingsville Citrus Center, U.S.A. Isolation of endophytic bacteria associated with roots from citrus infected with Phytophthora nicotianae A. OLIVARES (1), J. Hernandez-Mendoza (1), S. Nelson (2), V. Ancona (1) (1) Texas A&M Kingsville Citrus Center, U.S.A.; (2) Texas A&M Kingsville Citrus Center, U.S.A. Citrus orchards in Texas are greatly affected by Phytophthora foot and root rot. Because the presence of antagonistic bacteria associated with plant roots may play a crucial role in root health and disease development we decided to study the effect of Phytophthora infection on endophytic bacterial communities associated with citrus roots. Roots from three Phytophthora infected and three uninfected grapefruit trees were randomly selected to isolate culturable bacteria. Roots were collected from soil samples taken from each tree at four quadrants, 1 meter away from the trunk at a depth of 15 centimeters. Serial dilutions of macerated root tissues were plated onto nutrient agar, tryptone yeast, King’s B and actinomycetes isolation mediums and incubated at three different temperatures. A significantly higher number of cultivable bacteria were present in roots from infected trees versus uninfected trees. In addition, a total of 140 and 119 morphologically distinct bacterial colonies were recovered and isolated from Phytophthora infected and uninfected trees respectively. Evaluation of the biocontrol potential of different Pseudomonas species in suppressing Pythium root rot in petunia plugs D. MARTIN (1), M. Jones (2), C. Taylor (2), F. Peduto Hand (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A. Microbiome networks: A systems framework for identifying candidate microbial assemblages for disease management R. POUDEL (1), A. Jumpponen (2), D. Schlatter (3), T. Paulitz (3), B. McSpadden Gardener (4), L. Kinkel (5), K. Garrett (1) (1) Plant Pathology Department, Institute for Sustainable Food Systems, and Emerging Pathogens Institute, University of Florida, Gainesville, FL, U.S.A.; (2) Division of Biology and Ecological Genomics Institute, Kansas State University, Manhattan, KS, U.S.A.; (3) USDA-ARS, Wheat Health, Genetics, and Quality Research Unit, Washington State University, Pullman, WA, U.S.A.; (4) Department of Plant Pathology, The Ohio State University, Wooster, OH, U.S.A.; (5) Department of Plant Pathology, University of Minnesota, St. Paul, MN, U.S.A. Network models of soil and plant microbiomes present new opportunities for enhancing disease management, but also challenges for interpretation. We present a framework for interpreting microbiome networks, illustrating how the observed structure of networks can be used to generate testable hypotheses about candidate microbes affecting plant health. The framework includes four types of network analyses. “General network analysis” identifies candidate taxa for maintaining an existing microbial community. “Host-focused analysis” includes a node representing a plant response such as yield, identifying taxa with direct or indirect links to that node, interpreted as either beneficial or detrimental to plant health. “Pathogen-focused analysis” identifies taxa with direct or indirect links to taxon nodes known a priori to represent pathogens, which can be interpreted as agonists and antagonists, respectively. “Disease-focused analysis” identifies key nodes for both plant and pathogen responses. We illustrate the interpretation of network structure with analyses of two microbiomes: the oak phyllosphere and soil associated with the presence or absence of infection by Rhizoctonia solani. Such network analyses can be used to further characterize microbial communities and associated conditions involved in the suppression of plant pathogens, the biofertilization of crop plants, and/or the expression of host resistance in crop plants. Linking microbial taxa in SDS-suppressive soils of Soybean fields ( ), y ( ), ( ), ( ), ( ) (1) Southern Illinois University, U.S.A.; (2) Southern Illinois University, U.S.A.; (3) Iowa state University, U.S.A.; (4) Univesrity of Minnesota, U.S.A. Sudden Death Syndrome (SDS) is one of the most important diseases of soybean in the Midwest. SDS is caused by the soilborne fungus Fusarium virguliforme. Field Performance of Endophytic Actinomycetes in Relation to Plant Growth Promotion and Biological Control of Fusarium oxysporum a Pathogen of Tomato K. EL-TARABILY (1), A. Alkhajeh (1) (1) United Arab Emirates University Uae Four endophytic actinomycete strains identified as Actinoplanes missouriensis, Streptomyces olivaceiscleroticus, S. globosus and Dactylosporangium aurantiacum were obtained in a previous study from surface-disinfested tomato roots. The performance of these four strains was evaluated under field conditions to determine their potential to promote plant growth and to reduce the incidence of wilt disease of tomato caused by Fusarium oxysporum f.sp. lycopersici. When applied individually or in combination to tomato seedlings, the four strains significantly promoted growth and reduced the incidence of the disease. Individually the performance level of A. missouriensis was relatively the best followed by S. olivaceiscleroticus, then by S. globosus and then by D. aurantiacum. The combination of the four strains resulted in significantly better suppression of the disease and growth promotion, compared to individual strains. The ACC deaminase-producing isolates (ACC+) (A. missouriensis and S. olivaceiscleroticus) were significantly more effective in reducing the incidence of the disease compared to ACC deaminase-non-producing (ACC–) isolates (S. globosus and D. aurantiacum). The overall performance of the endophytic strains is promising and could replace the fungicides currently in use for the control of Fusarium diseases. The results also showed the potential to enhance the biocontrol agent’s performance by including the ACC deaminase ability into the biocontrol strains. S4.55 Assessment of three Trichoderma species to protect cabbage from Clubroot A. BOTERO (1), A. Cotes (2), C. García (3) (1) Universidad Nacional de Colombia, Colombia; (2) CORPOICA, Colombia; (3) Universidad Nacional deColombia, Colombia Assessment of three Trichoderma species to protect cabbage from Clubroot A. BOTERO (1), A. Cotes (2), C. García (3) (1) Universidad Nacional de Colombia, Colombia; (2) CORPOICA, Colombia; (3) Universidad Nacional Assessment of three Trichoderma species to protect cabbage from Clubroot A. BOTERO (1), A. Cotes (2), C. García (3) Assessment of three Trichoderma species to protect cabbage from Clubroot A. BOTERO (1), A. Cotes (2), C. García (3) (1) Universidad Nacional de Colombia, Colombia; (2) CORPOICA, Colombia; (3) Universidad Nacional deColombia, C Universidad Nacional de Colombia, Colombia; (2) CORPOICA, Colombia; (3) Universidad Nacional deColombia, Colombia Following reports that biocontrol agents are potential management tools for clubroot of crucifers, caused by Plasmodiophora brassicae, this research aimed at assessing one strain of each of three Trichoderma species: T. asperellum Th034, T. brevicompactum Th201 and T. koningiopsis Th003 with different number of applications in terms of disease reduction on ‘Delus’ cabbage. Experiments were conducted in growth chambers and infested fields. Seasonal dynamics of fungal communities in pistachio and almond in California: Implications for aflatoxin biocontrol management A. ORTEGA-BELTRAN (1), R. Puckett (2), T. Michailides (3), D. Morgan (4), J. Moral (3) (1) UC Davis, U.S.A.; (2) UC Davis -KARE, U.S.A.; (3) UC Davis, U.S.A.; (4) UC Davis -KARE, U.S.A. Evaluation of the biocontrol potential of different Pseudomonas species in suppressing Pythium root rot in petunia plugs D. MARTIN (1), M. Jones (2), C. Taylor (2), F. Peduto Hand (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A. Pythium spp., causing root rot and damping off, affects 300+ hosts including several ornamental plants, and has developed resistance to two widely used fungicides, mefanoxam and propamocarb. This poses a real threat to greenhouse growers’ efficiency in cultivating healthy plants. Ohio State researchers have identified several Pseudomonas strains that exhibit biological control potential for disease suppression in many crops, including corn and tomato. One strain of each of the species P. putida, P. brassicacearum, P. fluorescens, P. poae and P. protegens was tested for the ability to suppress the activity of different Pythium species in petunia plugs. Since the production of bacterial metabolites is higher in the transition between logarithmic and stationary phases, our first step included building growth curves of the five strains to identify the appropriate time needed for incubation of the bacterial inoculum prior to application to petunia plugs. Cultures were prepared in LB medium and the OD600 values were recorded using a spectrophotometer every two hours for up to 18 hours. Serial dilution plating at each time point provided correspondent CFUs. ‘Carpet White’ Petunia seeds were sown in 128-plug trays and immediately treated with a 107 CFU/mL concentration of each Pseudomonas strain. After 14 days, plugs were inoculated with the different Pythium species. Responses of disease incidence and severity were evaluated until plugs became shipment-ready. Pythium spp., causing root rot and damping off, affects 300+ hosts including several ornamental plants, and has developed resistance to two widely used fungicides, mefanoxam and propamocarb. This poses a real threat to greenhouse growers’ efficiency in cultivating healthy plants. Ohio State researchers have identified several Pseudomonas strains that exhibit biological control potential for disease suppression in many crops, including corn and tomato. Microbiome networks: A systems framework for identifying candidate microbial assemblages for disease management R. POUDEL (1), A. Jumpponen (2), D. Schlatter (3), T. Paulitz (3), B. McSpadden Gardener (4), L. Kinkel (5), K. Garrett (1) (1) Plant Pathology Department, Institute for Sustainable Food Systems, and Emerging Pathogens Institute, University of Florida, Gainesville, FL, U.S.A.; (2) Division of Biology and Ecological Genomics Institute, Kansas State University, Manhattan, KS, U.S.A.; (3) USDA-ARS, Wheat Health, Genetics, and Quality Research Unit, Washington State University, Pullman, WA, U.S.A.; (4) Department of Plant Pathology, The Ohio State University, Wooster, OH, U.S.A.; (5) Department of Plant Pathology, University of Minnesota, St. Paul, MN, U.S.A. Field Performance of Endophytic Actinomycetes in Relation to Plant Growth Promotion and Biological Control of Fusarium oxysporum a Pathogen of Tomato K. EL-TARABILY (1), A. Alkhajeh (1) (1) United Arab Emirates University Uae For growth chambers, two concentrations of the pathogen were used, and Trichoderma was applied once or twice; disease was measured 60 days after planting. For field experiments Trichoderma was applied once, seven or 15 times during the cycle, and evaluations were done quarterly. In growth chambers, disease reduction was observed only for the low pathogen concentration; the highest reduction was observed when T. koningiopsis and T. brevicompactum were applied once. Disease reduction by T. asperellum was proportional to the number of applications. Field results were not as clear cut as those from growth chambers; however, disease reduction was observed with T. asperellum, and again this reduction was proportional to the number of applications. In conclusion, T. asperellum shows potential for clubroot control when applied frequently, whereas one application of either T. brevicompactum or T. koningiopsis can be useful in disease management in low pathogen concentrations. Evaluation of the biocontrol potential of different Pseudomonas species in suppressing Pythium root rot in petunia plugs D. MARTIN (1), M. Jones (2), C. Taylor (2), F. Peduto Hand (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A. Reduction of soybean sudden death syndrome (Fusarium virguliforme) by seed treatment with Bradyrhizobium japonicum T. HUYNH (1), T. Huynh (1), X. Yang (1), S. Navi (1), X. Li (1) (1) Iowa State University, U.S.A. Soybean sudden death syndrome (SDS), caused by Fusarium virguliforme, is a major soil-borne disease in soybean production in America. In North America, epidemics of the disease occurred in years when soybeans were planted early in growing seasons. Previous study by Scherm and Yang (1996) showed that F. virguliforme infected soybean in cool soil temperatures. Because infection of symbiont Bradyrhizobium japonicum requires minimum temperature of 17.5°C, higher than the minimum for F. virguliforme infection, we hypothesize that infections of F. virguliforme prior to establishment of B. japonicum in soybean affects SDS occurrence in early planting. From 2013 to 2015, experiments were conducted in a greenhouse and fields to determine the effects of seed treatments with B. japonicum on the occurrence of soybean sudden death syndrome. Results of greenhouse and field studies showed that seed treatments with B. japonicum inoculum reduced SDS foliar symptom expression by 50% and 20% in greenhouse and field, respectively. Increase in soybean nodulation and increase in yield in field experiment were also observed. Our results suggest that seed treatment with B. japonicum can reduce SDS foliar symptoms. The mechanisms of the suppressive effects on F. virguliforme by B. japonicum are under investigation to improve the potential of using this measure for disease control. The use of fungal biological control agents to manage SDS, charcoal rot, and damping off in soybean A. WARNER (1), E. Arnao (2), A. Fakhoury (2) (1) Southern Illinois University Carbondale, U.S.A.; (2) Southern Illinois University Carbondale, U.S.A. The use of fungal biological control agents to manage SDS, charcoal rot, and damping off in soybean A. WARNER (1), E. Arnao (2), A. Fakhoury (2) (1) S th Illi i U i it C b d l U S A (2) S th Illi i U i it C b d l U S A Sudden death syndrome (Fusarium virguliforme), damping off (Rhizoctonia solani), and charcoal rot (Macrophomina phaseolina) are three important and detrimental diseases of soybean in soybean growing areas. There is a need for more options to control these pathogens; many of the available management options are costly or limited in their effectiveness. Additionally, many of the current control practices may not be sustainable given their unfavorable impact on the environment. The use of biological control agents (BCAs) provides a more natural alternative for controlling various plant diseases. We have evaluated the effectiveness of multiple BCAs against F. virguliforme, R. solani, and M. Reduction of soybean sudden death syndrome (Fusarium virguliforme) by seed treatment with Bradyrhizobium japonicum T. HUYNH (1), T. Huynh (1), X. Yang (1), S. Navi (1), X. Li (1) (1) Iowa State University, U.S.A. phaseolina. We have also investigated the mechanisms the BCAs use to control these pathogens. This was determined through the use of microscopy and the monitoring of the expression of a panel of eight mycoparasitism-induced genes. Biological control of soilborne diseases in organic potato production using hypovirulent strains of Rhizoctonia solani R. LARKIN (1) (1) USDA-ARS, U.S.A. Soilborne diseases are persistent problems in potato production and alternative management practices are needed, particularly in organic production, where control options are limited. Selected biocontrol organisms, including two naturally-occurring hypovirulent strains of Rhizoctonia solani (Rhs1a1 and Bs69) and a commercially available Bacillus subtilis (GB03), were evaluated, both individually and in combination, for control of soilborne diseases of potato under organic production practices over three field seasons in Maine. Overall, multiple biocontrol treatments resulted in modest but significant reductions in all three soilborne diseases observed (black scurf, common scab, and silver scurf), reducing incidence and severity of each by 10-32%. All biocontrol treatments reduced black scurf (by 15-32%), whereas only specific treatments reduced common scab and silver scurf (by 10-25%). Combinations including both a hypovirulent strain and GB03 tended to be the most effective treatments, although generally not significantly better than individual strains. Tuber yield varied somewhat by year, but Rhs1a1 and GB03 treatments, as well as combinations, increased yield by 10 to 24% overall. These results indicate that hypovirulent Rhizoctonia, along with other biocontrol organisms, may be useful for reducing certain soilborne diseases and enhancing yield in organic potato production. Evaluation of the biocontrol potential of different Pseudomonas species in suppressing Pythium root rot in petunia plugs D. MARTIN (1), M. Jones (2), C. Taylor (2), F. Peduto Hand (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A. These initial results indicate that citrus trees infected with Phytophthora spp. are more prone to develop bacteria-root associations. Characterization of the bacterial species associated with Phytophthora infection will lead to the identification of beneficial or antagonistic bacteria against Phytophthora spp. Seasonal dynamics of fungal communities in pistachio and almond in California: Implications for aflatoxin biocontrol management A. ORTEGA-BELTRAN (1), R. Puckett (2), T. Michailides (3), D. Morgan (4), J. Moral (3) (1) UC Davis, U.S.A.; (2) UC Davis -KARE, U.S.A.; (3) UC Davis, U.S.A.; (4) UC Davis -KARE, U.S.A. S4.56 Interactions between pathogenic and nonpathogenic fungal species in the tree canopy are complex and can determine if disease will manifest in the plant and in other organisms such as honey bees. Seasonal dynamics of fungi were studied in almond and pistachio in California. In an almond orchard, experimental release of the atoxigenic biopesticide Aspergillus flavus AF36 to displace toxigenic strains has been conducted for five years. Because A. flavus is a pathogen of honey bees, our emphasis was on the presence of A. flavus in the flowers and the honey bees that attend them. Fungal species most commonly recovered from developing pistachio fruits were: Cladosporium herbarum, Alternaria alternata, Penicillium spp., Botrytis cinerea, Aspergillus niger, A. flavus, and yeasts. The main seasonal changes were detected in P. expansum and A. niger, which markedly increased from April to September. Conversely, the B. cinerea population significantly decreased during this period. In the almond, the dominant genera were: Cladosporium, Aureobasidium, Alternaria, Aspergillus, and Fusarium. Surprisingly, the diversity index was greater in the summer for both crops. The incidence of almond flowers with A. flavus ranged from 0 to 4.5%, depending on the year of study, and the incidence of honey bees carrying A. flavus ranged from 6.5 to 10%. This study helps the understanding of seasonal dynamics of pathogenic and nonpathogenic fungi and the impact of AF36 to pollinator honey bees. Interactions between pathogenic and nonpathogenic fungal species in the tree canopy are complex and can determine if disease will manifest in the plant and in other organisms such as honey bees. Seasonal dynamics of fungi were studied in almond and pistachio in California. In an almond orchard, experimental release of the atoxigenic biopesticide Aspergillus flavus AF36 to displace toxigenic strains has been conducted for five years. Because A. flavus is a pathogen of honey bees, our emphasis was on the presence of A. Clonostachys rosea reduce the populations of Botrytis cinerea and changes the metabolic profiles of strawberry plant ges (2), W. Araujo (1) Leaf applications of Clonostachys rosea reduce the populations of Botrytis cinerea and changes the metabolic profiles of strawberry plants L. Maffia (1), A. Borges (2), W. Araujo (1) (1) Universidade Federal de Vicosa, Brazil; (2) Universidade Federal de Vicosa, Brazil Clonostachys rosea is an efficient antagonist of Botrytis cinerea. In a research program aiming at the biocontrol of gray mold of strawbwerry, we studied the effect of C. rosea application on both antagonist and B. cinerea leaf populations, as well as on plant metabolism at a commercial strawberry field production. The antagonist was applied at 7, 14, or 28-day intervals, B. cinerea was naturally inoculated, and the colonization of leaves by both fungi was assessed. The antagonist reduced B. cinerea colonization; most significant effects were found with week applications. The reduction in B. cinerea colonization was negatively correlated with the increase in C. rosea’s at all intervals. The metabolic profiles of treated strawberry plants were also most changed with week applications, and there was a significant increase of compounds related to the Krebs cycle, growth promotion and the activation of plant defense system. In conclusion, with week applications of C. rosea, there was a significant reduction on the population of B. cinerea in symptomless tissues and a change in the metabolic profile of strawberry plants. Sponsored by CNPq and FAPEMIG. Reduction of soybean sudden death syndrome (Fusarium virguliforme) by seed treatment with Bradyrhizobium japonicum T. HUYNH (1), T. Huynh (1), X. Yang (1), S. Navi (1), X. Li (1) (1) Iowa State University, U.S.A. Evaluation of the biocontrol potential of different Pseudomonas species in suppressing Pythium root rot in petunia plugs D. MARTIN (1), M. Jones (2), C. Taylor (2), F. Peduto Hand (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A. flavus in the flowers and the honey bees that attend them. Fungal species most commonly recovered from developing pistachio fruits were: Cladosporium herbarum, Alternaria alternata, Penicillium spp., Botrytis cinerea, Aspergillus niger, A. flavus, and yeasts. The main seasonal changes were detected in P. expansum and A. niger, which markedly increased from April to September. Conversely, the B. cinerea population significantly decreased during this period. In the almond, the dominant genera were: Cladosporium, Aureobasidium, Alternaria, Aspergillus, and Fusarium. Surprisingly, the diversity index was greater in the summer for both crops. The incidence of almond flowers with A. flavus ranged from 0 to 4.5%, depending on the year of study, and the incidence of honey bees carrying A. flavus ranged from 6.5 to 10%. This study helps the understanding of seasonal dynamics of pathogenic and nonpathogenic fungi and the impact of AF36 to pollinator honey bees. Evaluation of Management Practices on the Efficacy of Pasteuria nishizawae As a Biocontrol Agent of Soybean Cyst Nematode M. LUND (1), S. Conley (1), J. Ané (1) (1) University of Wisconsin-Madison U S A Evaluation of Management Practices on the Efficacy of Pasteuria nishizawae As a Biocontrol Agent of Soybean Cyst Nematode M. LUND (1), S. Conley (1), J. Ané (1) (1) University of Wisconsin-Madison, U.S.A. Soybean Cyst Nematode (Heterodera glycines) is the most economically significant pathogen on soybean (Glycine max), causing over $1.5 billion dollars in damages annually. Pasteuria nishizawae, an endospore-forming soil bacterium, is an obligate parasite of soybean cyst nematodes (SCN). P. nishizawae has the potential to provide season-long control of SCN in the form of a seed treatment called Clariva®Complete. In this study, we evaluated the efficacy of the active ingredient, P. nishizawae, for the management of SCN on soybean. The field design was a split-plot, randomized complete block design with sub-sampling for the 2014 and 2015 growing seasons. Each split-plot was randomly assigned a row spacing of 30-inch or 15-inch spacing. Each subplot within the split-plot was randomly assigned one of three seeding rates (75K seeds/acre, 150Kseeds/acre and 225K seeds/acre) and one of two seed treatments (Clariva®Complete and CruiserMaxx®/Vibrance®). Results showed no significant interaction between seed treatment and yield, but did show significant interactions between both seeding rate and row spacing with yield. No significant interaction was observed between the S4.57 SCN count and the yield. Because the soybean variety used had PI88788 resistance, the Hg-type of the SCN population may not have been able to develop on soybeans with this source of resistance. This may have masked the effects of the seed treatment and prevented accurate evaluation of its efficacy. SCN count and the yield. Because the soybean variety used had PI88788 resistance, the Hg-type of the SCN population may not have been able to develop on soybeans with this source of resistance. This may have masked the effects of the seed treatment and prevented accurate evaluation of its efficacy. Effect of biopestisides on Phytophthora Root Rot Disease of Oakleaf Hydrangea M. KABIR (1), P. Liyanapathiranage (1), T. Simmons (1), F. Baysal-Gurel (1) (1) Tennessee State University, U.S.A. Phytophthora root rot, caused by Phytophthora nicotianae, has been reported to infect almost all hydrangeas especially container-grown oakleaf hydrangea (Hydrangea quercifolia). The pathogen can be introduced into the nursery via Phytophthora-contaminated container stock or cuttings. Experiments were performed in a greenhouse to determine the efficacy of biopesticides and fungicide against Phytophthora root rot in oak leaf hydrangea cvs. Queen of Hearts and Munchkin. Evaluation of Management Practices on the Efficacy of Pasteuria nishizawae As a Biocontrol Agent of Soybean Cyst Nematode M. LUND (1), S. Conley (1), J. Ané (1) (1) University of Wisconsin-Madison U S A Treatments applied were the biopesticides RootShield WP (Trichoderma harzianum Rifai strain KRL- AG2) and RootShield Plus (T. harzianum Rifai strain T-22 and T. virens strain G-41) and fungicide Subdue Maxx (Mefenoxam). All the treatments reduced root rot disease severity compared to the inoculated, non-treated controls in both cultivars. There were no significant differences between the treatments. Root rot disease severity was significantly higher in the cultivar Queen of Hearts than cultivar Munchkin. There was no significant effect of treatment and cultivar on root biomass. This study shows that biopesticides, RootShield WP and RootShield Plus, should be considered to reduce the risk of Phytophthora root rot on container-grown oakleaf hydrangeas in the nursery. Putative biocontrol mechanisms of rice bacterial blight by Bacillus subtilis TKS1-1 T. HUANG (1), Y. Chen (1), Y. Jan (1), Y. Yeh (1), D. Tzeng (1) (1) Department of Plant Pathology, National Chung Hsing University, Taiwan Bacterial blight of rice caused by Xanthomonas oryzae pv. oryzae is one of the devastating diseases of rice plant. The main objectives of the research are to investigate the potential of using Bacillus subtilis TKS1-1 as a control agent of rice bacterial blight and the possible mechanisms involved in disease control. B. subtilis TKS1-1 showed antagonistic activity against X. oryzae WF6. Application of B. subtilis TKS1-1 at the time of or prior to inoculation with X. oryzae WF6 was found to suppress rice bacterial blight. Additionally, the lesion length of leaf blight was less by drenching application with B. subtilis TKS1-1 compared to the water control. Spray-application of B. subtilis TKS1-1 was shown to increase the expression of several plant defense- related genes including PR1a, PAL, NPR1, and WRKY45 at 16 hours post application compared to the water control. Furthermore, drenching application of strain TKS1-1 7 days prior to inoculation of the pathogen was found to increase the activity of phenylalanine ammonia lyase, peroxidase and polyphenol oxidase at 48 hours post-inoculation in comparison with the water treatment. Application of B. subtilis TKS1-1 on rice cultivar TNG67 showed significant promotion of plant growth. The findings suggested that B. subtilis TKS1-1 exhibited efficacy in suppression of rice bacterial blight and in growth promotion. The biocontrol potential is partly attributed to the induction of plant defense response by B. subtilis TKS1-1. Amino acids influence antibiotic production by Pantoea vagans strain C9-1 J. KLEIN (1), V. Stockwell (2), J. Evaluation of Management Practices on the Efficacy of Pasteuria nishizawae As a Biocontrol Agent of Soybean Cyst Nematode M. LUND (1), S. Conley (1), J. Ané (1) (1) University of Wisconsin-Madison U S A Loper (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, Horticultural Crops Research Unit, U.S.A. Pantoea vagans strain C9-1 is an effective biological control agent for fire blight caused by Erwinia amylovora. C9-1 is thought to suppress E. amylovora on stigmas via resource competition and production of the antibiotics pantocin A (formerly herbicolin O) and herbicolin I. We used a green fluorescent protein reporter construct in C9-1 to monitor the regulation of the promoter of the pantocin A operon (paaPABC) on solidified media and in broth culture with a Tecan Infinite M200PRO. In assays on solidified media, inhibition of E. amylovora by C9-1 and expression of the gfp reporter for pantocin A operon was detected on Neidhardt’s modified medium, but not on Luria Bertani (LB) medium. Addition of yeast extract or tryptone (components of LB) to Neidhardt’s broth medium suppressed the expression of the pantocin A promoter. We tested the effect of various amino acids (100 mM) added to Neidhardt’s broth medium on expression of the paaPABC promoter in C9-1. We found that arginine, histidine, or tryptophan reduced expression of the pantocin A promoter compared to expression in non-amended Neidhardt’s broth medium, whereas asparagine, aspartic acid, or proline upregulated expression. Asparagine and proline are two major amino acids reported on apple and pear flower stigmas. These results indicate that the chemical environment of stigmas likely would support expression of the paaPABC operon and pantocin A biosynthesis by the biological control agent C9-1. Microscopic analysis of parasitism mode of action by bacterial biological control agents on Cornus florida powdery mildew M. MMBAGA (1) (1) Tennessee State University U S A Microscopic analysis of parasitism mode of action by bacterial biological control agents on Cornus florida powdery mildew M. MMBAGA (1) (1) Tennessee State University, U.S.A. Three bacterial isolates, Baccillus( sp., Stenotrophomonassp. Serratia( sp. were previously identified as naturally occurring biological control agents (BCA) for powdery mildew in Cornus florida. The interaction between these BCA’s and powdery mildew fungus was studied using scanning electron microscopy (SEM) and detached leaf technique. Four replicates of similar leaves were infected with powdery mildew, sprayed with BCA’s, incubated for 3h, 6h, 15h, 48h and 5 days, then fixed overnight using formalin acetic acid alcohol and stored at 4°C untill SEM imaging was done. The SEM micrographs showed the three BCA’s colonizing powdery mildew spores and hyphae. The BCA’s displayed the ability to enter and lyse powdery mildew spores and hyphae; the (Baccillus( sp. lysed and destroyed mycelial structures completely within 48h; others took longer. The BCA’s multiplied on the leaf surface and inside powdery mildew spores and hyphae. The three BCA’s did not interfere with other microbes on the leaf surface; this displayed potential specificity to powdery mildew and potential preservation of other naturally occuring microflora. The collapsed conidia and lysed hyphae suggested antifungal compounds that caused structural damage may be involved. These observations confirm that the three naturally occuring BCA’s may provide a buffering effect against powdery mildew and a resource for powdery mildew management and reducing conventional fungicides usage. Investigation of antifungal and herbicide volatile terpenes in Streptomyces spp. G. CHO (1), H. Kim (1), J. Kim (2), J. Kim (1), Y. Kwak (2) (1) Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea; (2) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea Investigation of antifungal and herbicide volatile terpenes in Streptomyces spp. G. CHO (1), H. Kim (1), J. Kim (2), J. Kim (1), Y. Kwak (2) (1) Division of Applied Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea; (2) Institute of Agriculture & Life Science, Gyeongsang National University, Jinju 52828, Republic of Korea, South Korea Study on Colonization of Strain ZY-9-13 labeled with Green Fluorescent Protein(GFP) Gene within Roots of tobacco and its Biocontrol Effect J. XI (1), J. Song (1), M. Mo (2), C. Xue (1), W. Mou (1), Q. Yin (1) (1) Zhengzhou Tobacco Research Institute of CNTC, China; (2) Yunnan University, China Study on Colonization of Strain ZY-9-13 labeled with Green Fluorescent Protein(GFP) Gene within Roots of tobacco and its Biocontrol Effect J. XI (1), J. Song (1), M. Mo (2), C. Xue (1), W. Mou (1), Q. Yin (1) (1) Zhengzhou Tobacco Research Institute of CNTC, China; (2) Yunnan University, China Strain ZY-9-13 was isolated from fresh leaves of tobacco, which had considerably antagonism to Phytophthora parasiticavar. nicotianae. The stability of the control effect mainly depends on whether antagonistic microbes could effectively colonize in plant. The purpose of the study was to explore the colonization of stain ZY-9-13 in roots of tobacco and control effect to tobacco black shank. The pattern of colonization of the tobacco was observed with fluorescent confocal microscopy and control effect was calculated after the tobacco seedling being inoculated the GFP tagged stain on tobacco black shank. The results showed that there was no obvious difference between original strain ZY-9-13 and tagged strain in antibacterial activities testing. Fluorescence microscope observation indicated that the strain ZY-9-13 propagated in the zone of root cap, elongation, maturation, and the junctions of primary roots and lateral roots. The quantities of the stain in different parts were root cap> elongation > maturation > the junctions. The pot experiment data showed that application of ZY-9-13 significantly reduced the disease incidence of black shank on tobacco. Five doses of the strain ZY-9-13 (1 × 106 cfu·g−1?1 × 107 cfu·g−1?1 × 108 cfu·g−1?1 × 109 cfu·g−1?1 × 1010 cfu·g−1) showed different control effect on tobacco black shank, which was 42.43%, 54.70%, 68.80%, 73.03% and 77.60% respectively. Results provided scientific basis for the strain ZY-9-13 on controlling tobacco black shank. Developing a quantitative polymerase chain reaction protocol to quantitate root colonization by Bacillus amyloliquefaciens and Bacillus firmus H. MENDIS (1), L. De La Fuente (1), P. Schwientek (2), R. Salamzade (2), V. Thomas (2), J. Kloepper (3) (1) Auburn University, U.S.A.; (2) Bayer CropScience LP, U.S.A.; (3) Auburn University, U.S.A. Developing a quantitative polymerase chain reaction protocol to quantitate root colonization by Bacillus amyloliquefaciens and Bacillus firmus H. MENDIS (1), L. De La Fuente (1), P. Schwientek (2), R. Salamzade (2), V. Thomas (2), J. Kloepper (3) (1) Auburn University, U.S.A.; (2) Bayer CropScience LP, U.S.A.; (3) Auburn University, U.S.A. Bacillus amyloliquefaciens QST713 and B. Investigation of antifungal and herbicide volatile terpenes in Streptomyces spp. Functional analyses of the ribosomal and non-ribosomal processed antimicrobial peptides produced by biocontrol bacterium Bacillus pumilus PMB102 W. DENG (1), J. Wu (1), J. Huang (1) Functional analyses of the ribosomal and non-ribosomal processed antimicrobial peptides produced by biocontrol ba PMB102 W. DENG (1), J. Wu (1), J. Huang (1) (1) National Chung Hsing University, Taiwan Many Bacillus spp. are beneficial bacteria with potential applications in the integrated management of plant diseases, and the efficacy of biocontrol is mainly determined by the production of secondary metabolites, e. g. the ribosomal processed lantipeptides and non-ribosomal processed cyclic lipopeptides (CLPs). In this study, the biocontrol agent B. pumilus PMB102 was shown to reduce tomato Fusarium wilt disease in hydroponic seedling assays. Annotation of the draft genome identified one lantipeptide and two CLPs, surfactin and plipastatin (fengycin family), biosynthetic gene clusters. Partially purified proteins from the lantipeptide-containing culture filtrate were resolved by electrophoresis and assayed for antibacterial activity by overlay assays, and one small polypeptide was identified to inhibit the growth of B. cereus and B. mycoides. The culture filtrates containing CLPs were extracted by n-butanol and fractionated by C18 SPE column. The eluted fractions were resolved by TLC and assayed for antifungal activity by the overlay assays to show that the compounds sharing the same Rf value of fengycin could inhibit the mycelial growth of Alternaria brassicicola. The chemical compositions of the abovementioned antimicrobial compounds will be further verified by mass spectrometry. The expression of defense- associated genes will also be monitored to determine if the plant defense responses against fungal pathogens are modulated by the biocontrol bacterium PMB102. Investigation of antifungal and herbicide volatile terpenes in Streptomyces spp. Streptomyces spp. produce various secondary metabolites for interaction with environment. Some of these secondary metabolites have been reported as antibiotics or volatile compounds. In addition, Streptomyces spp. have been highly expectation for discovering new secondary metabolites. We observed that mycelia of various fungi were inhibited growth when these fungi were exposed to headspace of Streptomyces spp. Furthermore, germination and growth of Arabidopsis thaliana Col-2 were inhibited significantly by these headspaces. In these headspaces, three volatile terpenes that geosmin, caryolan-1-ol and unknown terpene were discovered in common. To confirm the bioactivity, we extracted mixtures from headspace of Streptomyces griseus S4-7. Mixture included geosmin didn’t inhibit mycelia growth of Botrytis cinera. A mixture included caryolan-1-ol and unknown terpene inhibited these mycelia. Furthermore, synthetic caryolan-1-ol caused dose-dependent inhibition of these mycelia and Saccharomyces cerevisiae growth. In addition, we found three terpene cyclase gene in S. griseus S4-7 genome. Geosmin and caryolan-1-ol synthases already had been reported but the other terpene synthase was unknown. Our present results show that caryolan-1-ol was discovered new bioactivity function. S4.58 Characterization and Expression of the Potent Antimicrobial Peptide LsGRP1C Originated from Lilium C. CHEN (1), C. Lin (1), Y. Pan (2), M. Chang (2), F. Liu (2) (1) National Taiwan University, Taiwan; (2) National Taiwan University, Taiwan LsGRP1 is a plant defense-related gene increasingly expressed in lily leaves exhibiting salicylic acid-induced systemic resistance against pathogenic fungus Botrytis elliptica. In vitro antimicrobial assay applied with synthetic peptides revealed that the 38-a.a. cysteine-rich C-terminal portion (LsGRP1C) of LsGRP1 inhibited kinds of bacterial and fungal species via the mechanisms of alteration of microbial membrane permeability and induction of fungal programmed cell death (PCD) as investigated by SYTOX Green staining and 4′,6′-diamidino-2-phenylindole staining/terminal deoxynucleotidyl transferase dUTP nick end labeling assays. Then the expression system for recombinant LsGRP1C production was established using an optimized Escherichia coli strain and an N-terminal fusion partner of small ubiquitin-like modifier protein (SUMO) Smt3 originated from yeast, conducting a high yield of SUMO-LsGRP1C fusion protein. According to the half inhibitory concentrations for spore germination and PCD occurrence of the assayed fungal species, SUMO-LsGRP1C was proven to exhibit higher antifungal activity and identical fungal PCD-inducibility as compared with synthetic LsGRP1C. Thus, we proposed that LsGRP1C would play a role in antimicrobial function of LsGRP1; moreover, efficient and effective E. coli expression strategy would enable the convenient production of antimicrobial LsGRP1C for practical use. Study on Colonization of Strain ZY-9-13 labeled with Green Fluorescent Protein(GFP) Gene within Roots of tobacco and its Biocontrol Effect J. XI (1), J. Song (1), M. Mo (2), C. Xue (1), W. Mou (1), Q. Yin (1) (1) Zhengzhou Tobacco Research Institute of CNTC, China; (2) Yunnan University, China firmus I-1582 are 2 key bacterial strains which are active ingredients in commercially-available biological products that colonize plant roots promoting plant growth and offering protection against pathogens. This study was carried out to develop a qPCR protocol to understand the dynamics of root colonization by these strains. Primers and TaqMan probes were designed based on genome comparisons of the 2 strains with other publicly available bacterial genomes. The specificity of the primers was tested in vitro and in growth chambers focusing on corn rhizosphere. An optimized qPCR protocol was developed to quantify the number of bacteria in batch cultures and in root samples using standard curves. Corn seeds were treated with 103, 105 and 107 spores of the 2 strains separately and planted in sterile sand. Whole root samples were taken at 2 and 3 weeks after germination and bacterial populations were assessed. QST713 and I-1582 primers did not amplify DNA extracted from untreated corn grown soil confirming that the primers are specific. The results also show that as low as 3.5 × 103 bacteria cells associated with 1g of corn roots can be detected using the current qPCR protocol. Furthermore, the number of bacteria cells associated with corn roots was quantified up to 3 weeks after germination. Our results show that the developed qPCR protocol can be used to understant dynamics of root colonization by plant growth promoting bacteria. The effects of seed coating “inert ingredients” in the virulence of Fusarium graminearum in soybean K. NAVARRO (1) (1) The Ohio State University, U.S.A. Effects of seed coating “inert ingredients” in the pathogenicity of Fusarium graminearum in soybean K. Navarro (1) (1) The Ohio State University U S A Fusarium graminearum, known to infect wheat, barley, corn, and other cereal crops has also been reported to cause seedling rot, root rot and pre- post emergence, damping off of soybean. Application of fungicides and other bioactive molecules are often combined with an “inert” ingredient such as sugar polymer dextrans as a seed coat to control seedling pathogens. Our study aims to investigate the effects of maltodextrin seed coating on the virulence of F. graminearum in soybean. Five soybean cultivars with different levels of resistance were coated with maltodextrin and inoculated with a microconidia suspension in a roll—towel assay. Our study demonstrates that maltodextrin coated soybean seeds had increase susceptibility to F. graminearum. Treated seedlings grew less vigorously in soil than non-treated seedlings. To address if fungal growth rates increased in the presence of maltodextrin, water agar plates amended with maltodextrin were prepared. Fungi grew faster in the presence of maltodextrin and exhibited a change in color as compared to non- amended water agar plates. Further experiments testing to address whether maltodextrin and other related sugar polymers used in seed coatings can affect fungal growth and toxin production are underway. Our study provides information about the potential for unintended adverse effects of specific “inert” ingredient in the formulation of seed coatings. The effects of seed coating “inert ingredients” in the virulence of Fusarium graminearum in soybean K. NAVARRO (1) d coating “inert ingredients” in the virulence of Fusarium The effects of seed coating “inert ingredients” in the virulence of Fusarium graminearum in soybean K. NAVARRO (1) (1) The Ohio State University, U.S.A. S4.59 Fusarium graminearum, known to infect wheat, barley, corn, and other cereal crops has also been reported to cause seedling rot, root rot and pre- post emergence, damping off of soybean. Application of fungicides and other bioactive molecules are often combined with an “inert” ingredient such as sugar polymer dextrans as a seed coat to control seedling pathogens. Our study aims to investigate the effects of maltodextrin seed coating on the virulence of F. graminearum in soybean. Five soybean cultivars with different levels of resistance were coated with maltodextrin and inoculated with a microconidia suspension in a roll—towel assay. Our study demonstrates that maltodextrin coated soybean seeds had increase susceptibility to F. graminearum. Treated seedlings grew less vigorously in soil than non-treated seedlings. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. ( ) (1) Center for Regulatory Research, LLC, U.S.A. The mycoparasites, Coniothyrium minitans and Trichoderma viride, of the plant pathogens, Sclerotinia sclerotiorum and Sclerotinia minor, offer a model system for the development of an approach for predicting their geographic distribution based on the physiological responses to the environmental variables of temperature and soil moisture. A biome analysis was performed to validate presence-absence data followed by a species response to environmental parameters using CLIMEX, a climate modeling and mapping software. Biome analysis identified the % of biome types capable of supporting Coniothyrium minitans (21.4%), Trichoderma viride (14.3%), Sclerotinia minor (28.6%) and Sclerotinia sclerotiorum (21.4%). Geographic distribution of Sclerotinia species was based on carpogenic germination of sclerotia in response to temperature and soil moisture parameters. Mycoparasite geographic distribution was determined by conidia germination with respect to the parameters. CLIMEX model analysis reports the US locations that are very favorable for establishment for Sclerotinia sclerotiorum (47%), Sclerotinia minor (27%), Coniothyrium minitans (53%) and Trichoderma viride (20%). Areas deemed very favorable for establishment of Coniothyrium minitans (53%) were larger than for Trichoderma viride (20%). Only 3% of the areas were classified as unsuitable for Coniothyrium minitans as compared to 17% for Trichoderma viride. Differential Colonization of Tomato Cultivars by the Biocontrol Agent Beauveria bassiana S. YERUKALA (1), M. Dee (1), B. Ownley (1) (1) University of Tennessee, U.S.A. Entomopathogenic Beauveria bassiana (Bb1198) is endophytic in tomato. Biocontrol of disease with Bb1198 is correlated with endophytic colonization. The aim of this study was to determine if colonization by Bb1198 is affected by tomato cultivar. Colonization of ‘Rutgers’ and ‘Mountain Spring’ by Bb1198 was tested in a gnotobiotic assay. Seed were treated with Bb1198 in 1% methylcellulose at 1.6 × 106 viable CFU/seed. Control seeds received methylcellulose. Replicated tests were factorials, in a CRD. Seed germination, BB1198 colonization, and plant growth were determined 17-21 days after treatment. Bb1198 was recovered from sections of root, stem, and leaf plated on semi-selective medium. Data were analyzed with mixed models ANOVA, and F-LSD (P<0.05). Germination was higher for ‘Mountain Spring’ seed treated with Bb1198 than control seed (94 vs 65%), germination of ‘Rutgers’ treated and control seed did not differ. Bb11-98 was not recovered from plants from control seed. For treated seed, colonization of ‘Rutgers’ was greater than ‘Mountain Spring’ (64 vs 29%). Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. For both cultivars from treated seed, colonization of leaf (50%) and stem sections (67%) was greater than roots (23%). Leaf number was greatest in ‘Rutgers’ from seed treated with Bb1198, followed by control plants for both cultivars, and least in ‘Mountain Spring’ treated with Bb1198. Understanding factors that affect colonization, i.e., cultivar, could increase consistency of biocontrol by B. bassiana. Effect of seedborne Xanthomonas translucens on wheat seed germination C. PEREZ (1), E. Juanicotena (2), O. Bentancur (2), C. Palladino (2) (1) EEMAC, Universidad de la Republica, Uruguay; (2) EEMAC, Universidad de la Repu Effect of seedborne Xanthomonas translucens on wheat seed germination C. PEREZ (1), E. Juanicotena (2), O. Bentancur (2), C. Palladino (2) (1) EEMAC, Universidad de la Republica, Uruguay; (2) EEMAC, Universidad de la Republica, Uruguay Bacterial leaf streak (BLS) has become an important disease on wheat production in Uruguay. However, very little is known about the epidemiology of this diseases. Seedborne pathogens may play an important role as primary source of inoculum but can reduce seed germination as well. The aim of this study was to investigate the effect of X. translucens on wheat seed germination. Seeds were collected from nine different commercial plots harvested in 2013, including different cultivars. Seeds lots were taken to the laboratory to test four seed treatments: i) check control (without treatment), ii) disinfectant (didecyldimethyl-ammonium chloride), iii) fungicide (iprodione + TMTD + carbendazim), vi) disinfectant + fungicide. Bacterial density was estimated by seed washing and dilution plating technique on Wilbrink´s agar. Seed germination was performed on paper towel. Significant differences on bacterial density were found among seed lots. Seed treatment significantly reduced the number of cfu.g–1 of seed, being disinfectant the most effective reducing bacterial population. However only the fungicide showed a significant effect on seed germination, indicating that germination was not affected by the bacterial population present at the seed surface. Based on this preliminary result, seedborne X. translucens might play an important role as source of inoculum but did not affect germination. Characterization and Application of Oil-In-Water Nanoemulsion for Controlling Citrus Huanglongbing M. ZHANG (1), C. Yang (2), C. Powell (1), Y. Duan (3), R. Shatters (3) (1) IRREC-IFAS, University of Florida, U.S.A.; (2) Guangxi University, China; (3) USHRL-USDA-ARS, U.S.A Candidatus Liberibacter asiaticus (Las), which can cause destructive citrus huanglongbing (HLB), resides in citrus phloem. Effects of seed coating “inert ingredients” in the pathogenicity of Fusarium graminearum in soybean K. Navarro (1) (1) The Ohio State University U S A To address if fungal growth rates increased in the presence of maltodextrin, water agar plates amended with maltodextrin were prepared. Fungi grew faster in the presence of maltodextrin and exhibited a change in color as compared to non- amended water agar plates. Further experiments testing to address whether maltodextrin and other related sugar polymers used in seed coatings can affect fungal growth and toxin production are underway. Our study provides information about the potential for unintended adverse effects of specific “inert” ingredient in the formulation of seed coatings. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. This study investigated the effects of Zinkicide™ in vitro on growth and biofilm formation of Las surrogate Liberibacter crescens and Xac in batch cultures and under flow conditions using microfluidic chambers. Xac was grown in 96-well plates in Nutrient Broth and treated with serial dilutions of Zinkicide™ to test inhibition of growth and biofilm formation. Xac was also introduced to microfluidic chambers under constant media flow, and inhibitory action of Zinkicide™ on biofilm formation was recorded using time-lapse video imaging microscopy. The Zinkicide™ formulation had better antimicrobial properties compared to similar formulations with the same active ingredients, and the minimum inhibitory concentration of Zinkicide™ for Xac was calculated at 10ppm. Time-lapse video imaging showed that untreated Xac grown in microfluidic chambers formed biofilm whereas Xac treated with Zinkicide™ did not form any biofilm. In vitro results confirmed that Zinkicide™ is effective against citrus bacterial pathogens and inhibits bacterial growth and biofilm formation under flow conditions. Progress towards a novel chemical control for common scab disease of potato R. TEGG (1), H. Thompson (2), C. Wilson (1) (1) Tasmanian Institute of Agriculture, University of Tasmania, Australia; (2) Department of Agriculture, Australia Common scab is a globally important potato disease caused by pathogenic Streptomyces spp. with no one effective control strategy. Essential for disease induction is the production by the pathogen of the toxin, thaxtomin A. This study sought to develop disease control options targeting the interaction of thaxtomin A with the host, using novel chemical applications. Trials identified two compounds, 2,4-dichlorophenoxyacetic acid (2,4-D) and 2,5- dibromobenzoic acid (2,5-DBB) that when applied to the foliage of newly emerged plants prior to tuber initiation, could significantly reduce common scab tuber disease incidence and severity by over 85%, compared to the untreated control. Seed tuber treatments with 2,4-D prior to planting were also effective at reducing disease although the level of control was not as great as from foliar treatments. Disease resistance was correlated with decreased sensitivity of the tuber material from treated plants to thaxtomin A. High application rates (>200mg/L) of 2,4-D were associated with negative traits including twisted foliage, tuber deformation and yield loss. Further trials demonstrated that greatly reduced 2,4-D application rates down to 1.7-25mg/L retained effective disease control but avoided detrimental phytotoxicity effects. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. Bacterial populations in petioles and root-rhizospheres initially increased after penicillin injections, possibly due to release of nutrients from dead bacteria, and then returned to control levels after one week. Penicillin resistance was common in penicillin-injected and control trees (30 to 94%). A few isolates (0 to 2%) were only resistant to glyphosate, while more isolates (29 to 30%) were only resistant to penicillin. Most isolates (54 to 71%) were resistant to both glyphosate and penicillin. Considering the widespread penicillin resistance in control soil, potential side effects of penicillin injection in citrus are small. grapefruit trees in field and greenhouse experiments. Trees were injected with penicillin G, using application rates of 0, 1000, or 6000 µg/ml. Bacteria were isolated on a low carbon medium from roots plus rhizosphere and surface-sterilized petioles at various times after penicillin injection. Selected isolates were tested for penicillin resistance (20 µg/ml) and glyphosate resistance (7000 µg/ml), because glyphosate is widely used and cross-resistance against antibiotics had been documented. Bacterial populations in petioles and root-rhizospheres initially increased after penicillin injections, possibly due to release of nutrients from dead bacteria, and then returned to control levels after one week. Penicillin resistance was common in penicillin-injected and control trees (30 to 94%). A few isolates (0 to 2%) were only resistant to glyphosate, while more isolates (29 to 30%) were only resistant to penicillin. Most isolates (54 to 71%) were resistant to both glyphosate and penicillin. Considering the widespread penicillin resistance in control soil, potential side effects of penicillin injection in citrus are small. Evaluation of a novel antimicrobial compound to control growth and biofilm formation in vitro of citrus bacterial pathogens H. MENDIS (1), E. Naranjo (1), S. Santra (2), M. Young (2), E. Johnson (3), P. Rajasekaran (2), L. De La Fuente (1) (1) Auburn University, Auburn, AL, U.S.A.; (2) University of Central Florida, Orlando, FL, U.S.A.; (3) Citrus Research and Education Center, University of Florida, Lake Alfred, FL, U.S.A. Zinkicide™ is a novel plant nutrient-based antimicrobial formulation developed for treating citrus bacterial pathogens including ‘Candidatus Liberibacter asiaticus’ (Las) and Xanthomonas citri subsp. citri (Xac). As per the design, Zinkicide™ is expected to enter phloem tissues of citrus plants when sprayed onto leaves for control of phloem-limited pathogens such as Las. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. Field trials within Australia, China and USA testing foliar and seed treatments are on-going, with initial results indicating useful field efficacy of these chemical treatments for disease control. Antibacterial potential of Magnesium oxide nanomaterial against Xanthomonas perforans causing bacterial spot of tomato Y. LIAO (1), A. Strayer (1), . White (2), A. Mukherjee (2), W. Elmer (3), L. Ritchie (4), D. Clark (4), J. Freeman (4), J. Jones (1), M. Paret (4) (1) Department of Plant Pathology, University of Florida, Gainesville, FL, U.S.A.; (2) Department of Analytical Chemistry, The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (3) Department of Plant Pathology and Ecology, The Connecticut Agricultural Experiment Station, New Haven, CT, U.S.A.; (4) North Florida Research and Education Center, University of Florida, Quincy, FL, U.S.A. Bacterial spot caused by Xanthomonas perforans can cause high disease severity on tomatoes in Florida. Due to the presence of copper (Cu)-tolerant X. perforans strains, copper-bactericides are of limited use; thus, there is a critical need for finding effective alternatives. In this study the antibacterial activity of magnesium oxide (MgO) and other metal oxide nanomaterials were evaluated against Cu-tolerant and sensitive X. perforans strains. In vitro assays demonstrated that MgO had high antibacterial ability compared to the equivalent concentration of a Cu-based bactericide against both strains. A greenhouse experiment demonstrated that disease severity was significantly reduced by MgO at 200 ppm compared to copper-mancozeb, the grower standard and the untreated control (p = 0.05). A field experiment demonstrated that the same treatment significantly reduced disease severity compared to the untreated control (p = 0.05). There was no significant difference in the yield between the treatments. Inductively Coupled Plasma Mass Spectrometry analysis of the fruits showed that MgO treatments did not lead to significant accumulation of Mg, Cu, Ca, K, Mn, P and S compared to the untreated. However, copper-mancozeb treatment led to significant accumulation of Cu and P compared to the untreated control (p = 0.05). This study shows the antibacterial potential of MgO nanomaterials against X. perforans and its potential use against bacterial spot of tomato. Adepidyn™: A new fungicide active ingredient for control of foliar diseases K. BUXTON (1), T. Harp (1), A. Tally (1), H. Mclean (1) (1) Syngenta Crop Protection, U.S.A. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. Based on various physiochemical characteristics of oils, surfactants, and organic solvents, oil-in-water (O/W) nanoemulsion was optimized to prepare the antimicrobial nano-formulation. The nanoemulsion was produced using a spontaneous emulsification method for the efficient delivery of antimicrobials into citrus phloem by bark application. The nanoemulsion prepared from cremophor® EL (viscous oil), acetone (water miscibility organic solvent), and Span 80/Tween 80 (surfactant) exhibited a small droplet size (17.33±0.52 nm). It also had an improved absorption rate, including 2-day peak concentration (tmax), 71.86±35.38 ng/g maximum concentration (Cmax), and 267.25%±44.1% relative bioavailability (RBA) of ampicillin in HLB-affected citrus compared to the Amp solution alone (tmax = 6 days, Cmax = 56.44±32.59 ng/g and RBA = 100%). The same nanoemulsion was used to deliver five antimicrobials to control citrus HLB by bark application. We found that droplet size of antimicrobial formulation prepared in nanoemulsion was significantly reduced. And the nanoemulsion also enhanced the efficacy against Las of validoxylamien A, combination of actidone and validoxylamine A, and sulfadimoethoxine sodium against Las. Therefore, this study provides an efficient self-nanoemulsifying delivery system for controlling citrus HLB. Effects of Penicillin Injection for Citrus HLB Control on Culturable Bacteria in Petioles and Rhizosphere, and Penicillin Resistance K. SHIN (1), M. Ascunce (1), H. Narouei-Khandan (1), X. Sun (2), D. Jones (2), E. Goss (1), A. van Bruggen (1) (1) University of Florida, U.S.A.; (2) Division of Plant Industry, U.S.A. Trunk injection with penicillin has been tested to control citrus huanglongbing (HLB), but environmental safety must be assured before approval of penicillin injection in groves. We investigated potential effects of penicillin injection on rhizospheric and endophytic bacterial populations using Trunk injection with penicillin has been tested to control citrus huanglongbing (HLB), but environmental safety must be assured before approval of penicillin injection in groves. We investigated potential effects of penicillin injection on rhizospheric and endophytic bacterial populations using S4.60 grapefruit trees in field and greenhouse experiments. Trees were injected with penicillin G, using application rates of 0, 1000, or 6000 µg/ml. Bacteria were isolated on a low carbon medium from roots plus rhizosphere and surface-sterilized petioles at various times after penicillin injection. Selected isolates were tested for penicillin resistance (20 µg/ml) and glyphosate resistance (7000 µg/ml), because glyphosate is widely used and cross-resistance against antibiotics had been documented. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. Adepidyn™ fungicide is a new active ingredient in the carboxamide chemical class (FRAC group 7) from Syngenta that provides excellent, long lasting control of powdery mildews, leaf spots, and other fungal pathogens on vegetables as well as row crops. The first wave crops to be registered include fruiting and leafy vegetables, cereals, peanut, grapes, potatoes, soybeans, and corn. Some of the key strengths of ADEPIDYN™ include early and late leaf spot on peanut, Alternaria spp., and powdery mildews. Field trials have shown the unparalleled residual control of ADEPIDYN™ fungicide, at low rates in comparison to commercial standards, providing more than three to four weeks control of A. solani on potato, Cercospora spp. on peanut, and powdery mildew on cucurbits. The high intrinsic activity and long-lasting duration of control of ADEPIDYN™ fungicide on these diseases, in rotation and in mixtures with other fungicides, will provide growers another tool for effectively managing leaf spot and powdery mildew diseases. Adepidyn™: A new fungicide active ingredient for control of Fusarium head blight on wheat T. HARP (1), K. Anaka (2), A. Tally (1) (1) Syngenta Crop Protection, U.S.A.; (2) Syngenta Crop Protection, Canada Adepidyn™ Fungicide is a new active ingredient in the carboxamide chemical class (Frac group 7) from Syngenta that provides broad spectrum disease control across many crops, including cereals. In addition to strong performance on various cereal leaf spots and powdery mildew, Adepidyn™ has also demonstrated efficacy on Fusarium head blight (FHB), a disease that is difficult to control and with limited management solutions. Adepidyn™ represents the only carboxamide that demonstrates good activity for control of FHB, offering growers a new tool for Fusarium control as well as providing an expanded window for application. In field trials, Adepidyn™ applied solo between BBCH 55 and BBCH 65 (Feekes 10.3 -10.52) at rates between 150 and 200 gai/ha, or at 125 to 150 gai/ha in mixture with propiconazole, provided superior efficacy to the current triazole solutions. Adepidyn™ provides excellent control of FHB and is a new mode of action for effective management for FHB in cereals. S4.61 Quantification of the mortality of conidia of Botrytis cinerea and Penicillium digitatum in peracetic acid J. Smilanick (1), L. Grant (2) Quantification of the mortality of conidia of Botrytis cinerea and Penicillium digitatum in peracetic acid J. Smilanick (1), L. Grant (2) (1) consultant, U.S.A.; (2) Jet Harvest Solutions, U.S.A. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. In both years, isolate populations collected from fungicide-treated fruit and from untreated control fruit were not statistically different in haploid gene diversity (p > 0.45 for 2013), (p > 0.46 for 2014), allele number (p = 0.88 for 2013), (p = 0.41 for 2014), and effective allele number (p > 0.88 for 2013), (p > 0.81 for 2014). Isolates from blossoms and corresponding cankers of fungicide treatments had no changes in simple sequence repeat size or evidence for induced transposon translocation. No emergence of reduced sensitivity to azoxystrobin, propiconazole, iprodione, and cyprodinil that would indirectly indicate increased genetic diversity was detected. Our results indicate that fungicide- induced, genetic changes may not occur as readily in field populations as they do in populations exposed continuously to sublethal doses in vitro. Citrus Canker as a bioassay for systemic bactericidal activity of Zinc nanoparticles S. COMMERFORD (1), K. Gerberich (1), P. Rajasekaran (2), M. Young (3), S. Das (3), J. Graham (4), S. Santra (3), E. Johnson (1) (1) Citrus Research Education Center, University of Florida, Lake Alfred, FL, U.S.A.; (2) University of Central Florida, Orlando, FL, U.S.A.; (3) University of Central Florida, U.S.A.; (4) Citrus Research Education Center, University of Florida, U.S.A. Huanglongbing (HLB) is caused by the phloem-limited bacterium, Candidatus Liberibacter asiaticus (Las), transmitted by the Asian citrus psyllid. Since August 2005, when HLB was first discovered in South Florida, citrus production has declined by more than 60%. HLB symptoms include vein yellowing, blotchy mottle, zinc deficiency, tree decline and fruit drop. Like most systemic diseases, HLB is difficult to control because foliar applied materials cannot enter the vascular tissue due to the leaf cuticle and selective permeability of cell membranes. Zinkicide™ is a plant nutrient-based nanoparticle with bactericidal activity that is designed to be translocated systemically. Prior to HLB, citrus canker was the most serious disease threat to Florida citrus. The causal agent, Xanthomonas citri subsp. citri (Xcc), incites leaf and fruit lesions within 2-3 weeks after infection. The goal was to investigate systemic movement of Zinkicide™ via soil drench using canker as a model for bactericidal activity because of the short evaluation time compared to HLB. In replicate greenhouse trials, soil drench and spray applications of Zinkicide™ were applied prior to leaf infiltration with 104 cfu ml–1 of Xcc. Two weeks post inoculation lesions per leaf were counted. Zinkicide™ spray and drench applications significantly reduced lesion number. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. Peracetic acid (PAA) is common sanitizer, but information about its fungal toxicity is sparse. PAA toxicity was assessed to conidia of Botrytis cinerea and Penicillium digitatum, causes of gray mold and citrus green mold, respectively. They were immersed in PAA, entrapped on a glass fiber filter, rinsed with dH2O, and placed on PDA to observe germination, then the time (LT95) or concentration (LD95) when 95% mortality occurred were estimated by probit analysis. Two 100 mg/l (= ppm) solutions were used, one from 4.9% PAA + 26.5% H2O2 and the other from 15.0% PAA + 22.0% H2O2. Mortality was similar in both therefore due to PAA with little or no H2O2 contribution. ET95 of B. cinerea and P. digitatum conidia in 100 mg/l PAA was 5.4 and 2.4 minutes, respectively. ET95 of B. cinerea and P. digitatum conidia in 500 mg/l PAA was 17.2 and 9.8 seconds, respectively. LD95 of B. cinerea and P. digitatum conidia during a 24-hour PAA exposure was 24.7 and 3.3 mg/l, respectively. LD95 of P. digitatum conidia during 10 or 30-minute PAA exposure was 50 and 41 mg/l, respectively. Although PAA resistance was markedly higher in B. cinerea than P. digitatum, label rates of PAA are adequate to control conidia of both pathogens. PAA can be mixed with fungicides, label rates are relatively high (650 mg/l preharvest and 85-100 mg/l postharvest), is USDA Organic Program organic rules, and its disinfection by-products are of minimal concern. Effect of fungicide applications on Monilinia fructicola population diversity and transposon movement Effect of fungicide applications on Monilinia fructicola population diversity and transposon movement M. DOWLING (1), H. Boatwright (1), G. Schnabel (1), P. Bryson (1), J. Wilson (1), Z. Fan (1), S. Everhart (3), P. Brannen (4) (1) Clemson University, U.S.A.; (2) Clemson University, Clemson, SC, U.S.A.; (3) University of Nebraska, Lincoln, U.S.A.; (4) University of Georgia, U.S.A. In this study we investigated whether previously reported, fungicide-induced mutagenesis observed in vitro in M. fructicola can also accelerate genetic changes in field populations. Azoxystrobin and propiconazole were applied at half label rate to nectarine trees for two consecutive years in weekly intervals for three months between bloom and harvest. Single spore isolates were obtained from blighted blossoms, corresponding cankers, and from fruit to investigate phenotypic and genotypic changes. Evaluation of conventional and biological pesticides for managing tomato bacterial spot during transplant production P. ABRAHAMIAN (1), J. Jones (2), G. Vallad (1) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Predicting the geographic establishment of introduced mycoparasites S. COHEN (1) (1) Center for Regulatory Research, LLC, U.S.A. Reduction of lesions by the drench treatment indicates that systemic activity of Zinkicide™ will be useful for further evaluation against HLB. Evaluation of the Spatiotemporal Dynamics of Oxytetracycline and Its Control Effect against Citrus Huanglongbing via Trunk Injection J. HU (1), J. Hu (2) (1) University of Florida/IFAS-CREC, U.S.A.; (2) University of Florida, U.S.A. Evaluation of the Spatiotemporal Dynamics of Oxytetracycline and Its Control Effect against Citrus Huanglongbing via Trunk Injection J. HU (1), J. Hu (2) (1) University of Florida/IFAS-CREC, U.S.A.; (2) University of Florida, U.S.A. Citrus Huanglongbing (HLB) or greening is a devastating bacterial disease that has destroyed millions of trees and is associated with phloem-residing ‘Candidatus Liberibacter asiaticus’ (Las) in Florida. In this study, we evaluated the spatiotemporal dynamics of oxytetracycline in planta and its control effect against HLB via trunk injection. Las-infected ‘Hamlin’ sweet orange trees on ‘Swingle’ citrumelo rootstock at the early stage of decline were treated with oxytetracycline hydrochloride (OTC) using trunk injection with varying number of injection ports. Spatiotemporal distribution of OTC and dynamics of Las populations were monitored by HPLC method and qPCR assay, respectively. Uniform distribution of OTC throughout tree canopies and root systems was achieved 2 days post injection. High levels of OTC (>850 µg/kg) were maintained in leaf and root for at least 1 month and moderate OTC (>500 µg/kg) persisted for more than 9 months. Reduction of Las populations in root systems and leaves of OTC-treated trees were over 95% and 99% (i.e. 1.76 and 2.19 log reduction) between 2 and 28 DPI. Conditions of trees receiving OTC treatment were improved, fruit yield was increased, and juice acidity was lowered than water-injected control even though their differences were not statistically significant during the test period. Our study demonstrated that trunk injection of OTC could be used as an effective measure for integrated management of citrus HLB. Managing QoI-resistant Cercospora sojina in Mississippi soybean and assessing the physiological impacts of foliar fungicide phytotoxicity J. MANSOUR (1), M. Tomaso-Peterson (2), A. Henn (2), J. Bond (1), T. Irby (2) (1) Mississippi State University, U.S.A.; (2) Mississippi State University, U.S.A. Following the widespread detection of quinone outside inhibitor (QoI) resistant frogeye leaf spot (FLS) (Cercospora sojina Hara) in Mississippi during 2013 and 2014, effective management of FLS in susceptible cultivars requires the use of fungicide products that contain alternative, non-QoI, modes of action (MOA). One drawback to applying alternative MOAs, such as demethylation inhibitors (DMI), can be phytotoxicity on treated soybean leaves. Field trials were conducted in 2015 to assess fungicide efficacy on QoI-resistant FLS severity and the resulting phytotoxicity on several soybean cultivars with differing FLS reactions. The fungicides chosen have previously been observed to produce moderate to severe phytotoxicity. Six different fungicide products, comprised of single or multiple MOAs, were applied at R2, R3, and R4 along with a non-treated. FLS and phytotoxicity were visually assessed pre- and several times post-application. Plant height, number of pods and nodes were recorded to determine physiological impacts. Analyzed data revealed that certain MOAs significantly reduced FLS severity compared to the non-treated. In addition, prothioconazole resulted in significantly greater phytotoxicity than the non-treated, a difference of 29.4%. However, yield was not significantly reduced as a result of fungicide phytotoxicity. In addition, plant heights and number of nodes per plant were not significantly different between fungicide treatments and the non-treated. Management of Fusarium Head Blight and DON in Alabama K. BOWEN (1), N. Sharma (1), N. McMaster (2), J. Jones (3), M. Pegues (3) (1) Auburn University, U.S.A.; (2) Virginia Tech, U.S.A.; (3) Alabama Agricultural Experiment Station, U.S.A. Management of Fusarium Head Blight and DON in Alabama K. BOWEN (1), N. Sharma (1), N. McMaster (2), J. Jones (3), M. Pegues (3) (1) Auburn University, U.S.A.; (2) Virginia Tech, U.S.A.; (3) Alabama Agricu Fusarium head blight of wheat (FHB), caused by Fusarium graminearum, was severe on winter wheat in southern Alabama in the spring of 2015. A fungicide trial with four replications, planted to ‘AGS 2060’, allowed evaluations of demethylation inhibitor (DMI) and DMI plus quinone outside inhibitor (QoI) fungicides for managing FHB and the mycotoxin, deoxynivalenol (DON). Fungicides were applied at Feekes stage 10.5 (inflorescence complete). FHB intensity across plots was rated on a scale of 0 to 9 on 27 Apr, where 0=no disease and 9=severe disease. Plots were machine-harvested 5 Jun and kernel samples assayed for DON content. Kasugamycin mixtures with copper, mancozeb, or a thiadiazole improve bactericidal inhibition and efficacy of treatments against walnut blight K. NGUYEN (1), H. Forster (1), L. Wade (2), J. Adaskaveg (3) Programs that included Actigard + Tanos + Agriphage and Tanos + K-Phite + Cueva also significantly reduced disease compared to the control. The antibiotics, oxytetracycline and streptomycin, and Agriphage were ineffective at reducing disease severity under high disease pressure. Severe phytotoxicity was associated with Quintec at rates greater than 1 fl. oz. The copper formulation in Cueva performed better than the copper hydroxide standard. Overall, Actigard provided significant disease reduction as a stand-alone or in a program with other compounds. Nanoparticle encapsulation of seed treatment active ingredients for protection of maize seeds against Fusarium graminearum L. WASHINGTON (1), A. Mullis (2), D. Mayfield (3), B. Narasimhan (2), G. Munkvold (3) (1) Department of Plant Pathology Iowa State University, Ames, IA, U.S.A.; (2) Department of Chemical and Biological Engineering Iowa State University, U.S.A.; (3) Department of Plant Pathology Iowa State University, U.S.A. Fungicide seed treatments have long been a viable and effective strategy for protecting against seedborne and soilborne pathogens. However, losses to seeding diseases are still an important issue and there is a need for improved seed treatment efficacy. Amphiphilic polyanhydride nanoparticles have been used to enhance efficacy for several active ingredients in animal systems and have potential for agricultural use. Nanoparticle encapsulation has the potential to reduce active ingredient dosage and to provide sustained release. In order to assess these potential benefits, we developed a method to load polyanhydride nanoparticles with fungicide active ingredients to protect maize seeds against seedling blight caused by Fusarium graminearum. The objectives of this study were to determine the efficacy of a reduced rate of nanoparticle-encapsulated thiabendazole for suppression of seedling blight on hybrid maize seeds. Loaded nanoformulations were applied at varying rates with a planting polymer. These nanoparticle treatments were compared to a non-encapsulated thiabendazole treatment and a control. Seeds were treated and placed in a rolled towel assay for one week, to evaluate the effects on root length, shoot length, plant weight, and disease severity. Preliminary results indicate that the nanoformulated thiabendazole had significantly higher plant weight (p<0.0001) and root length (p<0.0001) than traditional thiabendazole seed treatments and inoculated controls. Evaluation of host resistance and chemical control to manage white mold of soybean in Ohio J. HUZAR NOVAKOWISKI (1), J. Huzar Novakowiski (1), J. Winger (1), P. Paul (1), A. Dorrance (1) (1) The Ohio State University, U.S.A. White mold of soybean is caused by the fungus Sclerotinia sclerotiorum. Kasugamycin mixtures with copper, mancozeb, or a thiadiazole improve bactericidal inhibition and efficacy of treatments against walnut blight K. NGUYEN (1), H. Forster (1), L. Wade (2), J. Adaskaveg (3) Kasugamycin mixtures with copper, mancozeb, or a thiadiazole improve bactericidal inhibition and efficacy of treatments against walnut blight K. NGUYEN (1), H. Forster (1), L. Wade (2), J. Adaskaveg (3) (1) University of California Riverside, U.S.A.; (2) Arysta LifeScience, U.S.A.; (3) University of California Riverside, U.S.A. Resistance to copper is common in Xanthomonas arboricola pv. juglandis (Xaj) populations in California walnut orchards making disease management difficult when applying copper alone. The use of mancozeb in mixtures with copper led to improved disease control. The risk of resistance development to both compounds, however, is high because this has occurred with other Xanthomonas spp. This stresses the need for new bactericides. Kasugamycin is an effective antibiotic against several bacterial pathogens (e.g., Erwinia and Pseudomonas spp.) but strains of Xaj often exhibit higher minimal inhibitory concentrations (MICs) than those of other species. Our goal was to improve the activity of kasugamycin by mixing with copper, mancozeb, or a thiadiazole. In in vitro spiral gradient endpoint and dilution plate testing with copper-resistant strains of Xaj, the average MIC for kasugamycin was 27.5 mg/L. When kasugamycin was mixed with copper, mancozeb, or a thiadiazole, average MIC values were reduced to 11.2, 6.1, or 12.8 mg/L, respectively. In field trials, treatments of kasugamycin mixed with either copper, mancozeb, or a thiadiazole reduced natural disease incidence by 84.2, 81.7, or 87.8%, respectively, whereas kasugamycin alone lowered blight by 50.3%, indicating the importance of using this antibiotic exclusively in mixed treatments for increased performance. Kasugamycin is currently under federal and California regulatory review for full registration on walnut. S4.62 Bacterial spot caused by Xanthomonas perforans, is a ubiquitous disease of tomato in both field and transplant operations in Florida. The high prevalence of copper-tolerant bacterial strains has compromised the efficacy of traditional copper compounds. Fifteen treatments consisting of various chemical and biological pesticides alone or in programs, including a non-treated control and a copper hydroxide standard, were evaluated for their ability to control bacterial spot on tomato seedlings. Treatments were foliar applied to tomato seedlings grown in 141 cell Speedling trays maintained under conditions similar to a commercial transplant operation. Each treatment was tested at least twice in four, 3-week experiments. Disease severity was evaluated on the third week. Stand-alone applications of Actigard, Cueva, Quintec, Innovotech A, and Innovotech B significantly reduced disease compared to the inoculated controls. Kasugamycin mixtures with copper, mancozeb, or a thiadiazole improve bactericidal inhibition and efficacy of treatments against walnut blight K. NGUYEN (1), H. Forster (1), L. Wade (2), J. Adaskaveg (3) In Ohio, localized outbreaks of the disease have occurred in each of the past seven years. Chemical control has had inconsistent results for disease management, especially when applied to cultivars with moderate levels of resistance. Therefore, in order to improve disease management recommendations, the objective of this study was to evaluate the efficacy of chemical control on soybean cultivars with different levels of resistance. The experiment was carried out in two fields in 2014 and five fields in 2015, all with a long history of the disease. Ten chemical treatments (untreated check, four fungicides applied at R1 or R1+R2 growth stages and one herbicide applied at R1), and two to four soybean cultivars were arranged in a split strip randomized block design with eight replications. Two fields had white mold in the untreated plots. There was significant interaction between chemical treatments and cultivars (P<0.05). Disease severity index (DSI) in the trials ranged from 2 to 41 in 2014 and from 14 to 44 in 2015. Applications of Endura fungicide and Phoenix herbicide significantly reduced DSI compared to the untreated control in both years. The cultivars with the highest disease resistance ratings had the lowest DSI and the highest yields. Thus, based on our results, growers should focus on soybean cultivars with the highest resistance ratings to manage white mold of soybean. Managing QoI-resistant Cercospora sojina in Mississippi soybean and assessing the physiological impacts of foliar fungicide phytotoxicity J. MANSOUR (1), M. Tomaso-Peterson (2), A. Henn (2), J. Bond (1), T. Irby (2) (1) Mississippi State University, U.S.A.; (2) Mississippi State University, U.S.A. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. Ascospores are the primary inoculum source of Erysiphe necator in most regions that grapevine is grown and overwinter within cleistothecia that are formed prior to leaf drop. Preventative fungicides are concluded at véraison (BBCH 83), and E. necator has ample time to colonize large portions of the leaf tissue, mate, and produce cleistothecia. Interruption of E. necator sexual reproduction post-véraison using a fungicidal oil may reduce overwintering inoculum. Chardonnay vines were sprayed once with 1% Organic JMS Stylet Oil (Vero Beach, FL) in 2014 and 2015. Fungicide application treatments were made when cleistothecia primordia were observed on leaves (BBCH 89), then 7, 14 and 21 days after primordia were observed. Cleistothecia development was monitored by weekly sampling of 10 leaves, approximately 1.4 m from the ground, per plot beginning at harvest (BBCH 89). Cleistothecia were enumerated along a transect on the leaf, and treatments were assessed using pairwise comparisons. Stylet oil applications significantly reduced cleistothecia in both years (P < 0.05) with no effect of application timing in either year (P > 0.4). Despite a reduction in the number of cleistothecia produced, cleistothecia were present in all plots prior to leaf drop, indicating that there may be limited utility of a late season curative fungicide application for disease management. Etiology and management of sour rot in grapes M. HALL (1), M. Hall (1), G. Loeb (1), W. Wilcox (1) (1) Cornell University, U.S.A. Sour rot is a major challenge for affected grape growers and winemakers worldwide, yet basic disease etiology and management techniques are not well understood. Symptoms are characterized by fruit rot accompanied by the smell of acetic acid and presence of Drosophila (fruit fly) species. We’ve successfully reproduced these visual and olfactory disease symptoms and the accompanying characteristic production of ethanol and its conversion to acetic acid within affected berries, in the lab. Healthy fruit were wounded, inoculated with Saccharomyces cerevisiae and Acetobacter aceti, and exposed to D. melanogaster adults. Inoculation without exposure to flies significantly promoted ethanol production but not acetic acid generation, whereas concomitant exposure to flies resulted in both. In field trials conducted on cv. ‘Vignoles’ in 2013-15, both insecticide and antimicrobial treatments significantly reduced sour rot development. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. In 2015, untreated vines averaged 20.5% sour rot severity, and this was reduced by 73-81% on vines treated prophylactically post-veraison with weekly sprays containing a combination of the insecticide zeta-cypermethrin plus the antimicrobial potassium metabisulfite or hydrogen dioxide; severity was reduced by 49% on vines receiving only insecticide sprays. These trials further support the hypothesis that sour rot results from a complex of yeast, bacteria, and Drosophila, and that targeting these organisms can provide significant levels of control. Characterization of Diaporthe spp. Cadophora malorum and Neofusicoccum parvum causing cordon dieback in kiwi plant and their chemical control in Chile G. DIAZ (1), M. Lolas (1), B. Latorre (2), E. Ferrada (2), J. Zoffoli (2) porthe spp. Cadophora malorum and Neofusicoccum parvum causing cordon dieback in kiwi plant and their chemical Characterization of Diaporthe spp. Cadophora malorum and Neofusicoccum parvum causing cordon dieback in kiwi plant and their chemical control in Chile G. DIAZ (1), M. Lolas (1), B. Latorre (2), E. Ferrada (2), J. Zoffoli (2) (1) Universidad de Talca, Chile; (2) Pontificia Universidad Catolica de Chile, Chile Characterization of Diaporthe spp. Cadophora malorum and Neofusicoccum parvum causing cordon dieback in kiwi plant and their chemical control in Chile G. DIAZ (1), M. Lolas (1), B. Latorre (2), E. Ferrada (2), J. Zoffoli (2) (1) Universidad de Talca, Chile; (2) Pontificia Universidad Catolica de Chile, Chile racterization of Diaporthe spp. Cadophora malorum and Neofusicoccum parvum causing cordon dieback in kiwi plant rol in Chile Kiwifruits is the third largest exporter of fresh kiwifruit (Actinidia deliciosa) worldwide, with over 10,000 ha cultivated. Cordon dieback in kiwi plant has increased during the last decade in Chile. Recently, we have identified several species obtained from cordon dieback in kiwi plants. Therefore, the objectives of this work were i) to characterize by means of molecular and pathogenicity studies and ii) chemical control of Diaporthe ambigua, Diaporthe australafricana, Cadophora malorum and Neofusicoccum parvum. Pure cultures were characterized by phylogenetic analyses using three loci (ITS, β-tubulin and EF1-α). Pathogenicity test were conducted on kiwifruit canes and trunks. In vitro sensitivity and protection of pruning wounds with benomyl, pyraclostrobin and tebuconazole was evaluated. Managing QoI-resistant Cercospora sojina in Mississippi soybean and assessing the physiological impacts of foliar fungicide phytotoxicity J. MANSOUR (1), M. Tomaso-Peterson (2), A. Henn (2), J. Bond (1), T. Irby (2) (1) Mississippi State University, U.S.A.; (2) Mississippi State University, U.S.A. FHB in non-treated plots was rated at 7.2, which was significantly greater than FHB intensity in plots treated with propiconazole (Prop), tebuconazole (Tebu) plus trifloxystrobin (Trif), or Tebu plus prothioconazole (Proth), while Trif+Proth and metaconazole (Metc) plus pyraclostrobin (Pyra) had intermediate FHB levels. DON concentration, however, was > 5 ppm in grain samples from Trif+Proth- and Metc+Pyra-treated plots; only grain from Tebu+Proth-treated plots had lower DON concentrations, at 3.1 ppm, than from non-treated plots (4.6 ppm). Yield and test weight were low due to heavy disease pressure and lodging, and did not differ due to fungicide. These results confirm that a QoI fungicide, even when applied in combination with a DMI, can increase DON concentration under severe FHB pressure in wheat. Effect of leaf spot pathogens and fungicide application on Bacterial leaf streak development in wheat under field conditions in South Dakota S. ALI (1), S. Sehgal (1), K. Glover (1), N. Kaur (1) (1) South Dakota State University, Brookings, SD, U.S.A. Bacterial leaf streak (BLS), caused by Xanthomonas compastris (Xc) pv. Undulosa, is an important disease of wheat in South Dakota. The objective of this study was to see the effect of fungal leaf spot pathogen “Pyrenophora tritici-repentis (Ptr)” (tan spot) and fungicide application on BLS development. To obtain our objectives, two wheat cultivars Briggs (BLS susceptible) and Select (moderately resistant) were planted in a randomized complete block design with three replications at SDSU Volga Agricultural Experimental Station, Volga, SD in 2014. Three plots of each cultivar was S4.63 inoculated with; Xc cell suspension alone (T1), Ptr spores suspension and then with Xc (T2), Ptr, Xc and then applied fungicide “Prosaro” (T3) and Ptr alone (T4), and non-inoculated and untreated (T5). BLS disease data was recorded 12 days post-treatments. BLS disease severity was observed higher in T2 (50.5%) as compared to T1 (Briggs =23.33%) and T3 (41.83%) in both cultivars though the severity level was higher in Briggs as compared to Select. Our results indicate that Ptr infection may help in enhancing bacterial penetration. Also, fungicide application stops tan spot development and that may helps in increasing BLS development. inoculated with; Xc cell suspension alone (T1), Ptr spores suspension and then with Xc (T2), Ptr, Xc and then applied fungicide “Prosaro” (T3) and Ptr alone (T4), and non-inoculated and untreated (T5). BLS disease data was recorded 12 days post-treatments. Managing QoI-resistant Cercospora sojina in Mississippi soybean and assessing the physiological impacts of foliar fungicide phytotoxicity J. MANSOUR (1), M. Tomaso-Peterson (2), A. Henn (2), J. Bond (1), T. Irby (2) (1) Mississippi State University, U.S.A.; (2) Mississippi State University, U.S.A. BLS disease severity was observed higher in T2 (50.5%) as compared to T1 (Briggs =23.33%) and T3 (41.83%) in both cultivars though the severity level was higher in Briggs as compared to Select. Our results indicate that Ptr infection may help in enhancing bacterial penetration. Also, fungicide application stops tan spot development and that may helps in increasing BLS development. Control of apple rots caused by Phacidiopycnis washingtonensis, Phacidium lacerum y Diplodia seriata in postharvest in Chile G. A (1), M. Caceres (1), M. Lolas (1), C. Pacheco (1), T. Daza (1) (1) Universidad de Talca, Chile The apple rots caused by fungal species in pre-harvest and postharvest are an important problem for Chilean apple industry. Recently, we detected to Phacidiopycnis (Pha.) washingtonensis, Phacidium (P.) lacerum and Diplodia (D.) seriata associated with apple rot in pre-harvest and postharvest in Maule Region, Chile. The objective this study were determine i) to sensitivity in vitro to fludioxonil (phenyl pyrrole), pyrimethanil (aniline-pyrimidine) tebuconazole (DMI) and thiabendazole (benzimidazole), and ii) to reduce infection with fungicide applied prior to storage on apple cv. Cripps Pink. Apple fruits were protected with different fungicides and methods (fogging and drench), and after 24 h inoculated with each pathogen. All fruit were stored at 0°C for 60 d. The results indicate that the isolates of Pha. washingtonensis, P. lacerum and D. seriata were sensitive to all fungicides, obtaining differents EC50 values. Fludioxonil, pyrimethanil, and thiabendazole by thermo-fogging reduced significantly to infection during cold storage obtaining an efficacy of 75% to 96%. Fludioxonil and thiabendazole applied by drench reached between 84% and 100% of effectivity. Finally, fungicide applied prior to storage by fogging or drench can be an effective control of apple rots during cold storage in Chile. Reducing initial infections of apple scab and black rot with salicylic acid and Actigard applied as trunk injections or sprays P. ABBASI (1), G. Braun (2), E. Bevis (2), S. Fillmore (2) (1) Agriculture and Agri-Food Canada, Canada; (2) Agriculture and agri-Food Canada, Canada Reducing initial infections of apple scab and black rot with salicylic acid and Actigard applied as trunk injections or sprays P. ABBASI (1), G. Braun (2), E. Bevis (2), S. Fillmore (2) (1) Agriculture and Agri-Food Canada, Canada; (2) Agriculture and agri-Food Canada, Canada Apple scab [Venturia inaequalis] and black rot [Botryosphaeria obtusa] are two fungal diseases that affect apple leaves early in the growing season and also make fruit unmarketable at harvest. Salicylic acid has been found to induce plant defense mechanisms to reduce initial disease infections. In this study, salicylic acid and Actigard were applied as trunk injections and spray treatments to measure their effects on the development of apple scab and black rot on established apple trees (‘Honeycrisp’ and ‘Cortland’) in the field during 2014 and 2015. Treatments were applied at the tight cluster stage of bud development and again near bloom. Incidence of leaf infections was assessed in planta 7, 14, and 21 days after the second treatment and disease incidence on fruit was assessed at harvest. For injection treatments, the branch directly above the injection site was assessed. In 2014, salicylic acid and Actigard applied as trunk injection or spray treatments resulted in lowest black rot and apple scab incidences on leaves and fruit compared to the control treatment. In 2015, salicyclic acid and Actigard again showed a lower incidence of apple scab in the early stages of leaf infections only. No reduction in fruit infection was observed in 2015. Although salicylic acid and Actigard treatments showed a reduction in early leaf infections, apple trees must also be protected from secondary infections. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. However, the choice of the fungicide when weather conditions are highly favorable for disease development is the key for effective control. Interaction among leaf area index, spray volumes and fungicide doses in Asian soybean rust control A. CHECHI (1), W. Boller (1), C. Forcelini (1), R. Roehrig (1), E. Zuchelli (1) (1) UPF, Brazil Soybean cultivars vary in their leaf area index (LAI), which influences the distribution of the drops generated by the spray fungicides in plant canopy. The objective of this study was to investigate the interaction among the LAI, spray volumes and fungicide doses in controlling Asian soybean rust. The tests were conducted at UPF experimental field, greenhouse and growth chamber during 2014 to 2016. The dose was varied (75%,100% and 125% of the commercial dose) and the spray volume (100,150 and 200 L/ha) at application of fungicides on soybean plants with different LAIs. An assay was also conducted with the use of simulated rainfall at different times after the fungicide application. There were three cultivars in the experimental field: BMX Ponta (high LAI); BMX Vanguarda (medium), NS 5445 (low), and only the last one in the other trial. The applications were in V7 stages (trifloxystrobin+prothioconazole), R1 and R1+20 days (azoxystrobin+benzovindiflupir) for field trials and only in V8 stage for testing in the greenhouse. The severity of the rust, the number of spores/cm2, the LAI and the grain yield were quantified and analyzed by Anova and Skott-Knott test (0.05). The results showed that the spray volume and the fungicide dose should be appropriate according to the cultivar LAI. The closer the application when rain occurs, lower the control achieved by applying the fungicide. The cultivar LAI, spray volume and the fungicide dose influenced the disease control. Relationship between fungicide dose and Asian soybean rust control A. CHECHI (1), C. Forcelini (2), W. Boller (2), R. Roehrig (2), E. Zuchelli (2) (1) UPF, Brazil; (2) UPF, Brazil The diseases affect the soybean crop yield, and the main one is the Asian soybean rust (ASR) and the control of the disease is mainly based on fungicides. The objective of this study was to verify if the use of 0 to 100% of the commercial dose of fungicide affects the control of the disease. The experiment was conducted in a growth chamber at UPF, in a completely randomized design with six replications. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. Field trials conducted at two different locations in Central Florida evaluated Cabrio, Switch, Scala, Penncozeb, and Luna Experience for disease control on pomegranate. Fungicides were applied beginning in Early-March when > 5% flower buds were visible and reapplied every two weeks until the end of growing season in August. Disease severity was rated based on the Horsfall-Barratt scale, and values were used to calculate area under disease progress curves. Significant differences were observed between the treatments at both locations (P < 0.001). Cabrio, Luna Experience, and Penncozeb reduced the disease severity by 87, 84, and 67% respectively; while Switch and Scala reduced by 53 and 39% respectively compared to untreated control. Follow-up field studies are planned for 2016 to establish effective usage patterns. Effectiveness of fungicide treatments following the Strawberry Advisory System for control of Botrytis fruit rots in F L. CORDOVA (1), A. Amiri (2), N. Peres (3) (1) University of Florida, U.S.A.; (2) Washington State University, U.S.A.; (3) University of Florida, U.S.A. Effectiveness of fungicide treatments following the Strawberry Advisory System for control of Botrytis fruit rots in Florida L. CORDOVA (1), A. Amiri (2), N. Peres (3) (1) University of Florida, U.S.A.; (2) Washington State University, U.S.A.; (3) University of Florida, U.S.A. Botrytis fruit rot (BFR) of strawberry is caused by Botrytis cinerea. Its management was traditionally carried out by weekly fungicide applications using multi-site fungicides in early season and single-site fungicides during bloom when the environment is conducive for disease development. Recently, many Florida growers have been following the Strawberry Advisory System (SAS) to improve timing and reduce the number of fungicide applications. Our objective was to determine the efficacy of different fungicides for BFR control when following the SAS spray timing alerts compared to preventive Calendar-based applications, and to implement specific fungicide recommendations on SAS. Field trials conducted during two seasons on a commercial farm in Plant City-FL showed that during the highest disease incidence periods, treatments with cyprodinil + fludioxonil or fluopyram following SAS and Calendar-based applications were the most effective in controlling BFR. Pyrimethanil and boscalid + pyraclostrobin were not effective due to resistance problems and are no longer recommended in the SAS. For SAS alerts during absence of bloom, a multi-site fungicide such as captan or thiram is recommended. Treatments following SAS provided good control of BFR with about half the number of fungicide applications compared to Calendar- based applications. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. This study reveals that Diaporthe ambigua, Diaporthe australafricana, Cadophora malorum and Neofusicoccum parvum are the major fungal species causing cordon dieback, and that the protection of pruning wounds with chemical fungicide is a good measured to prevent infection in commercial orchards of kiwi plants in Chile. S4.64 Contribution of protectant fungicides applied as mid-season cover sprays to management of peach brown rot at harvest N. LALANCETTE (1), N. Lalancette (1), L. Blaus (1), J. Gager (1), K. McFarland (2) (1) Rutgers University, Agricultural Research & Extension Center, U.S.A.; (2) Rutgers University, Agricultural Research & Exten Protectant fungicides are routinely applied to peach trees during the period from shuck-split through early preharvest. These cover sprays are usually applied to control peach scab, rusty spot, and anthracnose. However, results from fungicide treatments in 2010, which lacked the typical preharvest applications, indicated that these mid-season sprays may also provide control of brown rot at harvest, caused by Monilinia fructicola. To confirm these observations and determine the mechanism of control, a field study was conducted from 2012-2015 in an experimental peach orchard. In each year, four protectant fungicides (captan, sulfur, ziram, and thiram) were applied from shuck-split through sixth cover. Captan and thiram in 2012 and captan in 2013 and 2015 significantly reduced brown rot at harvest. Estimation of fungicide levels on fruit in 2013 to 2015, using an in vivo bioassay based on M. fructicola spore germination, showed that residual activity was the primary mechanism for control. Results also showed that captan may have a higher intrinsic efficacy than the other protectants, thereby allowing extended control through the preharvest period. These findings indicate that residual activity from mid-season applications of protectant fungicides (i) can contribute significantly to the efficacy of preharvest programs and (ii) may play an important role in reducing selection for resistance among site-specific fungicides typically applied during the preharvest period. Fungicide efficacy for the control of pomegranate diseases in Florida A. KC (1), G. Vallad (2) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. A statewide survey identified six economically important pathogens of Pomegranate (Punica granatum) in Florida. Anthracnose fruit rot and leaf spot, Botryosphaeria shoot blight and stem canker, and Pilidiella fruit rot were commonly observed throughout the growing season resulting in more than 80% fruit loss. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. Further in vitro investigations are ongoing in Florida to examine the population diversity of these pathogens and their QoI fungicide resistance profiles. diseases. The study objectives were to examine the efficacy of commonly used QoI fungicides for managing these diseases and to collect isolates for future pathogen population studies. Field plots of the peanut cultivar Georgia-06G were established after June 1st during 2014 and 2015 growing seasons. The fungicides azoxystrobin (Abound 2.08 SC at 18 fl oz/A) and pyraclostrobin (Headline at 9 fl oz/A) were individually applied every 12 to 17 days starting about 30 days after planting for comparison with untreated controls. Disease severity and incidence were assessed using the Florida 1-10 scale and by sampling 12 leaflets per plot. The predominant disease in 2014 was LLS and ELS in 2015. Azoxystrobin and pyraclostrobin treated plots had similar AUDPC values in 2014 and were significantly less than those of untreated plots (P ≤0.05). Mean AUDPC values were not significantly different in 2015. These results suggest that QoI fungicides have greater efficacy for LLS, however, they do not provide adequate control of ELS. Further in vitro investigations are ongoing in Florida to examine the population diversity of these pathogens and their QoI fungicide resistance profiles. Effect of fungicide timing and frequency on reducing foliar diseases and yield loss on runner and valencia type peanuts in Haiti A. FULMER (1), T. Brenneman (1), R. Kemerait (1) (1) University of Georgia, U.S.A. Effect of fungicide timing and frequency on reducing foliar diseases and yield loss on runner and valencia type peanuts in Haiti A. FULMER (1), T. Brenneman (1), R. Kemerait (1) (1) University of Georgia, U.S.A. Peanut (Arachis hypogaea L.) yields in Haiti are often severely reduced by peanut rust (Puccinia arachidis) and late leaf spot (Cercosporidium personatum). Two trials were conducted in 2015 to evaluate the effect of 6 fungicide regimes on disease severity and yield for two market types. Both tests used a split plot design with 4 replications with variety as whole plot and fungicide as subplot. Runner (local landrace and Cv. Georgia-06G) and valencia (local landrace and Cv. N.M. Valencia A) market types were planted in northern and central Haiti, respectively. For runners, Muscle ADV (2.3 L/ha) was applied 2-6 times starting 30-60 days after planting (DAP) with 14-28 day intervals; valencias received 1-4 sprays starting 30-45 DAP with 14-28 day intervals. Evaluation of fungicide efficacy and cultivar susceptibility for the management of anthracose on celery M. MCDONALD (1) (1) University of Guelph, Canada Anthracnose on celery, also called celery leaf curl, is caused by Colletotricum fioriniae. The disease was first identified in Ontario, Canada, in 2011 and has been increasing in severity. Field trials were conducted to determine if there were differences in cultivar susceptibility and to identify effective fungicides. The celery cultivars were: TZ6010, TZ0295, TZ6200, TZ6029, TZ9779, Nero, Sabroso and Plato. Cv. TZ 6200 was used for the fungicide trial. Seedlings were transplanted on 24 June into ‘muck’ soil (organic matter ~70%, pH 6.5) in the Holland Marsh, Ontario. Each replicate consisted of 3 rows, 55 cm apart and 5 m in length. The trials were inoculated on 18 August with a suspension of 1 × 105 conidia ml–1, and harvested on 13 October. The fungicides evaluated were Omega (fluazinam 40.0%), Bravo (chlorothalonil 50%), and Flint (trifloxystrobin 50%). Fungicides were applied on 31 July, 13 August and 8 September, 2015. Disease incidence and marketable yield were assessed. Cultivars Plato, TZ 9779, 6200 and 0295 had high disease incidence (86, 66, 66, and 67%) compared to less susceptible cv.’s Sabroso (24%) and TZ 6010 (27%). Fungicides Flint and Bravo had low disease (34, 45%), compared to the untreated check (83%). Increased disease incidence in September was related to a decrease in the percent of marketable yield by weight (r2=0.45). Optimizing the systemicity of triazole formulations by enhancing leaf penetration G. POON (1), C. Irwin (2), J. Dinglasan (2), N. Loukine (1) (1) Vive Crop Protection, Canada; (2) Vive Crop Protection, Canada The systemicity of a fungicide plays a vital role in its performance. While the ability of a fungicide to penetrate the leaf cuticle and translocate through plant tissue is active specific, inert formulation ingredients can optimize these processes on target crops. Triazoles and strobilurins are broad spectrum fungicides with known systemicity; however, in lab testing only a small fraction of applied active ingredient was able to penetrate a synthetic membrane that mimics a leaf cuticle. Fungicide formulations that are optimized for membrane penetration may have enhanced disease control over existing formulations. Laboratory trials were conducted using a Franz cell fitted with a synthetic membrane to identify experimental formulations that may have better leaf cuticle penetration than the commercial product. Field trials were conducted in 2015 to evaluate the efficacy of experimental tebuconazole formulations on powdery mildew in acorn squash. Fungicides for management of Sclerotium rolfsii and enhanced overwintering survival of stevia A. KOEHLER (1), H. Shew (1) (1) NCSU, U.S.A. Fungicides for management of Sclerotium rolfsii and enhanced overwintering survival of stevia A. KOEHLER (1), H. Shew (1) (1) NCSU, U.S.A. Fungicides for management of Sclerotium rolfsii and enhanced overwintering survival of stevia A. KOEHLER (1), H. Shew (1) (1) NCSU, U.S.A. Stevia (Stevia rebaudiana) is an herbaceous perennial under evaluation as a new crop in NC. Stem rot caused by Sclerotium rolfsii results in wilting, rapidly followed by root and stem necrosis. Currently there are no fungicides labeled for use on stevia, which will limit expansion of commercial production. Initial fungicide efficacy trials were established in July 2014 in naturally infested soil in Kinston, NC. Treatments included azoxystrobin, flutolanil, and tebuconazole applied at 3 timings. This trial was repeated in May and July of 2015 in adjacent field space. Stand counts were taken to determine disease incidence and overwintering survival of stevia crowns. All fungicides reduced incidence of stem rot compared to the control. In 2015, azoxystrobin was correlated with enhanced overwintering survival (mean survival rate, 66%) compared to other fungicide treatments (19%) and the untreated control (16%). In 2015, a second field site was established in Rocky Mount, NC to evaluate the effect of late season applications of azoxystrobin and pyraclostrobin. Both fungicides resulted in higher root weights and greater new shoot production compared to the control in spring 2016. Final stand counts of all treatments will be taken in spring 2016 to establish overwintering survival rates. Successful overwintering of stevia crowns directly impacts the profitability of second and third year harvests and the likelihood of long term establishment of stevia in NC. Timing of fungicide applications is critical for blackleg control in canola L. DEL RIO MENDOZA (1), S. Ruud (1), S. Mansouripour (1) (1) North Dakota State University, U.S.A. Blackleg, caused by Leptosphaeria maculans, is an important disease affecting rapeseed/canola (Brassica napus) production worldwide. The genetic resistance currently available in most commercial cultivars is ineffective against strains of blackleg that have become prevalent in recent years and because of this, growers are relying more frequently on fungicide use to manage this disease. To optimize fungicide use, greenhouse studies evaluated the efficacy of different timings of application of azoxystrobin, pyraclostrobin, and fluxapyroxad on blackleg severity. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. For this, the central leaflets of soybean plants (NS 5445 cultivar) grown in a greenhouse were collected. The solutions from 0 to 100% of the dose were prepared using two fungicides, one consisting of trifloxystrobin + prothioconazole and other azoxystrobin + benzovindiflupir. The leaflets were dipped in appropriate fungicide solutions and placed in gerboxes, where humid chambers were set up. In the next day, a suspension of Phakopsora pachyrhizi (50,000 spores/mL) was prepared, which was sprayed on the leaflets. The gerboxes were left in the dark for 24 hours at a temperature of 23°C. After this, they were placed on benches in a growth chamber with a photoperiod of 12 hours light/dark and constant temperature of 23°C. After 22 days incubated, the number of spores/cm2 was assessed. The controls without application of fungicides showed an average of 85.4 spores/cm2 (T+P) and 83.5 spores/cm2 (A+B). There was a reduction in the number of spores/cm2 when increases the fungicide dose. The ASR control depends on the fungicide dose in the spray solution. Response of Early and Late Leaf Spot Diseases on Peanut to QoI Solo Spray Programs W. ELWAKIL (1), N. Dufault (1) (1) University of Florida, U.S.A. Foliar diseases including rust (Puccinia arachidis), early leaf spot (ELS; Cercospora arachidicola) and late leaf spot (LLS; Cercosporidium personatum) are annual problems for southeast peanut producers. Fungicides are a critical part of an integrated management program for these devastating peanut S4.65 diseases. The study objectives were to examine the efficacy of commonly used QoI fungicides for managing these diseases and to collect isolates for future pathogen population studies. Field plots of the peanut cultivar Georgia-06G were established after June 1st during 2014 and 2015 growing seasons. The fungicides azoxystrobin (Abound 2.08 SC at 18 fl oz/A) and pyraclostrobin (Headline at 9 fl oz/A) were individually applied every 12 to 17 days starting about 30 days after planting for comparison with untreated controls. Disease severity and incidence were assessed using the Florida 1-10 scale and by sampling 12 leaflets per plot. The predominant disease in 2014 was LLS and ELS in 2015. Azoxystrobin and pyraclostrobin treated plots had similar AUDPC values in 2014 and were significantly less than those of untreated plots (P ≤0.05). Mean AUDPC values were not significantly different in 2015. These results suggest that QoI fungicides have greater efficacy for LLS, however, they do not provide adequate control of ELS. Fungicides for management of Sclerotium rolfsii and enhanced overwintering survival of stevia A. KOEHLER (1), H. Shew (1) (1) NCSU, U.S.A. These active ingredients, present in four of the six fungicides registered to manage blackleg in North Dakota, were applied two days before and 0, 2, 4, 8, or 16 days after seedlings of cv. Westar were inoculated with an L. maculans spore suspension. Disease severity was evaluated 12 days after inoculation and before harvest. For each fungicide, trials were conducted separately using a randomized complete block design. Each study was replicated and conducted three times. All three fungicides were most effective when applied within eight days from inoculation and lost their efficacy when applied at a later time. This information highlights the need for early protection of seedlings as well as for the development of disease warning systems that could identify help growers optimize spraying schedules. Interruption and reduction of Erysiphe necator cleistothecia development utilizing fungicidal oil L. THIESSEN (1), W. Mahaffee (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS, U.S.A. There were no interactions between variety and fungicide regime. At each location, disease severity was similar between varieties, but Georgia-06G and the local valencia yielded 2500 and 430 kg/ha more, respectively. For valencias, at least 2 sprays were required to reduce severity and increase yield; however, 3 or more sprays gave the highest yields. For runners, 3 and 4 sprays were required to reduce rust and leafspot, respectively, but 6 sprays had the least disease and the only statistically different yield. Compared to untreated plots, the best treatments increased yields by 68% for both market types. These results are a significant step toward developing management recommendations for growers in Haiti. Greenhouse characterization of hydrazone-copper mixtures as low copper treatments to control early and late blight on tomato C. AVILA-ADAME (1), D. Young (2), J. Webster (2), O. Brian (2), D. Ouimette (2) (1) Dow AgroSciences LLC, U.S.A.; (2) Dow AgroSciences LLC, U.S.A. Copper-based fungicides remain critical tools in crop disease management programs to control a broad spectrum of fungal and bacterial plant pathogens. However, because of the high rate required to control plant pathogens, there is concern regarding their potential negative environmental impact. Certain salicylaldehyde benzoylhydrazone (SBH) compounds bind copper and show strong synergism with copper when tested for inhibition of fungal growth under in vitro conditions. Experiments were conducted to characterize synergism for Cu-hydrazone mixtures in controlling Phytophthora infestans and Alternaria solani on tomato under greenhouse conditions. Treatments included a SBH alone, three copper materials, and Cu-SBH mixtures. The SBH alone was weakly active against both pathogens with 26 and 33% disease control at 400 micromolar for P. infestants and A. solani, respectively. The copper materials, which included Cu(OH)2 in the form of Kocide® 2000, CuSO4, and CuCl2, provided a similar level of disease control (p > 0.3) when applied alone. The copper-SBH mixtures at various molar ratios produced synergistic interactions in nearly all combinations and were superior to the commercial fungicide Kocide® 2000 at equivalent rates of copper. Yield loss and management of orange cane blotch of blackberry, caused by the alga Cephaleuros virescens F. BROWNE (1), P. Brannen (1), H. Scherm (1), L. Fall (1), J. Taylor (2), J. Shealey (3), E. Beasley (1) (1) University of Georgia, U.S.A.; (2) University of Georgia, U.S.A.; (3) University of Georgia, U.S.A. The alga Cephaleuros virescens causes orange cane blotch (OCB), a serious disease of blackberries in the southeastern United States. OCB impact on blackberry growth and yield was assessed. Neither cane diameter nor fruit size were significantly impacted by severity of OCB; however, maximum fruit number per cane decreased by 80% with increasing OCB severity, thereby resulting in reduced yields. Field efficacy trials with diverse algicides, disinfectants, and fungicides were conducted over a 3-year period from 2013 to 2015 at two locations on a thornless blackberry cultivar, ‘Ouachita’. Treatments, applied during the summer and fall, included ametoctradin + dimethomorph, calcium polysulfide, calcium polysulfide + surfactants, captan, chlorothalonil, copper hydroxide, copper hydroxide + hydrogen dioxide, fluazinam, fluopicolide, hydrogen dioxide, mancozeb, mancozeb + copper hydroxide, mandipropamid, mefenoxam, mefenoxam + copper hydroxide, mefenoxam + mancozeb, potassium phosphite, potassium phosphite + captan, potassium phosphite + copper hydroxide, oxathiapiprolin and diluted sodium hypochlorite. Greenhouse characterization of hydrazone-copper mixtures as low copper treatments to control early and late blight on tomato C. AVILA-ADAME (1), D. Young (2), J. Webster (2), O. Brian (2), D. Ouimette (2) (1) Dow AgroSciences LLC, U.S.A.; (2) Dow AgroSciences LLC, U.S.A. Potassium phosphite, generally active on both oomycetes and fungi, was the only chemical that provided significant suppression of the disease, with 48-80% reduced lesion coverage of canes. Management of OCB will be difficult in light of the limited number of labeled applications of potassium phosphite allowed and the potentially long infection period of the pathogen. Evaluation of Nematicides for Control of Southern Root-Knot Nematodes in Lima Bean J. JONES (1), G. Johnson (1), N. Kleczewski (1), J. Desaeger (2) (1) University of Delaware, U.S.A.; (2) DuPont Crop Protection, U.S.A. Southern root-knot nematodes (Meloidogyne incognita-RKN) significantly reduce lima bean (Phaseolus lunatus) yields. Chemical control options for RKN are limited. We evaluated the efficacy of new nematicides on RKN in lima bean experiments conducted in greenhouse (GH) and microplot (MP) settings. Treatments included Nimitz at low and high labeled rates, Luna, Movento, Luna+Movento, Vydate, Mocap and an untreated, RKN infested control. GH treatments were arranged in a factorial design, with application of nematode eggs (0, 6,000, or 30,000 eggs pot-1) crossed with nematicide treatment. MP treatments were arranged in a randomized complete block design and all plots were infested with 4.80 kg of RKN infected tomato root tissue and soil. Root galling, RKN eggs and juveniles, and aboveground plant dry masses were determined and statistically analyzed using Mixed Model ANOVA. In the GH study, Nimitz at both rates provided the greatest reduction in RKN galling compared to the controls, whereas Movento treatments were not significantly different than the controls. In the MP study, all treatments except Movento significantly reduced RKN populations and had significantly greater yields relative to controls. Vydate, Mocap, and Nimitz (high and low rate) treatments had the highest yields in the MP study. Based on these results, several new nematicides show potential for managing RKN in lima beans. Evaluation of soil conditioner OR079 for the optimization of metalaxyl in control of Phytophthora nicotianae in Texas citrus orchards P. DUBERNEY (1), J. Hernandez (1), V. Ancona (1) (1) Texas A&M Kingsville Citrus Center, U.S.A. Evaluation of fungicide efficacy and cultivar susceptibility for the management of anthracose on celery M. MCDONALD (1) (1) University of Guelph, Canada All tebuconazole treatments were applied weekly at label rate following disease onset for a total of S4.66 four applications. While yield did not differ statistically between any of the treatments, an experimental formulation significantly reduced disease severity by 40.5% relative to the commercial product and 55.3% relative to the untreated control. From the positive field results achieved by enhancing leaf cuticle penetration, further field and lab testing to optimize performance will be completed. Season long management of potato silver scurf caused by Helminthosporium solani in Wisconsin S. MACCHIAVELLI-GIRÓN (1), S. Jordan (1), J. Crane (2), A. Gevens (1) 1) University of Wisconsin-Madison, U.S.A.; (2) University of Wisconsin-Madison, U.S.A. Silver scurf, caused by the fungus Helminthosporium solani, is a tuber blemish disease of increasing concern in potato production. The disease affects periderm quality and appearance, impacting marketability as well as storability. Cultural management strategies are limited and often impractical for large-scale growers, making fungicide applications an important component of integrated silver scurf management. Helminthosporium solani initially infects tubers in the field, and has secondary infection cycles during storage. Because inoculum can be reduced at multiple times during potato production, we conducted a full life cycle study in 2015 to evaluate the aggregated impact of fungicide selection and timing on silver scurf severity in ‘Dark Red Norland’ potatoes. Fungicides were applied at three times: at planting as seed or in-furrow treatments; in-field as a series of 3 foliar sprays; and post-harvest immediately before storage. Non-treated comparisons were included. The at-planting applications had the greatest impact on symptom severity, although the post-harvest treatments also contributed to disease reductions. The lowest severity resulted from the Maxim MZ (fludioxonil + mancozeb) seed treatment with the Stadium (fludioxonil + azoxystrobin + difenoconazole) post-harvest treatment. Along with cultural methods, at-plant and post-harvest fungicide treatments together were necessary for silver scurf control. Greenhouse characterization of hydrazone-copper mixtures as low copper treatments to control early and late blight on tomato C. AVILA-ADAME (1), D. Young (2), J. Webster (2), O. Brian (2), D. Ouimette (2) (1) Dow AgroSciences LLC, U.S.A.; (2) Dow AgroSciences LLC, U.S.A. Evaluation of soil conditioner OR079 for the optimization of metalaxyl in control of Phytophthora nicotianae in Texas citrus orchards P. DUBERNEY (1), J. Hernandez (1), V. Ancona (1) (1) Texas A&M Kingsville Citrus Center, U.S.A. The Texas citrus industry faces a great problem with the control of foot and root rot disease caused by Phytophthora nicotianae. P. nicotianae infects feeder roots and the trunk of trees causing poor water and nutrient uptake leading to yield loss, fruit size reduction and tree decline. Treatments for Phytophthora diseases are limited, therefore optimization of available products is of great importance. We hypothesized that the effectiveness of metalaxyl in reducing P. nicotianae soil propagules can be increased when applied in combination with the soil conditioner OR079. To test our hypothesis we applied metalaxyl, OR079, and metalaxyl plus OR079 to mature grapefruit trees with Phytophthora infection and quantified Phytophthora propagules in the soil. Phytophthora propagule counts were assessed at 4, 8, and 12 weeks post treatment and compared to an untreated control. Results showed that all treatments had significantly reduced Phytophthora counts in the soil at 4 and 8 weeks post application compared to that of the untreated control, but were not statistically different to the control by 12 weeks. Moreover, OR079 did not increase metalaxyl effectiveness in reducing Phytophthora propagules but reduced its activity when mixed. In addition, application of OR079 and metalaxyl significantly increased citrus fruit size. These results indicate that timely treatment for Phytophthora infection improves root health and water uptake leading to increase citrus fruit quality. S4.67 Efficacy of Oxathiapiprolin toward Phytophthora sojae and Phytophthora sansomeana A. VARGAS (1), M. Eyre (1), A. Dorrance (1) (1) The Ohio State University, U.S.A. Poor crop stand establishment caused by soil-borne pathogens is very common in soybean fields in Ohio. Metalaxyl has been the most commonly used fungicide to control Phytophthora and Pythium species in soybean. However, several studies have reported an increase of insensitivity to metalaxyl in Phytophthora and Pythium populations in Ohio. Therefore, alternative fungicides should be tested for efficacy towards Phytophthora and Pythium species. The objective of this experiment was to determine the efficacy of oxathiapiprolin in reducing the growth of Phytophthora sojae and Phytophthora sansomeana in soybean. A total of 28 isolates collected from infested fields were evaluated using amended agar Petri dishes with 5 different concentrations of oxathiapiprolin. Sensitivity of each isolate was evaluated by estimating the diameter of mycelial growth on each Petri dish and comparing it to the control. Results from preliminary data show that oxathiapiprolin treatments reduced P. sojae and P. sansomeana mycelial growth and mean values varied among isolates. Efficacy of three soybean fungicide seed treatments against Pythium species in seed plate and growth chamber assays K. SCOTT (1), A. Vargas (1), M. Eyre (1), A. Dorrance (1) (1) The Ohio State University, U.S.A. Efficacy of three soybean fungicide seed treatments against Pythium species in seed plate and growth chamber assays K. SCOTT (1), A. Vargas (1), M. Eyre (1), A. Dorrance (1) (1) The Ohio State University, U.S.A. Recent studies have reported a wide range of sensitivity to metalaxyl in Pythium species in Ohio, thus new active ingredients are important for management of seed and seedling blight. The objective of this study is to determine the efficacy of three seed treatments with a combination of active ingredients for oomycetes against a diverse population of Pythium species. Commercial formulations of Intego™ Suite, Cruiser Maxx® Advanced, and Acceleron® were applied to seed at labeled rates and evaluated with Pythium isolated from fields across Ohio in both a plate assay and a cup assay. In the cup assay, the mean root score was significantly lower in all fungicide seed treatments compared to the control, but there were no significant differences between treatments. In the seed plate assay, there was a significant difference for mean seed rot score between all treatments. Acceleron® had the lowest seed rot score towards all Pythium species, followed by Intego™ Suite, Cruiser Maxx® Advanced, and the non-treated check, respectively. These seed treatments may provide adequate protection against Pythium infection in soybean seed and seedling stages. In addition, the seed plate assay may be used to efficiently test for isolate pathogenicity and the efficacy of seed treatments. Evaluation of soil conditioner OR079 for the optimization of metalaxyl in control of Phytophthora nicotianae in Texas citrus orchards P. DUBERNEY (1), J. Hernandez (1), V. Ancona (1) (1) Texas A&M Kingsville Citrus Center, U.S.A. Eight isolates did not grow at 0.01 µg active ingredient/ml. Based on these results could be used to manage seed rot caused by P. sojae and P. sansomeana. Combined Application of Bacillus amyloliquefaciens ZY-9-13 with Fungicides for Control of Tobacco Black Shank W. MU (1), J. XI (1), C. XUE (1), L. HU (1), J. SONG (1) (1) Zhengzhou Tobacco Research Institute of CNTC, China Combined Application of Bacillus amyloliquefaciens ZY-9-13 with Fungicides for Control of Tobacco Black Shank W. MU (1), J. XI (1), C. XUE (1), L. HU (1), J. SONG (1) (1) Zhengzhou Tobacco Research Institute of CNTC, China Tobacco black shank is a devastating soil borne disease caused by Phytophthora parasitic var. nicotianae, which greatly reduces tobacco yield and quality. Fungicides, such as metalaxyl, dimethomorph and mancozeb, have been widely used to prevent and control this disease. Intergrated biological and chemical control can release pressures from environment and delay emergence of fungicide resistance. Bacillus amyloliquefaciens ZY-9-13 has good control effect to tobacco black shank in our previous studies. In this research, we 1) assessed sensitivities of P. nicotianae to metalaxyl and dimethomorph, 2) mixed B. amyloliquefaciens ZY-9-13 and fungicide metalaxyl, dimethomorph, mancozeb in order to provide an alternative disease control option. The baseline sensitivities of P. nicotianae to metalaxyl and dimethomorph were described by unimodal curves, with mean EC50 values of 0.0664(±0.0282) and 0.3502(±0.0332) µg/ml, respectively, which can be used for detection of resistance isolates. Laboratory tests showed that fungicide mixture of metalaxyl, dimethomorph and mancozeb had additive effect, and the inhibition ratio of ZY-9-13 to fungicide mixture was less than 10% even at high concentration. The intergrated biological and chemical control showed a synergistic action. In the field trial, disease control with the combination of B. amyloliquefaciens ZY-9-13 and fungicide mixture was 79.76%~86.90%, which was greater than with ZY-9-13 or fungicide alone. Preventative fungicide applications in production and their impact on residual efficacy against impatiens downy mildew in the landscape S. SUAREZ (1), P. Lopez (1), A. Chase (2), A. Palmateer (1) (1) University of Florida U S A ; (2) Chase Agricultural Consulting LLC U S A Preventative fungicide applications in production and their impact on residual efficacy against impatiens downy mildew in the landscape S. SUAREZ (1), P. Lopez (1), A. Chase (2), A. Palmateer (1) (1) University of Florida, U.S.A.; (2) Chase Agricultural Consulting, LLC, U.S.A. Since the emergence of impatiens downy mildew researchers have come to the conclusion that chemical control is the only effective short-term management option available. The greatest challenge is identifying cost effective management options for impatiens downy mildew in the landscape. Some of our first trials conducted early in the spring of 2012 revealed that impatiens transplanted into landscape beds incorporated with a granular form of mefenoxam remained healthy for 60 days. Subsequent experiments evaluating products for long residual (> 30 days) control have identified several additional chemistries including fluopicolide, various phosphonates and oxathiapiprolin as promising. Further studies investigated the potential for eradicative control and found phosphonate containing products effective even after infection. Most recent studies have focused on fungicide rotations targeting long term residual control of impatiens downy mildew. Weekly preventative fungicide spray rotations on potted impatiens in production have included cyazofamid, dimethomorph, fluopicolide, mefenoxam, potassium phosphite and oxathiapiprolin. To date, oxathiapiprolin has provided stellar control of impatiens downy mildew when used alone and in every rotation study. Results obtained from fungicide efficacy trials conducted over the past four years indicate that some chemistries provide long enough residual control for impatiens to remain untreated in the landscape. Combined Application of Bacillus amyloliquefaciens ZY-9-13 with Fungicides for Control of Tobacco Black Shank W. MU (1), J. XI (1), C. XUE (1), L. HU (1), J. SONG (1) (1) Zhengzhou Tobacco Research Institute of CNTC, China Phytophthora fruit rot (Phytophthora capsici) limits watermelon production in most states in the Southeastern US. We identified several effective fungicides (Revus, Presidio, and Zampro) to manage Phytophthora fruit rot in our previous studies. In this study, the effectiveness of fungicide rotations Fungicide rotation schemes and Melcast for managing Phytophthora fruit rot of watermelon in Southeastern United States C. KOUSIK (1), D. Egel (2), P. Ji (3), L. Quesada-Ocampo (4) (1) U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Purdue University, Vincennes, IN, U.S.A.; (3) University of Georgia, Tifton, GA, U.S.A.; (4) North Carolina State University, Raleigh, NC, U.S.A. Effect of kasugamycin application on bacterial resistance to kasugamycin and cross resistance with other antibiotics S. GEBBEN (1), S. Gebben (2), G. McGhee (2), G. Sundin (2) (1) Michigan State University, U.S.A.; (2) Michigan State University, U.S.A. Effect of kasugamycin application on bacterial resistance to kasugamycin and cross resistance with other antibiotics S. GEBBEN (1), S. Gebben (2), G. McGhee (2), G. Sundin (2) (1) Michigan State University, U.S.A.; (2) Michigan State University, U.S.A. Kasugamycin (Ks) is a an alternative option to the use of streptomycin for the control of bacterial plant diseases and it has been shown to be as effective as streptomycin in controlling fire blight, caused by Erwinia amylovora. However, there remains a concern that Ks application in orchards will select for Ks resistance that could be linked with other resistance genes that are active against antibiotics used in human medicine. To monitor for this possibility, we assessed the effect of the use of Ks (Kasumin 2L) in orchard systems on the level of resistance to Ks and five other antibiotics (streptomycin, ampicillin, gentamicin, cefotaxime, and tetracycline). Two sets of leaf and soil samples were collected (treated and non-treated with Ks) from a total of 23 orchards (5 different tree hosts from 7 states). Samples were processed in the laboratory and dilution plated onto King’s B medium with or without Ks amendment. Bacterial population sizes were determined per sample and up to 15 gram-negative colonies growing on Ks-amended medium per sample (a total of 1,038 isolates) were used in antibiotic resistance screening. Although the bacterial population sizes were larger on Ks-amended medium from soil compared to leaf samples, there were no differences in populations from Ks treated vs. non-treated sites. Similarly, there was no difference in levels of resistance to the five antibiotics tested between Ks-treated and non-treated sites. Kasugamycin (Ks) is a an alternative option to the use of streptomycin for the control of bacterial plant diseases and it has been shown to be as effective as streptomycin in controlling fire blight, caused by Erwinia amylovora. However, there remains a concern that Ks application in orchards will select for Ks resistance that could be linked with other resistance genes that are active against antibiotics used in human medicine. To monitor for this possibility, we assessed the effect of the use of Ks (Kasumin 2L) in orchard systems on the level of resistance to Ks and five other antibiotics (streptomycin, Fungicide sensitivity in the wild rice pathogen Bipolaris oryzae C. CASTELL-MILLER (1), D. Samac (2) (1) PLPA/UMN, U.S.A.; (2) USDA ARS, U.S.A. Laboratory and field evaluation of fungicides for management of Phytophthora blight of pumpkin, caused by Phytophthora capsici M. BABADOOST (1), J. de Souza (2), T. Reboucas (2), Y. Xiang (1) (1) Department of Crop Sciences, University of Illinois, U.S.A.; (2) Deptment of Plant Science, Southwest Bahia University State, Brazil de in plant tissues (1), M. Young (1), A. Towers (1), T. Washington (1), P. Rajasekaran (1), S. Das (1), E. Johnson (2), A. Gesquiere (1), S. p ARD (1), M. Soliman (1), M. Young (1), A. Towers (1), T. Washington (1), P. Rajasekaran (1), S. Das (1), E. Johnson (2), A ) ( ) 1) University of Central Florida, Orlando, FL, U.S.A.; (2) University of Florida, Lake Alfred, FL, U.S.A. The unprecedented crop losses caused by the high incidence of huanglongbing (HLB) vectored by the Asian citrus psyllid (ACP) in Florida citrus groves calls for the rapid development of an effective product available to growers for HLB management. Zinkicide™ is a prototype plant nutrient-based nanoparticle developed to provide growers with a new and effective systemic bactericide that will translocate into plant tissues to target the phloem- limited bacterium, Candidatus Liberibacter asiaticus (Las), responsible for infection and damage of internal phloem tissues in the plant associated with HLB. Unlike traditional bactericidal management, we show that the new Zinkicide™ treatment allows systemic movement to reach phloem tissue and interact with Las with ongoing assessment of tree health responses in field trials. To complement green house and field trial data, we explore the fundamental properties of Zinkicide™ formulations and their interactions with bacteria and citrus plant tissues. Morphology, structure and composition of the formulations were determined using a combination of electron microscopy, UV-Vis, fluorescence and infrared spectroscopy measurements. In addition, correlations with antibacterial efficacy, phytotoxicity, particle uptake and evolution in planta were established by monitoring the fluorescence, lifetime and infrared signatures of the particles as they penetrate into the plant and interact with the phloem tissues. Monitoring Pennsylvania orchards for the presence of streptomycin resistant Erwinia amylovora H. LAUKAITIS (1), B. Lehman (2), K. Peter (2), M. Baer-Lehman (1) (1) Shippensburg University, U.S.A.; (2) Pennsylvania State University Fruit Research and Extension Center, U.S.A. Monitoring Pennsylvania orchards for the presence of streptomycin resistant Erwinia amylovora H. LAUKAITIS (1), B. Lehman (2), K. Peter (2), M. Baer-Lehman (1) (1) Shippensburg University, U.S.A.; (2) Pennsylvania State University Fruit Research and Extension Center, U.S.A. Within the last 40 years, streptomycin resistant strains of Erwinia amylovora have emerged in apple orchards throughout the United States. To date, resistant strains of E. Laboratory and field evaluation of fungicides for management of Phytophthora blight of pumpkin, caused by Phytophthora capsici M. BABADOOST (1), J. de Souza (2), T. Reboucas (2), Y. Xiang (1) (1) Department of Crop Sciences, University of Illinois, U.S.A.; (2) Deptment of Plant Science, Southwest Bahia University State, Brazil Laboratory and field evaluation of fungicides for management of Phytophthora blight of pumpkin, caused by Phytophthora capsici M. BABADOOST (1), J. de Souza (2), T. Reboucas (2), Y. Xiang (1) (1) Department of Crop Sciences, University of Illinois, U.S.A.; (2) Deptment of Plant Science, Southwest Bahia University State, Brazil Phytophthora blight, caused by Phytophthora capsici, is a destructive disease of cucurbits and peppers in Illinois and worldwide. Fungicide use is an important component of management strategies of Phytophthora blight of cucurbits. Studies were conducted in laboratory and field to evaluate efficacy of selected fungicides for management of Phytophthora blight of pumpkin in Illinois. In the laboratory, sensitivity of five P. capsici isolates from Illinois was evaluated to three registered fungicides (cyazofamid, dimethomorph, mandipropamid) for use on cucurbits and two experimental fungicides (A20941OD, A20942SC) for control of Phytophthora spp. Development of colony and production of sporangia and zoospores of the isolates were assessed at different concentrations from 0 to 10 µg/ml for registered fungicides and from 0 to 200 µg/ml for experimental fungicides on V8 juice agar medium in Petri plates. The isolates were highly sensitive to mandipropamid, intermediately sensitive to cyazofamid and A20941OD, and less sensitive to dimetho- morph and A20942SC. In the field experiment in 2014, incidence of pumpkin fruit rot caused by P. capsici was 31% in control plots and 5, 10, 2, and 11% in the plots treated with cyazofamid, dimethomorph, mandipropamid, and A20942SC, respectively. In 2015, incidence of pumpkin fruit rot caused by P. capsici was 69% in control plots and 42, 21, and 18% in the plots sprayed with cyazofamid, dimethomorph, and mandipropamid, respectively. Fungicide rotation schemes and Melcast for managing Phytophthora fruit rot of watermelon in Southeastern United States C. KOUSIK (1), D. Egel (2), P. Ji (3), L. Quesada-Ocampo (4) (1) U.S. Department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Purdue University, Vincennes, IN, U.S.A.; (3) University of Georgia, Tifton, GA, U.S.A.; (4) North Carolina State University, Raleigh, NC, U.S.A. Phytophthora fruit rot (Phytophthora capsici) limits watermelon production in most states in the Southeastern US. We identified several effective fungicides (Revus, Presidio, and Zampro) to manage Phytophthora fruit rot in our previous studies. In this study, the effectiveness of fungicide rotations S4.68 and spray applications based on Melcast for managing Phytophthora fruit rot were conducted in P. Laboratory and field evaluation of fungicides for management of Phytophthora blight of pumpkin, caused by Phytophthora capsici M. BABADOOST (1), J. de Souza (2), T. Reboucas (2), Y. Xiang (1) (1) Department of Crop Sciences, University of Illinois, U.S.A.; (2) Deptment of Plant Science, Southwest Bahia University State, Brazil amylovora have not been identified within Pennsylvania; however, they have been found within neighboring states such as New York, raising concerns of resistant strains soon surfacing in Pennsylvania orchards. The purpose of this study was to determine if streptomycin resistant strains are present in apple orchards located throughout Pennsylvania. In 2015, isolates were collected from grower submitted fire blight samples to the Pennsylvania State University Fruit Research and Extension Center in Biglerville, Pennsylvania. Initial identification was performed using Crosse Goodman specific media and apple inoculation. Erwinia identification was confirmed with direct colony PCR using primers specific to plasmid Ea29. Positive isolates were screened for resistance using a disk diffusion assay (100 μg/ml and 2500 μg ml of streptomycin sulfate respectively). Resistance was evaluated by observation of the presence or absence of a zone of inhibition. Of the samples processed to date, none have demonstrated streptomycin resistance. Screening is ongoing and any resistant isolates obtained will be further evaluated for the presence of plasmid mediated and chromosomal genes involved in resistance. Effect of kasugamycin application on bacterial resistance to kasugamycin and cross resistance with other antibiotics S. GEBBEN (1), S. Gebben (2), G. McGhee (2), G. Sundin (2) (1) Michigan State University, U.S.A.; (2) Michigan State University, U.S.A. In recent years the occurrence of fungal brown spot, caused by Bipolaris oryzae has increased in cultivated wild rice (Zizania palustris) paddies in spite of the use of fungicides. To implement an efficient integrated disease management system, we are exploring whether field isolates have developed reduced sensitivity due to the recurrent use of strobilurin (i.e. inhibitors of the quinone outside site of the mitochondrial cytochrome b, QoI) and azole (i.e., C14 demethylation inhibitors, DMI) fungicides. We conducted in vitro spore germination assays to determine the EC50 of two sets of isolates collected in 2008 and 2015 using 0, 0.10, 0.15, 0.25, 0.35, 0.5, 1.0, 5.0, 10.0, and 100 ug/ml of the active ingredient azoxystrobin (QoI), and 0, 0.1, 0.15, 0.25, 0.5, 1, 5, 10, 35 ug/ml propiconazole (DMI). All isolates tested were found to express the mitochondrial alternative oxidation pathway (AOX) allowing the fungus to germinate at all the doses of azoxystrobin tested. When the AOX was blocked, an average EC50 value of 0.206 ug/ml was obtained for 2015 isolates and 0.158 ug/ml for isolates collected in 2008. Up to now, there is no evidence that isolates have become less sensitive to QoI fungicides. The EC50 obtained for propiconazole for all strains tested until now was lower than the dose-equivalent used in the field. Molecular and in planta tests are underway to test the efficacy of the AOX pathway in spore germination. High-throughput strobilurin sensitivity testing of R. solani on Louisiana rice confirms reduced azoxystrobin but continued trifloxystobin sensitivity High-throughput strobilurin sensitivity testing of R. solani on Louisiana rice confirms reduced azoxystrobin but continued trifloxystobin sensitivity Laboratory and field evaluation of fungicides for management of Phytophthora blight of pumpkin, caused by Phytophthora capsici M. BABADOOST (1), J. de Souza (2), T. Reboucas (2), Y. Xiang (1) (1) Department of Crop Sciences, University of Illinois, U.S.A.; (2) Deptment of Plant Science, Southwest Bahia University State, Brazil capsici infested fields at three locations in southeastern US (NC, SC, GA) in 2014 & 2015. The triploid seedless cv. Wonder was used at all locations with diploid cv. Mickylee as the pollenizer. Five weekly foliar application of fungicides were made at all locations. Significant fruit rot was observed in non-treated check plots in both years at all three locations (Mean=68%). All the fungicide rotation schemes significantly reduced Phytophthora fruit rot compared to non-treated check. Overall the rotation scheme of Zampro (dimethomorph+ametoctradin) alternated with Orondis (oxathiapiprolin) was very effective across three locations. Revus (mandipropamid) alternated with Presidio (fluopicolide) was similarly effective in SC. MELCAST reduced one spray application in SC in 2014 but not in NC or GA, and one spray in GA and SC in 2015 with reduction in fruit rot similar to other rotation schemes. Fungicides in different FRAC groups are currently available for use in rotations schemes to manage Phytophthora fruit rot and Melcast may also be useful for scheduling spray treatments. and spray applications based on Melcast for managing Phytophthora fruit rot were conducted in P. capsici infested fields at three locations in southeastern US (NC, SC, GA) in 2014 & 2015. The triploid seedless cv. Wonder was used at all locations with diploid cv. Mickylee as the pollenizer. Five weekly foliar application of fungicides were made at all locations. Significant fruit rot was observed in non-treated check plots in both years at all three locations (Mean=68%). All the fungicide rotation schemes significantly reduced Phytophthora fruit rot compared to non-treated check. Overall the rotation scheme of Zampro (dimethomorph+ametoctradin) alternated with Orondis (oxathiapiprolin) was very effective across three locations. Revus (mandipropamid) alternated with Presidio (fluopicolide) was similarly effective in SC. MELCAST reduced one spray application in SC in 2014 but not in NC or GA, and one spray in GA and SC in 2015 with reduction in fruit rot similar to other rotation schemes. Fungicides in different FRAC groups are currently available for use in rotations schemes to manage Phytophthora fruit rot and Melcast may also be useful for scheduling spray treatments. Characterization of Zinkicide™ in plant tissues L. TETARD (1), M. Soliman (1), M. Young (1), A. Towers (1), T. Washington (1), P. Rajasekaran (1), S. Das (1), E. Johnson (2), A. Gesquiere (1), S. Santra (1) (1) University of Central Florida, Orlando, FL, U.S.A.; (2) University of Florida, Lake Alfred, FL, U.S.A. Baseline sensitivities of Rhizopus stolonifer and first effective fungicides for management of almond hull rot in California S. HAACK (1), H. Förster (1), J. Adaskaveg (1) (1) University of California, Riverside, U.S.A. Baseline sensitivities of Rhizopus stolonifer and first effective fungicides for management of almond hull rot in California S. HAACK (1), H. Förster (1), J. Adaskaveg (1) (1) University of California, Riverside, U.S.A. Almond hull rot, caused primarily by Rhizopus stolonifer (Rs) and sometimes Monilinia fructicola (Mf), is an increasingly important disease in California almond production. These fungi infect the hull either during late fruit development (Mf) or at hull split (Rs). Fruit colonization leads to production of toxins that translocate into the fruiting wood, causing twig and branch dieback. Hull rot has been managed culturally by deficit irrigation and by limiting nitrogen application, but this opposes high-density, high-input farming that increases disease pressure. Fungicide treatments add to an integrated management approach. Baseline sensitivities to six fungicides representing DMI (difenoconazole, metconazole), SDHI (fluopyram, fluxapyroxad), and QoI (pyraclostrobin, trifloxystrobin) FRAC groups for inhibition of Rs mycelial growth or spore germination were determined using the spiral gradient dilution method. Mean EC50 values for these fungicides were 0.102, 0.082, 0.292, 0.850, 0.002, and 0.002 mg/L, respectively. Sensitivities to each fungicide were within a ten-fold range, and thus, all isolates are considered sensitive. In field efficacy and timing studies, a single application of these fungicides and selected pre-mixtures at the beginning of hull split timed with a standard insecticide application was sufficient to reduce the disease by 60 to 80% when Rs was the primary pathogen. This represents the first fungicide-based field management strategy for hull rot. Sensitivity of Neofabraea spp strain to newly registered fungicides P. SIKDAR (1), M. Mazzola (2) (1) Washington State University, U.S.A.; (2) USDA-ARS, U.S.A. Bull’s eye rot, incited by a group of closely related fungi in the genus Neofabraea, causes significant economic loss to the Washington State apple industry. The pathogen complex is a significant limitation as it has been listed as a quarantine pathogen with a zero tolerance level for fruit shipped to China and Israel. Access to export markets for Washington State apples relies on the development of effective bull’s eye rot control methods. Previous experiments indicate that fungicide treatments containing thiabendazole and thiophenate-methyl provide effective disease control, however use of this treatment is not recommended due to the potential for development of resistance in populations of Penicillium spp., which incites the common postharvest disease, blue mold. lurin sensitivity testing of R. solani on Louisiana rice confirms reduced azoxystrobin but continued trifloxystobin h-throughput strobilurin sensitivity testing of R. solani on Louisiana rice confirms reduced azoxystrobin but continue iti it A. LUNOS (1), C. Hollier (1), L. Brooks (1), S. Harding (1) (1) Dept. of Plant Pathology & Crop Physiology, LSU AgCenter, U.S.A. Azoxystrobin resistance in the rice sheath blight pathogen, Rhizoctonia solani AG 1-IA, has compromised a widely effective disease management tool for rice production in southwestern Louisiana. We previously characterized azoxystrobin, pyraclostrobin, and trifloxystrobin sensitivity of 40 R. solani isolates, one from each of 40 rice fields, to identify locations with azoxystrobin resistance and to assess cross-resistance. A high-throughput fungicide sensitivity assay was developed and used to characterize 122 additional isolates from these 40 fields. Serial dilutions of commercial grade fungicides were made in potato dextrose broth and added to 96-well microtiter plates. Fungal mycelial growth was measured based on optical density. Growth S4.69 curves were generated and used to determine EC50 values, resulting in sensitivity distributions that agree with previous results. The distributions of azoxystrobin and pyraclostrobin EC50 values were bimodal while trifloxystrobin was not. This and internal control isolates provide significant evidence of azoxystrobin resistance, reduced pyraclostrobin sensitivity, and continued sensitivity to trifloxystrobin in the sampled R. solani population. Growers need to prepare for the advent of azoxystrobin-resistant R. solani and could potentially substitute trifloxystrobin in fungicide rotations. Resistance to quinone outside inhibitor fungicides in Cercospora sojina in Ohio L. WEBER (1), A. Dorrance (1) (1) The Ohio State University, U.S.A. Quinone outside inhibitor (QoI) fungicides are a common class of fungicides used to manage the fungus Cercospora sojina, the causal agent of frogeye leaf spot in soybean. Soybean leaf samples exhibiting symptoms of frogeye leaf spot were sent to the Soybean Pathology lab from across the state of Ohio during the summer of 2015. Cercospora sojina conidia were recovered from lesions to test their sensitivity to QoI fungicides. Potential C. sojina samples were confirmed by ITS sequencing then tested for the presence of the G143A mutation in the cytochrome b gene, the most commonly observed mutation in QoI-resistant fungi. QoI fungicide resistance was detected in two C. sojina samples in a mixed population of sensitive and resistant spores, while all other samples were sensitive to QoI fungicides. Fast and efficient identification of fungicide resistant C. Baseline sensitivities of Rhizopus stolonifer and first effective fungicides for management of almond hull rot in California S. HAACK (1), H. Förster (1), J. Adaskaveg (1) (1) University of California, Riverside, U.S.A. Fludioxonil (Scholar) did not demonstrate consistently effective control of bull’s eye rot in fruit and did not limit in vitro growth of the pathogen. Additional analyses were conducted to screen Neofabraea spp. isolates for sensitivity to newer commercially available fungicides that include multiple active chemistries. Results from these trials suggest that fungicide containing fluopyram and trifloxystrobin inhibits in vitro growth of Neofabraea at a level equivalent to that of thiabendazole. In contrast, fungicide treatment containing cyprodinil with difenconazole, or solatenol alone failed to inhibit mycelial growth or spore germination in vitro. Baseline sensitivities to SDHI fungicides and characterization of the sdhB gene in Venturia inaequalis S. VILLANI (1), K. Ayer (2), K. Cox (2) (1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A. . S ( ), C. Cowge ( ) (1) North Carolina State University, U.S.A.; (2) USDA-ARS Plant Science Research Unit & North Carolina State University, U.S.A. ( ), g ( ) (1) North Carolina State University, U.S.A.; (2) USDA-ARS Plant Science Research Unit & North Carolina State University, U.S.A. Blumeria graminis f.sp. tritici (Bgt), the causal agent of wheat powdery mildew, is primarily controlled by resistant wheat varieties and foliar fungicides. However, both control strategies risk being defeated by the large, rapidly adapting pathogen population. In 2013 and 2014, over 350 Bgt isolates were sampled from 27 fields in 15 states. The fungicide sensitivity of these single-spored isolates was evaluated using a detached leaf assay of fungicide- treated susceptible wheat leaf pieces. Five different fungicides, representing three major fungicide classes, were evaluated using 11 concentrations per fungicide. Detached leaves were inoculated with individual isolates and after 10 days leaf pieces were rated for sporulation. Bgt isolates from Mid- Atlantic states (NC, VA), Great Lakes states (MI, NY, OH, PA), and Deep South states (AL, FL, GA, MS) exhibited decreased sensitivity to the Demethylation Inhibitor (DMI) fungicides tebuconazole and prothioconazole compared to isolates from MO, AR, and the Plains states (KS, NE, OK). Plains isolates exhibited decreased sensitivity to the succinate dehydrogenase inhibitor (SDHI) fungicide fluxapyroxad compared to Deep South isolates. Sensitivity to the Quinone outside Inhibitor (QoI) fungicides pyraclostrobin and picoxystrobin did not differ by region. Genotypic evaluation of phenotypically insensitive isolates will further clarify the extent and nature of potentially fungicide-resistant isolates in this collection. lurin sensitivity testing of R. solani on Louisiana rice confirms reduced azoxystrobin but continued trifloxystobin sojina in Ohio will be useful in minimizing potential soybean yield loss as frogeye leaf spot incidence in Ohio increases in the warm, moist conditions and mild winters that favor the pathogen. Evaluating fungicide sensitivity of regional populations of Blumeria graminis f.sp. tritici in the United States E. MEYERS (1), C. Cowger (2) (1) North Carolina State University U S A ; (2) USDA ARS Plant Science Research Unit & North Carolina State Uni Evaluating fungicide sensitivity of regional populations of Blumeria graminis f.sp. tritici in the United States E MEYERS (1) C Cowger (2) Evaluating fungicide sensitivity of regional populations of Blumeria graminis f.sp. tritici in the United States E. MEYERS (1), C. Cowger (2) (1) North Carolina State University, U.S.A.; (2) USDA-ARS Plant Science Research Unit & North Carolina State University, U.S.A. E. MEYERS (1), C. Cowger (2) (1) North Carolina State University, U.S.A.; (2) USDA-ARS Plant Science Research Unit & North Carolina State University, U.S.A. Baseline sensitivities to SDHI fungicides and characterization of the sdhB gene in Venturia inaequalis S. VILLANI (1), K. Ayer (2), K. Cox (2) 1) N th C li St t U i it U S A (2) C ll U i it U S A The succinate dehydrogenase inhibitor (SDHI) fungicides are a class of broad spectrum, single-site fungicides that interfere with fungal respiration in the mitochondrion. The recent registration of next-generation SDHIs for apple scab management prompted an investigation into the baseline sensitivity of Venturia inaequalis to penthiopyrad, fluopyram, and benzovindiflupyr. Thirty-five to 70 isolates with no prior exposure to single-site fungicides were used to determine the effective concentration at which conidial germination was inhibited by 50% (EC50). Mean EC50 values for penthiopyrad, fluopyram, and benzovindiflupyr were 0.086, 0.176 and 0.0016 μg ml–1, respectively. Linear correlation analysis revealed a significant and positive correlation in sensitivity between fluopyram and penthiopyrad (P ≤ 0.0001, r = 0.66) and fluopyram and benzovindiflupyr (P = 0.0014, r = 0.52). Genomic sequencing was performed using the Illumina MiSeq platform to identify and characterize the sdhB gene for 3 isolates collected from a research, baseline, and commercial orchard population. No common mutation sites commonly associated with SDHI resistance were discovered in these isolates, or in other isolates that were recovered following field applications of SDHIs. Results suggest that SDHIs are highly effective against conidial germination and provide a basis for phenotypic and genotypic monitoring of SDHI resistance in populations of V. inaequalis. S4.70 Assessment of QoI Resistance in Colletotrichum spp. Isolated from Boxthorn and Apple in Korea S. KIM (1), J. Min (1), D. Back (1), H. Kim (1), S. Lee (1), K. Kim (1) (1) Chungbuk National University, Korea Strobirulin fungicides including azoxystrobin, krezoxim-methyl, and pyraclostrobin are inhibitors of mitochondrial respiration; they inhibit the cytochrome bc1 enzyme complex at the Qo site (quinone outside inhibitors, QoIs). QoI resistance has been reported in many pathogens such as Blumeria gramins f. sp. tritici, Plasmopara viticola and so on. The primary molecular mechanism of QoI resistance is a point mutation at codon 143 based on the substitution of glycine by alanine(GGT to GCT, G143A) in the mitochondrial-encoded cytochrome b gene. We examined the sensitivity of Colletotrichum spp. isolated from boxthorn fields and apple orchards to pyraclostrobin through the mycelial growth inhibition test on PDA amended with the fungicide. The resistance of C. acutatum isolates from boxthorn was determined with values of EC50 (effective concentration reducing the mycelial growth by 50%). The EC50 of resistant isolates was more than 1.7µg/ml. On the other hand, The EC50 of sensitive isolates of C. Screening for QoI resistance among Colletotrichum species associated with ripe rot of grape in Virginia vineyards M. NITA (1), A. Bly (1) (1) Virginia Tech, U.S.A. Screening for QoI resistance among Colletotrichum species associated with ripe rot of grape in Virginia vineyards M. NITA (1), A. Bly (1) (1) Virginia Tech, U.S.A. Ripe rot of grape is a fungal disease caused by two Colletotrichum species complexes, C. acutatum and C. gloeosporioides. This disease can result in significant crop loss and off-flavors in wine. Among recommended fungicides against ripe rot are the QoI group, or strobilurins. We examined the prevalence of QoI insensitivity among Colletotrichum isolates collected throughout Virginia during 2013. For the fungicide-amended medium assay, petri dishes containing potato dextrose agar amended with 100 ppm azoxystrobin (Abound, 22.9% a.i. azoxystrobin, Syngenta Crop Protection) and 1,000 ppm SHAM (salicylhydroxamic acid) were inoculated with a mycelial plug of each of the isolates and then incubated at 25°C. The presence or absence of growth on azoxystrobin-amended medium was examined after 6 days. PCR-RFLP was utilized to detect a point mutation (G143A) of the cytochrome b gene. Primers GCCBF1 and RSCBR2 were used to amplify a 120-bp fragment, which was digested with restriction enzyme Fnu4HI to detect the G143A mutation. The results showed that among 283 isolates tested, 28% of isolates was insensitive to azoxystrobin in vitro, and 16% of the isolates had the G143A mutation. The higher percentage with the fungicide-amended medium assay was expected, since there are multiple mutations that can lead to insensitivity. High prevalence of QoI-insensitive isolates in Virginia vineyards warrants the modification of our management recommendations. Detection of quinone outside inhibitor resistant isolates of Erysiphe necator in Oregon vineyards B. WARNEKE (1), J. Yamagata (2), T. Neill (3), T. Miles (2), W. Mahaffee (3) (1) Oregon State University, U.S.A.; (2) California State University Monterey Bay, U.S.A.; (3) USDA-ARS, U.S.A. Grape Powdery Mildew (GPM, caused by Erysiphe necator (En)) is the most persistent disease in the west coast grape industry due to a long and dry growing season which favors its fecundity and inoculum dispersal via airborne conidia. GPM control failures in 2015 led to the exploration of the possibility of quinone outside inhibitor (QoI) fungicide resistance present in Oregon En populations. A survey of spatially distinct vineyards in Oregon was performed in which leaf and fruit samples infected with En were collected. Isolation and DNA extraction of En samples and subsequent quantitative PCR analysis using competitive TaqMan probes detected the presence of the G143A mutation. Isolates with the G143A mutation were considered resistant to QoIs. Characterization of resistance to DMI fungicides in Colletotrichum spp. isolates from peach S. CHEN (1), C. Luo (1), M. Hu (2), G. Schnabel (2) (1) Huazhong Agricultural University, China; (2) Clemson Universitiy, U.S.A. Characterization of fungicide resistance phenotypes in Botrytis cinerea populations from blueberry in Florida A. AMIRI (1), A. Zuniga (2), P. Harmon (2), N. Peres (2) (1) Washinton State University, U.S.A.; (2) University of Florida, U.S.A. Botrytis blossom blight caused by Botrytis cinerea is an important blueberry disease and is mainly controlled by fungicide applications prior and during bloom. In this study, we evaluated the sensitivity of 624 B. cinerea isolates collected from 11 blueberry fields in central Florida between February and May of 2013, 2014, and 2015. Six fields were near strawberry and 5 fields were located 40 km or more from strawberry fields. Isolates were grown on malt yeast extract agar and tested for their sensitivity to pyraclostrobin (Pyra), boscalid (Bosc), penthiopyrad (Penth), fludioxonil (Flud), fenhexamid (Fenh), cyprodinil (Cyp), and iprodione (Ipr) using a spore germination inhibition assay and previously determined discriminatory doses. Overall, disease incidence was higher in samples collected from blueberry fields planted next to strawberry fields compared to blueberry fields alone. In 2013, resistance frequencies were 1.7, 14.9, 46.3, 49.5, 57.8, 57.9 and 92.6% for Flud, Penth, Ipr, Cyp, Bosc, Fenh, and Pyra, respectively. Similar resistance frequencies were observed in 2014 and 2015 with some significant differences across locations and fungicides. Resistance frequencies were higher in blueberry fields next to strawberry fields for all fungicides except Flud, Penth and Ipr. Our findings indicate that resistance to multiple fungicides in B. cinerea is widespread in blueberry fields and that resistant populations selected on strawberry may spread to adjacent crops. Baseline sensitivity of Phyllosticta citricarpa isolates from Florida to succinate dehydrogenase inhibitor fungicides in vitro study K. NICOLETTA (1), M. Dewdney (1) (1) University of Florida Citrus Research and Education Center U S A Baseline sensitivity of Phyllosticta citricarpa isolates from Florida to succinate dehydrogenase inhibitor fungicides in vitro study K. NICOLETTA (1), M. Dewdney (1) (1) University of Florida, Citrus Research and Education Center, U.S.A. In 2010, Citrus Black Spot (CBS), caused by Phyllosticta citricarpa, was first found in Florida. CBS has a negative impact on fresh market fruit and yield production causing fruit lesions and premature fruit drop. This study assessed 116 monoconidial P. citricarpa isolates from ‘Valencia’ and ‘Hamlin’ with 39 isolates from Collier, 76 from Hendry and 1 from Polk Counties. Isolate sensitivity to the succinate dehydrogenase inhibitor (SDHI) fungicides: boscalid (0, 0.01, 0.1, 1, 5, 10 and 25 µg/ml), fluxapyroxad (0, 0.001, 0.01, 0.05, 0.1, 1 and 5 µg/ml), and fluopyram (0, 0.001, 0.01, 0.1, 0.5, 1 and 5 µg/ml) was evaluated by mycelial growth inhibition assays. The mean EC50 values for boscalid and fluxapyroxad were 0.179 and 0.026 µg/ml, respectively. These EC50 values were significantly different for each fungicide (P <0.001). It is expected the EC50 value of fluopyram will fall between boscalid and fluxapyroxad’s values. In the baseline assay all the isolates tested are sensitive to SDHI fungicides, therefore we expect no mutations in molecular analyses. Developing the baseline sensitivity of P. citricarpa to SDHI fungicides allow for resistance monitoring. In previous studies, P. citricarpa isolates were found to be sensitive to both quinone outside inhibitor (QoI) fungicides and demethylation inhibitor (DMI) fungicides. Thus, SDHI fungicides could potentially be used as rotational sprays with QoI and DMI fungicides to reduce the risk of resistance developing. Baseline sensitivities to SDHI fungicides and characterization of the sdhB gene in Venturia inaequalis S. VILLANI (1), K. Ayer (2), K. Cox (2) 1) N th C li St t U i it U S A (2) C ll U i it U S A gloeosporioides from apples was determined as less than 0.24µg/ml, whereas resistant isolates were more than 66.9ug/ml. The codon 143 of cytochrome b gene was substituted from GGT to GCT in all of resistant isolates regardless of Colletotrichum species. For the successful control of anthracnose in boxthorn fields or apple orchards in Korea, it is required a monitoring of the QoI resistance with an allele-specific PCR with primers generated from the 143 codon substitution. Characterization of fungicide resistance phenotypes in Botrytis cinerea populations from blueberry in Florida A. AMIRI (1), A. Zuniga (2), P. Harmon (2), N. Peres (2) (1) Washinton State University, U.S.A.; (2) University of Florida, U.S.A. Brazil P. LICHTEMBERG (1), Y. Luo (1), T. Michailides (1), L. May De Mio (2) (1) University of California - Davis, U.S.A.; (2) Universidade Federal do Paraná, Brazil The aim of this study was to investigate the mechanism associated with Monilinia fructicola resistance to demethylation inhibitor fungicide (DMI) tebuconazole in Brazil. In this study 10 isolates (8 DMI-R and 2 DMI-S) with broad tebuconazole sensitivity (0,003 - >10 µg/ml) were used to quantify the expression levels of the genes MfCYP51 and MfABC1, respectively encoding the target enzyme 14 α-demethylase and mediating the transport of substances across biological membrane. After the mycelial exposure to sub-lethal doses of tebuconazole, the 2–ΔΔCt method revealed increased MfCYP51 mRNA levels for 6 out of 7 DMI-R isolates (ranging from 1.06 to 9.08), while DMI-S revealed small expressions levels of 0.14 and 0.46. The induced expression of the MfABC1 gene with tebuconazole on isolate mycelium not always increased the levels of mRNA. The Mona element which likely trigger the elevated expression of MfCYP51 was not present on 58 DMI-R isolates from Brazil including those with increased expression, suggesting that other genetic change yet to be discovered may be triggering this mechanism. Full MfCYP51 sequencing is currently underway. This is the first report on molecular mechanisms for DMI resistance identification for M. fructicola isolates from Brazil; providing an important advancement for risk assessment of DMI fungicides used on brown rot management. Profiling of Resistance to SDHI Fungicides in Botrytis cinerea from Strawberry Fields M. HU (1), D. Fernández-Ortuño (2), G. SCHNABEL (1) (1) Clemson University, U.S.A.; (2) Universidad de Málaga, Spain From 2012-13 to 2014-15, a total of 2,570 B. cinerea isolates were collected from strawberry fields in the eastern United States and their sensitivity to the SDHI fungicides including boscalid, fluopyram, fluxapyroxad, and penthiopyrad was assessed. Assays were based on visual assessment of presence or absence of mycelial growth on media amended with discriminatory doses of fungicides in microtiter plates. Overall frequencies of isolates resistant to boscalid, fluopyram, fluxapyroxad and penthiopyrad increased over the three years to 30.0, 1.0, 5.5, and 7.4%, respectively. Four resistance patterns designated A, B, C or D were found. In-vitro azoxystrobin sensitivity of Colletotrichum gloeosporioides isolates from blueberry in north and central Florida M. VELEZ-CLIMENT (1), P. Harmon (1) (1) University of Florida, U.S.A. In-vitro azoxystrobin sensitivity of Colletotrichum gloeosporioides isolates from blueberry in north and central Florida M. VELEZ-CLIMENT (1), P. Harmon (1) (1) University of Florida, U.S.A. Anthracnose pathogens cause destructive diseases of blueberry fruits, leaves, and stems in Florida. Growers in central Florida have reported a lack of efficacy from QoI fungicides, specifically for anthracnose stem cankers on the southern highbush blueberry variety Flicker. Twenty-two single-spore isolates collected from symptomatic stems, leaves, and berries were identified and tested for sensitivity to QoI fungicides. Percent inhibition (PI) of conidial germination on PDA plates amended with 10µg mL–1 of azoxystrobin demonstrated all isolates from central Florida farms were insensitive to azoxystrobin. Isolates from north Florida farms were sensitive. Internal transcribed spacer region (ITS) sequence comparisons grouped all isolates within the C. gloeosporioides species complex, and the diverse morphology of the colonies and conidia agreed with this placement. These data suggest the fungicide failures reported by growers in central Florida were due to selection of QoI fungicide insensitivity in stem-infecting populations of C. gloeosporioides. Furthermore, insensitive isolates from fruits and leaves indicate that the fungicide azoxystrobin may no longer be a viable management option for ripe rot and anthracnose leaf spot, either. Additional efforts are needed to determine the nature and distribution of the resistance, to develop alternative management options, and to reinforce good resistance management practices for Florida’s blueberry industry. Fungicide sensitivity of Monilinia vaccinii-corymbosi in highbush blueberries in Michigan K. FITZGERALD (1), J. Gillett (1), A. Schilder (1) (1) Michigan State University, U.S.A. Mummy berry in blueberries, caused by the fungus Monilinia vaccinii-corymbosi (Mvc), is usually controlled with fungicides, in particular sterol inhibitors which have been used for almost 20 years. Fungicide sensitivity of Mvc isolates was studied across Michigan. Isolates were collected from various field sites and grown in pure culture: 72 isolates were tested in poison agar assays using six concentrations of three commonly used fungicides in the sterol inhibitor class (fenbuconazole, metconazole, and prothioconazole). Commercial formulations of these products were used. Colony diameter was measured after an incubation of 14 days at 22 degrees C in the dark and compared to growth on non-amended PDA. EC50 values were calculated for all isolates using a four-parameter logistical curve fit in the computer program R. EC50 values ranged from 0.0016 to 0.0146 ppm for fenbuconazole, 0.0007 to 0.0104 ppm for metconazole, and 0.0021 to 0.0986 ppm for prothioconazole. Screening for QoI resistance among Colletotrichum species associated with ripe rot of grape in Virginia vineyards M. NITA (1), A. Bly (1) (1) Virginia Tech, U.S.A. Conidia germination bioassays using Trifloxystrobin and Kresoxim-Methyl in 1.5% water agar confirmed genetic results of 28 isolates examined. Of the 28 isolates examined, 22 were resistant to both QoIs (EC50>100µg/ml), while 6 were sensitive (EC50 from 0.004 to 0.06µg/ml). Using a discriminatory dose of 0.1ug/ml, 19 of 19 more isolates were resistant to QoIs and agreed with results from the qPCR assay. S4.71 Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards. Phylogenetic analysis of the combined ITS, glyceraldehyde-3-phosphate dehydrogenase, and beta-tubulin gene sequences separated the isolates into two species, Colletotrichum nymphaeae and Colletotrichum fioriniae subgroups 1 and 2. The sensitivity of these and three other Colletotrichum species from peach (Colletotrichum fructicola, Colletotrichum siamense, and Colletotrichum truncatum) to demethylation inhibitor (DMI) fungicides difenconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole were determined. C. truncatum was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole. C. nymphaeae was resistant to flutriafol and fenbuconazole. C. fructicola and C. siamense were sensitive to all DMI fungicides, and C. fioriniae subgroup 2 was reduced-sensitive to DMI fungicides compared with C. fioriniae subgroup1. Difenconazole and propiconazole provided the best control efficacy in vitro to all five species. Tebuconazole and metconazole were effective against all Colletotrichum species, except for C. truncatum. The 14-α sterol demethylase genes CYP51A and CYP51B from resistant isolates revealed point mutations associated with resistance to DMI fungicides. The strong in vitro activity of some DMI fungicides against Colletotrichum species may be exploited for improved anthracnose disease management of peach. Isolates of the Colletotrichum acutatum species complex were collected from South Carolina and Georgia peach orchards. Phylogenetic analysis of the combined ITS, glyceraldehyde-3-phosphate dehydrogenase, and beta-tubulin gene sequences separated the isolates into two species, Colletotrichum nymphaeae and Colletotrichum fioriniae subgroups 1 and 2. The sensitivity of these and three other Colletotrichum species from peach (Colletotrichum fructicola, Colletotrichum siamense, and Colletotrichum truncatum) to demethylation inhibitor (DMI) fungicides difenconazole, propiconazole, tebuconazole, metconazole, flutriafol, and fenbuconazole were determined. C. truncatum was resistant to tebuconazole, metconazole, flutriafol, and fenbuconazole. C. nymphaeae was resistant to flutriafol and fenbuconazole. C. fructicola and C. siamense were sensitive to all DMI fungicides, and C. fioriniae subgroup 2 was reduced-sensitive to DMI fungicides compared with C. fioriniae subgroup1. Difenconazole and propiconazole provided the best control efficacy in vitro to all five species. Tebuconazole and metconazole were effective against all Colletotrichum species, except for C. truncatum. In-vitro azoxystrobin sensitivity of Colletotrichum gloeosporioides isolates from blueberry in north and central Florida M. VELEZ-CLIMENT (1), P. Harmon (1) (1) University of Florida, U.S.A. A slight shift towards reduced fungicide sensitivity was detected in more intensively managed fields compared to baseline sites; however, highly resistant isolates were not detected. The ITS region of the ribosomal DNA of the isolates was sequenced to confirm their identity and assess species-level genetic diversity. All isolates were nearly identical. Allthough the risk of sterol inhibitor resistance in Mvc appears to be relatively low, fungicide resistance management is still imperative. Induced overexpression of the gene MfCYP51 may reveal molecular mechanisms associated to tebuconazole resistanc of the gene MfCYP51 may reveal molecular mechanisms associated to tebuconazole resistance of Monilinia fructicola Induced overexpression of the gene MfCYP51 may reveal molecular mechan Brazil Brazil Pattern A isolates were resistant to boscalid with the allele H272R at locus sdhB; Pattern B isolates were resistant to boscalid and penthiopyrad with the allele H272R or H272Y at locus sdhB; Pattern C isolates were resistant to boscalid, fluxapyroxad, and penthiopyrad with the allele H272Y at locus sdhB; and pattern D isolates were resistant to boscalid, fluopyram, fluxapyroxad, and penthiopyrad with alleles P225F or N230I at locus sdhB. Isolates with alleles H272R, H272Y, N230I or P225F were sensitive to a new SDHIs, benzovindiflupyr, with mean EC50 values less than 0.5 μg/ml for each genotype, suggesting that this fungicide may be useful for resistance management. Screening for QoI resistance among Colletotrichum species associated with ripe rot of grape in Virginia vineyards M. NITA (1), A. Bly (1) (1) Virginia Tech, U.S.A. The 14-α sterol demethylase genes CYP51A and CYP51B from resistant isolates revealed point mutations associated with resistance to DMI fungicides. The strong in vitro activity of some DMI fungicides against Colletotrichum species may be exploited for improved anthracnose disease management of peach. Resistance of Colletotrichum gloeosporioides from strawberry to azoxystrobin and reduced-sensitivity to thiophanate-methyl M. OLIVEIRA (1), M. Chamorro (1), N. Peres (1) (1) University of Florida, U.S.A. This situation warrants immediate changes in anthracnose management strategies that integrate non-chemical strategies to limit selection and prevent spread of QoI-resistant populations. Sensitivity of Sclerotinia sclerotiorum to thiophanate-methyl, fluazinam, and procymidone M. Lehner (1), R. Silva (1), T. Paula Jr. (2), E. Mizubuti (1) (1) Universidade Federal de Vicosa, Brazil; (2) EPAMIG, Brazil The sensitivity of 282 Brazilian isolates of Sclerotinia sclerotiorum to thiophanate-methyl (TM), fluazinam, and procymidone was assessed using discriminatory doses and concentration that results in 50% mycelial growth inhibition (EC50). No isolate was resistant to fluazinam or procymidone. The EC50 values varied from 0.003 to 0.007 and from 0.11 to 0.72 mg/ml for fluazinam and procymidone, respectively. One isolate collected in 2010 was resistant to TM (EC50 > 100 mg/mL). The 2010 TM-resistant isolate had a L240F mutation in the b-tubulin gene. This is the first report of mutation at codon 240 conferring resistance to benzimidazole in S. sclerotiorum. The high-resolution melting analysis allowed the distinction of TM-sensitive and TM-resistant isolates by specific melting peaks and curves. The 2010 TM-resistant isolate had mycelial growth, sclerotia production, and aggressiveness comparable with that of the sensitive isolates. In 2015, 8 isolates were collected from the same region where the 2010 TM-resistant isolate was obtained. All 2015 isolates were sensitive to fluazinam and procymidone, but resistant to TM. The 2015 TM-resistant isolates exhibited considerable instability in mycelial growth rates after consecutive transferrings on PDA. Resistance to fluazinam and procymidone was nonexistent or rare. Resistance to TM may occur, but at this point no robust inference can be made about the persistence of TM-resistant isolates in the field. Sensitivity of Sclerotinia sclerotiorum to iprodione and propiconazole in Bangladesh M. ISLAM (1), C. Vrisman (1), S. Miller (1) (1) The Ohio State University, U.S.A. Sclerotinia sclerotiorum is a destructive plant pathogen causing white mold of mustard. The pathogen was first reported in Bangladesh in 2007. Iprodione and propiconazole have been used to control the disease, but the sensitivity of S. sclerotiorum isolates to these fungicides in Bangladesh has not been determined. One hundred eighteen S. sclerotiorum isolates from Bangladesh and six from Ohio were tested for sensitivity to the two fungicides. Isolates were collected from infected mustard, soybean and cauliflower plants, plated on Potato Dextrose Agar medium (PDA) and purified by mycelial tip culture. Resistance of Colletotrichum gloeosporioides from strawberry to azoxystrobin and reduced-sensitivity to thiophanate-methyl M. OLIVEIRA (1), M. Chamorro (1), N. Peres (1) (1) University of Florida, U.S.A. Resistance of Botrytis cinerea to fungicides in California strawberries S. COSSEBOOM (1), G. Schnabel (2), K. Ivors (1), G. Holmes (1) (1) Cal Poly State University, U.S.A.; (2) Clemson University, U.S.A. Botrytis gray mold, caused by Botrytis cinerea, is a very destructive pre- and post-harvest fruit rot of strawberry that affects production worldwide. Resistance to foliar applied fungicides has been reported in many regions, including California. The objective of this study is to characterize the fungicide resistance of B. cinerea isolates from California. Approximately 800 isolates of B. cinerea were collected from four strawberry fruit production regions of California (Oxnard, Santa Maria, Salinas, Watsonville) (10 isolates per ranch, 10 ranches per growing region). Isolates were collected early season (minimal selection pressure) and late season (maximum selection pressure) during the 2015-2016 growing season. Using a previously described technique, sensitivity of each isolate was determined with a discriminatory dosage of each active ingredient: boscalid, cyprodinil, fenhexamid, fludioxonil, fluopyram, iprodione, penthiopyrad, and thiophanate-methyl. Preliminary data show individual isolates with resistance to one or more active ingredients and modes of action. While previous work in California has documented resistance to a few active ingredients in a few fields, this is the first comprehensive study of B. cinerea resistance to commonly used fungicides and modes of action. Results of this study will help identify ineffective fungicides and improve gray mold management programs. Monitoring Colletotrichum acutatum Resistance to Quinone-outside Inhibitor Fungicides in Strawberry B. FORCELINI (1), B. Forcelini (1), N. Peres (1) (1) University of Florida, U.S.A. Colletotrichum acutatum, the causal agent of anthracnose fruit rot (AFR) and anthracnose root necrosis (ARN), is a major fungal pathogen affecting strawberry. Control of AFR and ARN has been dependent on the use of Quinone-outside Inhibitor (QoI) fungicides. Resistance of C. acutatum isolates to QoI fungicides was reported for the first time in 2013 in Florida, and was linked to five specific strawberry nurseries from Canada and North Carolina. In 2016, C. acutatum isolates were collected after outbreaks of ARN in Florida and coastal California. Ninety isolates from four nurseries were tested for QoI sensitivity using a discriminatory dose mycelial growth assay. Forty-two isolates were not inhibited at 3 nor at 100 µg azoxystrobin per ml and were considered QoI-resistant. Resistant isolates originated from two of the four nurseries tested. C. acutatum resistance to QoI fungicides is becoming a widespread problem in strawberry nurseries located in different regions. Resistance of Colletotrichum gloeosporioides from strawberry to azoxystrobin and reduced-sensitivity to thiophanate-methyl M. OLIVEIRA (1), M. Chamorro (1), N. Peres (1) (1) University of Florida, U.S.A. Resistance of Colletotrichum gloeosporioides from strawberry to azoxystrobin and reduced-sensitivity to thiophanate-methyl M. OLIVEIRA (1), M. Chamorro (1), N. Peres (1) (1) University of Florida, U.S.A. Colletotrichum gloeosporioides causes Colletotrichum crown rot (CCR) of strawberry in the southern United States. Symptoms start with wilting of the plants which consequently collapse. A discoloration can be observed when the crown is cut open. CCR is managed with the use of disease-free transplants and fungicide applications. Our objective was to evaluate the sensitivity of C. gloeosporioides to two common fungicides used to manage CCR in Florida strawberry fields. Sixty-three C. gloeosporioides isolated from strawberry crowns from 1995 to 2015 were evaluated in a conidial S4.72 germination assay for sensitivity to thiophanate-methyl and azoxystrobin at discriminatory concentrations of 3 and 100 μg/ml. An isolate collected in 1999 was found resistant to azoxystrobin. After that, resistance was observed sporadically only in isolates collected after 2007. Most of the isolates recently collected during the 2015-16 FL strawberry season were resistant to azoxystrobin. A reduced sensitivity to thiophanate-methyl was observed for all isolates in both fungicide concentrations tested, and none of the isolates was completely sensitive. We conclude that C. gloeosporioides isolates resistant to azoxystrobin have been selected after the introduction of this fungicide in the market in 2001. However, it appears that C. gloeosporioides isolates from strawberry have a natural reduced sensitivity to thiophanate-methyl. Increased frequency of multifungicide resistance in Botrytis cinerea M. HU (1), G. Schnabel (1) (1) Clemson University, U.S.A. Over four consecutive strawberry production seasons, resistance to 7 chemical classes of fungicides was determined for 2130 isolates from commercial strawberry fields located in South Carolina and 10 other states of the eastern United States. The overall frequencies of isolates resistant to thiophanate- methyl, pyraclostrobin, fenhexamid, boscalid, cyprodinil, iprodione, and fludioxonil increased over the four seasons to 84.7, 73.0, 32.2, 29.9, 29.7, 12.6, and 11.3%, respectively. Resistance to thiophanate-methyl was found virtually in every location. Resistance to fludioxonil was rarely found during the first three seasons, but increased to 11.3% in 2014-15. Our latest data from the 2015-16 season shows the frequency of resistance to fludioxonil has increased to 12.3%, indicating a strong selection for fludioxonil resistance in strawberry fields in the eastern United States. We hypothesize that the more FRAC codes are rotated into the spray program, the more we will select for isolates with increasing numbers of resistance determinants. Presence of fungicide resistant Botrytis strains in Norwegian forest nurseries G. STRØMENG (1), V. Talgø (1), I. Fløistad (1) (1) NIBIO, Norway Fungicide resistance in populations of Fusarium proliferatum causing bulb rot of onion in the Pacific Northwest K. Fairchild (1), S. Windes (1), A. Malek (1), J. Woodhall (2), P. Wharton (1) (1) University of Idaho, U.S.A.; (2) University of Idaho, U.S.A. Fusarium bulb rot, caused by the fungus Fusarium proliferatum, is an emerging disease on onion in Idaho. This bulb rot occurs in storage and shows symptoms similar to neck rot. Symptoms usually start at the neck and progress down along the fleshy scales, but the basal plate is not affected. Infected scales become translucent yellow and water-soaked, later turning tan-brown and soft. Frequently, the rot appears to progress down from the neck along a single fleshy scale in the bulb. In most cases the infected bulbs do not show any pronounced external symptoms or signs of infection. Currently, there are no control measures known for this disease as the mode of infection and conditions required for disease development are poorly understood. Fungicide sensitivity testing was done on a range of fungicides which have shown potential for the control of F. proliferatum using spiral plate dilution gradients. Results showed that there was a range of sensitivities in the populations of F. proliferatum to the fungicides tested. A knowledge of fungicides showing effective control of this pathogen in vitro is important in the development of effective management strategies to control this disease. Sensitivity of Podosphaera xanthii isolates from Illinois to fungicides Y. XIANG (1), M. Babadoost (2) Sensitivity of Podosphaera xanthii isolates from Illinois to fungicides Y. XIANG (1), M. Babadoost (2) (1) University of Illinois, Urbana-Champaign, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A. (1) University of Illinois, Urbana-Champaign, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A. Powdery mildew causes more than 50% yield losses in pumpkin and squash fields in Illinois, if it is not managed effectively. Since 2010, growers in Illinois have complained about failure of some fungicides to effectively control powdery mildew in cucurbit fields. This research was conducted to evaluate sensitivity of powdery mildew fungi from Illinois to major powdery mildew fungicide groups, including demethylation inhibitors (DMI), strobilurins or quinone outside inhibitor (QoI), succinate dehydrogenase inhibiting (SDHI), and cyflufenamid. In 2014 and 2015, 34 isolates of powdery mildew fungi were collected from cucurbits fields in 10 counties in Illinois. All of the collected isolates were identified as Podosphaera xanthii by using universal fungi primers ITS1F and ITS4 to amplify the internal transcribed spacer (ITS) region of nuclear ribosomal DNA (nrDNA). Fungicide resistance profiles and virulence of Stagonosporopsis species isolates from watermelon and melon in eastern China M. NEWARK (1), N. Dufault (1), M. Paret (2), L. Pingfang (3), X. Yang (3) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) Jiangsu Academy of Agricultural Science, China Fungicide resistance profiles and virulence of Stagonosporopsis species isolates from watermelon and melon in eastern China M. NEWARK (1), N. Dufault (1), M. Paret (2), L. Pingfang (3), X. Yang (3) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) Jiangsu Academy of Agricultural Science, China Stagonosporopsis spp. (syn: Didymella bryoniae) is a major pathogen on cucurbits. China is the largest producer of watermelon in the world with 22% of the seed imported to the US is coming from China. The purpose of this study was to examine fungicide resistance and virulence profiles of Stagonosporopsis spp. isolates in China as a first step in understanding potential global movement of novel strains. Sixty-nine mono-conidial isolates from 4 provinces in east China were collected between 2011 and 2015. Fungicides azoxystrobin, boscalid, difenoconazole, tebuconazole, and thiophanate M were tested using an amended agar assays at the rates of 0.096, 0.034, 0.032, 0.128, and 100mg/L, respectively. Virulence was examined using a detached leaf assay with the watermelon (Citrullus lanatus) var: ‘Sugar Baby’. Leaves were inoculated with the pathogen and evaluated using a disease scale. Statistical analysis was done using R version 3.2.3 examining frequencies and distributions of fungicide resistance and virulence. All isolates found in East China were pathogenic on a US variety with many isolates having moderate (67%) or even high virulence (29%). In total, 92% of the isolates were resistant to two or more classes of fungicides. Based on these findings, it is apparent that fungicide resistance is present in eastern China and could pose a risk to the global movement of resistant strains. Investigating dynamics of QoI fungicide resistance in potato early blight complex pathogens in Wisconsin S. DING (1), K. Meinholz (1), A. Gevens (1) (1) University of Wisconsin-Madison, U.S.A. Fungicide resistance in populations of Fusarium proliferatum causing bulb rot of onion in the Pacific Northwest K. Fairchild (1), S. Windes (1), A. Malek (1), J. Woodhall (2), P. Wharton (1) (1) University of Idaho, U.S.A.; (2) University of Idaho, U.S.A. Twenty of the isolates from different locations and different hosts were tested for their sensitivity to the four fungicides, including triflumizole (DMI), azoxystrobin (QoI), penthiopyrad (SDHI), and cyflufenamid. Our preliminary studies in the laboratory showed that the isolates were highly sensitive to triflumizole, penthiopyrad, and cyflufenamid, but less sensitive to azoxystrobin. The sensitivity of the isolates to azoxystrobin varied. Similarly, severity of powdery mildew in the field plots treated with azoxystrobin was significantly higher than those in the plots received the other three fungicides. Podosphaera xanthii strains with resistance and reduced sensitivity to several fungicides may challenge control of cucurbit powdery mildew M. McGrath (1) (1) Cornell University, U.S.A. Podosphaera xanthii strains with resistance and reduced sensitivity to several fungicides may challenge control of cucurbit powdery mildew M. McGrath (1) (1) Cornell University, U.S.A. Fungicides at risk for resistance development are important for effectively managing powdery mildew (PM) in cucurbit crops because they can move to lower leaf surfaces where the pathogen develops best. Isolates were collected from research and commercial plantings on Long Island, NY, on 16 Sep to 1 Oct 2015, which was after several fungicide applications for PM. A leaf disk bioassay was used to determine fungicide sensitivity. All but 1 of the 57 isolates tested were determined to be resistant to QoI fungicides (FRAC 11) and 79% of the isolates were resistant to boscalid, an SDHI fungicide (FRAC 7). Many grew on leaf disks treated with 500 ppm boscalid as well as on untreated disks. Isolates with very limited or no growth on 500 ppm boscalid disks tended to also be slightly tolerant or sensitive to 40 ppm myclobutanil (FRAC 3) and 40 ppm quinoxyfen (FRAC 13); most of these were collected from research plants at LIHREC not treated with any at-risk fungicides (organically-managed and non-treated plots in a fungicide evaluation). Many boscalid-resistant isolates also grew well on disks with 40 ppm myclobutanil and on disks with 40 ppm quinoxyfen. Some of these exhibit tolerance to higher doses. These results document strains of Podosphaera xanthii with resistance and reduced sensitivity to multiple fungicide chemistries can be selected during one growing season, including where not exposed to all 3 chemistries. Such strains could foil alternation fungicide programs. Resistance of Colletotrichum gloeosporioides from strawberry to azoxystrobin and reduced-sensitivity to thiophanate-methyl M. OLIVEIRA (1), M. Chamorro (1), N. Peres (1) (1) University of Florida, U.S.A. Five-millimeter mycelial discs from 2-day-old cultures were plated on non-amended PDA or PDA amended with iprodione (0.0001, 0.001, 0.01, 0.1, 0.3, 1.0 or 10.0 ppm) or propiconazole (0.001, 0.01, 0.1, 0.3, 1.0, 10.0 or 100.0 ppm). Cultures were incubated at room temperature and radial mycelial growth was measured after 48 hours of incubation. EC50 (50% mycelial growth inhibition) values were calculated by regression of mycelial growth relative to the control against log10 fungicide concentrations. EC50 values for iprodione and propiconazole ranged from 0.11 to 0.55 and 0.12 to 0.78 ppm respectively. Since S. sclerotiorum has appeared recently in Bangladesh and fungicide use has been relatively infrequent, these values will serve as baseline data for monitoring their efficacy going forward. Presence of fungicide resistant Botrytis strains in Norwegian forest nurseries G. STRØMENG (1), V. Talgø (1), I. Fløistad (1) (1) NIBIO, Norway S4.73 Approximately 30 million seedlings of Norway spruce (Picea abies) are planted in Norwegian forests each year, and the main part of these plants are produced by forest nurseries in Norway. Among the greatest challenges in the seedling production is the control of gray mold, caused by Botrytis. Fungicides used in Norwegian forest nurseries to control gray mold are thiophanate methyl, fenhexamid, iprodione, and a mixture of cyprodinil and fludioxonil. Increasing disease problems in newly planted seedlings over the last few years, made it necessary to investigate the possible presence of fungicide resistant Botrytis strains in forest nurseries. Isolates were collected from spruce seedlings and germ tube growth was measured on nutrient agar amended with different concentrations of the fungicides. We tested fludioxonil and cyprodinil separately, by using fungicides that contained fludioxonil or pyrimethanil (same chemical group as cyprodinil, cross resistance expected) as single active ingredients. Thus far, we have tested 35 isolates, and 54, 26 and 6% of these were resistant to thiophanate methyl, fenhexamid, and iprodione, respectively. Furthermore, 46, 26, 6, and 3% were moderately resistant to thiophanate methyl, pyrimethanil, iprodione, and fludioxonil, respectively. Nine isolates were resistant to two fungicides. We recommend that the nurseries avoid use of thiophanate methyl in the future, and that fenhexamid resistance is carefully monitored. Fungicide resistance in populations of Fusarium proliferatum causing bulb rot of onion in the Pacific Northwest K. Fairchild (1), S. Windes (1), A. Malek (1), J. Woodhall (2), P. Wharton (1) (1) University of Idaho, U.S.A.; (2) University of Idaho, U.S.A. Investigating dynamics of QoI fungicide resistance in potato early blight complex pathogens in Wisconsin S. DING (1), K. Meinholz (1), A. Gevens (1) 1) University of Wisconsin-Madison, U.S.A. The potato early blight complex (EBC), caused by Alternaria solani and A. alternata, is a perennial and destructive fungal disease that can lead to loss in yield and quality. Without cultivar resistance, management is with weekly-applied fungicides which can result in pathogen resistance in commercial fields treated with single site mode of action fungicides. One such group is the QoIs, which target the cytochrome b of pathogens. In this 2015 study, the F129L mutation which leads to QoI partial resistance was detected in all A. solani isolates collected from 4 farms representing unique potato production regions of Wisconsin. Three of the farms conducted conventional production practices; one farm was certified organic. QoI resistance in A. alternata was S4.74 due to the G143A mutation and was detected in 64% of the isolates in early July, and 91% in late August. At the organic farm, the percentage of A. alternata isolates with QoI resistance increased from 0 to 13% over the season. It is possible that A. alternata, with a broad host range, may have a wild type preserved in alternative hosts which can move into potato. Alternaria solani, which is more specifically a potato pathogen, may have been subjected to QoI selection more thoroughly. Our results suggested that there was rapid selection for QoI resistance within a single production season, indicating the need for modified and integrated management responses for long term management of the EBC. Sensitivity of Alternaria spp. causing diseases in tomato and potato to boscalid and azoxystrobin C. VRISMAN (1), M. Islam (1), S. Miller (1) (1) The Ohio State University, U.S.A. Tomato and potato growers in Ohio have recently reported reduced efficacy of fungicides used to control diseases caused by Alternaria. We aimed to assess the sensitivity of Alternaria isolates to two commonly used fungicides. Thirty isolates were collected from symptomatic tomato and potato plants in 2015, 28 of which were identified as A. alternata and two as A. solani. Isolates were screened for sensitivity to commercial formulations of azoxystrobin and boscalid. Mycelial discs were transferred to potato dextrose agar plates containing 0.0, 0.01, 0.1, 1, and 10 µg boscalid/mL; or 0.0, 0.01, 0.1, 1, 10, and 100 µg azoxystrobin/mL amended with SHAM (100 µg/mL). The diameter of fungal colonies was measured after 8 days of incubation. G143A mutation in cytochrome b is found in azoxystrobin resistant Phytophthora cactorum but not in resistant Phytop strawberry T. SEIJO (1), E. Zuchelli (2), N. Peres (1) (1) University of Florida - GCREC, U.S.A.; (2) Universidade de Passo Fundo, Brazil T. SEIJO (1), E. Zuchelli (2), N. Peres (1) (1) University of Florida - GCREC, U.S.A.; ( ) ( ) ( ) (1) University of Florida - GCREC, U.S.A.; (2) Universidade Phytophthora cactorum and P. nicotianae cause leather rot of fruit and Phytophthora crown rot (PhCR) in strawberry. Leather rot occurs sporadically, but can cause up to 50% fruit loss during conducive weather. In Florida’s annual strawberry winter production system, PhCR can be severe resulting in stunting, mortality and severe yield loss. Currently, azoxystrobin is labelled for control of leather rot but not for PhCR. P. cactorum and P. nicotianae isolates from both leather rot and PhCR were collected from multiple strawberry fields in Florida between 1997 and 2016. Isolates were tested for sensitivity to azoxystrobin at 0, 0.01, 0.1, 1.0, 10, and 50 mg/L amended with SHAM (100 mg/L). The isolates were separated into two groups, sensitive isolates, with EC50 values of less than 1.0 mg/L, and resistant isolates having EC50 values greater than 50 mg/L. Resistant P. cactorum and P. nicotianae isolates were found among both leather rot and PhCR isolates collected after 2010. The first 450 bases of the mitochondrial cytochrome b gene were sequenced from a selection of resistant and sensitive isolates of both species. The G143A mutation reported to cause azoxystrobin resistance was found in all three resistant P. cactorum isolates but not in resistant P. nicotianae isolates or in any sensitive isolate. This is also the first report of resistance to azoxystrobin in P. nicotianae from strawberry. Prevalence of metalaxyl resistance in Pythium populations in dryland agriculture in the US Pacific Northwest W. CHEN (1), S. Guy (2), R. McGee (3), T. Paulitz (1), L. Porter (4), K. Schroeder (5), G. Vandemark (1) (1) USDA ARS, Washington State University, Pullman, WA, U.S.A.; (2) Wasington State University, Pullman, WA, U.S.A.; (3) USDA ARS, Pullman, WA, U.S.A.; (4) USDA ARS, Washington State University, Prosser, WA, U.S.A.; (5) University of Idaho, Moscow, ID, U.S.A. Metalaxyl (mefenoxam) has been the primary fungicide used in managing Pythium damping off of chickpea and other pulse crops. Metalaxyl resistance among Pythiun spp. causing damping-off of alfalfa in Minnesota: Identification, pathogenicity and fungicide sensitivity (2) d (2) Pythium species causing damping-off of alfalfa in Minnesota: Identification, pathogenicity and fungicide sensitivity D. Samac (1), L. Berg (2), L. Radmer (2) (1) USDA-ARS, U.S.A.; (2) University of Minnesota, U.S.A. Damping-off and seed rot is an important disease of alfalfa, severely affecting stand establishment when conditions favor the disease. Globally, 15 Pythium species cause damping-off and seed rot of alfalfa, although surveys of species causing disease on alfalfa in Minnesota are lacking. Eight species were isolated by a seedling baiting technique from soil of five alfalfa fields in Minnesota with high levels of damping-off. Three species, P. sylvaticum, P. irregulare, and P. ultimum var. ultimum, were pathogenic on germinating alfalfa seedlings at 21 C. Strains of seven species isolated from infected soybean, P. irregulare, P. intermedium, P. sylvaticum, P. recalcitrans, P. conidiophorum, P. ultimum var. sporangiiferum, and P. ultimum var. ultimum, were pathogenic on alfalfa. Assays with Apron XL (mefanoxam) treated seed showed that fungicide sensitivity varied between and with species with approximately 56% of strains insensitive. In Apron XL amended medium, hyphal density was reduced at all concentrations but all strains had a similar growth rate as on non-amended medium. Insensitivity to Stamina seed treatments (pyraclostrobin) occurred in 94% of strains tested. The presence of broad host range species and fungicide resistance suggests that crop rotation and these widely used seed treatments are not effective tools for managing this disease. These results indicate that resistant cultivars are needed for managing damping-off in alfalfa production systems. G143A mutation in cytochrome b is found in azoxystrobin resistant Phytophthora cactorum but not in resistant Phytophthora nicotianae from strawberry Investigating dynamics of QoI fungicide resistance in potato early blight complex pathogens in Wisconsin S. DING (1), K. Meinholz (1), A. Gevens (1) 1) University of Wisconsin-Madison, U.S.A. The effective dose for 50% inhibition (EC50) of mycelial growth was calculated for each isolate by regressing the relative growth against the log10 of fungicide concentration. The A. alternata isolates were sensitive or slightly reduced in sensitivity to boscalid. One A. solani isolate was highly resistant to boscalid. More than 90% of the A. alternata isolates were resistant to some degree to azoxystrobin; both A. solani isolates were highly resistant. Overuse of azoxystrobin may have resulted in a shift in Alternaria populations to higher resistance to this fungicide. Resistance to boscalid was rare, but fungicide resistance management programs should be followed to reduce the risks of resistance development in Alternaria species. Fungicide sensitivity of Pythium species from the North Central Region of the United States R. MATTHIESEN (1), A. Robertson (1), M. Chilvers (2) (1) Iowa State University, U.S.A.; (2) Michigan State University, U.S.A. Pre- and post-emergence damping-off of soybean in the North Central Region (NCR) can be caused by several Pythium spp. Various fungicide seed treatments are used to manage damping-off. Little is known about the effect of temperature on fungicide sensitivity of Pythium spp. in the NCR. The goal of this study was to assess fungicide sensitivity of Pythium spp. from the NCR to metalaxyl, ethaboxam, and azoxystrobin by assessing mycelial inhibition in vitro at two temperatures. Isolates of four Pythium spp. (P. lutarium, P. oopapillum, P. sylvaticum, and P. torulosum) from the Midwest, collected through the USDA funded Oomycete Extension Network, were assessed for fungicide sensitivity using five concentrations (0, 0.1, 1.0, 10, and 100 μg a.i./ml) at 13°C and 23°C in an agar medium plate assay. All species from all states, except AR, were more sensitive (>50% mycelia inhibition) to metalaxyl and ethaboxam than to azoxystrobin. At 13°C isolates of P. oopapillum from all states, except AR, were less sensitive to all fungicides compared to 23°C. Isolates of P. sylvaticum from IA, ND, NE, SD, and WI were less sensitive to all fungicides at 23°C compared to 13°C. At 13°C isolates of P. torulosum from IA, IN, ND, NE, and WI were less sensitive to all fungicides tested compared to 23°C. An improved understanding of fungicide sensitivity of Pythium spp. could improve our ability to develop and implement effective fungicide seed treatments in the NCR. Assessment of acquired resistance of originally sensitive isolates of Phytophthora infestans to the systemic fungicides fluopicolide and propamocarb J. GONZÁLEZ-TOBÓN (1), J. Chirivi-Salomon (1), M. Parra-Bastidas (1), R. Childers (2), S. Restrepo (1), G. Danies (1) (1) Universidad de los Andes, Colombia; (2) Harvard University, U.S.A. Production of Phytophthora nicotianae isolates resistant to oxathiapiprolin R. BITTNER (1), A. Mila (1) (1) North Carolina State University U S A Production of Phytophthora nicotianae isolates resistant to oxathiapiprolin R. BITTNER (1), A. Mila (1) (1) North Carolina State University, U.S.A. Black shank, caused by Phytophthora nicotianae (Pn), is a devastating disease of tobacco throughout the world. Oxathiapiprolin (OXA), a newly discovered fungicide, has shown efficacy against numerous oomycetes including Pn, but the potential for OXA resistance is unknown. The generation of resistance to OXA was attempted using UV light mutagenesis and mycelial adaptation with four Pn isolates. UV mutagenesis generated two isolates (EdgeB7-M1 and YadA28-M1) with mycelial resistance to OXA. Sporangia production of both UV mutants also showed increased insensitivity to OXA. Resistance produced by UV mutagenesis was stable after five consecutive transfers on non-amended medium. In greenhouse studies, EdgeB7-M1 remained pathogenic to tobacco, while YadA28-M1 was nonpathogenic. For mycelial adaptation, 48 colonies, twelve replicates from each of the four isolates, were transferred fifteen times on OXA medium. After the 15th transfer, twelve colonies from the initial 48 had a significant increase in mycelial insensitivity to OXA, compared to the wild-type isolates. Increased insensitivity from mycelial adaptation was not stable. Sixty-six percent of the isolates lost their insensitivity after fifteen successive transfers on a non-amended medium. Of the twelve adapted isolates, one remained pathogenic on tobacco. Our results show that the generation of OXA resistant Pn isolates is possible; however resistance may be associated with fitness penalties. Management of mefenoxam-resistant isolates of Pythium ultimum causing pythium leak on potato in the Pacific Northwest I. CARRILLO (1), P. Wharton (1), K. Fairchild (1), L. Porter (2), K. Frost (3), P. Hamm (3) (1) University of Idaho, U.S.A.; (2) USDA-ARS, U.S.A.; (3) Oregon State University, U.S.A. Oxathiapiprolin baseline sensitivity distribution of Phytophthora capsici isolates G. OLAYA (1), R. Linley (2), K. Edlebeck (2), S. Kousik (3), P. Kuhn (4) (1) Syngenta, U.S.A.; (2) Syngenta, U.S.A.; (3) USDA-ARS, U.S. Vegetable Laboratory, U.S.A.; (4) Syngenta, U.S.A. Oxathiapiprolin baseline sensitivity distribution of Phytophthora capsici isolates G. OLAYA (1), R. Linley (2), K. Edlebeck (2), S. Kousik (3), P. Kuhn (4) (1) Syngenta, U.S.A.; (2) Syngenta, U.S.A.; (3) USDA-ARS, U.S. Vegetable Laboratory, U.S.A.; (4) Syngenta, U.S.A. Oxathiapiprolin is the only active ingredient in a new class of fungicides (piperidinyl-thiazole-isoxazoline) that is active against downy mildews and Phytophthora species, and generally weak against Pythium species. It is active against multiple developmental stages of Oomycete fungi including inhibition of mycelial growth, sporangial germination, zoospore release, and zoospore germination. The proposed site of action is the inhibition of an oxysterol binding protein (OSBP). FRAC has assigned oxyathapiproline to group U15 and is considered to have a medium to high risk for resistance development. The oxathiapiprolin baseline sensitivity of Phytophthora capsici has been constructed using 40 isolates never exposed to this fungicide. P. capsici isolates were obtained from pepper, zucchini, squash, tomato, watermelon, pumpkin, smooth gourd, bitter gourd and an unknown host. Sensitivity tests were conducted in vitro using V8 agar media amended with oxathiapiprolin. Mycelial growth inhibition was quantified using the following oxathiapiprolin concentrations: 0, 0.000001, 0.00001, 0.0001, 0.001, 0.01 and 0.1 mg.ai/L. The baseline sensitivity distribution (ED50 values) of the P. capsici isolates ranged from 0.00006090 to 0.0005169 with a geometric mean of 0.00019716 mg oxathiapiprolin/L and a range of 8.5X. Oxathiapiprolin baseline information is critical for the early detection of resistant isolates and the establishment of effective resistance management recommendations. Management of mefenoxam-resistant isolates of Pythium ultimum causing pythium leak on potato in the Pacific Northwest I. CARRILLO (1), P. Wharton (1), K. Fairchild (1), L. Porter (2), K. Frost (3), P. Hamm (3) (1) University of Idaho, U.S.A.; (2) USDA-ARS, U.S.A.; (3) Oregon State University, U.S.A. Mefenoxam is a fungicide used to manage oomycete pathogens on potato. Resistance to mefenoxam has developed in Phytophthora infestans in all potato growing regions of the U.S, and is becoming more common in P. erythroseptica. In a 2006 survey, many fields in the Columbia Basin were found to have mefenoxam-resistant isolates of P. ultimum. The percentage of resistant isolates to sensitive isolates for these fields ranged from 0 to 92.5%. Despite the risk of resistance and the cost of mefenoxam, it is still widely used in potato production in the Pacific Northwest (PNW) due to its efficacy in managing pythium leak and the lack of effective alternative compounds. In the PNW, pythium leak is most often seen at harvest or during storage. However in recent years, particularly in fields with high levels of resistance, extensive damage has been found weeks ahead of harvest, suggesting a higher disease risk for resistant isolates than sensitive ones. In this study, a field survey was undertaken to evaluate the extent and persistence of mefenoxam resistance in populations of Pythium in the PNW and determine potential alternative fungicides that can be used for the control of mefenoxam resistant P. ultimum isolates. Results showed that the levels of mefenoxam resistance in PNW Pythium isolates had decreased since 2006. Several new fungicides were also identified which had good efficacy against P. ultimum. Screening for phenylamide fungicide insensitivity in Wisconsin hop downy mildew (Pseudoperonospora humuli) populations M. MARKS (1), A. Gevens (1) Pseudoperonospora humuli is the oomycete pathogen causing hop downy mildew, an important disease concern in WI. The disease is primarily managed by fungicides since varietal resistance is unavailable. Phenylamides have been shown to be systemic and highly effective in control, but due to resistance, particularly in the Pacific Northwestern (PNW) hop producing region, their use has dwindled. To date, the use of phenylamides, namely mefenoxam, has been extremely limited in WI due to the high cost and uncertainty of efficacy. In 2015, nineteen P. humuli isolates were collected from a commercial hop yard in Dane County, WI and screened for mefenoxam insensitivity using a leaf disk assay. Fourteen leaf disks were placed onto either 1% water agar (WA) or 1% WA amended with 25 μg/ml of mefenoxam (Ridomil Gold SL, Syngenta). The disks were inoculated at 3 sites with sporangial suspensions from each isolate and the incidence of sporulation at each site was tallied after 5-7 days. The percentage of total sporulating sites was calculated and isolates were considered insensitive if the incidence of sporulation on amended media was at least 50% of that on nonamended media. All isolates showed no sporulation on WA plates amended with mefenoxam, and were sensitive. Mefenoxam may be highly effective in managing hop downy mildew in this location. Additional locations will be tested to better assess the phenylamide sensitivity status and aid in grower fungicide decision- making. G143A mutation in cytochrome b is found in azoxystrobin resistant Phytophthora cactorum but not in resistant Phytop strawberry has been reported from the irrigated Columbia basin in the state of Washington where metalaxyl has been applied multiple times annually to manage potato diseases, but not from dryland agriculture where metalaxyl has been used only once per year in seed treatments. However, in the spring of 2014 and 2015, severe seed rot and damping-off of metalaxyl-treated chickpea seeds were found in the dryland agriculture region in southeastern Washington and northern Idaho, and were associated with Pythium isolates with high levels of resistance to metalaxyl. High densities of metalaxyl-resistant Pythium populations were found in areas of severe chickpea seed rot and damping-off, and were observed in seven commercial chickpea fields in southeastern Washington and northern Idaho. P. ultimum var. ultimum dominated the populations that were highly resistant to metalaxyl. ED50 values for metalaxyl-resistant isolates were significantly higher than those of metalaxyl-sensitive isolates. Under controlled conditions, S4.75 metalaxyl treatments failed to protect chickpea seeds from seed rot and damping-off following inoculation with metalaxyl-resistant Pythium isolates. An effective alternative measure is urgently needed to successfully manage metalaxyl-resistant Pythium in chickpea production. metalaxyl treatments failed to protect chickpea seeds from seed rot and damping-off following inoculation with metalaxyl-resistant Pythium isolates. An effective alternative measure is urgently needed to successfully manage metalaxyl-resistant Pythium in chickpea production. Screening for phenylamide fungicide insensitivity in Wisconsin hop downy mildew (Pseudoperonospora humuli) populations M. MARKS (1), A. Gevens (1) (1) University of Wisconsin-Madison, U.S.A. Screening for phenylamide fungicide insensitivity in Wisconsin hop downy mildew (Pseudoperonospora humuli) populations M. MARKS (1), A. Gevens (1) Fungicide sensitivity of Phytophthora infestans isolates collected in Colombia M. PARRA-BASTIDAS (1), L. Cabrera-Villamizar (1), J. Chirivi-Salomon (1), C. Ñústez (2), S. Restrepo (1), G. Danies (1) (1) Universidad de los Andes, Colombia; (2) Universidad Nacional de Colombia, Colombia Phytophthora infestans, is a major constraint to potato production in Colombia. The disease is present in all regions where potatoes are grown and may cause losses of around 265,000 tons of potatoes per year. Control of late blight in Colombia is mainly achieved by the use of fungicides. Due to the specific mode of action of some of these fungicides and the high selection pressure exerted by the continued use of these products it is likely that some P. infestans strains in Colombia have already developed resistance against some of these systemic fungicides. Therefore, the aim of this study was to determine the sensitivity of isolates collected in potato-producing regions of Colombia to the systemic fungicides dimetomorph, fluopicolide, and propamocarb. Baseline sensitivity was tested in vitro on pea agar media amended with a range of concentrations of each fungicide tested. Dose-response curves and values for the median effective concentration, at which 50% of mycelial growth was inhibited, were determined for each isolate. Assessment of acquired resistance of originally sensitive isolates of Phytophthora infestans to the systemic fungicides fluopicolide and propamocarb J. GONZÁLEZ-TOBÓN (1), J. Chirivi-Salomon (1), M. Parra-Bastidas (1), R. Childers (2), S. Restrepo (1), G. Danies (1) (1) Universidad de los Andes, Colombia; (2) Harvard University, U.S.A. S4.76 Phytophthora infestans, a plant pathogenic oomycete, is the causal agent of late blight disease of potatoes and tomatoes. This disease causes devastating economical losses each year and control is mainly achieved by the use of fungicides. Previous studies have reported that sensitive isolates to the phenylamide fungicide, mefenoxam, become tolerant after a single passage on mefenoxam-containing medium. The acquired resistance of the sensitive isolates appeared to have a cost on the rate of mycelial growth and declined slightly with subsequent culturing on medium free of mefenoxam. The aim of this study was to investigate whether the same phenomenon is observed using two different systemic fungicides: fluopicolide and propamocarb. Susceptible isolates of P. infestans were exposed to three different concentrations of fungicide-amended medium: 0, 5, and 10 µg ml–1 for fluopicolide and 0, 1 and 100 µg ml–1 for propamocarb. To assess if the isolates had acquired resistance to each of the fungicides tested, mycelia from isolates growing at 5 µg ml–1 for fluopicolide and 1 µg ml–1 for propamocarb were transferred once again to media with the same concentrations mentioned above. Status of Huanglongbing (HLB) (citrus greening) Multi Agency Coordination (MAC) projects on thermal treatment of citrus trees with citrus greening D. KOMM (1), D. Komm (2), R. Ehsani (3) (1) USDA PPQ CPHST U S A ; (2) USDA U S A ; (3) University of Florida U S A g g g ( ) citrus greening) Multi Agency Coordination (MAC) projects on thermal treatment of citrus trees with citrus greenin D. KOMM (1), D. Komm (2), R. Ehsani (3) Citrus trees infected with ‘Candidatus Liberibacter asiaticus’, subjected to heat treatments, have shown an improvement in citrus yields and vigor. Thermal treatment equipment for all sizes of citrus trees is being developed and improved for citrus trees infected with ‘Ca. Liberibacter asiaticus’. Two MAC projects are contributing to the development and improvement of thermal treatment equipment for all sizes of citrus trees infected with this pathogen. Researchers involved in the first project have developed a mobile physical system using steam to prolong the productive life of citrus trees eight feet tall and smaller. This system uses a tent canopy that goes over the tree followed by steam treatment. Second project has developed a mobile thermotherapy system for citrus trees as tall as thirty feet. The entire system supports a tent canopy that continuously treats as it progresses in the row without stopping the steam treatment. Both projects have successfully heat-treated grove trees infected with HLB. There have been improvements in the thermal equipment to provide a fully functional mobile steaming system for immediate use on all sizes of trees for commercial treatments. Canopy and root response of HLB-affected citrus trees to steam-generated thermotheray N. Thapa (1), S. Commerford (1), R. Ehsani (1), E. Johnson (1), M. Dewdney (1) (1) CREC, University of Florida, U.S.A. Canopy and root response of HLB-affected citrus trees to steam-generated thermotheray N. Thapa (1), S. Commerford (1), R. Ehsani (1), E. Johnson (1), M. Dewdney (1) (1) CREC, University of Florida, U.S.A. Thermotherapy is an old technique used to control plant pathogens. Citrus huanglongbing (HLB) threatens Florida’s multibillion dollar citrus industry and has no effective control measures. The phloem-limited bacterium, Candidatus Liberibacter asiaticus (Las), is the causal agent of HLB and infects both canopy and root system. Thermotherapy can reduce Las titer in infected trees and this study explores steam as a heat source for rapid treatment to extend the productivity of HLB-affected citrus trees. Optimum temperature-time duration is important to reduce Las titer without affecting tree productivity. Status of Huanglongbing (HLB) (citrus greening) Multi Agency Coordination (MAC) projects on thermal treatment of citrus trees with citrus greening D. KOMM (1), D. Komm (2), R. Ehsani (3) (1) USDA PPQ CPHST U S A ; (2) USDA U S A ; (3) University of Florida U S A A randomized split-plot design field trial was laid out with the main plots at 3-month intervals to determine optimum time-of-year to reduce Las titer and the subplots at 6 temperature-time combinations (58°C/15s; 58°C/60s; 58°C/90s; 58°C/120s; 60°C/30s and untreated control, UTC) with 3 replications per treatment. Four leaves/tree from 10 trees/rep were collected quarterly and tested for Las titer via standard qPCR. Roots were assessed for Las titer as roots are an untreated reservoir. Preliminary data show an overall reduction in titer a month after the July treatment. Las titer increased in all leaves, but more in the UTC than the treatments, whereas root Las titer was reduced more in treatments than the UTC. This may be due to change of phloem flow from root to the treated canopy along with Las. Seasonality may be critical for successful suppression of Las. Inoculum sources of Xanthomonas fragariae in strawberry nursery packing houses: Presence, viability and transmission H. WANG (1), C. Gigot (2), N. McRoberts (2), W. Turechek (3) (1) EDISTO Research and Education Center, Clemson University, U.S.A.; (2) University of California, U.S.A.; (3) U.S. Horticultural Research Laboratory, USDA, U.S.A. Xanthomonas fragariae causes strawberry angular leaf spot, an important disease in strawberry nursery production. When plants are dug and harvested in the field, they are transported to a “trim shed” where the leaves are removed and the roots are trimmed with table-mounted steel blades. Maintaining constant and stringent phytosanitary conditions is nearly impossible because of the sheer numbers of plants handled each day as part of this sorting and packaging process. To determine the likelihood of X. fragariae transmission during this process, PBS-soaked cotton balls were used to swab the blades and tables at nursery trim shed stations after processing strawberry plants harvested from infected fields. X. fragariae presence and percent viability were determined by squeezing PBS from the cotton balls and testing it by PMA-qPCR. The likelihood of disease transmission was estimated by rub- inoculating strawberry plants with the cotton balls. X. fragariae was detected in ~70% of the samples and ~30% of these contained living bacteria. Disease symptoms were observed in 9 of 62 strawberry plants inoculated. Results indicate that contamination of healthy plants is likely to occur during the post-harvest trimming process in nurseries and identified points where sanitation can be improved for better disease control. Fungicide sensitivity of Phytophthora infestans isolates collected in Colombia M. PARRA-BASTIDAS (1), L. Cabrera-Villamizar (1), J. Chirivi-Salomon (1), C. Ñústez (2), S. Restrepo (1), G. Danies (1) (1) Universidad de los Andes, Colombia; (2) Universidad Nacional de Colombia, Colombia This study provides further insights into the in vitro adaptability of P. infestans against antifungal stress. Fertigation and soil acidification sustain root density of huanglongbing-infected trees in Florida citrus groves J. GRAHAM (1), E. Johnson (1), K. Gerberich (2), D. Bright (1) (1) University of Florida, U.S.A.; (2) University of Florida, Canada Status of Huanglongbing (HLB) Status of Huanglongbing (HLB) (citrus greening) Multi Agency Coordination (MAC) projects on thermal treatment of citrus trees with citrus greenin D. KOMM (1), D. Komm (2), R. Ehsani (3) (1) USDA PPQ CPHST, U.S.A.; (2) USDA, U.S.A.; (3) University of Florida, U.S.A. Fertigation and soil acidification sustain root density of huanglongbing-infected trees in Florida citrus groves J. GRAHAM (1), E. Johnson (1), K. Gerberich (2), D. Bright (1) (1) University of Florida, U.S.A.; (2) University of Florida, Canada Huanglongbing (HLB) is a devastating citrus disease caused by the phloem-limited bacterium Candidatus Liberibacter asiaticus (Las). Early symptoms of HLB include fibrous root loss and leaf blotchy mottle, followed by yield decline, leaf drop, and canopy dieback. As a consequence of initial bacterial infection of fibrous roots, a 30-50% reduction in fibrous root density and elevated soil Phytophthora populations were detected in field surveys. Continued sampling of Hamlin and Valencia orange trees on Swingle citrumelo rootstock in different stages of HLB decline revealed that root loss occurs in two stages. The second phase of root loss (70-80%) begins at the early stage of tree canopy thinning resulting from leaf drop and branch dieback. Managements have been implemented to reduce soil, nutrient and water stress, and Phytophthora root rot. They include frequent irrigation cycles, fertigation and acidification of irrigation water and soil to reduce rhizosphere pH, and fungicides. Root density of trees under these practices fluctuates seasonally and annually but has not declined over the last 3 years. Trees managed with soil acidification and fertigation have steadily recovered in health and yield. X. ZHANG (1), M. Babadoost (2) (1) Department of Crop Sceinces, University of Illinois, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A. ( ) ( ) Department of Crop Sceinces, University of Illinois, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A. (1) Department of Crop Sceinces, University of Illinois, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S Bacterial spot, caused by Xanthomonas cucurbitae, has become a serious disease of pumpkin in Illinois and other states in the Midwest. X. cucurbitae infects leaves and fruit. Fruit infection often results in fruit rot. More than 90% of fruit infection with X. cucurbitae in pumpkin fields has been reported. X. cucurbitae is considered as a seed-borne pathogen, hence developing an effective seed treatment to eradicate the pathogen in seed is essential. This study was conducted to develop reliable treatments for eradication of X. cucurbitae in seed. Hot-water, HCl, and NaClO treatments for eradication of X. cucurbitae in ‘Howden’ and ‘Dickinson’ pumpkins were assessed. Seed were treated in water with 49, 50, 51, 52, 53, 54, 55, and 56°C, for 5, 10, 15, 20, 25, and 30 min at each of the temperatures. Treatment at 55°C for 10 min eradicated X. cucurbitae in both naturally-infected and artificially-inoculated seed without significant adverse effects on seed germination or seedling vigor. HCl treatments included 0.5, 1.0, and 2.0% HCl solutions for 10, 20, 30, 40, 50 and 60 min. The most effected HCl-treatment was 0.5% concentration for 40 min, which eradicated X. cucurbitae in the seed without any negative effect on seed germination or seedling vigor. For NaClO treatment, pumpkin seeds were soaked in NaClO solution with concentration of 0.5, 1, 1.5 and 2% for 1, 2, 3 and 4 min. None of the NaClO treatments completely eradicated X. cucurbitae in pumpkin seed. Silicon Augments Xanthomonas gardneri symptoms in Tomato cv. Early Girl W. ZELLNER (1), C. Krause (2), J. Locke (1), J. Boldt (1), S. Leisner (3) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A.; (3) The University of Toledo, U.S.A. Silicon (Si) is a plant nutrient that enhances stress tolerance. Tomato accumulates a moderate amount of Si in foliar tissue, but its effect on Xanthomonas gardneri infection has not yet been determined. To test this, ‘Early Girl’ tomatoes were grown hydroponically in nutrient solution with (Si+) or without (Control) 2 mM potassium silicate. Plants were dip-inoculated with X. gardneri. Symptoms developed within 24 hrs post-inoculation. Total symptomatic leaf area was quantified 10 dpi, using the Assess 2.0 Program. X. ZHANG (1), M. Babadoost (2) (1) Department of Crop Sceinces, University of Illinois, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A. Si-treated plants had significantly more lesions and higher symptomatic leaf area (11.7%) compared to controls (6.5%). Symptoms present on Si+ plants were spread throughout the leaf, while lesions on control plants were concentrated around the leaf margins. This suggested entry by X. gardneri in Si+ plants was through both stomates and hydathodes, while control plants were infiltrated mainly via hydothodes. To determine if differences in symptom formation between Si+ and control plants was simply due to changes in stomatal opening, stomatal conductance was directly measured between Si+ and control plants that were removed from hydroponics solution for 2 min. At 570s, stomatal conductance in Si+ plants compared to control was slightly, but significantly lower. This suggests that differences in X. gardneri symptoms in ‘Early Girl’ are not due to changes in the stomatal conductance influenced by the dip-inoculation method. Study of the influence of winter rye on soybean seedling and root rot diseases G. ARALDI-DA-SILVA (1), T. Kaspar (2), M. Helmers (1), D. Mueller (1), L. Leandro (1) (1) Iowa State University, U.S.A.; (2) USDA, U.S.A. Cover crops can enhance or suppress plant diseases, but little is known about the effect of cover crops on soybean diseases. In 2015, the effect of winter rye (Secale cereale L.) cover crop on soybean seedling and root rot was studied at two experimental sites, Boyd and ISUAG-USB, in Ames, IA. Both sites were under a no-till corn-soybean rotation. Cover crop treatments were rye and no rye, at Boyd; and early termination rye, late termination rye, and no rye, at ISUAG-USB. Variables assessed were soybean stand, root rot severity, dry plant weight, healthy green leaf area (HGLA), and yield. Root rot severity was visually assessed on a percent scale, and HGLA was measured as canopy percent reflectance using a hand-held radiometer. At Boyd, soybean stand (P=0.68) and yield (P=0.61) were not affected by treatment, but healthy green leaf area (HGLA) was higher in rye plots (P<0.001) compared to plots with no rye. At ISUAG-USB, no rye plots had more soybean stand (P=0.08), greater yield (3105 ± 304 kg/ha) (P=0.07), and higher HGLA (P<0.001) compared to plots with late termination rye. Early termination rye did not differ from late termination and no rye treatments for any variables. No difference in dry plant weight and root rot severity were observed between treatments at both sites (P>0.2). The fungal disease citrus black spot (CBS) has become an increasing problem in Florida citrus groves. We conducted a 3-year study to evaluate the effect of 5% urea (9.4 kg/190 l water/0.4 ha), Soil-Set (142 ml/190 l water/0.4 ha), and a water control on the level of the P. citricarpa or the morphologically similar citrus endophyte P. capitalensis in the leaf litter and subsequent CBS incidence on fruit in a Valencia grove in Immokalee, FL. The experiment was a randomized complete block design with three rows per treatment (trt) and four blocks. Leaf litter samples were collected from arbitrarily selected trees at time of treatment and then three times biweekly post-treatment application. For every collection, 100 leaves/trt/block were inspected for fungal sporocarp presence and each fungus was quantified via qPCR. The disease incidence on fruit was collected for 50 fruit/tree on 125 trees/trt/block. Fungal DNA levels were analyzed with a 3-way ANOVA and fruit disease incidence by contingency table analysis and chi-square tests. Leaf litter DNA had a significant interaction (P < 0.0001) between block x trt x collection date. For the incidence of disease on fruit, significant X. ZHANG (1), M. Babadoost (2) (1) Department of Crop Sceinces, University of Illinois, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A. Site variables, as well as cover crop treatments, can influence soybean crop response. This study is going to be repeated in 2016, and additional research is underway to quantify Fusarium and Pythium populations at both sites. Reduced Macrophomina phaseolina colonization of soybean by supplementing with the secondary nutrients calcium and magnesium T. WILKERSON (1), M. Tomaso-Peterson (2), B. Golden (3), A. Brown (4), T. Allen (3) (1) Mississippi State University, Stoneville, MS, U.S.A.; (2) Mississippi State University, Starkville, MS, U.S.A.; (3) Mississippi State University Delta Research and Extension Center, Stoneville, MS, U.S.A.; (4) Mississippi State Cemical Lab/ Mississippi State University, Starkville, MS, U.S.A. Reduced Macrophomina phaseolina colonization of soybean by supplementing with the secondary nutrients calcium and magnesium T. WILKERSON (1), M. Tomaso-Peterson (2), B. Golden (3), A. Brown (4), T. Allen (3) (1) Mississippi State University, Stoneville, MS, U.S.A.; (2) Mississippi State University, Starkville, MS, U.S.A.; (3) Mississippi State University Delta Research and Extension Center, Stoneville, MS, U.S.A.; (4) Mississippi State Cemical Lab/ Mississippi State University, Starkville, MS, U.S.A. Macrophomina phaseolina (Tassi) Goid (Mp) is a ubiquitous soilborne fungal pathogen that causes charcoal rot (CR) of soybean. Charcoal rot annually limits profitability, especially in years when hot and dry environmental conditions persist. Throughout the southern United States, CR consistently remains one of the top five soybean diseases and has resulted in an estimated 11 million metric ton reduction in yield over the past 20 years. The objective of this research was to reduce Mp colonization of field grown soybean by supplementing secondary nutrients, specifically calcium (Ca) and magnesium (Mg). In 2014 and 2015 non-irrigated, Mp-inoculated field trials were conducted with a CR-susceptible and a CR-moderately-resistant cultivar. Treatment applications consisted of 1,120 kg/ha rate of Ca and Mg alone and in combination at each of three timings: pre-plant, at plant, and pre-plant fb at plant. Control plots consisted of a completely non-treated non-inoculated in addition to an Mp-inoculated non-treated. Root samples were taken at the R3, R5, R7, and R8 growth stages. Colony forming units (cfu) were enumerated from ground, disinfested root tissue using a semi-selective medium. Reductions in cfus were observed to be approximately 35% in the moderately-resistant cultivar and a 45 to 50% reduction in the susceptible cultivar, compared to the non-treated. Reduction in colonization was observed in some cases although not across all treatments. Status of Huanglongbing (HLB) (citrus greening) Multi Agency Coordination (MAC) projects on thermal treatment of citrus trees with citrus greening D. KOMM (1), D. Komm (2), R. Ehsani (3) (1) USDA PPQ CPHST U S A ; (2) USDA U S A ; (3) University of Florida U S A Assessment of in-vitro efficacy of different disinfectants against common bacterial diseases on woody ornamentals P. LIYANAPATHIRANAGE (1), S. Dawadi (1), M. Kabir (2), T. Simmons (1), F. Baysal-Gurel (1) (1) Tennessee State University, U.S.A.; (2) Tennessee State University, U.S.A. Plants are continuously exposed to disease causing agents, which enter to the nurseries in many ways. Cutting tools and other equipment that are used to work with infected plants is considered as a major source for bacterial disease spread. Various products are available in the market to use as disinfectants to clean cutting tools. This study was conducted to evaluate the effect of commercial disinfectants on Xanthomonas axonopodis, X. campestris and Pseudomonas syringae pv. syringae. We determined the contact time required to kill those bacterial pathogens by disinfectants in different chemical classes. The relative shelf-life stability of disinfectants was also determined. Replicated in-vitro experiments were conducted in which pure cultures of bacterial pathogens were exposed to disinfectants for 1, 30 and 60 sec, then 100 µL aliquots were withdrawn, plated on NBYA medium, and incubated at 25°C for 3 days. Clorox regular bleach (10%), Virkon-S (0.5, 1 and 2%), Last rinse sanitizer-beer clean, Seventh generation multi surface, Seventh S4.77 generation daily shower, Seventh generation all purpose, alcohol (70%), vinegar (100%), Lysol, ZeroTol 2.0 and Green-Shield killed all three pathogens at all exposure times during 3 weeks shelf-life of products. Further studies will be done to determine efficacy of these disinfectants in preventing plant- to-plant spread of bacterial diseases through cutting tools. generation daily shower, Seventh generation all purpose, alcohol (70%), vinegar (100%), Lysol, ZeroTol 2.0 and Green-Shield killed all three pathogens at all exposure times during 3 weeks shelf-life of products. Further studies will be done to determine efficacy of these disinfectants in preventing plant- to-plant spread of bacterial diseases through cutting tools. Effective treatments for eradication of Xanthomonas cucurbitae in pumpkin seed X. ZHANG (1), M. Babadoost (2) (1) Department of Crop Sceinces University of Illinois U S A ; (2) Department of Crop Effective treatments for eradication of Xanthomonas cucurbitae in pumpkin seed X. ZHANG (1), M. Babadoost (2) (1) Department of Crop Sceinces, University of Illinois, U.S.A.; (2) Department of Crop Sciences, University of Illinois, U.S.A. treatments for the reduction of inoculum and incidence of citrus black spot Phyllosticta citricarpa in Florida citrus Undiluted compost tea with NuFilm-P adjuvant applied every 7 days provided good control of black rot, powdery mildew, downy mildew, and Phomopsis cane and leaf spot. In general, less frequent and more dilute applications were less effective. ‘Niagara’ leaves were sampled daily for 7 days after application of aerated manure-based compost tea. Bacterial populations were higher on leaves sprayed with compost tea compared to non-treated leaves but declined to almost background levels after a week. Both yeast and fungal populations were reduced immediately after the application, but later increased over the control. Bacteria from various manure- and plant-based compost teas were isolated and evaluated for antagonism against Botrytis cinerea by dual plating. The majority of isolated bacteria belonged to the Bacillaceae family as determined by 16s rRNA sequencing. This study shows that compost tea may be used for disease suppression in grapes but further evaluation is warranted. Evaluation of disinfectant treatments to reduce transmission of Didymella bryoniae during watermelon grafting A. KEINATH (1), V. DuBose (2), C. Conrad (2) (1) Clemson University, U.S.A.; (2) Clemson University, U.S.A. Evaluation of disinfectant treatments to reduce transmission of Didymella bryoniae during watermelon grafting A. KEINATH (1), V. DuBose (2), C. Conrad (2) (1) Clemson University, U.S.A.; (2) Clemson University, U.S.A. Gummy stem blight can develop in greenhouses on watermelon seedlings grown as transplants or as scions and on cucurbit rootstock seedings used in grafting. When diseased seedlings are cut during grafting, the pathogen, Didymella bryoniae (Stagonosporopsis sp.), may be transferred to healthy seedlings. The objective of this study was to evaluate disinfectant treatments to prevent or reduce transmission. In a first experiment, Kleengro (didecyl dimethyl ammonium chloride), Physan 20 (benzyl ammonium chloride) and Virkon S (potassium peroxymonosulfate) were ineffective when sprayed onto entire watermelon seedlings before or after cutting hypocotyls with a blade contaminated with mycelium or conidia. In a second experiment, applying 0.6% sodium hypochlorite, 0.4% Physan 20 or 70% and 95% ethanol to a contaminated blade or heating it before cutting watermelon hypocotyls reduced incidence of gummy stem blight (P=0.01); water, 3% hydrogen peroxide, 1% Virkon S and 7.5% Kleengro were ineffective. In a third experiment, both the watermelon scion and the interspecific hybrid squash rootstock were cut with contaminated and treated blades before grafting. treatments for the reduction of inoculum and incidence of citrus black spot Phyllosticta citricarpa in Florida citrus K. RODRIGUES (1), T. Hobbs (1), M. Dewdney (1) (1) CREC, University of Florida, U.S.A. The fungal disease citrus black spot (CBS) has become an increasing problem in Florida citrus groves. We conducted a 3-year study to evaluate the effect of 5% urea (9.4 kg/190 l water/0.4 ha), Soil-Set (142 ml/190 l water/0.4 ha), and a water control on the level of the P. citricarpa or the morphologically similar citrus endophyte P. capitalensis in the leaf litter and subsequent CBS incidence on fruit in a Valencia grove in Immokalee, FL. The experiment was a randomized complete block design with three rows per treatment (trt) and four blocks. Leaf litter samples were collected from arbitrarily selected trees at time of treatment and then three times biweekly post-treatment application. For every collection, 100 leaves/trt/block were inspected for fungal sporocarp presence and each fungus was quantified via qPCR. The disease incidence on fruit was collected for 50 fruit/tree on 125 trees/trt/block. Fungal DNA levels were analyzed with a 3-way ANOVA and fruit disease incidence by contingency table analysis and chi-square tests. Leaf litter DNA had a significant interaction (P < 0.0001) between block x trt x collection date. For the incidence of disease on fruit, significant S4.78 treatment effects were seen in 2015 (P = 0.002) and 2016 (P < 0.0001) with Soil-Set reducing disease incidence by 29.6% and 26.5%, respectively but not with urea. Soil-Set may enhance the efficacy of fungicide applications for the management of CBS. treatment effects were seen in 2015 (P = 0.002) and 2016 (P < 0.0001) with Soil-Set reducing disease incidence by 29.6% and 26.5%, respectively but not with urea. Soil-Set may enhance the efficacy of fungicide applications for the management of CBS. Utility of compost tea for disease management in grapes G. KOTAMRAJU (1), R. Sysak (1), J. Gillett (1), A. Schilder (1) (1) Michigan State University, U.S.A. Compost tea is a fermented watery extract of compost used for disease suppression and plant nutrition. A replicated trial was conducted in a ‘Niagara’ vineyard in Clarksville, Michigan to evaluate the efficacy of compost tea for disease control. Non-aerated manure-based compost tea was prepared over a 2-wk period, and foliar applications were made with a handheld sprayer every 7 or 14 days from 10 June through 27 Aug, 2016. Potential of heat treatment for management of Botrytis cinerea resistance on strawberry A ZUNIGA (1) N Peres (1) Potential of heat treatment for management of Botrytis cinerea resistance on strawberry A. ZUNIGA (1), N. Peres (1) (1) University of Florida, U.S.A. Potential of heat treatment for management of Botrytis cinerea resistance on strawberry A. ZUNIGA (1), N. Peres (1) (1) University of Florida, U.S.A. Botrytis cinerea is the causal agent of gray mold, an important disease of strawberry in Florida. The use of fungicides to control gray mold has been exploited over the past years and as a result, resistance to many fungicide groups has been reported. In this study, we are aiming to determine the effect of a heat treatment that has proven to reduce systemic infection with Xanthomonas fragarie in strawberry propagating material on the survival of fungicide-resistant B. cinerea isolates. For this, four different B. cinerea isolates were used to evaluate survival at four temperatures (44, 48, 52 and 56°C) and seven different duration times (1, 5, 10, 30, 60, 120 and 240 min). After treatment, the spores were transferred to a growth medium to determine the percentage of spore survival. Although, B. cinerea spores survived for 1 min for all temperatures, germination was reduced by 28 to 50% at the highest temperature. Spores of all isolates exposed to 44°C for 30 min did not survive and spore germination was reduced by 7 to 16% between 48 and 52°C for 1 min, with no spores survived for all other duration times. Previous experiments have determined that most strawberry cultivars can tolerate 44°C for up to 4 h. Thus, heat treatment of strawberry plants is a feasible strategy to produce disease-free strawberry plants and to reduce fungicide-resistant inoculum. Eradication of the boxwood blight pathogen, Calonectrua pseudonaviculata, in compost R. HARVEY (1), D. Davis (1), J. Pecchia (1) (1) Penn State University, U.S.A. Calonectria pseudonaviculata, the causal agent of boxwood blight, is a serious threat to boxwood growers across the country. Boxwood blight is currently present in at least 15 US states, however this number is likely to increase as more infections are found. Due to the production of microsclerotia by the pathogen, there is concern that if infected plants are composted, the pathogen could survive the composting process and possibly cause further infections if the compost is used around healthy boxwoods, or other susceptible plants. treatments for the reduction of inoculum and incidence of citrus black spot Phyllosticta citricarpa in Florida citrus After 7 days in a humid healing chamber, heating the contaminated blade reduced incidence (11%) more than ethanol (45%), and both treatments reduced incidence compared to Physan 20 (100%) and water (100%) (P=0.01). Using heat or ethanol to disinfest cutting tools may reduce transmission of D. bryoniae during grafting. Scab severity in relation to hedge pruning pecan trees in the Southeastern USA C. BOCK (1), M. Hotchkiss (1), T. Brenneman (2), K. Stevenson (2), B. Goff (3), M. Smith (4), L. Wells (5), B. Wood (1) (1) USDA-ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A.; (2) Department of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.; (3) Department of Horticulture, Auburn University, Auburn, AL, U.S.A.; (4) Department of Horticulture, Oklahoma State University, Stillwater, OK, U.S.A.; (5) Department of Horticulture, University of Georgia, Tifton, GA, U.S.A. Scab is the most damaging disease of pecan in the Southeastern USA. Pecan trees are tall (up to 30+ m), and managing disease in the upper canopy is problematic. Hedge pruning trees to ~12 m is being explored to facilitate efficacy of ground-based fungicide sprays, but resulting vigorous shoot growth could result in more severe scab. Nine experiment compared scab severity on trees receiving hedge pruning regimes vs no hedging. All trees received fungicide treatments (ground-based air-blast sprays and up to 3 aerial applications). Results showed that different hedging methods either had no effect, increased or decreased scab severity on immature fruit in one experiment (P<0.0001, assessed before mid-August [immature fruit were assesses in 8 of the 9 experiments]), and on mature fruit in 6 of 9 experiments (P<0.0001, assessed after mid-August), but in all cases differences were small. However, height in the canopy invariably had a significant effect on scab severity, with up to 18-fold more severe scab on fruit at ≥12 m compared to 5 m. Immature fruit high in the canopy had more severe scab in all experiments (P<0.0001-0.03), and mature fruit had more severe scab in 6 of 9 experiments (P<0.0001). In trees that received the same fungicide program the effect of hedging in increasing scab severity was negligible and not consistent, but scab was consistently more severe at greater heights in the canopy of both hedge pruned and non-hedged trees. treatments for the reduction of inoculum and incidence of citrus black spot Phyllosticta citricarpa in Florida citrus g py y g y p p Immature fruit high in the canopy had more severe scab in all experiments (P<0.0001-0.03), and mature fruit had more severe scab in 6 of 9 experiments (P<0.0001). In trees that received the same fungicide program the effect of hedging in increasing scab severity was negligible and not consistent, but scab was consistently more severe at greater heights in the canopy of both hedge pruned and non-hedged trees. Potential of heat treatment for management of Botrytis cinerea resistance on strawberry A ZUNIGA (1) N Peres (1) To determine survivability during the composting process, discreet microsclerotia were inoculated into compost within a bioreactor system and subsequently sampled at set time/temperature intervals. Survival was rated as a plus/minus system based on the presence of microsclerotia germination. The data presented here are a refinement of data previously presented previously, and suggest that C. pseudonaviculata microsclerotia will not survive composting at greater than 50°C for over 24 hours. Further work needs to be done to define the minimum time/temperature that is sufficient to ensure complete eradication. In addition, similar research is needed to assess pathogen survival within infected plant material and to confirm the time/temperature combinations in vivo. S4.79 Thermal inactivation of Calonectria pseudonaviculata, the causal agent of boxwood blight M. MILLER (1), M. Cubeta (2) Thermal inactivation of Calonectria pseudonaviculata, the causal agent of boxwood blight M. MILLER (1), M. Cubeta (2) (1) North Carolina State University, Department of Plant Pathology, U.S.A.; (2) North Carolina ( ) ( ) (1) North Carolina State University, Department of Plant Pathology, U.S.A.; (2) North Carolina State University, U.S.A. (1) North Carolina State University, Department of Plant Pathology, U.S.A.; (2) North Carolina State University, U.S.A Thermotherapy is an effective method for eliminating viruses and microorganisms from seeds and bulbs, but little information is available on the use of this approach to inactivate pathogenic fungi in leaves of woody plants. In this study, we investigated the utility of hot water treatments for killing Calonectria pseudonaviculata (Cps), a fungal, foliar pathogen of boxwood (Buxus) during propagation. Experiments predicting the thermal death kinetics of Cps conidia, and experiments determining the ability of commonly grown, commercial boxwood cultivars to survive exposure to hot water were performed. Conidia of three field isolates of Cps were suspended in water and exposed to temperatures ranging from 45°C to 55°C for 0 to 20 min. Suspensions were then spread on potato dextrose agar, and percent spore germination was assessed after 24 h of incubation. Approximately 16 min were required to kill 90% of conidia exposed to 45°C, but only 4.5 min were required to kill 90% of spores when heated to 55°C. Cuttings from six partially resistant boxwood cultivars were submerged in water heated to 45°C, 50°C, and 55°C for 0 to 60 min. Treated cuttings were placed under mist and their rooting responses were rated 86 to 91 d later. Nutrients and in vitro growth of Phaeocryptopus gaeumannii, a fungus causing the Swiss needle cast disease on Pseudotsuga menziesii M. Yáñez-Morales (1), O. Salgado-Feregrino (1), J. Velázquez-Mendoza (1), M. Jiménez-Casas (1) (1) Colegio de Postgraduados, Mexico The hyphae intercellular development of Phaeocryptopus gaeumannii, a foliage pathogen in Douglas-fir trees, is affected by the nutritional composition of the needles. The objective was to examine the effect of different nutrients and silicon, Si, on the in vitro growth of the pathogen. Two experiments were established in a completely random design with a fungus isolate (JN204508). The first experiment was by plating the isolate aliquot (1 mm, 0.002 g) in 2% MEA amended with different nutrients, 10 treatments, and incubated at 21°C in darkness for 42 days; diameter colony growth was measured and instantaneous relative rate growth was estimated. The second experiment was in liquid medium, potato extract-dextrose, and amended with nutrients, 21 treatments; incubation was under shaking conditions at 250 rpm at 23°C for 35 days. Mycelium growth was measured by their weight in wet and dry stage. The macronutrients (N P K Ca Mg S) favored the growth of P. gaeumannii; in contrast the micronutrients (Fe Cu Zn Mn Mo B) severely inhibited the development of the fungus. The complete nutrient solution plus Si at 1000 ppm reduced colony growth in the agar medium, and at 250 ppm in the liquid medium. Thus, the addition of Si inhibited growth of the fungus and this was attributed to a possible toxic effect of the element. The results provide a basis for designing a fertilization formula for Douglas-fir and allowing to partially inhibit intercellular fungal growth in needles. Adapting to water insecurity: Co-managing oomycete pathogens and water use using deficit irrigation sensor networks J. DEL CASTILLO MÚNERA (1), C. Swett (1), B. Belayneh (1), A. Ristvey (2), J. Lea-Cox (1) (1) University of Maryland, U.S.A.; (2) University of Maryland, U.S.A. Adapting to water insecurity: Co-managing oomycete pathogens and water use using deficit irrigation sensor networks J. DEL CASTILLO MÚNERA (1), C. Swett (1), B. Belayneh (1), A. Ristvey (2), J. Lea-Cox (1) (1) University of Maryland, U.S.A.; (2) University of Maryland, U.S.A. Circumglobally, the agricultural sector consumes the highest percentage of available fresh water, and with increases in water insecurity, agricultural demands are becoming unsustainable. Sensor-driven deficit irrigation networks use less water by allowing a mild stress of the crop. The objectives of this study were to evaluate (i) deficit irrigation (DI) as means to reduce root disease in annual and perennial nursery/greenhouse crops; and (ii) effects of DI under various infection scenarios using tomato and P. Assessment of genetic diversity and virulence spectrum of Magnaporthe oryzae population from sub-Saharan Africa DEVI GANESHAN (1), S. Mutiga (2), F. Rotich (3), D. Mwongera (4), J. Harvey (5), B. Zhou (6), L. Wasilwa (7), G. Wang (1), D. Silue (8), O. Ibrahima (9), J. Correll (3), N. Talbot (10), T. Mitchell (1) (1) The Ohio State University, Department of Plant, (2) Biosciences eastern and central Africa (BecA) Hub, Nairobi, Kenya; (3) University of Arkansas, Department of Plant Pathology, AR, U.S.A.; (4) University of Exeter, United Kingdom; (5) Biosciences eastern and central Africa (BecA) Hub, Kenya; Nutrients and in vitro growth of Phaeocryptopus gaeumannii, a fungus causing the Swiss needle cast disease on Pseudotsuga menziesii M. Yáñez-Morales (1), O. Salgado-Feregrino (1), J. Velázquez-Mendoza (1), M. Jiménez-Casas (1) (1) Colegio de Postgraduados, Mexico capsici as pathosystem model, to address grower risks hindering technology adoption. In a two year study, Rhododendron were inoculated with P. cinnamomi and placed under four different irrigation treatments. Mortality incidence after 18 months of inoculation was significant (P = 0.0084), reflecting significantly lower mortality (21%) at 33.5% VWC compared to the 43, and 28% VWC treatments (29, 51% mortality). Preliminary studies on Poinsettia indicate a higher root rot incidence in the 48% WVC deficit irrigation treatment compared to saturated conditions (60% VWC), but differences were not significant. Studies underway in the model pathosystem are evaluating whether DI strategies can predispose plants to pathogen infection, and use of DI as a tool to prevent latent infection and disease. These studies will be used to develop recommendations for using deficient irrigation to co-manage water use, and disease losses in nursery and greenhouse production. Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota U S A Potential of heat treatment for management of Botrytis cinerea resistance on strawberry A ZUNIGA (1) N Peres (1) The ability of boxwood cuttings to produce roots after hot water treatment at 45°C and 50°C varied by cultivar, whereas no cultivars survived treatment at 55°C. Further experiments will determine whether hot water can eliminate the pathogen in planta. Using soil amendments to reduce survival of Botrytis cinerea sclerotia in Mid-Atlantic diversified farming systems E. KOIVUNEN (1), E. Koivunen (1), C. Swett (1) (1) University of Maryland, U.S.A. Using soil amendments to reduce survival of Botrytis cinerea sclerotia in Mid-Atlantic diversified farming systems E. KOIVUNEN (1), E. Koivunen (1), C. Swett (1) (1) University of Maryland, U.S.A. Botrytis cinerea causes major pre-harvest losses in a wide range of crops in the Mid-Atlantic, including strawberries, blueberries, raspberries, and grapes. Management challenges have escalated with the loss of soil fumigants as well as increased land-use intensity associated with crop diversification. In a summer 2015 field survey, Maryland fields had up to 350 B. cinerea colony forming units per gram of soil and 20% yield losses from gray mold. The objective of this project was to evaluate the efficacy of soil amendments in degrading B. cinerea sclerotia – the most resilient source of inoculum. In potted studies, sclerotia viability decreased in crab meal and leaf compost treatments by 91.7% (± 5%) and 96.7% respectively, compared to 71.7% (± 5%) in untreated soil sixty days after burial. Percent of sclerotia colonized by hyphal fungi was highest in the crab meal (86.7% ± 3.3%) and leaf compost treatments (93.8% ± 6.3%). Similar trends between sclerotia viability and colonization were seen in Maryland field trials: sixty days after burial, the leaf compost + crab meal treatment had the lowest sclerotia viability and the highest colonization by bacteria or yeasts. These studies suggest that soil amendments may hold promise in reducing B. cinerea survival and dispersal in diversified farming systems in the Mid-Atlantic, in part by promoting microbial colonization of sclerotia. Nutrients and in vitro growth of Phaeocryptopus gaeumannii, a fungus causing the Swiss needle cast disease on Pseudotsuga menziesii M. Yáñez-Morales (1), O. Salgado-Feregrino (1), J. Velázquez-Mendoza (1), M. Jiménez-Casas (1) (1) Colegio de Postgraduados, Mexico Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota, U.S.A. Chen (3) (1) Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou, China; (2) Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, China; (3) South China Agricultural University, China To understand the genetic mechanisms of space-induced rice mutants’ resistance to blast, T2, an indica rice mutant from TaiHang68, was subjected to resistance spectrum using 39 blast isolates collected from Guangdong Province, China along with main cultivars from Guangdong province. The results showed that T2 conferred broader resistance spectrum than most of main cultivars from Guangdong province. Genetic analysis and gene mapping were also applied in this study. Isolate GD0193 was used for inoculating a population of F2 derived from the cross between T2 and highly susceptible variety LTH. The results showed that the segregation of resistant (R) and susceptible (S) progenies matched a 3:1 ratio in the F2 population (1585R:537S, χ2= o.77, P>0.75), indicating that this R gene was controlled by a single dominant gene, temporarily designed as PiTH gene. The R gene was mapped between T5 (1.4 cM) and K10 (0.29 cM) at chromosome 11 using SSR and InDel markers, and it was co-segregating with marker K7. PiTH may be a novel resistance gene in the Pik cluster, which contributes greatly to the blast resistance of T2. Thus, T2 could be used as a new germplasm to facilitate rice blast resistance breeding. Exploring gene loci controlling bakanae disease resistance by genome-wide association mapping Exploring gene loci controlling bakanae disease resistance by genome-wide association mapping S. CHEN (1), K. Huang (2), Y. Kuo (2), M. Lai (3), D. Wu (3), C. Chung (1) (1) Department of Plant Pathology and Microbiology, National Taiwan University, Taipei, Taiwan; (2) Department of Bio-Industrial Mechatronics Engineering, National Taiwan University, Taipei, Taiwan; (3) Crop Science Division, Taiwan Agricultural Research Institute, Taichung, Taiwan Bakanae disease, caused by Fusarium fujikuroi, is a rice seed-borne disease and widely distributed in rice growing areas. Infected rice seedlings may show complex symptoms including abnormal elongation of stem or internodes, wider leaf angle, slenderness, and even die, causing considerable yield and quality loss. In recent years, studies have reported the rising incidence and severity of the disease in Asia. Even though the disease has been known over a century, little is known about the resistance genetics and resources in rice. In this study, we used genome-wide association mapping approach to identify gene loci controlling bakanae disease resistance. Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota, U.S.A. Legge (2) (1) U i i f M i b C d (2) B d R h d D l C A i l d A i F d ( ) ( ) ( ) gg ( ) University of Manitoba, Canada; (2) Brandon Research and Development Centre, Agriculture and Agri-Food Canada, Canad Fusarium head blight (FHB) incited by Fusarium graminearum is a major disease of barley (Hordeum vulgare) in Canada, which significantly impacts seed quality through production of mycotoxins in the grain. While breeding efforts have been ongoing at the Agriculture and Agri-Food, Brandon Research and Development Centre for a couple decades, progress has been limited by requirements for labour-intensive screening nurseries and quantitative based resistance. With advent of affordable genomic tools, breeding for crop characters that are difficult to measure and/or display low heritability are suitable for development through application of genomic selection techniques. A large population of 400 two-row barley lines has been assembled based on diverse reaction to FHB. This collection has been phenotyped in 2014 and 2015 at three locations in Manitoba (Brandon, Carberry, and Carman), and deoxynivalenol content in matured grains was measured by enzyme linked immunosorbent assay (ELISA). This data will be used in combination with genomic information to make predictions of genomic estimated breeding values. Genomic markers will be validated in breeding populations segregating for FHB resistance. Analysis of Resistance to Blast Isolates and Mapping of Rice Blast Resistance Gene in T2 Analysis of Resistance to Blast Isolates and Mapping of Rice Blast Resistance Gene in T2 Q. YANG (1), D. Sun (2), G. Chen (2), J. Zhang (2), D. Zhou (3), X. Zhu (2), H. Wang (3), Z. Chen (3) (1) Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, Guangzhou, China; (2) Institute of Plant Protection, Guangdong Academy of Agricultural Sciences, China; (3) South China Agricultural University, China y pp g Q. YANG (1), D. Sun (2), G. Chen (2), J. Zhang (2), D. Zhou (3), X. Zhu (2), H. Wang (3), Z. Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota, U.S.A. Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota, U.S.A. (1) University of Minnesota, U.S.A. Application of sugar beet factory spent lime to soil in fields infested with the soilborne oomycete pathogen Aphanomyces cochlioides has been shown to decrease disease and increase sugar beet yield in Minnesota and North Dakota. Sugar beet growers are inquiring about the need to reapply lime in fields where lime has previously been applied. In a field trial where lime had originally been applied in April 2004 at 0, 6, 12, 18 and 24 Mg dry wt ha–1, plots were split and an additional 7.8 Mg dry wt lime ha–1 was added to half of each plot on October 31, 2014. The following spring, sugar beet was sown and data was collected on early season stand, Aphanomyces root rot and sugar beet yield. There was significant interaction between supplemental and original rates of lime for early season stand, root rot ratings, and yield (P ≤ 0.05). At 4 and 7 weeks after planting, sugar beet stands were higher in plots that received supplemental lime where original lime rates were 0 and 6 Mg ha–1 (P ≤ 0.05), but not where original lime rates were 12 Mg ha–1 or higher. Supplemental lime reduced Aphanomyces root rot and increased sugar beet yield only in plots where lime had not been previously applied (P ≤ 0.05). Results demonstrate a benefit from adding supplemental lime to fields previously limed at low rates, but do not support a need to add supplemental lime to fields previously limed with at least 12 Mg dry wt ha–1 within the past ten years. S4.80 (6) International Rice Research Institute, Philippines; (7) Kenya Agricultural and Livestock Research Organization, Kenya; (8) AfricaRice Center, Ivory Coast; (9) INERA, Burkina faso; (10) University of Exeter, Department of Biosciences, United Kingdom (6) International Rice Research Institute, Philippines; (7) Kenya Agricultural and Livestock Research Organization, Kenya; (8) AfricaRice Center, Ivory Coast; (9) INERA, Burkina faso; (10) University of Exeter, Department of Biosciences, United Kingdom Rice Blast Disease (RBD), caused by the fungus Magnaporthe oryzae is a devastating disease found throughout the world and is particularly damaging to rice production in sub-Saharan Africa (SSA). QTL Mapping of Resistance to Tan Spot in a Winter Recombinant Inbred Line Population Derived from Cross between Harry and Wesley G. KARIYAWASAM (1), W. Hussain (2), A. Easterly (2), M. Guttieri (2), V. Belamkar (3), J. Venegas (2), S. Baenziger (2), J. Poland (4), J. Faris (5), S. Xu (5), J. Rasmussen (1), Z. Liu (1) Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota, U.S.A. Yield losses of 50-80% are common not only leading to net imports from abroad but also invasion of new pathotypes. An effective strategy to control RBD is to utilize resistant cultivars, but M. oryzae evolves rapidly due to genome instability, frequent loss/break of resistance genes occurs, overcoming resistance within few years of deployment. Breeding for durable resistance requires knowledge of pathogen’s genetic diversity and virulence spectrum. In our study, genetic diversity of M. oryzae field isolates from 9 SSA countries (Benin, Burkina Faso, Ghana, Kenya, Nigeria, Tanzania, Mali, Togo, and Uganda) were assessed by SNPs using GBS technology. Phylogenetic analysis of SNPs from 78 isolates, revealed 7 distinct clades differing in virulence. PCA showed that isolates from East were genetically distinct from West Africa. Further, standard least square regression analysis of virulence spectrum in the 7 clades revealed 4 major groups which differed in disease scores highlighting the association between genetic diversity and virulence. GWAS will be performed to further strengthen the association between genetic diversity and virulence and map the putative avirulence (AVR) genes for functional characterization facilitating an informed deployment of RBD resistance programs. Quantitative resistance loci identified for the bacterial blight causative agent, Xanthomonas oryzae pv. oryzae A. HUERTA (1), E. Delorean (2), A. Bossa-Castro (2), C. Raghavan (3), R. Corral (2), V. Verdier3 (4), H. Leung (5), J. Leach (2) (1) Colorado State University, Fort Collins, CO, U.S.A.; (2) Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, U.S.A.; (3) International Rice Research Institute (IRRI), Los Baños, Philippines; (4) Institut de Recherche pour le Développement IRD-CIRAD-UM2, Montpellier, France; (5) 2 International Rice Research Institute (IRRI), Los Baños, Philippines Quantitative resistance loci identified for the bacterial blight causative agent, Xanthomonas oryzae pv. oryzae Qu ve es s ce oc de ed o e b c e b g c us ve ge , a t o o as o yzae pv. o yzae A. HUERTA (1), E. Delorean (2), A. Bossa-Castro (2), C. Raghavan (3), R. Corral (2), V. Verdier3 (4), H. Leung (5), J. Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota, U.S.A. Leach (2) (1) Colorado State University, Fort Collins, CO, U.S.A.; (2) Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, U.S.A.; (3) International Rice Research Institute (IRRI), Los Baños, Philippines; (4) Institut de Recherche pour le Développement IRD-CIRAD-UM2, Montpellier, France; (5) 2 International Rice Research Institute (IRRI), Los Baños, Philippines ( ) ( ) ( ) g ( ) ( ) ( ) g ( ) ( ) (1) Colorado State University, Fort Collins, CO, U.S.A.; (2) Department of Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, U.S.A.; (3) International Rice Research Institute (IRRI), Los Baños, Philippines; (4) Institut de Recherche pour le Développement IRD-CIRAD-UM2, Montpellier, France; (5) 2 International Rice Research Institute (IRRI), Los Baños, Philippines Any factor that limits or reduces attainable agricultural yield, including plant disease, will impact global food security. Xanthomonas oryzae pv. oryzae (Xoo) causes the most important bacterial disease of rice, bacterial blight (BB). Deployment of resistance varieties is the gold standard for managing plant diseases, having the strongest impact with minimal environmental effects and cost. However, resistance breeding depends on harnessing genetic diversity in available germplasm for identification of effective and durable disease resistance. To identify durable resistance, we screened 330 advanced inbred lines (AILs) from the indica Multi-Parent Advanced Generation Inter-Cross (MAGIC) rice population for resistance to Xoo strains, PXO99A and PXO99A harboring TAL-effector TAL7b (+TAL7b). TAL7b, a virulence factor for Xoo, allowed us to test the hypothesis that resistance targeted to important virulence factors will be more durable when deployed. Transgressive segregation for resistance was observed to both PXO99A and PXO99A +TAL7b, suggesting the presence of resistance QTL for BB. Genome-wide association (GWA) and interval mapping analysis, revealed 13 disease resistance QTL, six specific to PXO99 +TAL7B and seven to PXO99A. Future exploitation of the MAGIC AILs and screening a new subset of lines selected based on the knowledge acquired from this study will refine the resistance loci, and help elucidate the molecular basis for the resistance phenotypes identified. Accelerating the pace of developing FHB resistant two-row malting barley: Genomic selection approach D. FERNANDO (1), J. Tucker (2), A. Badea (2), W. Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota, U.S.A. A set of 413 diverse rice varieties from 82 countries (rice diversity panel; containing 44,100 SNPs available from open-source) was artificially inoculated and disease levels were evaluated by visual observation using a 0-3 rating scale, detailed image analysis using a newly developed MATLAB-based software and quantification of F. fujikuroi colonization in rice seedlings with a selective medium. A total of 24 QTLs associated with disease severity and/or pathogen colonization were mapped by the mixed linear model in TASSEL software. Defense- and hormone-related candidate genes identified within the QTLs will be further validated. QTL Mapping of Resistance to Tan Spot in a Winter Recombinant Inbred Line Population Derived from Cross between Harry and Wesley G. KARIYAWASAM (1), W. Hussain (2), A. Easterly (2), M. Guttieri (2), V. Belamkar (3), J. Venegas (2), S. Baenziger (2), J. Poland (4), J. Faris (5), S. Xu (5), J. Rasmussen (1), Z. Liu (1) S4.81 (1) North Dakota State University, Fargo, U.S.A.; (2) University of Nebraska, Lincoln, U.S.A.; (3) University of Nebraska, U.S.A.; (4) Kansas State University, Manhattan, KS, U.S.A.; (5) USDA-ARS, Northern Crop Science Laboratory, Fargo, ND, U.S.A. (1) North Dakota State University, Fargo, U.S.A.; (2) University of Nebraska, Lincoln, U.S.A.; (3) University of Nebraska, U.S.A.; (4) Kansas State University, Manhattan, KS, U.S.A.; (5) USDA-ARS, Northern Crop Science Laboratory, Fargo, ND, U.S.A. (1) North Dakota State University, Fargo, U.S.A.; (2) University of Nebraska, Lincoln, U.S.A.; (3) University of Nebraska, U.S.A.; (4) Kansas State University, Manhattan, KS, U.S.A.; (5) USDA-ARS, Northern Crop Science Laboratory, Fargo, ND, U.S.A. Tan spot, caused by the Pyrenophora tritici-repentis, is an important disease on both common and durum wheat worldwide. The disease system is known to involve three necrotrophic effectors (NEs) that interact with corresponding host sensitivity genes, including Ptr ToxA-Tsn1, Ptr ToxB-Tsc2 and Ptr ToxC-Tsc1. To investigate the role of NE-host sensitivity interactions in disease, we conducted the QTL mapping of tan spot resistance in a recombinant inbred line population derived from a cross between two US winter wheat cultivars ‘Harry’ and ‘Wesley’. Harry was sensitive to Ptr ToxA and highly susceptible to tan spot while Wesley was insensitive and highly resistant. QTL mapping revealed two major loci: the one corresponding to the Tsn1 locus, and one on 1AS, which likely corresponded to the Tsc1 locus. Development of molecular makers for spinach resistance genes to downy mildew disease C. FENG (1), J. Correll (1) (1) University of Arkansas, U.S.A. Spinach is an increasingly popular vegetable crop. Downy mildew, caused by Peronospora farinosa f. sp. spinaciae (Pfs), is the most economically important disease of spinach and the main focus of all spinach breeding programs. Resistant cultivars are the most economical way to manage downy mildew, particularly for organic spinach production, which is approaching 50% in the US. Resistance to downy mildew is typically controlled by single dominant genes, and 11 resistant loci have been hypothesized in spinach. Three resistant loci, RPF1, 2 and 3 have been genetically characterized; each provides resistance to more than 10 of the 16 described races of Pfs. A molecular SCAR marker, linked to RPF1, has been shown to be useful in selecting this locus for resistance. Bulked segregant analysis and genotyping by sequencing approaches were employed to develop markers for RPF2 and 3. One out of 900 10-mer random primers examined generated a fragment specific to resistant plants, and the derived SCAR marker co-segregated with the RPF2 locus. For RFP3, a SNP linkage map was constructed with two SNP markers closely linked (1.1 and 4 cM) to the RPF3 locus. The RPF2 and RPF3 markers have been shown to be effective in selecting the respective resistance loci outside of the original mapping populations demonstrating their utility. These markers could be used to pyramid resistant loci to improve the durability of downy mildew resistance in spinach. Genome-wide association study oF transmission of Soybean mosaic virus through seed Q. Liu (1), H. Hobbs (1), L. Domier (2) (1) University of Illinois, U.S.A.; (2) USDA-ARS, University of Illinois, U.S.A. Soybean mosaic virus (SMV), an aphid- and seed-transmitted member of the Potyviridae, causes significant reductions in soybean yield and seed quality worldwide. In North America, seedborne infections serve as the primary sources of inoculum for SMV infections. Therefore, host-plant resistance to seed transmission of SMV provides a means to limit the impact of SMV on soybean production. In this study, populations of 200 and 477 plant introductions from the United States Department of Agriculture Soybean Germplasm Collection were evaluated for their transmission of SMV through seed. To identify loci associated with resistance to SMV seed transmission, the phenotypic data and single nucleotide polymorphism (SNP) information for each line from the SoySNP50K resource were combined and analyzed using a compressed mixed linear model as implemented in the GAPIT package in R. Identification of resistance genes and resistance gene analogs by si-RNA sequencing of the sweet potato leaf phytobiome A. ALLEYNE (1), A. Alleyne (2), M. James (3), D. Gutierrez (4), S. Fuentes (4) (1) The University of the West Indies- Cave Hill Campus, Bridgetown, Barbados; (2) The University of the West Indies-Cave Hill Campus, Bridgetown, Barbados; (3) Ministry of Agriculture, Food and Water Resources, Plant Pathology Section, Graeme Hall, Barbados, Barbados; (4) International Potato Center (CIP), Virology Laboratory, Lima, Peru Molecular mapping of effective stripe rust resistance genes in wheat germplasm PI 182126 X CHEN (1) J F (2) M W (3) Molecular mapping of effective stripe rust resistance genes in wheat germplasm PI 182126 X. CHEN (1), J. Feng (2), M. Wang (3) (1) USDA-ARS and Washington State University, U.S.A.; (2) Department of Plant Pathology, Washington State University, U.S.A.; (3) Department of Plant Pathologist, Washington State University, U.S.A. Molecular mapping of effective stripe rust resistance genes in wheat germplasm PI 182126 X. CHEN (1), J. Feng (2), M. Wang (3) (1) USDA-ARS and Washington State University, U.S.A.; (2) Department of Plant Pathology, Washington State University, U.S.A.; (3) Department of Plant Pathologist, Washington State University, U.S.A. ( ) g ( ) g ( ) (1) USDA-ARS and Washington State University, U.S.A.; (2) Department of Plant Pathology, Washington State University, U.S.A.; (3) Department of Plant Pathologist, Washington State University, U.S.A. Stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most damaging diseases of wheat. Spring wheat landrace PI 182126, originally from Sind, Pakistan, has shown a high level of resistance to stripe rust in field experiments for more than 10 years and in greenhouse tests with several predominant races. The objective of this study was to map the resistance gene(s) in PI 182126. A population of 120 F5 recombinant inbred lines was developed from the cross between PI 182126 and susceptible genotype Avocet S through single-seed decent. The population was tested with US predominant races PSTv-14 and PSTv-37 at seedling stage under greenhouse conditions and at adult-plant stage in fields under natural infection of the pathogen near Pullman and Mount Vernon, Washington in 2014. The population was genotyped using microsatellite markers. Two resistance genes were mapped to chromosomes 5B and 7B. Using Chinese Spring nulli-tetrasomic and 7B deletion lines, the gene on 7B was mapped to the 0.14 bin of the long arm. Based on the chromosomal location, tightly linked markers, and effective throughout growth stages against all races tested, the gene on 7B is different from Yr39, Yr52, Yr59, YrZH84, and YrC591 previously mapped to the long arm of chromosome 7B, and is useful for diversifying stripe rust resistance genes used in breeding programs. SNP variation in switchgrass disease resistance genes SNP variation in switchgrass disease resistance genes B. BAHRI (1), G. Daverdin (2), J. Cheng (3), K. Barry (3), S. Smith (4), K. Devos (5) (1) Dept. of Plant Protection, National Agronomic Institute of Tunisia/ Dept. of Crop and Soil Sciences, University of Georgia, U.S.A.; (2) Marucci Center for Blueberry & Cranberry Research and Extension, Rutgers University, Chatsworth, NJ, U.S.A.; (3) DOE Joint Genome Institute, Walnut Creek, California, CA, U.S.A.; (4) Dept. of Plant Pathology, University of Georgia, Athens, GA, U.S.A.; (5) Institute of Plant Breeding, Genetics and Genomics, Dept of Crop and Soil Sciences, University of Georgia, Athens, GA, U.S.A. ( ) ( ) g ( ) y ( ) ( ) ( ) (1) Dept. of Plant Protection, National Agronomic Institute of Tunisia/ Dept. of Crop and Soil Sciences, University of Georgia, U.S.A.; (2) Marucci Center for Blueberry & Cranberry Research and Extension, Rutgers University, Chatsworth, NJ, U.S.A.; (3) DOE Joint Genome Institute, Walnut Creek, California, CA, U.S.A.; (4) Dept. of Plant Pathology, University of Georgia, Athens, GA, U.S.A.; (5) Institute of Plant Breeding, Genetics and Genomics, Dept of Crop and Soil Sciences, University of Georgia, Athens, GA, U.S.A. Switchgrass (Panicum virgatum L.), an allotetraploid perennial C4 grass native to North America, is an important feedstock for cellulosic biofuel production. Its high genotypic and phenotypic variation yield substantial potential for genetic improvement of bioenergy-related traits, including disease resistance. We studied SNP variation in 276 switchgrass genotypes, belonging to 36 accessions from mainly the Southern USA, across ~1 kb NBS-LRR regions of 4 classes of putative resistance (R) genes. PCR amplicons for an R gene with homology to the barley powdery mildew resistance gene Mla from 150 switchgrass accessions mapped to two homoeologous regions in the allotetraploid switchgrass genome. Reads from individual accessions aligned preferentially against only one locus. Fitfty-eight SNP positions and 78 haplotypes were recorded. Twelve SNPs were present in more than 35% of the accessions. Although switchgrass is largely an obligate outcrossing species, 95% of the SNPs were homozygous. The results indicate that selection for some potentially beneficial SNPs occurred in natural switchgrass population which were subsequently fixed in homozygous condition at the locus level. Addition of supplemental spent lime to previously limed soils for control of Aphanomyces root rot on sugar beet J. BRANTNER (1) A. Chanda (1) (1) University of Minnesota, U.S.A. Both the tsn1 and tsc1 loci were significantly associated with resistance to race 1, but only tsn1 was significantly associated with race 2, and only tsc1 was associated with race 3. Neither locus was significantly associated with disease caused by a race 2 ToxA knockout strain. Additional QTL were also identified in this population, but their effects on disease were relatively minor. This work indicates that the Ptr ToxA-Tsn1 and Ptr ToxC-Tsc1 interactions play important roles in conferring tan spot susceptibility in winter wheat, and breeding programs should remove these susceptibility loci in order to improve tan spot resistance. Two SREBP Transcription Factors are Required for Ergosterol Biosynthesis and DMI Resistance in Penicillium digitatum R. RUAN (1) (1) Institute of Biotechnology, Zhejiang University, China SREBP Transcription Factors are Required for Ergosterol Biosynthesis and DMI Resistance in Penicillium digitatu UAN (1) Two SREBP Transcription Factors are Required for Ergosterol Biosynthesis and DMI Resistance in Penicillium digitatum R. RUAN (1) (1) Institute of Biotechnology, Zhejiang University, China ( ) 1) Institute of Biotechnology, Zhejiang University, China Application of imazalil is a common method for control of citrus green mold caused by Penicillium digitatum. As a consequence of continuous use, imazalil-resistant P. digitatum strains have emerged worldwide. Our previous study showed that overexpression of imazalil target genes CYP51A and CYP51B, induced by the insertion mutations in their promoter regions, is responsible for the resistance. However, the regulators inducing overexpression of CYP51s are unclear. In this study, we demonstrated two SREBP homologs, designated as PdSrbA and PdSrbB. Both ?PdsrbA and ?PdsrbB showed markedly reduced ergosterol contents and increased sensitivities to imazalil and other DMIs, while double deletion led to hypersensitive to DMIs. ?PdsrbA but not ?PdsrbB is hypersensitive to terbina?ne and tridemorph, two SBIs targeting to ERG1 and ERG24, respectively. In addition, PdsrbA also mediated responses to iron-depletion and hypoxia. However, no change was observed in growth rate or virulence for those mutants. Expression of CYP51s and other genes involved in ergosterol biosynthesis was significantly down-regulated in ?PdsrbA and ?PdsrbB. These results suggested that these two SREBP homologs co-regulate ergosterol biosynthesis as well as DMIs resistance in P. digitatum, and PdsrbA may be with wider functions. cal association of potato susceptibility to Rhizoctonia solani ZHANG (1, 2), H. H. Jiang (2), J. J. Hao (2) Chemical association of potato susceptibility to Rhizoctonia solani X. Y. ZHANG (1, 2), H. H. Jiang (2), J. J. Hao (2) (1) College of Agronomy, Inner Mongolia Agricultural University, Hohhot, China; (2) School of Food and Agriculture, University of Maine, Orono, ME, U.S.A. 1) College of Agronomy, Inner Mongolia Agricultural University, Hohhot, China; (2) School of Food and Agriculture, Univ ME, U.S.A. Rhizoctonia solani is the causal agent of stem canker and black scurf of potato. To investigate chemical factors related to potato resistance to R. solani, 17 cultivars of potato were selected for a two-year field trial. Sixty-day-old plants were dug from field, and wax content of underground stems was measured. For other chemical measurement, potato seedlings grown in Murashige and Skoog medium to an approximate height of 12 cm, were transferred into greenhouse pots containing vermiculite and soil (v:v = 1:1). Two SREBP Transcription Factors are Required for Ergosterol Biosynthesis and DMI Resistance in Penicillium digitatum R. RUAN (1) (1) Institute of Biotechnology, Zhejiang University, China After 30 days, plants were dug and the underground stems were used to determine the silicon content. Leaves were used to determine the content of soluble sugar, soluble starch, free amino acid and soluble protein. Black scurf was measured based on 0 to 5 scale, which was used for disease index calculation. Wax content of underground stems of field-grown potato was negatively correlated with disease index (r = -0.532 P < 0.05), but silicon content did not have a significant correlation (r = 0.042 P > 0.05), indicating wax-enhanced potato resistance to R. solani. The correlation between soluble starch, soluble protein, soluble sugar and free amino acid and disease index were 0.476 (P < 0.05), 0.458 (P < 0.05), 0.300 (P > 0.05) and 0.219 (P > 0.05), respectively. This showed that lower levels of soluble starch and higher levels of soluble protein enhanced disease resistance, while soluble sugar and free amino acid had no significant effects on disease resistance. Differential effects of stalk rot diseases on photosystem II photochemical efficiency among resistant and susceptible sorghum lines A. BANDARA (1), D. Weerasooriya (1), T. Tesso (1), C. Little (1) (1) Kansas State University, U.S.A. Differential effects of stalk rot diseases on photosystem II photochemical efficiency among resistant and susceptible sorghum lines A. BANDARA (1), D. Weerasooriya (1), T. Tesso (1), C. Little (1) (1) Kansas State University, U.S.A. Stalk rots are among the most injurious sorghum diseases worldwide. Understanding the plant physiological changes that occur during these diseases will help to uncover potential resistance mechanisms. The objective of this study was to test the effect of stalk rots on sorghum (Sorghum bicolor (L.) Moench) leaf chlorophyll content and photosystem II photochemical efficiency (Fv/Fm). Two resistant (SC599, SC35) and susceptible (Tx7000, BTx3042) lines were field evaluated in 2014 and 2015. Plants were inoculated with Fusarium thapsinum (Fusarium stalk rot), Macrophomina phaseolina (charcoal rot), and phosphate-buffered saline (control) at 14 d after flowering. Flag leaf chlorophyll and Fv/Fm were measured at soft-dough, hard- dough, and physiological maturity using a Soil and Plant Analytical Development meter (SPAD) and a chlorophyll fluorometer, respectively. ANOVA was performed with appropriate modeling for repeated measures. Compared to control, both pathogens significantly reduced SPAD (P < 0.0001) across genotypes and growth stages and the reduction was greater in 2014. Across years and growth stages, both pathogens significantly reduced the Fv/Fm (P < 0.016) of susceptible lines while no such effect was evident for the resistant lines. Despite the apparent chlorophyll reduction during disease development, the ability to maintain higher photosynthetic activity at the tested growth stages appeared to contribute to physiological resistance against stalk rot diseases. Development of molecular makers for spinach resistance genes to downy mildew disease C. FENG (1), J. Correll (1) (1) University of Arkansas, U.S.A. The loci identified were compared to the results from previous biparental mapping studies of resistance to SMV seed transmission. This study provides a comprehensive analysis of loci associated with seed transmission of an economically important plant virus. Identification of resistance genes and resistance gene analogs by si-RNA sequencing of the sweet potato leaf phytobiome A. ALLEYNE (1), A. Alleyne (2), M. James (3), D. Gutierrez (4), S. Fuentes (4) (1) The University of the West Indies- Cave Hill Campus, Bridgetown, Barbados; (2) The University of the West Indies-Cave Hill Campus, Bridgetown, Barbados; (3) Ministry of Agriculture, Food and Water Resources, Plant Pathology Section, Graeme Hall, Barbados, Barbados; (4) International Potato Center (CIP), Virology Laboratory, Lima, Peru S4.82 Sweet potato (Ipomoea batatas L. (Lam) is affected by single and synergistic virus co-infections manifested as biotic stress and severe disease. Consequently, breeding for disease resistance is used in disease management. However, there are few reports of resistance genes (R-genes) against specific viruses in sweet potato virus disease complex (SPVD). This study sought to identify R- genes in the sweet potato leaf phytobiome using Next Gen-sequencing. RNA-seq has been used to characterize viruses causing SPVD while discovery of R-genes traditionally involves cloning and characterization of resistance proteins and resistance gene analogs (RGA). In this study, RNA was extracted from three sweet potato cultivars (CBS 49, CBS 32 and C-104) symptomatic with Sweet potato feathery mottle virus (SPFMV), Sweet potato leaf curl virus (SPLCV) and Sweet potato badnavirus A and B. These were then subjected to RNA-seq analysis and bioinformatics analysis of the assembled contigs for resistance genes. We report the presence of transcripts of a total of thirty-one such genes, and putative proteins representing several classes of resistance genes including nucleotide- binding site leucine-rich repeat (NBS-LRR) and RGA from sweet potato such as AB125881, DQ903328, and DQ903597 in the sweet potato phytobiome. These results provide a genome resource of R-genes for development of tagged molecular markers useful for mapping disease resistance in specific sweet potato cultivars. Genetic characterization of quantitative resistance to Bremia lactucae, the causal organism of lettuce downy mildew L. Parra (1), I. Simko (2), R. Michelmore (3) (1) UC Davis- Genome center U S A ; (2) USDA-ARS U S A ; (3) UC Davis-Genome Center U S A Genetic characterization of quantitative resistance to Bremia lactucae, the causal organism of lettuce downy mildew Genetic characterization of quantitative resistance to Bremia lactucae, the causal organism of lettuce downy mildew L. Parra (1), I. Simko (2), R. Michelmore (3) (1) UC Davis- Genome center, U.S.A.; (2) USDA-ARS, U.S.A.; (3) UC Davis-Genome Center, U.S.A. Genetic characterization of quantitative resistance to Bremia lactucae, the causal organism of lettuce downy mildew L. Parra (1), I. Simko (2), R. Michelmore (3) Lettuce (Lactuca sativa) is one of the most valuable vegetable crops in the United States. Downy mildew (DM), caused by Bremia lactucae, is the most important foliar disease of lettuce worldwide, which decreases the quality of the marketable portion of the crop. The use of resistant varieties carrying dominant genes (Dm genes) is the most effective method for controlling this disease. However, the high pathogen variability leads to the defeat of cultivars containing resistance genes by new isolates of the pathogen. Some lettuce varieties such as Iceberg and Grand Rapids exhibit field resistance that cannot be attributed to Dm genes. This resistance trait, which is manifested in adult stages of development, has shown to be quantitatively inherited. To determine the genetic basis of field resistance, two populations of recombinant inbred lines originated from crosses between a field resistant cultivar and a susceptible cultivar (Grand Rapids x Salinas and Iceberg x PI491224) have been evaluated for DM severity and genotyped using Genotyping-By- Sequencing for quantitative trait loci (QTL) analysis. Marker-assisted gene pyramiding of multiple Dm genes in combination with QTLs for field resistance provides the opportunity for more durable resistance to B. lactucae. Effectiveness of adult plant resistance to wheat stem rust is specific to Puccinia graminis f. sp. tritici race M ROUSE (1) J Briggs (2) Adult plant resistance (APR) in wheat to stem rust caused by Puccinia graminis f. sp. tritici is considered to be conferred by multiple loci that are race non-specific. Our objective was to test the race-specificity of individual and combinations of wheat stem rust APR loci. We evaluated a biparental wheat population (Thatcher/McNeal) that is segregating for six APR loci with minor effects in response to four races of the stem rust pathogen (QTHJC, TPMKC, RCRSC, and QFCSC), in single-race field nurseries over three years. QTL mapping and ANOVA identified the significance and effectiveness of the six minor effect loci across the years and races. The QTL with the largest effect, coincident with the Sr12 locus, was effective to virulent races QTHJC and TPMKC, but conferred a variable, sometimes nonsignificant, response to race RCRSC. The other five QTL varied in effectiveness between years and were generally less effective in response to race RCRSC in particular. The data from QTL mapping suggested that some of the QTL could be race specific, such as QSr.cdl.1AL, because they were detected in response to races such as TPMKC and QTHJC, but not to race RCRSC. This observation has implications specifically for wheat stem rust resistance breeding because it suggests that resistance observations to a single race may not be indicative of resistance to another race, even if the mechanism of resistance is multiple minor effect loci. Lack of interaction between Fusarium oxysporum f. sp. niveum and Meloidogyne incognita on cucurbit rootstocks resistant to Fusarium wilt of watermelon Lack of interaction between Fusarium oxysporum f. sp. niveum and Meloidogyne incognita on cucurbit rootstocks resi watermelon A. KEINATH (1), P. Agudelo (1) A. KEINATH (1), P. Agudelo (1) (1) Clemson University, U.S.A. Interspecific hybrid squash (Cucurbita maxima × C. moschata ‘Strong Tosa’) and bottle gourd (Lagenaria siceraria ‘Macis’) rootstocks are resistant to Fusarium oxysporum f. sp. niveum (FON) but susceptible to Meloidogyne incognita, Southern root-knot nematode. Co-infection with root-knot nematode has rendered Fusarium resistance ineffective in other hosts. The objectives of this study were to determine if such an interaction occurred between M. incognita and FON races 1 and 2 on ‘Strong Tosa,’ ‘Macis,’ and watermelon ‘Fascination’ (resistant to race 1) and ‘Tri-X 313’ (susceptible to both races). The four hosts were inoculated in a greenhouse with one of four pathogen treatments: FON applied as 2 × 107 microconidia per pot, M. Effectiveness of adult plant resistance to wheat stem rust is specific to Puccinia graminis f. sp. tritici race M ROUSE (1) J Briggs (2) incognita applied as 2000 J2 per pot, both pathogens, or neither pathogen. Each race was tested twice with 10 replicate plants per host and inoculation treatment. Plants not inoculated with FON did not wilt. After 4 weeks, severity of Fusarium wilt and recovery of F. oxysporum did not differ when hosts were inoculated with FON alone or with FON and M. incognita together (host-by-treatment interactions not significant, P≥0.08 and P≥0.17 for races 1 and 2, respectively). Galling was present on ≥10% of the root system of 90% of the plants inoculated with M. incognita, and no non-inoculated plants had galls. In conclusion, resistance to FON in cucurbit rootstocks is not compromised when M. incognita infects the roots. Adaptation to quantitative resistance in the hop cultivar Cascade by Podosphaera macularis D. GENT (1), D. Gent (1), M. Twomey (2), S. Wolfenbarger (2) (1) USDA Agricultural Research Service, U.S.A.; (2) Oregon State University Department of Botany and Plant Pathology, U.S.A. In the hop powdery mildew pathosystem, the cultivar Cascade has not been substantially affected by powdery mildew despite being produced on a relatively broad scale for decades. Beginning in 2012, development of powdery mildew on this cultivar under field conditions has become increasingly common in Washington State. Surveys conducted during 2013 to 2015 indicated increasing occurrence and severity of powdery mildew on Cascade, concomitant with increases in the number of fungicide applications made growers. Nearly all isolates of Podosphaera macularis sporulated when inoculated to Cascade, although the latent period was shortest, infection frequency greatest, and the fungus most fecund on Cascade when inoculum was originally derived from this cultivar as compared to other cultivars. Only 4.9% of isolates obtained from Cascade were able to infect cv. Nugget, which possess the resistance gene termed R6, indicating that Cascade-adapted strains of the fungus are distinct from strains that attack cultivars possessing R6. In growth chamber experiments, powdery mildew levels on various cultivars were similar when inoculated with Cascade-adapted strains of P. macularis as compared to non-adapted strains, further supporting a specific adaptation by the fungus to the quantitative resistance in Cascade. Multiple R-genes are predicted to provide resistance to Cascade-adapted strains of the fungus, as is quantitative resistance found in other germplasm. Exserohilum turcicum races causing northern leaf blight of corn in the North Central United States J. Weems (1), C. Bradley (1) (1) University of Kentucky, U.S.A. (1) University of Kentucky, U.S.A. Northern leaf blight of corn, caused by Exserohilum turcicum, is a yield- reducing foliar disease that commonly occurs across the North Central U.S. Previous race population distribution studies identified five physiological races in the U.S. prior to 1995. For this study, 156 E. turcicum isolates from Illinois, Indiana, Iowa, Minnesota, North Carolina, Ohio, and Wisconsin were screened against corn differential lines containing Ht1, Ht2, Ht3, Htm1, S4.83 and Htn1 resistance genes. Isolates were collected between 1979 and 1985 (n=13) and between 2007 and 2014 (n=143). Twenty physiological races were observed based on the responses of the differential corn lines. Races 0, 1, and 1mn were the most prevalent races, comprising 21%, 27%, and 13% of the isolates, respectively. Races were diverse within all states and years. Virulence to multiple Ht resistance genes within individual isolates was observed in 47% of those tested, with 3% of the isolates conferring virulence to all Ht resistance genes tested. Virulence to the Ht1, Ht2, Ht3, Htm1, and Htn1 resistance genes was present in 64%, 20%, 18%, 32%, and 27% of the E. turcicum isolates, respectively. Virulence to Ht resistance genes was fairly evenly distributed across states in isolates collected after 2008. Although Ht genes were still effective against several isolates, the use of quantitative resistance in conjunction with Ht genes would likely improve management of northern leaf blight in the North Central U.S. Wheat yellow rust dynamics in Tunisia since 2013 and resistance genes in durum wheat B. BAHRI (1), M. Leconte (2), S. Hamza (1), C. de Vallavieille Pope (2) (1) National Agronomic Institute of Tunisia, Tunisia; (2) UMR BIOGER, INRA, AgroParisTech, Universi Wheat yellow rust dynamics in Tunisia since 2013 and resistance genes in durum wheat B. BAHRI (1), M. Leconte (2), S. Hamza (1), C. de Vallavieille Pope (2) (1) National Agronomic Institute of Tunisia, Tunisia; (2) UMR BIOGER, INRA, AgroParisTec Wheat yellow rust dynamics in Tunisia since 2013 and resistance genes in durum wheat B. BAHRI (1), M. Leconte (2), S. Hamza (1), C. de Vallavieille Pope (2) (1) National Agronomic Institute of Tunisia, Tunisia; (2) UMR BIOGER, INRA, AgroParisTec Wheat yellow rust epidemics, caused by Puccinia striiformis f. sp. tritici (PST) have increased worldwide with the emergence in Western Europe and North Africa of an aggressive strain and tolerant to high temperature (PstS2) since 2004 and exotic strains (Triticale 2006, 2015, Warrior and Kranich) recently. Even though resistant varieties bring to agriculture effective solutions to reduce the use of pesticides, races of the pathogen quickly overcome introduced resistance genes. In this study we determined the virulence combinations using the European and world differential sets and the simple sequence repeat diversity of 94 PST isolates collected in Tunisia since 2013 from the major wheat growing areas. In addition, 39 local Tunisian durum wheat accessions and 109 recombinant inbred lines RILs (F9) were evaluated at the seedling stage against old and new races of PST in order to identify resistant genotypes and markers associated with resistance genes. The results underlined the rapid change in wheat yellow rust population where 3 predominant races, Warrior1 (W1), Warrior- (W-) and Triticale 2006 were detected since 2013. In addition, 10% of the durum wheat accessions were resistant to W1, W- and PstS2. The evaluation of the RILs issued from a cross between Tunisian durum wheat Khiar and Agili39 revealed one major resistance gene against W1 and two minor genes against PstS2. The use of these genes in breeding can improve durability of the genetic control against PST. Gene-For-Gene in Fusiform Rust Disease of Loblolly Pine Using an improved Cqf assembly and re-sequencing of field isolates, we are attempting define the Avr1 gene and the causative mutation that led to virulence in the Avr1 mapping population. Melampsora rust running amuck: Exploring willow rust population diversity in the Northeast United States C. CROWELL (1), M. Bekauri (1), C. Carlson (2), F. Gouker (2), L. Smart (2), C. Smart (1) (1) Cornell University Plant Pathology and Plant-Microbe Biology Section, U.S.A.; (2) Cornell University Horticulture Shrub Willow (Salix spp.) has proved to be a reliable short-rotation coppice (SRC) biofuel feedstock in the Northeast United States. However, the willow rust pathogen Melampsora spp. can cause up to a 50% reduction in yield, making some growing operations economically limited. This has spurred investigation into both the identification of resistance within in Salix spp. and the diversity of Melampsora spp. in the Northeast. Approximately 200 single pustule isolates were collected from MI, NY, PA, VT, and WV, and are being characterized by rDNA sequencing. Preliminary data shows that both M. americana and M. paradoxa are present in our regions of interest. Further investigation of Melampsora spp. diversity throughout the Northeast will be conducted using genotyping-by-sequencing, a genome wide high-throughput SNP identification method, providing thousands of markers for population studies targeting un-methylated coding sequences. Additionally, we explored disease resistance by characterizing association and linkage mapping populations of Salix purpurea with the aim of identifying QTL and tightly-linked SNPs for marker-assisted selection. Preliminary data show a continuum of resistance, suggesting promising breeding opportunities. This information will lead to further understanding of the population biology of Melampsora willow rust, while providing crucial information necessary for the success of future willow breeders. Marker development and fine-mapping for the Phytophthora crown rot resistance locus, Pc1, in strawberry Y. NOH (1), J. Mangandi (1), S. Verma (1), V. Whitaker (1), S. Isobe (2), J. Cha (3), S. Lee (1) (1) University of Florida, U.S.A.; (2) Kazusa DNA Research Institute, Japan; (3) Chungbuk National University, South Korea Phytophthora crown rot (PhCR) caused mainly by Phytophthora cactorum is a destructive disease and can cause economic losses to strawberry growers in US. In our previous study, a major QTL for PhCR resistance, Pc1, located on the linkage group 7D, was discovered in complex and multi-family populations using the whole-genome SNP genotyping (IStraw90 Axiom® SNP Array) and pedigree-based marker-trait association analysis (FlexQTL™). Induction of resistance in cultivated peanut against peanut early leaf spot by salicylic acid K. BOWEN (1), L. Gong (2) (1) Auburn University, U.S.A.; (2) Auburn University, U.S.A. Induction of resistance in cultivated peanut against peanut early leaf spot by salicylic acid K. BOWEN (1), L. Gong (2) (1) Auburn University, U.S.A.; (2) Auburn University, U.S.A. Cultivated peanut (Arachis hypogaea L.) is an economically important crop for the United States and throughout the world. Cercospora arachidicola (Ca), the causal agent of early leaf spot (ELS) on cultivated peanut, can cause yield losses of up to 50%. Since earlier work reported inconsistent results with salicylic acid (SA) relative to peanut pathogens, we sought to examine the ability of SA to induce resistance against ELS on a high-oleic cultivar. We evaluated the effect of spraying three rates of SA (100, 200, and 300 µM) at 3, 5, and 7 days before inoculation (DBI) with ELS in whole peanut plants (cultivar GA-09B) in a growth chamber. Each treatment was replicated three times. At 15 days after inoculation (DAI), all SA treatments had significantly fewer lesions (3.1, 2.8, and 2.5 lesions with increasing SA rates; and 1.9, 3.0, and 3.6 lesions with increasing DBI), compared with the control (12.7). At 42 DAI, all sporulating lesions from SA treated plants tended to produce fewer spores compared with the control (170.0); for example, 27.2 with 300 µM SA, 54.5 with 3 DBI, and 31.0 with 7 DBI. Although there were fewer lesions on SA treated plants, these lesions were larger than those on the control. Results of this study suggest that SA plays at least a transient role in ELS resistance in peanut cultivar GA-09B. Development of a molecular marker platform for the breeding of blast resistance varieties in Taiwan W. CHEN (1), W. Shen (1), F. Chang (2), W. Chang (3), Z. Yu (1), J. Liao (1), M. Lai (4), C. Wu (2), C. Chung (1) (1) National Taiwan University, Taiwan; (2) Kaohsiung District Agricultural Research and Extension Station, Taiwan; (3) Taoyuan District Agricultural Research and Extension Station, Taiwan; (4) Taiwan Agricultural Research Institute, Taiwan Development of a molecular marker platform for the breeding of blast resistance varieties in Taiwan Rice blast is the most important disease commonly occurring in the first cropping season in Taiwan. To facilitate marker-assisted backcrossing, this study aims to identify effective blast resistance (R) genes in Taiwan and develop R gene and background selection markers for the use of IRRI-bred blast resistant lines (IRBLs) as donors. Gene-For-Gene in Fusiform Rust Disease of Loblolly Pine Gene-For-Gene in Fusiform Rust Disease of Loblolly Pine K. SMITH (1), D. Ence (2), D. Nelson (3), M. Yandell (2), J. Davis (4) (1) USDA Forest Service U S A ; (2) University of Utah U S A ; (3) US K. SMITH (1), D. Ence (2), D. Nelson (3), M. Yandell (2), J. Davis (4) (1) USDA Forest Service, U.S.A.; (2) University of Utah, U.S.A.; (3) USD ), ( ), ( ), ( ), ( ) orest Service, U.S.A.; (2) University of Utah, U.S.A.; (3) USDA Forest Service, U.S.A.; (4) University of Florida, U.S.A. The rust fungus Cronartium quercuum f.sp. fusiforme (Cqf) incites fusiform rust disease on southern pines causing loss of stem quality or death. Given that fusiform rust is a coevolved, gene-for-gene pathosystem there is potential for resistance to be overcome in the field. Nine resistance genes (Fr1-Fr9) and one avirulence gene (Avr1) have been placed on the genetic maps of loblolly pine and Cqf. More recently genomic references have become available for both the host and the fungus. Allelic-level sequence data for resistance genes and their corresponding avirulence genes would make it possible to monitor virulence in the field, guide deployment and understand local outbreaks. Toward that end work is underway in both the loblolly host and Cqf pathogen. A candidate for the Fr1 resistance gene was identified in the 22 gigabase pair, loblolly pine reference genome. This gene remains a candidate S4.84 and fine mapping is currently being done to improve the loblolly genetic map in this region, as well as, identify additional resistance gene candidates from elite pine families. Research on the Cqf fungus has located a genomic interval that contains the Avr1 gene using both traditional marker sequences and high-throughput re-sequencing of progeny from of an Avr1 segregating cross. Using an improved Cqf assembly and re-sequencing of field isolates, we are attempting define the Avr1 gene and the causative mutation that led to virulence in the Avr1 mapping population. and fine mapping is currently being done to improve the loblolly genetic map in this region, as well as, identify additional resistance gene candidates from elite pine families. Research on the Cqf fungus has located a genomic interval that contains the Avr1 gene using both traditional marker sequences and high-throughput re-sequencing of progeny from of an Avr1 segregating cross. Mitigating the effects of shoot blight caused by Erwinia amylovora by enhancing plant systemic resistance B. LEHMAN (1), C. Bower (1), K. Peter (1) (1) Penn State University, U.S.A. Mitigating the effects of shoot blight caused by Erwinia amylovora by enhancing plant systemic resistance B. LEHMAN (1), C. Bower (1), K. Peter (1) (1) Penn State University, U.S.A. Fire blight, caused by Erwinia amylovora, is one of the most devastating diseases of apple and pear and continues to cause large economic losses for tree fruit producers. Antibiotics are largely used to control blossom blight where bacterial resistance is absent, but the need exists for alternative control strategies where resistance occurs, and where shoot and canker blight are prevalent. One strategy is through controlled activation of the plant immune system by the application of materials containing plant defense activating compounds. Our goal was to test the effectiveness of an experimental resistance activating compound in reducing shoot blight severity. We applied three different concentrations of the chemical as foliar sprays on potted ‘Gala’ apple trees grafted on M.26 rootstocks. Three concentrations were applied at three, two, and one week before shoot tip inoculation and three, two and one day before shoot tip inoculation; Acibenzolar-S-methyl (Actigard) was also applied at three, two, and one week before shoot tip inoculation. Evaluations of shoot severity were made at one and two weeks after inoculation. Treatments started three days before inoculation reduced severity by 9, 26, and 35% for the three concentrations tested. Treatments started three weeks before inoculation reduced severity by 27, 21, and 44%, for the concentrations tested. Acibenzolar-S-methyl applications reduced severity by 75%. The testing of other alternatives is ongoing. Gene-For-Gene in Fusiform Rust Disease of Loblolly Pine For the fine-mapping of Pc1, a total of 25 SNP-based high resolution melting (HRM) markers and 63 simple sequence repeat (SSR) markers were selected in the Pc1 region. All markers were tested with five strawberry varieties and 20 advanced breeding selections that inherited putative functional allele combinations of Pc1pc1 (heterozygous resistant) or pc1pc1 (susceptible). Total five markers were found to co-segregate with the resistance locus, Pc1. In addition, we developed three populations (n=339) by crossing the resistance parent (Pc1pc1), ‘12.55-220’, with three susceptible accessions (pc1pc1), ‘12.82-44’, ‘11.116-56’, and ‘11.98-41’. All populations were planted to the field (Wimauma, FL) and evaluated for the PhCR disease resistance. The HRM and SSR markers linked to the Pc1 resistance locus were tested for the PhCR fine-mapping population, and the linkage analysis was conducted. The precise location of Pc1 and DNA markers developed in this study will be utilized for the PhCR resistance breeding program. Evaluating soybean breeding lines developed from different sources of resistance to Phomopsis seed decay S LI (1) J Smith (1) Phomopsis seed decay (PSD) causes poor soybean seed quality worldwide. The primary causal agent of PSD is Phomopsis longicolla (syn. Diaporthe longicolla). Breeding for PSD-resistance is the most effective long-term strategy to control this disease. To develop soybean lines with resistance to PSD, multiple breeding populations were developed from crosses between PSD-resistant and high-germination lines, as well as between PSD-resistant lines. Based on the seed plating assay, the range of percent seed infected by P. longicolla among all lines tested was from 0 to 52%, with an overall line mean of 7.5. There were significant differences in the reaction to PSD among lines derived from the same source of resistance, as well as among lines from different sources of resistance. More important for making future populations, the mean PSD score of the lines derived from the PI 424324B source was significantly lower than that of those derived from the PI 80837/SS93-6181 source. The PSD of lines from PI 417050 source were not significantly different from those from the other sources. These results indicate an apparent superiority for using the PI 424324B source of resistance for breeding improved lines. Seeds for many of the lines derived from both PI 424324B and PI 417050 had no incidence of PSD. Therefore, it is likely that some of them will be released in the future as resistant to PSD and thereafter utilized in breeding programs to develop PSD-resistant cultivars. Transgenic expression of pattern recognition receptor EFR in tomato leads to effective field resistance to bacterial wilt S. KUNWAR (1), S. Kunwar (1), E. Evaristo da Silva (2), F. Iriarte (2), L. Ritchie (2), D. Clark (2), J. Freeman (2), R. Stall (3), J. Jones (3), J. Minsavage (3), C. Zipfel (4), D. Horvath (5), M. Paret (2) (1) University of Florida, Gainesville, FL, U.S.A.; (2) North Florida Research and Education Center, University of Florida, U.S.A.; (3) University of Florida, U.S.A.; (4) The Sainsbury Laboratory, Nowich, UK, United Kingdom; (5) Two Blades Foundation, U.S.A. ( ) ( ) nesville, FL, U.S.A.; (2) North Florida Research and Education Center, University of Florida, U.S.A.; (3) University of sbury Laboratory, Nowich, UK, United Kingdom; (5) Two Blades Foundation, U.S.A. Bacterial wilt of tomato caused by Ralstonia solancearum is a major problem in tomato production in the tropics and sub-tropics worldwide and can cause 50-80% yield losses. Induction of resistance in cultivated peanut against peanut early leaf spot by salicylic acid K. BOWEN (1), L. Gong (2) (1) Auburn University, U.S.A.; (2) Auburn University, U.S.A. The resistance spectra of known R genes were evaluated by inoculating 31 IRBLs with 18 M. oryzae isolates collected from six rice-growing areas in Taiwan in 2009-2013. The R alleles at Pi2/9, Pik, and Pita/Pita2 were resistant to more isolates. Genotyping-by- sequencing technology was used to analyze 13 IRBLs with broader-spectrum resistance and 20 Taiwan high-quality rice varieties. Based on the 131,942 SNPs between IRBLs and Taiwan varieties, a set of 11 polymorphic R gene markers and 96 Fluidigm markers evenly distributed on rice genome were developed. Two M. oryzae isolates 12YL-TT4-1 and 13TN-HB1-3 were used to determine which Taiwan varieties would be suitable recipients for the 13 R alleles. The result showed that Taikeng 4, Taikeng 9, Taikeng 10, Taikeng 14, Taikeng 15, Tainung 71, Taiyuan 3, Tainan 11, Kaohsiung 145, and Kaohsiung 147 do not carry the R alleles in IRBL9-W, IRBLz5-CA, IRBLz5-CA (R), IRBLz-Fu, IRBL1-CL, IRBL7-M, IRBLk-Ka, IRBLkp-K60, IRBLKm-Ts, IRBLkh-K3, IRBL20-IR24, IRBLta2-Pi, and IRBLta2-Re. The 13 IRBLs and newly developed markers can be used to improve blast resistance of the 10 high-quality rice varieties. S4.85 Introgression of crown rot resistance into cultivated squash Cucurbita moschata, C. maxima and C. agyrosperma S. ZHANG (1), Y. Fu (1), P. Moon (1), C. Waddill (1) (1) Tropical Research and Education Center, University of Florida, IFAS, U.S.A. Introgression of crown rot resistance into cultivated squash Cucurbita moschata, C. maxima and C. agyrosperma S. ZHANG (1), Y. Fu (1), P. Moon (1), C. Waddill (1) Phytophthora capsici incites crown rot in squash and often causes significant losses to growers worldwide. No resistance is available in commercial squash varieties, but resistance has been found in the wild species Cucurbita lundelliana and introduced into C. moschata, resulting in several resistant lines. However, the yields of these resistant lines are too poor to warrant their commercial release. To overcome this, four of these resistant lines were crossed with commercial varieties of C. moschata, C. maxima and C. agyrosperma in single or three-way crosses. The F1 progeny of C. moschata ‘Butterbush’ and C. moschata resistant lines were resistant to P. capsici and showed improved yield. Interspecific cross of ‘Buttercup’ (C. maxima) x 399 (432) (a resistant line) was made. The F1 progeny were completely resistant to P. capsici, and showed strong heterosis in plant growth and fruit size, but were male sterile. Backcross progeny exhibited low seed settings. Three-way crosses of C. agyrosperma x F1 (C. Evaluating soybean breeding lines developed from different sources of resistance to Phomopsis seed decay S LI (1) J Smith (1) We investigated whether transgenic tomato (Solanum lycopersicum) expressing the resistance gene EFR (Elongation Factor Tu-Receptor) from Arabidopsis thaliana or Bs2 gene from Capsicum annuum or both provides improved resistance to bacterial wilt disease in a field with R. solanacearum. The presence of EFR and Bs2 or EFR alone on the highly susceptible FL 8000 background, significantly reduced the bacterial wilt incidence compared to Bs2 line (carrying only Bs2) or non-transformed control. Although yields of marketable fruit from EFR or EFR/Bs2 lines were statistically not different than those from Bs2 lines or non-transformed control, the total yield was significantly improved in the EFR and EFR/Bs2 lines. Following harvest, the basal and middle stem sections of the remaining living plants in the field were assessed by diluting plating on modified SMSA media. The results revealed no significant differences in the populations at the basal stem. Interestingly, in the middle stem, no bacteria could be recovered from EFR lines or EFR/Bs2 lines but R. solanacearum populations were recovered from Bs2 and non-transformed control lines at 104 CFU/g and 103 CFU/g tissue, respectively. This finding indicates the potential of EFR gene for field management of tomato bacterial wilt. Plant defensins inhibit growth of pathogens in the alfalfa crown rot disease complex A. SATHOFF (1), D. Samac (2) (1) University of Minnesota, U.S.A.; (2) USDA-ARS, U.S.A. Alfalfa crown rot is a disease complex that severely limits alfalfa stand density and productivity in all alfalfa-producing areas. Currently, there are no viable methods of control. Plant defensins are small cationic antimicrobial peptides with a conserved signature of cysteines. Defensins have a gamma- core motif, a cluster of positively charged residues, which is essential for antimicrobial activity. The core motifs of five synthetic defensins were tested for antimicrobial activity against the pathogens in the alfalfa crown rot disease complex. In a 96-well microplate, each well contained half strength potato dextrose broth, approximately 2000 spores, and concentrations of defensin peptide up to 30 ug/mL in a total volume of 100 uL. After 48 h of incubation at 25°C in the dark, absorbance of the wells was measured at 595 nm on a microplate reader to quantify the inhibition of fungal growth. The amount of defensin needed to inhibit growth of pathogen strains by 50% (EC50) was calculated. Evaluating soybean breeding lines developed from different sources of resistance to Phomopsis seed decay S LI (1) J Smith (1) The core motif of MtDef4 was shown to be the most effective peptide with EC50 values of 5.3 uM against Phoma medicaginis and 6.9 uM against Fusarium oxysporum f.sp. medicaginis. In addition, MtDef4 had activity against Pseudomonas syringae pv. syringae and Xanthomonas alfalfa but not the oomycete Aphanomyces euteiches in in vitro assays. These results indicate that transgenic expression of plant defensins in alfalfa has the potential to lead to improved crown rot resistance. A novel vector system to engineer begomovirus resistance in transgenic plants by transcriptional and post-transcriptional gene-silencing mechanisms Induction of resistance in cultivated peanut against peanut early leaf spot by salicylic acid K. BOWEN (1), L. Gong (2) (1) Auburn University, U.S.A.; (2) Auburn University, U.S.A. moschata x C. moschata) were obtained. The hybrids showed improved seed settings with higher percentage of normal seeds (41.2%) compared to the F1 interspecific progeny of C. maxima x C. moschata (8.8%). Embryo rescue was employed to overcome interspecific barriers. Further backcrossing with recurrent parents and pedigree selection for P. capsici resistance, high fruit yield, and flesh quality will be performed. Evaluating soybean breeding lines developed from different sources of resistance to Phomopsis seed decay S LI (1), J Smith (1) Evaluating soybean breeding lines developed from different sources of resistance to Phomopsis seed decay S. LI (1), J. Smith (1) (1) USDA-ARS, Crop Genetics Research Unit, U.S.A. A novel vector system to engineer begomovirus resistance in transgenic plants by transcriptional and post-transcripti mechanisms C. TAI (1), J. Chen (2), F. Jan (1) . Jan (1) Pathology, National Chung Hsing University, Taiwan; (2) Department of Agronomy, National Chung Hsing University, Taiw C. ( ), J. C e ( ), . Ja ( ) (1) Department of Plant Pathology, National Chung Hsing University, Taiwan; (2) Department of Agronomy, National Chun To generate transgenic plants that are highly resistant to whitefly-transmitted Tomato leaf curl Taiwan virus (ToLCTWV) and Tomato yellow leaf curl Thailand virus (TYLCTHV), plasmid vectors were constructed to induce transcriptional gene silencing (TGS) and/or post-transcriptional gene silencing (PTGS) in a transgenic plant. The intergenic region (IR) of begomovirus containing inverted repeat sequences was inserted into the intron of a tomato gene encoding a RNA-directed RNA polymerase 1 (RDR1) or a proline dehydrogenase (PDH). Expression of a GFP-RDR in construct carrying a TYLCTHV IR (IRhp), tomato intron and GFP led to nuclear localization of IRhp RNAs in plant cells after intron was spliced out and induced TGS. A C1C2C3 construct carrying the 3’end of C1 ORF and the overlapping region of C2 and C3 ORFs of TYLCTHV triggered PTGS in the cytoplasm. Infection assays revealed that constructs inducing TGS or PTGS alone displayed moderate resistance to TYLCTHV. However, C-RDRin-IR constructs carrying an IRhp, intron and C1C2C3 ORFs induced both TGS and PTGS and displayed strong resistance to TYLCTHV. Constructs carrying RDR1 introns could be easily spliced out and triggered TGS after transient expression in tobacco plants. Constructs carrying PDH intron3 had lower efficiencies in splicing and in triggering TGS. Our results indicated that a combination of inducing both TGS and PTGS in a construct shows great promise in developing resistance against plant viruses. S4.86 Exploiting the approaches of biotechnology in sweetpotato for virus diseases resistance Y. MENG (1), Y. Meng (2), C. Zhang (2), V. Njiti (2) (1) Alcorn State University, U.S.A.; (2) Alcorn State University, U.S.A. ( ) g ( ) g ( ) j ( ) lcorn State University, U.S.A.; (2) Alcorn State University, U. Sweetpotato (Ipomoea batatas (L.) Lam.; Convolvulaceae), a plant widely grown in all tropical and subtropical areas, is among the 10 most important food crops worldwide. As a crop produced by vegetative propagation, “cultivar decline” due to viral infections significantly reduces sweetpotato yield and storage root quality. A novel vector system to engineer begomovirus resistance in transgenic plants by transcriptional and post-transcripti mechanisms Evaluations in 2016 will further investigate cultivars that show promise in sweet onion production. Early detection of adult plant resistance to oat crown rust using a qPCR assay B. YIMER (1), B. Yimer (2), T. Gordon (2), J. Bonman (2) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. Oat crown rust caused by the fungus Puccinia coronata f. sp. avenae is a major production constraint of oat in North America. There are over 100 major genes identified from various oat accessions, but their effectiveness is generally short lived. Adult plant resistance is the alternative and best option for durable crown rust resistance in oats. In field grown small grain cereals, adult plant rust resistance is usually measured at the flag leaf stage using a visual assessment of disease severity and infection type. Quantitative PCR-based assays have the potential to provide a less subjective measure of host resistance at an earlier stage in plant development. Such quantitative assay techniques provide a measure of the amount of fungus within infected plants which, for some rusts, can be directly extrapolated to host resistance. Here we report the use of a qPCR assay to detect adult plant resistance to crown rust in oats before the flag leaf stage in an experiment conducted in a growth chamber environment. Response of sweet sorghum lines to stalk pathogens Fusarium thapsinum and Macrophomina phaseolina D. FUNNELL-HARRIS (1), P. O’Neill (1), S. Sattler (1) (1) USDA-ARS; Grain, Forage and Bioenergy Research Unit, U.S.A. Response of sweet sorghum lines to stalk pathogens Fusarium thapsinum and Macrophomina phaseolina D. FUNNELL-HARRIS (1), P. O’Neill (1), S. Sattler (1) (1) USDA-ARS; Grain, Forage and Bioenergy Research Unit, U.S.A. Sweet sorghum [Sorghum bicolor (L.) Moench] has potential for bioenergy. It is adapted to a variety of U.S. locations and the extracted juice can be directly fermented into ethanol. However, little research on fungal stalk rots has been reported, even though these diseases pose serious constraints for yield, juice quality and biomass usability. A greenhouse bioassay was designed to assess charcoal rot (Macrophomina phaseolina) and Fusarium stalk rot (Fusarium thapsinum) susceptiliby at maturity, when these diseases are manifested in the field. Multiple plantings of a susceptible grain line, RTx430, were used as a control for variation in flowering times amongst sweet sorghum lines. Lesion length measurements in inoculated peduncles were used to quantify disease severity. Sweet sorghum lines ‘Rio’ and M81E exhibited resistance to F. A novel vector system to engineer begomovirus resistance in transgenic plants by transcriptional and post-transcripti mechanisms In the United States, Sweet potato feathery mottle virus (SPFMV) and Sweet potato leaf curl virus (SPLCV) are two of the most prevalent sweetpotato viruses that cause devastating diseases and yield reductions. Even though resistance to virus infections remains the most cost effective way of disease management, to date, immune resistance to these viruses has not been identified in I. batatas. In this study, we explored a novel biotechnological method to develop transgenic sweetpotato plants with resistance to these two viruses. We engineered part of coat protein (CP) gene of SPFMV and the replication origin region of SPLCV into a binary vector for Agrobacterium-mediated genetic transformation for transgene expression. The SPFMV CP gene and SPLCV replication origin sequence are designed in antisense for the optimal induction of RNA silencing in transgenic sweetpotato. Research results showed that expression of foreign genes has been achieved by using Agrobacterium tumefaciens strain EHA105 harboring the expression cassette. Investigations on transgenic plant regeneration and resistance to virus infection under control conditions will be discussed. Identifying onion cultivars with reduced susceptibility to center rot J. MAZZONE (1), B. Gugino (2), M. Mansfield (2) (1) The Pennsylvania State University, U.S.A.; (2) The Pennsylvania State University, U.S.A. Onion growers in Pennsylvania (PA) are continually challenged by losses from center rot disease caused by the bacterial pathogens Pantoea ananatis and P. agglomerans. The objective of this research is to identify onion cultivars that are less susceptible to center rot than the commercial standard cv. Candy but still meet the criteria of the PA Simply Sweet Onion Marketing Program. Thirteen onion cultivars were evaluated between 2015 and 2016 for center rot susceptibility and select horticultural characteristics, including neck and bulb diameter. Cultivars were arranged in a randomized complete block design and plots were split by proximity to inoculated plants. Two onion leaves of select plants were inoculated with a mixture of P. ananatis and P. agglomerans isolates. At harvest, onions were graded by size, marketability and disease incidence. Sub-samples of asymptomatic onions were evaluated for soluble solids, pungency and postharvest disease incidence. In 2015, six cultivars had marketable yields comparable to cv. Candy. Center rot incidence significantly differed between cultivars at harvest (P=0.002) but not after 3-months in storage (P=0.241). A positive, linear relationship was observed between late season onion neck diameter and center rot incidence. All cultivars met the minimum soluble solids and pungency criteria for the marketing program. A novel vector system to engineer begomovirus resistance in transgenic plants by transcriptional and post-transcripti mechanisms thapsinum and M. phaseolina, respectively, and in contrast, line ‘Colman’ exhibited susceptibility to both pathogens. Lesion development over time was monitored in Colman. These results will enhance molecular and biochemical analyses of sweet sorghum responses to pathogens, and breeding stalk rot resistant sweet sorghum. Development and evaluation of germplasm lines resistant to Sclerotinia stem rot M. MCCAGHEY (1), M. McCaghey (1), J. Willbur (1), C. Grau (2), S. Chapman (2), B. Diers (3), C. Groves (2), A. Ranjan (2), M. Kabbage (2), D. Smith (1) Development and evaluation of germplasm lines resistant to Sclerotinia stem rot M. MCCAGHEY (1), M. McCaghey (1), J. Willbur (1), C. Grau (2), S. Chapman (2), B. Diers (3), C. Groves (2), A. Ranjan (2), M. Kabbage (2), D. Smith (1) Development and evaluation of germplasm lines resistant to Sclerotinia stem rot M. MCCAGHEY (1), M. McCaghey (1), J. Willbur (1), C. Grau (2), S. Chapman (2), B. Diers (3), C. Groves (2), A. Ranjan (2), M. Kabbage (2), D. Smith (1) (1) University of Wisconsin- Madison, Madison, WI, U.S.A.; (2) University of Wisconsin- Madison, U.S.A.; (3) University of Illinois- Urbana- Champaign U S A sconsin- Madison, Madison, WI, U.S.A.; (2) University of Wisconsin- Madison, U.S.A.; (3) University of Illinois- Urbana- ( ) 1) University of Wisconsin- Madison, Madison, WI, U.S.A.; (2) University of Wisconsin- Madison, U.S.A.; (3) University Champaign, U.S.A. Sclerotinia sclerotiorum, the causal agent for Sclerotinia stem rot (SSR), is a devastating fungal pathogen in soybean and reduced yield by 59 million bushels in 2009. Incomplete resistance in commercial cultivars can be improved through additional breeding efforts and understanding the genetic basis of resistance. SSR-resistant soybean germplasm was developed by crossing two sources of resistance, W04-1002 and AxN-1-55, with lines exhibiting resistance to other pathogens such as Heterodera glycines. After multiple screenings, 31 lines were selected for advanced SSR in field screens in 2014 and were down-selected to 16 lines with a high-level of multi-disease resistance in 2015. Germplasm lines 91-38, 51-23, and SSR51-70 exhibited SSR resistance with minimal lodging. Line 91-38 yielded 2605.5 kg/ha, 51-23 yielded 3156.2 kg/ha, and SSR51-70 yielded 2801.8 kg/ha. The highest yield achieved was 3748.8 kg/ha for 52-82B. While many of these experimental lines have excellent physiological resistance, yield and agronomic performance of these lines are not at the level of an elite commercial cultivar. A novel vector system to engineer begomovirus resistance in transgenic plants by transcriptional and post-transcripti mechanisms For example, 91-38 yielded over 1000 kg/ha less than the best line in the trial, however it exhibited one of the lowest disease severity rankings. Germplasm lines such as 91-38, 51-23, SSR51-70, or 52-82B could be improved through further breeding to generate high-yielding commercial soybean cultivars with a high level of physiological resistance to SSR. g y p p y g y g performance of these lines are not at the level of an elite commercial cultivar. For example, 91-38 yielded over 1000 kg/ha less than the best line in the trial, however it exhibited one of the lowest disease severity rankings. Germplasm lines such as 91-38, 51-23, SSR51-70, or 52-82B could be improved through further breeding to generate high-yielding commercial soybean cultivars with a high level of physiological resistance to SSR. Exploring the slow rusting potential of wheat genotypes against leaf and stripe rust of wheat M RAZA (1) M Kh (2) M Y (3) F N tt (4) M. RAZA (1), M. Khan (2), M. Yaseen (3), F. Nutter (4) (1) Department of Plant Pathology and Microbiology, Iowa State University of Science and Technology, Ames, IA, U.S.A.; (2) Department of Plant Pathology, University of Agriculture Faisalabad, Pakistan; (3) Department of Math & Stat, University of Agriculture Faisalabad, Faisalabad, Pakistan; (4) Department of Plant Pathogy and Microbiology, Iowa State University of Science and Technology, Ames, IA, U.S.A. The focus of current wheat research is to achieve durable rust resistance in Pakistan. We explored slow leaf and stripe rusting potential in 37 wheat genotypes by studying different slow rusting parameters, e.g., average coefficient of infection (ACI), latent period, pustule size, pustule density, infection S4.87 rate (IR), area under disease progress curve (AUDPC) and yield assessment (1000 grain weight and yield/ha). Multivariate statistical analysis techniques (Biplots and Heatmaps) were used to analyze and visualize the slow rusting parameters for identification of slow rusting genotypes. Genotypes including Lasani-2008, Chris, Crow, AS-2002, Punjab-2011, Frontana and FSD-85 demonstrated lower ACI, pustule density, IR, AUDPC, small pustule size, longer latent period, and higher 1000 grain weight and yield/ha against leaf rust. Genotypes encompassing Chris, AS-2002, Punjab-85, Yecora and Lasani-2008 revealed lower ACI, pustule density, IR, AUDPC, small pustule size, longer latent period, and higher 1000 grain weight and yield/ha against stripe rust. Combined analysis recognized genotypes including Uqab-2000, Yecora, Lasani-2008, Chris, Crow, Frontana and AS-2002 as slow leaf and stripe rusters. Hence, these slow rusters can be cultivated in fields for sustainable wheat production, and can be included in further breeding programs to produce durable rust resistant varieties. rate (IR), area under disease progress curve (AUDPC) and yield assessment (1000 grain weight and yield/ha). Multivariate statistical analysis techniques (Biplots and Heatmaps) were used to analyze and visualize the slow rusting parameters for identification of slow rusting genotypes. Genotypes including Lasani-2008, Chris, Crow, AS-2002, Punjab-2011, Frontana and FSD-85 demonstrated lower ACI, pustule density, IR, AUDPC, small pustule size, longer latent period, and higher 1000 grain weight and yield/ha against leaf rust. Genotypes encompassing Chris, AS-2002, Punjab-85, Yecora and Lasani-2008 revealed lower ACI, pustule density, IR, AUDPC, small pustule size, longer latent period, and higher 1000 grain weight and yield/ha against stripe rust. Combined analysis recognized genotypes including Uqab-2000, Yecora, Lasani-2008, Chris, Crow, Frontana and AS-2002 as slow leaf and stripe rusters. Exploring the slow rusting potential of wheat genotypes against leaf and stripe rust of wheat M RAZA (1) M Kh (2) M Y (3) F N tt (4) Hence, these slow rusters can be cultivated in fields for sustainable wheat production, and can be included in further breeding programs to produce durable rust resistant varieties. Resistance to Claviceps purpurea (Fr.) Tul. in the winter wheat lines “Robigus” and “Solstice” J. MENZIES (1), A. Gordon (2), D. O’Sullivan (3) Resistance to Claviceps purpurea (Fr.) Tul. in the winter wheat lines “Robigus” and “Solstice” J. MENZIES (1), A. Gordon (2), D. O’Sullivan (3) (1) Agriculture and Agri-Food Canada, Canada; (2) National Institute of Agricultural Botany, United Kingdom; (3) University of Reading, United Kingdom ( ), ( ), ( ) (1) Agriculture and Agri-Food Canada, Canada; (2) National Institute of Agricultural Botany, United Kingdom; (3) University of Reading, United Kingdom Ergot, caused by Claviceps purpurea, is an important disease of cereals which can result in extensive financial losses to producers because of the presence of sclerotia containing toxic alkaloids in the grain. The severity of ergot in Canada and parts of Europe has increased over the past 20 years, and the development of wheat lines with resistance is being explored. The objective of this work was to determine if the winter wheat varieties “Robigus” and “Solstice” possess physiologic resistance to C. purpurea. Two winter wheat lines (“Robigus” and “Solstice”), two spring wheat lines ((“Paragon” and “Cadillac”) and one durum wheat line (“Avonlea”) were inoculated with 5 Canadian and 4 UK isolates of C. purpurea. Nine spikes per wheat line were needle inoculated prior to anthesis for each pathogen isolate using a conidial suspension. Each spike was assessed for honeydew production 14 days after inoculation using a scale of 1 to 4; 1 = no honeydew to 4 = large drops running down the spike. The weight and number of sclerotia per spike were determined at plant maturity. “Robigus” had significantly lower honeydew production than “Solstice”, which had lower production than the other three lines. “Robigus” and “Solstice” also had significantly lower sclerotial weight and number per spike than the other three lines. “Robigus” and “Solstice” showed resistance to infection by C. purpurea compared to the other three wheat lines. Monitoring of Leaf rust in seventeen Egyptian wheat cultivars during 2013/2014 and 2014/2015 seasons A. ABDELRHIM (1), M. Abou-Zeid (2), H. Abd-Alla (3), C. Cowger (4), A. El-Bana (3), M. Exploring the slow rusting potential of wheat genotypes against leaf and stripe rust of wheat M RAZA (1) M Kh (2) M Y (3) F N tt (4) Boykin (4), B. Wood (1) (1) USDA/ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A.; (2) USDA/ARS Pecan Breeding and Genetics, Somerville, TX, U.S.A.; (3) University of Georgia, Tifton, GA, U.S.A.; (4) USDA-ARS, Stoneville, MS, U.S.A. (1) USDA/ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A.; (2) USDA/ARS Pecan Breeding and Genetics, Somerville, TX, U.S.A.; (3) University of Georgia, Tifton, GA, U.S.A.; (4) USDA-ARS, Stoneville, MS, U.S.A. Scab (caused by Fusicladium effusum) is the most economically destructive disease of pecan in the Southeast US. Epidemics are favored by rainfall and high humidity. A provenance collection of ~950 pecan trees from 19 locations representing the native range of the species is located in Byron, Georgia, and was assessed for pecan scab severity in 1998, 2013 and 2014. Foliar scab severity was estimated visually. There were consistent significant differences among the 19 provenances (F = 5.6 to 62.5, P<0.0001), with those from wetter locations (generally north of Texas) with the greatest proportion of scab resistant trees, while those provenances from the drier southern areas (Texas and Mexico) tended to be the most susceptible. However, within each provenance there was a range in susceptibility of trees. An association of provenance susceptibility with precipitation at the provenance source location bore out the relationship (r=-0.625 to -0.823 (P<0.0001 to 0.004)). Estimates of heritability were not entirely consistent among years, but different methods were used to assess scab severity in 1998 (a 1-5 category scale) compared to 2013 and 2014 (the percent ratio scale). Despite using different methods, there was generally good agreement among years in regard to severity of disease on individual trees. In conclusion, trees from more northern populations (in areas with greater annual rainfall) are most likely to provide valuable and diverse sources of resistance to scab. Exploring the slow rusting potential of wheat genotypes against leaf and stripe rust of wheat M RAZA (1) M Kh (2) M Y (3) F N tt (4) Ismail (3) (1) North Carolina State University, Raliegh, NC, U.S.A.; (2) Agriculture research center, Giza, Egypt; (3) Minia University, El-Minya, Egypt; (4) North Carolina State University, Raleigh, NC, U.S.A. g gyp g A. ABDELRHIM (1), M. Abou-Zeid (2), H. Abd-Alla (3), C. Cowger (4), A. El-Bana (3), M. Ismail (3) (1) North Carolina State University, Raliegh, NC, U.S.A.; (2) Agriculture research center, Giza, Egypt; (3) Minia University, El-Minya, Egypt; (4) North Carolina State University, Raleigh, NC, U.S.A. Wheat cultivars’ response to leaf rust caused by Puccinia triticina Eriks. vary between seasons; some cultivars lose resistance over time due to factors such as appearance of new pathogen races that have new virulence genes which overcome Lr-genes. This study monitored leaf rust development for adult plant stage of 17 Egyptian wheat cultivars, expressed in terms of infection type, final rust severity, area under disease progress curve and rate of disease increase (r- value) during 2013/2014 and 2014/2015 growing season in Sids research stations, Egypt. According to the results, only two varieties (Sakha94 and Misr1) exhibited adult plant resistance, three varieties (Gemmiza9, and 10, and Misr2) were moderately susceptible, and twelve varieties (Sakha93, Giza168, and 171, Gemmiza7, and 11, Sids1, 12, and 13, Shandwile1, and Bani-Swif1, 5, and 6) were susceptible during 2013-2014 and 2014-2015 growing seasons. However, nine varieties (Sakha94, Misr1, and 2, Giza 168, and 171, Gemmiza 9, and 10, Sids 12 and 13) recorded low AUDPC up to 310 and the rest varied between 320:1120. Only five varieties (Sakha94, Misr1, Giza 168, and 171, and Gemmiza 9) recorded r-value up to 0.092, while the rest ranged from 0.11 to 0.195 in both seasons. These data may help researchers and breeders by shedding light on the response of some Egyptian varieties to leaf rust in the last two growing seasons, and helping track the virulence genes in P. triticina during the same seasons. Scab susceptibility of a provenance collection of pecan Scab susceptibility of a provenance collection of pecan C. BOCK (1), L. Grauke (2), P. Conner (3), S. Burrell (1), M. Hotchkiss (1), D. Boykin (4), B. Wood (1) (1) USDA/ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A.; (2) USDA/ARS Pecan Breeding and Genetics, Somerville, TX, U.S.A.; (3) University of Georgia, Tifton, GA, U.S.A.; (4) USDA-ARS, Stoneville, MS, U.S.A. C. BOCK (1), L. Grauke (2), P. Conner (3), S. Burrell (1), M. Hotchkiss (1), D. Development of an in vitro bioassay to screen perennial Phlox germplasm for susceptibility to Golovinomyces cichorac cichoracearum) Development of an in vitro bioassay to screen perennial Phlox germplasm for susceptibility to Golovinomyces cichoracearum (Erysiphe cichoracearum) C. FARINAS (1), P. Jourdan (1), F. Peduto Hand (1) (1) The Ohio State University, U.S.A. Phlox is a genus of perennial and annual plants with colorful, long-lasting flowers, and a common inhabitant of gardens worldwide. In humid and warm areas, its cultivation is severely limited by powdery mildew. Published studies on resistance of Phlox to powdery mildew have mainly focused on the species subulata, paniculata and drummondii. However, the genus includes many more species, some of which could be valuable from both an ornamental and resistance standpoint. This project aims to develop a reliable laboratory bioassay to conduct in vitro screenings of Phlox germplasm for susceptibility to powdery mildew. The project is in collaboration with the OSU Ornamental Plant Germplasm Center, which allows access to more than 65 species of Phlox. For this purpose, detached leaves of healthy Phlox were disinfected and plated on water-agar medium. Variations of the host species, media type, disinfection process, and inoculation technique were evaluated. Disinfected leaf tissues remained in good status and contaminant- free for up to one month. Infection and growth of the pathogen on the host required specific conditions of light, temperature and relative humidity. To the best of our knowledge, this study represents the first attempt to develop a laboratory bioassay to screen Phlox resistance to powdery mildew. This assay has the potential to represent a useful tool for screening resistant lines and for phytopathological studies on the biology of the pathogen. Virulence of Calonectria pseudonaviculata and C. henricotiae isolates and screening for resistant boxwood cultivars in detached leaf assays H. Guo (1), J. Crouch (2), M. Pooler (3) (1) USDA-ARS and Rutgers University, U.S.A.; (2) USDA-ARS Systematic Mycology and Microbiology Laboratory, U.S.A.; (3) USDA-ARS US National Arboretum, U.S.A. V u e ce o Calonect ia pseudonaviculata a d C. hen icotiae so ates a d sc ee g o es sta t bo wood cu t va s detac ed ea assays H. Guo (1), J. Crouch (2), M. Pooler (3) (1) USDA-ARS and Rutgers University, U.S.A.; (2) USDA-ARS Systematic Mycology and Microbiology Laboratory, U.S.A.; (3) USDA-ARS US National Arboretum U S A , ( ) gers University, U.S.A.; (2) USDA-ARS Systematic Mycology and Microbiology Laboratory, U.S.A.; (3) USDA-ARS US Boxwood blight caused by the two Calonectria species, C. Effectiveness of a seed plate assay evaluating charcoal rot resistance in soybean M. DA SILVA (1), P. Chen (1), J. Rupe (1) (1) University of Arkansas, U.S.A. Effectiveness of a seed plate assay evaluating charcoal rot resistance in soybean M. DA SILVA (1), P. Chen (1), J. Rupe (1) (1) University of Arkansas, U.S.A. Charcoal rot (Macrophomina phaseolina) of soybean (Glycine max (L.) is a disease of economic significance in the United States. While there is moderate host resistance in soybean cultivars, identifying and quantifying that resistance are difficult. Current assay methods, such as cut-stem inoculation and colony-forming unit index (CFUI), can be time consuming, and data may vary between tests. The objective of this research was to compare results of a seed plate assay to published results of the cut-stem and CFUI assays. Ten surface disinfested seeds of eight differential soybean genotypes, including LS94-3207, LS98-0358 and Pharaoh, susceptible and DT97-4290, DT98-7553, DT99-16864, DT99-17554 and DT99-17483 as resistant, were placed on water agar plates that had been inoculated with an isolate of M. phaseolina five days before. Plates were incubated at room temperature in the dark for 7 days and the number of seeds that germinated were determined. Noninoculated plates served as controls. In general, germination results from the plate assay were in agreement with the cut-stem and CFUI assays. Germination of resistance lines range from 58% to 84% and susceptible lines from 46% to 54%. Addition data will be presented later. Screening soybean germplasm for resistance to multiple Fusarium species P. OKELLO (1), L. Hyronimus (1), A. Weber (1), A. Singh (2), F. Mathew (1) (1) South Dakota State University, U.S.A.; (2) Iowa State University, U.S.A. Fusarium root rot caused by multiple Fusarium spp. continue to cause increased soybean [Glycine max L.] yield losses in the United States. Commercial soybean varieties with complete resistance to Fusarium spp. are not available, thus necessitating the need for continued search for sources of resistance. The objective of this study was to identify screen soybean germplasm for resistance to four Fusarium species (F. proliferatum, F. graminearum, F. sporotrichioides, and F. subglutinans), which were identified as aggressive Fusarium spp. based on a preliminary study on soybean in South Dakota. To evaluate soybean germplasm for resistance to the four Fusarium spp., 44 genotypes belonging to maturity groups I from 10 different countries were S4.88 screened in replicated tests using the inoculum-layer method. Disease severity was evaluated at 14 days after inoculation as a percentage of lesion produced on soybean roots by the causal pathogen. Susceptibility of Buxus accessions to the boxwood blight pathogen Calonectria pseudonaviculata J. LAMONDIA (1) (1) The Connecticut Agricultural Experiment Station, U.S.A. ( ) ) The Connecticut Agricultural Experiment Station, U.S.A. Forty Buxus accessions from the National Boxwood Collection of the U.S. National Arboretum provided as potted plants were evaluated for susceptibility to boxwood blight caused by Calonectria pseudonaviculata (Cps). Two experiments were conducted with 3 to 5 replicate plants each. Plants were inoculated with 1.5 × 105 Cps conidia in 2 ml coarse spray per plant and grown under high humidity with overhead irrigation. Leaf and stem lesions were recorded 3 or 4 wks after inoculation. B. harlandii (1 accession), B. sinica (3), B. microphylla (7) and B. bodinieri (1) had fewer lesions per plant than B. microphylla × sempervirens (6), which had fewer lesions than B. sempervirens (20 accessions). B. wallichiana (1 accession) was the most susceptible species (>160 lesions). Variation within species was also observed. B. sinica var. aemulans (accession 60705H), B. sempervirens (36365J) and B. harlandii (18834H) were the least susceptible with < 10 lesions per plant. B. sempervirens ‘Scupi’ (9548H), B. microphylla ‘Compacta’ (4899CH), B. sempervirens ‘Arborescens’ (57953H), B. sinica var. insularis ‘Pincushion’ (51898H) and B. microphylla var. japonica ‘Jim Stauffer’ (72213H) each had < 20 lesions. These data differ from previous detached leaf and unrooted cutting assays. Plant size may affect inoculation and disease, normalizing did not result in significant differences in susceptibility ranking. These results may aid in the development of boxwood resistance to Cps. Evaluating nine boxwood cultivars for susceptibility to Calonectria pseudonaviculata and C. henricotiae N. SHISHKOFF (1) (1) Foreign Disease Weed Science Research Unit/ARS/USDA U S A Boxwood blight is caused by two species of Calonectria: C. pseudonaviculata (Cps) and C. henricotiae (Che). In North America, only Cps is known to occur, but the 2nd species is of concern since there is evidence that it differs in its temperature optimum, pesticide sensitivity and tolerance to sanitizers. US boxwood cultivars are currently being evaluated for tolerance to Cps and it would be useful to know if their reaction to Che is similar. Based on a previous assessment of susceptibility of 42 boxwood cultivars to Cps, nine were selected on a spectrum from highly susceptible to relatively tolerant. Cultivar sets were inoculated with spore suspensions (mixtures of 4 representative isolates) of each species and the experiment was repeated three times. While results showed some differences in cultivar behavior from trial to trial, there were cultivars that were consistently susceptible (‘Dee Runk’, ‘Vardar Valley’) or consistently tolerant (‘SB17’, ‘Little Missy’) and the interaction ‘pathogen type‘’cultivar’ was not significant. This suggests that breeding for tolerance for one species will confer tolerance for the other. Development of an in vitro bioassay to screen perennial Phlox germplasm for susceptibility to Golovinomyces cichorac cichoracearum) pseudonaviculata and C. henricotiae, is a recently emerged threat to the annual $1.3 million boxwood industry. While fungicidal control can be effective, breeding resistant or tolerant cultivars is the preferred long-term strategy to control the disease in the landscape. Developing an effective assay to quickly screen potentially thousands of boxwood genotypes is an essential first step in such a breeding program. Therefore, this study was conducted to evaluate the virulence of different Calonectria isolates on a susceptible boxwood host. The virulence of 24 isolates was determined by inoculating detached leaves of Buxus sempervirens ‘Suffruticosa’ using two different inoculation techniques. All 24 isolates were virulent on ‘Suffruticosa’, but the virulence varied among isolates. The isolate NCBB-1 was the most virulent, and was used to compare relative resistance among eight diverse boxwood taxa. Preliminary results indicate that two boxwood taxa, B. harlandii and B. sinica var. insularis ‘Nana’, are the most tolerant to this isolate in detached leaf assays. Effectiveness of a seed plate assay evaluating charcoal rot resistance in soybean M. DA SILVA (1), P. Chen (1), J. Rupe (1) (1) University of Arkansas, U.S.A. Preliminary results suggests one genotype (PI 603371) had significantly lower disease severity (P ≤ 0.05) than the susceptible check for all the four Fusarium species. This genotype will be useful as a parental material to develop commercial soybean cultivars with resistance to Fusarium species. screened in replicated tests using the inoculum-layer method. Disease severity was evaluated at 14 days after inoculation as a percentage of lesion produced on soybean roots by the causal pathogen. Preliminary results suggests one genotype (PI 603371) had significantly lower disease severity (P ≤ 0.05) than the susceptible check for all the four Fusarium species. This genotype will be useful as a parental material to develop commercial soybean cultivars with resistance to Fusarium species. Susceptibility of Buxus accessions to the boxwood blight pathogen Calonectria pseudonaviculata J. LAMONDIA (1) Susceptibility of Buxus accessions to the boxwood blight pathogen Calonectria pseudonaviculata J. LAMONDIA (1) (1) Th C i A i l l E i S i U S A Susceptibility of Buxus accessions to the boxwood blight pathogen Calonectria pseudonaviculata J. LAMONDIA (1) (1) The Connecticut Agricultural Experiment Station, U.S.A. Identification of Brassica napus plant introductions with resistance to pathogenicity group 4 of Leptosphaeria maculans S. MANSOURIPOUR (1), L. del Rio Mendoza (1) (1) North Dakota State University, U.S.A. Reliable and rapid stem cutting-based screening technique to evaluate hydrangea cultivars for resistance to Phytophthora nicotianae is needed toward disease management. Hydrangea cvs. Queen of Hearts, Ruby Slippers and Munchkin stem cuttings were screened in a laboratory procedure by point inoculation with 5-day-old culture, #1 size (outside diameter 5 mm) plugs of P. nicotianae. Each stem cutting was evaluated for lesion area and lesion severity after 3, 5 and 7 days of incubation at 25°C with 16-hr-light/8-hr- dark cycle. The experiment was repeated twice. After comparing the lesion development on three hydrangea cultivars, the results showed that Queen of Hearts is most susceptible to P. nicotianae than other cultivars and five days observation is the optimal time for stem cutting-screening technique. This study will facilitate the ongoing large-scale screening for Phytophthora root rot resistance among hydrangea cultivars very easily and it is clear that described stem cutting-inoculation technique has the potential to play a significant role for simple, efficient and reproducible screening. Cavity spot of carrots: Progress in screening for resistance M. MCDONALD (1), M. McDonald (2) (1) University of Guelph, Canada; (2) University of Guelph, Canada Cavity spot of carrot is caused by several Pythium species, especially Pythium violae and P. sulcatum. Field trials were conducted from 2013 to 2015 to screen 60 carrot lines from the USDA breeding program at the University of Wisconsin lines for differences in susceptibility to cavity spot. Cultivars Atomic Red, Cellobunch and Envy were included as commercial checks. Carrots were seeded into ‘muck’ soil (pH 5.7- 6.5, organic matter 65- 78%) where the cavity spot pathogens occur naturally. Carrots were direct seeded (≈75 seeds/m) in early June, onto raised beds, 66 cm apart and 6 m in length. Fifty carrots per replicate were harvested in late October, placed in cold storage for 6 – 8 weeks and washed and assessed for cavity spot incidence and disease severity (DSI). Cavity spot incidence and severity were very high in 2013 (97% and 69 DSI) and 2014 (96%, 54 DSI) and lower in 2015 (50%, 26 DSI). A wide range of susceptibility to cavity spot was found each year. Purple lines 6139B, 6244B, 7262B and 3497B were highly resistant (1- 8%, 1-7 DSI). Two orange carrot lines, in the trials in 2014 and 2015, had consistently low disease. Identification of Brassica napus plant introductions with resistance to pathogenicity group 4 of Leptosphaeria maculans S. MANSOURIPOUR (1), L. del Rio Mendoza (1) (1) North Dakota State University, U.S.A. These resistant cultivars may be used to provide a higher degree of protection against infection from Pythium species in soybean seeds and seedlings. Disease susceptibility screening for cold-climate wine grape cultivars D. JONES (formerly Schreiner) (1), P. McManus (1) (1) University of Wisconsin, Madison, Dept. of Plant Pathology, U.S.A. The wine grape industry in the Upper Midwest has expanded rapidly in the past 15 years. Inconsistent reports on disease susceptibility in cold-climate wine grape cultivars may be leading to overspraying and underutilization of host resistance in the region. Inconsistencies in reports may stem from a lack of study on adult plants in a randomized, replicated, and fungicide free vineyard environment. Two vineyards containing eight cultivars were left unsprayed by fungicides in 2015. Severity of downy mildew, powdery mildew, and black rot was measured from bud break until 2 weeks post-harvest. There was a significant difference among cultivars for all three diseases at both sites. A second assay was conducted using potted grape vines as “trap plants” to capture inoculum present in the field and gain further information on disease susceptibility. Using trap plants and artificial humidification did not generate results consistent with the disease severity and progress observed in the field during the growing season. These preliminary data suggest that selection of certain cultivars has the potential to reduce sprays needed to manage diseases during the growing season in the Upper Midwest. Results further indicate differences exist between foliar and fruit susceptibility for all three diseases, a distinction that is not made in current rating systems. This work will continue in 2016 to gain additional susceptibility data and observe differences between growing seasons. Evaluation of stem inoculation technique for assessing resistance to Phytophthora nicotianae in hydrangea cultivars M. KABIR (1), T. Simmons (1), P. Liyanapathiranage (1), C. Curry (1), L. Alexander (2), F. Baysal-Gurel (1) (1) Tennessee State University, Nashville, TN, U.S.A.; (2) USDA-ARS, U.S. National Arboretum, McMinnville, TN, U.S.A. Evaluation of stem inoculation technique for assessing resistance to Phytophthora nicotianae in hydrangea cultivars M. KABIR (1), T. Simmons (1), P. Liyanapathiranage (1), C. Curry (1), L. Alexander (2), F. Baysal-Gurel (1) 1) Tennessee State University, Nashville, TN, U.S.A.; (2) USDA-ARS, U.S. National Arboretum, McMinnville, TN, U.S.A Almost all hydrangeas, especially container-grown oakleaf hydrangeas (Hydrangea quercifolia) are more susceptible to Phytophthora root rot disease. Most economic and trading hydrangea cultivars are propagated as rooted cuttings. Identification of Brassica napus plant introductions with resistance to pathogenicity group 4 of Leptosphaeria maculans S. MANSOURIPOUR (1), L. del Rio Mendoza (1) (1) North Dakota State University, U.S.A. Identification of Brassica napus plant introductions with resistance to pathogenicity group 4 of Leptosphaeria maculans S. MANSOURIPOUR (1), L. del Rio Mendoza (1) (1) North Dakota State University, U.S.A. Blackleg caused by Leptosphaeria maculans (Desmaz) Ces. & de Not is a serious disease of canola (Brassica napus L.) worldwide. In North Dakota, L. maculans isolates that overcome resistance genes Rlm1, Rlm2, and Rlm3 are the most prevalent and have been classified in pathogenicity group (PG) 4 using a set of three differentials. The objective of this study was to identify sources of resistance against PG-4. Replicated greenhouse trials using mixtures of five PG-4 isolates were used to evaluate the reaction of B. napus plant introductions (PI) at the seedling stage. Twenty one PI materials identified as resistant were evaluated in field trials in 2014 and 2015 using a randomized complete block design with four replications. Cultivar “Westar” and two commercial canola hybrids were used as controls. Seedlings were inoculated repeatedly with a mixture of spores from the same five isolates used in the greenhouse and supplemented with naturally infested residues. Disease severity was estimated at harvest as percentage of internal stem crown tissue discoloration. Five PIs were considered highly resistant and behaved consistently better (P < 0.05) than the hybrids in both field trials with an average disease severity < 4% compared to 25% of the hybrids. Crosses between these PIs and canola breeding lines have been made to transfer the resistance into advanced germplasm. In addition, efforts to produce doubled haploid mapping populations from these sources are under way. S4.89 Evaluating chemical fingerprinting as a tool to rapidly screen hybrid chestnut for resistance to pathogens A. CONRAD (1), J. Westbrook (2), T. Zhebentyayeva (3), L. Rodriguez-Saona (4), P. Bonello (4), C. Nelson (5), A. Abbott (1) (1) University of Kentucky, Lexington, KY, U.S.A.; (2) The American Chestnut Foundation, Asheville, NC, U.S.A.; (3) Clemson University, Clemson, SC, U.S.A.; (4) The Ohio State University, Columbus, OH, U.S.A.; (5) USDA Forest Service, Lexington, KY, U.S.A. Chestnut blight, caused by the introduced pathogen Cryphonectria parasitica, continues to plague American chestnut (Castanea dentata) in the eastern US. Restoration is focused on breeding hybrids that morphologically resemble American chestnut but contain blight resistance from Chinese chestnut (C. mollissima). Compounding the problem for restoration efforts, Phytophthora cinnamomi, an agent of root rot, has emerged as a major pathogen in chestnut’s southern range. Identification of Brassica napus plant introductions with resistance to pathogenicity group 4 of Leptosphaeria maculans S. MANSOURIPOUR (1), L. del Rio Mendoza (1) (1) North Dakota State University, U.S.A. Time and resource intensive inoculation-based screening methods are used currently to identify chestnuts resistant to these pathogens. Therefore, we evaluated if chemical fingerprinting (CF), a potentially less resource-intensive tool for phenotyping resistant/susceptible trees, can be used to screen chestnut for disease resistance. A Varian 3100 Fourier-transform infrared (FT-IR) spectrometer equipped with an attenuated total reflectance accessory was used to fingerprint methanol extracts from 0-1 year old chestnut stem tissue that differed in susceptibility to chestnut blight. Using Soft Independent Modeling of Class Analogy, chemical differences among disease rating classes were identified and used to develop class discriminating models. Early results suggest that CF may be a useful tool for screening seedlings for blight resistance; however, we are currently testing additional samples to refine, optimize, and validate models for both diseases. results suggest that CF may be a useful tool for screening seedlings for blight resistance; however, we are currently testing optimize, and validate models for both diseases. Screening soybean germplasm for resistance towards Pythium species K. SCOTT (1), M. Eyre (1), A. Dorrance (1) (1) The Ohio State University, U.S.A. Pythium species are a major cause of soybean seedling root rot and seed rot in Ohio. One method of management is the use of resistant cultivars. Previous studies identified a “differential” resistance response to Pythium ultimum var. ultimum and P. ultimum var. sporangiiferum among soybean cultivars. The purpose of this experiment was to quantify the resistance level of several germplasm lines and to identify any potential “differential” effect. To do this, several soybean germplasm lines were evaluated for resistance to Pythium isolates collected from fields in Ohio using a seed plate assay. Seed plate assay results indicated that Conrad, Dennison, Lorain, Sloan, and Kottman all had varying levels of resistance towards 13 species of Pythium; more importantly, there was a highly significant (P<0.0001) cultivar x Pythium spp. interaction. All cultivars were very susceptible to P. ultimum var. ultimum, P. ultimum var. sporangiiferum, and P. sylvaticum. When inoculated with P. conidiophorum, P. attrantheridium, and P. dissotocum, Conrad, Dennison, and Lorain had a lower degree of seed rot than either Sloan or Kottman. These resistant cultivars may be used to provide a higher degree of protection against infection from Pythium species in soybean seeds and seedlings. dissotocum, Conrad, Dennison, and Lorain had a lower degree of seed rot than either Sloan or Kottman. Identification of Brassica napus plant introductions with resistance to pathogenicity group 4 of Leptosphaeria maculans S. MANSOURIPOUR (1), L. del Rio Mendoza (1) (1) North Dakota State University, U.S.A. Line 1137B had 8% cavity spot in both years, with DSI of 7 in 2014 and 4 in 2015. Line 5367 B had 14 and 21% cavity spot in 2014 and 2015, respectively, with a DSI of 8 and 7. Field screening has identified breeding lines with promising levels of resistance to cavity spot. Resistance in pea to the Bean leaf roll virus L. PORTER (1) (1) USDA ARS, U.S.A. Nelson (5) (1) Texas A&M University - Kingsville/Texas A&M AgriLife Extension, Kingsville, TX, U.S.A.; (2) Texas A&M AgriLife Extension Service, Brownfield, TX, U.S.A.; (3) Texas A&M University-Kingsville Citrus Center, Weslaco, TX, U.S.A.; (4) Texas A&M AgriLife Extension Service, Lubbock, TX, U.S.A.; (5) Texas A&M University-Kingsville, Kingsville, TX, U.S.A. Mycotoxins are secondary fungal metabolites that have adverse health effects on animals and humans. Lepidopteran damage to corn is suspected to facilitate fungal colonization and infection to kernels and to increase the risk of mycotoxin production and accumulation in corn. This study was initiated to evaluate the application of AF-36 and Afla-Guard at two growth stages in reducing mycotoxin levels in corn. Treatments included an untreated control, insect control, AF-36 and Afla-Guard applied at the V8 or VT growth stage, and Afla-Guard applied at the V8 or VT growth stage with insect control. The application of AF-36 or Afla-Guard applied at the V8 or VT growth stage did not significantly reduce the concentration of either aflatoxin or fumonisin. In 2013, the insect control significantly reduced the fumonisin concentration, and in 2014 application of insecticide alone or with Afla- Guard applied at the V8 or VT growth stage reduced the concentration of fumonisin. Aflatoxin levels were not significantly reduced by the application of AF-36 or Afla-Guard at either application time or by the insecticide applications. Fumonisin concentrations in both years showed a positive relationship with the amount of ear damage caused by Helicoverpa zea and Spodoptera frugiperda. Aflatoxin concentrations in 2013 were positively correlated to the amount of ear damage, but no such correlation was observed between aflatoxin and insect damage in 2014. Deciphering soil and plant microbiomes associated with suppression of soybean diseases H. PFAFFE (1), S. Marzano (1), L. Domier (1), A. Davis (1), D. Eastburn (1) 1) University of Illinois, U.S.A. General disease suppression of soilborne pathogens can add to the toolbox of strategies for disease management. This study was undertaken to identify the microbiome indicators for soil suppressiveness to sudden death syndrome (SDS) of soybeans. Five cover crop treatments (rye, vetch, mustard, rye+vetch, fallow) and two tillage methods (chisel plow, ridge till) were compared. Each cover crop plot was divided to receive both tillage treatments, and there were four replications of this experiment. Soybean plants were evaluated for naturally occurring foliar and root diseases at the R6-R7 growth stage. Resistance in pea to the Bean leaf roll virus L. PORTER (1) (1) USDA ARS, U.S.A. Resistance in pea to the Bean leaf roll virus L. PORTER (1) (1) USDA ARS, U.S.A. Bean leaf roll virus (BLRV) is a major luteovirus impacting dry pea production on an annual basis in Idaho and Washington. Symptomatic plants are often chlorotic, stunted and distorted. The virus is transmitted by aphids in a persistent manner and cannot be mechanically transmitted. Host resistance is an important economic and environmentally favorable means to manage this pathogen. Pea aphids were used to phenotype the genetic resistance of 36 pea genotypes, mostly consisting of dry pea cultivars, under controlled greenhouse conditions. Two pea genotypes, Journey and Stirling, were determined to have complete resistance to BLRV. Journey is a commercial forage green dry pea and Stirling is a formerly grown commercial green dry pea. Three commercial pea cultivars, Majoret, Granger and Spector were determined to be highly susceptible to BLRV with a 100% incidence of infection across three tests. Mapping populations will be established between the resistant and susceptible pea genotypes to identify molecular markers that can be used in marker-assisted selection of pea germplasm for genetic resistance to BLRV. The SimpleSynteny Server: Genome Comparison Simplified D. VELTRI (1), M. Malapi-Wight (2), J. Crouch (1) (1) USDA-ARS, U.S.A.; (2) USDA-APHIS, U.S.A. Visualizing syntenic relationships amongst gene clusters is a common task for studying organismal evolution. Here we present SimpleSynteny, a new comparative genomics web server which includes a number of helpful tools for the analysis of microsynteny across multiple genome assemblies. Requiring only FASTA files for genomes and gene targets of interest as input, SimpleSynteny’s easy interface helps the user explore and customize individual genomes before combining them into a final high-resolution figure. The approach gives users the freedom to incorporate additional knowledge when editing contigs/scaffolds after computational predictions are made. A range of image formats and customization options are also provided to give additional control when saving results. In this presentation, we provide a quick overview of the tools provided and generate example figures from fungal genomes via a live demonstration of the site. SimpleSynteny stands to be a powerful new tool in a researcher’s toolbox of comparative genomics software. The server is free to use and is currently available at: http://www.simplesynteny.com. Reducing Mycotoxin Levels in Corn via Lepidopteran Insect Control and Application of Atoxigenic Fungi G. SCHUSTER (1), T. Mays (2), M. Sétamou (3), P. Porter (4), S. Screening of Wild Hop (Humulus lupulus) Germplasm for Identification of Resistance to Pseudoperonospora humuli J. HAVILL (1), A. Orshinsky (1), D. Carter (1), S. Poulose (1) (1) University of Minnesota, U.S.A. We identified two geographically distinct locations in Minnesota containing previously unreported germplasm possessing high levels of foliar resistance that may be useful for breeding locally-adapted varieties. Work is currently underway to characterize the morphological characters and chemical attributes of the hop inflorescences of selected resistant wild plants. Barley yellow dwarf incidence and bird cherry-oat aphid preference in four wheat varieties in Idaho M. Rashidi (1), J. Marshall (1), N. Bosque-Perez (2), A. Rashed (1) (1) University of Idaho, Aberdeen Research and Extension Center, U.S.A.; (2) University of Idaho, Department of Plant, Soil and Entomological Sciences, U.S.A. Barley/Cereal yellow dwarf viruses (B/CYDV) are aphid-borne pathogens that are transmitted by several species of cereal aphids, chief among them the bird cherry-oat aphid (BCOA), Rhopalosiphum padi (L.). This species has also been linked to the recent consecutive outbreaks of BYDV-PAV, between 2013 and 2015, in southern Idaho. The present study was conducted to evaluate the relative susceptibility of four commonly planted winter wheat varieties in Idaho, namely, SY Ovation, Brundage, Stephens, and WB-Junction, to BYDV and its BCOA vector. In choice experiments, viruliferous aphid preference for the four varieties was assessed by counting the number of aphids per plant 24 hours after infestation. BYDV titer within seedlings was quantified using qPCR, three weeks after inoculation. During a two-year field-cage study, BYDV symptom severity, aphid pressure, and root and shoot biomass were assessed for the four varieties. Root/shoot biomass data are currently being analyzed. BYDV in experimental cages was confirmed using ELISA. Although Stephens and SY Ovation were preferred relatively less frequently than WB-Junction and Brundage by BCOA, no significant differences were detected in aphid preference among the four varieties. Likewise, BYDV incidence in field cages was not statistically different among the four varieties, however SY Ovation had the lowest BYDV incidence, an observation consistent across both years of the study. Screening of Wild Hop (Humulus lupulus) Germplasm for Identification of Resistance to Pseudoperonospora humuli J. HAVILL (1), A. Orshinsky (1), D. Carter (1), S. Poulose (1) (1) University of Minnesota, U.S.A. Screening of Wild Hop (Humulus lupulus) Germplasm for Identification of Resistance to Pseudoperonospora humuli J. HAVILL (1), A. Orshinsky (1), D. Carter (1), S. Poulose (1) (1) University of Minnesota, U.S.A. Pseudoperonospora humuli, the causal organism of hop downy mildew, is considered the most economically-damaging pathogen to hop producti Host resistance, in addition to intensive cultural management practices and fungicide use, is the primary control measure. Wild hops (Humulus sp Pseudoperonospora humuli, the causal organism of hop downy mildew, is considered the most economically-damaging pathogen to hop production. Host resistance, in addition to intensive cultural management practices and fungicide use, is the primary control measure. Wild hops (Humulus spp.) are S4.90 distributed across the North America and Eurasia. They may provide a resource for novel resistance genes and germplasm diversity for local hop breeding programs. Sixteen sites in Minnesota with wild populations of hops were identified. At 14 of these sites individual rhizomes were recovered from 1 – 27 unique individuals. One-hundred six wild and cultivated hop genotypes originating from the USA, England, Japan, and Kazakhstan were screened for foliar resistance to downy mildew (Pseudoperonospora humuli) using a detached-leaf assay. Each genotype was tested with three replicates. In total, 33 genotypes were as resistant as the current commercial standard (cv. ‘Teamaker’) which included five other cultivars, five Japanese hop (H. japonicus) genotypes, two experimental breeding lines, and 21 wild H. lupulus genotypes. We identified two geographically distinct locations in Minnesota containing previously unreported germplasm possessing high levels of foliar resistance that may be useful for breeding locally-adapted varieties. Work is currently underway to characterize the morphological characters and chemical attributes of the hop inflorescences of selected resistant wild plants. distributed across the North America and Eurasia. They may provide a resource for novel resistance genes and germplasm diversity for local hop breeding programs. Sixteen sites in Minnesota with wild populations of hops were identified. At 14 of these sites individual rhizomes were recovered from 1 – 27 unique individuals. One-hundred six wild and cultivated hop genotypes originating from the USA, England, Japan, and Kazakhstan were screened for foliar resistance to downy mildew (Pseudoperonospora humuli) using a detached-leaf assay. Each genotype was tested with three replicates. In total, 33 genotypes were as resistant as the current commercial standard (cv. ‘Teamaker’) which included five other cultivars, five Japanese hop (H. japonicus) genotypes, two experimental breeding lines, and 21 wild H. lupulus genotypes. Effect of root pathogens Bipolaris sorokiniana and Fusarium graminearum on germination and seedling blight in wheat in South Dakota S. ALI (1), N. Kaur (1), S. Sehgal (1), K. Glover (1), S. Kumar (1) (1) South Dakota State University, Brookings, SD, U.S.A. Effect of root pathogens Bipolaris sorokiniana and Fusarium graminearum on germination and seedling blight in wheat in South Dakota S. ALI (1), N. Kaur (1), S. Sehgal (1), K. Glover (1), S. Kumar (1) (1) South Dakota State University, Brookings, SD, U.S.A. Bipolaris sorokiniana (Bs) and Fusarium graminearum (Fg) are important wheat root pathogens that effect seed germination and seedlings establishment thus ultimately impact the crop productivity. The objective of this study was to explore the effect of Bs and Fg infested seed on germination and seedlings establishment (blight) of 7 wheat cultivars, Advance, Briggs, Forefront, Oxen, Russ, Prevail, and SD4215 which were planted at Volga, SD in 2015. The treatments included, uninfested + untreated, uninfested + treated with fungicide, infested (Bs) + treated, infested (Bs) + untreated, infested (Fg) + treated, infested (Fg) + untreated. Seed germination and seedling blight data was recorded after the germination for 3 consecutive weeks. Wheat cultivars varied in seed germination and seedling blight to both pathogens; low seed germination (ranging from 35-71%) and high seedling blight (30- 65%) was observed in Fg infested seed, whereas, in Bs infested seed the germination ranged 58-100% and seedling blight ranged 0-42%. The cultivars Oxen and Prevail had the lowest (33%) and the highest (71%) seed germination respectively. Also the treatments including the fungicide significantly increased the germination (ranging from 5-44%) and the seedling blight got reduced in almost all the cultivars. Evaluation of management programs of yellowing and wilting of blackberry (Rubus sp.) caused by Fusarium oxysporum in Michoacan Mexico A. REBOLLAR-ALVITER (1), A. Hernandez-Cruz (2), A. Saldivia-Tejeda (3) (1) Universidad Autonoma Chapingo, Mexico; (2) Instituto Tecnologico del Valle de Morelia, Mexico; (3) Universidad Autonoma Chapingo, Posgrado en Proteccion Vegetal, Mexico Yellowing and wilting of blackberry (Rubus sp.), caused by F. oxysporum is one of the most devastating diseases in Michoacan, Mexico. The objective of this research was to evaluate different disease management programs using biological control agents (BCA), plant activators, and fungicides. The experiment was established in 2014 and 2015 in a plantation of blackberry cv. Tupy. A randomized complete block design with 4 blocks and 13 treatments and a control were used. Treatments consisted of sequential applications of fungicides (prochloraz, difenoconazole, thiabendazole, azoxystrobin and methyl thiophanate), plant activators (acibenzolar-S-methyl, potassium phosphite, oligosaccharin-glutathione), BCA (Trichoderma harzianum, Streptomyces lydicus strain WYEC 108) and microbial mixtures (Maya Magic® and Microsoil®). Resistance in pea to the Bean leaf roll virus L. PORTER (1) (1) USDA ARS, U.S.A. Root, rhizospheric soil, and bulk soil samples were taken early in the season, and DNA was extracted for PCR amplification, targeting 16s bacteria and archaeal, and ITS genes, for subsequent high throughput sequencing. Our results have shown no significant differences in microbial populations of the cover crop treatments. However, there are several notable differences with the tillage treatments. Chisel plowed plots had increased levels of the S4.91 genera, Verrucomicrobia, Glomeromycota, Proteobacteria, and Actinobacteria. Ridge tilled plots showed increased levels of Streptophyta, Firmicutes, and Crenarchaeota. These results are being verified with a greenhouse assay to determine if it is possible to delineate microbiome profiles that correspond to general disease suppression. genera, Verrucomicrobia, Glomeromycota, Proteobacteria, and Actinobacteria. Ridge tilled plots showed increased levels of Streptophyta, Firmicutes, and Crenarchaeota. These results are being verified with a greenhouse assay to determine if it is possible to delineate microbiome profiles that correspond to general disease suppression. Effects of fungicide application timing and cultivar resistance on Fusarium head blight and deoxynivalenol in winter wheat C. BOLANOS-CARRIEL1 (1), S. Wegulo (1), H. Hallen-Adams (1), D. Funnell-Harris (2), P. Baenziger (1) (1) University of Nebraska, U.S.A.; (2) USDA-ARS, U.S.A. Fusarium graminearum causes Fusarium head blight (FHB) in wheat. FHB reduces yield and quality and contaminates grain with the mycotoxin deoxynivalenol (DON). Effective management strategies are needed. The objectives of this research were to 1) Determine the effect of fungicide application timing at anthesis (the standard timing) and 6 and 12 days later on FHB and DON in the winter wheat cultivars Overley (susceptible) and Overland (moderately resistant) and 2) Compare the effects of a triazole and a strobilurin fungicide on FHB and DON in Overley and Overland. In 2015 two field trials (irrigated and rain-fed) were conducted in Nebraska, USA. The triazole Prosaro (prothioconazole + tebuconazole) and the strobilurin Headline (pyraclostrobin) were applied with a CO2-powered backpack sprayer at anthesis and 6 and 12 days later. A split plot design in randomized complete blocks with 4 replications was used. Main plots were cultivars and subplots were fungicide treatments. FHB index and DON were significantly (P < 0.05) lower in Overland than in Overley. The window of fungicide application to control FHB and DON was widened from anthesis to 6 days later without loss of efficacy. Headline was less effective than Prosaro in controlling FHB and DON. Resistance in pea to the Bean leaf roll virus L. PORTER (1) (1) USDA ARS, U.S.A. Moderate resistance combined with a triazole fungicide most effectively reduced FHB and DON. The results indicate a wider fungicide application window and the effectiveness of combining resistance with a triazole fungicide. Impact of Agronomic Practices and Diseases on Wheat Profitability J. SALGADO (1), P. Paul (1), L. Lindsey (1), R. Minyo (1) (1) The Ohio State University, U.S.A. Changes in nitrogen fertilizer rates, row spacing, and fungicide programs have been proposed as strategies for increasing wheat productivity and profitability in Ohio. The main goal of this study was to evaluate the economic benefit of these practices as influenced by diseases. Field experiments were conducted in 2013, 2014 and 2015 using a split-split-plot design with nitrogen rates (N-rate) as whole-plot, row spacing (19 and 38 cm) as sub-plot, and Prosaro fungicide treatments (TRT) as sub-sub-plot. Combinations of these factors represented separate management programs (PROG), and an economic analysis was conducted using yield (YLD), test weight (TW), grain prices and price discounts due to poor grain quality, as well as fertilizer, seed and fungicide costs to estimate net cash income for each PROG. Linear mixed model analyses showed that leaf rust and Fusarium head blight (FHB) were higher and YLD and TW lower in wide than in narrow rows. Leaf rust, YLD, and TW increased with increasing N-rates, but these effects varied with TRT. FHB severely affected the economic benefit of the different PROG. Compared to the standard PROG used in Ohio (100.9 kg N ha–1, narrow rows and untreated), several of the modified PROGs only resulted in greater economic benefit ($3 to 215 ha–1) when FHB levels were low. PROGs with narrow rows plus high N rates (> 134 kg N ha–1) and a Prosaro treatment were among the most profitable, while wide-row PROGs were among the least profitable. Effect of cropping rotation and input level over 18 years on the northern Great Plains B. GOSSEN (1), K. Bassendowski (1), E. Johnson (2), R. Lemke (1) (1) Agriculture and Agri-Food Canada, Canada; (2) University of Saskatchewan, Canada A cropping rotation study was conducted from 1994-2012 (three 6-yr cycles) at the Agriculture and Agri-Food Canada Research Farm at Scott, Saskatchewan, Canada in a split-split-plot design with four replicates. Resistance in pea to the Bean leaf roll virus L. PORTER (1) (1) USDA ARS, U.S.A. The main plots were three levels of inputs; high (HIGH), reduced (RED), and organic (ORG) The subplots were three levels of cropping diversity (rotations); fallow-annual grains (LOW), diversified annual grains (DAG), and diversified annuals and perennials (DAP). The sub-sub plots (each 13 × 40 m) were phases of the crop rotation. Foliar disease assessments were made shortly before crop maturity each year. Across treatments and years, there was no consistent pattern of response of final disease severity to treatment. For example, level of input and cropping diversity did not affect severity on the penultimate or flag leaf of wheat in 2007?2013. In those years where there were differences in severity, severity was often highest in the high-input treatments, but organic rotations were highest in some years. However, foliar disease severity differed substantially among years. This study demonstrated that weather conditions on the northern Great Plains had a much larger impact on foliar disease severity than input levels or cropping rotation in the moderate to highly diverse rotations assessed in this 18-year study. Novel approaches for the integrated control of the soilborne strawberry pathogens Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae M Carter (1) H Smith (2) G Holmes (3) K Ivors (1) Macrophomina phaseolina (Mp) and Fusarium oxysporum f. sp. fragariae (Fof) are emerging soilborne pathogens causing crown rot and Fusarium wilt in commercial strawberry fields in California. Fungicides representing eight active ingredients from four different FRAC groups were evaluated for their efficacy against each pathogen in greenhouse bioassays. Twenty-four fungicide treatments (eight fungicides at three rates) were drench applied to pathogen infested potting media two days prior to planting and 19 days after planting. Tea bags of buried inoculum were recovered two days after each fungicide application and plated on semi-selective medium to quantify each pathogen; disease severity was assessed weekly. Differences in the colony forming units (CFUs) and disease severity (AUDPC) for all treatments and the inoculated water-drench control for both Mp and Fof were not statistically significant, although there were differences among the CFUs for Mp. In addition, a strawberry plant extract was assessed for its ability to stimulate germination of Mp microsclerotia in vitro. Preliminary results showed that the number of germinating microsclerotia was significantly higher after the application of the strawberry extract. Germinated microsclerotia may be more sensitive to fungicides and offer a means of increasing fungicide efficacy against soilborne pathogens. Correlating the Effects of Glyphosate on Soybean Nutrition with Cercospora Leaf Blight and Septoria Brown Spot T. GARCIA AROCA (1), E. Silva (1), B. Ward (1), C. Robertson (1), P. Price (1), R. Schneider (1), R. Levy (2) (1) Louisiana State University, Baton Rouge, LA, U.S.A.; (2) Louisiana State University, Alexandria, LA, U.S.A. Glyphosate is linked to detrimental effects on soil microbiota and reduced levels of metal cations available for plant uptake. There is no agreement in literature regarding effects of glyphosate on plant diseases or micronutrient uptake. Some authors claim the use of glyphosate in Roundup Ready (glyphosate-resistant) soybeans leads to increased disease severity due to negative effects on uptake of manganese (Mn), iron (Fe) and other micronutrients, while others dispute these findings. This controversy is relevant to findings made by our laboratory regarding tissue concentrations of certain micronutrients, e.g. Mn and Fe, which substantially affect Cercospora leaf blight (CLB) in soybean. Therefore, we tested the hypothesis that glyphosate is affecting disease severity in soybean. Twelve Roundup Ready soybean varieties were planted at two locations in Louisiana in randomized complete block designs. Plots were either not sprayed or sprayed with Roundup. CLB and Septoria brown spot disease assessments were conducted in 2014 and 2015. Proactive Strategy for Management of Seedborne Pathogens of Pulse Crops in Montana B. AGINDOTAN (1), M. BURROWS (1) (1) Montana State University, U.S.A. Novel approaches for the integrated control of the soilborne strawberry pathogens Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae M Carter (1) H Smith (2) G Holmes (3) K Ivors (1) Samples were taken for micronutrient analyses and real-time quantitative PCR (qPCR) for the CLB pathogen. Results showed significant increases of some elements, e. g. N and Mn, for Roundup-treated samples. CLB and Septoria brown spot severities were higher in non-treated plots. Results from qPCR analyses indicated more pathogen DNA (biomass) in Roundup-treated samples, however, these findings were not correlated with disease severity. Effect of root pathogens Bipolaris sorokiniana and Fusarium graminearum on germination and seedling blight in wheat in South Dakota S. ALI (1), N. Kaur (1), S. Sehgal (1), K. Glover (1), S. Kumar (1) (1) South Dakota State University, Brookings, SD, U.S.A. Novel approaches for the integrated control of the soilborne strawberry pathogens Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae M. Carter (1), H. Smith (2), G. Holmes (3), K. Ivors (1) (1) California Polytechnic State University, U.S.A.; (2) California Polytechnic State University, Statistics, U.S.A.; (3) California Polytechnic State University Strawberry Center, U.S.A. Novel approaches for the integrated control of the soilborne strawberry pathogens Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae M. Carter (1), H. Smith (2), G. Holmes (3), K. Ivors (1) (1) C lif i P l t h i St t U i it U S A (2) C lif i P l t h i St t U i it St ti ti U S A (3) C lif i P l t h i St t Decision models for fungicide applications for frogeye leaf spot in soybean H Kelly (1) Frogeye leaf spot (FLS), caused by Cercospora sojina, is a common foliar pathogen of soybean in the United States and other soybean production areas in the world. While foliar fungicides can reduced disease severity and yield loss caused by FLS on susceptible cultivars, the appropriate application timing is essential for optimal fungicide efficacy and yield protection. To better manage FLS epidemics in Tennessee, fungicide application decision models were developed and evaluated across locations in 3 different counties in 2013 and 2014. Application models were developed based on growth stage of soybean and rain/irrigation events or temperature and relative humidity parameters. The number and timing of fungicide applications varied by location, year, and application model. All applications reduced disease severity, but only applications under moderate to severe disease pressure were necessary to protect yield. Results indicate models for fungicide application timing for FLS management need to incorporate additional disease parameters to more accurately trigger fungicide applications based on environmental factors and soybean growth stage. Effect of root pathogens Bipolaris sorokiniana and Fusarium graminearum on germination and seedling blight in wheat in South Dakota S. ALI (1), N. Kaur (1), S. Sehgal (1), K. Glover (1), S. Kumar (1) (1) South Dakota State University, Brookings, SD, U.S.A. Area Under Disease Progress (AUDPC) of incidence, severity and plant mortality were assessed. There were significant differences between treatments. Treatments with the lowest AUDPC were the mixtures of microorganisms (Maya Magic®) without significant differences with treatments beginning with 2 to 3 applications of prochloraz or difenoconazole followed by Glutathione-oligosaccharins, and azoxystrobin, followed by acibenzolar S methyl applications. These results indicate that it is feasible to significantly reduce the severity and mortality in F. oxysporum-infected blackberry plantings, if disease management programs begin early in the growing season. S4.92 Response of grafted seedless watermelon to root regeneration and inoculation with Fusarium oxysporum f. sp. niveum Q. Liu (1), X. Zhao (1), T. Sanchez (1), Z. Black (1), N. Dufault (1), Q. Liu (1) (1) University of Florida, U.S.A. Grafting can be a successful tool in an integrated approach to manage soilborne diseases such as Fusarium wilt of watermelon caused by Fusarium oxysporum f. sp. niveum (FON). In cucurbit grafting, excision and regeneration of grafted seedling roots is often practiced by nurseries. The objectives of this greenhouse experiment were to assess the performance of grafted seedless watermelon plants under inoculation with FON, and compare grafted plants with original rootstock roots and with root regeneration. ‘Melody’ watermelon was grafted onto two squash rootstocks including ‘Marvel’ and ‘Super Shintosa’. Various plant growth parameters were measured. All the plants were plated on PDA to confirm the presence of FON colonies 32 days after transplanting. Non-grafted watermelon plants showed a significantly (P ≤ 0.05) higher percentage of recovered FON colonies (75%) compared to grafted treatments (0-8%). Plant growth measurements indicated less vigorous growth for non-grafted plants when compared to the grafted treatments. Root regeneration treatments had a numerically higher percentage of FON colonies (0-8%) compared with the regular grafting treatments (0-3%), while no differences were observed in plant growth measurements. These results showed that grafting can effectively prevent FON colonization in seedless watermelon, and supported the use of this practice as part of a watermelon production integrated management program. Novel approaches for the integrated control of the soilborne strawberry pathogens Macrophomina phaseolina and Fusarium oxysporum f. sp. fragariae M. Carter (1), H. Smith (2), G. Holmes (3), K. Ivors (1) (1) California Polytechnic State University, U.S.A.; (2) California Polytechnic State University, Statistics, U.S.A.; (3) California Polytechnic State University Strawberry Center, U.S.A. Controlling Asian Soybean Rust and Phytophthora Late Blight by Biologicals Alone and in Combination with Fungicides M. TWIZEYIMANA (1), E. Gachango (1), B. Espejo (1), K. Craig (1), P. Hammer (1) (1) AgBiome, Inc., Research Triangle Park, NC, U.S.A. The most prevalent fungi isolated were Alternaria (71 - 83%), Cladosporium (53 - 67%), Ascochyta (18-56%), Penicillium (46 - 57%), Rhizopus (20-40%), and Nigospora (6-20%; 1st report). Others were Fusarium (14- 17%), Aureobasidium (7.4-11%; 1st report), Botrytis (8-14%), Stemphylium (9 - 16%), Collectotrichum (1-9%), and Diaporthe spp. (4-14%; 1st report). Growers can manage these fungal diseases if they know the health status of their seedlots, use seed treatment fungicides, and prevent planting seed lots infested with pathogens with long-lived survival structures on organic and conventionally managed acres. Production practices impact yield parameters and disease severity on sweet and forage sorghum grown as biomass feedstock A. HAGAN (1), S. Scott (1), K. Bowen (1) (1) Auburn University, U.S.A. Sweet and forage sorghum are potential feedstocks for energy and chemical industries but the impact of production practices on diseases is largely unknown. Planting date (PD), nitrogen (N) rate, and variety impact were assessed in 2011 and 2012 on plant populations, above ground dry matter (ADM) yield, total fermentable sugar concentration (°Bx), and anthracnose severity. A factorial with year as the main plot, PD as the split-plot, N rate as the split split-plot, and sweet and forage sorghum variety as the split split split-plot was used. Disease assessment and harvest was at soft dough. Plant population was not impacted by any variable. ADM yield segregated by year, PD, and variety. Generally, similar ADM yield was noted at all N rates. M81-E sweet sorghum ADM yield trended higher than forage sorghum. °Bx varied by year, PD, and variety with values declining at the later PD. M81-E had a higher °Bx than either forage sorghum with 1990 having a higher °Bx in 2011 than SS405 in 2012. Anthracnose differed by year, PD, N rate and variety where disease severity was highest in 2012, when sorghum followed sorghum, and summer weather was wetter than 2011. In both years, M81-E had less anthracnose damage than either forage sorghum with SS405 having a higher disease rating in 2012 than 1990 in 2011 due to rotation and drier summer in latter year. Higher °Bx and anthracnose ratings were seen at 88 than 22 kg N ha–1. Anthracnose ratings were lower at the April than June PD. Anthracnose impacts biomass and sugar yield of forage but not sweet sorghum A. HAGAN (1), K. Bowen (1), H. Miller (1) (1) Auburn University, U.S.A. Controlling Asian Soybean Rust and Phytophthora Late Blight by Biologicals Alone and in Combination with Fungicides M. TWIZEYIMANA (1), E. Gachango (1), B. Espejo (1), K. Craig (1), P. Hammer (1) (1) AgBiome, Inc., Research Triangle Park, NC, U.S.A. Controlling Asian Soybean Rust and Phytophthora Late Blight by Biologicals Alone and in Combination with Fungicides M. TWIZEYIMANA (1), E. Gachango (1), B. Espejo (1), K. Craig (1), P. Hammer (1) (1) AgBiome, Inc., Research Triangle Park, NC, U.S.A. Tremendous losses are associated with Asian soybean rust (Phakopsora pachyrhizi) and Phytophthora late blight (Phytophthora infestans) yearly. At AgBiome, isolates from our large collection of proprietary environmental microbes are screened in high-throughput assays against a broad range of fungal pathogens, insect pests, and plant-parasitic nematodes. Genome sequences from these isolates are analyzed for known families of proteins active against plant pests, and these proteins are assayed in bacterial expression systems and transgenic crops. Successful biological and trait candidates are tested, and confirmed active biologicals proceed to the fermentation and formulation. Nearly 1200 and 500 isolates have been screened against P. pachyrhizi and P. infestans, respectively. For both pathogens, the initial screening is done using leaf disks and hits are confirmed on whole plants in growth chambers. Selected leads undergo mouse toxicity testing and scale up by fermentation and formulation before they are evaluated in greenhouse and field. From all isolates screened for the control of Asian soybean rust, seven were selected and were evaluated in the greenhouse in Florida and Illinois and in the field in Florida and Argentina. Additional selected leads with activity against P. infestans will be sent to the field this year. Proactive Strategy for Management of Seedborne Pathogens of Pulse Crops in Montana B. AGINDOTAN (1), M. BURROWS (1) (1) Montana State University, U.S.A. S4.93 Pulse crops (chickpea, lentil, and field pea) are grown for their health and nutritional benefits. In 2015, Montana led the U.S. in field pea and lentil production at 48% acreage. The pulse crop acreage projection by 2019 is 1.4 mil acres. Growers in Montana are aware of the fungal disease Ascochyta/Mycospharella blight and have been testing their seeds exclusively for this disease complex. Due to limited seed availability, seed is coming into the state from other long-term pulse producing areas with pathogens. To determine the prevalence of seedborne fungi in pulse crops from the 2015 growing season, seed samples of field pea (256), lentil (170), and chickpea (35) were tested by plating 400-600 seeds per sample on potato dextrose agar and incubating at 20⁰C (12h light: dark) for 10-14 d. Fungi were identified to genus level using microscopy and DNA sequencing. ( ), ( ) (1) University of Nebraska Panhandle Research & Extension Center, U.S.A.; (2) University of Nebraska Panhandle REC, U.S.A. ( ), ( ) (1) University of Nebraska Panhandle Research & Extension Center, U.S.A.; (2) University of Nebraska Panhandle REC, U.S.A. In Nebraska, Rhizoctonia root and crown rot, caused by Rhizoctonia solani, is generally considered to be the most commonly occurring and damaging sugar beet disease. Previous studies have demonstrated that applications of azoxystrobin will effectively reduce disease and improve yield parameters. However, the almost exclusive use of Quadris (azoxystrobin) for more than a decade raises concern for resistance development by the pathogen to this fungicide. Studies were conducted from 2013-2015 to evaluate the performance of alternative fungicides currently registered for this disease in sugar beets (Priaxor, Proline, Headline, and Vertisan) and compare their performance to Quadris. The study consisted of 16 treatments: 1) untreated control, 2- 6) applications applied in-furrow at planting, 6-10) foliar applications after 4 inch soil temperatures averaged 65° F for three consecutive days, and 11- 15) both in-furrow and foliar applications. Data collected included multiple disease counts during the season, and sucrose and root yield determinations at harvest. Our results indicated that Quadris, Priaxor, and Proline all performed similarly, significantly reducing disease incidence and increasing sugar yields compared with untreated controls. We further determined that better results were obtained with the combination of in-furrow and foliar applications later in the season than either treatment alone. Controlling Asian Soybean Rust and Phytophthora Late Blight by Biologicals Alone and in Combination with Fungicides M. TWIZEYIMANA (1), E. Gachango (1), B. Espejo (1), K. Craig (1), P. Hammer (1) (1) AgBiome, Inc., Research Triangle Park, NC, U.S.A. Impact of anthracnose on aboveground dry matter (ADM), estimated sugar yield (SY), and total fermentable sugar concentration (°Bx) of energy feedstock forage and sweet sorghum is undefined. A factorial study with irrigated SS405 forage sorghum and Topper 76-6 (2014) or M81-E (2015) sweet sorghums as whole plots, and 1, 2, 3, 5, 6, or 7 (2014 only) broadcast applications of Headline (12 fl oz A–1) at 28, 35, 42, 49, 56, 64, and 72 DAP [days after planting] as the split-plot treatment was used. A non-fungicide treated control was included. Anthracnose intensity was recorded at 7 to 14 d intervals from 30 DAP until harvest at soft dough; areas under the disease progress curves (AUDPC) for anthracnose were calculated from these ratings. On SS405, anthracnose intensified from onset at 30 to 45 DAP through harvest at 111 DAP when > 75% leaf area loss was noted for all but the 6- or 7- application programs. While Topper 76-6 was anthracnose-free, this disease intensified on M81-E from onset at 90 DAP until harvest at 133 DAP when > 75% leaf area loss was observed for all Headline programs and the non-fungicide treated control. In 2015, anthracnose AUDPC increased as Headline application number declined on SS405 but not M81-E. AUDPC was regressed against ADM, SY, and °Bx. In both years, ADM, SY, and °Bx declined by up to 30% as anthracnose intensified on SS405. Headline application number did not impact ADM, SY, and °Bx of the disease-free Topper 76-6. Managing Rhizoctonia crown and root rot of sugar beets utilizing azoxystrobin-alternative fungicides in Nebraska R HARVESON (1) R Harveson (2) Managing Rhizoctonia crown and root rot of sugar beets utilizing azoxystrobin-alternative fungicides in Nebraska R. HARVESON (1), R. Harveson (2) (1) University of Nebraska Panhandle Research & Extension Center, U.S.A.; (2) University of Nebraska Panhandle REC, U.S.A. R. HARVESON (1), R. Harveson (2) (1) University of Nebraska Panhandle Research & Extension Center, U.S.A.; (2) University of Nebraska Panhandle REC, U.S.A. R. HARVESON (1), R. Harveson (2) (1) University of Nebraska Panhandle Research & Extension Center, U.S.A.; (2) University of Nebraska Panhandle REC, U.S.A. Effect of temperature and sugar beet growth stage on yellowing decline caused by Fusarium secorum M. KHAN (1) (1) North Dakota State University and University of Minnesota U S A ( ) (1) North Dakota State University and University of Minnesota, U.S.A. Fusarium yellowing decline caused by Fusarium secorum was recently reported in Minnesota. Conditions favorable for disease development were determined using three isolates (12-4P, 12-2P and 1189), three temperatures (55°C, 65°C, and 75°C), and three growth stages (3, 5 and 7 weeks old plants [WOP]). One F. oxysporum f. sp. betae isolate was used for comparison. Plants were inoculated using root dip (105 microconidia or macroconidia ml–1) for two minutes. Evaluation for foliar symptoms was done every two days until harvest 35 days after inoculation, and area under disease progress curve (AUDPC) was calculated. Temperatures of 75 and 65°C were more favorable for disease development than 55°C. The mean AUDPC were 38, 29, and 4 for 12-4P, 12-2P and 1189 isolates, respectively. 12-4P isolate had significantly higher AUDPCs at 75 and 65°C for 3 WOP compared to both 5 and 7 WOP. 12-2P isolate resulted in no significant differences between AUDPCs at 3, 5, and 7 WOP within each temperature. However, 12-2P isolate caused significantly more disease at 75°C and 65°C compared to 55°C for 3 and 5 WOP. Isolate 1189 was least aggressive with no significant differences in AUDPC among all combinations of temperature and growth stage. Isolates 12-4P and 12-2P resulted in higher AUDPC than F. oxysporum f. sp. betae at the same temperature and sugar beet growth stage. Results suggest early planting in cool soils may help to manage the disease. g y p gg g differences in AUDPC among all combinations of temperature and growth stage. Isolates 12-4P and 12-2P resulted in higher AUDPC than F. oxysporum f. sp. betae at the same temperature and sugar beet growth stage. Results suggest early planting in cool soils may help to manage the disease. Management of wilt diseases on tomato by organically acceptable methods M. RAHMAN (1), M. Rahman (1), L. Jett (1) (1) West Virginia University, U.S.A. Soilborne wilt disease caused by fungal pathogen Verticillium spp, Fusarium spp and bacterial pathogen Ralstonia solanacearum often cause serious losses to tomato growers either by stunting or totally killing the plants prematurely. Rotation and chemical fumigation of soil is not feasible to many growers either due to limited farm size or organic growing conditions. Biofumigation and/or biologically based options for managing these pathogens are critical needs to these growers. Monitoring pomegranate pathogens towards developing effective disease management program A. KC (1), G. Vallad (1) Bacterial (Ralstonia) and oomycete (Pythium S4.94 and Phytophthora) pathogens were highly suppressed by ASD treatment (80%-88%), whereas across fungal pathogens (Cylindrocarpon, Fusarium, Macrophomina, Rhizoctonia, Sclerotium, Verticillium) suppression ranged from 59%-86%, with the exception of Sclerotinia (15%). Among different host specific F. oxysporum (Fo) pathogens (237 studies), spinach and tomato wilt caused by Fo f. sp. spinaciae (87%) and Fo f. sp. lycopersici (76%) were significantly suppressed by ASD. Under most environmental conditions, suppression of pathogen inoculum due to ASD treatment ranged from 62%-94%, that included study types, soil temperature, soil types and incubation period. Our meta-analysis indicates that ASD is effective against a broad range of soil borne pathogens and optimization to environmental conditions and cropping systems is likely to improve treatment consistency. and Phytophthora) pathogens were highly suppressed by ASD treatment (80%-88%), whereas across fungal pathogens (Cylindrocarpon, Fusarium, Macrophomina, Rhizoctonia, Sclerotium, Verticillium) suppression ranged from 59%-86%, with the exception of Sclerotinia (15%). Among different host specific F. oxysporum (Fo) pathogens (237 studies), spinach and tomato wilt caused by Fo f. sp. spinaciae (87%) and Fo f. sp. lycopersici (76%) were significantly suppressed by ASD. Under most environmental conditions, suppression of pathogen inoculum due to ASD treatment ranged from 62%-94%, that included study types, soil temperature, soil types and incubation period. Our meta-analysis indicates that ASD is effective against a broad range of soil borne pathogens and optimization to environmental conditions and cropping systems is likely to improve treatment consistency. Evaluation of the weather-based spray advisory program MELCAST, fungicide alternations and mulch to manage watermelon diseases in Missouri Z. MERSHA (1), M. O’Connor (1) (1) Lincoln University - MO U S A Melons are economically important crops in Missouri. Each year, Alternaria leaf blight (ALB), anthracnose (ANT) and gummy stem blight (GSB) cause yield losses in melons. Most commercial growers use calendar-based fungicide spray schedule. MELCAST (MELon disease foreCASTer) is a weather- based disease-forecasting and spray advisory program that allows cantaloupe and watermelon growers to schedule sprays based on weather. From 2013– 2015, there were 3-6 MELCAST sites in central, east and south east Missouri. Research plots were established at one of the sites in central Missouri at G. W. Carver Farm to examine MELCAST, mulches and fungicides in reducing diseases of watermelon using variety ‘Crimson Sweet’. Fungicide sprays significantly (P<0.05) reduced diseases compared to non-sprayed plots. Monitoring pomegranate pathogens towards developing effective disease management program A. KC (1), G. Vallad (1) Pomegranate (Punica granatum L.) is becoming a popular alternative crop in the Southeastern United States due to its nutritional benefit and emerging production potential in the area. Studies were conducted in 2015 to monitor pathogens prevalence from bud break to fruit maturity. Buds were collected from late January through March from three locations in Florida, two in Georgia, and one in South Carolina and were divided into six different stages from an unopened flower bud to fruit initials. The first, second and third stage buds were cut into halves whereas fourth, fifth, and sixth stages were cut into six longitudinal sections and were individually plated in V8 agar media and incubated at 21°C. Plates were monitored daily and the pathogen was identified based on morphology. Three orchards in Central Florida were also monitored every other week from late February to August and samples were collected to monitor pathogen prevalence. Several pathogens were recovered in the absence of any symptoms during the bud stages. The first symptoms observed on fruits were anthracnose caused by Colletotrichum sp. With increasing precipitation, the disease spread leading to spotting and blighting of leaves, stems and fruits. Further disease development, including the blighting of shoots and fruit mummification became more prevalent towards the mid to late season and was caused by combination of several pathogens. A meta-analysis of efficacy of anaerobic soil disinfestation on soil borne pathogen suppression U. SHRESTHA (1), R. Auge (1), A. Saxton (2), D. Butler (1) (1) Department of Plant Sciences, (2) Department of animal sciences, University of Tennessee, Knoxville, U.S.A. A meta-analysis of efficacy of anaerobic soil disinfestation on soil borne pathogen suppression U. SHRESTHA (1), R. Auge (1), A. Saxton (2), D. Butler (1) ( ), g ( ), ( ), ( ) (1) Department of Plant Sciences, (2) Department of animal sciences, University of Tennessee, Knoxville, U.S.A. Anaerobic soil disinfestation (ASD) is a widely applied pre-plant soil treatment practice to control soil borne pathogens under variable environmental conditions. A meta-analysis on multiple ASD studies (533 studies from 46 published/unpublished articles) on soil borne pathogens including bacterial, oomycete and fungal pathogens was conducted to determine the effect sizes and efficacy of ASD on pathogen control. We also examined various environmental conditions as moderator variables to explore the effectiveness of ASD against pathogens. Effect of temperature and sugar beet growth stage on yellowing decline caused by Fusarium secorum M. KHAN (1) (1) North Dakota State University and University of Minnesota U S A A study was conducted with heirloom tomato ‘Mortgage lifter’ at the WVU Organic Farm to investigate the efficacy of bio-fumigants, biological antagonists, and resistant rootstock in managing wilt disease. Thirty five days after planting in contaminated soil, grafted (on resistant rootstock “Maxifort”) tomato plants showed significantly higher plant vigor compared with non-treated check. All treatments had significantly lower symptomatic leaves than that of non-treated check at 35 days after planting. Cumulative harvests for six weeks showed that yield from all treatments except mustard cover crop were significantly (P<0.001) higher compared with non-treated check. Grafted plants produced 20 lb tomatoes/plant compared with only 11 lbs by non-treated check. Results suggest that organically acceptable methods can provide significant yield advantage to heirloom tomatoes in soilborne pathogen infested soil. Cryotherapy and Mini-plant Biological Indexing: New Tools for use in Citrus Clean Stock/Certification Programs C. RAMADUGU (1), M. Keremane (2), G. Volk (3), J. Hartung (4), G. McCollum (5), R. Lee (2) (1) University of California Riverside, Riverside, CA, U.S.A.; (2) USDA ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, U.S.A.; (3) USDA ARS National Center for Genetic Resources Preservation, Fort Collins, CO, U.S.A.; (4) USDA ARS Molecular Plant Pathology, Beltsville, MD, U.S.A.; (5) USDA ARS US Horticultural Research Laboratory, Fort Pierce, FL, U.S.A. Cryotherapy and Mini-plant Biological Indexing: New Tools for use in Citrus Clean Stock/Certification Programs C. RAMADUGU (1), M. Keremane (2), G. Volk (3), J. Hartung (4), G. McCollum (5), R. Lee (2) (1) University of California Riverside, Riverside, CA, U.S.A.; (2) USDA ARS National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, U.S.A.; (3) USDA ARS National Center for Genetic Resources Preservation, Fort Collins, CO, U.S.A.; (4) USDA ARS Molecular Plant Pathology, Beltsville, MD, U.S.A.; (5) USDA ARS US Horticultural Research Laboratory, Fort Pierce, FL, U.S.A. Shoot tip grafting (STG) followed by biological indexing using 10-14 month old indicator plants has been the mainstay for eliminating graft transmissible pathogens (GTP) from citrus accessions. We report the application of cryotherapy (freezing vegetative buds in liquid nitrogen followed by grafting onto seedling rootstocks) as an alternate, effective method for the elimination of certain GTPs. Because larger vegetative buds are used for cryotherapy, up to 1.5 mm, as compared to 0.1 mm of apical meristem buds used for STG, personnel become proficient after 1-2 weeks of training as compared to 6-12 months training needed for STG. Temporal variation of soil biota in fumigated and non-fumigated potato fields T. SMART (1), B. Geary (1) (1) Brigham Young University, U.S.A. Temporal variation of soil biota in fumigated and non-fumigated potato fields T. SMART (1), B. Geary (1) (1) Brigham Young University, U.S.A. Soil biota plays a prominent role in the regulation of soilborne pathogen incidence and severity. Thus, microbial community structure is of particular interest in commercial potato production as fumigation practices have been shown to alter soil biota. Despite long-term use of fumigation in potato production, there is still a limited understanding of soil biota and pathogen recovery and regeneration. In this study, soil cores were taken from neighboring fumigated and non-fumigated potato fields located in five different states: Washington, Oregon, Idaho, North Dakota, and Minnesota. Each core was assessed for pathogen incidence and severity and changes in soil biota over a temporal scale. Fungal and bacterial soil microbial diversity was assessed using meta-barcoded primers on an Illumina MiSeq platform. Our results demonstrate that fumigation creates disparity between pathogen populations and other soil biota which causes disequilibrium when compared to non-fumigation. Future research may establish predictive models in evaluating pathogen incidence and severity as part of an integrated disease management system that seeks to improve sustainable soil health for high- quality potato production. Monitoring pomegranate pathogens towards developing effective disease management program A. KC (1), G. Vallad (1) Alternating Bravo WeatherStik and Inspire Super or Quadris Opti and Luna Experience significantly reduced ALB, ANT and GSB. No significant difference was detected between these two plans. In 2014, total foliar biomass was significantly (P<0.05) higher with fungicide sprays (34.7 lb/plot) than without (9.1 lb/plot) for the plastic mulch plots. Disease suppression was not significantly different either between MELCAST and weekly sprays or between rye-vetch and plastic mulch. Use of MELCAST saved 1-2 fungicide sprays per season. Growers are encouraged to use mulch and to alternate fungicides in order to overcome risk of fungicide resistance and to improve productivity of watermelons. nce to mefenoxam in Pythium aphanidermatum and its impact on managing Pythium root rot in poinsettia cultivars Practical resistance to mefenoxam in Pythium aphanidermatum and its impact on managing Pythium root rot in poinsettia cultivars E. Lookabaugh (1), B. Shew (1) (1) NC State University, U.S.A. Mefenoxam (mef) resistance is widespread among populations of Pythium in NC greenhouses, but resistant (MR) and sensitive (MS) isolates often are found concurrently. The combined utility of host resistance and mef to manage root rot in plants exposed to mixed populations of P. aphanidermatum was tested on seven poinsettia cultivars (cvs). One MR and one MS isolate were used, giving four inoculum/fungicide combinations: MR+mef, MR-mef, MS+mef, MS-mef. Plants were treated twice with the labeled rate of Subdue Maxx. Plants were scored for root rot (RR, 1 = 0% rot; 5 = 100%) and aboveground disease severity (DS, 1 = healthy; 5 = dead plant) 10 wk after inoculation. MS-mef treated plants had the highest RR (mean = 4.0) and DS (mean = 3.9) in all cvs. Mef controlled RR in all cvs inoculated with the MS isolate (mean = 1.6) but not in 5 of 7 cvs inoculated with the MR isolate (mean = 3.4), demonstrating practical resistance to mef in P. aphanidermatum. MR-mef treated plants had lower DS severity than MS-mef treated plants (3.3 vs. 3.9), suggesting that the MS isolate was more aggressive than the MR isolate. Treatment with mef lowered DS in MR inoculated plants only slightly (mean = 2.6). If confirmed with other MS and MR isolates, this may explain why MS isolates persist in facilities that regularly use mef. Resistant poinsettia cvs could be used to offset loss of fungicide efficacy and reduce losses to Pythium root rot in facilities with mixed Pythium populations. Evaluation of Commercial Hot and Bell Pepper Cultivars for Resistance to Phytophthora capsici C. PARADA ROJAS (1), C. Parada Rojas (2), L. Quesada Ocampo (2) (1) North Carolina State Universtity, Colombia; (2) North Carolina State University, U.S.A. Phytophthora blight (caused by Phytophthora capsici) is an important disease with a broad host range including Solanaceous, Cucurbitaceous, and Fabaceous crops. In pepper, P. capsici causes crown, root, and fruit rot as well as foliar lesions. Disease management relies primarily on the application of fungicides, planting of tolerant cultivars, and cultural practices. Field trials were conducted over the summers of 2015 and 2016 to evaluate 32 commercial hot and bell pepper cultivars in North Carolina (NC). Bacillus mycoides isolate J (BmJ): Plant protection through Systemic Acquired Resistance. A new option for plant disease management S. OCKEY (1), B. Highland (2), B. Jacobsen (3), M. Dimock (2) (1) Certis USA, U.S.A.; (2) Certis USA, U.S.A.; (3) Montana State University, U.S.A. Bacillus mycoides isolate J (BmJ): Plant protection through Systemic Acquired Resistance. A new option for plant disease management S. OCKEY (1), B. Highland (2), B. Jacobsen (3), M. Dimock (2) (1) Certis USA, U.S.A.; (2) Certis USA, U.S.A.; (3) Montana State University, U.S.A. Bacillus mycoides isolate J (BmJ) induced resistance provides control of bacterial, fungal, oomycete and viral pathogens on a wide range of crop plants. BmJ induced resistance is signaled through the NPR -1 gene and involves several PR proteins and ethylene. Optimal systemic resistance induction occurs 3-5 days after application of live cells and provides disease control for 14-21 days depending on the plant induced. BmJ is compatible with pesticides including; triazole, EDBC, and QoI class fungicides and a wide range of insecticides. Disease control equal to commercial standards has been achieved with BmJ alone or in combination with fungicides where BmJ replaces half the fungicide used in fungicide alone programs. BmJ has also been demonstrated to be of benefit to triazole and QoI fungicide resistance management programs in Cercospora leaf spot of sugarbeet control programs. BmJ used in combination with insecticides and rouging has produced >50% control of potato PVY in multiple years in the field. Research results also support disease control in cucurbit crops, tomato, pepper, potato, pecans, spinach, lettuce and sugarbeet with comparisons to commercial pesticide standards. BmJ is licensed to CERTIS USA by Montana State University and will be sold as a WDG formulation. Application rates are based on 1 × 107cfu/ml ( 60-240 gm/A depending on spray volume). Registration is expected with Canada’s Pest Management Registration Authority (PMRA), the USEPA. Comparison of carbon quantity in anaerobic soil disinfestation J. HONG (1), F. Di Gioia (2), M. Ozores-Hampton (2), E. Rosskopf (1) (1) USDA ARS, U.S.A.; (2) University of Florida, Institute of Food and Agricultural Sciences, South West Florida Research and Education Center, U.S.A. U.S.A.; (2) University of Florida, Institute of Food and Agricultural Sciences, South West Florida Research and Education C 1) USDA ARS, U.S.A.; (2) University of Florida, Institute of Food and Agricultural Sciences, South West Florida Research U.S.A. Anaerobic soil disinfestation (ASD) is a pre-plant alternative to chemical fumigation, used to manage soil plant pathogens, nematodes, and weeds. Compared to chemical fumigation, ASD was as effective for controlling these pests, and fruit yield often exceeded. Bacillus mycoides isolate J (BmJ): Plant protection through Systemic Acquired Resistance. A new option for plant disease management S. OCKEY (1), B. Highland (2), B. Jacobsen (3), M. Dimock (2) (1) Certis USA, U.S.A.; (2) Certis USA, U.S.A.; (3) Montana State University, U.S.A. Application of ASD in Florida includes incorporating composted broiler litter, 22 Mg ha–1, and molasses, 13.9 m3 ha–1, tarping the soil, and saturating to capacity. The environment under the tarp becomes anaerobic during the treatment, allowing anaerobic microbes, which contribute to pathogen management, to thrive. The objective of this study was to compare various rates of molasses, 0-4 times the standard rate (SR), in greenhouse conditions. Treatments were compared using the following variables: soil conditions, plant vigor, soil microbiome, and weed count. Soil microbiome was investigated by extracting microbial DNA from the soil, taken periodically during the treatment, and using length-heterogeneity PCR. Shifts in the microbiome correlated to changes in soil pH and reduction potential. The microbiome fluctuated slightly in the no molasses treatment, while the microbiome of the SR changed throughout the treatment, and the pH temporarily decreased. Bacterial diversity decreased 7 day post treatment (dpt) for the treatments with molasses rates greater than the SR. The soil pH for these treatments dropped from pretreatment levels and remained acidic; pH for 4 times the SR was 1 dpt = 6.9 and 21 dpt = 5.3. Effect of temperature and sugar beet growth stage on yellowing decline caused by Fusarium secorum M. KHAN (1) (1) North Dakota State University and University of Minnesota U S A We also have developed a biological indexing method using very young (mini) indicator plants, 60- 90 days old. Side by side comparisons of 30 accessions using traditional and mini-plant biological indexing indicate the same level of sensitivity between S4.95 the two methods. The use of mini-plants for biological indexing requires much less time and greenhouse space, also GTP requiring cooler temperatures to produce symptoms may be indexed year-round by placing the mini-plants near the cooling pads in summer. The use of cryotherapy and mini-plant biological indexing will enable citrus clean stock and/or certification programs to be more productive while using fewer resources. the two methods. The use of mini-plants for biological indexing requires much less time and greenhouse space, also GTP requiring cooler temperatures to produce symptoms may be indexed year-round by placing the mini-plants near the cooling pads in summer. The use of cryotherapy and mini-plant biological indexing will enable citrus clean stock and/or certification programs to be more productive while using fewer resources. nce to mefenoxam in Pythium aphanidermatum and its impact on managing Pythium root rot in poinsettia cultivars Cultivars Martha-R and Meeting were found to be the most resistant to a group of isolates from NC. Ebano-R, Revolution, Paladin, and Vanguard showed intermediate levels of resistance. Bastille, Red Knight, and Plato were highly susceptive to P. capsici. Greenhouse experiments were conducted to determine the virulence of three individual P. capsici isolates across 48 commercial hot and bell pepper cultivars. The isolates exhibited different levels of virulence to the pepper cultivars screened for crown and root rot resistance. Cultivars CM334, Martha-R, Meeting, and Intruder were resistant to the isolates tested. In addition, we phenotypically characterized a P. capsici isolate collection from NC for fungicide sensitivity and mating type. This information will be useful for NC growers in selecting resistant cultivars and for future population genetics analyses. Managing Potato Pink Rot Using Chemical and Non-Chemical Strategies X. ZHANG (1), J. Hao (1), H. Jiang (1), N. Marangoni (1), X. Zhang (1) (1) University of Maine, U.S.A. Pink rot (Phytophthora erythroseptica) is a widespread disease on potatoes (Solanum tuberosum). Controlling pink rot becomes a challenge owing to the fast development of a fungicide-resistant P. erythroseptica population. To determine effective strategies for pink rot control, field trials were conducted in 2014 and 2015 using fungicides or crop rotation. Pink-rot-susceptible seed tubers (cv. ‘Russet Norkotah’) were used. Phytophthora erythroseptica inoculum was distributed in-furrow at planting prior to soil treatments. In fungicide trials, chemical and biological products were applied in-furrow at planting or through soil drenching after plant emergence. The crop rotation trial was conducted with “rotation crop-potato” sequences. Tuber yield was measured and disease severity was rated based on a “0 (healthy tuber) to 5 (>50% rot area)” scale at harvest. Fluopicolide, mefenoxam–oxathiapiprolin combinations, and fluopicolide/oxathiapiprolin plus Bacillus combinations significantly reduced pink rot. None of the tested biocontrol agents significantly reduced pink rot alone. Mefenoxam was effective when there was no mefenoxam-resistant population of P. erythroseptica. Oats, clover, and barley underseeded with ryegrass lowered pink rot severity levels, although it was not statistically significant. Soil microbial communities in rotation plots are being investigated using next generation sequencing technology. S4.96 The Nærstad model for potato late blight is available on the open source license technology platform VIPS B. NORDSKOG (1), A. Hermansen (1), V. Le (1), H. Eikemo (1), A. Hjelkrem (1), T. Skog (1), R. Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic M. KAZUMI (1) Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic M KAZUMI (1) ( ) (1) Toyama Prefectural Agricultural, Forestry and Fisheries Research Center, Japan Tulip mild mottle mosaic disease, cause of Tulip mild mottle mosaic virus, TMMMV and Tulip streak disease, cause of Tulip streak virus, TuSV transmitted by Olpidium virulentus cause chronic damage and become one of the factors of avolition on produce of tulip bulb in Toyama, Japan. The management of both viral diseases depends on use of fungicide, a fertilizing efficiency-control type fertilizer and cultivation of late time. However, it is necessary to choose the appropriate control measures according to the level of both viral disease occurrence risks on farmer’s field. In this study, we developed management of both viral disease consisting of risk evaluation of both viral diseases on tulip field by the quantification of TMMMV and TuSV from soil using real-time PCR, evaluation of individual control measures for both viral diseases by meta-analysis and selection of appropriate control measures according to the level of both viral diseases occurrence risks on tulip field. Management of Tomato chlorotic spot virus, an emerging tospovirus of tomato causing severe losses in south Florida S. ZHANG (1), Y. Fu (2), X. Fan (2), D. Seal (2), Q. Wang (3), E. McAvoy (4) (1) Tropical Research and Education Center, University of Florida, IFAS, Homestead, FL, U.S.A.; (2) Tropical Research and Education Ce University of Florida, IFAS, U.S.A.; (3) UF/IFAS Miami-Dade County Extension, U.S.A.; (4) UF/IFAS Hendry County Extension, U.S.A ( ) ( ) Q g ( ) y ( ) d Education Center, University of Florida, IFAS, Homestead, FL, U.S.A.; (2) Tropical Research and Education Center, AS, U.S.A.; (3) UF/IFAS Miami-Dade County Extension, U.S.A.; (4) UF/IFAS Hendry County Extension, U.S.A. Tomato chlorotic spot virus (TCSV) is a devastating tospovirus transmitted by thrips. TCSV was first reported in the USA from tomato plants in Homestead, FL in 2012. Outbreaks of TCSV in Homestead area since 2014 have caused significant losses to growers. TCSV has become established in south Florida and it occurs more widely with greater frequency across this region, bringing a serious concern that TCSV may spread to other tomato production regions. Field trials have conducted in Homestead since 2015 to develop management strategies for TCSV. Fourteen tomato varieties resistant to Tomato spotted wilt virus (TSWV) were evaluated. Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic M. KAZUMI (1) In the spring trial in 2015, all tested varieties exhibited varying levels of resistance to TCSV (10-40% vs 100% infection in the commercial standard cv. ‘FL 47’) two months after transplanting. In the two repeated trials carried out in the fall of 2015, all these varieties had lower than 8% disease incidence 16 weeks after transplanting, whereas 50% plants of cv. ‘FL 47’ showed TCSV symptoms. In an insecticide trial conducted in the spring of 2015, only spinetoram (Radiant®) and cyantraniliprole (Exirel®) significantly (P<0.05) reduced TCSV incidence compared to the untreated control. In the fall trials of 2015, plant activator acibenzolar-S-methyl (Actigard®) numerically reduced TCSV incidence. The use of metalized UV-reflective plastic mulch failed to suppress the disease. Results from this study will be reported and discussed in detail. Variation within and among laboratories in detection of Candidatus Liberibacter asiaticus using qPCR G. MCCOLLUM (1), C. Levesque (2), M. Keremane (3), M. Kunta (4) (1) USDA-Agricultural Research Service, U.S.A.; (2) Citrus Research Board, U.S.A.; (3) USDA, ARS, U.S.A.; Huanglongbing (HLB) disease has ravaged the Florida citrus industry during the last decade and poses a serious threat to citrus production worldwide. Candidatus Liberibacter asiaticus (CLas) is the presumed causal agent of HLB in the United States. Diagnosis of CLas infection is based on PCR; qPCR for wide-scale surveys and conventional PCR for confirmation. The objective of our research was to compare results among multiple laboratories using slightly different qPCR protocols to confirm the sensitivity and diagnostic reliability of qPCR. An end point dilution series of CLas-positive nucleic acid was prepared and distributed to 4 laboratories in which CLas diagnostics are routinely performed. In each laboratory qPCR diagnostics were performed using the dilution series as template, however, specific protocols typical for each laboratory varied slightly. Results were compared within and between laboratories to determine linearity, amplification efficiency, end point and consistency. Excellent agreement in results was seen among protocols within individual laboratories as well as between individual laboratories. Regression analysis indicated that regardless of protocol, PCR efficiencies were between 95 and 105%. In addition, results confirmed a Ct value of 38 for the endpoint (i.e. a single copy of target). Our results support the robustness and diagnostic reliability of the currently approved qPCR protocol. Regulatory implications of a newly discovered transmission mechanism for the pathogens that cause huanglongbing (citrus greening disease) S. HALBERT (1), M. Keremane (2), C. Ramadugu (3), W. nce to mefenoxam in Pythium aphanidermatum and its impact on managing Pythium root rot in poinsettia cultivars Nærstad (1) (1) Norwegian Institute of Bioeconomy Research, Norway The Nærstad model for potato late blight (Phytopthora infestans) is a weather driven forecast model based on validation trials with spore traps and trap plants under Norwegian field conditions. The model combines sub models for spore production, release, survival, and infection, with calculations derived from hourly values of temperature, relative humidity, global radiation, leaf wetness (LW), and precipitation. LW may also be calculated with additional wind and net radiation parameters. The model output is presented as hourly risk values for infection of P. infestans. The Nærstad model is currently provided to users in Norway, Sweden and Latvia through the open source licensed technology platform VIPS (www.vips-landbruk.no). Data from most online weather stations or public weather data networks can be implemented into the VIPS system, including virtual weather stations based on weather forecasts. In VIPS, models such as the Nærstad model can easily be tested and validated under local conditions. Model outputs from multiple weather stations can be displayed in a map to visualize regional differences, in addition to more detailed outputs for each location. The open source license allows users of VIPS to make adaptations and changes to accomodate local needs, test and validate models locally or as part of an international network, and eventually provide the models directly to end users through the same system. Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic M. KAZUMI (1) Dawson (4), J. Lee (5), J. Keesling (5), B. Singer (6), R. Lee (7) (1) Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, FL, U.S.A.; (2) USDA/ARS, National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, U.S.A.; (3) University of California, Riverside, Riverside, CA, U.S.A.; (4) Citrus Research and Education Center, University of Florida, Lake Alfred, FL, U.S.A.; (5) University of Florida Mathematics Department, Gainesville, FL, U.S.A.; (6) Emerging Pathogens Institute, University of Florida, Gainesville, FL, U.S.A.; (7) Retired, USDA/ARS, Peyton, CO, U.S.A. Regulatory implications of a newly discovered transmission mechanism for the pathogens that cause huanglongbing (citrus greening disease) S. HALBERT (1), M. Keremane (2), C. Ramadugu (3), W. Dawson (4), J. Lee (5), J. Keesling (5), B. Singer (6), R. Lee (7) (1) Florida Department of Agriculture and Consumer Services, Division of Plant Industry, Gainesville, FL, U.S.A.; (2) USDA/ARS, National Clonal Germplasm Repository for Citrus and Dates, Riverside, CA, U.S.A.; (3) University of California, Riverside, Riverside, CA, U.S.A.; (4) Citrus Research and Education Center, University of Florida, Lake Alfred, FL, U.S.A.; (5) University of Florida Mathematics Department, Gainesville, FL, U.S.A.; (6) Emerging Pathogens Institute, University of Florida, Gainesville, FL, U.S.A.; (7) Retired, USDA/ARS, Peyton, CO, U.S.A. Huanglongbing (HLB, citrus greening disease) is one of the most devastating citrus diseases in the world. In Florida, it is associated with a bacterium Candidatus Liberibacter asiaticus (Las) and transmitted by a psyllid, Diaphorina citri Kuwayama. Lee et al. (2015; PNAS 112: 7605–7610) documented a new transmission mechanism for Las. An adult female psyllid, or her mate, infects the new growth where she lays eggs. The localized infection becomes a source for developing nymphs. Progeny can repeat the process as soon as they become adults. Since each female can lay 750 or more eggs, and psyllids can complete development in 15 days, the potential for increase of infected insects is rapid. In the laboratory, plants colonized by infected psyllids usually develop disease, but symptoms may be delayed for months or years. Catastrophic spread of Las can occur long before symptoms manifest. This delayed expression of disease symptoms could allow for the disease to spread undetected. Known unknowns and unknown unknowns: Fungi intercepted at the U.S. borders M. ROMBERG (1), J. McKemy (1) (1) USDA APHIS PPQ, U.S.A. Known unknowns and unknown unknowns: Fungi intercepted at the U.S. borders M. ROMBERG (1), J. Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic M. KAZUMI (1) An overview of intercepted fungi will be presented including fungi intercepted for the first time from various ports and not known to occur in the U.S. Functional genomic analysis of Botrytis cinerea isolates from Ohio V. VIJAYAKUMAR (1), B. Cottrell (1), H. Reynolds (1), J. Slot (1), F. Hand (1), G. Valero (1), C. Tomashuk (1) (1) The Ohio State University, U.S.A. Botrytis cinerea is one of the most widely spread phytopathogenic fungi displaying a broad host-range. While Botrytis is generally considered a model necrotroph, systemic symptomless infections have also been reported. Previous studies have shown significant genomic variation between gray and rare bikaverin producing pink isolates, but their contribution to diversity and host/pathogen relationships is understudied. We aim to elucidate genomic features of symptomless and host specific infections, and biochemically characterize virulence determinants. For this, we sequenced the genomes of 4 isolates from Ohio: 1 gray, isolated as asymptomatic endophyte of apple blossoms; 1 pink and 1 gray, isolated from infected flowers of Matthiola incana; and 1 purple, obtained from distinct sectors of pink isolate. We compared all 4 genomes to B05.10 and T4 reference genomes. Results showed large polymorphisms within coding regions of predicted genes (52,710, missense; 418 nonsense and 80,430 silent) and deletion of gene clusters (GC) associated with virulence. In both pink and purple isolates the botcinic acid GC was deleted, while in the gray asymptomatic isolate the abscisic acid GC was deleted. Investigation of virulence determinants among isolates showed a fully functional sclerotia formation, while the pink and purple strains alone were impaired in oxalic acid formation. Our results suggest that genetic diversity plays a significant role in species complexity and lifestyle. Virus testing in certified strawberry nursery plants in Oregon D. SHARMA-POUDYAL (1), S. Lane (1), J. Grant (1), N. Osterbauer (1) (1) Oregon Department of Agriculture, U.S.A. Strawberry crinkle virus (SCV), Strawberry mottle virus (SMoV), Strawberry mild yellow edge virus (SMYEV), and Strawberry vein banding virus (SVBV) are aphid-borne strawberry viruses frequently found in the Pacific Northwest. As such, they are excellent indicators for the effectiveness of nursery certification programs in maintaining virus free plants. Oregon nurseries import certified strawberry plants primarily from California and multiply these plants prior to sale to the final consumer. Testing for viruses in certified plants reveals the virus status of the plants and measures the effectiveness of the originating state’s certification program. Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic M. KAZUMI (1) McKemy (1) (1) USDA APHIS PPQ, U.S.A. Global trade in plants and plant products continues to increase yearly and has the potential to move fungi associated with poorly studied hosts around the world. USDA Animal and Plant Health Inspection Service (APHIS) plant pathology identifiers at ports across the U.S. are responsible for identification S4.97 of fungi on products arriving by sea, land and air. In 2015, over 18,000 interceptions were examined by USDA APHIS plant pathology identifiers. These interceptions covered about 600 genera of hosts, with more than 250 genera of fungi identified on these hosts. The inspected hosts comprise ornamental, forestry, and horticultural plants and plant products. Nearly one quarter of the interceptions were from cargo awaiting entry decisions and required identifications in less than 48 hours. The rest were from plants and plant products seized from passenger baggage, mail, ship stores and other sources and represent some of the as yet unknown fungal/host combinations that could be arriving in the U.S. via these pathways. If a fungus not known to be present in the U.S. is intercepted on a shipment, that shipment is either destroyed or re-exported, which underscores the need to know which fungi have been reported in the US and also the need for systematic research in widespread fungal groups. An overview of intercepted fungi will be presented including fungi intercepted for the first time from various ports and not known to occur in the U.S. of fungi on products arriving by sea, land and air. In 2015, over 18,000 interceptions were examined by USDA APHIS plant pathology identifiers. These interceptions covered about 600 genera of hosts, with more than 250 genera of fungi identified on these hosts. The inspected hosts comprise ornamental, forestry, and horticultural plants and plant products. Nearly one quarter of the interceptions were from cargo awaiting entry decisions and required identifications in less than 48 hours. The rest were from plants and plant products seized from passenger baggage, mail, ship stores and other sources and represent some of the as yet unknown fungal/host combinations that could be arriving in the U.S. via these pathways. If a fungus not known to be present in the U.S. is intercepted on a shipment, that shipment is either destroyed or re-exported, which underscores the need to know which fungi have been reported in the US and also the need for systematic research in widespread fungal groups. Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic M. KAZUMI (1) A survey for these four viruses was performed in a Klamath County nursery multiplying certified strawberry plants. A total of 59 plant samples were collected and tested for SCV and SMYEV while 118 samples were tested for SMoV and SVBV. SVBV was detected using PCR with SVBV-specific primers. SCV, SMoV, and SMYEV were not detected using RT-PCR with virus-specific primers. SVBV was detected in one out of 118 samples tested. These results demonstrate the continued need for virus testing of strawberries throughout the plants’ entire life cycle in an official certification program. National Seed Health Accreditation Pilot Program: Monitoring seed health of seeds imported into the United States T. BRUNS (1), G. Munkvold (2), A. McMellen-Brannigan (3), R. Dunkle (4) (1) Iowa State University Seed Science Center, U.S.A.; (2) Iowa State University, U.S.A.; (3) USDA-APHIS, U.S.A.; (4) American Seed Trade Association, U.S.A. The risk of introducing plant pathogens into new areas by seed movement is a critical issue in agriculture. Part of the mission of USDA-APHIS is to protect U.S. agriculture from foreign pathogens. In order to help fulfill this mission, a pilot program, the U.S. National Seed Health Accreditation Pilot Program (NSHAPP), has been authorized by USDA-APHIS. NSHAPP is a voluntary program for seed importers that is administered by the National Seed Health System at Iowa State University Seed Science Center in Ames, IA. NSHAPP aims to prevent the introduction of plant pathogens into the United States by organizing the testing of imported seed. The scope of program focuses on cucumber green mottle mosaic virus (CGMMV) in cucumber, melon and watermelon seed. CGMMV causes severe disease of cucurbit crops, and was first detected in the U.S. in 2013 in California field production. Testing for CGMMV under NSHAPP is done by an ELISA method validated by the Int. Seed Testing Assoc. Participants report data on tested seedlots monthly, but positive seed tests must be reported immediately. Positive seedlots must be destroyed or re-exported. This program represents a proactive approach to reducing the risk of pathogen introduction in imported seed and demonstrates effective collaboration between seed importers and USDA- APHIS. Successful implementation of the pilot program may lead to expansion of voluntary testing and reporting to include other pathogens in imported seed. Assessing Goss’s bacterial wilt and leaf blight of corn in the High Plains of Texas with infrared aerial imagery J. WOODWARD (1), R. French-Monar (2), J. Todd (3), O. Aspergillus flavus can colonize a wide range of environments. The advent of effective biocontrol agents makes it important to know where fungi overwinter, because the amount and toxigenicity in each reservoir may determine the most effective site(s) to apply biocontrol. Aspergillus propagules on leaf, husk, silk and tassel of Bt and non-Bt corn, and from soil and air/dust were quantified using dilution plating on dichloronitroaniline rose Bengal agar. There was no effect of Bt corn on the amount of Aspergillus propagules. Aspergillus propagules in air/dust varied dramatically from year to year (0- 278 cfu/1000L). We studied corn hulls, cob and kernel fragments that accumulated near on-farm grain storage bins in normal farm operations and were Control of tulip mild mottle mosaic disease and tulip streak disease based on soil diagnostic M. KAZUMI (1) Moore (4) (1) Texas A&M AgriLife Extension, U.S.A.; (2) Texas A&M AgriLife Extension, U.S.A.; (3) Todd Ag Consulting, U.S.A.; (4) High Plains Consulting, U.S.A. Outbreaks of Goss’s bacterial wilt and leaf blight of corn, caused by Clavibacter michiganensis subsp. nebraskensis, were reported in the Texas High Plains from 2013 to 2015. The disease was identified based on symptomology, bacterial streaming, colony characteristics and Immunostrip test results. Impact of the disease on corn is poorly understood in this region, thus the objective of this study was to evaluate disease development using aerial imagery. Fields were photographed from a fixed-wing aircraft from mid-July to late-September of each year. Images were effective at differentiating resistant and susceptible hybrids and improved scouting efficiency when assessing incidence and severity. Disease onset differed by location but initial infections were easily identifiable. In general, infection foci were non-random or aggregated; however, edge effects were observed in two areas adjacent to fields where the disease was observed the previous year and reduced tillage was implemented. Yield reductions ranging from 12 to 53% were observed in areas of some fields; whereas, other fields exhibiting >85% incidence were abandoned for grain production and cut for silage. These results indicate that Goss’s wilt has the potential to negatively affect corn production under favorable conditions. Increased awareness through educational materials, including information on hybrid selection, crop rotation and residue management has allowed for better management of the disease. Understanding fungal reservoirs: Tracking Aspergillus populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1) (1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A. Understanding fungal reservoirs: Tracking Aspergillus populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1) (1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A. Understanding fungal reservoirs: Tracking Aspergillus populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1) (1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A. Understanding fungal reservoirs: Tracking Aspergillus populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1) (1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A. Fusarium stalk rot (Fusarium thapsinum, FT) and charcoal rot (Macrophomina phaseolina, MP) are high priority fungal diseases of sorghum, which can potentially affect sweet sorghum biofuel traits. The objective of this study was to test the effects of FT and MP on sweet sorghum sugar concentration and content. Seven parental lines and 12 hybrids were field evaluated in 2014 and 2015. At 14 d after flowering, plants were inoculated with FT, MP, and phosphate- buffered saline (control). Plants were harvested at 35 d after inoculation and measured for disease severity using stem lesion length. Juice volume (per plant) extracted from stalks was measured and subjected to HPLC to determine sugar concentration (mg/mL). ANOVA revealed non-significant pathogen main or simple effects on glucose and fructose concentration and significant genotype × pathogen effect on sucrose concentration. Pathogens did not significantly affect the total sugar concentration (= ∑ sucrose, glucose, fructose). However, when analyzed for content (g/plant), genotype × pathogen and environment × pathogen effects were evident for total sugars and sucrose. Pathogens affected glucose and fructose content in an environment-specific manner. Therefore, in general, stalk rot pathogens do not affect the concentration of major sweet sorghum sugars, but do affect sugar content. Effect of planting date and peanut cultivar on leaf spot epidemics and pod yield B. JORDAN (1), A. Culbreath (2), W. Branch (3) (1) University of Georgia, Dept. of Plant Pathology, U.S.A.; (2) University of Georgia, Dept of Plant Pathology, U.S.A.; (3) University of Georgia, Dept. of Crop and Soil Science, U.S.A. Planting date can affect the risk of losses to early and late leaf spot caused by, Cercospora arachidicola and Cercosporidium personatum, respectively, of peanut, Arachis hypogaea, in both conventional and organic systems. The objective of this study was to characterize the effect of planting date on leaf spot epidemics and yield in new cultivars with moderate tolerance to these diseases. A field trial was conducted in 2015 in Tifton, GA. Treatments were six planting dates (24 and 27 April, 4, 11, 19, and 26 May) arranged factorially with two cultivars, Georgia-06G and Georgia-12Y. Experimental design was a randomized complete block design with 4 replications. No foliar fungicides were applied. Late leaf spot was the predominant disease. Epidemics were severe in plots planted at the later dates. Final leaf spot ratings (Florida 1-10 scale) and AUDPC increased linearly with later planting date (Julian day) for both cultivars. Understanding fungal reservoirs: Tracking Aspergillus populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1) (1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A. Aspergillus flavus can colonize a wide range of environments. The advent of effective biocontrol agents makes it important to know where fungi overwinter, because the amount and toxigenicity in each reservoir may determine the most effective site(s) to apply biocontrol. Aspergillus propagules on leaf, husk, silk and tassel of Bt and non-Bt corn, and from soil and air/dust were quantified using dilution plating on dichloronitroaniline rose Bengal agar. There was no effect of Bt corn on the amount of Aspergillus propagules. Aspergillus propagules in air/dust varied dramatically from year to year (0- 278 cfu/1000L). We studied corn hulls, cob and kernel fragments that accumulated near on-farm grain storage bins in normal farm operations and were S4.98 periodically moved to the edge of an adjacent field and disked into soil in fall tillage. The following March, A. flavus propagules in soil samples ~20, ~30 and ~40 meters from the bins were 1,500-28,000 CFU/g, with ~3 times as many at 20m as at 40m, but 24% aflatoxigenic at all distances. In January 2016 similar debris and dust accumulating near on-farm grain storage bins contained 14,500±5,400 CFU/g A. flavus propagules. Additional studies on A. flavus propagules from rice, sesame and Illinois corn field soil and plant debris will be discussed. The findings are consistent with a substantial plant debris-associated soil populations. These findings are very useful in selecting optimal biological control strategies for aflatoxin. periodically moved to the edge of an adjacent field and disked into soil in fall tillage. The following March, A. flavus propagules in soil samples ~20, ~30 and ~40 meters from the bins were 1,500-28,000 CFU/g, with ~3 times as many at 20m as at 40m, but 24% aflatoxigenic at all distances. In January 2016 similar debris and dust accumulating near on-farm grain storage bins contained 14,500±5,400 CFU/g A. flavus propagules. Additional studies on A. flavus propagules from rice, sesame and Illinois corn field soil and plant debris will be discussed. The findings are consistent with a substantial plant debris-associated soil populations. These findings are very useful in selecting optimal biological control strategies for aflatoxin. Impact of stalk rot diseases on sweet sorghum sugar concentration and content A. BANDARA (1), T. Tesso (1), D. Wang (1), K. Zhang (1), C. Little (1) (1) Kansas State University, U.S.A. The sugar-rich nature of sweet sorghum [Sorghum bicolor (L.) Moench] makes it an attractive bioethanol feedstock. Understanding fungal reservoirs: Tracking Aspergillus populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1) (1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A. Paul (3) (1) The Ohio State University, U.S.A.; (2) United States Department of Agriculture-Agricultural Research Service, U.S.A.; (3) The Ohio State University, U.S.A. Wheat streak mosaic virus differentially affects soft red winter wheat cultivars B. HODGE (1), L. Stewart (2), P. Paul (3) (1) The Ohio State University, U.S.A.; (2) United States Department of Agriculture-Agricultural Research Service, U.S.A.; (3) The Ohio State University, U.S.A. ( ) ( ) niversity, U.S.A.; (2) United States Department of Agriculture-Agricultural Research Service, U.S.A.; (3) The Ohio State Wheat viruses are known to impact production throughout the world. A statewide survey conducted in 2012 identified several viruses in wheat samples taken from fourteen Ohio counties. Wheat streak mosaic virus (WSMV) was identified in samples from two counties, Auglaize and Champaign. WSMV is a positive sense, single-stranded RNA virus transmitted by wheat curl mites (Aceria tosichella), and is known to cause severe yield loss and stunting in hard red winter wheat. However, there is little information available about WSMV specific to soft red winter wheat cultivars grown in Ohio. To assess its potential impact, nineteen Ohio cultivars were mechanically inoculated with WSMV at Feekes stage 1. All cultivars supported systemic infection. Over 21 days post-inoculation, individual plants were rated for symptom severity, final height, and final wet weight. Across cultivars, inoculated plants exhibited an average 13.2% decrease in plant height, and a 32.1% decrease in wet weight. The cultivars differed in virus responses, as determined by generalized linear mixed model analysis. This experiment demonstrates that WSMV infection affects soft red winter wheat cultivars differentially, which may lead to the identification of cultivars that minimize yield loss. Occurrence of a Citrus canker strain of limited host specificity in Texas M. KUNTA (1), J. da Graça (1), B. Salas (2), D. Bartels (2), J. Park (1), G. Santillana (3), V. Mavrodieva (3) (1) Texas A&M University-Kingsville Citrus Center, Weslaco, TX, U.S.A.; (2) USDA APHIS PPQ CPHST Mission Laboratory, Edinburg, TX, U.S.A.; (3) CPHST Beltsville Laboratory USDA APHIS PPQ, Beltsville, MD, U.S.A. M. KUNTA (1), J. da Graça (1), B. Salas (2), D. Bartels (2), J. Park (1), G. Santillana (3), V. Mavrodieva (3) (1) Texas A&M University-Kingsville Citrus Center, Weslaco, TX, U.S.A.; (2) USDA APHIS PPQ CPHST Mission Laboratory, Edinburg, TX, U.S.A.; (3) CPHST Beltsville Laboratory USDA APHIS PPQ, Beltsville, MD, U.S.A. Understanding fungal reservoirs: Tracking Aspergillus populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1) (1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A. Yield of Georgia-06G decreased linearly, and yield of Georgia-12Ydecreased according to a quadratic function with later planting. For most planting dates, final leaf spot severity and AUDPC were lower, and yield was higher for Georgia-12Y than for Georgia-06G. The combination of early planting with Georgia-12Y shows potential for reducing risks of losses, leaf spot, and maximizing yield in situations such as organic production where fungicide use would be minimal. Fungal profiles in ginger root and cayenne pepper powders from U.S. retail V. TOURNAS (1), J. Kohn (2), E. Katsoudas (2) (1) Center for Food Safety and Applied Nutrition/FDA, U.S.A.; (2) Northeast Regional Lab/Office of R Fungal profiles in ginger root and cayenne pepper powders from U.S. retail V. TOURNAS (1), J. Kohn (2), E. Katsoudas (2) ( ) ( ) Safety and Applied Nutrition/FDA, U.S.A.; (2) Northeast Regional Lab/Office of Regulatory Affairs/FDA, U.S.A. (1) Center for Food Safety and Applied Nutrition/FDA, U.S.A.; (2) Northeast Regional Lab/Office of Regulatory Affairs/FD A limited survey was conducted to determine the mycological quality (mould and yeast levels and profiles) of ginger and cayenne pepper powders sold in local stores in the Washington, D.C. area. Ginger powder samples were purchased from four companies (A, B, C, and D), while cayenne pepper was obtained from two companies (A and B). Powders packed in capsules as well as in bulk bottles and jars were examined. All ginger samples tested during this study contained live fungi. Mould levels ranged between 2.4 × 103 and 6.2 × 104 colony forming units/g (cfu/g). The most common species were Aspergillus flavus and Aspergillus niger followed by eurotia and Fusarium spp. No yeasts were found in any of the samples tested. Some variation in fungal species and contamination levels were apparent among different companies. Higher levels of fungal infestation were observed in cayenne pepper samples. Seventy one percent of the analyzed samples were carrying live moulds while only one sample tested positive for yeasts. Mould counts ranged between <100 and 6.9 × 105 cfu/g. The most frequent fungal contaminants were A. niger, A. flavus, and eurotia (E. chevalieri and E. rubrum) followed by Aspergillus spp. and members of the Mucorales family. Variation in mould levels existed between the two companies and among lots of the same company. Wheat streak mosaic virus differentially affects soft red winter wheat cultivars B. HODGE (1), L. Stewart (2), P. Three races of Xanthomonas campestris pv. vitians causing bacterial leaf spot on lettuce identified C BULL (1) M Trent (2) R Hayes (3) Three races of Xanthomonas campestris pv. vitians causing bacterial leaf spot on lettuce identified C. BULL (1), M. Trent (2), R. Hayes (3) (1) Department of Plant Pathology and Environmental Microbiology, Penn State University, U.S.A.; (2) USDA/ARS, U.S.A.; (3) USDA/ARS, U.S.A. C. BULL (1), M. Trent (2), R. Hayes (3) (1) Department of Plant Pathology and Environmental Microbiology, Penn State University, U.S.A.; (2) USDA/ARS, U.S.A.; (3) USDA/ARS, U.S.A. . Hayes (3) ology and Environmental Microbiology, Penn State University, U.S.A.; (2) USDA/ARS, U.S.A.; (3) USDA/ARS, U.S.A. C. U ( ), . e t ( ), . ayes (3) 1) Department of Plant Pathology and Environmental Microbiology, Penn State University, U.S.A.; (2) USDA/ARS, U.S.A Xanthomonas campestris pv. vitians (Xcv), the causal agent of bacterial leaf spot on lettuce, reduces lettuce quality and yield worldwide. Although strain specific, host resistance is the most feasible method to reduce losses. Therefore, we screened lettuce germplasm for resistance using the broadest diversity within the pathogen population. A collection of 120 strains were assigned to one of six sequence types (A, B1, B2, C, D and E) by multilocus sequence typing (MLST) using concatenated sequences of rpoD, dnaK, fyuA, gyrB, and gap1. The abaxial sides of lettuce leaves were infiltrated with single bacterial isolates (1 × 108 CFU/ml) representing each sequence type. Tissue collapse beginning at 30 hr and resulting in light-brown papery lesions at 48 hr was considered an incompatible reaction. Replicated assays with strains were conducted with germplasm for which resistance was identified. This method was used to determine that the single dominant gene, named Xanthomonas resistance 1 (Xar1), confers complete resistance to MLSTs B, D, and E. Subsequently, germplasm was identified with complete resistance to strains from sequence type A and a separate source of resistance was identified to strains from sequence type C. The genetics of resistance to sequence types A and C is being elucidated and we are completing analysis of all strains on resistant germplasm. However, the data gathered to-date indicate that there are at least three races of Xcv. Candidatus Liberibacter solanacearum associated to Physalis philadelphica, a new solanaceous host H. SILVA-ROJAS (1), A. Contreras-Rendon (2), J. Sanchez-Pale (2) (1) Colegio de Postgraduados, Mexico; (2) Universidad Autonoma del Estado de Mexico, Mexico Candidatus Liberibacter solanacearum (CaLs) bacterium is an economically important plant pathogen of solanaceous is vectored by the psyllid Bactericera cockerelli. Three races of Xanthomonas campestris pv. vitians causing bacterial leaf spot on lettuce identified C BULL (1) M Trent (2) R Hayes (3) Recently, were observed in husk tomato (Physalis philadelphica) fields in Toluca Valley in the state of Mexico, Mexico. Plants exhibiting stunting, severe leaf chlorosis, shortened internodes, curling of leaves, aborted flowers, deformation and reduced fruit size were observed, affecting the productive period. Large numbers of the psyllid B. cockerelli were observed on the crop. The objective of this research was to detect CaLs in husk tomato plants with similar symptoms as described above and whether this bacterium was present in B. cockerelli populations in the field. In 2015, we collected 250 samples from symptomatic plants and five psyliid B. cockerelli per plant in commercial plots in Toluca Valley. The molecular detection of CaLs was performed using OA2/OI2c, CLipoF/OI2c and Lp Frag 1-25F/427R primers. The amplified fragments were sequenced using Sanger methodology. Sequence alignment was carried out with the sequences belonging to the haplotypes currently recognized in GenBank database. The SNPs were identified visually. Haplotype B of CaLs was found in 100% of the plants tested, whereas in the insect found A and B haplotypes. This information will enable the approach of management strategies to husk tomato production. of an unmanned aerial vehicle to assess tomato spotted wilt severity in large peanut mapping populations Use of an unmanned aerial vehicle to assess tomato spotted wilt severity in large peanut mapping populations S. PELHAM (1), C. Holbrook (2), B. Guo (2), Y. Chu (3), P. Ozias-Akins (3), C. Li (4), R. Xu (4), A. Cubreath (1) (1) University of Georgia - Department of Plant Pathology, Tifton, GA, U.S.A.; (2) United States Department of Agriculture - Agricultural Research Service, Tifton, GA, U.S.A.; (3) University of Georgia - Department of Horticulture, Tifton, GA, U.S.A.; (4) University of Georgia - College of Engineering, Athens, GA, U.S.A. ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) (1) University of Georgia - Department of Plant Pathology, Tifton, GA, U.S.A.; (2) United States Department of Agriculture - Agricultural Research Service, Tifton, GA, U.S.A.; (3) University of Georgia - Department of Horticulture, Tifton, GA, U.S.A.; (4) University of Georgia - College of Engineering, Athens, GA, U.S.A. Recombinant inbred line (RIL) populations of peanut (Arachis hypogaea L.) are being used to develop markers for resistance to several diseases, including tomato spotted wilt, caused by Tomato spotted wilt virus (TSWV). A new ‘Candidatus Phytoplasma pini’-related strain associated with witches’-broom of Pinus spp. is detected in Maryland S. COSTANZO (1), J. Rascoe (1), Y. Zhao (2), R. Davis (3), M. Nakhla (1) (1) USDA-APHIS-PPQ-CPHST, U.S.A.; (2) USDA-ARS, U.S.A.; (3) USDA-ARS A new ‘Candidatus Phytoplasma pini’-related strain associated with witches’-broom of Pinus spp. is detected in Maryland S. COSTANZO (1), J. Rascoe (1), Y. Zhao (2), R. Davis (3), M. Nakhla (1) (1) USDA-APHIS-PPQ-CPHST, U.S.A.; (2) USDA-ARS, U.S.A.; (3) USDA-ARS Phytoplasmas are insect transmitted specialized bacteria that cause devastating diseases in crops and natural ecosystems. Pine trees (Pinus spp.) showing abnormal shoot branching and witches’ broom symptoms in a wooded area of Laurel, Maryland, were examined for the presence of phytoplasmas. Nested PCR assays for amplification of the 16S ribosomal RNA gene (rDNA) sequences were used on pine tree DNA samples to confirm possible association of a phytoplasma with the disease. Comparison of restriction fragment length polymorphism (RFLP) patterns and nucleotide sequence analysis of 16S rRNA gene fragments revealed that several trees were infected by a ‘Candidatus Phytoplasma pini’ -related strain (MDPP). The 16S rDNA sequence of MDPP shares 98.4% similarity with that of the ‘Candidatus Phytoplasma pini’ reference strain reported from Spain (isolate P127). The collective 16Sr DNA F2nR2 RFLP pattern is distinct from the reference patterns of all previously delineated subgroups, and is most similar to that of subgroup 16SrXXI-A with a pattern similarity coefficient of 0.79. Since the similarity coefficient value is lower than the threshold (0.97) for new subgroup recognition, MDPP may represent a new subgroup within the group 16SrXXI. The new subgroup is hereby designated as 16SrXXI-B. To our knowledge, this is the first report of a ‘Ca. Phytoplasma pini’-related strain associated with pine trees in North America and possibly the entire western hemisphere. Understanding fungal reservoirs: Tracking Aspergillus populations in air, soil, crop debris and the reproductive organs of Bt and non-Bt corn H. ABBAS (1), M. Weaver (1), C. Accinelli (2), W. Shier (3), R. Zablotowicz (1) (1) USDA-ARS, U.S.A.; (2) University of Bologna, Italy; (3) University of Minnesota, U.S.A. In October 2015, a Mexican lime exhibiting citrus canker-like symptoms was found by the USDA-APHIS-PPQ citrus disease survey in a residential property in Cameron County, Texas. The Texas A&M University-Kingsville Citrus Center detected citrus canker using a real-time PCR analysis. The USDA-APHIS-PPQ Lab in Beltsville, MD confirmed that it was a Xanthomonas citri subsp. citri isolate. A delimiting survey was initiated by USDA and by February 2016, 169 suspect leaf samples were sent to the PPQ Beltsville lab for analysis. Of 118 samples positive for citrus canker, all within a 5- mile radius, 116 were from Mexican lime, 1 was from Kaffir lime and 1 from Ponderosa lemon. Survey data collected on stem lesions suggested the oldest lesions to be between 4 – 6 years old. To examine the host range of the TX citrus canker strain, four pots each of 11 different citrus varieties, including the major commercial varieties grown in TX, were inoculated by syringe infiltration of symptomatic crude leaf extracts. The only citrus plants S4.99 that developed clear canker lesions were Mexican limes. A combination of serological and molecular tests also suggested that the Texas citrus canker isolate was different from the typical Asiatic strain (Xac A) and more similar to other known X. citri subsp. citri strains with limited host range such as Xac AW or A. Xac AW strain was previously reported from Florida on Mexican lime and Alemow, a close relative of Kaffir lime. that developed clear canker lesions were Mexican limes. A combination of serological and molecular tests also suggested that the Texas citrus canker isolate was different from the typical Asiatic strain (Xac A) and more similar to other known X. citri subsp. citri strains with limited host range such as Xac AW or A. Xac AW strain was previously reported from Florida on Mexican lime and Alemow, a close relative of Kaffir lime. The phytoplasma associated with purple woodnettle witches’-broom disease in Taiwan represents a new subgroup of the aster yellows phytoplasma group F. JAN (1), Y. Tseng (1), C. Chang (2) (1) Department of Plant Pathology/ National Chung Hsing University, Taiwan; (2) Department of Plant Pathology/ University of Georgia, Taiwan The phytoplasma associated with purple woodnettle witches -broom disease in Taiwan represents a new subgroup of the aster yellows phytoplasma group F. JAN (1), Y. Tseng (1), C. Chang (2) (1) Department of Plant Pathology/ National Chung Hsing University, Taiwan; (2) Department of Plant Pathology/ University of Georgia, Taiwan ( ), g ( ), g ( ) 1) Department of Plant Pathology/ National Chung Hsing University, Taiwan; (2) Department of Plant Pathology/ Universit In October 2013, a new disease affecting purple woodnettle (Oreocnide pedunculata) plants was found in Miaoli County, Taiwan. Diseased plants exhibited leaf yellowing and witches’-broom symptoms. Molecular diagnostic tools and electron microscopic cell observation were used to investigate the possible cause of the disease with a specific focus on phytoplasmas. The result of PCR with universal primer pairs indicated that phytoplasmas were strongly associated with the symptomatic purple woodnettles. The virtual restriction fragment length polymorphism (RFLP) patterns and phylogenetic analysis based on 16S rDNA and ribosomal protein, rplV-rpsC region revealed that purple woodnettle witches’-broom phytoplasma (PWWB) belongs to a new subgroup of 16SrI and rpI group and was designated as 16SrI-AH and rpI-Q, respectively, herein. RFLP analysis based on tuf gene region revealed that the PWWB belongs to tufI-B, but phylogenetic analysis suggested that PWWB should be delineated to a new subgroup under the tufI group. Taken together, our analyses based on 16S rRNA and rplV-rpsC region gave a finer differentiation while classifying the subgroup of aster yellows group phytoplasmas. To our knowledge, this is the first report of a Candidatus Phytoplasma asteris-related strain in 16SrI-AH, rpI-Q and tufI-B subgroup affecting purple woodnettle, and of an official documentation of purple woodnettle as being a new host of phytoplasmas. Identification of the causal agents of soft rot of potato in Bangladesh and activity of biocontrol agents against the pathogens F. ELAHI (1) (1) The Ohio state University, U.S.A. (1) The Ohio state University, U.S.A. Potato is a staple food crop in Bangladesh. Bacterial soft rot is a major postharvest threat to potato, reducing yield approximately 37% in storage systems. Limited research has been conducted to identify and manage the causal agents of soft rot in Bangladesh. Eighteen bacterial isolates were collected from twelve potato-growing regions of Bangladesh in 2015. Sixteen of the isolates produced deep pits on Crystal-Violet Pectate (CVP) medium, were gram negative, oxidase negative, and hypersensitive response (HR) positive on tobacco, characteristic of Pectobacterium. Two isolates produced shallow pits on CVP medium, were gram negative, oxidase positive, fluorescent, and weakly HR positive, characteristic of Pseudomonas. All isolates caused soft rot on potato tubers. Partial 16s ribosomal DNA sequences from 16 presumptive Pectobacterium isolates exhibited >85% identity with Pectobacterium ssp. sequences and two presumptive Pseudomonas isolates exhibited 90% identity with P. fluorescens and P. putida sequences previously deposited in GenBank. Fifteen Pseudomonas sp. strains with known biocontrol activity were tested for in vitro antibiosis against the soft rotting isolates. The biocontrol agents inhibited the growth of all Pectobacterium isolates but neither of the Pseudomonas isolates. Biological control of potato soft rot by Pseudomonas spp. is promising but additional research is needed to identify potential biocontrol agents for soft rotting Pseudomonas spp. Race characterization of Pyrenophora tritici-repentis population from Lithuania and Romania A. SIDRAT (1), S. Ali (2), Z. Liatukas (3), M. Ittu (4), S. Sehgal (2), N. Kaur (2) Race characterization of Pyrenophora tritici-repentis population from Lithuania and Romania A. SIDRAT (1), S. Ali (2), Z. Liatukas (3), M. Ittu (4), S. Sehgal (2), N. Kaur (2) (1) South Dakota State University, Brookings, SD, U.S.A.; (2) South Dakota State Universty, Brookings, SD, U.S.A.; (3) Institute of Agriculture, Lithuania Research Center for Agriculture and Forestry, Instituto al., Akademija 58344, Lithuania; (4) National Agricultural Research and Development Institute Fundulea, Calarasi, Romania y p p p p A. SIDRAT (1), S. Ali (2), Z. Liatukas (3), M. Ittu (4), S. Sehgal (2), N. Kaur (2) (1) South Dakota State University, Brookings, SD, U.S.A.; (2) South Dakota State Universty, Brookings, SD, U.S.A.; (3) Institute of Agriculture, Lithuania Research Center for Agriculture and Forestry, Instituto al., Akademija 58344, Lithuania; (4) National Agricultural Research and Development Institute Fundulea, Calarasi, Romania Tan spot, caused by Pyrenophora tritici-repentis (Ptr) is an economically important disease of wheat. Eight races exist of this fungal population. Three races of Xanthomonas campestris pv. vitians causing bacterial leaf spot on lettuce identified C BULL (1) M Trent (2) R Hayes (3) In efforts to develop molecular markers to assist in selection for resistance to TSWV, several populations have been developed from parents with varying levels of TSWV resistance. Mapping programs are faced with the challenge of phenotyping large numbers of genotypes in a timely manner with a limited number of people. Through the use of an unmanned aerial vehicle, imaging techniques, and ground truthing, we developed an analysis method to quickly assess spotted wilt severity in the field using selected RILs from four mapping populations of peanut. With adequate ground truthing, spotted wilt is easily differentiated from other diseases in the field by the color and the visible stunting of the plant. By using a greenness equation in Matlab to identify yellow pixels we can rate the severity of the disease in S4.100 many small plots in a short amount of time. Both Matlab greenness values and severity values from APS Assess analysis of aerial images correlated well with field disease ratings. This technique allows for quick acquisition of field response data from large mapping populations. many small plots in a short amount of time. Both Matlab greenness values and severity values from APS Assess analysis of aerial images correlated well with field disease ratings. This technique allows for quick acquisition of field response data from large mapping populations. Reporting the occurrence of Ralstonia based wilts of chillies in major vegetable growing areas of Punjab A. HAMEED (1), S. Jameel (1), N. Liaqat (1), M. Ashfaq (2), R. Riaz (3), K. Riaz (1) (1) Department of Plant Pathology, University of Agriculture Faisalabad, Pakistan; (2) Department of Plant Pathology, PMAS-Arid Agriculture University Rawalpindi, Pakistan; (3) Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad-38040, Pakistan, Faisalabad, Pakistan A. HAMEED (1), S. Jameel (1), N. Liaqat (1), M. Ashfaq (2), R. Riaz (3), K. Riaz (1) (1) Department of Plant Pathology, University of Agriculture Faisalabad, Pakistan; (2) Department of Plant Pathology, PMAS-Arid Agriculture University Rawalpindi, Pakistan; (3) Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad-38040, Pakistan, Faisalabad, Pakistan ( ), q ( ), q ( ), ( ), ( ) Pathology, University of Agriculture Faisalabad, Pakistan; (2) Department of Plant Pathology, PMAS-Arid Agriculture akistan; (3) Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad-38040, Pakistan, Ralstonia solanacearum (Rs), is one of the most severe pathogens of solanaceous crops with a very wide host range. A metagenomic study was conducted to identify viruses present in 180 leaf samples of Saccharum species collected in 2013/2014 from plants in commercial fields in the Everglades Agricultural Area and the germplasm collection of the USDA-ARS Sugarcane Field station at Canal Point, FL. The objective of this study was to identify known and potentially new sugarcane viruses. In addition to previously reported sugarcane viruses in Florida, we identified the presence of a potential new Mastrevirus species. Since there are no Mastrevirus species reported from Florida, and species in this genus such as Sugarcane streak virus and Maize streak virus are known to cause diseases in sugarcane, efforts were made to obtain full length viral genome sequences from individual plants of Saccharum spp. Rolling circle amplification followed by enzymatic restriction and cloning was used to obtain nine full genome length sequences (each approximately 2.8 kb): three from S. barberi, two from S. officinarum and four from S. spontaneum, all from the germplasm collection. All nine genome sequences appeared to represent a new species of Mastrevirus after their pairwise comparison using the species Morphological and molecular identification of Phoma medicaginis var. medicaginis causing spring black stem and leaf spot of alfalfa in Nevada R. Bomberger (1), S. Wang (1) (1) Nevada Department of Agriculture, U.S.A. (1) Nevada Department of Agriculture, U.S.A. (1) Nevada Department of Agriculture, U.S.A. In June 2015, an outbreak of foliar blight disease was observed in an alfalfa (Medicago sativa L.) field with more than 50% of plants affected. Individual plants exhibited rapid death of foliage with sporadic or coalesced lesions on both lower leaves and stems. To isolate the causative agent, necrotic stem and leaf tissue, as well as pycnidia, were surface sterilized and then placed onto potato dextrose agar (PDA) amended with streptomycin sulfate and incubated at 22ºC in the dark. Colonies of Phoma species were obtained from all samples collected from 5 separate field locations. Ten single spore cultures (SSCs) were obtained from 10 isolates and compared morphologically. All SSCs and isolates produced almost black colonies with abundant hyaline, oval to cylindrical, single-celled conidia with no significant morphological variation observed. A 540 bp DNA fragment was amplified from the ITS regions of rRNA genes and it was sequenced directly from both strands. Sequence analysis revealed that all 10 SSCs had the same amplicon sequence (Accession No. KU695570), with 100% homology to the corresponding regions of P. medicaginis var. medicaginis (Accession No. AY504634). Thus, the pathogen isolated from diseased alfalfa plants in Nevada was determined to be P. medicaginis var. medicaginis, the only pathogen associated with the outbreak. This is the first confirmed outbreak of spring black stem disease in Nevada since 1977 when it was initially reported in the state. q ( ), gy p g g g g ( AY504634). Thus, the pathogen isolated from diseased alfalfa plants in Nevada was determined to be P. medicaginis var. medicaginis, the only pathogen associated with the outbreak. This is the first confirmed outbreak of spring black stem disease in Nevada since 1977 when it was initially reported in the state Identification of a new Mastrevirus of Saccharum barberi, Saccharum officinarum and Saccharum spontaneum in Florida W. BOUKARI (1), R. Alcala-Briseño (2), S. Kraberger (3), E. Fernandez (4), D. Filloux (4), J. Comstock (5), A. Varsani (3), P. Roumagnac (4), J. Polston (1), P. Identification of the causal agents of soft rot of potato in Bangladesh and activity of biocontrol agents against the pathogens F. ELAHI (1) (1) The Ohio state University, U.S.A. In this study, 223 single spore isolates from Lithuania (n=114), and Romania (n=109) were evaluated for their race structure through genotypic and phenotypic characterization. Molecular variation was identified through PCR with Ptr ToxA and Ptr ToxB genes specific primer. Of these 223 isolates genotyped, 110 isolates were further phenotyped by inoculating them individually on two-week old seedlings of tan spot wheat differentials Glenlea, 6B365, 6B365, and Salamouni. Nearly 86% (191/223) isolates genotyped contained ToxA genes. Phenotypic characterizations on 110 isolates identified 69, 6, 13, and 4 isolates Ptr race 1, 2, 3, and 4, respectively. Eighteen of 223 isolates lacking in Ptr ToxA gene could not fit into currently identified 8 races as they produced necrosis on Glenlea and chlorosis on 6B365. These results indicate that a diverse Ptr population exists in Lithuania and Romania. This information would help the regional wheat breeding programs in the development of durable tan spot resistance cultivars. Three races of Xanthomonas campestris pv. vitians causing bacterial leaf spot on lettuce identified C BULL (1) M Trent (2) R Hayes (3) Recently, a survey was conducted in districts of Punjab Province including Okara, Chiniot and Faisalabad to determine the incidence of bacterial wilt of chillies. The maximum disease incidence (58%) was recorded in district Okara followed by Chiniot (43%). The least disease incidence was found in district Faisalabad with 20% occurrence. The samples were collected and after surface sterilization, placed on casamino acid-peptone-glucose-agar (CPG) medium. The colonies obtained on CPG medium were transferred on 2,3,5-triphenyltetrazolium chloride (TZC) medium. It was observed that the colonies on TZC were irregular, mucoid and white with pink centers. Out of all the isolates tested, 72% showed positive hypersensitive reaction (HR) with variable response. Only 40% of these HR positive isolates were confirmed as R. solanacearum through simple staining, streaming, KOH solubility, biochemical, pathogenicity and Plate Trapped Antigen-Enzyme Linked Immunosorbent Assay (PTA-ELISA) tests. Molecular confirmation of R. solanacearum was also done through genomic DNA using Rs-specific primers. Fusarium canker on holly in Norway Fusarium canker on holly in Norway G. STRØMENG (1), V. Talgø (1), A. Stensvand (1), J. Razzaghian (1) (1) NIBIO, Norway Holly (Ilex aquifolium) thrives well in the mild and wet costal climate of southwestern Norway, where it is commonly harvested for Christmas decorations. During the last decade, a Fusarium sp. has frequently been found to damage both natural and cultivated stands, causing leaf drop, dead twigs, canker wounds and berry rot. The most severe outbreaks of the disease ever occurring were observed in late autumn 2015. Isolates of the fungus are most similar to F. acuminatum (97% identical TEF sequences). On one occasion, the fungus was detected on dead shoots of Nordmann fir (Abies nordmanniana). Inoculation tests proved it pathogenic to both host plants. Many holly stands are no longer intensively cultivated, and they have become very dense and overgrown by other vegetation, thus creating an ideal microclimate for fungal pathogens. In an investigation in a natural stand in 2006- 2009, the quality of the holly improved by letting more air and light into the dense plantation by different combinations of pruning, thinning, clearance of unwanted trees and bushes, and grazing with sheep. Thus, it is possible to gain control with the disease by sanitation and proper management, and thereby increase both the quality and quantity for marketing. There is a potential market in other Nordic countries where the climate is too harsh for holly, but earlier attempts of export fell through due to quality issues. Fungal Diseases of Stevia rebaudiana Grown in Eastern Tennessee T. COLLINS (1), M. Dee (1), H. Korotkin (1), D. Hensley (1), B. Ownley (1) (1) University of Tennessee, U.S.A. Extract of Stevia rebaudiana, a plant native to Brazil and Paraguay, is a substitute for sugar to sweeten food and drink products. In the last five years, acreage of stevia for commercial production has increased significantly in the southeastern USA. Crops produced in large acreage monoculture are more susceptible to pathogens; potentially destroying whole crops or limiting crop yields. The aim of this study was to identify fungi recovered from diseased stevia field plants grown in Eastern Tennessee. Fungi were screened for pathogenicity in detached stevia leaf assays. Isolates that caused significant necrosis on detached leaves were identified and evaluated in replicated, greenhouse plant disease assays. Pathogens with significant potential to cause disease were identified as Fusarium armeniacum, Botrytis cinerea, and Alternaria alternata. Morphological and molecular identification of Phoma medicaginis var. medicaginis causing spring black stem and leaf spot of alfalfa in Nevada R. Bomberger (1), S. Wang (1) (1) Nevada Department of Agriculture, U.S.A. Rott (6) (1) U i it f Fl id G i ill FL U S A (2) U i it f Fl id G i ill U S A (3) U i it f C t b Ch i t h h N Identification of a new Mastrevirus of Saccharum barberi, Saccharum officinarum and Saccharum spontaneum in Florida W. BOUKARI (1), R. Alcala-Briseño (2), S. Kraberger (3), E. Fernandez (4), D. Filloux (4), J. Comstock (5), A. Varsani (3), P. Roumagnac (4), J. Polston (1), P. Rott (6) (1) University of Florida, Gainesville, FL, U.S.A.; (2) University of Florida, Gainesville, U.S.A.; (3) University of Canterbury, Christchurch, New Zealand; (4) CIRAD, Montpellier, France; (5) USDA-ARS, Canal Point, FL, U.S.A.; (6) University of Florida, Belle Glade, FL, U.S.A. A metagenomic study was conducted to identify viruses present in 180 leaf samples of Saccharum species collected in 2013/2014 from plants in commercial fields in the Everglades Agricultural Area and the germplasm collection of the USDA-ARS Sugarcane Field station at Canal Point, FL. The objective of this study was to identify known and potentially new sugarcane viruses. In addition to previously reported sugarcane viruses in Florida, we identified the presence of a potential new Mastrevirus species. Since there are no Mastrevirus species reported from Florida, and species in this genus such as Sugarcane streak virus and Maize streak virus are known to cause diseases in sugarcane, efforts were made to obtain full length viral genome sequences from individual plants of Saccharum spp. Rolling circle amplification followed by enzymatic restriction and cloning was used to obtain nine full genome length sequences (each approximately 2.8 kb): three from S. barberi, two from S. officinarum and four from S. spontaneum, all from the germplasm collection. All nine genome sequences appeared to represent a new species of Mastrevirus after their pairwise comparison using the species S4.101 demarcation tool (SDTversion1.2). Sequence similarity among sequences also suggested the presence of two strains of this new virus. This new species was found in plants that were introduced to Florida more than six decades ago. demarcation tool (SDTversion1.2). Sequence similarity among sequences also suggested the presence of two strains of this new virus. This new species was found in plants that were introduced to Florida more than six decades ago. Mycosphaerellaceae associated with greasy spot of citrus in different climatic regions A. VICENT (1), V. Aguilera-Cogley (2), J. Armengol (3), M. Morphological and molecular identification of Phoma medicaginis var. medicaginis causing spring black stem and leaf spot of alfalfa in Nevada R. Bomberger (1), S. Wang (1) (1) Nevada Department of Agriculture, U.S.A. First report of Botrytis pseudocinerea in blueberry fields in southern Chile E. BRICEÑO (1), E. Briceño (2), S. Aguirre (3), A. Behn (2) (1) Universidad Austral de Chile, Chile; (2) Universidad Austral Chile, Chile; (3) Univ austral Chile, Chile First report of Botrytis pseudocinerea in blueberry fields in southern Chile E. BRICEÑO (1), E. Briceño (2), S. Aguirre (3), A. Behn (2) (1) Universidad Austral de Chile, Chile; (2) Universidad Austral Chile, Chile; (3) Univ austral Chile, Chile Botrytis cinerea is the most important pathogen in blueberry crops (Vaccinium spp.) in southern Chile, where the rainy and windy weather are highly favorable to its reproduction. Due we had observed a high morphological variability on Botrytis isolates from different commercial fields, the aim of this work was to perform a molecular study of isolates of Botrytis associated with blueberry gray mold. To estimate the diversity of this pathogen, 39 monosporic cultures of Botrytis obtained from mature blueberry fruits from eight locations (Lat. 39º06’S to 41º28’S) and eight cultivars were genetically characterized. Their genotype was determinate by PCR detection of transposons Boty and Flipper. In addition, coding fragment for the MS457 gene was sequenced and a phylogenetic tree was built using the Neighbor-joining and UPGMA clustering method. The genetic studies showed the presence of four genotypes, predominating Vacuma (57%) in the geographic region, then Boty (18%), Flipper (15%) and finally Transposa (10%). MS457 gene only was present in 10/39 isolates, its homology and phylogenetic analysis showed the presence of two sympatric populations of Botrytis on blueberry, allowed us to identify eight isolates as B. cinerea and two isolates as Botrytis pseudocinerea. In our knowledge this is the first report de B. pseudocinerea in blueberries in Chile. Morphological and molecular identification of Phoma medicaginis var. medicaginis causing spring black stem and leaf spot of alfalfa in Nevada R. Bomberger (1), S. Wang (1) (1) Nevada Department of Agriculture, U.S.A. Berbegal (3) (1) Instituto Valenciano de Investigaciones Agrarias (IVIA). Moncada 46113, Valencia, Spain; (2) Laboratorio de Protección Vegetal, Instituto de Investigaciones Agropecuarias de Panamá (IDIAP), Los Canelos – Santa María, Panamá., Panama; (3) Instituto Agroforestal Mediterráneo, Universitat Politècnica de València. 46022, Valencia, Spain., Valencia, Spain Mycosphaerellaceae associated with greasy spot of citrus in different climatic regions A. VICENT (1), V. Aguilera-Cogley (2), J. Armengol (3), M. Berbegal (3) (1) Instituto Valenciano de Investigaciones Agrarias (IVIA). Moncada 46113, Valencia, Spain; (2) Laboratorio de Protección Vegetal, Instituto de Investigaciones Agropecuarias de Panamá (IDIAP), Los Canelos – Santa María, Panamá., Panama; (3) Instituto Agroforestal Mediterráneo, Universitat Politècnica de València. 46022, Valencia, Spain., Valencia, Spain ( ), g g y ( ), g ( ), g ( ) (1) Instituto Valenciano de Investigaciones Agrarias (IVIA). Moncada 46113, Valencia, Spain; (2) Laboratorio de Protección Vegetal, Instituto de Investigaciones Agropecuarias de Panamá (IDIAP), Los Canelos – Santa María, Panamá., Panama; (3) Instituto Agroforestal Mediterráneo, Universitat Politècnica de València. 46022, Valencia, Spain., Valencia, Spain Citrus greasy spot is distributed in humid areas in the Caribbean and Central America. The disease is characterized by yellow-brown spots on leaves and premature defoliation. Symptoms similar to greasy spot were observed in semi-arid areas in the Mediterranean Basin, although with low severity. The fungus Zasmidium citri-griseum (= Mycosphaerella citri) has been identified as the causal agent of greasy spot in the Caribbean and Central America, but disease etiology in the Mediterranean is unknown. A selection of 42 isolates of Mycosphaerellaceae obtained from greasy spot symptoms in humid areas in Ghana and Panama and arid regions in Spain and Morocco was studied. Isolates were characterized based on morphology, growth rate at different temperatures, multi-locus analysis (ITS, EF-1α and RPB2), and pathogenicity tests. Isolates from Panama and Ghana (n=24) were identified as Z. citri-griseum whereas isolates from Spain and Morocco (n=20) were identified as Amycosphaerella africana. The optimum growth temperature of Z. citri-griseum was 27.9ºC while A. africana grew better at 22.8ºC. Isolates of Z. citri-griseum from Panamá were pathogenic to ‘Valencia’ sweet orange plants. None of the A. africana isolates from Spain induced symptoms in ‘Ortanique’ plants. These results confirmed Z. citri-griseum as the causal agent of greasy spot in Panama, being associated also with the disease in Ghana. Greasy spot symptoms in Spain and Morocco were associated with A. africana. Identifying and characterizing fungal pathogens causing seedling diseases of soybean through a multi-state surve A. WARNER (1), J. Bond (2), A. Fakhoury (2) (1) Southern Illinois University Carbondale, U.S.A.; (2) Southern Illinois University Carbondale, U.S.A. Identifying and characterizing fungal pathogens causing seedling diseases of soybean through a multi-state survey A. WARNER (1), J. Bond (2), A. Fakhoury (2) (1) Southern Illinois University Carbondale, U.S.A.; (2) Southern Illinois University Carbondale, U.S.A. Seedling diseases are common and very detrimental to soybean, Glycine max. They are caused by a variety of plant pathogens including fungi, bacteria, and oomycetes. The central goal of this project was to identify fungal pathogens causing seedling diseases of soybean in various soybean producing states. Two approaches were used for genus and species determination, including morphological features using microscopy and the sequencing of specific genetic loci. For sequencing, up to three different barcodes were targeted, the internal transcribed spacer (ITS), the elongation factor EF-1α, and the intergenic spacer (IGS). The data produced from this project included the identification of approximately 150 fungal isolates per state from eight different states: Arkansas, Indiana, Illinois, Iowa, Nebraska, Michigan, Minnesota, and Kansas. Isolates were collected in 2012 and 2013 to help determine the predominant causal agents of seedling diseases of soybean in those surveyed locations. A variety of genera were isolated from the fields surveyed, with Fusarium species constituting approximately 74% of the isolates. Data analyses also involved determining associations and correlations between different factors, such as cultural practices, soil physical and chemical properties, irrigation practices, cropping history, and the frequency of the incidence of the species in the surveyed locations. Fusarium canker on holly in Norway The resistance of the 11 most important table cultivars to Neofusicoccum mediterraneum and Botryosphaeria dothidea, the causal agent of “escudete” (small shield) of fruit, was also studied by inoculation of branches and immature fruits, respectively. The species Cytospora pruinosa, N. mediterraneum, Nothophoma quercina, Phoma incompta, and Phomopsis sp. were identified. Only N. mediterraneum and P. incompta were able to induce the typical dieback symptoms and cankers that affected plants development. N. mediterraneum was the most virulent of the evaluated species although difference of virulence among their isolates were observed. The remaining fungal species were weakly pathogenic on potted plants. According to resistance tests, cv. Gordal Sevillana followed by the cvs. Manzanilla Cacereña and Manzanilla de Sevilla were the most susceptible to branch dieback caused by N. mediterraneum. Furthermore, the fruits of cv. Aloreña de Atarfe followed by those of cvs. Ocal and Manzanilla de Sevilla were the most susceptible to B. dothidea. Knowledge of the etiology and cultivar resistance of these diseases will help to establish better control measures. Identification of Alternaria species associated with tomato and potato early blight disease in Oman Identification of Alternaria species associated with tomato and potato early blight disease in Oman H. MAYTON (1), A. Al-Rubaii (2), S. Al-Kaabi (2), M. Al-Jabri (2), R. Al-Maqbali (2), A. Al-Adawi (2) (1) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman; (2) Ministry of Agriculture and Fisheries Sultanate of Oman, Oman Identification of Alternaria species associated with tomato and potato early blight disease in Oman H. MAYTON (1), A. Al-Rubaii (2), S. Al-Kaabi (2), M. Al-Jabri (2), R. Al-Maqbali (2), A. Al-Adawi (2) 1) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman; (2) Ministry of Agriculture and Oman H. MAYTON (1), A. Al-Rubaii (2), S. Al-Kaabi (2), M. Al-Jabri (2), R. Al-Maqbali (2), A. Fusarium canker on holly in Norway Fungal identification was based on morphological charact- eristics and sequence identity of the internal transcribed spacer region of 18S ribosomal DNA, and translation elongation factor 1-alpha. Nucleotide amplicons obtained with PCR, were compared with published sequences in Fusarium-ID and the National Center for Biotechnology Information. In greenhouse assays, inoculated plants exhibited necrotic leaf lesions, similar to those observed in field plants. The three fungi were re-isolated from inoculated plants and identity was re-confirmed. Early identification and management of known stevia pathogens will help reduce crop loss. S4.102 Interaction between a rust fungus and Colletotrichum truncatum J. DIANESE (1), É. Souza (1), H. Vale (1), R. Pereira-Carvalho (1) (1) Departamento de Fitopatologia, Universidade de Brasília, Brazil Mixed natural infection by Uromyces and Colletotrichum species in Euphorbia hirta lead to characteristic symptoms with rust pustules surrounded by black setose acervuli, formed exclusively in the presence of the rust fungus. Besides morphologically identified with precision, U. euphorbiae, was now characterized comparing sequences of the 28S rDNA with GenBank accesses of Uromyces species. Bayesian Inference (BI) and Maximum Likelihood (ML) analyses showed that U. euphorbiae is phylogenetically close to the type species, U. appendiculatus, and well distinguished from the remaining species considered, including those pathogenic to Euphorbiaceae. The U. euphorbiae type species reported on E. hypericifolia was collected in Albany, New York, USA in 1873. As the sequences from our specimen from Brazil, far from the type location, are not suited for its epitypification, it is here designated as a reference specimen, obtained from genomic DNA extracted from teliospores, and deposited in GenBank under number: KU133293, corresponding to our herbarium voucher: UB Mycol. Col. 22311. Two Colletotrichum isolates, one endophytic from symptomless areas of the leaves, and another from an acervulus close to rust pustules were isolated. For both isolates, sequences of the ITS, GAPDH, BTUB, and HIS were obtained and subjected to BI and ML analyses. The result clearly indicated, confirming the morphological analyses, that both isolates belong to the same species, C. trucatum. Thielaviopsis punctulata Causes Black Scorch Disease on Date Palm in the United Arab Emirates K. EL-TARABILY (1), S. AbuQamar (1), E. Saeed (1), A. Sham (1) (1) United Arab Emirates University, UAE Date palm (Phoenix dactylifera L.) is one of the most important plants grown in the arid and semi-arid land that tolerates extreme environmental desert conditions. Fusarium canker on holly in Norway The main associated pathogens causing palm diseases are attributed to fungi and phytoplasma. In the United Arab Emirates (UAE), the causal agent of black scorch on date palm was found to be the fungal pathogen, Thielaviopsis punctulata. The pathogen was isolated from all tissues of diseased trees with virulent Thielaviopsis punctulata. Depending on the severity of the infection, symptoms included tissue necrosis, wilting, neck bending, death of terminal buds, and eventually plant mortality. This fungus, which was consistently isolated on potato dextrose agar from infected tissues, produced two types of conidia: the thick-walled aleuroconidia (chlamydospores) and phialoconidia (endoconidia). In addition, all target regions of 5.8S rRNA (ITS), 28S rDNA, β-tubulin and transcription elongation factor 1-α (TEF1-α) genes were amplified using polymerase chain reaction. We also found that the fungicide Score® inhibited the mycelial growth of Thielaviopsis punctulata both in vitro and in vivo. Altogether, the morphology of the fruiting structures, pathogenicity tests and molecular identification confirmed that the causal agent of symptomatic tissues is Thielaviopsis punctulata. This is the first report of the black scorch disease and the fungus Thielaviopsis punctulata on date palm in the UAE. Fungal Species Associated with Olive Dieback of Branches and Evaluation of Cultivar Resistance to Botryosphaeriaceae species J. MORAL (1), C. Agustí-Brisach (2), M. Pérez-Rodríguez (2), C. Xavíer (2), A. Rhouma (3), A. Trapero (2) (1) University of California, Davis, Kearney Agricultural Research and Extension Center, Parlier, CA, U.S.A.; (2) Departamento de Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C4, 14071, Cordoba, Spain; (3) Lab. Improvement and protection of olive genetic resources, Olive Tree Institute, BP 208 Mahrajene 9 City– 1082, Tunis, Tunisia Fungal Species Associated with Olive Dieback of Branches and Evaluation of Cultivar Resistance to Botryosphaeriaceae species J. MORAL (1), C. Agustí-Brisach (2), M. Pérez-Rodríguez (2), C. Xavíer (2), A. Rhouma (3), A. Trapero (2) (1) University of California, Davis, Kearney Agricultural Research and Extension Center, Parlier, CA, U.S.A.; (2) Departamento de Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C4, 14071, Cordoba, Spain; (3) Lab. Improvement and protection of olive genetic resources, Olive Tree Institute, BP 208 Mahrajene 9 City– 1082, Tunis, Tunisia Over two consecutive seasons, 16 olive orchards with trees exhibiting dieback symptoms of branches were surveyed in Southern Spain. The six most frequent fungal species recovered were morphologically, molecularly (by sequencing of the ITS, TUB, and LSU regions), and pathogenically characterized. Fusarium canker on holly in Norway Al-Adawi (2) (1) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman; (2) Ministry of Agriculture and Fisheries Sultanate of Oman, Oman ( ), ( ), ( ), ( ), q ( ), ( ) 1) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman; (2) Ministry of Agriculture and Fis Oman ( ), ( ), ( ), ( ), q ( ), ( ) (1) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman; (2) Ministry of Agriculture and Fisheries Sultanate of Oman, O In Oman, approximately 1400 hectares of tomatoes are planted each year with an annual production that exceeds 74000 tons. Tomatoes are ranked as the most important vegetable crop grown by local farmers. Early blight is a devastating disease in Oman and affects both tomato and potato production. In order to manage the disease more effectively, the Ministry of Agriculture and Fisheries in Oman initiated a project to characterize the pathogen population. Samples were collected from infected tomato and potato plants in commercial production fields during the 2014 and 2015 growing seasons from states in the north and south Al-Batinah governates in Oman. Alternaria spp. were isolated from symptomatic tissues. Microscopic examination of cultures from single spore isolations revealed three distinct species: Alternaria tenuissima, A, alternata and A. solani. Molecular identification was achieved through amplification and sequencing of internal transcribed spacer (ITS) and histone 3 gene regions. Phylogenetic analysis of the ITS gene clearly separated Alternaria solani from the other two species. Partial coding of histone 3 gene revealed three clades representing A. alternata (440bp), A. solani (489bp) and A. tennusima (546bp). Pathogenicity tests of the sample isolates on detached tomato leaves of cultivar ‘Ginan’ showed brown lesions similar to symptoms observed in field. No symptoms were observed in water-inoculated control tomato leaves. Vermiform plant-parasitic nematodes on soybean in North Dakota and their relationship with soybean cyst nematode G. YAN (1), G. Yan (2), A. Plaisance (3) Caceres (1) (1) Universidad de Talca, Chile; (2) Univerisdad de Talca, Chile CChilean apple industry is one of the major exporter of fresh apples worldwide with an export volume of 628.247 ton. Maule Region (35°25´) is the most important apple production region in Chile with a 60% of production of fresh apple. The fungal diseases in postharvest, is one of the major problems for Chilean industry of fresh apple. Recently was described speck rot caused by Phacidiopycnis washingtonensis in apple in Chile. During survey for postharvest diseases of apple in 2014-2015, a postharvest rot was observed on stored apple cv. Jonagold and Pink Lady collected in packinghouses. Symptomatic apples (n=40) were characterized by an initial light brown skin discoloration with spongy to firm flesh texture. With the objective to isolate the causal agent, small fragments were collected from diseased tissues and aseptically placed on Corn Meal Agar and PDA. A Phytophthora sp. was consistently isolated. Eight representative Phytophthora isolates were characterized for morphological and molecular identification, pathogenicity tests and sensitivity to fungicide. Based on morphology and phylogenetic analyses, this fungus was identified as Phytophthora syringae. All isolates grew at 0°C. Inoculated apple fruits developed necrotic lesions. The isolates were sensitive to dimethomorph. To our knowledge, this study is the first description of P. syringae causing Phytophthora fruit rot on apple during postharvest in Chile. Phytophthora root and crown rot on field-grown lavender plants in 2015 S. JEFFERS (1), S. Sharpe (1), M. Williamson (2) (1) Clemson University, U.S.A.; (2) Clemson University, U.S.A. Phytophthora nicotianae has a very wide host range, including numerous ornamental plants. In Jun 2015, P. nicotianae was isolated from lavender plants and rooted cuttings (Lavandula angustifolia and L. ×intermedia) with symptoms of Phytophthora root and crown rot (PRCR). The plants recently had been planted in a field in northwestern SC. When this was brought to the attention of the US Lavender Growers Association (USLGA), it was discovered that other growers suspected problems with PRCR—particularly on recently-planted, nursery-grown plants. USLGA members were encouraged to send samples to Clemson University to test for Phytophthora spp. Between Jul and Nov 2015, plant and soil samples were received from 12 states; all plants were cultivars of L. angustifolia or L. ×intermedia. Samples from 11 states were diagnosed with PRCR; Phytophthora spp. were recovered from roots and crowns by isolation on selective media and from soil with a baiting bioassay. Four species of Phytophthora recovered from roots of American and hybrid chestnut seedlings and associated soils in the southeastern US S. SHARPE (1), S. Jeffers (1), S. Clark (2) (1) Clemson University, U.S.A.; (2) USDA Forest Service, U.S.A. Four species of Phytophthora recovered from roots of American and hybrid chestnut seedlings and associated soils in the southeastern US S. SHARPE (1), S. Jeffers (1), S. Clark (2) (1) Clemson University, U.S.A.; (2) USDA Forest Service, U.S.A. Since Phytophthora root rot (PRR) was first described on American chestnut in 1932, the only species of Phytophthora reported as a causal agent has been P. cinnamomi. In Europe, however, multiple species of Phytophthora are involved in PRR of native chestnut trees. In 2009, the USDA Forest Service began evaluating American, Chinese, and hybrid chestnut seedlings in forest test plots in NC, TN, and VA, but PRR caused some seedlings to die prematurely. The objective of this research was to determine if multiple species of Phytophthora may be causing PRR of American and hybrid chestnut seedlings. A total of 260 root and 454 soil samples were assayed from 2010 to 2014. Phytophthora spp. were recovered by isolation from roots on PARPH-V8 selective medium and baiting soil with rhododendron and camellia leaf pieces. All isolates were tentatively identified based on morphology; species identifications will be confirmed by DNA sequences. P. cinnamomi was recovered most frequently—from 41 (16%) root and 109 (24%) soil samples. However, P. cambivora and P. heveae were also recovered—from 18 (7%) and 2 (<1%) root samples and 18 (4%) and 17 (4%) soil samples, respectively. At a separate test site in SC, P. drechsleri was isolated from roots of several hybrid chestnut seedlings. This is the first report of Phytophthora species other than P. cinnamomi associated with PRR of pure and hybrid American chestnut. These species are being tested for pathogenicity to American chestnut. Two new races and several novel strains of the spinach downy mildew pathogen Peronospora farinosa f. sp. spinaciae C. FENG (1), J. Correll (1), K. Saito (1), K. Kammeijer (2), S. Koike (3) (1) University of Arkansas, U.S.A.; (2) University of California, U.S.A.; (3) University of California Cooperative Extension—Mon Downy mildew disease, caused by Peronospora farinosa f. sp. spinaciae (Pfs), is the most economically important disease of spinach. Current high- density spinach production provides conducive conditions for disease development, particularly for the increasing acreages of organic spinach. The use of resistant cultivars to manage downy mildew exerts strong selection pressure on the pathogen and many new races of Pfs have emerged in recent years threatening spinach production worldwide. Vermiform plant-parasitic nematodes on soybean in North Dakota and their relationship with soybean cyst nematode G. YAN (1), G. Yan (2), A. Plaisance (3) Vermiform plant-parasitic nematodes on soybean in North Dakota and their relationship with soybean cyst nematode G. YAN (1), G. Yan (2), A. Plaisance (3) Vermiform plant-parasitic nematodes on soybean in North Dakota and their relationship with soybean cyst nematode G. YAN (1), G. Yan (2), A. Plaisance (3) (1) North Dakota State University, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A.; (3) North Dakota State University, Department of Plant Pathology, U.S.A. ( ), ( ), ( ) (1) North Dakota State University, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A.; (3) North Dakota State University, Department of Plant Pathology, U.S.A. ( ), ( ), ( ) (1) North Dakota State University, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A.; (3) North Dakota State University, Department of Plant Pathology, U.S.A. Plant-parasitic nematodes (PPN) are an important group of pests on soybean. The occurrence and population densities of PPN other than soybean cyst nematode (SCN; Heterodera glycines) are unknown in soybean fields of North Dakota. A survey was conducted in 2015 in soybean fields or fields with history of SCN to ascertain the incidence and abundance of vermiform PPN and their possible association with SCN. Vermiform and cyst nematodes were extracted separately from each sample (100 cm3 soil) and then identified and quantified. Out of the 155 soil samples collected from 14 counties in North Dakota, 144 were found to be infested with PPN. Eight genera of vermiform PPN were detected including Helicotylenchus (incidence: 49%; highest density: 1,800; average density: 174), Tylenchorhynchus (41%; 340; 30), Paratylenchus (37%; 2,480; 151), Pratylenchus (19%; 245; 9), Xiphinema (7%; 180; 4), Paratrichodorous (4%; 60; 1), Hoplolaimus (3%; 140; 2), and Mesocriconema (1%; 300; 2). Both SCN juveniles (24%; 1,200; 46) from soil and SCN eggs (56%; 21,540; 501) from cysts were counted, but weak correlation was found between them for all samples (r = 0.489; p <0.0001). Poor or no correlations between population densities of each genus of vermiform nematodes and SCN were observed with r ranging from - 0.307 to 0.248. More soil samples will be collected in 2016 to confirm the occurrence and abundance of nematodes in soybean fields and the relationship of vermiform nematodes with SCN. Occurrence of Phytophthora syringae causing Phytophthora fruit rot in apple in Chile G. DÍAZ (1), M. Lolas (1), R. Méndez (1), J. Contreras (2), M. Vermiform plant-parasitic nematodes on soybean in North Dakota and their relationship with soybean cyst nematode G. YAN (1), G. Yan (2), A. Plaisance (3) Isolates were tentatively identified by morphology, but species identification will be confirmed by DNA sequences. Three species of Phytophthora were isolated: P. nicotianae was isolated most frequently—from samples from 11 states; P. palmivora was found in samples from 3 states; and Phytophthora sp. was found in one sample. Previously, PRCR has been reported on English lavender, L. angustifolia, but not on hybrid lavender, L. ×intermedia. Lavender plants infected with Phytophthora spp. may have come from the nursery. Authors acknowledgeCONICYT PhD fellowship (MV), CONICYT-GO (MV), USM 131562, FONDECYT 1151174& CBDAL grants(MS). Morphological and Molecular Characterization of Plant-Parasitic Nematodes Populations from Corn Fields in Ohio A. SIMON (1), T. Niblack (1), P. Paul (2), H. Lopez-Nicora (1) (1) Ohio State University, U.S.A.; (2) Ohio State University, U.S.A. Morphological and Molecular Characterization of Plant-Parasitic Nematodes Populations from Corn Fields in Ohio A. SIMON (1), T. Niblack (1), P. Paul (2), H. Lopez-Nicora (1) (1) Ohio State University, U.S.A.; (2) Ohio State University, U.S.A. Results from an Ohio survey in 2013 and 2014 showed that root-lesion nematode, among the most economically important plant-parasitic nematodes, was detected in more than 80% of the 425 fields sampled. Other plant-parasitic nematodes extracted were the lance, stunt, dagger, pin and spiral nematodes, found in 48, 37, 57, and 94% of fields, respectively; however, the impact of these nematodes on corn in Ohio remains unclear. Because damage caused by nematodes varies among genera and species within genera, it is necessary to characterize these nematodes to species. Nematodes were killed, fixed, and mounted before examination with a compound microscope, and identified to species based on quantitative and qualitative morphological characteristics with the aid of diagnostic keys. Three species with distinct morphological features of the genus Pratylenchus were identified as P. penetrans, P. neglectus, and P. thornei. Two species of Tylenchorhynchus were identified as T. silvaticus and T. nudus. Species of the lance, pin, spiral and dagger nematodes, namely Hoplolaimus galeatus, Paratylenchus neoamblycephalus and Helicotylenchus pseudorobustus were also identified based on morphology. Species identity will be further confirmed based on analyses of the D2-D3 regions of 28S rRNA gene and ITS rRNA gene. Findings from this study will be useful for establishing experiments to develop damage relationships and management programs for corn nematodes in Ohio. S4.103 Raspberry leaf curl disease y P. DI BELLO (1), A. Diaz-Lara (2), R. Martin (3) ( ), ( ), ( ) (1) Oregon State University, U.S.A.; (2) Oregon State University`, U.S.A.; (3) USDA-ARS Horticultural Crops Research Unit, U.S.A. Raspberry leaf curl disease (RLCD) was first reported in the late 19th century in eastern Canada and northeastern U.S. and is a sporadic destructive disease of red raspberry. Symptoms include leaf curling, leaf distortion, leaf chlorosis, shoot dwarfing, and shoot proliferation. Infected plants produce crumbly, unmarketable fruit, show severe decline within a few years and are much more susceptible to winter injury, which can kill infected plants. RLCD is vectored by the aphid Aphis rubicola in a persistent manner. Based on transmission properties it is suspected RLCD is caused by a virus or virus complex. To determine the viral etiology of RLCD, total nucleic acids were extracted from two red raspberry plants showing typical symptoms of RLCD, digested with DNase, rRNA removed and finally subjected to next generation sequencing on the Illumina Miseq platform using the paired end 150 bp kit. Sequences were analyzed with the VirFind pipeline and specific primers were designed to validate potential virus-like sequences. RLCD infected plants were grafted to blackberries and raspberries, and vector studies with aphids were undertaken. The virome of the plants exhibiting RLCD- like symptoms will be presented. Current Status of Grapevine Virus Diseases in British Columbia S. POOJARI (1), J. Boulé (1), N. DeLury (1), T. Lowery (1), M. Rott (2), A. Schmidt (2), J. Úrbez Torres (1) (1) Summerland Research and Development Centre, Canada; (2) Canadian Food Inspection Agency, Canada Grapevine leafroll disease (GLRD), caused by Grapevine leafroll associated viruses (GLRaVs), is considered the most wide spread virus disease of grapevine (Vitis vinifera L.) worldwide. British Columbia (BC) is the second largest grape-growing region in Canada with most of the acreage planted in the Okanagan Valley. To understand the epidemiology of GLRaVs in BC vineyards, a comprehensive survey was conducted during the 2014 and 2015 growing seasons throughout all wine growing regions of BC. The status of GLRaVs in BC was determined by analyzing a total of 2740 random composite samples representing 12 white and 9 red cultivars. Both ELISA and PCR based diagnostic test results showed GLRaV-3 to be the most prevalent (23.9%) followed by GLRaV-2 (7.1%), GLRaV-1 (2.7%) and GLRaV-4 (2.7%). Four species of Phytophthora recovered from roots of American and hybrid chestnut seedlings and associated soils in the southeastern US S. SHARPE (1), S. Jeffers (1), S. Clark (2) (1) Clemson University, U.S.A.; (2) USDA Forest Service, U.S.A. Internal transcribed spacer (ITS) and cytochrome c oxidase 1 (Cox1) gene regions were amplified and sequenced. BLAST search for ITS and Cox1 sequences of isolates collected from Oman showed 100% similarity with sequences of P. infestans isolates available in genebank (NCBI). To confirm pathogencity of P. infestans in tomato, three isolates were selected to fulfill Koch’s postulates. A spore suspension with concentration of 2.5 × 104 sporangia/ml of each isolate was inoculated on tomato leaves of the cultivar “Ginan” and incubated at 18 Co for 12 days. Symptoms of water soaked and greyish black lesions were observed on inoculated leaves while tomato leaves inoculated with sterile water remained healthy. Red clover vein mosaic virus (RCVMV), a recently reported disease agent from alfalfa, cultivated crops and weeds in Saudi Arabia I. ALSHAHWAN (1), I. AlShahwan (2) (1) King Saud University, Saudi Arabia; (2) College of Food and Agric. Sciences, King Saud University, Saudi Arabia Red clover vein mosaic virus (RCVMV), a recently reported disease agent from alfalfa, cultivated crops and weeds in Saudi I. ALSHAHWAN (1), I. AlShahwan (2) (1) King Saud University, Saudi Arabia; (2) College of Food and Agric. Sciences, King Saud University, Saudi Arabia During a two years survey (2012-2013), 1166 samples were collected from symptomatic alfalfa plants in the major alfalfa-producing regions in Saudi Arabia to detect RCVMV and other viruses. Fourteen samples were positive by DAS-ELISA to RCVMV. In 2014, a detailed study was performed to determine the status and to characterize RCVMV where 315 samples were collected from the three utmost producing regions. Fifty six samples were positive, which were confirmed by RT-PCR. Re-PCR products of 932 bp were obtained from the cDNA of RCVMV-infected plants. In the host range study five (alfalfa, black eye pea, cowpea, Gomphrena and glutinosa tobacco) out of 17 plant species resulted in variable symptoms which were confirmed by ELISA, PCR and nucleic acid hybridization assay. This study reports for the first time that cowpea, black eye pea and Nicotiana glutinosa are hosts for RCVMV. This is the first detailed report regarding RCVMV infecting alfalfa in Saudi Arabia and its characterization. Data regarding infection of field crops and weeds with this virus will be included. Sequence Homology tree revealed high CP sequence similarity between the Saudi isolates of RCVMV (99-100%), but low CP sequence similarity between the Saudi isolates and other foreign isolates of RCVMV (35%). Four species of Phytophthora recovered from roots of American and hybrid chestnut seedlings and associated soils in the southeastern US S. SHARPE (1), S. Jeffers (1), S. Clark (2) (1) Clemson University, U.S.A.; (2) USDA Forest Service, U.S.A. To monitor race diversity of Pfs, downy mildew samples were collected from major spinach production areas and tested for race identification with a standard set of differentials. Two new races (races 15 and 16, sanctioned by the IWGP) and several novel strains have been identified in the past 3 years. The disease reactions of Pfs15 (isolate UA4712) was identical to that of race 4 except the race 4 could not overcome the RPF9 locus resistance and race 15 could. The RPF1, 2, 4, and 6 loci provided resistance to Pfs15. Pfs16 (isolate UA201519B) could overcome the resistance of RPF2, 4, 5, 9, and 10 but not RPF1, 3, and 6. One novel strain (UA1014APLP) could overcome all known spinach resistant loci, but it only infects cotyledons and not true leaves of some cultivars. None of the 360 USDA spinach germplasm accessions tested has resistance to Pfs16 or UA1014APLP. Improved resistance is needed for protection of spinach to the highly variable spinach downy mildew pathogen. S4.104 First report of tomato late blight disease in Oman First report of tomato late blight disease in Oman H. MAYTON (3), S. Al-Kaabi (1), M. Al-Jabri (1), W. Al-Shibili (1), W. Fry (2), A. Al-Adawi (1) (1) Ministry of Agriculture and Fisheries Sultanate of Oman, Oman; (2) Cornell University, Ithaca, NY, U.S.A.; (3) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman p g H. MAYTON (3), S. Al-Kaabi (1), M. Al-Jabri (1), W. Al-Shibili (1), W. Fry (2), A. Al-Adawi (1) (1) Ministry of Agriculture and Fisheries Sultanate of Oman, Oman; (2) Cornell University, Ithaca, NY, U.S.A.; (3) Ministry of Agriculture and Fisheries Sultanate of Oman, Cornell University, Oman Prior to 2013, tomato late blight had not been reported from the Sultanate of Oman. Tomato is an important vegetable crop, with approximately 1400 ha of tomatoes in Oman. However, in 2013 a disease resembling late blight was observed in several states, with some farms reporting destruction of 100% of the area planted with tomatoes and decrease in production of 30-90% for other farms. The causal agent of late blight, Phytophthora infestans, was successfully isolated from infected plant tissues in February and March 2013. Identification of P. infestans was achieved through microscopic observation and molecular analyses. Sporangia were lemon shaped, semipapillate and caduceus ranging from 36 to 41 µm in length and 13 to 30 µm in width. The prevalence of grapevine leafroll and red blotch diseases in Washington vineyards J. ADIPUTRA (1), P. Swamy (1), B. Donda (1), B. Bagewadi (1), N. Natra (1), R. Naidu (1) (1) Washington State University, U.S.A. Four species of Phytophthora recovered from roots of American and hybrid chestnut seedlings and associated soils in the southeastern US S. SHARPE (1), S. Jeffers (1), S. Clark (2) (1) Clemson University, U.S.A.; (2) USDA Forest Service, U.S.A. Phylogenetic analysis of sequences of the Saudi isolates of RCVMV and 40 other CP sequences of Carlavirus species from GenBank grouped in the same clade though they had low homology percentages. Rubus yellow net virus: An integrated element in the raspberry genome A. Diaz-Lara (1), N. Mosier (2), K. Keller (2), R. Martin (2) (1) Oregon State University, U.S.A.; (2) USDA-ARS Horticultural Crops Rese Rubus yellow net virus (RYNV) infects Rubus spp. causing a serious decline when present in mixed infections and a vein chlorosis in indicator plants. RYNV belongs to the family Caulimoviridae, which can exist as infectious particles (exogenous) or integrated elements (endogenous). It was observed that RYNV PCR-positive plants often did not induce symptoms in bioassays, suggesting an integrated element. Molecular techniques were used to discriminate between exogenous and endogenous RYNV sequences. Reverse transcription-PCR using RYNV specific primers after DNase treatment produced positive results for the virus in graft transmissible isolates only. To confirm these results, rolling circle amplification on DNA preparations from these plants resulted in amplicons whose sequence belonged to RYNV. This suggested that graft-transmissible isolates contained an exogenous version of the virus. In Southern blot analysis using Rubus genomic DNA digested with BamHI (one recognition site in the RYNV genome), samples that were positive in bioassays for RYNV gave a single band the size of the RYNV genome, while plants with non-transmissible isolates produced three extra bands. Next generation sequencing and appropriate bioinformatics programs are being used to identify the specific integration sites. These results show that RYNV can be integrated into the raspberry genome and highlight the need to consider this phenomenon in future Rubus quarantine and certification programs. Survey of Arabis Mosaic and Grape Leaf Roll Associated virus in Maryland vineyards R POKHAREL (1) Total nucleic acids were isolated from leaves of three symptomatic watermelon plants, and subjected to rolling circle amplification (RCA). Libraries prepared for the RCA amplicons were subjected to Illumina MiSeq, and reads were assembled using SeqMan NGen software. BLASTn analysis of assembled contigs revealed the presence of the DNA- A and DNA-B genomic components most closely related to the Old World bipartite WmCSV. The three DNA-A components, at 2752 bp, shared 99- 100% nucleotide (nt) identity with each other, and 99% nt with a WmCSV isolate from Leith, Saudi Arabia [KJ939448]. The three DNA-B component sequences, at 2757 bp, shared 96-97% nt identity with each other, and 96% nt identity with the DNA-B component of an isolate, also from Leith [KJ939447]. This is the first report of WmCSV in the Leith, a farming community separated from Usfan by a vast, inhospitable desert. Necrotic Streaking of Asiatic Lilies caused by Plantago asiatica mosaic virus in Chile X. BESOAIN (1), A. Vidal Takasaki (2), R. Camps (2) (1) Pontificia Universidad Católica de Valparaíso, Chile; (2) Pontificia Universidad Católica Necrotic Streaking of Asiatic Lilies caused by Plantago asiatica mosaic virus in Chile X. BESOAIN (1), A. Vidal Takasaki (2), R. Camps (2) (1) Pontificia Universidad Católica de Valparaíso, Chile; (2) Pontificia Universidad Católica de Valparaíso, Chile During the spring of 2015 a survey was done in Quillota, in greenhouses with Asiatic hybrid lilies for cut flower. Symptomatic plants were found in four different cultivars. Symptoms were brown necrotic streaking associated with irregular leaf chlorosis, and severely infected plants also showed brown streaks in the stem and irregular necrosis in tepal tips. Only in cv. Litouwen it was observed that microbulbs replaced flower buds. Tissue extracts from symptomatic plants were obtained and RT-PCR of gene RdRp was performed employing specific primers for Potexvirus. The obtained sequences revealed 99% identity with the consensus sequences of PlAMV. After positive results for PlAMV, a pathogenicity test was performed on plants of two cultivars of Asiatic lilies. Two isolates of PlAMV were inoculated in three plants for each isolate, and other three plants were left as controls. Forty-five days after inoculation, plants from Asiatic cv. Indian Summerset showed a shorter stem, necrotic streaks in leaves and in the stems and chlorosis in irregular pattern. Litouwen plants showed symptoms of decreased growth, brown necrotic streaks in the leaves associated with chlorosis and microbulbs instead of flower buds. Survey of Arabis Mosaic and Grape Leaf Roll Associated virus in Maryland vineyards R POKHAREL (1) Survey of Arabis Mosaic and Grape Leaf Roll Associated virus in Maryland vineyards R. POKHAREL (1) (1) Maryland Dept of Agriculture, U.S.A. Arabis mosaic virus (ArMV) and grape leaf roll associated virus (GRLaV) cause significant crop loss in grapes. However, their natural incidence in Maryland vineyards is not known, and several growers have reported symptoms similar to those caused by these viruses. To find their distribution, 15 vineyards were surveyed during August to September 2015 in Maryland. Up to six varieties per vineyard were examined for virus symptoms. Up to five samples (symptomatic, if present) were collected from each variety. Two hundred seventy samples were tested for each virus group, including the three most symptomatic samples per variety. These samples were tested using a double-antibody sandwich (DAS)-enzyme-linked immunosorbent assay (ELISA) kit developed by Bioreba for GRLaV, and by Agdia for ArMV. Three groups of GRLaV (GRLaV- 1+3 and GRLaV- 2) were tested in two groups. Forty percent of vine samples collected were symptomatic for GRLaV and 29% were symptomatic for ArMV. Only ten percent of samples were found positive for ArMV. Similarly, only 1.1% of samples tested were positive for (GRLaV 1 + GRLaV 3) and 1.5% of samples were positive for GRLaV-2. This result indicated that many plants exhibiting symptoms similar to GRLaV and ArMV were negative for these viruses. This may be because symptoms observed in the field were produced by causes other than the virus tested. Further testing of such vines for other virus and virus-like pathogens may help growers to manage this problem. First report of Watermelon chlorotic stunt virus in rain-fed farms in Usfan, Saudi Arabia N. CHINGANDU (1), M. Al-Saleh (2), A Idris (3), J. Brown (1) (1) University of Arizona, Tucson, AZ, U.S.A.; (2) Department of Plant Protection, King Saud University, Saudi Arabia; (3) University of Arizona, School of Plant Sciences, Tucson, AZ, U.S.A. Watermelon (Citrullus vulgaris L.), an important crop in Saudi Arabia, is cultivated in the lowlands and wadis following seasonal winter rainfall, along the western coast. Production of watermelon faces constraints imposed by abiotic stresses and biotic diseases, and insects, including whiteflies. During spring 2015, >95% of watermelon plants growing on a farm near Usfan exhibited foliar chlorosis and mottling, and overall stunting symptoms, reminiscent of those caused by Watermelon chlorotic stunt virus (WmCSV) (Begomovirus, Gemniviridae). Raspberry leaf curl disease GLRaVs mixed infections were detected in different combinations from composite samples with GLRaV-1 + GLRaV-3 (2.5%) to be the most common, followed by GLRaV-2 + GLRaV-3 (2.1%) and GLRaV-3 + GLRaV-4 (0.9%). Our results also revealed the presence of Grapevine fan leaf virus, Grapevine fleck virus, Grapevine Pinot gris virus, Grapevine red blotch associated virus and Rupestris stem pitting associated virus (RSPaV). These results along with information generated on the disease progress and insect vectors in BC will emphasize the importance of a clean plan network program for sustainability of Canadian wine grapes. S4.105 Grapevine leafroll disease (GLD) is a significant constraint to the production of wine grapes (Vitis vinifera) in Washington State. Grapevine leafroll associated virus (GLRaV) 1, 2, 3, and 4 have so far been documented, with GLRaV-3 being the most widely distributed in Washington vineyards. However, recent reports of the occurrence of grapevine red blotch disease (GRBD) have raised concerns about the extent of its distribution in Washington vineyards. Since the symptomatology of GLD and GRBD is highly similar in wine grape cultivars, reliable information on the prevalence of these two diseases is necessary for deploying appropriate disease mitigation strategies. Towards this objective, surveys of vineyards were conducted during 2014 and 2015 seasons in seven American Viticultural Areas of Washington. A total of 1,469 samples collected from fourteen red-berried and five white-berried wine grape cultivars were tested for the presence of GLRaV-3 and Grapevine red blotch-associated virus (GRBaV) by polymerase chain reaction (PCR)-based diagnostic assays. The PCR results were further confirmed by cloning and sequencing of virus-specific amplicons. The combined data showed that 83.7% and 13.7% of samples were positive for GLRaV-3 and GRBaV, respectively, indicating that GLD is predominant relative to GRBD in Washington vineyards. A multiplex PCR assay has been optimized for high throughput testing of grapevine samples for the presence of GLRaV-3 and GRBaV. Association of Grapevine rupestris stem pitting-associated virus and Grapevine Pinot gris virus in declining grapevines on Schwarzman rootstock H. MCCOWN (1), S. Bag (1), M. Eng (1), M. Sudarshana (2) (1) University of California, U.S.A.; (2) USDA-ARS, U.S.A. Association of Grapevine rupestris stem pitting-associated virus and Grapevine Pinot gris virus in declining grapevines on Schwarzman rootstock H. MCCOWN (1), S. Bag (1), M. Eng (1), M. Sudarshana (2) (1) University of California, U.S.A.; (2) USDA-ARS, U.S.A. In the fall of 2014, in Napa County, California, four-year-old grapevines of Cabernet Sauvignon (Vitis vinifera) on Schwarzmann (V. riparia X V. rupestris) rootstock were found to exhibit red canopy and vine decline. Upon examination of the woody cylinder, the rootstocks, but not the scions, had extensive stem pitting. To investigate the causal agent(s), nucleic acid extracts from affected grapevines were analyzed for viruses by RT-PCR assays. These vines tested positive for the Syrah strain of Grapevine rupestris stem pitting-associated virus (GRSPaV-Syrah) and negative for five leafroll- associated viruses, three vitiviruses, and Grapevine red blotch-associated virus. Further examination by deep sequencing of a cDNA library, prepared from the dsRNA fraction of cambial scrapings from the affected trunks, and subsequent bioinformatics analysis, revealed the presence of Grapevine Pinot gris virus (GPGV) in addition to GRSPaV-Syrah. Canopy surveys conducted in the fall of 2015 indicated that >40% (n=342) of the vines had declined. It is likely that the association of GPGV and GRSPaV may have a synergistic effect resulting in the stem pitting symptom. Infectivity analysis of strain L of chickpea chlorotic dwarf virus, a cotton mastrevirus, in Nicotiana benthamiana plants M. MANZOOR (1), A. Bibi (2) ( ) ( ) (1) Institute of agricultural sciences, University of the punjab, Pakistan; (2) institute of agricultural sciences, university of the Cotton is damaged by a group of viruses, responsible for cotton leaf curl disease complex, belongs to the genus Begomovirus; family Geminiviridae. The present study unveils another virus which belongs to genus of the same family causing leaf curl disease in cotton. Agrobacterium-mediated inoculation, of strain L of the chickpea chlorotic dwarf virus (CpCDV) (mastrevirus) isolated from cotton plants, to Nicotiana benthamiana, alone or in combination with other begomovirus showed a remarkable difference in symptoms from very mild symptoms to severe cup shaped with stunted growth. The variability in symptoms and their development in other parts of tested plants and subject viruses, belonging to different genera, perhaps owing to difference in ORFs of subject viruses. Nucleotide sequence and genome organization of a novel Panicovirus from Bermuda grass M. TAHIR (1), B. Lockhart (2), S. Grinstead (1), D. Mollov (1) (1) USDA-ARS, U.S.A.; (2) University of Minnesota, Department of Plant Pathology, U.S.A. Bermuda grass (Cynodon dactylon) is widely used in tropical and subtropical areas for golf greens, athletic fields, and landscapes. Samples of Bermuda grass displaying symptoms of decline were tested at the University of Minnesota plant virology lab. Spherical virus particles (28-30 nm) were observed by transmission electron microscopy. Total RNA was extracted and sent for Next Generation Sequencing (NGS) on an Illumina platform. Contigs were assembled using CLC Workbench, and analyzed using BLAST searches against reference genomes. A contig of about 4 kb contained four predicted ORFs in an arrangement typical of viruses in the family Tombusviridae. Overlapping primer pairs were designed from the consensus sequence for RT- PCR verification and sequence confirmation. All amplicons were cloned and the resulting sequences matched the original NGS contig. Amino acid sequence comparisons of the putative replicase (RdRp) and movement and coat proteins (CP) showed sequence similarities in the range of 37-66% compared to Thin paspalum asymptomatic virus, Cocksfoot mild mosaic virus, and Panicum mosaic virus. Phylogenetic analyses using the CP and RdRP of this newly identified virus place it in the genus Panicovirus. The sequence of this Bermuda grass virus is sufficiently different from other viruses to conclude it is a new member of the genus Panicovirus, family Tombusviridae. Survey of Arabis Mosaic and Grape Leaf Roll Associated virus in Maryland vineyards R POKHAREL (1) All symptomatic plants tested positive with specific primer for PlAMV and control plants tested negative. Koch’s postulates were fulfilled for PlAMV on Lilium plants and the disease was called Necrotic Streaking of Lily caused by PlAMV affecting Asiatic cultivars. Trichosanthes latent virus (TrLV), a novel potyvirus from Trichosanthes cucumeroides in Japan O. KIM (1), K. Kobayashi (2), K. Natsuaki (2), H. Shinohara (2), H. Negishi (2) (1) Tokyo University of Agriculture (TUA), Japan; (2) Tokyo University of Agriculture (TUA), Japan Only a few studies have been conducted on incidence of plant viruses on wild cucurbit weeds in Japan. As wild and horticultural cucurbits often share same viruses, epidemiological survey as well as precise identification of causal virus(es) is required for the efficient control strategy. We once reported un-common potyvirus tentatively named as JK virus isolated from Trichosanthes cucumeroides (Kim et al., 2011). By additional surveys, most of S4.106 collected T. cucumeroides in Kanto region were found to be infected with JK virus showing high co-incidence with Papaya ringspot virus (PRSV). On the contrary, as far as examined, JK virus was not detected from any wild cucurbit samples including Trichosanthes spp. in Okinawa Prefecture where Papaya leaf distortion mosaic virus (PLDMV) infection on cucurbit weeds have been reported (Maoka and Hataya, 2005). Mechanical inoculation of JK virus indicated a narrow host range limited to family Cucurbitaceae and showed symptomless or mild symptom when back-inoculated to T. cucumeroides. Compared with sequences of CI or CP regions from other potyviruses, JK virus shared less than 80% identity with PLDMV which was the most closely related potyvirus. Therefore, JK virus can be considered as a novel potyvirus species, for which the name as Trichosanthes latent virus (TrLV) is proposed. collected T. cucumeroides in Kanto region were found to be infected with JK virus showing high co-incidence with Papaya ringspot virus (PRSV). On the contrary, as far as examined, JK virus was not detected from any wild cucurbit samples including Trichosanthes spp. in Okinawa Prefecture where Papaya leaf distortion mosaic virus (PLDMV) infection on cucurbit weeds have been reported (Maoka and Hataya, 2005). Mechanical inoculation of JK virus indicated a narrow host range limited to family Cucurbitaceae and showed symptomless or mild symptom when back-inoculated to T. cucumeroides. Compared with sequences of CI or CP regions from other potyviruses, JK virus shared less than 80% identity with PLDMV which was the most closely related potyvirus. Survey of Arabis Mosaic and Grape Leaf Roll Associated virus in Maryland vineyards R POKHAREL (1) Therefore, JK virus can be considered as a novel potyvirus species, for which the name as Trichosanthes latent virus (TrLV) is proposed. g y y p y p cucumeroides. Compared with sequences of CI or CP regions from other potyviruses, JK virus shared less than 80% identity with PLDMV which was the most closely related potyvirus. Therefore, JK virus can be considered as a novel potyvirus species, for which the name as Trichosanthes latent virus (TrLV) is proposed. Identification and characterization of Zucchini tigre mosaic virus on creeping cucumber in Japan K. KOBAYASHI (1), H. Shinohara (2), H. Negishi (2), O. Kim (2) (1) Tokyo University of Agriculture (TUA), Japan; (2) Tokyo University of Agriculture (TUA), Japan Creeping cucumber (Melothria pendula), a wild cucurbit weed, showing mottle and distortion symptoms was observed in Okinawa main island, Japan in September 2015. Total RNA using TRIzol reagent extracted from collected samples was used for reverse transcription-polymerase chain reaction (RT- PCR) with degenerate primers (CIFor and CIRev) for potyviruses (Ha et al., 2008) followed by cloning and sequencing. Obtained 637 nucleotides (nts) sequence of partial CI region from isolate Oki-47 showed 86% with corresponding sequence from Zucchini tigre mosaic virus (ZTMV; accession no. KC345554) as a highest identity. When the whole coat protein (CP) coding region was considered, the nt and amino acid (aa) sequence identities shared 94% and 97% with Papaya ringspot virus (PRSV; accession no. AB583208) respectively. The compared PRSV isolate was re-classified as one of ZTMV isolates by Romay et al. (2014). Serological test by western blotting assay revealed that isolate Oki-47 was closely related to PRSV, but not to Papaya leaf distortion mosaic virus (PLDMV). Absence of PRSV, PLDMV, and Zucchini yellow mosaic virus (ZYMV) were also confirmed which are known as major causal potyviruses infecting to cucurbits in Japan. This is the first report of ZTMV incidence in Japan as well as creeping cucumber for its natural host. Complete nucleotide sequences of a new bipartite begomovirus from Malvastrum sp. plants with bright yellow mosaic symptoms in South Texas O. ALABI (1), C. Villegas (2), L. Gregg (3) (1) Dept. of Plant Pathology & Microbiology, Texas A&M University AgriLife Research & Extension Center, U.S.A.; (2) South Texas College, U.S.A.; (3) Texas A&M AgriLife Research and Extension Center, U.S.A. Complete nucleotide sequences of a new bipartite begomovirus from Malvastrum sp. plants with bright yellow mosaic symptoms in South Texas O. Survey of Arabis Mosaic and Grape Leaf Roll Associated virus in Maryland vineyards R POKHAREL (1) ALABI (1), C. Villegas (2), L. Gregg (3) (1) Dept. of Plant Pathology & Microbiology, Texas A&M University AgriLife Research & Extension Center, U.S.A.; (2) South Texas College, U.S.A.; (3) Texas A&M AgriLife Research and Extension Center, U.S.A. Two isolates of a new bipartite Begomovirus, tentatively named as Malvastrum bright yellow mosaic virus (MaBYMV), were molecularly characterized from naturally-infected Malvastrum sp. plants showing bright yellow mosaic disease symptoms in South Texas. The host plant genus identity was determined by DNA barcoding of the non-coding trnH-psbA intergenic spacer region and the matK plastid gene. Restriction enzyme-digested rolling cycle amplification products from symptomatic plants were used as template in PCR to amplify complete virus genome segments with pairs of newly- designed abutting primers. The obtained ~2.7 Kb fragments were cloned and sequenced. Six complete DNA-A and five DNA-B genome sequences of MaBYMV obtained from the two isolates ranged in length from 2,608-2,609 nucleotides (nt) and 2,578-2,605 nt, respectively. Both genome segments share a 178-180 nt common region. In pairwise comparisons, complete DNA-A and DNA-B of MaBYMV were most identical (87-88% and 79-81%, respectively) and phylogenetically related to corresponding sequences of Sida mosaic Sinaloa virus-[MX-Gua-06]. Further analysis showed that MaBYMV is a putative recombinant virus, thus supporting the notion that malvaceous hosts may be influencing the evolution of several begomoviruses. The design of new diagnostic primers enabled the detection of MaBYMV in cohorts of Bemisia tabaci collected from symptomatic Malvastrum sp. plants, thus implicating whiteflies as potential vector of the virus. Infectivity analysis of strain L of chickpea chlorotic dwarf virus, a cotton mastrevirus, in Nicotiana benthamiana plants M. MANZOOR (1), A. Bibi (2) Infectivity analysis of strain L of chickpea chlorotic dwarf virus, a cotton mastrevirus, in Nicotiana benthamiana plant M. MANZOOR (1), A. Bibi (2) Infectivity analysis of strain L of chickpea chlorotic dwarf virus, a cotton mastrevirus, in Nicotiana benthamiana plants M. MANZOOR (1), A. Bibi (2) g ( ), ( ) (1) Plant Protection Research Institute, Guangdong Academy of Agricultural Sciences, China Viral disease is one of main diseases of pepper production in Guangdong province of China. During 2013-2015, we surveyed the occurrence of the viral disease and identified the viruses infecting pepper. The diseased samples of pepper were collected randomly from the main pepper-growing areas of Guangdong. The diseased samples of the same survey location were mixed and extracted total RNA for deep sequencing of small RNA. The viruses were verified by RT-PCR, respectively using the specific primers designed according to the assembling sequences. The diseased plants in fields exhibited diversiform symptoms, including stunting, mosaic, chlorosis, leaf narrowing and distortion, and mottled. The sequence analysis results showed that there were 16 species viruses infecting pepper, including Chilli ringspot virus, Broad bean wilt virus, Cucumber mosaic virus, Pepper vein yellows virus, Chilli veinal mottle virus, Pepper veinal mottle virus, Pepper yellow leaf curl virus, Capsicum chlorosis virus, Tobacco mild green mosaic virus, Pepper cryptic virus, Potato virus Y, Pepper mild mottle virus, Watermelon silver mottle virus, Tobacco mosaic virus, Bell pepper endornavirus, Nicotiana tabacum pararetrovirus-like. The co-infection with two, three and more than three viruses accounted for 6.02%, 21.29% and 69.88% in one S4.107 diseased sample, respectively. It is concluded that the number of virus species infecting pepper crop was abundant and co-infection was common in Guangdong of China. diseased sample, respectively. It is concluded that the number of virus species infecting pepper crop was abundant and co-infection was common in Guangdong of China. Beet curly top virus strains associated with sugar beet in Idaho, Oregon, and a survey collection C. STRAUSBAUGH (1), I. Eujayl (1), B. Wintermantel (2) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. Curly top of sugar beet is a serious yield limiting disease in semi-arid production areas caused by Beet curly top virus (BCTV) and vectored by the beet leafhopper. The primary means of control for BCTV is host resistance, but effectiveness of resistance can vary with BCTV strain. BCTV variation was last investigated in Idaho and Oregon during a 2006-2007 survey, but changes in disease severity suggested a need for reevaluation. Therefore, 406 leaf samples symptomatic for curly top were collected from sugar beet plants in commercial sugar beet fields in Idaho and Oregon from 2012 to 2015. Infectivity analysis of strain L of chickpea chlorotic dwarf virus, a cotton mastrevirus, in Nicotiana benthamiana plants M. MANZOOR (1), A. Bibi (2) DNA was isolated and the BCTV strain composition was investigated based on polymerase chain reaction (PCR) assays with strain specific primers for Severe (Svr) and California/Logan (CA/Logan) strains and nonspecific primers that amplified Worland (Wor)-like strains. The 2006-2007 ID/OR BCTV positive samples from sugar beet included the following strains: 87% Svr, 7% CA/Logan, and 60% Wor-like. BCTV strain distribution in the new survey averaged 2% Svr, 30% CA/Logan, and 87% Wor-like. Whole genome sequencing (GenBank accessions KT276895 to KT276920) with overlapping primers, suggests that the Wor-like strains included Wor, Colorado (CO), and a previously undescribed strain designated Kimberly1 (Kim1). These data indicate there was a shift from Svr being one of the dominant BCTV strains in commercial sugar beet fields in 2006-2007 to being undetected at times in recent years. Virus diseases: A major threat to high value vegetable crops in Bangladesh and Nepal A. FAYAD (1), A. Fayad (1), R. Naidu (2) Virus diseases: A major threat to high value vegetable crops in Bangladesh and Nepal A. FAYAD (1), A. Fayad (1), R. Naidu (2) (1) Virginia Polytechnic Institute and State University, U.S.A.; (2) Washington State University, U.S.A. ( ), y ( ), ( ) (1) Virginia Polytechnic Institute and State University, U.S.A.; (2) Washington State University, U.S.A. The Feed the Future Innovation Lab for Integrated Pest Management (IPM IL), a USAID-funded program managed by Virginia Tech, has been working on identifying major threats to vegetable production in Bangladesh and Nepal. Surveys have been conducted in major production areas including those of smallholder farmers. Results show a trend of emerging and re-emerging virus disease in cucurbits including cucumber, squash, pumpkin and other gourds, tomato, pepper, potato, and okra. Research is focused on identifying sources of virus, with a special emphasis on insect vector and seed transmission pathways. The IPM IL is working with farmers, researchers, and local and international NGOs in Bangladesh and Nepal to address management of these virus diseases, and developing ecologically-based and environmentally friendly IPM strategies that reduce the reliance on synthetic pesticides and improve yield while at the same time reducing damage to the environment and improving nutrition and health. IPM packages have been developed and are being disseminated through farmer participatory approaches including IPM demonstration plots and farmer groups. Tomato yellow mottle virus (ToYMoV): Characterization of a newly emergent begomovirus associated with tomato disease in Costa Rica M. MALIANO (1), M. Rojas (1), T. Validation of a novel gene target for detection and quantification of Clavibacter michiganensis subsp. nebraskensis, the causal agent of Goss’s wilt R. MCNALLY (1), C. Ishimaru (2), D. Malvick (2) ( ) i i f i ( ) i i f i R. MCNALLY (1), C. Ishimaru (2), D. Malvick (2) (1) University of Minnesota, U.S.A.; (2) University of Minnesota, U.S.A. R. MCNALLY (1), C. Ishimaru (2), D. Malvick (2) (1) University of Minnesota, U.S.A.; (2) University of Minnesota, U.S.A. Goss’s wilt of maize is a reemerging and significant disease caused by the bacterium Clavibacter michiganensis subsp. nebraskensis (Cmn). Despite its expansion in North America, molecular tools for diagnosing and studying the epidemiology of this disease remain limited. Here we report the identification of CMN_01184 as a novel gene target and demonstrate its use for development of conventional PCR (cPCR) and quantitative PCR (qPCR) assays for detection and quantification of Cmn. Primers designed for cPCR and qPCR were evaluated for specific amplification of CMN_01184 using a diverse collection of 129 bacterial and fungal isolates, including multiple corn pathogens, environmental organisms from agricultural fields, and all known subspecies of C. michiganensis. Of these, only isolates of Cmn tested positive for CMN_01184. To evaluate the utility of CMN_01184 for in situ detection of Cmn, cPCR and qPCR based on this target gene was performed on infested field soil and naturally infected corn leaves containing Cmn. CMN_01184 was amplified only in soil samples and leaves containing Cmn. Finally, in greenhouse studies, systemic infection of corn leaves by Cmn was quantified over a two-week infection period using the qPCR assay based on CMN_00184. The results demonstrate collectively that specific and sensitive PCR assays based on CMN_01184 can aid in diagnosis as well as studies of the epidemiology and ecology of Goss’s wilt. In-field detection of the select agent Rathayibacter toxicus using loop-mediated isothermal amplification M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Stack (5) M. ARIF (1), G. Busot (2), R. Mann (3), B. Rodoni (4), J. Infectivity analysis of strain L of chickpea chlorotic dwarf virus, a cotton mastrevirus, in Nicotiana benthamiana plants M. MANZOOR (1), A. Bibi (2) Melgarejo (2), M. Macedo (3), N. Barboza (4), A. Inoue Nagata (3), R. Gilbertson (5) (1) UC Davis, davis, CA, U.S.A.; (2) Universidad Nacional Agraria La Molina, Peru; (3) University of Brasilia, Brazil; (4) Universidad de Costa Rica, Costa rica; (5) UC Davis, CA, U.S.A. Tomato yellow mottle virus (ToYMoV): Characterization of a newly emergent begomovirus associated with tomato disease in Costa Rica M. MALIANO (1), M. Rojas (1), T. Melgarejo (2), M. Macedo (3), N. Barboza (4), A. Inoue Nagata (3), R. Gilbertson (5) (1) UC Davis, davis, CA, U.S.A.; (2) Universidad Nacional Agraria La Molina, Peru; (3) University of Brasilia, Brazil; (4) Universidad de Costa Rica, Costa rica; (5) UC Davis, CA, U.S.A. Tomato production in Costa Rica is affected by diseases caused by whitefly-transmitted begomoviruses. Since the early 1990s, the main virus involved was the New World (NW) bipartite Tomato yellow mottle virus (ToYMoV), which was associated with stunting and yellow mottling. To fulfill Koch’s postulates, putative full-length were obtained. Nicotiana benthamiana and tomato seedlings inoculated with the ToYMoV DNA-A and DNA-B clones developed stunting and yellow mottling of leaves, confirming the infectivity of the clones. Sequence analysis revealed that ToYMoV is closely related to bipartite tomato-infecting begomoviruses from Mexico and Guatemala. Multimeric clones were generated and used to assess the host range of ToYMoV. More recently, the NW bipartite Tomato leaf curl Sinaloa virus (ToLCSiV) and the invasive Old World (OW) monopartite Tomato yellow leaf curl virus (TYLCV) were introduced to Costa Rica where they have caused yield losses. Pseudorecombinants made between infectious DNA-A and DNA-B clones of ToYMoV and ToLCSiV did not induce symptoms. Tomato plants inoculated with cloned DNAs of TYLCV, ToLCSiV and ToYMoV alone or in mixtures revealed that symptoms of ToLCSiV and ToYMoV appeared earlier than those of TYLCV. However, within 14 days after inoculation, TYLCV symptoms became dominant, although ToLCSiV and ToYMoV were still detected at high levels. These results will be discussed in terms of synergy among these viruses. Development of recombinase-polymerase amplification assay for the detection of Western X phytoplasma (‘Candidatu sweet cherry VqPCR assay results obtained using two different chemistries including SYBR® and TaqMan® will be presented. Development of a multiplex real-time PCR for the improved detection of citrus canker-causing Xanthomonas V. MAVRODIEVA (1), G. Santillana (1), G. Dennis (2) (1) USDA APHIS PPQ S&T Beltsville Laboratory, U.S.A.; (2) USDA APHIS PPQ S&T, U.S.A. To improve on the current assay for the detection of all citrus canker causing Xanthomonas which targets the pthA gene, a multiplex assay was developed to incorporate the 16S rRNA gene as a second target. Two new pthA amplisets designed to have better sensitivity than the current one, and two new 16S amplisets were evaluated individually, and in multiplex format. The PCR product of each ampliset was sequenced and verified to be the correct targets. Primers and probes interaction was determined and optimal concentrations selected followed by dNTP and MgCl2 concentrations optimization for the different ampliset combinations. In silico and experimental specificity analyses were performed. All amplisets were found to have a 100% match with the target organism. The pth-targeted amplisets showed cross-reactivity to some non-citrus Xanthomonas but not to other citrus-infecting genera. The method being developed targets symptomatic citrus therefore cross-reactions of the primers with Xanthomonas spp. of other crops should not interfere because most xanthomonads exhibit highly restricted host range (Delcourt et al., 2013). One of the 16S amplisets showed insufficient specificity thus was excluded from further optimization studies. Initial comparability studies suggest that the new pthA amplisets (used in multiplex with 16S rRNA gene) may be more sensitive than the currently used uniplex (pthA), having lower Ct values for the sets of archived diagnostic samples tested. Development of a rapid and reliable isothermal AmplifyRP diagnostic assay for specific detection of Xylella fastidiosa R. LI (1), P. Russell (2), N. Mcowen (2), B. Davenport (2), S. Zhang (2) (1) Agdia Inc., U.S.A.; (2) Agdia Inc, U.S.A. Development of a rapid and reliable isothermal AmplifyRP diagnostic assay for specific detection of Xylella fastidiosa R. LI (1), P. Russell (2), N. Mcowen (2), B. Davenport (2), S. Zhang (2) (1) Agdia Inc., U.S.A.; (2) Agdia Inc, U.S.A. Xylella fastidiosa, infecting 309 plant species belonging to 63 families and 193 genera, is a regulated bacterial pathogen in many countries. Recently X. fastidiosa was associated with the devastating olive quick decline syndrome (OQDS) in southeastern Italy, resulting in an unprecedented concern to its spread in Mediterranean countries. Development of recombinase-polymerase amplification assay for the detection of Western X phytoplasma (‘Candidatu sweet cherry Development of recombinase-polymerase amplification assay for the detection of Western X phytoplasma (‘Candidatus Phytoplasma pruni’) in sweet cherry Resurgence of Western X disease (WX) of sweet cherry (P. avium), caused by WX phytoplasma (‘Candidatus Phytoplasma pruni’), has been observed in Washington State (WA) in recent years. The disease presents a major economic impact in WA sweet cherry production as affected trees not only decline but produce unmarketable small, insipid fruits. In this study, an isothermal recombinase-polymerase amplification assay (RPA) for the detection of WX phytoplasma was developed. Using sequence data generated by high-throughput sequencing of three isolates of WX phytoplasma from WA along with sequence available in the GenBank database, primers and probe were selected within the immunodominant protein (idpA) coding region. The RPA assay system has three attributes that make it a very useful tool for detection of WX phytoplasma by cherry growers: (1) it is fast and simple – results are obtained in 20 minutes and because it uses only crude sap preparation, no sophisticated equipment for nucleic acid extraction is necessary; (2) it is sensitive – the RPA assay showed similar sensitivity with PCR both in temporal detection throughout the growing season and the amount of bacterial DNA detected; and (3) it is specific only to WX phytoplasma. Additionally, RPA detected WX phytoplasma from total DNA extracts of three known leafhopper vectors of the pathogen; this makes it a valuable tool in pathogen vector research. p M. KUNTA (1), E. Louzada (1), P. Vedasharan (1) (1) Texas A&M University-Kingsville Citrus Center, Citrus Huanglongbing, associated with Candidatus Liberibacter asiaticus (CLas), is the most serious disease in the USA causing substantial economic losses. The real-time PCR (qPCR) assays used to detect CLas have a limitation that they cannot differentiate live and dead bacterial cells. Accurate quantification of live CLas cells in the plant tissue is essential to devise effective disease management strategies and to test the efficacy of new disease control methods. Several optimized quantitative real-time PCR methods using propidium monoazide (VqPCR), a DNA-binding dye, to quantify live CLas populations were described. However, it is unclear if live CLas populations vary with age of the leaf and varying degrees of HLB disease symptom development. To address this question, we have quantified the levels of live CLas populations in grapefruit leaves at different maturity stages and leaves with varying levels of symptoms collected from naturally infected field trees. Development of recombinase-polymerase amplification assay for the detection of Western X phytoplasma (‘Candidatu sweet cherry In order to meet the needs for quarantine detection and field diagnostics, Agdia developed an advanced AmplifyRP assay for the detection of X. fastidiosa. The assay combined both endpoint (Acceler8) and real-time (XRT) detection in a single reaction PCR tube. This characteristic allows users to either collect the quantitative fluorescence data using a portable fluorometer or observe the qualitative test result in a disposable detection chamber. The assay is as sensitive as that of the published quantitative real-time PCR and has no cross-reaction to the closely related Xanthomonas species and numerous tested host plants including grape, citrus, and olive. The assay uses crude plant extracts and is applicable for use in both laboratories and fields. This assay provides users a fast and reliable tool to detect X. fastidiosa carried by plant materials and insect vectors from the contaminated regions. Recombinase polymerase-based diagnostics for in-field detection of Pseudomonas syringae pv. actinidiae J. STACK (1), M. Arif (2), J. Rascoe (3), M. Nakhla (3), G. Busot (1) (1) Kansas State University, U.S.A.; (2) Kansas State Univesrity, U.S.A.; (3) USDA APHIS PPQ CPHST, U.S.A. In-field detection of the kiwifruit pathogen, P. syringae pv. actinidiae (Psa), will support plant biosecurity decisions regarding trade and production. To detect and discriminate among Psa strains (including the 2008 global outbreak strain, Psa-V), a Recombinase Polymerase Amplification (RPA) assay was developed for laboratory and field use. Primer sets targeting two effector genes (hopZ3 and hopZ5) were designed and screened against isolates of highly virulent Psa and closely related P. syringae pathovars. All Psa strains can be detected by the presence of hopZ3 amplicons but only the virulent type, Psa-V or biovar 3, is detectable by hopZ5 amplicons. Because RPA assays were coupled to lateral flow device (LFD), reverse primer were labeled with either biotin or digoxigenin and the reaction was developed in the presence of a carboxyfluorescein (FAM) labeled probe. RPA assays had an amplification time of 10 minutes. Sensitivity determinations indicated a detection limit of 1 pg or 100 fg depending of the targeted gene. Isothermal assays are a rapid diagnostic assay that enables the early detection of pathogens and a valuable tool for in-field pathogen detection to support plant biosecurity decisions. Evidence for seed transmission of Xylella fastisiosa in pecan (Carya illinoinensis) K. CERVANTES (1), D. Ray (1), R. Stamler (2), J. French (1), J. Soneji (3), R. Heerema (4), L. Grauke (5), J. Evidence for seed transmission of Xylella fastisiosa in pecan (Carya illinoinensis) K. CERVANTES (1), D. Ray (1), R. Stamler (2), J. French (1), J. Soneji (3), R. Heerema (4), L. Grauke (5), J. Randall (4) (1) New Mexico State University, Las Cruces, NM, U.S.A.; (2) New Mexico State University, U.S.A.; (3) NMSU, Las Cruces, NM, U.S.A.; (4) New Mexico State Universitye, Las Cruces, NM, U.S.A.; (5) USDA-ARS Pecan Breeding, Somerville, TX, U.S.A. Validation of a novel gene target for detection and quantification of Clavibacter michiganensis subsp. nebraskensis, the causal agent of Goss’s wilt R. MCNALLY (1), C. Ishimaru (2), D. Malvick (2) ( ) i i f i ( ) i i f i Stack (5) (1) Kansas State University, Manhattan, KS, U.S.A.; (2) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.; (3) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Bundoora, Australia; (4) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Australia; (5) Department of Plant Pathology, Kansas State University, Australia (1) Kansas State University, Manhattan, KS, U.S.A.; (2) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A.; (3) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Bundoora, Australia; (4) Department of Economic Development, Jobs, Transport and Resources, Latrobe University, Australia; (5) Department of Plant Pathology, Kansas State University, Australia Early, accurate and sensitive detection of Rathayibacter toxicus by plant health officials would improve inspections of imported annual ryegrass hay and seed at ports of entry and enhance in-field detection by biosecurity surveillance personnel. The gram positive bacterium and select agent R. toxicus produces a toxin in annual ryegrass (Lolium rigidum). Livestock deaths in Australia due to toxin contaminated ryegrass have raised concerns about the global spread of R. toxicus in ryegrass hay and seed. Genomes from all available R. toxicus populations were explored to design target-specific loop- mediated isothermal amplification (LAMP) primers. Gene rpoD was selected for primers design. Internal LAMP primers targeting the ITS gene of host plant L. rigidum were also designed to enhance reliability and accuracy. Genie II, Sybr Green I and lateral flow device were used for LAMP assays. The developed assays were validated in silico against the NCBI GenBank nucleotide database and in vitro against the inclusivity and exclusivity panels for specificity. In-field assays were performed in South Australia; accurately detected R. toxicus from infected annual ryegrass samples. The developed S4.108 LAMP assays are sensitive, accurate and easy to implement. The proposed LAMP method will be helpful in diagnostics, surveys, disease management, and biosecurity decisions to support export/import of annual ryegrass. Real-time PCR detection and discrimination of the parsley pathogens Pseudomonas syringae pv. apii and Pseudomonas syringae pv. coriandricola J. REINTKE (1), G. Hiddink (2), D. Sanchez Mendez (2) (1) Enza Zaden BV Netherlands; (2) Enza Zaden BV Netherlands Real-time PCR detection and discrimination of the parsley pathogens Pseudomonas syringae pv. apii and Pseudomonas syringae pv. coriandricola J. REINTKE (1), G. Hiddink (2), D. Sanchez Mendez (2) (1) Enza Zaden BV, Netherlands; (2) Enza Zaden BV., Netherlands detection and discrimination of the parsley pathogens Pseudomonas syringae pv. apii and Pseudomonas syringae pv. J. REINTKE (1), G. Hiddink (2), D. Sanchez Mendez (2) (1) Enza Zaden BV, Netherlands; (2) Enza Zaden BV., Netherlands J. REINTKE (1), G. Hiddink (2), D. Sanchez Mendez (2) (1) Enza Zaden BV, Netherlands; (2) Enza Zaden BV., Netherlands Bacterial leaf spot in parsley is caused by two different Pseudomonas syringae pathovars, Pseudomonas syringae pv. apii and Pseudomonas syringae pv coriandricola (Koike, Bull, 2011 Bull et al, 2011). Both were identified as causal agents in outbreaks of bacterial leaf spot in parsley. Seed was claimed to be the initial source of infection. Current seed test methods include a pathogenicity tests but lack confirmation methods. Therefor a realtime PCR assay was developed to detect and discriminate the two pathovars causing leaf spot disease in parsley. The primers and probes were developed on sequence variation in the gap1-gene of 35 closely related Pseudomonas syringae pathovars. Both P.s. apii and P.s. coriandricola were amplified with the forward primer 5’-ACCCAGTCGATGATCCCGA-3’, reverse primer 5’-AGCGAAACGTTGATCACCGG-3’, while probes differentiated P.s. apii (5’- 6FAM-CAGGCAAACTGACCGGCATGGCCGTT-BHQ1-3’) from P.s. coriandricola (5’- HEX-CGGGCAAGCTCACGGGGATGGCTGTT-BHQ1- 3’). Primers and probe showed sufficient discriminative power at the genomospecies level when tested on 52 closely or non-related strains. Primers were used in an improved seed health tests for the detection of P.s. apii and P.s. coriandricola on parsley seeds. The seed health test was validated and test results were confirmed for biological relevance using grow-outs of all used seed lots. PCR Assays for the Detection of Xanthomonas euvesicatoria, Causal Agent of Bacterial Spot on Pepper and Tomato Plants R. JEONG (1), S. Son (2), M. Kyeon (2), H. Lee (3), J. Cha (2) (1) Chungbuk National University, Korea; (2) Department of Plant Medicine, Chungbuk National University, Korea; (3) Animal and Plant Quarantine Agency, Korea Xanthomonas euvesicatoria (Xev) causes bacterial spot disease on pepper and tomato plants. A specific detection technique for this pathogen is important for the management of the disease and an efficient plant quarantine services. To obtain species-specific primers for PCR assays, low homologous ORFs of Xanthomonas euvesicatoria 85-10 to genes in NCBI GenBank were selected by BLASTn. Development of recombinase-polymerase amplification assay for the detection of Western X phytoplasma (‘Candidatu sweet cherry Randall (4) (1) New Mexico State University, Las Cruces, NM, U.S.A.; (2) New Mexico State University, U.S.A.; (3) NMSU, Las Cruces, NM, U.S.A.; (4) New Mexico State Universitye, Las Cruces, NM, U.S.A.; (5) USDA-ARS Pecan Breeding, Somerville, TX, U.S.A. S4.109 Xylella fastidiosa is a gram-negative bacterial phytopathogen found exclusively in the xylem tissue of host plants. In pecan (Carya illinoinensis), X. fastidiosa was previously shown to be the causal agent of pecan bacterial leaf scorch (PBLS) in the United States and was shown to result in yield losses and a reduction in overall plant health. We observed that X. fastidiosa in pecan is difficult to detect through PCR due to non-specific amplification and inhibition from phenolic compounds, additional efforts are being made to improve PCR diagnosis of this pathogen in pecan tissue and to determine the location of bacterial populations in infected trees. In previous studies on pecan, X. fastidiosa was presumed to be solely transmitted via xylem-feeding insect vectors such as sharpshooters and spittlebugs. However, in this study, PCR and sequence analysis identified the presence of X. fastidiosa in pecan seed from multiple cultivars. Currently, greenhouse studies are in progress to determine if X. fastidiosa in pecan seeds is viable and able to colonize seedling tissues. In addition, a microbiome enrichment kit will be used to identify microbial populations present in the samples using next generation sequencing (NGS). Real-time PCR detection and discrimination of the parsley pathogens Pseudomonas syringae pv. apii and Pseudomonas syringae pv. coriandricola J. REINTKE (1), G. Hiddink (2), D. Sanchez Mendez (2) (1) Enza Zaden BV Netherlands; (2) Enza Zaden BV Netherlands Xev60 F/R primers that were designed from one of the select ORFs amplified specific DNA from all of tested strains of Xev including the 9 internationally collected strains and 67 Korean strains while the primers did not amplified any DNA from the 20 internationally collected strains of 3 other bacterial spot-causing Xanthomonads, X. vesicatoria, X, perforans, and X. gadneri and the other plant pathogenic bacteria. Taqman probe, Xev60-Tq was designed from the inside the PCR amplicon. Real-time PCR with Xev60 F/R and the Xev60-Tq probe generated Ct values with dose-dependent of target DNA () and Ct values of Xev strains were above of threshold Ct value for positive detection. PCR assays developed in this study can reliably detect and identify Xev. Loop-mediated isothermal amplification for the detection of soft rot causing Dickeya spp. G. MARRERO (1), J. Yasuhara-Bell (2), M. Melzer (2), A. Alvarez (2) 1) University of Hawaii at Manoa, U.S.A.; (2) University of Hawaii at Manoa, U.S.A. Dickeya spp. cause soft rot, wilt and blackleg diseases in a wide range of plants and crops of agricultural importance. The emerging pathogen D. solani may be a contributing factor to the increased incidence of blackleg and soft rot diseases on potato in Europe, whereas D. dianthicola, a close relative, is currently listed on the European and Mediterranean Plant Protection Organization’s list of pests recommended for regulation as quarantine pests. Presently, the North American Plant Protection Organization has placed D. solani on the U.S. list for emerging pest alerts. Symptoms caused by Dickeya spp. can be confused with those produced by Pectobacterium spp. in the field; differentiation of these two soft rot causing groups is time-consuming and requires multiple assays for confirmation. Therefore a robust rapid diagnostic test for the differentiation and detection of Dickeya spp. was developed using loop-mediated isothermal amplification (LAMP). In this study, a genus-specific LAMP assay was developed using whole genome data. The specificity of the assay was evaluated on six of the seven recognized Dickeya species and Pectobacterium spp. associated with potato; the LAMP assay only showed positive reactions with Dickeya spp. Direct detection of Dickeya from infected plant tissue was assessed and provided a result in less than 30 minutes. This LAMP assay offers a rapid and sensitive way for testing of Dickeya spp. from plant samples. A computer program for fast and easy typing of Ralstonia solanacearum species complex strains into genospecies and sequevars 1&2 M. STULBERG (1), Q. Huang (2) (1) FNPRU, USDA/ARS & ORISE, U.S.A.; (2) FNPRU, USDA/ARS, U.S.A. The phytopathogen Ralstonia solanacearum is a species complex that contains race 3 biovar 2 strains belonging to phylotype IIB sequevars 1 and 2 that are quarantined or select agent pathogens. Recently, the R. solanacearum species complex strains have been reclassified into three genospecies: R. solanacearum, R. pseudosolanacearum and R. sygyzii. An unidentified R. solanacearum strain is considered a select agent in the US until proven to be a non-race 3 biovar 2 (non-phylotype IIB sequevars 1&2). We wrote a computer program that analyzes a minimum 400-bp user-input egl sequence from a R. solanacearum species complex strain for egl homology and SNP content to determine 1) whether it belongs to the R. solanacearum species complex, 2) if so, to which genospecies, and 3) whether it is of the sequevar type (sequevars 1 and 2) associated with the select agent/quarantined R. solanacearum strain. The program correctly typed all 371 tested egl sequences with known sequevars. It also successfully typed 25 R. solanacearum strains with no prior sequevar information, as well as 4 strains from infected plant samples, using their partial egl sequences amplified and sequenced with primers designed in this study. The Ralstonia solanacearum typing program does not require expertise or specific knowledge to use, gives results in seconds, and provides data interpretation for the user regarding the genospecies and sequevar 1 or 2 status of the strain in question. Real-time PCR detection and discrimination of the parsley pathogens Pseudomonas syringae pv. apii and Pseudomonas syringae pv. coriandricola Development of a loop-mediated isothermal amplification assay for the detection of Harpophora maydis N. GONZALEZ (1), G. Munkvold (1) (1) Iowa State University, U.S.A. Development of a loop-mediated isothermal amplification assay for the detection of Harpophora maydis N. GONZALEZ (1), G. Munkvold (1) (1) Iowa State University, U.S.A. Harpophora maydis, a seedborne fungal plant pathogen that causes late wilt of maize, is an important pathogen that occurs in Africa, Europe and Asia, but not in the Americas. It is a high-priority quarantine pathogen listed on the APHIS-PPQ OPIS pest list. Thus, there is a need for effective pathogen detection assays that can be used to identify contaminated seeds. We have developed a loop-mediated isothermal amplification (LAMP) assay to detect H. maydis based on a specific AFLP sequence from GenBank. H. maydis isolates were grown on PDA and DNA was extracted with the Extract-N-Amp plant kit. A primer set (AFLP 17) consisting of 5 primers recognizing different regions of the target sequence was designed using Primer Explorer V4 software. The LAMP reactions were performed in a 25 µl reaction mix (1X Isothermal master mix) and incubated at 67°C for 25 minutes. The amplified products were detected by fluorescence and results were confirmed by gel electrophoresis. The LAMP assay showed specificity to the pathogen as shown by negative amplification when tested with other fungal species and was developed without the need to standardize DNA concentrations. The assay was effective for detecting the pathogen in seed samples containing inoculated seeds. This LAMP assay should be suitable as a fast, easy to perform and reliable method to detect H. maydis, providing a tool to help prevent the introduction of this potentially devastating pathogen into the USA. Using Color Spectrophotometry to Evaluate Disease Severity of Macrophomina phaseolina in Soybean J. JORDAN (1), A. Mengistu (2), H. Kelly (1) (1) University of Tennessee, U.S.A.; (2) USDA, U.S.A. Charcoal rot is a soilborne disease of soybean caused by the fungus Macrophomina phaseolina (Tassi) Goid. The fungus is associated with hot dry weather and can dramatically reduce yields. Two current methods are used to evaluate disease severity in root tissues: colony forming units (CFU) and visual severity ratings. These methods are time consuming and expensive. Therefore, a more efficient methodology is needed to improve the screening process. We evaluated the use of color spectrophotometry as an alternative method for screening disease severity in 2006-2010 using 340 conventional and 893 roundup ready cultivars. Relative post-inoculation detection frequencies of ‘Candidatus Liberibacter solancearum’ haplotypes in three solanaceous hosts C. RUSH (1), L. Paetzold (1), F. Workneh (1) (1) T A&M A iLif R h B hl d TX U S A Relative post-inoculation detection frequencies of ‘Candidatus Liberibacter solancearum’ haplotypes in three solanaceous hosts C. RUSH (1), L. Paetzold (1), F. Workneh (1) (1) Texas A&M AgriLife Research, Bushland, TX, U.S.A. Relative post-inoculation detection frequencies of ‘Candidatus Liberibacter solancearum’ haplotypes in three solanaceous hosts C. RUSH (1), L. Paetzold (1), F. Workneh (1) (1) Texas A&M AgriLife Research, Bushland, TX, U.S.A. ‘Candidatus Libreibacter solanacearum’ (Lso), vectored by the potato psyllid (Bactericera cockerelli), is a putative bacterial pathogen, which causes potato zebra chip disease. The pathogen also infects all known solanaceous plants including peppers and tomatoes. Currently there are two haplotypes (A and B) of the pathogen identified from potatoes, and individual psyllids can carry either A or B or both. Greenhouse experiments were conducted to investigate post-inoculation relative incidences of the haplotypes in host plants (pepper, potato, and tomato) and in successive psyllid generations reared on the hosts. Plants were infested with psyllids in 5 cages each (12 psyllids/cage) with each cage containing 2 plants. Psyllids were obtained from a S4.110 colony in which nearly 90% were positive for A+B (the rest B). Plant and psyllid samples from the cages were collected 3 times (about every 2 weeks) beginning 6 weeks after infestation and tested for Lso haplotypes using qPCR and SYBR analysis. There was a highly significant difference among the hosts in incidence of the haplotypes (Chisq=15.3, P<0.0001). In peppers, 70% of the haplotypes were A, and 30% A+B. Haplotype B by itself was not detected. In potatoes, 75, 18, and 7% of haplotypes were A+B, B, and A, respectively. However, nearly all samples from tomatoes (96.7%) had haplotype A+B (the rest B) but A as a single haplotype was not detected. Haplotype frequencies in psyllids followed a similar detection pattern. colony in which nearly 90% were positive for A+B (the rest B). Plant and psyllid samples from the cages were collected 3 times (about every 2 weeks) beginning 6 weeks after infestation and tested for Lso haplotypes using qPCR and SYBR analysis. There was a highly significant difference among the hosts in incidence of the haplotypes (Chisq=15.3, P<0.0001). In peppers, 70% of the haplotypes were A, and 30% A+B. Haplotype B by itself was not detected. In potatoes, 75, 18, and 7% of haplotypes were A+B, B, and A, respectively. Relative post-inoculation detection frequencies of ‘Candidatus Liberibacter solancearum’ haplotypes in three solanaceous hosts C. RUSH (1), L. Paetzold (1), F. Workneh (1) (1) T A&M A iLif R h B hl d TX U S A However, nearly all samples from tomatoes (96.7%) had haplotype A+B (the rest B) but A as a single haplotype was not detected. Haplotype frequencies in psyllids followed a similar detection pattern. Early detection of Leifsonia xyli subsp. xyli, the causal agent of ratoon stunting disease, in sugarcane seedlings D. OLIVERA (1), A. Urashima (1) (1) Federal University of Sao Carlos, Brazil The first major change of the Brazilian sugarcane industry over the last decades occurred when mechanical harvest replaced the manual cuttings of burned canes. The second transformation has started with mechanization of planting materials thanks to new methodologies such as pre-sprouted cane (PSC). To ensure the success of this innovation, however, is paramount the use of clean seed cane especially against ratoon stunting disease, caused by the fastidious bacterium Leifsonia xyli subsp. xyli (Lxx), which can only be spread over new areas by contaminated propagative materials. Therefore, the purpose of this work was to develop a routine diagnostic test for early detection of Lxx in PSC seedlings. In the first assay, four previously published pair of primers, Cxx ITSf#5/Cxx ITSr#5, Cxx1/Cxx2, C2F/C2R, LX23SF/LX23SR, was challenged under the original conditions, as well as, our own modifications against adult diseased sugarcane with various concentrations of Lxx. In the second assay, two of the best primers/conditions were tested against PSC of 45 and 60 days of age derived from propagative materials with different levels of Lxx (106 to 109 UFC/ml). Our result showed primers Cxx ITSf#5/Cxx ITSr#5 run under our modified conditions was the most efficient since Lxx was detected in all 52 PSC seedlings of 45 days, including all 18 resulted from propagative materials contaminated with 106 UFC/ml. Part of MSc dissertation by the second author at PPGPVBA, CAPES Scholarship. Development of a loop-mediated isothermal amplification assay for the detection of Harpophora maydis N. GONZALEZ (1), G. Munkvold (1) (1) Iowa State University, U.S.A. Field plants were sampled at the R7 growth stage and evaluated using CFU, visual ratings (1-5 scale), and a color spectrophotometer. As previously reported, CFU values and visual ratings were predictive of each other across all years (F=7.66, p<0.05). Color data was not predictive of severity ratings (F = 2.70, p = 0.10), however they were negatively correlated across all years (r = -0.62, p<0.001). This association suggests that this method may be used as alternative in disease severity quantification of charcoal rot. Detection of Erysiphe necator fungicide-resistant alleles in leaf and air samples using novel molecular diagnostic techniques J. YAMAGATA (1), B. Warneke (2), T. Neill (3), W. Mahaffee (4), L. Miles (5), A. Schilder (6) (1) CSU Monterey Bay, Seaside, CA, U.S.A.; (2) Oregon State University, Corvallis, OR, U.S.A.; (3) USDA ARS, Corvallis, RI, U.S.A.; (4) USDA ARS, Corvallis, CA, U.S.A.; (5) Hartnell College, Salinas, CA, U.S.A.; (6) Michigan State University, Department of Plant, Soil and Microbial Sciences, East Lansing, MI, U.S.A. Erysiphe necator (En) the causal agent of grapevine powdery mildew is managed using fungicides such as quinone outside inhibitors (QoIs). Unfortunately, En is known to develop resistance to QoIs by a single nucleotide polymorphism, resulting in an amino acid substitution (G143A) in cytochrome b. To monitor this allele, a TaqMan assay was developed and validated against powdery mildew DNA from strawberry, blueberry, snap pea, squash, Gerbera daisy, rose, and oak plants. Single spored DNA isolates (n = 103) were used to verify the accuracy of the TaqMan assay by comparing it to previous trifloxystrobin germination tests. In-vitro tests of single spored isolates identified 20% as the necessary proportion of the mutant allele in a mixed DNA sample to test positive for resistance. Leaf and air samples from Oregon (n = 130) were utilized in field validations. Digital droplet PCR was also utilized to quantify the allele distribution in a mixed DNA sample and lowered the detection limit the mutant allele for positive resistance to <5% of the total E. necator DNA present. This study resulted in novel allele-specific TaqMan and Digital droplet PCR assays that detect G143A in En. This assay could be used to significantly reduce the labor required in dealing with leaf and air samples of En in the laboratory and could be used to more closely monitor resistance development for a standard management program of grapevine powdery mildew. Inmunochemical detection of the Silvelrleaf disease of fruit trees caused by Chondrostereum pupureum D. GRINBERGS (1), J. Chilian (1), A. France (1) (1) Instituto de Investigaciones Agropecuarias, Chile The Silverleaf disease affects numerous species, including important fruit crops such as apple, blueberry, cherry, kiwi, peach, pear and plum. The leaves silvering is caused by the acumulation of an enzyme (endoPG) produced by Chondrostereum purpureum, however, foliar symptoms are not visible untill the third year after the orchard plantation. Curative control is not effective, thus, an early detection is essential to avoid the fungus dissemination. Therefore, the objective of this work was to develop an early and non destructive method to detect the disease. Using the endoPG’s aminoacidic sequence, monoclonal antibodies were synthesized and DAS-ELISA protocol developed. Mature leaves showing different silvering intensity were assessed, using healthy plants as negative control and purified endoPG as positive control. The presence of C. purpureum in the plants was confirmed through its isolation on 25% potato dextrose agar (PDA) and by the amplification of the large intergenic spacer (IGS) región, located between 5S and 18S rDNA. Results indicated that there was a positive correlation between foliar symptoms intensity and endoPG concentration, and that the method was able to discriminate among leaves from healthy and diseased but asymptomatic plants. This detection method was validated in apple, blueberry, peach and plum, as far, becoming a useful tool to prevent the disemination of the Silverleaf disease. Evaluation of molecular diagnostic methods for Monilinia species of regulatory concern S. HAYMES (1), K. Zeller (1), Z. Abad (1), L. Knight (1) (1) USDA APHIS PPQ S&T Center for Plant Science and Technology, U.S.A. Brown rot caused by Monilinia species is an extremely destructive disease of pome and stone fruits in many producing areas of the world, resulting in significant pre- and post-harvest losses. Diagnostics of species is difficult due to overlapping morphology, host-ranges, and symptoms. There are currently no molecular diagnostics methods that clearly discriminate all closely related species. Monilinia fructicola and M. laxa are native to or established in the US. Monilinia fructigena and M. polystroma are widely distributed in Europe and Asia and are exotic for the US. Both are listed as level “A” pests of economic importance to US agriculture in the 2015 Prioritized Offshore Pest List and the 2016 Cooperative Agricultural Pest Survey (CAPS) List. Monilinia mumecola and M. yunnanensis have been described recently (in 2004 and 2011, respectively) but are of less known impact, and neither of these species is known from the United States. Development of a loop-mediated isothermal amplification assay for the detection of Harpophora maydis N. GONZALEZ (1), G. Munkvold (1) (1) Iowa State University, U.S.A. Investigation of primary infection period of apple blotch and circular leaf spot based on loop-mediated isothermal amplification assay in Korea H. JUNG (1), S. Lee (2), S. Lee (2), J. Keum (2), S. Park (2), H. Woo (3), Y. Lim (4), I. Kang (2) (1) College of Agricultural and Life Sciences, Kyungpook National University, Daegu, South Korea; (2) College of Agricultural and Life Sciences, Kyungpook National University, South Korea; (3) Bayer Crop Science, South Korea; (4) Gyeongsangbuk-do Agricultural Research & Extension Services, South Korea Investigation of primary infection period of apple blotch and circular leaf spot based on loop-mediated isothermal amplification assay in Korea H. JUNG (1), S. Lee (2), S. Lee (2), J. Keum (2), S. Park (2), H. Woo (3), Y. Lim (4), I. Kang (2) (1) College of Agricultural and Life Sciences, Kyungpook National University, Daegu, South Korea; (2) College of Agricultural and Life Sciences, Kyungpook National University, South Korea; (3) Bayer Crop Science, South Korea; (4) Gyeongsangbuk-do Agricultural Research & Extension Services, South Korea Loop-mediated isothermal amplification (LAMP) method is widely used to detect various plant pathogens. In this study, we surveyed primary infection period of apple blotch (AB) on apple and circular leaf spot (CLS) on persimmon using developed LAMP method. The causal agent of AB and CLS; Marssonina coronaria and Mycosphaerella nawae have long incubation period to appear the typical symptoms. Up to now, primary infection period was predicted through conidia scattering period or measurement of number of conidia. To control the AB and CLS properly, primary infection periods should to be clear. Following the M. coronaria and M. nawae have long incubation period, we developed two different LAMP assay which can apply to apple and persimmon, and surveyed primary infection periods of AB and CLS in Korea using developed LAMP assay. As the results, not only infection was confirmed in healthy apple and persimmon leaves, but also the infection periods were gradually earlier than before. Although the relationship between S4.111 primary infection period and fungicide program should to be studied, it is assumed that, to control and prevent economical losses of AB and CLS, fungicide spray program should be change. primary infection period and fungicide program should to be studied, it is assumed that, to control and prevent economical losses of AB and CLS, fungicide spray program should be change. A new paradigm for early diagnosis tool for major fungal disease in apple and persimmon S. LEE (1), M. Jeon (2), J. Kim (3), H. Jung (1) A new paradigm for early diagnosis tool for major fungal disease in apple and persimmon S. LEE (1), M. Jeon (2), J. Kim (3), H. Jung (1) (1) College of Agricultural and Life Sciences, Kyungpook National University, Daegu, South Korea; (2) School of Electrical Engineering and Computer Science, Kyungpook National University, Daegu, South Korea; (3) School of Electrical Engineering and Computer Science, Kyungpook National University, South Korea S. LEE (1), M. Jeon (2), J. Kim (3), H. Jung (1) (1) College of Agricultural and Life Sciences, Kyungpook National University, Daegu, South Korea; (2) School of Electrical Engineering and Computer Science, Kyungpook National University, Daegu, South Korea; (3) School of Electrical Engineering and Computer Science, Kyungpook National University, South Korea ( ), ( ), ( ), g ( ) (1) College of Agricultural and Life Sciences, Kyungpook National University, Daegu, South Korea; (2) School of Electrical Engineering and Computer Science, Kyungpook National University, Daegu, South Korea; (3) School of Electrical Engineering and Computer Science, Kyungpook National University, South Korea To diagnose the fungal disease, it can be diagnose when the visible symptoms or sign are appeared. Or molecular biological method, such as polymerase chain reaction (PCR) is widely used, however, it is hard to apply in field condition and takes long time. To overcome these problem, we applied optical coherence tomography (OCT) tool for diagnosis the major fungal disease; apple blotch (AB) and circular leaf spot (CLS) in apple and persimmon. The casual agents of AB and CLS is Marssonina coronaria and Mycosphaerella nawae, which has long incubation period in their host. Using these characteristics, we tried to find the differences between healthy and infected leaves in the incubation period stage. OCT is non-invasive method which can examine the leaf samples without treatment. A-scan analysis which showing signal intensity showed that there was difference between healthy and infected leaves. Furthermore, infected leaves which had any visible symptoms were appeared similar pattern of signal intensity with diseased leaves. And infection was re-confirmed using species-specific PCR assay. Based on these results, it is assumed that OCT can be a useful diagnostic tool for early diagnose of two major fungal disease in apple and persimmon. Inmunochemical detection of the Silvelrleaf disease of fruit trees caused by Chondrostereum pupureum D. GRINBERGS (1), J. Chilian (1), A. France (1) (1) Instituto de Investigaciones Agropecuarias, Chile Inmunochemical detection of the Silvelrleaf disease of fruit trees caused by Chondrostereum pupureum D. GRINBERGS (1), J. Chilian (1), A. France (1) (1) Instituto de Investigaciones Agropecuarias, Chile The objective of this study is to evaluate two existing conventional PCR assays for distinguishing between native and exotic Monilinia for regulatory purposes. We have verified analytical sensitivity and specificity of these assays and determined the functional sensitivity with field-derived or spiked samples. A two-stage molecular diagnostic protocol that incorporates both assays allows for discrimination among all six of these Monilinia species, and should facilitate the accurate diagnosis for CAPS surveys. Identification of genomics-based detection assay for Rhizoctonia solani anastomosis groups T. MILES (1), J. Cerda (1) (1) CSU Monterey Bay, U.S.A. Identification of genomics-based detection assay for Rhizoctonia solani anastomosis groups T. MILES (1), J. Cerda (1) (1) CSU Monterey Bay, U.S.A. Rhizoctonia is an important plant pathogen for many crops throughout the world causing plant tissue rot. Typically, diagnoses is accomplished using pure culture “tester” isolates and determining an anastomosis group. In order to develop new diagnostic tools, three mitochondrial genomes of Rhizoctonia solani were aligned with Mauve using the software Geneious 7.1. AG1 and AG3 had dramatically different mitochondrial genome sizes (160 Kb versus 250 Kb, respectively) due to the presence of introns. A region of the rRNA large subunit from the mitochondria was identified and several primers were designed. DNA was extracted from 5 isolates of R. solani followed by the polymerase chain reaction and sequencing. Sequence length for the large subunit of rRNA for each tested isolate varied significantly in size to what was expected. Difference in size is due to the presence and length of introns in the mitochondrial genome. Sequences were aligned for homology and differences. We are in the process of extracting purified genomic mitochondrial DNA from type isolates of R. solani to get a more complete picture of the significant diversity between AG groups. Even further research will be done to convert this method into a rapid field detection tool via recombinase polymerase amplification. Development, testing and pending deployment of a LAMP diagnostic screening method for the Citrus Black Spot pathogen, Guignardia citricarpa Phytogenetic identification of pathogenic and endophytic fungal populations in west coast Douglas-fir foliage D. DANIELS (1), J. Kiser (1) (1) Oregon State University U S A ( ) ( ) (1) Oregon State University, U.S.A. Douglas-fir provides social, economic and ecological benefits in the Pacific Northwest (PNW). Minor defoliation has been linked to Swiss Needle Cast, associated with Phaeocryptopus gaeumannii. Unprecedented defoliation has increased since the 1990s, leading to decreased growth and yield. Affected areas exceed 500,000 acres in Oregon. Recent symptoms are inconsistent with predicted effects of P. gaeumannii. We hypothesize that the entire endophytic community is relevant to disease ecology, though this has not been investigated to date. Dynamics of needle cast are explored and baseline inventories of endophytes established by Sanger and Next-gen Sequencing (NGS). Endophytes were evaluated along environmental gradients at three sites in the PNW. Probability of occurrence (presence or absence of any endophyte in needle) was hypothesized to be higher in cooler, wetter climates. Preliminary results suggest that the probability of endophyte occurrence at the cool, wet site was 2.6 (p<0.0007) times higher than at the warm, dry site, and 3.7 (p<0.0001) times higher than at the warm, wet site. Despite advances in technology, few studies have identified endophytes in Douglas-fir by molecular means. Traditional culturing is hypothesized to be less effective than NGS. More inclusive identification could lead to greater efficiency in forest management. It is vital to understand the complete etiology of SNC and the ecological implications of disease in this important species. apid diagnostic assay development for detecting patch diseases on turf . KARAKKAT (1), M. Franchett (1), M. Olson (1), C. Mullenberg (1), P. Koch ) Department of Plant Pathology, University of Madison, WI, U.S.A., (2) Department oxicology Center, University of Wisconsin, Madison, Wisconsin, U.S.A. Rapid diagnostic assay development for detecting patch diseases on turf B. KARAKKAT (1), M. Franchett (1), M. Olson (1), C. Mullenberg (1), P. Koch (1) Department of Plant Pathology, University of Madison, WI, U.S.A., (2) Department of Plant Pathology and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin, U.S.A. B. KARAKKAT (1), M. Franchett (1), M. Olson (1), C. Mullenberg (1), P. Koch (1) Department of Plant Pathology, University of Madison, WI, U.S.A., (2) Department of Plant Pathology and Molecular and Environmental Toxicology Center, University of Wisconsin, Madison, Wisconsin, U.S.A. Necrotic Ring Spot, Summer Patch, and Take-all Patch are turf root diseases caused by soil-borne plant pathogenic fungi. BioPCR: An improved method for detection of Colletotrichum acutatum in asymptomatic strawberry nursery plants P. SUDARSHANA (1), K. Mani (1), P. Randhawa (1), T. Walters (2) (1) California Seed and Plant Labs, U.S.A.; (2) Walters Ag Research, U.S.A. Colletotrichum acutatum infections in strawberry nurseries often stay symptomless. Infected plants usually collapse upon transplanting in grower fields resulting in economic losses to the growers. A routine method to detect C. acutatum in asymptomatic plants is to culture root and crown tissue on semi- selective agar media such as TPDA and BR and confirm identification by microscopic observations of spore morphology and PCR. In our experience, the culture method often fails to detect C. acutatum when non-target fungi such as ?Trichoderma sp., ?Alternaria sp. and Aspergillus sp. are present in a sample. The rapid growth of non-target fungi mask the pathogen in culture. To overcome this problem, we investigated BioPCR as an alternative method to detect infections in asymptomatic tissue. In BioPCR, tissue was cultured for four days on agar media to enrich C. acutatum in the plant tissue. After 4 days, tissue was removed from the culture plates and tested by PCR using primers specific for C. acutatum (1). Culture plates were still available for reading. During 2015, we analyzed 106 commercial samples from strawberry nurseries in California by culture and BioPCR method. BioPCR detected C. acutatum in 83% plants compared to 42% by culture method. We conclude that BioPCR method is superior to culture method and can be used to assay strawberry nursery plants for detection of C. acutatum in asymptomatic plants. Development of a quantitative PCR assay to quantify resistance to Diaporthe helianthi and Diaporthe gulyae in sunflower T. OLSON (1), F. Mathew (1), A. Adhikari (1), B. Kontz (1), L. Marek (2) (1) South Dakota State University, Brookings, SD, U.S.A., (2) Iowa State University, Ames, IA, U.S.A. / USDA-ARS In the U.S., Phomopsis stem canker was first detected in 1983, but it was not until the 2010 epidemic in Minnesota, North Dakota, and South Dakota that two pathogens, Diaporthe helianthi and Diaporthe gulyae were identified as the causal agents. At this time, no commercial hybrids are available that have complete resistance to the two pathogens. The objectives of this study were to (1) develop quantitative polymerase chain reaction (qPCR) assays for quantification of D. helianthi and D. gulyae from sunflower and (2) evaluate sunflower germplasm for resistance to the two pathogens using qPCR assays. The primers and probes for the two qPCR assays were designed from the translation elongation factor region for amplification of D. helianthi and D. gulyae. Rapid differentiation of Claviceps species occurring in Oregon and Washington using high resolution melting curve analysis N. KAUR (1), R. Cating (2), J. Dung (3), S. Alderman (4), D. Walenta (5), P. Hamm (1), K. Frost (1) (1) Oregon State University, Hermiston, OR, U.S.A.; (2) Oregon State University, Corvallis, OR, U.S.A.; (3) Oregon State University, Madras, OR, U.S.A.; (4) USDA-ARS National Forage Seed production Research Center, Corvallis, OR, U.S.A.; (5) Oregon State University, La Grande, OR, U.S.A. g and pending deployment of a LAMP diagnostic screening method for the Citrus Black Spot pathogen, Guignardia citricarpa K. ZELLER (1), K. Levin (2), G. Wei (2), G. Abad (2), H. Gomez (3), T. Riley (3), Z. Liu (2) (1) USDA-APHIS-PPQ, CPHST, U.S.A.; (2) USDA-APHIS-PPQ, CPHST, U.S.A.; (3) USDA-APHIS-PPQ, CHRP, U.S.A. p K. ZELLER (1), K. Levin (2), G. Wei (2), G. Abad (2), H. Gomez (3), T. Riley (3), Z. Liu (2) (1) USDA-APHIS-PPQ, CPHST, U.S.A.; (2) USDA-APHIS-PPQ, CPHST, U.S.A.; (3) USDA-APHIS-PPQ, CHRP, U.S.A. p K. ZELLER (1), K. Levin (2), G. Wei (2), G. Abad (2), H. Gomez (3), T. Riley (3), Z. Liu (2) (1) USDA-APHIS-PPQ, CPHST, U.S.A.; (2) USDA-APHIS-PPQ, CPHST, U.S.A.; (3) USDA- Guignardia citricarpa, the causal agent of Citrus Black Spot (CBS) was discovered in Florida during 2010. Today, three FL counties remain under quarantine. G. citricarpa causes unsightly lesions that reduce fruit marketability, and CBS is subject to national and international quarantine restrictions. The affected packing houses must monitor all fruits coming into the facility before they ship to the EU. If CBS symptoms are found on just a single fruit, entire shipments are put on hold where this perishable commodity’s time is extremely valuable and a decision must be made. Current protocol requires that suspect fruit be shipped to PPQ or State Labs for confirmatory diagnosis which include a conventional and qPCR assay. We have developed a diagnostic tool that will enable inspectors to turn a 2-3 day decision into less than 1 hour using a LiNK extraction method followed by Loop-Mediated Isothermal AMPlification (LAMP). The LiNK column allows for rapid DNA extraction for use on the Genie III, a durable, sensitive, and user-friendly isothermal platform. Our LAMP screening assay is able to detect down to 30 fg of DNA with amplification times between 5-20 minutes. The Genie has been able to reproduce qPCR results on all 55 citrus samples tested from Florida as well as 32/33 CBS DNA diagnostic samples submitted to CPHST. Further testing on environmental samples and non-target hosts are underway. We will discuss the advantages and limitations of the method and deployment. S4.112 Rapid diagnostic tools for soilborne pathogens of strawberry A. BURKHARDT (1), T. Miles (2), M. Ramon (1), S. Koike (3), F. Martin (1) (1) USDA, U.S.A.; (2) CSUMB, U.S.A.; (3) Cooperative Extension Monterey County, U.S. Rapid diagnostic tools for soilborne pathogens of strawberry A. BURKHARDT (1), T. Miles (2), M. Ramon (1), S. g and pending deployment of a LAMP diagnostic screening method for the Citrus Black Spot pathogen, Guignardia Koike (3), (1) USDA, U.S.A.; (2) CSUMB, U.S.A.; (3) Cooperative Extensi pid diagnostic tools for soilborne pathogens of strawberry The ability to rapidly identify and quantify strawberry pathogens in the soil allows growers to make faster decisions about what to plant and how to manage diseases a field. Therefore, we are developing rapid assays using TaqMan real-time PCR and recombinase polymerase amplification (RPA) to diagnose a variety of pathogens commonly infecting strawberry fields, including Verticillium dahliae, Fusarium oxysporum, Phytophthora spp., and Macrophomina phaseolina. These assays can provide results in hours (TaqMan) or in as little as 20 minutes (RPA) using DNA extracted from either infected plant tissue or soil. Species-specific TaqMan and RPA assays have been developed for Phytophthora and are very sensitive (0.2 pg) and specific. For V. dahliae, a species-specific TaqMan assay with a sensitivity of 1-2 microsclerotia per gram of soil is being converted to an RPA assay. Host-specific markers for F. oxysporum f. sp. fragariae are currently being developed for both TaqMan and RPA using a locus previously published by Suga et al. (Plant Dis. 2013). Additional loci for F. oxysporum f. sp. fragariae are being identified through sequencing and comparative genomics of multiple isolates in order to expand the breadth of detection. For M. phaseolina, sequencing of multiple isolates has identified a genotype-specific locus, and primers are being developed for RPA and TaqMan assays to detect isolates which have been found to only infect strawberry. BioPCR: An improved method for detection of Colletotrichum acutatum in asymptomatic strawberry nursery plants P. SUDARSHANA (1), K. Mani (1), P. Randhawa (1), T. Walters (2) 1) California Seed and Plant Labs, U.S.A.; (2) Walters Ag Research, U.S.A. BioPCR: An improved method for detection of Colletotrichum acutatum in asymptomatic strawberry nursery plants P. SUDARSHANA (1), K. Mani (1), P. Randhawa (1), T. Walters (2) (1) California Seed and Plant Labs, U.S.A.; (2) Walters Ag Research, U.S.A. To evaluate sunflower germplasm for resistance to Diaporthe spp., 54 sunflower genotypes from 9 different countries were screened for resistance using the stem-wound method in the greenhouse. Disease severity was evaluated 14 days after inoculation using a 0-5 rating scale from Mathew et al. (2015). Preliminary results suggest significant differences among genotypes in their resistance to D. helianthi and D. gulyae; PI 561918 (HA 378) appeared to be less susceptible to the two pathogens than the susceptible check ‘HA 288’. The qPCR assays developed in this study will be used to compare genotypes for resistance to D. helianthi and D. gulyae by quantifying the levels of pathogen DNA present in them after inoculation. Phytogenetic identification of pathogenic and endophytic fungal populations in west coast Douglas-fir foliage D. DANIELS (1), J. Kiser (1) (1) O St t U i it U S A Phytogenetic identification of pathogenic and endophytic fungal populations in west coast Douglas-fir foliage D. DANIELS (1), J. Kiser (1) (1) Oregon State University U S A Yan (2) (1) North Dakota State University, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A. Real-time and conventional PCR assays for identifying the stubby root nematode Paratrichodorus allius G. YAN (1), D. Huang (2), G. Yan (2) (1) North Dakota State University, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A. Stubby root nematode (SRN), Paratrichodorus allius, is an important pest on many crops, particularly, on potato due to its ability to transmit Tabacco rattle virus causing corky ringspot disease on tubers. Nematode surveys revealed that SRN is present in many fields on different crops in North Dakota and Minnesota. Detection and identification of SRN are important for effective disease management. Protocols of P. allius identification by real-time and conventional PCR with species-specific primers were developed. Primers were designed from internal transcribed spacer of rDNA based on sequences of P. allius and six closely related Paratrichodorus spp. A single amplicon (246 bp) was produced from DNA of P. allius only, but not from DNA of two other SRN species and 21 non-SRN species. Detection sensitivity analysis indicated that an equivalent of 1/20 of a nematode could be detected by conventional PCR and an equivalent of 1/1000 of a nematode by real-time PCR with Cq value at 32.6±0.5. The PCR assays were tested with SRN from 20 soil samples collected from various fields with five crops in ND and MN, and they successfully amplified DNA from all samples. These SRNs were confirmed to be P. allius by sequencing of three genomic regions. The PCR assays are rapid and reliable and can be used in P. allius identification using nematode individuals. This finding will help develop a real-time PCR assay for quantification of P. allius directly from DNA extracts of field soils. Development of species-specific qPCR tests for detection and quantification of Meloidogyne hapla A. GORNY (1), X. Wang (2), S. Pethybridge (1) (1) Cornell University, School of Integrative Plant Sciences, Plant Pathology & Plant-Microbe Biology Section, U.S.A.; (2) USDA-ARS, Plant Protection Research Unit, Cornell University, U.S.A. A. GORNY (1), X. Wang (2), S. Pethybridge (1) (1) Cornell University, School of Integrative Plant Sciences, Plant Pathology & Plant-Microbe Biology Section, U.S.A.; (2) USDA-ARS, Plant Protection Research Unit, Cornell University, U.S.A. Phytogenetic identification of pathogenic and endophytic fungal populations in west coast Douglas-fir foliage D. DANIELS (1), J. Kiser (1) (1) Oregon State University U S A The infected turf shows aboveground symptoms that are broadly categorized as ‘patch’ diseases that ruin the aesthetic value of cool-season turfgrass on lawns/fields in North America from late spring to early summer. These fungal pathogens are notoriously difficult to isolate from roots. Therefore, we decided to develop a rapid detection assay that can be performed on suspected root samples. We are testing two assays: Loop mediated isothermal amplification (LAMP) and Recombinase polymerase amplification (RPA). Both LAMP and RPA do not require pure genomic DNA but only a solution of root sample. LAMP and RPA rely on DNA strand displacement activity for its rapid amplification and can be completed at 65oC in an hour and 39oC in 20 minutes incubation, respectively. Initial LAMP screening gave false positives repeatedly in almost all assays indicating that very pure conditions are needed, which is not ideal in a diagnostic laboratory. Hence, we have started to work on RPA methodology available as Agdia’s discovery kit to eliminate any false positives. Once we achieve a consistent and specific amplification with these assays, we will proceed to screen samples that will be arriving in late Spring 2016. Assay development, primer-probe design and samples surveyed will be reported in this study. Rapid differentiation of Claviceps species occurring in Oregon and Washington using high resolution melting curve analysis N. KAUR (1), R. Cating (2), J. Dung (3), S. Alderman (4), D. Walenta (5), P. Hamm (1), K. Frost (1) (1) Oregon State University, Hermiston, OR, U.S.A.; (2) Oregon State University, Corvallis, OR, U.S.A.; (3) Oregon State University, Madras, OR, U.S.A.; (4) USDA-ARS National Forage Seed production Research Center, Corvallis, OR, U.S.A.; (5) Oregon State University, La Grande, OR, U.S.A. S4.113 Ergot disease of grass seed crops can be caused by Claviceps purpurea and C. humidiphila, a species recently reported to occur in certain regions of OR and WA. Routine identification of Claviceps species involves conventional PCR amplification and sequencing of ITS, β-tubulin or EF-1α genes. In this study, a high resolution melting (HRM) assay was developed to rapidly identify and differentiate C. purpurea and C. humidiphila based on the melting temperatures (Tm) of partial β-tubulin amplicons. PCR primers were designed to target and amplify a 110 bp region of the β-tubulin gene containing multiple single nucleotide polymorphisms generating amplicons with distinct melting profiles for each species. Phytogenetic identification of pathogenic and endophytic fungal populations in west coast Douglas-fir foliage D. DANIELS (1), J. Kiser (1) (1) Oregon State University U S A Forty-six fungal cultures, previously classified based on RAPD profiles, and ITS, β-tubulin, and EF-1α gene sequences, were used to validate the HRM assay. The Tm for C. purpurea and C. humidiphila ranged between 79.8 to 80.3 and 78.9 to 79.3, respectively. Classification of Claviceps species based on Tm using linear contrasts (P < 0.05) correctly grouped 96% (N=46) of samples previously classified using sequence data. In two cases, C. purpurea was misclassified as a mixture of the two Claviceps species. Therefore HRM analysis allows rapid detection and differentiation of these two Claviceps species and will be useful to study their distributions within OR and WA cool-season grass seed growing regions. Specific detection of the root-lesion nematode Pratylenchus scribneri using conventional and real-time PCR G. YAN (1), D. Huang (2), G. Yan (3) (1) North Dakota State University, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A.; (3) North Dakota State University, Department of Plant Pathology, U.S.A. Pratylenchus scribneri is a plant-parasitic root-lesion nematode causing economic damage to various crops worldwide. Identifying root-lesion nematodes to species using traditional morphological method is an arduous task requiring extensive training on nematode taxonomy. Consequently, molecular methods were developed to identify P. scribneri by conventional and real-time PCR using a species-specific primer set that was designed based on the alignment of 20 sequences of internal transcribed spacer of rDNA of seven Pratylenchus spp. The primer set specifically amplified DNA of P. scribneri but not DNA from five other Pratylenchus spp. or ten non-Pratylenchus spp. A single melt curve was observed indicating a high specificity according to real-time PCR analysis. The capability of specific PCR was tested using DNA extracts of root-lesion nematodes from 23 soil samples collected from various fields with five crops in North Dakota and Minnesota. The PCR primers and protocols were successfully used in P. scribneri identification. The conventional PCR could detect DNA of 1:8 dilution of two nematodes equivalent to 1/4 of a nematode. The real-time PCR was more sensitive and able to amplify DNA of serial dilutions (2 nematodes - 1/128 nematode) with Cq values ranging from 25.4±0.0 to 33.6±0.4. The developed PCR assays provide a rapid and reliable identification of P. scribneri, suitable for use in diagnostic laboratories for detection of field infestations with this nematode. Real-time and conventional PCR assays for identifying the stubby root nematode Paratrichodorus allius G. YAN (1), D. Huang (2), G. Phytogenetic identification of pathogenic and endophytic fungal populations in west coast Douglas-fir foliage D. DANIELS (1), J. Kiser (1) (1) Oregon State University U S A The Northern root-knot nematode, Meloidogyne hapla, is an important soilborne pathogen of potatoes and other vegetables grown in the United States and is common within New York State. Effective management of M. hapla and other plant-parasitic nematodes relies on the accurate identification and timely quantification of pathogen populations prior to planting. Identification and quantification may be substantially improved by pre-plant soil tests which utilize quantitative PCR (qPCR) techniques to target DNA sequences specific to the pathogen of interest. Here, qPCR primers were designed to target unique regions of an effector gene 16D10 in M. hapla and assayed for specificity against plant-parasitic nematodes using qualitative PCR. The primers developed in this study were shown to be specific to M. hapla. A qPCR tests was developed and the sensitivity of the primers determined. DNA-nanosensor based diagnostics of plant pathogens A. BAETSEN-YOUNG (1), M. Vasher (1), E. Alocilja (1), B. Day (1) (1) Michigan State University, U.S.A. Crop loss from plant pathogens is a leading threat to food security in the United States. Current pathogen detection assays have limited field operability, leading to improper disease management decisions. Recently, rapid diagnostic DNA-nanosensors have increased perception and aided precision care of human pathogens in resource limited locations. Here we demonstrate the application of DNA-based nanosensor to detect an obligate biotroph, Pseudoperonospora cubensis. The detection of P. cubensis is based upon primers highly specific to a targeted gene, and the physical properties of DNA and nanoparticles with sodium chloride. Diluted DNA was denatured and cooled in a solution with the primers. Then nanoparticles were added to the solution followed by sodium chloride to reveal primer-target interaction. Target DNA presence was quantified colorimetrically (i.e. naked eye) and by absorbance on a spectrophotometer over a 400 to 700 nm range. Current PCR amplified target can be detected to 5 pg/mL by the naked eye. Optimization of this DNA-nanosensor to detect extracted genomic DNA, and within environmental matrices will be discussed. The deployment of this rapid bioassay has the potential to assist growers in developing sustainable disease management decisions in a field based setting. Optimization of a fluorescence in situ hybridization assay for detection and visualization of Plasmopara obducens from plants and soil C. SALGADO-SALAZAR (1) (1) USDA-ARS, Rutgers University, U.S.A. Evaluation of Recombinase Polymerase Amplification (RPA) Isothermal Amplification Diagnostic Assay for Phytophthora ramorum J. BIENAPFL (1), L. Knight (1), Z. Abad (1) Evaluation of Recombinase Polymerase Amplification (RPA) Isothermal Amplification Diagnostic Assay for Phytophthora ramorum J. BIENAPFL (1), L. Knight (1), Z. Abad (1) (1) USDA APHIS PPQ S&T, U.S.A. Phytophthora ramorum was found as the causal agent of ‘Sudden Oak Death’ oaks and tanoaks in the coastal counties of California in the 1990s. Since then, the pathogen has been confirmed on numerous native hosts and nursery plants in California, Oregon, and Washington, with sporadic occurrences in other regions of the USA. The current methods of testing for P. ramorum include conventional and real-time PCR which are expensive and timely. The primary purpose of this work is to enhance Plant Protection and Quarantine efforts in the detection of P. ramorum by evaluating a field-deployable, simple, and rapid detection method. A previously published Recombinase Polymerase Amplification (RPA) Isothermal Amplification Assay for detection of P. ramorum was evaluated for both sensitivity and specificity using the Twista™ apparatus. DNA extracted from pure culture, infected and healthy rhododendron tissue, as well as environmental samples were used to validate the P. ramorum protocol for regulatory use. Preliminary data comparing the isothermal amplification assay to current confirmatory P. ramorum diagnostic assays, evaluating ITS and Elicitin qPCRs shows that this protocol was not as sensitive as the ITS qPCR, but was comparable to the Elicitin qPCR. We will present the value of RPA isothermal amplification as a regulatory diagnostic tool for the rapid detection of P. ramorum. Extracting DNA for qPCR amplification from plant tissue in ELISA buffer N. OSTERBAUER (1), S. Navarro (2) (1) Oregon Dept. of Agriculture, U.S.A.; (2) Oregon Dept. of Forestry, U.S.A. USDA APHIS mandates testing protocols for Phytophthora ramorum. Diagnostic labs use an ELISA kit to screen for Phytophthora infection in leaves. If a leaf tests ELISA-positive, a second sample is collected from the leaf for DNA extraction and molecular testing. This may delay detection by a day. DNA extraction directly from macerated leaf tissue in ELISA buffer would speed testing. To determine if DNA extracted from such tissue would be adequate for molecular testing, 198 leaf samples from a nursery were tested with ELISA, with 30 testing positive. For each ELISA-positive sample, leaf tissue was collected and DNA extracted following WI-B-T-2-3 Rev. 1; DNA was also extracted from the macerated leaf tissue in ELISA buffer. Tissue- DNA and ELISA-DNA was then tested for P. ramorum following N-R-WI-008-0 using the reporter dyes FAM (P. Optimization of a fluorescence in situ hybridization assay for detection and visualization of Plasmopara obducens from plants and soil C. SALGADO-SALAZAR (1) (1) USDA-ARS Rutgers University U S A Optimization of a fluorescence in situ hybridization assay for detection and visualization of Plasmopara obducens from plants and soil C. SALGADO-SALAZAR (1) (1) USDA-ARS, Rutgers University, U.S.A. Plasmopara obducens is the biotrophic oomycete responsible for downy mildew of ornamental impatiens, a destructive disease that significantly impacts the production of Impatiens walleriana in the United States. To date, there are no methods to detect P. obducens from asymptomatic plants, which may be contributing to the spread of the disease. Furthermore, the extent that P. obducens uses oospores to colonize and persist in the soil is undetermined. Fluorescence in situ hybridization (FISH) is a sensitive and robust method that uses sequence-specific, fluorescence-labeled oligonucleotide probes to detect target organisms from the environment. A FISH assay for the impatiens downy mildew pathogen was designed by preparing DNA sequence alignments of the rDNA-ITS region of P. obducens and other related taxa to identify suitable regions for species-specific probe development. Because P. obducens cannot be propagated in vitro, we developed a custom E. coli expression vector that transcribes the rDNA-ITS gene sequence of P. obducens S4.114 for use as a control and to allow for standardized fluorescence signal quantification (clone-FISH). The optimized probe hybridization stringency resulted in good fluorescence signal compared to those of the negative controls. The standardized protocol developed using the clone-FISH reagents will be used in subsequent experiments to directly visualize P. obducens in situ from plant and soil samples. for use as a control and to allow for standardized fluorescence signal quantification (clone-FISH). The optimized probe hybridization stringency resulted in good fluorescence signal compared to those of the negative controls. The standardized protocol developed using the clone-FISH reagents will be used in subsequent experiments to directly visualize P. obducens in situ from plant and soil samples. Evaluation of Recombinase Polymerase Amplification (RPA) Isothermal Amplification Diagnostic Assay for Phytophthora ramorum J. BIENAPFL (1), L. Knight (1), Z. Abad (1) ramorum-specific) and JOE (internal control); all DNA was tested undiluted and diluted 1:10. For all samples, the tissue-DNA and ELISA-DNA tested negative for P. ramorum (FAM Cq = 0.00) at both dilutions. For the internal control, the tissue-DNA tested positive for amplifiable DNA with a mean Cq of 27.30, whereas the ELISA-DNA tested positive with a mean Cq of 26.41. For diluted DNA, the mean Cq for tissue-DNA was 20.45 and for ELISA-DNA was 20.79. The results suggest amplification of the internal control was comparable between DNA extracted from leaf tissue and DNA extracted from macerated leaf tissue in ELISA buffer. Utilizing mitochondrial loci to develop TaqMan and recombinase polymerase amplification assays for the genus Pythium T. MILES (1), A. Burkhardt (2), F. Martin (2) (1) California State University Monterey Bay, U.S.A.; (2) USDA-ARS, U.S.A. Mitochondrial marker systems have been utilized in other oomycete systems to develop novel molecular diagnostic tools in both laboratory and field settings. For Pythium a mitochondrial gene order difference was identified that allowed for genus specific amplification of more than 75 species across a wide range of clades within the genus. PCR results for this locus tested negative on Phytophthora and several Phytopythium spp. A TaqMan diagnostic assay was developed using a FAM labeled probe and validated on environmental samples from over 40 host plants (nursery and field samples). A sequencing protocol was also developed to confirm a positive, however, if multiple species are present amplicon cloning will be needed to get clean data. In addition to the TaqMan assay an assay using the isothermal technology known as recombinase polymerase amplification (RPA) was developed. Using the same purified and field samples the RPA technique was effective at detecting Pythium species in less than 20 minutes without a laborious DNA extraction. The use of this marker system will not only help confirm Pythium infection in plants, it may also be useful in environmental samples and it will complement the other marker system developed for Phytophthora. Sequencing of additional species is in progress to evaluate species specific detection/identification capabilities. Due to the amplicon size, this locus may also be useful in future metagenomic studies of Pythium diversity. Development of molecular diagnostic tools for the invasive oomycete pathogen Phytophthora tentaculata N. Luecke (1), S. Koenig (1), T. Developing an innovative molecular toolbox for identification of Phytophthora species with emphasis on species of regulatory concern Z. Abad (1), J. Bienapfl (1), L. Knight (1), K. Jennings (1), M. Galvez (1), L. Schena (2) (1) USDA APHIS PPQ S&T, U.S.A.; (2) Mediterranean University of Reggio Calabria, Italy Developing an innovative molecular toolbox for identification of Phytophthora species with emphasis on species of regulatory concern Z. Abad (1), J. Bienapfl (1), L. Knight (1), K. Jennings (1), M. Galvez (1), L. Schena (2) (1) USDA APHIS PPQ S&T, U.S.A.; (2) Mediterranean University of Reggio Calabria, Italy Developing an innovative molecular toolbox for identification of Phytophthora species with emphasis on species of regulatory concern Z. Abad (1), J. Bienapfl (1), L. Knight (1), K. Jennings (1), M. Galvez (1), L. Schena (2) (1) USDA APHIS PPQ S&T, U.S.A.; (2) Mediterranean University of Reggio Calabria, Italy The genus Phytophthora consists of 155 species, many of which cause significant economic impact to crops, ornamentals, and forest ecosystems worldwide. Many species are also exotic and of regulatory importance for the USA. Accurate species identification can be extremely challenging due to the need for extensive training and experience, as well as many misidentified DNA sequences deposited in public databases. In order to facilitate correct identification of Phytophthora species, we are pioneering work with international collaborators for the implementation of a “Molecular Toolbox” containing DNA sequences generated from the type specimens used for the original descriptions. Ten nuclear genes and five mitochondrial genes currently used for identification and phylogenic studies have been evaluated. Six nuclear genes, including the YPT1 gene that is amplified with new primers developed by Schena, and one mitochondrial gene have been selected for use in our “Molecular Toolbox” based on their utility in the robust identification and phylogenetic studies of Phytophthora species. However, many of the types lack sequences for these different genes (over 50%) and predominantly only have ITS sequences available. We will provide all files generated from the DNA sequencing, which will also demonstrate the high quality of data generated, to be used for identification purposes. Emphasis of this project is for species of high economic impact and regulatory concern for the USA. Early detection and quantification of Pseudoperonospora cubensis airborne sporangia using real-time PCR A. RAHMAN (1), L. Quesada-Ocampo (1) (1) NCSU U S A Early detection and quantification of Pseudoperonospora cubensis airborne sporangia using real-time PCR A. RAHMAN (1), L. Quesada-Ocampo (1) (1) NCSU, U.S.A. Pseudoperonospora cubensis, the causal agent of cucurbit downy mildew, recently reemerged as a major destructive pathogen of a broad range of crops belonging to the Cucurbitaceae. Airborne sporangia of P. cubensis are the most important source of inoculum to initiate disease as well as cause secondary plant infections. Furthermore, airborne sporangia of this obligate oomycete are known to be able to travel long distances and survive for prolonged periods of time to solar radiation. Therefore, early and rapid detection of the airborne sporangia could serve as a warning for potential disease outbreaks. By designing genome specific primers and using SYBR green-based qPCR method, a preliminary threshold detection level of target pathogen DNA was determined in extracted DNA from P. cubensis sporangia and infected plant tissue. While the method successfully detected up to 5.3 pg/ml of sporangia DNA, the minimum DNA concentration required from infected plant material for successful detection was found to be almost 10-fold higher (69 pg/ml). Using Next Generation Sequencing (NGS) and bioinformatics tools, several unique and conserved candidate genomic regions were also generated to serve as diagnostic genomic markers for P. cubensis. These candidate genomic regions were tested for their suitability as diagnostic markers for detecting and monitoring airborne P. cubensis sporangia inoculum level in the field using real-time qPCR following entrapment with spore traps. On-site detection of Pythium ultimum in potatoes using loop-mediated isothermal amplification (LAMP) J. WOODHALL (1), P. Wharton (2), S. Dangi (2), K. Perkins (3), J. Azcona (3) 1) University of Idaho, U.S.A.; (2) University of Idaho, U.S.A.; (3) Fera Science Ltd, United Kingdom Pythium ultimum is a soil-borne plant pathogen that causes damping off and root rot diseases in a wide range of crops. In potatoes, Pythium leak caused by P. ultimum has been a major storage disease problem in the Pacific Northwest potato production areas for many years, but in recent years reported losses have increased. Since similar symptoms can be caused by other pathogens, rapid and robust tests are required to detect the pathogen. Loop- mediated isothermal amplification (LAMP) has great potential for diagnostic use offering fast, specific and sensitive detection. In this study a new LAMP assay was designed for P. ultimum and evaluated in terms of specificity and sensitivity. Evaluation of Recombinase Polymerase Amplification (RPA) Isothermal Amplification Diagnostic Assay for Phytophthora ramorum J. BIENAPFL (1), L. Knight (1), Z. Abad (1) Miles (2) (1) CSU M t B U S A (2) C lif i St t U i it M t B U S A Development of molecular diagnostic tools for the invasive oomycete pathogen Phytophthora tentaculata N. Luecke (1), S. Koenig (1), T. Miles (2) (1) CSU Monterey Bay, U.S.A.; (2) California State University Monterey Bay, U.S.A. (1) CSU Monterey Bay, U.S.A.; (2) California State University Monterey Bay, U.S.A. Phytophthora basal rot (PBR) of plants is a disease recently observed in Central California Native plant nurseries and restoration locations. The primary causal agent of this disease is the oomycete pathogen Phytophthora tentaculata. Prior to 2012, P. tentaculata was among the ~30 Phytophthora species not known to be present in the US. Previous research had developed an isothermal diagnostic tool known as recombinase polymerase amplification (RPA), which had Phytophthora genus specific detection capability with results obtainable within as little as 15 minutes directly in the field without conventional DNA extraction. A P. tentaculata species-specific RPA assay was developed and specificity validated against pure DNA from 135 Phytophthora taxa. To test this technique for detection of the pathogen in PBR, 113 symptomatic samples were collected and evaluated with the new RPA assay as well as the previously validated TaqMan assay, Immunostrip and conventional culturing and baiting techniques. Results were similar across the various amplification platforms, with qPCR being the most sensitive in general. Finally, spatial models were created to test if elevation correlated with the presence of the pathogen. These results indicate that P. tentaculata is often present in low-lying areas when infected plant material is used for restoration. This information will assist in more efficient, rapid, specific pathogen detection for management decisions in a field. S4.115 A CANARY Assay for Phytophthora spp. A CANARY Assay for Phytophthora spp. Z. LIU (1), D. Kalkofen (1), A. Flannery (1), G. Wei (2), F. Nargi (3), Z. Liu (2), G. Abad (2), J. Bienapfl (2), M. Nakhla (2) (1) PathSensors, Inc., Baltimore, MD, U.S.A.; (2) USDA-APHIS-PPQ-S&T-Center for Plant Health Science and Technology (CPHST), U.S.A.; (3) Bioengineering Systems & Technologies, MIT Lincoln Laboratory (MIT-LL), U.S.A. Phytophthora is a genus of Oomycetes (water molds) that can cause serious plant diseases and considerable economic loss. A notable example is P. Early detection and quantification of Pseudoperonospora cubensis airborne sporangia using real-time PCR A. RAHMAN (1), L. Quesada-Ocampo (1) (1) NCSU U S A Choi (1) (1) N i l I i f H i l l d H b l S i S h K (2) N i l I i f H i l l d H b l S i S h Evaluation of Recombinase Polymerase Amplification (RPA) Isothermal Amplification Diagnostic Assay for Phytophthora ramorum J. BIENAPFL (1), L. Knight (1), Z. Abad (1) infestans, which was responsible for the potato late blight that caused the Great Irish Famine in the 1840s, and is still the most destructive pathogen of solanaceous crops. Another example is P. ramorum, the cause of Sudden Oak Death which has had devastating effects on forests in California and Oregon. Efficient management of Phytophthora diseases requires early detection and accurate identification of the pathogen. CANARY® (Cellular Analysis and Notification of Antigen Risks and Yields) is a fast, sensitive, and easy to use technology that has been successfully utilized for the detection of bacteria, viruses, and toxins. We are developing a CANARY® assay for Phytophthora spp. Using pure culture preparations, the assay was able to detect all species of Phytophthora tested, including P. cactorum, P. cambivora, P. capsici, P. cinnamomi, P. citrophthora, P. kernoviae, P. infestans, P. nicotianae, P. niederhauserii, P. ramorum, and P. sojae. Importantly, exclusivity studies showed that the assay did not cross-react with Pythium spp. Preliminary testing with infected plant material indicates that the assay takes less than 20 minutes to complete. A user-friendly software interface guides the user through all steps of the assay, providing a simple and rapid screening method for Phytophthora spp. Early detection and quantification of Pseudoperonospora cubensis airborne sporangia using real-time PCR A. RAHMAN (1), L. Quesada-Ocampo (1) (1) NCSU U S A The assay was highly specific when tested with DNA from 30 other isolates consisting of closely related Pythium species and other potato pathogens. The assay was also highly sensitive detecting as little as 17.5 fg of DNA in less than 30 minutes when used with a Genie® II for fluorescence measurement. A simple, rapid on site DNA extraction method for tubers was developed for use with the assay which resulted in a highly sensitive test capable of detecting the pathogen in range of both naturally infected asymptomatic and symptomatic tubers. In conclusion, LAMP assays can offer a rapid, sensitive, on-site test for the presence of P. ultimum in potato tubers. Sensitive detection and discrimination of HPWMoV, WSMV and TriMV using multiplex RT-PCR and High Resolution Melting: Part II A. LARREA-SARMIENTO (1), M. Arif (2), F. Ochoa-Corona (3), A. Olmedo-Velarde (4), J. Olson (3) (1) Universidad de las Américas, Quito, OK, Ecuador; (2) Kansas State University, Manhattan, KS, U.S.A.; (3) Oklahoma State University, Stillwater, OK, U.S.A.; (4) Universidad de las Fuerzas Armadas ESPE, Sangolqui, Ecuador High Plains wheat mosaic virus (HPWMoV), Wheat streak mosaic virus (WSMV) and Triticum mosaic virus (TriMV) cause disease in wheat crops in Central and Western USA, including Oklahoma and Kansas. Although these viruses are from different families, they are transmitted by Aceria tosichella, the wheat curl mite (Prostigmata: Eriophyidae). These viruses may infect simultaneously single plants. Melting curve analysis of RT-PCR products obtained by High Resolution Melting (HRM) enables to detect and to discriminate different virus species. The method is rapid, sensitive and allows discrimination among multiple samples. Specific primers for original single endpoint RT-PCR and reference positive controls from Agdia® were used as previously reported (Part I this research, 2013) to generate cDNA. uMELTSM analysis of expected PCR products was used to predict HRM scenarios. Special attention was given to PCR product size, GC percentage and melting temperature (Tm). Tm derived from dF/dT plots in vitro were similar to predictions by uMELTSM in silico. All primers sets showed a sensitivity down to 1 fg/uL from cDNA in single-endpoint RT-PCR reactions. HRM assays are sensitive and allows detection and discrimination of viruses causing diseases and significant economic losses in wheat and corn. Detection of Apple scar skin viroid in apple fruits by reverse transcription loop-mediated isothermal amplification (RT-LAMP) S. KWON (1), I. Cho (2), S. Choi (1), J. Yoon (1), G. Development and characterization of polyclonal and monoclonal antibodies to Rose rosette virus R. JORDAN (1), M. Guaragna (1), J. Hammond (1) 1) US National Arboretum, USDA-ARS, U.S.A. Garden roses, which form the cornerstone of the multi-billion dollar landscape industry, annually generate wholesale US domestic production valued at ~ $400 million. Over the past few decades Rose rosette disease, caused by Rose rosette virus (RRV; genus Emaravirus), has become a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early identification and eradication of the infected plants, thereby limiting its potential spread. Key to this effort is the development of efficient diagnostic tools to enable sensitive, rapid, user-friendly and accurate detection of the virus; currently limited to nucleic acid-based methods, such as PCR. With the aim of developing a serological diagnostic tool, rabbit polyclonal and several mouse monoclonal antibodies have been developed to the nucleocapsid protein (NP) of RRV. These antibodies were evaluated against NP peptides, cloned NP expressed in bacteria and plants, and RRV-infected plants. These results and those using the antibodies in various serological assay formats, including double- and triple-antibody sandwich ELISA and membrane-based assays, for lab and field detection of Rose rosette virus in infected sources, will be presented. Validation of RT-PCR High Resolution Melting as a detection method of Tombusvirus F. OCHOA-CORONA (1), Y. Carrillo Tarazona (1) (1) Oklahoma State University, U.S.A. Plant waterborne viruses, such as some in the genus Tombusvirus, spread as colloids in water and also by mechanical transmission. Broad detection and simultaneous species discrimination of viruses in this genus is needed for testing field samples. RT-PCR coupled to High Resolution Melting (HRM) has been used for different forensic applications and allows species differentiation as well as SNP detection. Tombusvirus. complete genomic sequences retrieved from the NCBI-Genbank were used to design primers for broad detection using this method. Ten different virus species, Carnation italian ringspot virus (CIRV), Cucumber necrosis virus (CNV), Cymbidium ringspot virus (CymRSV), Grapevine Algerian latent virus (GALV), Neckar river virus (NRV), Pelargonium leaf curl virus (PLCV), Pelargonium necrotic spot virus (PNSV), Petunia asteroid mosaic virus (PetAMV), Tomato bushy stunt virus (TBSV) and Pelargonium line pattern virus (PLPV) were sourced by the DSMZ (Germany) and AGDIA (USA) and used for nucleic acid extraction. All viruses tested positive to broad detection endpoint RT-PCR with primers designed to react when coupled to HRM. Melting curve predictions calculated by uMeltSM algorithms were compared with data obtained using three DNA dyes (SYBR® Green, SYTO®9 and LCGreen®). Detection of Apple scar skin viroid in apple fruits by reverse transcription loop-mediated isothermal amplification (RT-LAMP) S. KWON (1), I. Cho (2), S. Choi (1), J. Yoon (1), G. Choi (1) (1) National Institute of Horticultural and Herbal Science South Korea; (2) National Institute of Horticultural and Herbal Scicence Sout CLD is also caused by Citrus leprosis virus nuclear type (CiLV-N) a possible Dichorhavirus (Dichorhabdovirus) and Hibiscus green spot virus 2 (HGSV 2) a Higrevirus S4.116 infecting citrus trees in Hawaii. The presence of CiLV-C in the western plains of Colombia was reported in 2005 using reverse transcription polymerase chain reaction (RT-PCR). A second report of CiLV-C2 sequenced from symptomatic citrus samples using next generation sequencing was made in 2013. These two reports had raised questions about distribution of CiLV-C and CiLV-C2 in Colombia and whether both viruses coinfect citrus groves. This study analyzed genomic sequences of CiLV-C and CiLV-C2 seeking target sequences for detection and discrimination using reverse transcription multiplex PCR. The developed assay is fast, sensitive and specific for detection and discrimination of these two species of Cilevirus infecting citrus. Only CiLV-C2 and no coinfections were found in samples collected in the citrus growing region of Meta and Casanare, Colombia. infecting citrus trees in Hawaii. The presence of CiLV-C in the western plains of Colombia was reported in 2005 using reverse transcription polymerase chain reaction (RT-PCR). A second report of CiLV-C2 sequenced from symptomatic citrus samples using next generation sequencing was made in 2013. These two reports had raised questions about distribution of CiLV-C and CiLV-C2 in Colombia and whether both viruses coinfect citrus groves. This study analyzed genomic sequences of CiLV-C and CiLV-C2 seeking target sequences for detection and discrimination using reverse transcription multiplex PCR. The developed assay is fast, sensitive and specific for detection and discrimination of these two species of Cilevirus infecting citrus. Only CiLV-C2 and no coinfections were found in samples collected in the citrus growing region of Meta and Casanare, Colombia. Development of conventional and Real-time RT-PCR assays for Strawberry necrotic shock virus T. THEKKE VEETIL (1), T. Ho (2), V. Whitaker (3), I. Tzanetakis (2) (1) University of Arkansas, U.S.A.; (2) University of Arkansas, U.S.A.; (3) University of Florida, U.S.A. Strawberry necrotic shock virus (SNSV) is one of the several viruses reported in strawberry and Rubus as a component of viral complexes that cause plant decline. Highly sensitive conventional and real-time RT-PCR detection assays were developed; able to detect all sequenced isolates of the virus. Primers and probes were designed from conserved regions of the virus coat protein gene and reactions were optimized to ensure both specificity and sensitivity. Detection of Apple scar skin viroid in apple fruits by reverse transcription loop-mediated isothermal amplification (RT-LAMP) S. KWON (1), I. Cho (2), S. Choi (1), J. Yoon (1), G. Choi (1) (1) National Institute of Horticultural and Herbal Science South Korea; (2) National Institute of Horticultural and Herbal Scicence Sout ection of Apple scar skin viroid in apple fruits by reverse transcription loop-mediated isothermal amplification (RT-LA WON (1), I. Cho (2), S. Choi (1), J. Yoon (1), G. Choi (1) Detection of Apple scar skin viroid in apple fruits by reverse transcription loop-mediated isothermal amplification (RT-LAMP) S. KWON (1), I. Cho (2), S. Choi (1), J. Yoon (1), G. Choi (1) (1) National Institute of Horticultural and Herbal Science South Korea; (2) National Institute of Horticultural and Herbal Scicence South Korea , ( ), ( ), ( ) Horticultural and Herbal Science, South Korea; (2) National Institute of Horticultural and Herbal Scicence, South Korea ( ) ( ) ( ) ( ) ( ) (1) National Institute of Horticultural and Herbal Science, South Korea; (2) National Institute of Horticultural and Herbal Sc Apple scar skin viroid (ASSVd) is one of the most important pathogen causing scar skin, dappling or cracking on the surface of apple fruits. A reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for detection of ASSVd. A set of two primers were designed inner primers (FIP and BIP) and outer primers (F3 and B3) from sequences of the Korean strain of ASSVd. The LAMP reaction was optimized using Bst DNA polymerase for 30 min at 65°C. The RT-LAMP products were evaluated as ladder-like bands by electrophoresis and visualized in the reaction tube by staining with SYBR Green I dye. RT-LAMP was more sensitive than that of conventional RT-PCR for the detection of ASSVd. The RT-LAMP assay developed in this study is a simple, rapid and sensitive and can be applied as a practical molecular diagnostic tool. Three genomic regions of Cilevirus allows detection and discrimination of two related species infecting citrus W. TURIZO (1), O. Oliveros (1), F. Ochoa-Corona (2) Three genomic regions of Cilevirus allows detection and discrimination of two related species infecting citrus W. TURIZO (1), O. Oliveros (1), F. Ochoa-Corona (2) (1) Universidad Nacional de Colombia, Colombia; (2) Oklahoma State University, U.S.A. Citrus leprosis disease (CLD) is caused by diverse etiological agents such as Citrus leprosis virus C (CiLV-C) and a newly proposed Citrus leprosis virus cytoplasmic type 2 (CiLV-C2), both in the genus Cilevirus and transmitted by Tenuipalpidae (false spider mites) Brevipalpus spp. Detection of Apple scar skin viroid in apple fruits by reverse transcription loop-mediated isothermal amplification (RT-LAMP) S. KWON (1), I. Cho (2), S. Choi (1), J. Yoon (1), G. Choi (1) (1) National Institute of Horticultural and Herbal Science South Korea; (2) National Institute of Horticultural and Herbal Scicence Sout Ochoa-Corona (3), G. Knox (1) (1) North Florida Research and Education Center, University of Florida, Quincy, FL, U.S.A.; (2) Department of Biological Science, Florida State University, Tallahassee, FL, U.S.A.; (3) Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, U.S.A. (1) North Florida Research and Education Center, University of Florida, Quincy, FL, U.S.A.; (2) Department of Biological Science, Florida State University, Tallahassee, FL, U.S.A.; (3) Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK, U.S.A. Rose rosette disease, caused by Rose rosette virus (genus Emaravirus) is a major threat to the rose industry in the U.S. The only strategy currently available for disease management is early identification and eradication of the infected plants, thereby limiting its potential spread. Current RT-PCR based diagnostic methods for RRV are time consuming and is inconsistent in detecting the virus even from symptomatic plants. Hence a novel probe- based isothermal reverse transcription-recombinase polymerase amplification (RT-exoRPA) assay, based on the nucleocapsid gene of the RRV, has been developed. The assay is highly specific as tested against other common rose infecting viruses (both inclusive and exclusive). Dilution assays using the in vitro transcript showed that the assay is highly sensitive, with a detection limit of 1 fg. Moreover a rapid technique for the extraction of viral RNA (< 5 min) has been standardized from RRV infected tissue sources, using a single buffer which facilitates virus adsorption, followed by denaturation to release the RNA. RT-exoRPA analysis of the infected plants indicated that the virus could be detected from leaves, stems, petals, primary roots and secondary roots. The entire process including the extraction can be completed in less than 15 min, with less sophisticated equipments. The developed assay is currently evaluated for efficiency in large scale field testing for rapid detection of RRV in commercial nurseries and landscape. Development and characterization of polyclonal and monoclonal antibodies to Rose rosette virus R. JORDAN (1), M. Guaragna (1), J. Hammond (1) (1) US National Arboretum, USDA-ARS, U.S.A. Detection of Apple scar skin viroid in apple fruits by reverse transcription loop-mediated isothermal amplification (RT-LAMP) S. KWON (1), I. Cho (2), S. Choi (1), J. Yoon (1), G. Choi (1) (1) National Institute of Horticultural and Herbal Science South Korea; (2) National Institute of Horticultural and Herbal Scicence Sout Comparison between the two assays revealed an at-least 100-fold higher sensitivity for the latter. Both tests were successfully validated using samples collected from several areas in the United States as well as a collection of isolates from around the world. These assays could be integrated into virus screening programs in certification, nursery or commercial field settings in both strawberry and Rubus. Rose rosette virus detection using loop-mediated isothermal amplification (LAMP) A. SALAZAR AGUIRRE (1), S. Molina Cárdenas (1), F. Ochoa-Corona (2), J. Olson (2) (1) Universidad de las Fuerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A. Rose rosette virus (RRV) is an Emaravirus transmitted by Phyllocoptes fructiphilus strongly associated with Rose rosette disease (RRD). Visual diagnosis maybe confusing and specific, sensitive, easy to use with visual detection methods are required. LAMP is an isothermal amplification that combines these criteria. Alignment of 20 RRV P4 (RNA 4) accessions allowed primer design seeking broad detection of reported isolates. Optimal amplification was obtained with Bst 2.0 WarmStart® DNA polymerase (0.32 u), outer primers F3 and B3 (0.2 µM), internal primers FIP and BIP (0.8 µM) and MgSO4 (4 mM) at 66.5°C for 1 hour. Bovine serum albumin (BSA) (4 mg/mL) and polyvinylpyrrolidone (PVP) (1%) were added as isothermal amplification enhancers. The detection limit was 1 pg/µL with plasmid carrying the targeted sequence. Products were visualized by electrophoresis. The visual detection limit of plasmid in colorimetric reactions using Hydroxynaphthol blue (HNB) (120 µM) without BSA and PVP was 0.01 ng/µL. No cross-reactions with cDNA from ten frequently rose co-infecting or related viruses (HPV, MSV, INSV, TSWV, GRSV, ApMV, ArMV, PNRSV, TRSV and TMV) was detected. Healthy tissue and non-template controls were included in all reactions. RRV-LAMP tested successfully with 25 tissue samples of symptomatic RRD roses from Oklahoma. The method has potential applications in biosecurity, microbial forensics and nursery virus-free monitoring of germplasm. nt of a field-based detection technique for Rose rosette virus using isothermal reverse transcription-recombinase polym Development of a field-based detection technique for Rose rosette virus using isothermal reverse transcription-recombinase polymerase amplification Development of a field-based detection technique for Rose rosette virus using isothermal reverse transcription-recomb amplification Development of a field-based detection technique for Rose rosette virus using isothermal reverse transcription-recombinase polymerase amplification p B. BABU (1), B. Washburn (2), S. Miller (2), F. Ochoa-Corona (3), G. Knox (1) B. BABU (1), B. Washburn (2), S. Miller (2), F. Multiplex RT-PCR detection of eight viruses infecting cucurbits and discrimination using High Resolution Melting analysis L. PEÑA ZUNIGA (1), F. Ochoa-Corona (1), A. Ali (2) (1) Oklahoma State University, U.S.A.; (2) University of Tulsa, U.S.A. Cucurbits are one of the largest horticultural commodities in United States and Oklahoma largely contributes with watermelon production. However, the cucurbit cultivated acreage of Oklahoma had decreased, in part, due to viral infections. This research targets eight disease-causing viruses: Cucumber mosaic virus (CMVI and CMVII), Cucurbit yellow stunting disorder virus (CYSDV), Melon necrotic spot virus (MNSV), Papaya ringspot virus (PRSV), Squash mosaic virus (SqMV), Watermelon mosaic virus (WMV), and Zucchini yellow mosaic virus (ZYMV). A rapid detection and discrimination method is needed for biosecurity and breeding for resistance against these viruses. Sequences from the polymerase protein gene (RNA) from the six genera were retrieved from NCBI and aligned using MEGA 6. Primer sets were designed from conserved regions using the web application PrimerQuest® and the thermodynamic features of each primer were tested using Oligonalyzer 3.1. A Multiplex RT-PCR was developed to be coupled to High Resolution Melting (HRM) analysis for rapid detection of the targeted viruses. The obtained melting profiles allowed a clear discrimination among these viruses. This Multiplex RT-PCR High Resolution Melting method has potential to be applied in epidemiological studies of viral diseases in cucurbit crops, microbial forensics and biosecurity. Toward broad detection of emaraviruses: Endpoint RT-PCR A. OLMEDO-VELARDE (1), F. Ochoa-Corona (2), T. Elbeaino (3) (1) Universidad de las Fuerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A ( ) ( ) ( ) Universidad de las Fuerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A.; (3) Istituto Agronomico Medite uerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A.; (3) Istituto Agronomico Mediterraneo di Bari, Italy Emaravirus has six confirmed species and two unclassified species. Rapid detection and an effective screening of existing and new emaraviruses is needed for detection, identification, plant virus discovery and biosecurity applications. Available sequences of RNA dependent RNA polymerase (RNA 1) for European mountain ash ringspot-associated virus (EMARaV), Fig mosaic virus (FMV), Pigeonpea sterility mosaic virus (PPSMV), Pigeonpea sterility mosaic virus 2 (PPSMV2), Rose rosette virus (RRV), High Plains wheat mosaic virus (HPWMoV) formerly High plains virus (HPV), Raspberry leaf blotch virus (RLBV) and Redbud yellow ringspot-associated virus (RYRSaV), were aligned using Mega 6. A primer set (EMARA F&R7/8) was designed from conserved domains after strand sense normalization. The sensitivity of EMARA F&R7/8 was found to be 100 fg/reaction assessed using a reference positive control of HPWMoV (Agdia). Virus detection of Tobamovirus with wide spectrum degenerate oligonucleotides by TD RT-PCR High Resolution Melting J. GARCIA SUAREZ (1), S. Dobhal (1), F. Ochoa-Corona (1) (1) Oklahoma State University, U.S.A. Tobamoviruses cause notable plant diseases worldwide in Solanaceae, Cucurbitaceae, orchids and some are reported to be waterborne. Molecular tools exist for detection of this genus, however, a detection method combining genus detection and species discrimination is needed. Eleven reference positive controls from the DMSZ repository (Germany) and AGDIA (USA), were studied. Complete Tobamovirus genomes were aligned and a single antisense primer was designed to target the RNA dependent RNA polymerase (RdRp). This antisense primer co-reacted with five sense primers for selective subgroup detection by RT-PCR and HRM discrimination of eleven species within five subgroups. A protocol for detection and discrimination based on Touchdown Reverse transcription-Polymerase Chain Reaction (TD RT-PCR) coupled to High Resolution Melting (HRM) was developed. Species specific dF/dT Tm curves ranged 75-85°C and were generated for each primer groups as follow: I (BpEMV, PMMoV, TMV, ToMV), II (PaMMV, TMGMV), III (SFBV, RMV), IV (ORSV, CGMMV) and V (SHMV). The method allows the incorporation of new groups of tobamoviruses to the assay by designing additional group specific sense primers. The resulting 11 diagnostic sequences showed 98-100% identity regarding the targeted viruses. HRM facilitates a reliable discrimination of species specific profiles for each virus species. The method is amenable to microbial forensics diagnosis and biosecurity applications. Discriminating Potexvirus species by RT-PCR coupled to High Resolution Melting A. OLMEDO-VELARDE (1), F. Ochoa-Corona (2) (1) Universidad de las Fuerzas Armadas ESPE, Ecuador; (2) Oklahoma State University, U.S.A. Potexvirus is a cosmopolitan plant virus genus and most of its thirty-seven confirmed species are mechanically transmitted, causing mosaic and ringspot symptoms on herbaceous hosts. High Resolution Melting analysis (HRM) allows the discrimination at species and strain levels and has application in diagnostics. Complete genome sequences for Alternanthera mosaic virus (AltMV), Clover yellow mosaic virus (ClYMV), Lettuce virus X (LeVX), Opuntia virus X (OpVX), Papaya mosaic virus (PapMV), Pepino mosaic virus (PepMV), Potato aucuba mosaic virus (PAMV), Potato virus X (PVX), Schlumbergera virus X (SchVX), Strawberry mild yellow edge virus (SMYEV) and Hosta virus X (HVX), were retrieved from NCBI Genebank and aligned using Mega 6 and screened for conserved domains. Primer set Potex F3/R3 was selected to discriminate among eleven potexviruses by a melting temperature prediction using uMeltSM, a melting curve prediction software program. Reference positive controls for these eleven viruses were obtained from DMSZ (Germany) and Agdia, Inc. (USA) and tested using RT-PCR coupled to HRM with Potex F3/R3. Development and characterization of polyclonal and monoclonal antibodies to Rose rosette virus R. JORDAN (1), M. Guaragna (1), J. Hammond (1) 1) US National Arboretum, USDA-ARS, U.S.A. Results with LCGreen® are closer to the predicted data calculated by uMeltSM when the amplicon size is between 50-200bp. S4.117 Virus detection of Tobamovirus with wide spectrum degenerate oligonucleotides by TD RT-PCR High Resolution Melting J. GARCIA SUAREZ (1), S. Dobhal (1), F. Ochoa-Corona (1) (1) Oklahoma State University, U.S.A. Virus detection of Tobamovirus with wide spectrum degenerate oligonucleotides by TD RT-PCR High Resolution Melting J. GARCIA SUAREZ (1), S. Dobhal (1), F. Ochoa-Corona (1) (1) Oklahoma State University, U.S.A. All viruses tested positive and showed different melting temperatures ranging from 77.9°C to 82.4°C allowing for their discrimination through HRM analysis. The combination of RT-PCR- HRM can be used in diagnostic laboratories to discriminate eleven Potexviruses with high confidence. A preemptive detection system to screen water for the presence of plant viruses J. Daniel (1), B. Gallucci Mazziero (1), B. Dunn (1), F. Ochoa-Corona (1) (1) Oklahoma State University, U.S.A. Growers are expanding cultivation into non-favorable cropping areas pumping water from nearby lakes, rivers, wells, aquifers, and runoff, increasing the need for waterborne plant pathogen monitoring. Particulate matter dilution factors due to high water volumes, make detection and identification of potential pathogens a challenge because of the extremely low virion numbers. Current plant pathogen detection methods are passive, and sampling and testing occurs after disease symptoms appear. In the event of highly virulent pathogen incursions, detection may be too late to limit losses. This project seeks to develop a preemptive detection system that will easily sample large volumes of water to screen for the presence of plant pathogens. Three waterborne plant viruses: Tomato bushy stunt virus, Pepino mosaic virus, and Pepper mild mottle virus were used as models for this purpose. Previously developed multiplex detection protocols, were modified to detect these three waterborne viruses. Recovery of viruses using polyethylene glycol from 250 ml, 500 ml, and 1 L of water was demonstrated. Adaptation of an inexpensive, durable, and field-ready sampling device is presented. The testing of waterborne virus movement in two different microcosms is described. Monitoring of microbial loads in agricultural irrigation systems, and other water sources, is essential for effective surveillance and disease prevention for plant health. Multiplex RT-PCR detection of eight viruses infecting cucurbits and discrimination using High Resolution Melting analysis L. PEÑA ZUNIGA (1), F. Ochoa-Corona (1), A. Ali (2) (1) Oklahoma State University, U.S.A.; (2) University of Tulsa, U.S.A. Multiplex RT-PCR detection of eight viruses infecting cucurbits and discrimination using High Resolution Melting analysis L. PEÑA ZUNIGA (1), F. Ochoa-Corona (1), A. Ali (2) (1) Oklahoma State University, U.S.A.; (2) University of Tulsa, U.S.A. The multiple Emaravirus detection was confirmed with three rose samples showing symptoms caused by RRV from three locations in Stillwater, OK, a HPWMoV reference positive control, and cDNA of EMARaV and FMV. All samples were screened by endpoint RT-PCR and tested positive for HPWMoV, RRV, EMARaV and FMV. Other emaraviruses (RLBV, RYRSaV, PPSMV and PPSMV2) were detected in-silico using Primer-Blast. This method addresses the need for sensitive molecular detection methods and discovery of novel emaraviruses. Waterborne plant virus sampling and detection in aqueous environment Waterborne plant virus sampling and detection in aqueous environment B. GALLUCCI MAZZIERO (1) (1) Oklahoma State University, U.S.A. S4.118 S4.118 The aim of this research studying water sampling as a critical tool for detection of unwanted plant pathogenic virus that can seriously damage agriculture and environment. Water sources, such as reservoirs, lakes, farm ponds, tanks, and irrigation systems are vulnerable to inadvertent (most frequent) and intentional (rare) contamination by microbial pathogens. Pathogen detection in large bodies of water is complex because volume, dilution and water dynamics. This project assesses the development of a method for water sampling and detection of waterborne plant viruses of concern for water-security. Rapid detection and monitoring of high consequence plant pathogens in water systems, where may be present at extremely low titers present a significant challenge. The project seeks statistically validation of a water sampling model that uses quantitative-PCR as the method to detected Potexvirus, a genera of plant virus reported to have water borne members. In order to determine the minimum amount detectable in water, sensitivity tests will be conducted by ELISA and qPCR. Different pH will be tested during a process of virus filtration using a filtration system based on glass-wool. The capture is based on the isoelectric point affinity of the virus capsid protein with glass wool at an acid-neutral pH. This research is needed to support decision-making and will be useful for survey analysis and prevention, detection, and mitigation of unwanted waterborne plant viruses. Occurrence of endornaviruses in non-cultivated plant species Occurrence of endornaviruses in non-cultivated plant species R. HERSCHLAG (1), E. Rodrigues de Souto (2), R. Valverde (1) (1) Louisiana State University Agricultural Center, U.S.A.; (2) Universida Occurrence of endornaviruses in non-cultivated plant species R. HERSCHLAG (1), E. Rodrigues de Souto (2), R. Valverde (1) (1) Louisiana State University Agricultural Center U S A ; (2) Uni p p R. HERSCHLAG (1), E. Rodrigues de Souto (2), R. Valverde (1) (1) L i i St t U i it A i lt l C t U S A (2) U Large dsRNAs have been extracted from plants infected with acute and persistent viruses and from fungi infected with mycoviruses. These extracted dsRNAs have been used for viral disease diagnosis and virus identification. We used a modified version of the non-phenol batch method, in combination with reverse-transcription PCR (RT-PCR), to test symptomless, non-cultivated, plant species for endornaviruses. Foliar tissues were collected from different plant species from various locations within the city of Baton Rouge, Louisiana. S4.118 Tissues were desiccated with silica gel and used for dsRNA extractions. After testing 132 plant species, belonging to 60 families, six yielded dsRNAs similar in size (14-15 kb) to reported endornaviruses. Putative endornaviruses were detected in Alternanthera philoxeroides (alligator weed), Geranium carolinianum (wild geranium), Sonchus asper (spiny sow thistle), Sorghum halepense (Johnson grass), and Hydrocotyle umbellata (dollar weed). The same endornaviruses were also detected in at least 10 individual plants of the same species. RT-PCR tests using endornavirus-degenerate primers were conducted with the dsRNAs of the six putative endornaviruses. Results of this investigation suggest that endornaviruses occur at a rate of approximately 5% in non-cultivated plant species and that like endornaviruses of cultivated plants, their vertically transmission rate is high. Development and validation of a quadruplex real-time RT-PCR assay for simultaneous detection of three Citrus leprosis viruses in plants G. WEI (1), A. Roy (2), W. Schneider (3), R. Brlansky (2), M. Nakhla (1) (1) USDA-APHIS-PPQ-S&T-CPHST, U.S.A.; (2) University of Florida, IFAS, Plant Pathology Department, Citrus Research and Education Center, U.S.A.; (3) USDA-ARS-FDWSRU, U.S.A. Citrus leprosis virus (CiLV) is the causal agent of Citrus leprosis - one of the most destructive diseases of citrus. CiLV is an RNA virus transmitted by Brevipalpus mite species. There are two major types of CiLV being described – one is the cytoplasmic type residing in the cytoplasm of infected cells and the other is the nuclear type primarily residing in the nucleus of infected cells. In the last decade CiLV spread swiftly from South America to Central America and recently has been detected in Mexico. The proximity and the potential threat to the multi-billion dollar US citrus industry have caused concerns in the United States where the disease has not been found since the 1960s. Molecular identification and early detection of each the cytoplasmic and nuclear types are essential to prevent CiLV from introduction and establishment in the US. In order to reduce testing time, cost and labor, we are developing and validating a quadruplex real-time RT-PCR assay for simultaneous detection of cytoplasmic CiLV-C, CiLV-C2, and nuclear CiLV-N with a Nad5 plant RNA internal control. From our repeated experiments, the results showed that sensitivity and specificity for the quadruplex RT-PCR assay were comparable to the level of monoplex RT-PCR detecting CiLV-C, CiLV-C2, and CiLV-N respectively. Further validation for the quadruplex RT- PCR assay including tests with additional field samples is in progress. S4.118 al assessment of Potato virus Y in western Washington: Barriers and bridges to adopting new management practices GER (1), C. Benedict (2), J. Goldberger (3), D. Inglis (1) A sociological assessment of Potato virus Y in western Washington: Barriers and bridges to adopting new management practices A. BEISSINGER (1), C. Benedict (2), J. Goldberger (3), D. Inglis (1) (1) Washington State University Northwestern Washington Research and Extension Center, Mount Vernon, WA, U.S.A.; (2) Washington State University Whatcom County Extension, Bellingham, WA, U.S.A.; (3) Washington State University, Pullman, WA, U.S.A. ( ) ( ) g ( ) g ( ) (1) Washington State University Northwestern Washington Research and Extension Center, Mount Vernon, WA, U.S.A.; (2) Washington State University Whatcom County Extension, Bellingham, WA, U.S.A.; (3) Washington State University, Pullman, WA, U.S.A. Seed and commercial potatoes are grown in separate geographic areas of western Washington (WWA). In 2012, recombinant strains of Potato virus Y (PVY), PVYNTN and PVYN-Wi, were reported in WWA potato fields. The strains are largely asymptomatic on potato foliage and difficult to detect by visual field inspections and winter greenhouse grow-out tests. The problem led to tuber yield and quality losses of ~40% for commercial producers who purchased inaccurately certified seed. In 2015-16, most (N=6) of the seed potato growers in WA and approximately one-third (N=6) of the large-scale commercial potato growers in WWA were interviewed about the barriers and bridges to adopting updated PVY detection approaches such as ELISA and PCR-based laboratory tests, tropical winter field grow-outs, and PVY recognition trainings. The major barriers to adoption were logistical learning curves (67%), uncertainty that updated techniques would be advantageous (67%), and distrust of laboratory results (50%). The major bridges included premiums paid by customers for reliable PVY-free seed (75%), customer demand for more sensitive PVY testing methods (67%), and faster grow-out results (33%). The study results can guide researchers and extension educators who make PVY management recommendations that must be relevant to the needs and perceptions of growers. Further, they suggest that sociological studies of plant pathogens can provide a valuable tool for assisting in plant disease management. stripe disease pathogen Stenotrophomonas maltophilia strain Sia5 in rice fields of Punjab under the climate change Objectives include determination of the susceptibility of peach/almond rootstock to PBTS isolates and the influence of root and shoot infections on symptom development and host colonization. Either roots or shoots of clonal ‘Hansen 536’ rootstock were dipped in a bacterial suspension containing both PBTS isolates. Uninoculated control plants were dipped in buffer solution. Plant growth metrics and shoot and root biomass were determined 8 weeks after inoculation, and leaves were sampled for endo- and epiphytic populations of Rf and Rc. Root-inoculated plants were stunted and exhibited increased node density in comparison to control plants. Foliar-inoculated plants remained asymptomatic, but foliar inoculation resulted in endophytic colonization of leaves. Root-inoculations resulted in aboveground plant colonization. The results demonstrate that ‘Hanson 536’ rootstock is susceptible to the PBTS isolates. The results indicate a need for field evaluation for disease in PBTS-affected orchards replanted to almond. Xylella fastidiosa isolates differ in the ability to undergo genetic recombination P. KANDEL (1), R. Almeida (2), L. De La Fuente (1) (1) Department of Entomology and Plant Pathology, Auburn University, U.S.A.; (2) Department of Environmental Science, Policy and Management, UC Berkeley, U.S.A. Xylella fastidiosa isolates differ in the ability to undergo genetic recombination P. KANDEL (1), R. Almeida (2), L. De La Fuente (1) (1) Department of Entomology and Plant Pathology, Auburn University, U.S.A.; (2) Department of Environmental Science, Policy and Management, UC Berkeley, U.S.A. (1) Department of Entomology and Plant Pathology, Auburn University, U.S.A.; (2) Department of Environmental Science, Policy and Management, UC Berkeley, U.S.A. Xylella fastidiosa (Xf) is a bacterium causing incurable diseases in economically important crops such as grapevines, citrus, and olives. Historically assumed to be restricted to the Americas, Xf diseases have been reported in both new locations and new crops in recent years. Inter-subspecific recombination (ISR) has been proposed to contribute to host shifts based on genetic diversity studies. Natural competence, as a mode of recombination, has been shown to occur in vitro in systems mimicking natural habitats. However, little is known about the recombination potential of Xf isolates, which are believed to be somewhat host specific. Therefore, in this study we compared the recombination frequencies of twelve Xf isolates belonging to two subspecies (fastidiosa and multiplex), using plasmids containing antibiotic markers flanked on either side by Xf homologous regions. The recombination frequency varied greatly (3.14 × 10–2 – 2.3 × 10–8) among isolates. Generally, subsp. tive detection of tomato chlorotic dwarf viroid in crude plant extracts using isothermal RT-recombinase polymerase Rapid and sensitive detection of tomato chlorotic dwarf viroid in crude plant extracts using isothermal RT-recombinase polymerase amplification R. HAMMOND (1), S. Zhang (2) (1) USDA ARS, U.S.A.; (2) Agdia, Inc., U.S.A. Tomato chlorotic dwarf viroid (TCDVd) is a serious threat to tomato production worldwide. Infection leads to reduced plant vigor, small and deformed fruit, and yield loss. TCDVd is easily transmitted mechanically from plant to plant once introduced and through seed; seed treatments do not control transmission of disease. The most effective control is the use of certified viroid-free planting materials. We developed a molecular diagnostic assay utilizing reverse transcription-recombinase polymerase amplification (RT-RPA) at an isothermal constant temperature of 39°C and target-specific primers and probe for the rapid, sensitive, and specific detection of TCDVd in crude extracts of infected leaf and seed tissues. The performance of the AmplifyRP® Acceler8™ RT-RPA diagnostic assay, utilizing a lateral flow strip contained within an amplicon detection chamber, was evaluated and the results were compared with a standard RT-PCR assay. The AmplifyRP® Acceler8™ assay was specific for TCDVd and its sensitivity was comparable to conventional RT-PCR. The assay does not require extensive sample purification, specialized equipment, or technical expertise. It is easy to perform and can be used under both laboratory and field conditions. Distribution and vertical transmission of Southern tomato virus in tomato S. COSKAN (1), R. Alcalá-Briseño (1), J. Polston (1) (1) University of Florida, U.S.A. Southern tomato virus (STV) is a recently identified virus that infects tomato cultivars worldwide. STV has is a monopartite virus with a double-stranded RNA genome (3.5 kb) containing two overlapping open reading frames (ORFs) and is the type species of the genus Amalgavirus (family Southern tomato virus (STV) is a recently identified virus that infects tomato cultivars worldwide. STV has is a monopartite RNA genome (3.5 kb) containing two overlapping open reading frames (ORFs) and is the type species of the genus Amalgav S4.119 Amalgaviridae). A reverse-transcription polymerase chain reaction (RT-PCR) using virus-specific primers was used to detect STV in 21 tomato cultivars. STV was detected in plants produced by a number of seed companies which indicates that the virus is widely distributed in the vegetable seed industry. STV was detected for the first time in four cultivars: ‘Agriset 761’, ‘Mexico Midget’, ‘Roma’ and ‘Sweet Hearts’. ‘Sweet Hearts’ tomato plants were evaluated for the presence of STV using RT-PCR at different developmental stages of tomato plants. stripe disease pathogen Stenotrophomonas maltophilia strain Sia5 in rice fields of Punjab under the climate change Moilno-Lova (5) (1) American University of Beirut, Beirut, Lebanon, (2) FAFS, AUB, (3) LARI, (4) UNIMI, Milan, Italy, (4) IDISAFA, Univ. Di Torino, Torino, Italy, (5) NNIMI, Milan, Italy Introduced into North Lebanon in the early 1990s, almond witches’ broom (AlmWB) disease spread rapidly in Lebanon killing about 200,000 almond, peach and nectarine trees. The disease is associated with ‘Candidatus Phytoplasma phoenicium’ that belongs to 16S rRNA subgroup IX-B. The major symptoms are proliferation, small chlorotic leaves, development of witches’ broom followed by dieback and death of almond trees. So far, AlmWB has been reported in Lebanon and Iran. In Lebanon, AlmWB was considered of quarantine importance and official containment measures were initiated in major stone fruit production areas. Specific, sensitive and reliable PCR and real-time PCR detection methods were developed. Preliminary data revealed that a leafhopper and planthoppers are vectors of the disease and two weed species are suspected to act as alternative hosts. An integrated disease management appraoch is considered for highly infested regions. Grafting experiements are in progress and preliminary results look encouraging, a recovery phenomemon was observed. Finally, regional and international cooperation are highly recommended to continue studies on the epidemiology of this invasive disease and to adopt phytosanitary control measures and strategies in order to contain the further spread of the disease. Root and shoot susceptibility of peach/almond rootstock to pistachio bushy top syndrome isolates of Rhodococcus spp E. Fichner (1), S. Dhaouadi (2) Root and shoot susceptibility of peach/almond rootstock to pistachio bushy top syndrome isolates of Rhodococcus spp. E. Fichner (1), S. Dhaouadi (2) (1) University of California Division of Agriculture and Natural Resources, U.S.A.; (2) University of California Division of Agriculture and Natural Resources, U.S.A. E. Fichner (1), S. Dhaouadi (2) (1) University of California Division of Agriculture and Natural Resources, U.S.A.; (2) University of California Division of Agriculture and Natural Resources, U.S.A. ( ), ( ) (1) University of California Division of Agriculture and Natural Resources, U.S.A.; (2) University of California Division of Agriculture and Natural Resources, U.S.A. Approximately 30,000 acres of UCB-1 clonal pistachio rootstock planted in California from 2011- 2014 were affected with pistachio bushy top syndrome (PBTS) caused by two phytopathogenic bacteria highly related to Rhodococcus fascians (Rf) and Rhodococcus corynebacteriodes (Rc). Orchards with high disease incidence were removed and replanted to pistachio or almond. The susceptibility of almond rootstock to PBTS isolates is unknown. Emergence of a novel white stripe disease pathogen Stenotrophomonas maltophilia strain Sia5 in rice fields of Punjab under the climate change i Emergence of a novel white stripe disease pathogen Stenotrophomonas maltophilia strain Sia5 in rice fields of Pun i tive detection of tomato chlorotic dwarf viroid in crude plant extracts using isothermal RT-recombinase polymerase STV could be detected in the 1 to 2 week-old germinated seedlings at a frequency similar to that of older plants from the same seed lot. The presence or absence of STV was consistent across all four developmental stages. Seed transmission studies of progeny produced from crosses of different combinations of parents that were STV-positive or negative, revealed that STV could be transmitted to the next generation by either ovule or pollen. Emergence of a novel white stripe disease pathogen Stenotrophomonas maltophilia strain Sia5 in rice fields of Punjab under the climate change scenario stripe disease pathogen Stenotrophomonas maltophilia strain Sia5 in rice fields of Punjab under the climate change K. RIAZ (1), K. Riaz (2), A. Dilferoze (3), M. Alam (3), A. Riaz (4), S. Jameel (5), S. Khan (6), A. Hannan (7) (1) Department of Plant Pathology, University of Agriculture Faisalabad, Pakistan; (2) University of Agriculture Faisalabad, Pakistan; (3) University of Agriculture Faisalabad, Pakistan; (4) Plant Protection Division NIAB, Faisalabad, Pakistan; (5) Department of Plant Pathology, University of Agriculture, Faisalabad-38040, Pakistan; (6) Centre of Agricultural Biochemistry and Biotechnology, University of Agriculture, Faisalabad-38040, Pakistan; (7) Department of Botany, Ghazi University, Dera Ghazi Khan, Dera Ghazi Khan, Pakistan Under severe fluctuations in temperature and rainfall patterns several previously unimportant pathogens are gaining importance especially in chief cash crops in many parts of the world. Recent surveys over the last three years in the rice Kallar belt districts of Punjab (Kala Shah Kaku, Hafizabad, Sheikhupura, Narowal, Shakargarh, and Sialkot) depicted the presence of a novel disease and its associated pathogen which is responsible for the appearance of white stripe symptoms on leaves that start from the leaf tip and extend along the midrib through the leaf lamina towards the base of the leaf sheath. Under severe infection leaf size got reduced, whole leaves got dried and panicles matured very late. Diseases incidence ranged from 25-30% inflicting 20-25% yield losses. Pathogen was isolated on nutrient agar that formed dark to pale yellow raised colonies with fluffy appearance and was reconfirmed through pathogenicity testing on susceptible rice variety. The most aggressive and most virulent isolate belonged to Sialkot region and was characterized as Stenotrophomonas maltophilia strain Sia5 through the BLAST searches and phylogenetic analysis of its PCR amplified 16srDNA sequence (GB: KT924441.1) and other biochemical characteristics. This is the first report on the occurrence of white stripe disease in rice from Pakistan. An invasive lethal phytoplasma disease threatening stone fruit production: Detection, epidemiology and management Y. A. JAWDAH (1), M Jawhari (2), P. Tawidian (2), E. Choueiri (3), F. Quaglino (4), P. Bianco (4), A. Alma (4), M. Moilno-Lova (5) (1) American University of Beirut, Beirut, Lebanon, (2) FAFS, AUB, (3) LARI, (4) UNIMI, Milan, Italy, (4) IDISAFA, Univ. Di Torino, Torino, Italy, (5) NNIMI, Milan, Italy An invasive lethal phytoplasma disease threatening stone fruit production: Detection, epidemiology and management Y. A. JAWDAH (1), M Jawhari (2), P. Tawidian (2), E. Choueiri (3), F. Quaglino (4), P. Bianco (4), A. Alma (4), M. Soil survival potential of pistachio bushy top syndrome isolates of Rhodococccus spp. E. FICHTNER (1), S. Dhaouadi (1), E. Molina (2), R. Stamler (2), J. Randall (2) (1) University of California Division of Agriculture and Natural Resources, U.S.A.; (2) New Mexico State University, U.S.A. Soil survival potential of pistachio bushy top syndrome isolates of Rhodococccus spp. stripe disease pathogen Stenotrophomonas maltophilia strain Sia5 in rice fields of Punjab under the climate change fastidiosa isolates had higher recombination frequencies than multiplex isolates, and within a subspecies, the most virulent isolates had higher frequencies. When combinations of marker-tagged, heat-killed donor and live recipient cells from two subspecies were mixed, ISR occurred with a genomic region of ~10kb transferred from the donor to the recipient cell. This study demonstrates different recombination abilities, and therefore suggests different evolutionary potentials among Xf isolates. Bacterial black spot of mango, Mangifera indica, caused by Xanthomonas citri pv mangiferaeindicae, is confirmed in the Western Hemisphere G. SANAHUJA (1), R. Ploetz (1), P. Lopez (1), J. Konkol (1), A. Palamateer (1) (1) University of Florida U S A Thus, the most sustainable way to reduce yield losses is using resistant cultivars. Preliminary studies showed that different isolates did not share the same lettuce host range. Therefore, different X. hortorum pathotypes should be considered in a breeding program for resistance. The objective was to determine which pathotypes are present in Canada by sampling diseased lettuce plants in commercial production. In 2014 and 2015, 376 lettuce samples were collected in the main lettuce production area. Xanthomonas isolates were recovered from leaf lesions and characterized based on DNA sequences of four housekeeping genes (dnaK, fyuA, gyrB and rpoD). Concatenated DNA sequences of these genes for the isolates and reference strains were analyzed with Neighbor-Joining method using MEGA v6.04 to generate a phylogenetic tree. The 363 isolates formed two major clusters. Cluster I includes 17 strains representing 358 isolates and grouped with the Xhv reference strain BS3046. This Cluster can be separated into two sub-groups and preliminary evidence shows that they shared different lettuce host range. The Cluster II, a distinct group of X. hortorum, contains eight new strains, but the pathotype has to be determined. Further investigation is needed on the epidemiology of this unknown pathotype. Wheat blast is caused by multiple Pyricularia species, including Pyricularia graminis-tritici sp. nov. V. CASTROAGUDIN (1), S. Moreira (2), D. Pereira (3), S. Moreira (4), P. Brunner (3), J. Maciel (5), P. Crous (6), B. McDonald (3), E. Alves (7), P. Ceresini (4) ( ) (1) University of Sao Paulo State, Ilha Solteira, Brazil; (2) Federal University of Lavras, Lavras, Brazil; (3) Institute of Integrative Biology, ETH Zurich, Zurich, Switzerland; (4) University of Sao Paulo State, Ilha Solteira, Brazil; (5) Brazilian Agriculture Research Corporation-Wheat (EMBRAPA-Trigo), Passo Fundo, Brazil; (6) CBS-KNAW Fungal Biodiversity Centre, Utrech, Netherlands; (7) Federal University of Lavras, Lavas, Brazil Presently, Pyricularia oryzae pathotype Oryza (Po-O) is considered the widely distributed rice blast pathogen, whereas P. oryzae pathotype Triticum (Po-T) is the South American wheat blast pathogen. In this study, we investigated whether Po-O and Po-T were distinct at species level. We also described a new Pyricularia species causing blast on several Poaceae, including wheat, from Brazil. Bayesian phylogenetic analyses were conducted on 10 genes from an extensive sampling (N = 128) of populations of Pyricularia adapted to rice, wheat and other Poaceae. Isolates were grouped into two major clusters (Clusters I and II, P=0.99). Bacterial black spot of mango, Mangifera indica, caused by Xanthomonas citri pv mangiferaeindicae, is confirmed in the Western Hemisphere G. SANAHUJA (1), R. Ploetz (1), P. Lopez (1), J. Konkol (1), A. Palamateer (1) (1) University of Florida U S A During the summer of 2015, symptoms of what appeared to be bacterial black spot (BBS) were observed on mature fruits of mango, Mangifera indica, i the vicinity of Boynton Beach, FL. Star-shaped lesions, up to 2 cm in diameter, erupted from the surface of ‘Keitt’ and other cultivars, and oozed a sticky, clear exudate. From lesions, a cream-colored bacterium was recovered on YPGA and characterized with housekeeping genes used by Bui Thi Ngoc et al. (2010) (efp and dnaK). Amplicons from a representative isolate were sequenced (KU746336 and KU746337) and were 100% homologous with strains of Xanthomonas citri pv mangiferaeindicae (Xcm) in GenBank. ML analysis (Tamura-Nei model) with MEGA 6.06 placed the isolate among other strains of Xcm. In incubator studies (30°C day, 26°C night, 12h light), the mesophyll of leaves on potted ‘Keitt’ and ‘Haden’ plants were injected with a hypodermic needle and 1 × 105 CFU mL–1 of the isolate. After 7 days, black lesions developed on inoculated leaves, but not on leaves injected with sterile deionized H2O. Diagnostic amplicons for Xcm were generated for isolates recovered from the lesions, and the experiment was conducted twice. BBS is widespread in the Eastern Hemisphere, but this is the first report of the disease in the Western Hemisphere. Environmental conditions in southern Florida appear to be ideal for an increased importance of BBS in the region. Survey on pathogenic Xanthomonas recovered from different lettuce cultivars in Canada V. TOUSSAINT (1), D. Xu (1), P. Hébert (1), M. Cadieux (1), M. Ciotola (1), H. Van der Heyden (2), D. Rekika (3), S. Jenni (1) (1) Agriculture and Agri-Food Canada, St-Jean-sur-Richelieu, QC, Canada; (2) Compagnie de recherche Phytodata, Sherrington, QC, Canada; (3) Fondation pour l’amélioration génétique de la laitue et des légumes feuilles, Napierville, QC, Canada Survey on pathogenic Xanthomonas recovered from different lettuce cultivars in Canada V. TOUSSAINT (1), D. Xu (1), P. Hébert (1), M. Cadieux (1), M. Ciotola (1), H. Van der Heyden (2), D. Rekika (3), S. Jenni (1) (1) Agriculture and Agri-Food Canada, St-Jean-sur-Richelieu, QC, Canada; (2) Compagnie de recherche Phytodata, Sherrington, QC, Canada; (3) Fondation pour l’amélioration génétique de la laitue et des légumes feuilles, Napierville, QC, Canada Bacterial leaf spot, caused by X. hortorum pv. vitians (Xhv), is an important lettuce disease. In Canada, the main difficulty for disease control is the absence of registered chemicals. Soil survival potential of pistachio bushy top syndrome isolates of Rhodococccus spp. Bacterial black spot of mango, Mangifera indica, caused by Xanthomonas citri pv mangiferaeindicae, is confirmed in the Western Hemisphere G. SANAHUJA (1), R. Ploetz (1), P. Lopez (1), J. Konkol (1), A. Palamateer (1) (1) University of Florida, U.S.A. Bacterial black spot of mango, Mangifera indica, caused by Xanthomonas citri pv mangiferaeindicae, is confirmed in the Western Hemisphere G. SANAHUJA (1), R. Ploetz (1), P. Lopez (1), J. Konkol (1), A. Palamateer (1) (1) University of Florida, U.S.A. Bacterial black spot of mango, Mangifera indica, caused by Xanthomonas citri pv mangiferaeindicae, is confirmed in the Western Hemisphere G. SANAHUJA (1), R. Ploetz (1), P. Lopez (1), J. Konkol (1), A. Palamateer (1) (1) University of Florida U S A Cluster I contained isolates from wheat and another Poaceae (P=0.98). Cluster II was sub-divided in three clades (P>0.75). Clades 1 and 2 grouped wheat-derived isolates, and Clade 3 contained rice-derived isolates. The Clusters I and II corresponded to two distinct species: Pyricularia graminis-tritici sp. nov. (Pgt), newly described here, and Pyricularia oryzae (Po). The clades of Po concurred with Po-T and Po-O, and were not distinct at species level. Among species, morphological differences were not recorded; however, a distinctive pathogenicity spectrum was observed. Pgt and Po-T were highly virulent on wheat, barley, signal grass, and oats. Po-O was highly virulent on rice, wheat, barley, and oats, but not on signal grass. We demonstrated that blast disease on wheat and other Poaceae from Brazil is caused by multiple Pyricularia species. Soil survival potential of pistachio bushy top syndrome isolates of Rhodococccus spp. Over 35,000 acres of pistachio planted from 2011-2014 in California were affected by pistachio bushy top syndrome (PBTS), a disease caused by bacteria related to Rhodococcus corynebacteriodes (Rc) and Rhodococcus fascians (Rf). Because the survival potential of PBTS isolates in soil is unknown, growers are reluctant to replant pistachios in affected orchards. The goal of this work was to assess the longevity of Rc and Rf in field soils S4.120 exposed to environmental conditions and in potting medium under greenhouse conditions. An in situ field study was established in a PBTS orchard with each isolate embedded on wooden sticks and buried at various depths. Another study utilized excavated field soils incubated outdoors to investigate the influence of soil type and moisture on survival. In a greenhouse study, potting medium was infested with Rf in the presence and absence of dried pistachio plant material. After three months, populations of Rf in potting medium remained similar to initial populations, regardless of incorporation of plant tissue. After one month, Rf populations increased in moist, non-saline field soil, but Rc was almost eliminated. Both isolates were inhibited by salinity and drying, but depth did not influence survival. The results suggest saprophytic competence of Rf, and a consequent concern for persistence of Rf in affected orchards and nurseries and illustrate the need for vigilance in sanitation from the nursery to the field. exposed to environmental conditions and in potting medium under greenhouse conditions. An in situ field study was established in a PBTS orchard with each isolate embedded on wooden sticks and buried at various depths. Another study utilized excavated field soils incubated outdoors to investigate the influence of soil type and moisture on survival. In a greenhouse study, potting medium was infested with Rf in the presence and absence of dried pistachio plant material. After three months, populations of Rf in potting medium remained similar to initial populations, regardless of incorporation of plant tissue. After one month, Rf populations increased in moist, non-saline field soil, but Rc was almost eliminated. Both isolates were inhibited by salinity and drying, but depth did not influence survival. The results suggest saprophytic competence of Rf, and a consequent concern for persistence of Rf in affected orchards and nurseries and illustrate the need for vigilance in sanitation from the nursery to the field. Unraveling Silverleaf disease: Reversion of foliar symptoms and plants recovery D. GRINBERGS (1), A. France (1), J. Chilian (1) (1) Instituto de Investigaciones Agropecuarias, Chile Unraveling Silverleaf disease: Reversion of foliar symptoms and plants recovery D. GRINBERGS (1), A. France (1), J. Chilian (1) (1) Instituto de Investigaciones Agropecuarias, Chile Silverleaf, caused by the Basidiomycete Chondrostereum purpureum, is an important disease in fruit trees which produce wood necrosis and leaves silvering. In apple has been increasing due to changes in management, reducing orchards viability. Plants deteriorate their physiological condition, being water uptake and chlorophyll content the most affected parameters. The latter alters photosynthesis, reducing fruit yield and quality. Field studies have shown the spontaneous reversion of foliar symptoms, thus, the objective was to compare the physiological and sanitary condition of Brookfield plants before and after reversion. The sanitary condition was determined assessing symptoms severity, isolating the fungus on PDA, amplificating the large intergenic spacer (IGS) rDNA región and measuring foliar endopoligalacturonase concentration using DAS-ELISA. Physiological condition was measured through xylem water potential, chlorophyll fluorescence, a, b and a + b chlorophyll content and stomatal conductance. Besides, yield and fruit quality parameters such as color and starch were assessed. Isolation, PCR and ELISA indicated that the fungus was present in symptomatic plants and after reversion. Symptomatic plants presented alterations in all physiological parameters (P<0,05%), reduction of yield (38%), maduration disorders and lack of color. However, after reversion of silvering, physiological condition, yield and fruit quality were restored. Fungi in the wood: The fungi associated with the ambrosia beetle, Xylosandrus germanus and its galleries in Malus domestica S. VILLANI (1), K. Ayer (2), D. Breth (3), K. Cox (2) (1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell Cooperative Extension, U.S.A. The Botrytis cinerea species complex comprises two cryptic species, originally referred as Group I and Group II based on Bc-hch gene RFLP haplotyping. Group I was described as a new cryptic species B. pseudocinerea. During a survey of Botrytis spp. causing gray mold in blueberries and table grapes in the Central Valley of California, six isolates, three from blueberries and three from table grapes, were placed in Group I but showed a distinct morphological character with conidiophores significantly longer than those of B. cinerea and B. pseudocinerea. We compared these isolates with B. cinerea and B. pseudocinerea by examining morphological and physiological characters, sensitivity to fenhexamid, and phylogenetic analysis inferred from sequences of three nuclear genes. Phylogenetic analysis using the three partial gene sequences encoding glyceraldehyde-3-phosate dehydrogenase (G3PDH), heat-shock protein 60 (HSP60), and DNA-dependent RNA polymerase subunit II (RPB2), supported the proposal of a new Botrytis species, B. californica, which is genetically closely related to B. cinerea, B. pseudocinerea, and B. sinoviticola, all known as causal agents of gray mold of grapes. B. californica caused decay on blueberry and table grape fruit inoculated with the fungus. This study suggests that B. californica is a cryptic species sympatric with B. cinerea on blueberries and table grapes in California. Multiple Pestalotiopsis and Neopestalotiopsis species cause flower bight of macadamia in Australia O. AKINSANMI (1), S. Nisa (1), O. Jeff-Ego (1), A. Drenth (2) (1) University of Queensland, Brisbane, Australia; (2) University of Queensland, Australia Incidence of ‘dry flower’ the disease, has been increasing and now occurs in different production regions in Queensland and New South Wales, Australia. Although yield losses range between 10% and 30%, total crop failures have occurred in certain cultivars. Therefore, this study examined the etiology of the disease in Australia. Results from a survey of over 240 macadamia racemes in different orchards in Australia revealed that all the four developmental stages of macadamia racemes including the florets and the rachis were affected. Several fungi genera including Alternaria, Epicoccum, Fusarium, Botrytis, Cladosporium, Penicillium, Aspergillus and Diaporthe were obtained from tissue samples, however, their occurrence was irregular between samples and significantly (P <0.05) less than Pestalotiopsis -like isolates, which constituted 41% of the total isolates. Fungi in the wood: The fungi associated with the ambrosia beetle, Xylosandrus germanus and its galleries in Malus domestica S. VILLANI (1), K. Ayer (2), D. Breth (3), K. Cox (2) (1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell Cooperative Extension, U.S.A. We collected decayed mandarin fruit from three citrus packinghouses in the Central Valley of California in 2015 and identified this disease as Mucor rot caused by Mucor spp. Mucor rot occurred in 11 of the 15 grower lots sampled, and the incidence of the disease in the total decayed fruit varied among affected grower lots, ranging from 3.3 to 93.1% with an average of 49.2%. In total, 197 isolates of Mucor spp. were obtained from decayed mandarin fruit and identified based on ITS sequence and morphological characteristics. Of the 197 isolates, 92.4%, 3.6%, 2.0%, 1.5%, and 0.5% were identified as M. piriformis, M. circinelloides, M. racemosus, M. hiemalis, and M. mucedo, respectively. Pathogenicity tests on mandarin fruit showed that M. piriformis was the most virulent among all tested species. Our results indicated that Mucor rot of mandarins in California is caused by Mucor species consisting of M. piriformis, M. circinelloides, M. racemosus, M. hiemalis, and M. mucedo, with M. piriformis being the most prevalent species. Previously, M. racemosus was reported on citrus. This is the first report of Mucor rot on citrus caused by M. piriformis, M. circinelloides, M. hiemalis, and M. mucedo. Botrytis californica, a new Botrytis species causing gray mold in blueberries and table grapes in California S. SAITO (1), D. Margosan (2), T. Michailides (3), C. Xiao (1) (1) USDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (2) USDA-Agricultural Re Agricultural Sciences Center, Parlier, CA, U.S.A.; (3) Kearney Agricultural Research & Extension Center, Parlier, Botrytis californica, a new Botrytis species causing gray mold in blueberries and table grapes in California S. SAITO (1), D. Margosan (2), T. Michailides (3), C. Xiao (1) (1) USDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (2) USDA-Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (3) Kearney Agricultural Research & Extension Center, Parlier, CA, U.S.A. S. SAITO (1), D. Margosan (2), T. Michailides (3), C. Xiao (1) (1) USDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (2) USDA-Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (3) Kearney Agricultural Research & Extension Center, Parlier, CA, U.S.A. ( ), g ( ), ( ), ( ) (1) USDA-ARS, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (2) USDA-Agricultural Research Service, San Joaquin Valley Agricultural Sciences Center, Parlier, CA, U.S.A.; (3) Kearney Agricultural Research & Extension Center, Parlier, CA, U.S.A. Fungi in the wood: The fungi associated with the ambrosia beetle, Xylosandrus germanus and its galleries in Malus domestica S. VILLANI (1), K. Ayer (2), D. Breth (3), K. Cox (2) (1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell Cooperative Extension, U.S.A. germanus, while Ambrosiella xylebori, another common food source of ambrosia beetles, was only isolated from the interior and exterior of one beetle. Interestingly, however, A. xylebori was predominantly recovered from infested wood tissue. Our results suggest that X. germanus can establish fungal gardens of N. haematococca and A. xylebori within vascular tissue of apple trees, however it is unclear if tree decline is likely due to direct tunneling or colonization of A. xylebori. Additional investigations are ongoing to understand the implications of A. xylebori colonization of apple. High diversity in the population of an emerging blackberry pathogen, Fusarium oxysporum A. PASTRANA (1), S. Kirkpatrick (1), T. Gordon (1) (1) University of California, Davis, U.S.A. A wilt disease of blackberry, caused by the soilborne fungus Fusarium oxysporum, has become a more widespread problem in California over the past two years. This emerging pathogen is severely reducing fruit production in affected fields. To improve our understanding of the structure of pathogen population, isolates collected from symptomatic blackberry plants in California and Mexico were evaluated for morphology, pathogenicity and vegetative compatibility. The translation elongation factor 1 alpha (TEF1-α), the intergenic spacer (IGS) and the β-tubulin regions were also sequenced. The combination of TEF1-α, β-tubulin and IGS sequences identified clusters of isolates that corresponded to vegetative compatibility groups (VCGs). A high number of distinct populations were founded both in California and Mexico. However, one VCG included isolates from both countries. No relation was found between VCG and cultivar or geographic origin. Diversity in the pathogen population suggests that the capacity to cause disease on blackberry is not a recent development. Emerging Postharvest Disease of Mandarin Fruit Caused by Mucor piriformis and other Mucor spp. in California Michailides (2), C. Xiao (1) Mucor Rot – An Emerging Postharvest Disease of Mandarin Fruit Caused by Mucor piriformis and other Mucor spp. in California S. SAITO (1), T. Michailides (2), C. Xiao (1) (1) USDA-ARS, San Joaquin Valley Agricultural Sciences Center, U.S.A.; (2) Kearney Agricultural Research & Extension Center, UC-Davis, Division of Agriculture and Natural Resources, U.S.A. S. SAITO (1), T. Michailides (2), C. Xiao (1) (1) USDA-ARS, San Joaquin Valley Agricultural Sciences Center, U.S.A.; (2) Kearney Agricultural Research & Extension Center, UC-Davis, Division of Agriculture and Natural Resources, U.S.A. In recent years, an emerging, undescribed postharvest disease was observed on mandarin fruit after extended storage in California. Fungi in the wood: The fungi associated with the ambrosia beetle, Xylosandrus germanus and its galleries in Malus domestica S. VILLANI (1), K. Ayer (2), D. Breth (3), K. Cox (2) (1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell Cooperative Extension, U.S.A. the wood: The fungi associated with the ambrosia beetle, Xylosandrus germanus and its galleries in Malus domestica ANI (1), K. Ayer (2), D. Breth (3), K. Cox (2) h C li St t U i it U S A (2) C ll U i it U S A (3) C ll C ti E t i U S A Fungi in the wood: The fungi associated with the ambrosia beetle, Xylosandrus germanus and its galleries in Malus domestica S. VILLANI (1), K. Ayer (2), D. Breth (3), K. Cox (2) (1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell Cooperative Extension, U.S.A. The ambrosia beetle, Xylosandrus germanus, is considered an emerging threat to Western New York apple orchards. There are concerns that fungal farms established by X. germanus within vascular tissue play a role in the collapse of young apple trees. To gain further insight into the identity and relationship between fungi associated with X. germanus and decline of Malus x domestica, fungal isolations were performed from X. germanus wood S4.121 galleries and adjacent vascular tissue, and beetles recovered from galleries. Fungal identity was confirmed by sequencing the 2 internal transcribed spacer regions and the 5.8S gene of ribosomal DNA. Five distinct fungal species were recovered from the exterior of 36 beetles, while only 2 fungal species were recovered from the beetles’ interior. Nectria haematococca (an. Fusarium solani) was most frequently isolated from the exterior and interior of X. germanus, while Ambrosiella xylebori, another common food source of ambrosia beetles, was only isolated from the interior and exterior of one beetle. Interestingly, however, A. xylebori was predominantly recovered from infested wood tissue. Our results suggest that X. germanus can establish fungal gardens of N. haematococca and A. xylebori within vascular tissue of apple trees, however it is unclear if tree decline is likely due to direct tunneling or colonization of A. xylebori. Additional investigations are ongoing to understand the implications of A. xylebori colonization of apple. galleries and adjacent vascular tissue, and beetles recovered from galleries. Fungal identity was confirmed by sequencing the 2 internal transcribed spacer regions and the 5.8S gene of ribosomal DNA. Five distinct fungal species were recovered from the exterior of 36 beetles, while only 2 fungal species were recovered from the beetles’ interior. Nectria haematococca (an. Fusarium solani) was most frequently isolated from the exterior and interior of X. Cytospora canker on peaches (Prunus persica (L.) Batsch) is known to be caused by three species, Cytospora cinctum, C. leucostoma, and C. paraleucostoma within growing areas of the United States. In Colorado, recent surveys show that over 90% of the trees on the Western Slope are infected with Cytospora canker, causing significant economic impacts to peach growers. Understanding how these species differ phenotypically and in disease cycle and progression is important for disease management. The objectives of this study were to identify Cytospora isolates collected from high and low elevation sites, examine population structure and recombination, and assess each species/population for phenotypic characters such as virulence, temperature growth and culture morphology. The ITS region was sequenced to identify 106 isolates. Both C. leucostoma and C. paraleucostoma were recovered, although Cytospora leucostoma was the most prevalent. Additional sequencing of 4 genetic loci (ca. 1950 bps), suggested that C. leucostoma and C. paraleucostoma recombine and that well-supported clades do not exist for 3 of the 5 loci. Phenotypic characterization indicates overlapping Fungi in the wood: The fungi associated with the ambrosia beetle, Xylosandrus germanus and its galleries in Malus domestica S. VILLANI (1), K. Ayer (2), D. Breth (3), K. Cox (2) (1) North Carolina State University, U.S.A.; (2) Cornell University, U.S.A.; (3) Cornell Cooperative Extension, U.S.A. Based on the concatenated partial sequence of three gene loci (Internal transcribed spacer, β-tubulin and translation elongation factor 1-alpha) the phylogenectic analysis of 32 Pestalotiopsis -like isolates showed they formed two novel taxa in the Neopestalotiopsis and Pestalotiopsis genera. The grouping of the isolates was regardless of location or plant part. Koch’s postulates were confirmed with the different Neopestalotiopsis and Pestalotiopsis species and this is the first report of these two genera as causal agents of raceme blight of macadamia. Cytospora leucostoma is the most virulent and prevalent causal agent of Cytospora Canker on peaches on the Western Slope of Colorado J. Stewart (1), K. Otto (1), I. Minas (2), S. Miller (1) Powdery mildews on native plants in Utah E. LAURITZEN (1) (1) Utah State University, U.S.A. Many people want to grow more native plants in their yards. However, there is little information on plant diseases of native plants of the western United States. Powdery mildew is very common in Utah on many cultivated plants. The purpose of this project was to determine the presence of powdery mildew on native plants and identify the powdery mildew species using morphological and molecular techniques. Plants from the families Asteraceae, Geraniaceae and Polemoniaceae with powdery mildew were collected from a high elevation mountain valley (2,750 meters) in Northern Utah in fall 2015. Chasmothecia and conidia were collected from the plant tissue for morphological identification using a compound microscope. Identification to genuse and/or species was confirmed using polymerase chain reaction (PCR) amplifying the internal transcribed spacer (ITS) region and DNA sequencing. The resulting sequences were compared in a BLAST search to powdery mildew sequences in the NCBI GenBank database. Several species of powdery mildew including Podosphaera sp. on sticky purple geranium (Geranium viscosissimum) which to our knowledge is the first report of powdery mildew on this species in the United States and Golovinomyces magnicellulatus on Polemonium sp., the first report of this powdery mildew in Utah. Diaporthe and spruce decline: Incidence, pathogenicity, and population genetics C. MCTAVISH (1), D. Fulbright (2), A. Jarosz (1) (1) Michigan State University, U.S.A.; (2) Michigan State University, U.S.A.; (3) Michigan State University, U.S.A. Diaporthe and spruce decline: Incidence, pathogenicity, and population genetics C. MCTAVISH (1), D. Fulbright (2), A. Jarosz (1) (1) Michigan State University, U.S.A.; (2) Michigan State University, U.S.A.; (3) Michigan State University, U.S.A. Spruce decline, characterized by needle loss and branch dieback, is found throughout Michigan. A survey of fungi from cankers in the Lower Peninsula of Michigan found Diaporthe (formerly Phomopsis) to be the most common, disease-causing fungus. Sequencing Diaporthe isolates using ITS1 and ITS4 established 5 different groups. Pathogenicity studies were performed on these groups by inserting mycelial agar plugs containing a representative isolate of each group into a small wound, sealing the wound with Parafilm, and measuring the canker area 9 weeks post-inoculation. Groups 4 and 5 were the most virulent, while group 3 was the least virulent group. Groups 1 and 2 were intermediate on the virulence spectrum. Colorado blue spruce (Picea pungens) was the most susceptible species, and 9 different seed sources of P. pungens did not differ in susceptibility. Cytospora leucostoma is the most virulent and prevalent causal agent of Cytospora Canker on peaches on the Western Slope of Colorado J. Stewart (1), K. Otto (1), I. Minas (2), S. Miller (1) Further sequencing of RPB2, Hsp60, NEP1, and NEP2 genes for three representative isolates revealed that these isolates were identical to isolates of Botrytis fragariae, a species recently discovered infecting strawberry in Germany, but never before observed in the United States. In order to determine the distribution and prevalence of this species in the eastern United States, primers were developed that distinguish Botrytis cinerea and Botrytis fragariae. Preliminary data suggest that Botrytis fragariae isolates are more likely to have reduced sensitivity to the new fungicide polyoxin D. The discovery of a new species of Botrytis causing gray mold of strawberry may require adjustments in gray mold management strategies. Laurel wilt, caused by Raffaelea lauricola, is detected for the first time outside the southeastern USA R. PLOETZ (1), Y. Thant (2), M. Hughes (3), T. Dreaden (4), J. Konkol (1), A. Kyaw (2), J. Smith (3), C. Harmon (3) (1) University of Florida, Homestead, FL, U.S.A.; (2) Winrock International, Farmer-to-Farmer Program, Yangon, Myanmar, Yangon, Burma; (3) University of Florida, Gainesville, FL, U.S.A.; (4) U.S. Forest Service, Lexington, KY, U.S.A. Agriculture is an important industry in Myanmar (aka Burma), accounting for 60% of the GDP and employing 65% of the work force. In October 2014, a survey for diseases and pests of an emerging fruit crop in the country, avocado (Persea americana), was conducted in Southern Shan State. Around Tuanggyi, monocultures of up to 20 hectares have been established, whereas avocado is an over-story tree where coffee is grown in the Ywangan area. Symptoms of laurel wilt, including sapwood discoloration, leaf necrosis, defoliation, and tree mortality were observed during the survey in both areas. In Myanmar, isolates that resembled the laurel wilt causal agent, Raffaelea lauricola, were recovered from symptomatic sapwood on ½ strength potato dextrose agar + streptomycin sulfate. In the USA, R. lauricola-specific amplicons were generated for isolates from eight different trees with PCR primers for two taxon-specific microsatellite loci. In a quarantine greenhouse in Gainesville, FL, three of the isolates caused laurel wilt in each of two tests on avocado and swamp bay (P. palustris), with disease severities similar those caused by an isolate of R. lauricola from the USA. Although R. lauricola was probably introduced from Asia to the southeastern USA with its ambrosia beetle symbiont, Xyleborus glabratus, it had previously been reported only in the southeastern USA. Cytospora leucostoma is the most virulent and prevalent causal agent of Cytospora Canker on peaches on the Western Slope of Colorado J. Stewart (1), K. Otto (1), I. Minas (2), S. Miller (1) ( ) ( ) ( ) ( ) (1) Bioagricultural Sciences and Pest Management, Colorado State University, Fort Collins, CO, U.S.A.; (2) Department of H Architecture; Western Colorado Research Center Colorado State University, Grand Junction, CO, U.S.A. Cytospora canker on peaches (Prunus persica (L.) Batsch) is known to be caused by three species, Cytospora cinctum, C. leucostoma, and C. paraleucostoma within growing areas of the United States. In Colorado, recent surveys show that over 90% of the trees on the Western Slope are infected with Cytospora canker, causing significant economic impacts to peach growers. Understanding how these species differ phenotypically and in disease cycle and progression is important for disease management. The objectives of this study were to identify Cytospora isolates collected from high and low elevation sites, examine population structure and recombination, and assess each species/population for phenotypic characters such as virulence, temperature growth and culture morphology. The ITS region was sequenced to identify 106 isolates. Both C. leucostoma and C. paraleucostoma were recovered, although Cytospora leucostoma was the most prevalent. Additional sequencing of 4 genetic loci (ca. 1950 bps), suggested that C. leucostoma and C. paraleucostoma recombine and that well-supported clades do not exist for 3 of the 5 loci. Phenotypic characterization indicates overlapping S4.122 variation in temperature, growth, culture morphology, and virulence. These data suggest that isolates are genetically distinct populations of C. leucostoma rather than cryptic species. variation in temperature, growth, culture morphology, and virulence. These data suggest that isolates are genetically distinct populations of C. leucostoma rather than cryptic species. New Botrytis species affecting strawberry on the east coast G. SCHNABEL (1), M. Dowling (1), M. Hu (1) (1) Clemson University, U.S.A. We detected a new species of Botrytis infecting strawberries in the United States. While monitoring fungicide resistance of 459 Botrytis isolates collected from South Carolina (211), Maryland (123), North Carolina (101), Virginia (17), and Ohio (7), we observed isolates with phenotypes different from typical Botrytis cinerea. These isolates produced fluffy, white mycelium and sporulated poorly on potato dextrose agar, minimal medium, strawberries, apples, and tomatoes. These isolates were found in 11 different locations across South Carolina (20), and North Carolina (4). The G3PDH gene was sequenced for these isolates and nucleotide BLAST search revealed that they formed a cluster separate from Botrytis species previously described. Pathogenicity tests confirm Macrophomina phaseolina as an aggressive fungal pathogen of flowering dogwood (Cornus florida L.) L. MACKASMIEL (1) (1) Tennessee State University, Nashville, TN, U.S.A. (1) Tennessee State University, Nashville, TN, U.S.A. Pathogenicity tests where seedlings of flowering dogwood (C. florida L.) were challenged with extracted spores of Macrophomina phaseolina from 10 day-old cultures, while control were mocked inoculated with plain malt extract agar in sterile deionized water, demonstrated the capability of the pathogen to cause severe root rot in C. florida. Roots of juvenile seedlings vernalized in sterilized vermiculite; then grown on large water agar petri plates overlaid with sterile, moisten-filter paper, after being inoculated with the pathogen, showed high percentage of necrotic tissue in the first 10 mm of root tips. Concurrently, juvenile seedlings grown in large containers after similar treatment exhibited presence of fungal structures in roots from challenged plants that were not observed in the control. On 35 day-young dogwood seedlings grown in sterile soil under greenhouse conditions and inoculated on wounded stems, fungus-treated plants produced decay and large canker-like lesions that were not on the control. The extent of fungal growth in millimeter (mm) as the index to evaluate the host’s response(s) showed marked difference between treatments and control. The level of aggressiveness of the pathogen was determined by measuring the duration and number of dead tissues or plants per given time, and cell death confirmed the amount of infection inside the roots. Powdery mildews on native plants in Utah E. LAURITZEN (1) (1) Utah State University, U.S.A. Cytospora leucostoma is the most virulent and prevalent causal agent of Cytospora Canker on peaches on the Western Slope of Colorado J. Stewart (1), K. Otto (1), I. Minas (2), S. Miller (1) This is the first report of laurel wilt in the Asian homeland of R. lauricola and X. glabratus. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. Pondberry is a rhizomatous, clonally reproducing, woody shrub species that is federally endangered. Like other members of the Lauraceae that occur in the United States (US), pondberry is highly susceptible to laurel wilt, a disease caused by Raffaelea lauricola and vectored by the redbay ambrosia beetle (Xyleborus glabratus). A study was initiated in 2012 to assess the potential impact of the disease on clonal populations of pondberry. Individual pondberry plants were planted in nursery beds near Athens, Georgia and grown for approximately 3 years, and during this time individual plants produced many ramets. The original plant in two populations were inoculated with R. lauricola in July, 2015 and plants were monitored regularly. After 12 wk, 55 ramets died in one population at distances up to 172 cm from the original inoculated plant, and in the other population 22 ramets died at distances up to 337 cm. R. lauricola was reisolated from 84% of the wilted ramets. The study confirms that pondberry is highly susceptible to laurel wilt and that the disease can spread rapidly through rhizomes of infected plants. Although pondberry is a small shrub that is not often attacked by X. glabratus, when infections occur they could be detrimental to populations. Additional studies are being conducted to examine the feasibility of using trenching around infected plants to prevent disease transmission, and the use of chemicals to protect plants from infection. The laurel wilt story: Introduction and impact of an exotic vector (Xyleborus glabratus) and pathogen (Raffaelea lauricola) M. HUGHES (1), J. Riggins (2), A. cognato (3), F. Koch (4), C. Anderson (5), T. Dreaden (6), J. Formby (2), R. Ploetz (7), J. Smith (8) (1) University of Florida, School of Forest Resources and Conservation, Gainesville, FL, U.S.A.; (2) Mississippi State University, Department of Biochemistry, Molecular Biology, Entomology, and Plant Pathology, U.S.A.; (3) Michigan State University, Department of Entomology, MI, U.S.A.; (4) U.S. Forest Service, Southern Research Station, Eastern Forest Environmental Threat Assessment Center, NC, U.S.A.; (5) Research School of Biology, Australian National University, Australia; (6) U.S. Forest Service, Southern Research Station, U.S.A.; (7) University of Florida, Department of Plant Pathology, FL, U.S.A.; (8) University of Florida, School of Forest Resources and Conservation, U.S.A. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. Laurel wilt is a lethal vascular disease that affects plants in the Lauraceae family. Since the exotic vector (Xyleborus glabratus) and its pathogenic fungal symbiont (Raffaelea lauricola) were introduced to the USA in 2002, laurel wilt has killed redbay (Persea borbonia) and related trees in southern forests. Many questions remain about the disease in the USA, including the number of introduction events, the genetic diversity of the vector and pathogen, and how many host trees have died. To infer the number of introduction events and assess genetic variability, Amplified Fragment Length Polymorphism (AFLP) markers were used to screen a collection of R. lauricola isolates, and a portion of the cytochrome oxidase I (COI) gene was sequenced and compared for X. glabratus samples. Results suggest that R. lauricola and X. glabratus are both clonal organisms in the USA that were likely introduced in a single event. Forest Inventory and Analysis (FIA) data were used to obtain pre- and post-epidemic redbay population levels, which were then used to calculate redbay mortality due to laurel wilt. The FIA data suggest that laurel wilt has spread over 250,000 km2 and killed nearly half a billion redbay trees. These data highlight the devastating impact that invasive organisms can have on native forests. Susceptibility of eight untested species of Cucurbitaceae to gummy stem blight under field conditions G. RENNBERGER (1), A. Keinath (1) Susceptibility of eight untested species of Cucurbitaceae to gummy stem blight under field conditions G. RENNBERGER (1), A. Keinath (1) (1) Coastal Research and Education Center, Clemson University, U.S.A. Susceptibility of eight untested species of Cucurbitaceae to gummy stem blight under field conditions G. RENNBERGER (1), A. Keinath (1) (1) Coastal Research and Education Center, Clemson University, U.S.A. To date, 23 species of 12 genera in the Cucurbitaceae are known to be affected by the cucurbit disease gummy stem blight (GSB), caused by the fungus Stagonosporopsis spp. (Didymella bryoniae). Eight different species representing seven genera of Cucurbitaceae were selected for taxonomic and geographic diversity. Muskmelon (Cucumis melo ‘Athena’), which is highly susceptible, was included as a control. A field trial was set up as a randomized complete block design with four replications and seven inoculated plants per replication to assess the susceptibility of these species. The severity of the foliar blight phase and the incidence of the crown canker phase of GSB were rated. Powdery mildews on native plants in Utah E. LAURITZEN (1) (1) Utah State University, U.S.A. Other tree taxa tested, in order from most to least susceptible, were Norway (P. abies), white (P. glauca), Black Hills (P. glauca var. densata), Serbian (P. omorika), and Meyer (P. meyeri) spruce. Group 1 was more common in the eastern Michigan, and fell into a Diaporthe eres clade. Group 3 was more common in the central region, and also fell into a D. eres clade. Group 2 was most common in the central region, and was unresolved taxonomically. Groups 4 and 5 were common throughout the Lower Peninsula and were also unresolved. Future work includes sequencing more gene regions to resolve Diaporthe groups. S4.123 New canker disease of Incense-cedar in Oregon caused by Phaeobotryon cupressi J. WEILAND (1), R. Sniezko (2), M. Wiseman (3), M. Serdani (3), M. Putnam (3) (1) USDA-ARS, Horticultural Crops Research Laboratory, Corvallis, OR, U.S.A.; (2) USDA Forest Service, Dorena Genetic Resource Center, Cottage Grove, OR, U.S.A.; (3) Oregon State University, Botany and Plant Pathology Department, Corvallis, OR, U.S.A. Incense-cedar (Calocedrus decurrens) is a tree native to Oregon and California. Since the early 2000’s, a canker disease has been observed with increasing frequency on ornamental and windbreak trees planted in the Willamette Valley of Oregon. Symptoms appear as dead, flagging, small- diameter (<1 cm) branches that are scattered throughout the crown. Phaeobotryon cupressi was consistently isolated from symptomatic trees located along the length of the Willamette Valley. Identification was based on morphology and on sequences from the internal transcribed spacer (ITS) and the translation elongation factor 1-α (EF1-α) region. Six isolates were tested for pathogenicity on potted 0.6-1 m tall incense-cedar saplings. Approximately 1 to 1.5 months after inoculation, inoculated branches began to turn brown and die, while control branches that received no inoculum remained healthy. P. cupressi was described as a new species in 2009, and was originally found causing cankers on Italian cypress (Cupressus sempervirens) in Iran. The pathogen was also detected once in the U.S. from Juniperus squamosus in Kansas. It is unknown whether P. cupressi affects native stands of incense- cedar, where similar symptoms have been observed, but studies are underway to evaluate the extent of this disease in Oregon. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. This survey effort will continue through 2016, focusing on PPV, ESFY and the Light Brown Apple Moth (LBAM). This survey is monitor for possible introduction of these pests and allows for early detection. Red rust of bromeliads caused by a parasitic alga in Florida G. Sanahuja (1), P. Lopez (1), A. Palmateer (1) (1) University of Florida, U.S.A. Cephaleuros is a genus of plant parasitic thalloid green algae containing at least two species C. virescens and C. parasiticus that are reported to cause disease on ornamental plants. Florida has 165 plant species belonging to 53 families that have been reported as hosts of Cephaleuros. Recent outbreaks of a “red rust” disease affecting bromeliads in production nurseries and landscapes led to an investigation of the causal agent, which was determined to be a Cephaleuros species. Morphological characteristics from Cephaleuros isolates recovered from Neoregelia hybrids match with isolates of C. parasiticus collected from leaves of Psidium guajava (guava). Molecular analyses were performed from 18S ribosomal RNA and ribulose-1,5- bisphosphate carboxylase/oxygenase large subunit (rbcL) genes. Pathogenicity tests were conducted using Cephaleuros isolates collected from leaves of Neoregelia hybrids and isolates of C. parasiticus from guava. Both produced symptoms consistent with those originally described as “red rust” on Neoregelia hybrids and the causal agent of red rust on Neoregelia hybrids provisionally will be Cephaleuros parasiticus. First detection of the stubby root nematode Paratrichodorus allius on potato in North Dakota and on sugarbeet in Minnesota G. Yan (1), A. Plaisance (2), D. Huang (1), Z. Handoo (3) (1) North Dakota State University, Department of Plant Pathology, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A.; (3) USDA-ARS, Nematology Laboratory, U.S.A. G. Yan (1), A. Plaisance (2), D. Huang (1), Z. Handoo (3) (1) North Dakota State University, Department of Plant Pathology, U.S.A.; (2) North Dakota State University, Department of Plant Pathology, U.S.A.; (3) USDA-ARS, Nematology Laboratory, U.S.A. Stubby root nematodes (SRN) are migratory ectoparasites that feed on roots, transmit tobraviruses and cause significant crop loss. In 2015, 49 soil samples were collected from a potato field in Sargent County, ND of which seven contained SRN with population densities ranging from 135 to 300/kg soil. Subsequently, 13 soil samples were collected from a sugarbeet field in Clay County, MN and six of them had SRN from 200 to 1,000/kg soil. Individual SRN were examined morphologically and molecularly for species identification. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. The specimens from both potato and sugarbeet fields were identified as Paratrichodorus allius. Observations of female morphological characteristics critical for identification included rod like/oval and well separated vaginal sclerotization, onchiostyle 42-45μm, absence of lateral body pores, and small ventral pharyngeal overlap. DNA was extracted from single nematodes. D2/D3 region of 28S rRNA, partial 18S rRNA and ITS1 rDNA were amplified and PCR products were cloned and sequenced. The consensus sequence from each region was deposited into GenBank and compared with known sequences. The sequence information supported the identity as P. allius. P. allius is known to be the prevalent vector of Tobacco rattle virus causing potato corky ringspot disease. Until now, problems with infestations of SRN on sugarbeet have been confined to parts of Europe, California and Idaho. This represents the first occurrence of P. allius in North Dakota and Minnesota. Phytophthora nicotianae causing foliar blight of Codiaeum variegatum and Zamioculcas zamiifolia in Florida G. Sanahuja (1), P. Lopez (1), A. Palmateer (1) (1) University of Florida, U.S.A. Phytophthora is an oomycete plant pathogen, commonly known as a ‘water mold’ that attacks all parts of the host plant, causing root, crown and stem rot and foliar blight. Phytophthora diseases affect numerous ornamentals in nurseries, garden centers and landscapes generally during the hot and humid rainy season in south Florida. Phytophthora nicotianae was isolated for the first time from diseased Codiaeum variegatum (‘variegated’ croton) and Zamioculcas zamiifolia (‘ZZ’ plant) plants. Initially the leaves appeared water soaked and eventually turned dark brown to black in both plants. Morphological characteristics, molecular analysis of the ITS region, and pathogenicity test revealed that P. nicotianae was the causal agent. Tropical foliage plants typically account for the greatest percentage of sales within the ornamental industry and we anticipate these diseases will have a substantial impact on tropical foliage in south Florida. Local tropical foliage producers lost 10% and 35% of croton and ZZ plant production, respectively. Therefore, it is important to enlist the use of effective, registered pesticides to provide preventative control of Phytophthora blight during the periods where consistent outbreaks occur. Characterization of viral particles formed by Grapevine vein clearing virus (GVCV) Y. ZHANG (1), M. Adhab (1), J. Schoelz (1) (1) University of Missouri, U.S.A. Grapevine vein clearing virus (GVCV) is closely associated with a grapevine vein clearing syndrome observed in Missouri and surrounding states. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. Matheron (2) (1) University of Arkansas, U.S.A.; (2) University of Arizona, U.S.A. A unique foliar disease of spinach caused by P. aphanidermatum was observed in spinach in the Yuma, AZ/Imperial Valley, CA spinach production areas in October of 2015. The foliar symptoms of the disease included water soaked foliage, rapid collapse of young plants, with evidence of white cottony mycelium. The disease was associated with hot and wet conditions under high density (>3.0 M seed/A) plantings. Pure cultures were isolated from symptomatic spinach leaves and isolates were sequenced using ITS1/ITS4 primers. BLAST searches showed 100% homology with ITS sequences of P. aphanidermatum in GenBank. The objective of this research was to determine the effect of the P. aphanidermatum recovered on damping-off and foliar web blight on a broad range of hosts. Pathogenicity was tested in the greenhouse on spinach, cotton, soybean, pepper, tomato, cucumber, melon, squash, lettuce, corn, wheat, and rice, by either infesting the soil or doing foliar inoculations with colonized plugs of PDA. P. aphanidermatum caused severe damping off of all dicots tested, but only caused some seedling size reduction in corn and wheat. Web blight symptoms were severe with spinach, and all other dicot hosts. No web blight symptoms were observed on lettuce, corn, wheat, or rice. Metalaxyl provided complete protection for damping off of spinach and protected spinach plants for 10-20 days for U.S. treatment rates or 20-30 days after planting for E.U treatment rates, respectively. Stone fruit survey efforts in Texas monitoring for Plum Pox Virus, European Stone Fruit Yellows & Light Brown Apple Moth: 2015-2016 K. ONG (1), S. Rhodes (1) (1) Texas A&M AgriLife Extension Service, U.S.A. The Texas Plant Disease Diagnostic Lab continues participation in the National Stone Fruit Survey in FY15, monitoring for pathogens of concern for the stone fruit industry in Texas. The Texas A&M stone fruit breeding plots and four (4) commercial peach orchards in 3 counties were tested for the presence of Plum Pox Virus (PPV) and European Stone Fruit Yellows (ESFY) phytoplasma in 2015. A total of 667 foliar peach and plum samples were tested for PPV. For virus detection, each sample was tested by ELISA using Agdia ELISA kits and following USDA APHIS PPQ and National Plant Diagnostic Network protocols. All 667 samples tested negative for all five strains of PPV. ESFY was not observed or detected in any of the surveyed peach orchards. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. Ecballium elaterium showed the highest severity to foliar blight, followed by Apodanthera sagittifolia and Kedrostis leloja. These species were more susceptible than the control. A group of five species, Acanthosicyos horridus, Cucurbita digitata, Cucurbita ecuadorensis, Cucumis zambianus and Melothria scabra, were as susceptible to foliar blight as the control. The highest incidence of cankers was observed on crowns of K. leloja and ‘Athena’ muskmelon, followed by C. zambianus, A. sagittifolia and E. elaterium in decreasing order. The pathogen was recovered from crowns of all species. This is the first report of susceptibility of these eight cucurbit species to GSB. Raffaelea arxii may be the primary symbiont of Xyleborus affinis J. SAUCEDO (1), R. Ploetz (2), D. Carrillo (2), J. Konkol (2), J. Smith (1), J. Rollins (1), S. Ochoa (3) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A.; (3) Universidad Michoacana de San Nicolás de Hidalgo, Mexico Symbioses between fungi and ambrosia beetles are poorly understood. Most of the ambrosia beetles that have been studied have a primary fungal symbiont that predominates in a species’ mycangium and natal gallery, where it is cultivated as a food source. However, there is increasing evidence that species of fungal symbionts can be laterally transferred to other beetle species (are not uniquely associated with a single species). For example, the primary symbiont of Xyleborus glabratus, Raffaelea lauricola (cause of laurel wilt), has been found in at least nine other species of ambrosia beetle. Symbiont composition was examined in several of the latter species. Mandibular mycangia of one of the species, X. affinis reared from laurel wilt- affected logs of avocado (Persea americana), were assayed for fungal communities. Partial sequences of LSU of rDNA regions identified high concentrations of phenotypically variable populations of R. arxii in 95%, whereas Raffaelea sp., R. lauricola, and Ambrosiozyma platypodis were recovered from, respectively, 45, 20 and 15% of 20 assayed individuals. Although R. arxii was first reported in South Africa as the primary symbiont of X. torquatus (syn. X. volvulus), the abundance and persistent association between R. arxii and X. affinis suggests that R. arxii may also be the primary symbiont of X. affinis. Primary and secondary symbionts of X. affinis, X. volvulus and other potential vectors of R. lauricola will be discussed. S4.124 Web blight of spinach in the desert southwest caused by Pythium aphanidermatum C. FENG (1), B. Liu (1), M. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. Pairwise comparisons of the polymerase conserved domains of BGMV and established members of the genus showed amino acid identity ranging from 57-61% whereas phylogenetic analysis placed the virus in a distinct clade within the genus. The virus has been found in all plants showing green mosaic symptoms and its distribution and role in symptomology are currently being evaluated. Genome sequence analysis of a legume-infecting Tomato chlorotic spot virus isolate R. ADEGBOLA (1), R. Kemerait (2), S. Adkins (3), R. Naidu (1) (1) Washington State University, U.S.A.; (2) Department of Plant Pathology, The Univer iversity, U.S.A.; (2) Department of Plant Pathology, The University of Georgia, U.S.A.; (3) USDA ARS USHRL, U.S.A. Tomato chlorotic spot virus (TCSV, genus Tospovirus, family Bunyaviridae) has emerged as a significant constraint to economically important horticultural crops in Florida and the Caribbean region. In this study, we have determined the full genome sequence of a TCSV isolate from peanut (Arachis hypogea) collected in Haiti. Total RNA isolated from virus-infected leaves was used to generate sequences of the S-, M-, and L-RNA genomic segments by a combination of conventional Sanger sequencing and next-generation sequencing (NGS). The consensus sequences generated from Sanger sequencing were compared against those derived from de novo assembly of the filtered NGS reads to confirm the quality and accuracy of the complete genome sequences of the three RNA segments. The 8,873 nucleotide (nt) L-RNA shared 98% nt identity with the L-RNA sequence of a TCSV isolate from Brazil. The 4,847 nt M-RNA shared 99% nt identity with the M-RNA sequence of a Groundnut ringspot virus and TCSV reassortant from Florida. The 3,310 nt S-RNA shared 32.5 to 73.8% nt identity with S-RNA sequences of other tospoviruses. Phylogenetic analysis of proteins encoded by individual TCSV genomic RNA segments with corresponding proteins encoded by other tospoviruses is in progress. This study demonstrates advantages of NGS methods for high throughput, insightful analysis of genome segment sequences of a multi-partite negative-stranded RNA virus directly from field infected tissue with no viral enrichment. Viral metagenomics of Caladium x hortulanum (Araceae) in Florida R. ALCALA BRISEÑO (1), M. Londoño (1), J. Polston (1) (1) University of Florida, U.S.A. Viral metagenomics and high-throughput sequencing were used to identify novel viruses present caladiums (Caladium x hortulanum, Araceae), collected from Lake Placid, Florida in 2013. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. Survey for viruses of grapevine (Vitis vinifera L.) in coastal vineyards of Croatia D. VONCINA (1), M. Al Rwahnih (2), A. Rowhani (2), R. Almeida (3) (1) Department of Plant Pathology, University of Zagreb Faculty of Agriculture, Zagreb, Croatia; (2) Department of Plant Pathology, University of California, Davis, Davis, CA, U.S.A.; (3) Department of Environmental Science, Policy and Management, University of California, Berkeley, Berkeley, CA, U.S.A. Viticulture in Croatia has a very long tradition representing an important branch of the national economy. Approximately 70 grapevine (Vitis vinifera L.) cultivars are considered to be autochthonous and are mostly grown in Croatian coastal region. Virus diseases of grapevines generally have a worldwide distribution and some have significant impact on vineyard longevity as well as quality and quantity of the fruit. This survey was initiated due to the limited data about the presence of economically most important viruses in Croatia. A survey was done on 10 autochthonous grapevine cultivars on a total of 48 vines from 22 vineyards. Extraction of total RNA was done from leaf petioles collected in August 2015 and vines were tested on the presence of 32 viruses by RT-qPCR. The most common virus found was GLRaV-3 (91,7%), followed by GVA and GRSPaV (81,3%), GFkV (68,8%), ArMV and GLRaV-1 (33,3%), GVB (10,4%), GPGV (8,3%) and GLRaV-2 (4,2%). Six viruses were detected for the first time in Croatia: GRVFV (14,6%), GRGV (12,5%), viruses from GLRaV-4 group, GVD and GVF (6,3%) and GSyV-1 (2,1%). Mixed infections were also common in individual grapevines. The survey indicates a poor sanitary status of autochthonous grapevine cultivars, demonstrating the necessity to extend work on sanitary selection programs. Blueberry green mosaic symptoms are associated with the presence of a new vitivirus T. THEKKE-VEETIL (1), T. Ho (1), J. Polashock (2), I. Tzanetakis (1) (1) University of Arkansas, U.S.A.; (2) USDA-ARS, U.S.A. A virus was discovered in blueberry plants showing green mosaic symptoms, distinct symptomology from that associated with mosaic disease and was provisionally named as Blueberry green mosaic virus (BGMV). The genome was amplified using overlapping PCRs and has the typical organization of members of the genus Vitivirus, family Betaflexiviridae including five open reading frames (ORFs) and a 3’ polyadenylated terminus. The ORFs code for the viral replicase, a 16 kDa protein of unknown function, the movement, coat and nucleic acid binding proteins, the latter presumably involved in RNA interference. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. Caladium cultivars showing viral-like symptoms were sequenced, reads were assembled and contigs were analyzed using BLASTX showing homology with viral species in the Potyviridae, Rhabdoviridae and Caulimoviridae. The bioinformatic results were validated by reverse transcription (RT)-PCR for RNA viruses and PCR for DNA viruses. Only two potyvirus species, Dasheen mosaic virus (DsMV), and Konjac mosaic virus (KoMV) were able to be confirmed by RT-PCR amplification and sequencing of the coat protein region (CP). DsMV was previously reported from caladium in Florida, however this is the first report of KoMV in Florida as well as North America. In order to explore the abundance and diversity of these viruses, 315 plants from 15 caladium cultivars were screened using RT-PCR with CP primers. DsMV was present in 69.5% of tested plants and in all cultivars. KoMV was present in 11% of tested plants, and in eight out of 15 cultivars. Sixty-one and thirteen CP sequences of DsMV and KoMV were sequenced, respectively. Pairwise comparison analysis indicated sequence similarity of 87.4% and 96.5% among isolates of DsMV and KoMV, respectively. The low diversity, abundance and phylogenetic analysis are consistent with a single introduction event for both DsMV and KoMV. Sequence variation between isolates of Plantago asiatica mosaic virus from lily and other hosts J HAMMOND (1) M Reinsel (1) Sequence variation between isolates of Plantago asiatica mosaic virus from lily and other hosts J. HAMMOND (1), M. Reinsel (1) (1) USDA ARS USNA Floral and Nursery Plants Research Unit U S A uence variation between isolates of Plantago asiatica mosaic virus from lily and other hosts Sequence variation between isolates of Plantago asiatica mosaic virus from lily and other hosts J. HAMMOND (1), M. Reinsel (1) (1) USDA-ARS, USNA, Floral and Nursery Plants Research Unit, U.S.A. Sequence variation between isolates of Plantago asiatica mosaic virus from lily and other hosts J. HAMMOND (1), M. Reinsel (1) (1) USDA-ARS, USNA, Floral and Nursery Plants Research Unit, U.S.A. Plantago asiatica mosaic virus (PlAMV) was reported from Plantago asiatica in the Russian Far East, and Nandina mosaic virus was reported from heavenly bamboo (Nandina domestica) in California, both in the 1970’s. Nandina mosaic virus has not been reported to naturally infect any other hosts and was recognized as a strain of PlAMV (PlAMV-NMV) only when the full sequence was determined. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. The virus was initially characterized through deep sequencing of small RNAs isolated from symptomatic grape tissue. Three overlapping fragments of GVCV genomes were cloned and sequenced, from which the GVCV genome was assembled. The nucleotide sequence revealed that GVCV belonged to the Badnavirus genus. However, GVCV virions have never been observed under electron microscope. A GVCV infectious clone has been constructed in order to characterize GVCV virions and to complete Koch’s postulates. The GVCV infectious clone was infiltrated into Nicotiana benthamiana young S4.125 leaves, and leaves were collected at 7 dpi, followed by a virion purification procedure involving ultracentrifugation through sucrose gradients. In contrast to the expected bacilliform virions, a flexuous rod structure was observed that had a 15nm diameter and varied in length from 83 to 930 nm. The same virion purification procedure was applied to GVCV-infected grape leaves showing typical vein clearing symptoms and two types of virion particles were observed from a single virion prep: a flexuous rod 15nm in width and up to 8,025 nm in length, as well as the typical bacilliform structure. Antibodies targeting the GVCV coat protein were designed from the GVCV coding sequence and will be applied to test the nature of these two potential GVCV particles. leaves, and leaves were collected at 7 dpi, followed by a virion purification procedure involving ultracentrifugation through sucrose gradients. In contrast to the expected bacilliform virions, a flexuous rod structure was observed that had a 15nm diameter and varied in length from 83 to 930 nm. The same virion purification procedure was applied to GVCV-infected grape leaves showing typical vein clearing symptoms and two types of virion particles were observed from a single virion prep: a flexuous rod 15nm in width and up to 8,025 nm in length, as well as the typical bacilliform structure. Antibodies targeting the GVCV coat protein were designed from the GVCV coding sequence and will be applied to test the nature of these two potential GVCV particles. Survey for viruses of grapevine (Vitis vinifera L.) in coastal vineyards of Croatia D. VONCINA (1), M. Al Rwahnih (2), A. Rowhani (2), R. Almeida (3) (1) Department of Plant Pathology, University of Zagreb Faculty of Agriculture, Zagreb, Croatia; (2) Department of Plant Pathology, University of California, Davis, Davis, CA, U.S.A.; (3) Department of Environmental Science, Policy and Management, University of California, Berkeley, Berkeley, CA, U.S.A. Molecular Detection of Ti ringspot associated virus, a novel emaravirus associated with ti ringspot disease of Cordyline fruticosa (L.) in Hawai’i M. MELZER (1), A. Park (1) (1) University of Hawaii, U.S.A. Molecular Detection of Ti ringspot associated virus, a novel emaravirus associated with ti ringspot disease of Cordyline fruticosa (L.) in Hawai’i M. MELZER (1), A. Park (1) (1) University of Hawaii, U.S.A. Molecular Detection of Ti ringspot associated virus, a novel emaravirus associated with ti ringspot disease of Cordyline fruticosa (L.) in Hawai’i M. MELZER (1), A. Park (1) (1) University of Hawaii, U.S.A. An emerging virus-like disease of ti plant (Cordyline fruticosa L.) is spreading quickly throughout the Hawaiian Islands. Foliar symptoms include chlorotic lesions constricted by secondary veins or circular ring-spots that can coalesce into amorphous lesions. Tissue samples were collected from various locations across Hawaii and RNA was extracted. Degenerate RT-PCR primers targeting the polymerase gene of known emaraviruses amplified a ~400 bp product from the RNA of symptomatic plants. The RT-PCR products were sequenced and found to be >97.5% identical, indicating they represent a single virus species, designated Ti ringspot associated virus (TiRaV). When translated into amino acid sequences, this virus was found to be 70% identical to Raspberry leaf blotch virus, 55% to European mountain ash ringspot-associated virus, 53% to Redbud yellow ringspot virus, 50% to Rose rosette virus, 49% to Fig mosaic virus (FMV), and 46% to Pigeon pea sterility mosaic virus. These sequences and the C. fruticosa large rubisco subunit sequence were used to create specific primers and probes for a multiplex RT-qPCR detection assay. This assay was able to reliably detect TiRaV in symptomatic samples collected from various locations across Hawaii, and did not react to asymptomatic samples or cross react with an isolate of FMV. Is there resistance to Rose Rosette Disease among cultivated roses? Is there resistance to Rose Rosette Disease among cultivated roses? g K. ONG (1), J. Olson (2), T. Evans (3), M. Windham (4), E. Roundey (5), J. Lau (5), D. Byrne (5) ( ), ( ), ( ), ( ), y ( ), ( ), y ( ) (1) Texas A&M AgriLife Extension Service, College Station, TX, U.S.A.; (2) Oklahoma State University, Stillwater, OK, U.S.A.; (3) Department of Plant and Soil Sciences, University of Delaware, Newark, DE, U.S.A.; (4) Entomology and Plant Pathology Department, University of Tennessee, Knoxville, TN, U.S.A.; (5) Department of Horticultural Sciences, Texas A&M University, College Station, TX, U.S.A. Rose Rosette Disease (RRD) is caused by the Rose Rosette Virus (RRV) and transmitted by a wind-blown, eriophyid mite (Phyllocoptes fructiphilus). The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. In contrast, PlAMV was reported to affect lilies (Lilium spp.) and primrose (Primula seiboldii) in Japan, and in lilies in the Netherlands, Italy, Hungary, Spain, the UK, Taiwan, Chile, and the USA. Cut- flower Oriental and Asiatic hybrid lilies suffer significant losses of foliar and flower quality, or are even rendered unmarketable by PlAMV infection, showing foliar mosaic and necrosis, and streaking or distortion of the flowers. We have sequenced the coat protein (CP) gene and 3’ non-coding region of PlAMV isolates from multiple imported lily cultivars, and compared these sequences to isolates from other countries and hosts, including PlAMV- NMV. Sequences from all imported lilies fall within the same clade as PlAMV isolates from Europe; lily and primula isolates from Japan fall into another clade; whereas PlAMV-type (P. asiatica) and PlAMV-NMV form monophyletic clades. PlAMV-type and PlAMV-NMV share multiple CP S4.126 amino acids differentiating them from other isolates; other residues are uniquely conserved within the ‘European’ clade, or within the ‘Japanese’ (lily and primrose) clade. amino acids differentiating them from other isolates; other residues are uniquely conserved within the ‘European’ clade, or within the ‘Japanese’ (lily and primrose) clade. Detection and characterization of a novel potyvirus, Crinum mosaic virus, in ornamental Crinum R. JORDAN (1), A. Khawaja (1), M. Guaragna (1) (1) US National Arboretum, USDA-ARS, U.S.A. Ornamental flower bulbs (including true bulbs, bulbils, corms, tubers and rhizomes) are increasingly important floriculture crops. Crinum is a large genus of herbaceous perennial flowering bulbs in the family Amaryllidaceae and most of the species are grown in the summer and have large, showy, fragrant flowers. Only two viruses have been reported to infect cultivated Crinum, both potyviruses - Nerine yellow stripe virus (NeYSV; in India) and Crinum mosaic virus (CriMV; in Australia, FL and HI). CriMV has not been characterized and infection has only been noted by the presence of potyvirus particles or characteristic potyviral inclusions. Leaf samples from several Crinum plants growing at the US National Arboretum exhibiting mosaic symptoms tested positive for the presence of Potyvirus in an RT-PCR screen using several universal potyvirus primers yielding the expected 1,600 bp product corresponding to the partial NIb gene, full-length coat protein (CP) gene and 3’UTR. The RT-PCR amplicon was cloned and sequenced and NCBI BLAST analysis of the consensus sequence revealed highest similarities, but not identity, with isolates of NeYSV. Molecular Detection of Ti ringspot associated virus, a novel emaravirus associated with ti ringspot disease of Cordyline fruticosa (L.) in Hawai’i M. MELZER (1), A. Park (1) (1) University of Hawaii, U.S.A. Little is known about resistance of roses to the virus or the mite vector. A few rose species, such as Rosa palustris, R. setigera, R. carolina, R. virginiana, and R. spinosissima have been reported to be resistant to RRD, but resistance in cultivated roses is unknown. Although it is possible to genetically introgress disease resistance from a wild species into the cultivated rose, the development of commercial roses would be quicker if there was a good source of resistance within the cultivated germplasm. Preliminary observational data on over 400 rose accessions indicates that there is resistance to RRD among the cultivated rose germplasm. Large field trials to assess RRD resistance of about 300 rose accessions have been planted in Tennessee and Delaware. The disease pressure in these trials is being augmented by inter-planting RRD infected roses among and by placing mite infested shoot tips from infected plants onto the plants to be tested for their resistance. In addition, smaller trials are being planted in Oklahoma and Texas to assess the level of RRD resistance of selected roses. Since disease symptoms may not appear for many weeks to months after inoculation with the virus, we anticipate this will be a long term study and it may be 1 or more years before we know if resistance occurs in cultivated roses. Survey for Grapevine Pinot gris virus infecting Grapevine in the Foundation Plant Services Vineyards at the University of California, Davis M. AL RWAHNIH (1), N. Westrick (2), D. Golino (2), A. Rowhani (2) (1) University of California, Davis, U.S.A.; (2) University of California, Davis, U.S.A. The impact of laurel wilt caused by Raffaelea lauricola on clonal populations of pondberry (Lindera melissifolia) S. BEST (1), S. Fraedrich (1) (1) USDA Forest Service, Athens, GA, U.S.A. Pair-wise analyses of the 258 amino acid sequence of the predicted CP had 65-68% sequence identity with the NeYSV isolates from Crinum, Hymenocallis, Nerine, Stenomesson, and Vallota; whereas the seven NeYSV isolates share 85-98% CP identity. We propose to associate this unique potyvirus sequence with the aforementioned CriMV. Grapevine Pinot gris virus (GPGV) is a new member of the genus Trichovirus in the family Betaflexiviridae. The virus was first discovered by high- throughput sequencing from an Italian Pinot gris grapevine (Vitis vinifera) showing chlorotic mottling, leaf deformation and stunting symptoms. Since then, GPGV has been reported in symptomatic and asymptomatic plants of other cultivars in many countries. Recent transmission studies indicated that the eriophyid mite Colomerus vitus (Pagenstecher) is the suspected vector. This vector infests grapevine wherever they are grown and it has been reported in California. To address the possible presence and prevalence of GPGV in the collections of the Foundation Plant Services (FPS, University of California, Davis), 2,014 vines from the collection were screened including 23 vines of Pinot gris. Among all the vines tested, only one asymptomatic vine cv. Touriga Nacional was positive for GPGV. This grapevine cultivar has been imported from Portugal in 1981and planted in the FPS collection in 2001. This is believed to be the first reported detection of GPGV in the U.S. Twenty samples in close proximity to the GPGV-positive vine were also collected and tested. These vines were all tested negative for GPGV. As a leading source of orchard and vineyard planting stock in the U.S., in addition to being a central component of the Grape Clean Plant Network, these negative results indicate that FPS stock is not a major source of GPGV. Viruses of brugmansia in Mississippi g pp N. ABOUGHANEM-SABANADZOVIC (1), A. Lawrence (1), S. Sabanadzovic (1) (1) Mississippi State University, U.S.A. Brugmansia (Brugmansia spp.), or Angel’s trumpet, is an ornamental woody plant/shrub originating from South America and belonging to the family Solanaceae. Because of production of attractive large and fragrant flowers, they are now commonly grown in the temperate climate worldwide, including USA. As any other plant, it is susceptible to different pathogens including viruses. During the late summer/fall of 2015, several symptomatic brugmansia samples of different cultivars were brought to our attention by a local plant enthusiast. Symptoms varied among samples and, generally speaking, consisted of leaf mosaics and distortion. Samples were examined separately by biological, serological and molecular methods, including deep sequencing, and resulted in identification of several potyviruses, such as Colombian datura virus (CDV) and Brugmansia mosaic virus (BruMV). CDV was previously reported from brugmansia collected in Florida and few other states in the USA; however, this study represents the first state record for this virus in Mississippi. BruMV was not previously known to occur in the United States and was probably recently introduced into the country by infected plant material. Survey for Grapevine Pinot gris virus infecting Grapevine in the Foundation Plant Services Vineyards at the University of California, Davis M. AL RWAHNIH (1), N. Westrick (2), D. Golino (2), A. Rowhani (2) (1) University of California, Davis, U.S.A.; (2) University of California, Davis, U.S.A. of Plant Pathology & Microbiology, Texas A&M University AgriLife Research & Extension Center, Weslaco, TX, U.S.A.; (2) Department of Plant Pathology, University of California, Davis, Davis, CA, U.S.A.; (3) School of Plant Sciences, University of Arizona, Tucson, AZ, U.S.A.; (4) Department of Horticultural Sciences, Texas A&M AgriLife Research and Extension Center, Weslaco, TX, U.S.A.; (5) Texas A&M University, Kingsville Citrus Center, Weslaco, TX, U.S.A.; (6) Texas A&M AgriLife Research and Extension Center, Weslaco, TX, U.S.A.; (7) Department of Agriculture, Agribusiness, and Environmental Sciences, Texas A&M University, Kingsville Citrus Center, Weslaco, TX, U.S.A. Association of a mixed infection of Lettuce chlorosis virus, Papaya ringspot virus, and Tomato yellow leaf curl virus-IL in a Texas papaya orchard O. ALABI (1), M. Al Rwahnih (2), J. Brown (3), J. Jifon (4), J. Park (5), L. Gregg (6), M. Sétamou (7), A. Idris (3) (1) Dept. of Plant Pathology & Microbiology, Texas A&M University AgriLife Research & Extension Center, Weslaco, TX, U.S.A.; (2) Department of Plant Pathology, University of California, Davis, Davis, CA, U.S.A.; (3) School of Plant Sciences, University of Arizona, Tucson, AZ, U.S.A.; (4) Department of Horticultural Sciences, Texas A&M AgriLife Research and Extension Center, Weslaco, TX, U.S.A.; (5) Texas A&M University, Kingsville Citrus Center, Weslaco, TX, U.S.A.; (6) Texas A&M AgriLife Research and Extension Center, Weslaco, TX, U.S.A.; (7) Department of Agriculture, Agribusiness, and Environmental Sciences, Texas A&M University, Kingsville Citrus Center, Weslaco, TX, U.S.A. Severe virus-like symptoms consisting of mosaic, distortion, yellowing, and brittleness were observed on papaya plants in a 50-ha orchard in South Texas during the 2014-2015 growing season. Disease incidence increased from ~40 to 100% within six-months of the outbreak and severely affected plants were stunted with reduced fruit loads and poor fruit quality. cDNA library constructed from DNAse-treated total nucleic acids from symptomatic plants was subjected to high-throughput sequencing (HTS). Based on BLASTn analysis of the assembled HTS contigs, the plant viruses detected were validated by Sanger sequencing of the complete genome sequences. The collective sequencing results indicated the presence of Papaya ringspot virus (PRSV; Potyvirus), Lettuce chlorosis virus (LCV; Crinivirus), and Tomato yellow leaf curl virus (TYLCV-IL; Begomovirus). RT-PCR analyses of leaves from 51 randomly sampled papaya plants indicated the presence of PRSV, LCV, and TYLCV-IL in 100, 39.2 and 15.7% of the samples, respectively. Seed-borne virome in cucurbits S. SABANADZOVIC (1), N. Aboughanem-Sabanadzovic (1) (1) Mississippi State University, U.S.A. In this work we investigated the ‘community’ of seed-borne viruses in more than 50 cultivars and PI lines/accessions belonging to different botanical species of cucurbits by the next-generation sequencing approach. To this aim, double stranded RNAs (dsRNAs) were extracted from plant tissue collected at the first true leaf stage from a pool of young seedlings belonging to the same genotype and grown under greenhouse conditions in order to prevent contamination by horizontally transmitted viruses. Polyacrylamide gel electrophoreses (PAGE) revealed the presence of single or multiple dsRNA bands in the majority of tested plants indicating ongoing infections by vertically transmitted viruses. The dsRNA extracts were reverse transcribed, tagged, and submitted to Illumina sequencing. Results of the sequence analyses demonstrated the widespread presence of persistent viruses in tested cucurbits, mainly those belonging to the families Partitiviridae, Amalgaviridae, Endornaviridae and Totiviridae. In this study, we identified and characterized numerous viruses, representatives of new species and confirmed the prevalence of Cucumis melo endornavirus and Lagenaria siceraria endornavirus in tested melons and bottle gourds, respectively. The importance of widespread presence of persistent viruses in some cucurbits and their possible effects to the host are yet to be understood. Reemergence of the torradovirus, Tomato necrotic dwarf virus, in processing tomatoes in the Central Valley of California O. BATUMAN (1), M. Vasquez-Mayorga (1), J. Nunez (2), L. Hladky (3), W. Wintermantel (3), R. Gilbertson (1) (1) University of California Davis, Davis, CA, U.S.A.; (2) University of California Cooperative Extension, Bakersfield, CA, U.S.A.; (3) United States Department of Agriculture, Salinas, CA, U.S.A. Reemergence of the torradovirus, Tomato necrotic dwarf virus, in processing tomatoes in the Central Valley of California O. BATUMAN (1), M. Vasquez-Mayorga (1), J. Nunez (2), L. Hladky (3), W. Wintermantel (3), R. Gilbertson (1) (1) University of California Davis, Davis, CA, U.S.A.; (2) University of California Cooperative Extension, Bakersfield, CA, U.S.A.; (3) United States Department of Agriculture, Salinas, CA, U.S.A. In the fall of 2015, processing tomato plants (Solanum lycopersicum) in a late-planted field in Kern County, California had high populations of whiteflies and developed virus-like symptoms including stunting and distorted growth; leaf curling, crumpling and necrosis; and stem and shoot necrosis. The symptoms resembled those induced by Tomato spotted wilt virus (TSWV), but immunostrip and RT-PCR tests were negative for TSWV and other tospoviruses. Survey for Grapevine Pinot gris virus infecting Grapevine in the Foundation Plant Services Vineyards at the University of California, Davis M. AL RWAHNIH (1), N. Westrick (2), D. Golino (2), A. Rowhani (2) (1) University of California, Davis, U.S.A.; (2) University of California, Davis, U.S.A. Augustine, Trinidad; (4) Cocoa Research Center, The University of the West Indies, St. Augustine, Trinidad; (5) Centre National de Recherché Agronomique (CNRA) Programme Cacao, Divo, Ivory Coast; (6) Cocoa Research Institute of Ghana, Tafo, Ghana; (7) USDA-ARS Subtropical Horticultural Research Station, Miami, FL, U.S.A. ( ), ( ), ( ) (1) University of Arizona, Tucson, AZ, U.S.A.; (2) IAGS, University of Punjab, Lahore, Pakistan; (3) Cocoa Research Centre, The University of the West Indies, St. Augustine, Trinidad; (4) Cocoa Research Center, The University of the West Indies, St. Augustine, Trinidad; (5) Centre National de Recherché Agronomique (CNRA) Programme Cacao, Divo, Ivory Coast; (6) Cocoa Research Institute of Ghana, Tafo, Ghana; (7) USDA-ARS Subtropical Horticultural Research Station, Miami, FL, U.S.A. ( ) ( ) ( ) (1) University of Arizona, Tucson, AZ, U.S.A.; (2) IAGS, University of Punjab, Lahore, Pakistan; (3) Cocoa Research Centre, The University of the West Indies, St. Augustine, Trinidad; (4) Cocoa Research Center, The University of the West Indies, St. Augustine, Trinidad; (5) Centre National de Recherché Agronomique (CNRA) Programme Cacao, Divo, Ivory Coast; (6) Cocoa Research Institute of Ghana, Tafo, Ghana; (7) USDA-ARS Subtropical Horticultural Research Station, Miami, FL, U.S.A. Sustained production of cacao (Theobroma cacao) in West Africa, which supplies over 70% of the world’s bulk cacao, is hindered by virus-like diseases. To date, Cacao swollen shoot virus (CSSV) (Caulimoviridae, Badnavirus), endemic to West Africa, is the only known cacao-infecting badnavirus. In West Africa, CSSV-like symptoms became prevalent during 2000-2003, and continue to persist. Since the 1940s, virus-like symptoms in cacao of suspect badnaviral etiology have been recognized in Trinidad at the International Cocoa Genebank, one of the custodians of cacao germplasm resources. To identify the suspect viruses, total DNA isolated from symptomatic cacao leaves collected in Ghana, Cote d’Ivoire and Trinidad was subjected to rolling circle amplification and sequenced using Illumina HiSeq. DNA sequences were assembled using DNASTAR, and annotated using the NCBI ORF Finder, and the results were validated by sequencing PCR-amplified full-length genomes. Genome characterization revealed seven previously unidentified badnavirus isolates, each with four open reading frames, characteristic of other badnaviruses. Pairwise comparisons using Standard Demarcation Tool (SDT) of the RT-RNaseH sequence (80% nucleotide identity) with analogous badnavirus sequences indicated that the isolates represented three new badnavirus species. However, SDT analysis of the full-length genome sequences suggested at least five previously unidentified badnavirus species are associated with cacao. Survey for Grapevine Pinot gris virus infecting Grapevine in the Foundation Plant Services Vineyards at the University of California, Davis M. AL RWAHNIH (1), N. Westrick (2), D. Golino (2), A. Rowhani (2) (1) University of California, Davis, U.S.A.; (2) University of California, Davis, U.S.A. First report and characterization of Taro bacilliform virus in the USA M. MELZER (1), K. Dey (1), A. Chan Borges (1), M. Long (1), W. Borth (1), N. Wichitrnithed (1), J. Hu (1), R. Li (2) (1) University of Hawaii, U.S.A.; (2) USDA-ARS, U.S.A. Taro bacilliform virus (TaBV) is a member of the genus Badnavirus (Family Caulimoviridae) and a pathogen of taro (Colocasia esculenta), an important starch staple in tropical regions. TaBV is transmitted by mealybugs and appears to be widely distributed throughout the tropical Pacific. Taro has cultural, economic and nutritional importance in Hawaii, but only Dasheen mosaic virus and Taro vein chlorosis virus have been reported in the state. We surveyed 328 taro plants from 35 sites on the islands of Kauai, Oahu, Molokai, Maui, and Hawaii for the presence of TaBV by PCR using degenerate badnavirus and TaBV-specific primers. Of these, 171 samples (52%) tested positive using the degenerate primers, but only 153 tested positive using TaBV-specific primers (47%), indicating the presence of novel badnaviruses and/or strains of TaBV in Hawaii’s taro. The 7825 bp genome of one isolate of TaBV was sequenced and found to be 80% identical and 367 bp larger than the only complete TaBV genome in GenBank (accession AF357836), indicating that considerable genetic diversity exists for this virus. PCR, RT-PCR and Southern hybridization assays were conducted and provided evidence that TaBV is integrated into the genome of some Hawaiian taro varieties. TaBV was mechanically inoculated onto eight herbaceous plant species, and after 25 days, TaBV could be detected by PCR in Cucurbita pepo, Pisum sativum, Cucumis sativus, Vigna unguiculata, and Phaseolus vulgaris. Association of a mixed infection of Lettuce chlorosis virus, Papaya ringspot virus, and Tomato yellow leaf curl virus-IL in a Texas papaya orchard O. ALABI (1), M. Al Rwahnih (2), J. Brown (3), J. Jifon (4), J. Park (5), L. Gregg (6), M. Sétamou (7), A. Idris (3) (1) Dept. Survey for Grapevine Pinot gris virus infecting Grapevine in the Foundation Plant Services Vineyards at the University of California, Davis M. AL RWAHNIH (1), N. Westrick (2), D. Golino (2), A. Rowhani (2) (1) University of California, Davis, U.S.A.; (2) University of California, Davis, U.S.A. Plants with mixed infections of PRSV, in combination with LCV and/or TYLCV-IL, exhibited more severe symptoms compared to plants with PRSV alone. The association of LCV and TYLCV-IL with symptomatic plants represents first report of these whitefly-transmitted viruses in papaya. Papaya, a perennial crop, could serve as an over-seasoning reservoir for TYLCV-IL and LCV presenting new challenges to disease management in papaya and other crop hosts of these viruses. Survey for Grapevine Pinot gris virus infecting Grapevine in the Foundation Plant Services Vineyards at the University of California, Davis M. AL RWAHNIH (1), N. Westrick (2), D. Golino (2), A. Rowhani (2) (1) University of California, Davis, U.S.A.; (2) University of California, Davis, U.S.A. Survey for Grapevine Pinot gris virus infecting Grapevine in the Foundation Plant Services Vineyards at the University of California, Davis M. AL RWAHNIH (1), N. Westrick (2), D. Golino (2), A. Rowhani (2) (1) University of California, Davis, U.S.A.; (2) University of California, Davis, U.S.A. Grapevine Pinot gris virus (GPGV) is a new member of the genus Trichovirus in the family Betaflexiviridae. The virus was first discovered by high- throughput sequencing from an Italian Pinot gris grapevine (Vitis vinifera) showing chlorotic mottling, leaf deformation and stunting symptoms. Since then, GPGV has been reported in symptomatic and asymptomatic plants of other cultivars in many countries. Recent transmission studies indicated that the eriophyid mite Colomerus vitus (Pagenstecher) is the suspected vector. This vector infests grapevine wherever they are grown and it has been reported in California. To address the possible presence and prevalence of GPGV in the collections of the Foundation Plant Services (FPS, University of California, Davis), 2,014 vines from the collection were screened including 23 vines of Pinot gris. Among all the vines tested, only one asymptomatic vine cv. Touriga Nacional was positive for GPGV. This grapevine cultivar has been imported from Portugal in 1981and planted in the FPS collection in 2001. This is believed to be the first reported detection of GPGV in the U.S. Twenty samples in close proximity to the GPGV-positive vine were also collected and tested. These vines were all tested negative for GPGV. As a leading source of orchard and vineyard planting stock in the U.S., in addition to being a central component of the Grape Clean Plant Network, these negative results indicate that FPS stock is not a major source of GPGV. S4.127 Identification and proposed species demarcation for Cacao swollen shoot virus and previously unidentified badnaviruses of Theobroma cacao N. CHINGANDU (1), M. Zia-Ur-Rehman (2), T. Sreenivasan (3), S. Surujdeo-Maharaj (4), P. Umaharan (4), K. Kouakou (5), R. Aka (5), G. Ameyaw (6), O. Gutierrez (7), J. Brown (1) Identification and proposed species demarcation for Cacao swollen shoot virus and previously unidentified badnaviruses of Theobroma cacao N. CHINGANDU (1), M. Zia-Ur-Rehman (2), T. Sreenivasan (3), S. Surujdeo-Maharaj (4), P. Umaharan (4), K. Kouakou (5), R. Aka (5), G. Ameyaw (6), O. Gutierrez (7), J. Brown (1) (1) University of Arizona, Tucson, AZ, U.S.A.; (2) IAGS, University of Punjab, Lahore, Pakistan; (3) Cocoa Research Centre, The University of the West Indies, St. Seed-borne virome in cucurbits The severity is estimated as an interval scale value and is used to determine a disease severity index (DSI). However, very few studies have investigated the effects of different scales on accuracy of the DSI. Using an interval scale designed for pecan scab (Fusicladium effusum), we investigated performance of the DSI. We used interval scale values, the scale data converted to midpoint values of each category, and nearest percent estimates (NPEs). Results of the pecan scab data indicate that there is a consistent bias of the mean value when applying the interval scale values directly. The mean value of the DSI per cultivar is underestimated as compared with that of NPEs; results of the mid-point conversion are akin to those of the NPEs. Furthermore, based on the data used, the simulation studies were executed to explore the mean value of DSI for three different parameters. The results of these simulation studies are consistent with those of studies which used real data. Finally, a new disease index, which uses equal intervals with additional grades at low severities, was developed to minimize the effect of the scale on error. Predicting Robust Candidates for a Boxwood Blight Immunoassay: An Automated Computational Workflow with Broad Applications for Phytopathology D. VELTRI (1), D. Luster (2), M. McMahon (2), J. Crouch (1) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. Predicting Robust Candidates for a Boxwood Blight Immunoassay: An Automated Computational Workflow with Broad Applications for Phytopathology D. VELTRI (1), D. Luster (2), M. McMahon (2), J. Crouch (1) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. The boxwood blight fungal pathogens Calonectria pseudonaviculata and C. henricotiae have emerged as a serious threat to natural and cultivated plant populations around the world. These pathogens can persist as latent infections in asymptomatic plant tissue even after fungicide treatment. Therefore, a fast and field-deployable diagnostic test based on immunological detection would provide a useful tool to help minimize spread of the pathogen. Although several nucleic acid-based approaches are available to detect boxwood blight pathogens, none are field-deployable solutions. We are developing an integrated computational pipeline using recent genomic and RNA-Seq data to identify quality candidate sequences for use in designing a diagnostic immunoassay. The pipeline first identifies proteins likely to be secreted using SECRETOOL. To robustly test the putative secreted proteins for solubility, we developed a new classifier with improved recognition performance over other methods using a publicly-available dataset of secreted proteins. Seed-borne virome in cucurbits RT-PCR tests were then performed for Tomato mosaic virus (ToMV), Alfalfa mosaic virus (AMV), Tomato necrotic spot virus (ToNSV), S4.128 and torradoviruses (family Secoviridae) were performed. The expected size DNA fragments were amplified for ToMV and torradovirus (degenerate primers for RNA-1 and RNA-2), whereas negative results were obtained for AMV and ToNSV. Analysis of the DNA sequences of the PCR-amplified torradovirus RNA-1 and RNA-2 fragments revealed that these plants were infected with a torradovirus that was most similar (~94%) to RNA-1 and RNA-2 of Tomato necrotic dwarf virus (ToNDV). This is the first report of ToNDV in the Central Valley of California, although it was previously reported from the Imperial Valley of California in the 1980s. Surveys to determine inoculum sources and further sequencing and biological characterization of ToNDV isolates in California are in progress. and torradoviruses (family Secoviridae) were performed. The expected size DNA fragments were amplified for ToMV and torradovirus (degenerate primers for RNA-1 and RNA-2), whereas negative results were obtained for AMV and ToNSV. Analysis of the DNA sequences of the PCR-amplified torradovirus RNA-1 and RNA-2 fragments revealed that these plants were infected with a torradovirus that was most similar (~94%) to RNA-1 and RNA-2 of Tomato necrotic dwarf virus (ToNDV). This is the first report of ToNDV in the Central Valley of California, although it was previously reported from the Imperial Valley of California in the 1980s. Surveys to determine inoculum sources and further sequencing and biological characterization of ToNDV isolates in California are in progress. Discussion on calculation of disease severity index values from scales with unequal intervals K. CHIANG (1), H. Liu (1), C. Bock (2) (1) Division of Biometrics, Department of Agronomy, National Chung Hsing University, Taiwan; (2) USDA-ARS-SEFTNRL, U.S.A. Discussion on calculation of disease severity index values from scales with unequal intervals K. CHIANG (1), H. Liu (1), C. Bock (2) ( ), ( ), ( ) (1) Division of Biometrics, Department of Agronomy, National Chung Hsing University, Taiwan; (2) USDA-ARS-S ( ) ( ) ( ) of Biometrics, Department of Agronomy, National Chung Hsing University, Taiwan; (2) USDA-ARS-SEFTNRL, U.S.A. When estimating severity of disease, a disease interval (or category) scale comprises a number of categories of known numeric values – with plant disease this is generally the percent area with symptoms (e.g., the Horsfall-Barratt (H-B) scale). Studies in plant pathology and plant breeding often use quantitative interval scales. Seed-borne virome in cucurbits Gevens (1) (1) University of Wisconsin-Madison, U.S.A.; (2) University of Wisconsin-Madison, U.S.A. Comparing epidemics of unique Phytophthora infestans clonal lineages with biometrically enhanced spatio-temporal modeling tools K. MOREY GOLD (1), Z. Zhang (2), D. Rouse (1), A. Gevens (1) (1) University of Wisconsin-Madison U S A ; (2) University of Wisconsin-Madison U S A The oomycete Phytophthora infestans causes late blight, a plant disease notorious for giving rise to the Irish Potato Famine of the 1840s. In 2009, a pandemic caused by a newly emerged clonal lineage of P. infestans (US-22) destroyed gardens and small farm tomatoes and potatoes across the US. In the following years, three clonal lineages (US-22, -23, -24) comprised national epidemics, with US-23 predominating from 2013 onward. US-23 is of noteworthy aggressiveness, has small sporangial size compared to previous lineages, and is highly pathogenic on both potato and tomato. Previously, two disease dispersal models were fit using Markov chain Monte Carlo (MCMC) for late blight epidemics in central Wisconsin caused by clonal lineage US- 8. In our current work, we validated and enhanced these models using the late blight epidemic caused by US-23 in this region for 2009-2015 and we fit a spatial-temporal semivariogram for both epidemics. Disease data were collected from scouting records of a cooperating crop consultant and were manipulated using ArcGIS and RStudio. The power law model predicted disease dispersion accurately with R2 values between 0.2 and 0.9. Sporangial size greatly influenced dispersion rate which played a role in disease progression. Modeling of specific clonal lineage epidemics in high-risk production regions can aid in developing prescriptive, risk-averse late blight management plans to reduce disease perpetuation and economic loss. Impacts of climate change on wheat powdery mildew epidemics in China Y. Zhou (1), X. Tang (1) (1) Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Chin Seed-borne virome in cucurbits The candidate proteins are further narrowed down through a comparative genomics analysis that identifies proteins with predicted antigenicity that are specific to the target pathogens yet share low similarity to outgroup species. This pipeline stands to help speed up immunoassay development and lower costs by providing a starting set of candidate proteins with a higher likelihood of success in further wet laboratory experiments. Identification of conducive temperatures for decision support modeling of sugarcane rusts in Florida B. CHAULAGAIN (1), M. Hincapie (1), S. Sanjel (1), C. Fraisse (2), R. Raid (1), P. Rott (1) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Most sugarcane varieties grown in Florida are currently susceptible to either brown (Puccinia melanocephala) or orange (Puccinia kuehnii) rust, two diseases that have been reported to cause economic losses in sugarcane industries worldwide. In Florida, sugarcane rusts are controlled using fungicides and application schedules are solely based on disease scouting. In order to develop a forecasting system to optimize fungicide applications, disease severity data were collected during the entire 2014 crop season. Percent rust affected area of the top visible dewlap leaf was recorded every two weeks from seven sugarcane varieties susceptible to brown or orange rust and planted in October, November and December 2013. Brown and orange rust progress curves were generated using maximum disease severity values at each scoring date, whatever variety and planting date. Because relative humidity and leaf wetness does not appear to be a limiting factor for development of sugarcane rusts in Florida, efforts were focused on temperature that is also known to affect rust progress. Curves of cumulative average daily temperatures between 17°C and 29°C were compared with disease progress curves. Temperature ranges of 17-25.5°C and 17-26.5°C appeared to be the best fit for progress of brown and orange rust, respectively. Weather and disease data from additional crop seasons need to be analyzed to confirm these first results and to develop a decision support model for rusts in Florida. Comparing epidemics of unique Phytophthora infestans clonal lineages with biometrically enhanced spatio-temporal modeling tools K. MOREY GOLD (1), Z. Zhang (2), D. Rouse (1), A. Gevens (1) (1) University of Wisconsin-Madison, U.S.A.; (2) University of Wisconsin-Madison, U.S.A. Comparing epidemics of unique Phytophthora infestans clonal lineages with biometrically enhanced spatio-temporal modeling tools K. MOREY GOLD (1), Z. Zhang (2), D. Rouse (1), A. Distribution models of common bean dry root rot in Brazil: Current and predicted disease distribution in climate change scenarios R. Macedo (1), L. Abud (1), F. Yoshida (1), L. Sales (1), E. Barbosa (2), M. Lobo (2), R. Macedo (1) (1) Universidade Federal de Goiás, Brazil; (2) Brazilian Corporation for Agricultural Research (Embrapa), Brazil Distribution models of common bean dry root rot in Brazil: Current and predicted disease distribution in climate change scenarios R. Macedo (1), L. Abud (1), F. Yoshida (1), L. Sales (1), E. Barbosa (2), M. Lobo (2), R. Macedo (1) (1) Universidade Federal de Goiás, Brazil; (2) Brazilian Corporation for Agricultural Research (Embrapa), Brazil Dry root rot is an ubiquitous problem on common beans in Brazil, with yearly yield losses attributed to the Fusarium solani species complex. Species distribution models (SDMs) based on 21 field disease records were developed to assess dry root rot current geographical distribution, and estimate disese range expected in IPCC scenarios A1B and A2 by the year 2030. Projections were adjusted by an ensemble of three modelling methods: Generalized Adittive Models, Surface Range Envolop and Artificial Neural Networks, built with “biomod” and “raster” packages from R statistical software. Also, we verified if three inoculum threshholds (1200, 3700 and 4500 propagules of F. solani per soil, in 2623 records) could work as disease spatial proxies, supported by Sperman’s correlations between inoculum thresholds and disease records. The best disease × soil inoculum density correlation was found with 3700 progules per soil gram (r=0.85, P<0.001). The current projection shows high climatic suitability for common bean dry root rot in the Brazilian central region. According to IPCC scenarios, the geographical area affected by the disease will be reduced by almost 23%, but highly favorable areas will move towards Brazil South Region, which represents 40% of the country’s common bean yields. Implications and consequences of these results will be discussed to support future plans on root rot management. Discovery of biological drivers of pitch canker disease in a changing climate T. QUESADA (1), T. Quesada (1), C. Staub (1), M. Marsik (1), K. Shin (1), K. Smith (2), J. Hughes (1) (1) University of Florida, U.S.A.; (2) US Forest Service / University of Florida, U.S.A. Natural and agricultural ecosystems are affected by climate change through increased temperature, humidity, and unusual weather patterns. They are increasingly threatened by invasions by non-native pests and pathogens. The fungus Fusarium circinatum causes pitch canker disease in several pine species, leading to high economic losses in the timber industry. Favored by high temperatures and humidity, future outbreaks may become more frequent under predicted changes in environmental conditions over the next 50-100 years. A better understanding of F. Quantifying the potential effects of regional climate change on wheat leaf rust disease in the Grand-Duchy of Luxembourg M. EL JARROUDI (1), L. Kouadio (2), J. Junk (3), B. Tychon (1), P. Delfosse (3) (1) University of Liege, Belgium; (2) University of Southern Queensland, Australia; (3) Luxembourg Institute of Science & Technology ( ) ( ) y ( ) ( ) (2) University of Southern Queensland, Australia; (3) Luxembourg Institute of Science & Technology, Luxembourg Climate change will affect wheat crop production both in the main processes of plant growth and development and in the occurrences and severities of plant diseases. We assessed the potential infection periods of wheat leaf rust (WLR) in a changing climate at two climatologically different sites in Luxembourg using a threshold-based model for WLR. Field experiments were conducted during 2003–2013 at the selected sites to test the model. Projected climate data, from a multi model ensemble of regional climate models (spatial resolution 25 km) as well as an additional projection with a higher spatial resolution of 1.3 km, were used to investigate the potential WLR infection periods for two future time spans. The model simulated satisfactorily WLR infection periods at both sites during 2003–2013: probabilities of detection were close to 1 and the critical success index ranged from 0.80 to 0.94. Regional climate projections indicate an increase in temperatures over the 2014-2050 and 2091-2100 periods compared to the reference period 1991–2000. Annual precipitation is also expected to increase slightly in the future. Moreover, trends in increased favourable days of WLR infection would likely occur at both sites due to projected climatic conditions more conducive than in the reference period. Our methodology can be easily transferred to other regions and other fungal diseases by adjusting the meteorological threshold values essential for a specific disease development. Screening some rust universal-susceptible wheat varieties for ozone tolerance and some applications A. MASHAHEET (1), D. Marshall (2), K. Burkey (2), A. Abdelrhim (1), F. Ullah (1) (1) North Carolina State University, U.S.A.; (2) USDA-ARS, North Carolina State University, U.S.A. Screening some rust universal-susceptible wheat varieties for ozone tolerance and some applications A. MASHAHEET (1), D. Marshall (2), K. Burkey (2), A. Abdelrhim (1), F. Ullah (1) (1) North Carolina State University, U.S.A.; (2) USDA-ARS, North Carolina State University, U.S.A. Elevated ground-level ozone associated with climate change and the rapidly evolving rust races are major challenges to wheat production worldwide. Because of higher susceptibility, rust universal-susceptible varieties are used as susceptible hosts to maintain and increase inoculum for most of the rust races. These varieties could also be used to study the interactive effects of ozone on different rust races. A first step is to assess the O3 responses of these wheat varieties. Pseudotsuga menziesii and Armillaria solidipes are native forest host/pathogen species of the interior western USA that have co-evolved over millions of years. Armillaria root disease caused by A. solidipes is a major limitation for growth, survival, and economic/environmental benefits of P. menziesii. Historically, A. solidipes played a beneficial ecological role by preferentially attacking maladapted trees. However, under increasing climate change, more forest trees will likely become progressively maladapted, leading to increased damage from Armillaria root disease. We developed species- J. HANNA (1), M. Warwell (2), H. Maffei (3), M. Fairweather (4), J. Blodgett (5), P. Zambino (6), J. Worrall (7), K. Burns (8), J. Jacobs (9), S. Ashiglar (2), J. Lundquist (10), M. Kim (11), A. Ross-Davis (2), C. Hoffman (12), R. Mathiasen (12), R. Hofstetter (12), J. Shaw (13), E. Pitman (2), E. Nelson (12), G. McDonald (2), M. Cleary (14), S. Brar (15), B. Richardson (16), N. Klopfenstein (2) (1) USDA Forest Service - RMRS, Moscow, ID, U.S.A.; (2) USDA Forest Service, RMRS, Moscow, ID, U.S.A.; (3) USDA Forest Service, FHP Region 6, Bend, OR, U.S.A.; (4) USDA Forest Service, Region 5 Regional Office, Vallejo, CA, U.S.A.; (5) USDA Forest Service, FHP Region 2, Rapid City, SD, U.S.A.; (6) USDA Forest Service, FHP Region 1, Coeur d’Alene, ID, U.S.A.; (7) USDA Forest Service, FHP Region 2, Gunnison, CO, U.S.A.; (8) USDA Forest Service, FHP Region 2, Lakewood, CO, U.S.A.; (9) USDA Forest Service, FHP Region 3, Albuquerque, NM, U.S.A.; (10) USDA Forest Service, FHP Region 10/PNWRS, Anchorage, AK, U.S.A.; (11) Department of Forestry, Environment and Systems, Kookmin University, Seoul, South Korea; (12) School of Forestry, Northern Arizona University, Flagstaff, AZ, U.S.A.; (13) USDA Forest Service, Interior West Forest Inventory and Analysis, Ogden, UT, U.S.A.; (14) Swedish University of Agricultural Sciences, Uppsala, Sweden; (15) SCION/Massey University, New Zealand; (16) USDA Forest Service, RMRS, Provo, UT, U.S.A. Impacts of climate change on wheat powdery mildew epidemics in China Y. Zhou (1), X. Tang (1) ( ), g ( ) (1) Institute of Plant Protection, Chinese Academy of Agricultural Sciences, China Epidemics of wheat powdery mildew (Blumeria graminis f. sp. tritici) have been highly destructive to winter wheat in China. We analyzed trends in the regions of wheat powdery mildew epidemics in China from 1970 to 2012 and PA (percent area affected by wheat powdery mildew) in response to climate change. Using PA and temperature for pathogen oversummering and overwintering periods, a model was constructed and subsequently used to predict changes in PA for 15 Global Climate Models under low (B1), medium (A1B), and high (A2) climate emission scenarios for the 2020s, 2050s, and 2080s relative to the 1970–2009 period. Our results showed a significant increase in T (mean monthly air temperature) (P < 0.001), while SH (sunshine hours) and RH (relative humidity) decreased. Using the original value method, there was a significant positive relationship between T and PA (P < 0.05). Using the first-differences time-series value method, ΔT (first-differences in T) in oversummering and late spring periods were negative for ΔPA (first- differences in PA) while positive relationships were recorded in the rest of the periods. The results using the first-differences time-series value method S4.129 were more precise for disease epidemics. The results of model prediction indicate that the values for the percent area affected by powdery mildew will increase in future. These findings will aid in decision making for management strategies of wheat powdery mildew. Quantifying the potential effects of regional climate change on wheat leaf rust disease in the Grand-Duchy of Luxembourg M. EL JARROUDI (1), L. Kouadio (2), J. Junk (3), B. Tychon (1), P. Delfosse (3) (1) University of Liege, Belgium; (2) University of Southern Queensland, Australia; (3) Luxembourg Institute of Science & Technology, Luxembourg Distribution models of common bean dry root rot in Brazil: Current and predicted disease distribution in climate change scenarios R. Macedo (1), L. Abud (1), F. Yoshida (1), L. Sales (1), E. Barbosa (2), M. Lobo (2), R. Macedo (1) (1) Universidade Federal de Goiás, Brazil; (2) Brazilian Corporation for Agricultural Research (Embrapa), Brazil circinatum biology is needed to inform predictions of which pathogen variants would likely cause outbreaks in future climate settings. We evaluated growth, spore production and germination among F. circinatum isolates cultured at various temperatures (25, 28, and 31°C). We also implemented spore trapping experiments in three north- Florida sites to survey spore release throughout the spring and summer, and to test if weather events trigger increased spore discharge. Results show significant temperature effects (p<0.05) in mycelium growth and sporulation among isolates. Though spore trapping experiments need to be followed across several growth seasons, we present preliminary trends on spore release in the environment. These results will constitute the baseline for future research on fungal diversity, spatial distribution patterns and disease prediction, aimed at mitigating adverse effects of climate change on forest health. Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior Quantifying the potential effects of regional climate change on wheat leaf rust disease in the Grand-Duchy of Luxembourg M. EL JARROUDI (1), L. Kouadio (2), J. Junk (3), B. Tychon (1), P. Delfosse (3) (1) University of Liege, Belgium; (2) University of Southern Queensland, Australia; (3) Luxembourg Institute of Science & Technology In this research, we screened the wheat lines Chinese Spring, CSA, Line E, Little Club, LMPG-6, McNair 701, Morocco, Rusty and Thatcher for O3 tolerance at four O3 concentrations. Results showed that Thatcher is the most sensitive and Chinese Spring is the most tolerant among the nine lines tested. For future studies of the effects of ozone on different stem rust races, LMPG-6, Rusty and McNair 701 could be used as O3- sensitive if compared to the more ozone-tolerant CSA or Chinese Spring. Similarly, Thatcher is a suitable O3-sensitive line that could be used with either Little Club or Morocco for studying O3 effects on different races of leaf rust. Identification of lines having differential ozone response will enable further genetic studies on ozone tolerance. Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior Resende (3) (1) UFLA, Brazil; (2) Lavras Federal University, UFLA, Brazil; (3) Lavras Federal University, UFLA, Brazil The brown eye spot of coffee (Cercospora coffeicola), reduces both productivity and quality of the beverage. Thus, the objective was to evaluate the spatio-temporal disease progress in coffee farm irrigated by center pivot. The pivot area, located in the southern state of Minas Gerais in Brazil, was georeferenced with 50 points in 17 ha to proceed the geostatistical analysis. The disease incidence was evaluated in 60 leaves, in five plants/point at 60- days intervals, from August 2012 to December 2014, totaling 15 evaluations. Climatic variables were obtained by weather station installed in the area and the data were correlated with disease incidence. The production of the years 2013 and 2014 were correlated with the area under the incidence progress curve (AUIPC). The disease progress curves were plotted and the adjustment of semivariogram models was done. Then, the data were interpolated by ordinary kriging, and the maps of disease were constructed (ArcGIS 9.2®). The incidence reaching in August 2013 and 2014, 23 and 21%, respectively. The exponential model was adjusted to the semivariogram. In over 50% of the area, the intensity was high, exceeding 20%, with spatial gradient. There was negative correlation between incidence of the disease with minimum and medium temperatures and AUIPC and productivity in 2014. Effect of pre-anthesis rainfall patterns on fusarium head blight and deoxynivalenol in wheat: A multi-state study W. BUCKER MORAES (1), K. F. Andersen (1), C. Cowger (2), R. Dill-Macky (3), L. V. Madden (1), P. Anderson Paul (1) (1) Department of Plant Pathology, The Ohio State University, Wooster, OH, U.S.A.; (2) Department of Plant Pathology, North Carolina State University, Raleigh, NC, U.S.A.; (3) Department of Plant Pathology, University of Minnesota, Saint Paul, MN, U.S.A. Although there is often a correlation between visual symptoms of Fusarium head blight (FHB) and deoxynivalenol (DON) in wheat, under certain conditions, DON levels may be higher than one would expect based on visual symptoms. Between 2012 and 2015, field experiments were conducted at three locations (Minnesota, North Carolina, and Ohio), representing different wheat growing regions and market classes, to investigate the effects of intermittent rainfall during the 7-8-day pre-anthesis window on FHB and DON in wheat. Plots were inoculated with corn kernels colonized by Fusarium graminearum at jointing (Feekes GS 6). Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior solidipes and P. menziesii are highly correlated, and the predicted suitable climate space moves dramatically northward for year 2070 under the RCP8.5 scenario. In many areas of the inland west, A. solidipes is predicted to persist under climate change, and it will likely cause major damage where maladapted forest trees remain. distribution models using Maximum Entropy (MaxEnt) to predict area suitable for A. solidipes and P. menziesii under changing climatic conditions. We compared contemporary (1950-2000) predicted suitable climate for A. solidipes and P. menziesii versus year 2070 (2061-2080) predictions using the “business as usual” greenhouse gas-emissions pathway 8.5 (RCP8.5) paired with the HadGEM2-ES global circulation model. Our predictions of suitable climate for A. solidipes and P. menziesii are highly correlated, and the predicted suitable climate space moves dramatically northward for year 2070 under the RCP8.5 scenario. In many areas of the inland west, A. solidipes is predicted to persist under climate change, and it will likely cause major damage where maladapted forest trees remain. Effects of vernalization on the differential ozone response of two winter wheat genotypes A. MASHAHEET (1), D. Marshall (2), K. Burkey (2) (1) North Carolina State University, U.S.A.; (2) USDA-ARS, North Carolina State University, U.S.A Effects of vernalization on the differential ozone response of two winter wheat genotypes A. MASHAHEET (1), D. Marshall (2), K. Burkey (2) Elevated ground-level ozone, associated with climate change is a limiting factor to wheat production. Evaluating winter wheat for ozone responses could be accomplished more rapidly at the seedling stage using unvernalized seedlings. However, there is no information available on the effects of vernalization on the expression of ozone injury responses, and the association of these responses to yield losses at adult plant stage. Twenty five winter wheat genotypes (including historical and recent varieties and breeding lines) were evaluated for ozone tolerance (at CF, 50, 70, 90 and 110 ppbv, 8hrs/day for 1 week), and two lines with differential ozone response were selected, Coker-9553 (O3-sensitive) and MD01W28-08-11 (O3-tolerant). These two genotypes were used to study the effects of vernalization on the differential ozone response, and the validity of results if unvernalized seedlings are used. Foliar Injury results at the seedling stage showed that vernalization has no significant effect on the differential ozone responses, and the two genotypes could be separated using vernalized or non-vernalized plant material. The differential response obtained at the seedling stage was validated at the adult-plant stage. Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior Yield losses under elevated ozone treatments in open-top chambers were associated with increased foliar injury observed at the seedling stage. Screening the monosomic lines of the Chinese Spring wheat variety for ozone tolerance A. MASHAHEET (1), D. Marshall (2), K. Burkey (2), R. Ullah (1), A. Abdelrhim (3) 1) North Carolina State University, U.S.A.; (2) USDA-ARS, North Carolina State University, U.S. Screening the monosomic lines of the Chinese Spring wheat variety for ozone tolerance A. MASHAHEET (1), D. Marshall (2), K. Burkey (2), R. Ullah (1), A. Abdelrhim (3) (1) North Carolina State University, U.S.A.; (2) USDA-ARS, North Carolina State University, U.S.A.; (3) North Carolina State University, U.S.A. Screening the monosomic lines of the Chinese Spring wheat variety for ozone tolerance A. MASHAHEET (1), D. Marshall (2), K. Burkey (2), R. Ullah (1), A. Abdelrhim (3) (1) North Carolina State University, U.S.A.; (2) USDA-ARS, North Carolina State University, U.S.A.; (3) North Carolina State University, U.S.A. versity, U.S.A.; (2) USDA-ARS, North Carolina State University, U.S.A.; (3) North Carolina State University, U.S.A. Elevated tropospheric ozone is currently a major challenge to wheat production worldwide and is anticipated to be more challenging in the future. Breeding wheat for ozone tolerance is needed to maintain or increase wheat yield. However, there is limited information on the genetic control of ozone tolerance in wheat. The current understanding is that genome A and genome B are responsible for tolerance, whereas genome D is responsible for ozone sensitivity of modern wheat. In this study, we investigated the contribution of each of the 21 chromosomes of the Chinese Spring variety towards its ozone tolerance. We screened the 21 monosomic lines of Chinese Spring, at four ozone concentrations (CF, 50, 70 and 90 ppbv, 12 hr daily average, for 2 weeks). Ozone-induced foliar injury results showed that the absence of chromosome 7A reduces ozone tolerance. In contrast, the absence of chromosome 4A increases tolerance. None of the chromosomes of the genome D were found to individually reduce ozone tolerance. Future studies are needed to identify the genetic basis of ozone tolerance and the associated DNA markers to facilitate the incorporation of ozone tolerance into wheat breeding programs. Spatio-temporal brown eye spot progress of irrigated coffee by central pivot M. SILVA (1), M. Silva (2), E. Pozza (2), E. Chaves (3), G. Vasco (2), P. Paula (3), G. Dornelas (3), M. Silva (3), M. Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing cli tributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interio J. HANNA (1), M. Warwell (2), H. Maffei (3), M. Fairweather (4), J. Blodgett (5), P. Zambino (6), J. Worrall (7), K. Burns (8), J. Jacobs (9), S. Ashiglar (2), J. Lundquist (10), M. Kim (11), A. Ross-Davis (2), C. Hoffman (12), R. Mathiasen (12), R. Hofstetter (12), J. Shaw (13), E. Pitman (2), E. Nelson (12), G. McDonald (2), M. Cleary (14), S. Brar (15), B. Richardson (16), N. Klopfenstein (2) (1) USDA Forest Service - RMRS, Moscow, ID, U.S.A.; (2) USDA Forest Service, RMRS, Moscow, ID, U.S.A.; (3) USDA Forest Service, FHP Region 6, Bend, OR, U.S.A.; (4) USDA Forest Service, Region 5 Regional Office, Vallejo, CA, U.S.A.; (5) USDA Forest Service, FHP Region 2, Rapid City, SD, U.S.A.; (6) USDA Forest Service, FHP Region 1, Coeur d’Alene, ID, U.S.A.; (7) USDA Forest Service, FHP Region 2, Gunnison, CO, U.S.A.; (8) USDA Forest Service, FHP Region 2, Lakewood, CO, U.S.A.; (9) USDA Forest Service, FHP Region 3, Albuquerque, NM, U.S.A.; (10) USDA Forest Service, FHP Region 10/PNWRS, Anchorage, AK, U.S.A.; (11) Department of Forestry, Environment and Systems, Kookmin University, Seoul, South Korea; (12) School of Forestry, Northern Arizona University, Flagstaff, AZ, U.S.A.; (13) USDA Forest Service, Interior West Forest Inventory and Analysis, Ogden, UT, U.S.A.; (14) Swedish University of Agricultural Sciences, Uppsala, Sweden; (15) SCION/Massey University, New Zealand; (16) USDA Forest Service, RMRS, Provo, UT, U.S.A. Pseudotsuga menziesii and Armillaria solidipes are native forest host/pathogen species of the interior western USA that have co-evolved over millions of years. Armillaria root disease caused by A. solidipes is a major limitation for growth, survival, and economic/environmental benefits of P. menziesii. Historically, A. solidipes played a beneficial ecological role by preferentially attacking maladapted trees. However, under increasing climate change, more forest trees will likely become progressively maladapted, leading to increased damage from Armillaria root disease. We developed species- S4.130 distribution models using Maximum Entropy (MaxEnt) to predict area suitable for A. solidipes and P. menziesii under changing climatic conditions. We compared contemporary (1950-2000) predicted suitable climate for A. solidipes and P. menziesii versus year 2070 (2061-2080) predictions using the “business as usual” greenhouse gas-emissions pathway 8.5 (RCP8.5) paired with the HadGEM2-ES global circulation model. Our predictions of suitable climate for A. Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior In this study, airborne spores were trapped on the tape with a Burkard sporewatch spore samplers mounted in a rice field in Panjin, Liaoning province, China from July 1 to October 13, 2015. The spores were washed off the tape and followed by DNA extraction by using Mobio Power Soil DNA Isolation Kit, and quantified by TaqMan Real-time PCR. Specific primers and probe were designed to target the 28S rDNA of Magnaporthe oryzae and reaction system was optimized. qPCR results of field samples revealed two sporepeaks during the study period. They were 9.46 spores /m3 from July 18 to 20 and 2.95 spores /m3 from August 15 to 17, which were followed by a high incidence of leaf blast on August 4 and first observation of neck blast on August 22. This suggested that quantity of airborne spores of Magnaporthe oryzae could be useful in disease forecasting of rice blast. The qPCR system combined with spore samplers provided an efficient way to quantify airborne pathogen for disease forecasting and guiding disease management of rice blast. The seasonal dynamics of Phyllosticta spp. in Floridian citrus grove leaf litter K. ZHANG (1), J. Rollins (2), M. Dewdney (3) The seasonal dynamics of Phyllosticta spp. in Floridian citrus grove leaf litter K. ZHANG (1), J. Rollins (2), M. Dewdney (3) (1) Plant Pathology Department, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, U.S.A.; (2) Plant Pathology Department, University of Florida, Gainesville, FL, U.S.A.; (3) Plant Pathology Department, Citrus Research and Education Center. University of Florida, Lake Alfred, FL, U.S.A. ( ), ( ), y ( ) (1) Plant Pathology Department, Citrus Research and Education Center, University of Florida, Lake Alfred, FL, U.S.A.; (2) Plant Pathology Department, University of Florida, Gainesville, FL, U.S.A.; (3) Plant Pathology Department, Citrus Research and Education Center. University of Florida, Lake Alfred, FL, U.S.A. The causal agent of Citrus Black Spot (CBS), Phyllosticta citricarpa, is a restricted pathogen in Europe and quarantined in the US after it was found in Florida in 2010. Additionally, an endophytic fungus P. capitalensis, associated with a wide range of hosts including citrus, is globally distributed and commonly found in Floridian citrus. These two species cannot be distinguished morphologically; therefore, previous CBS epidemiology studies included both endophyte and pathogen. Both fungi overwinter and produce ascospores and conidia in leaf litter. Field observations of ascospore discharge of Monilinia vaccinii-corymbosi in northern highbush blueberries G. Dabbah (1), A. Schilder (2) Field observations of ascospore discharge of Monilinia vaccinii-corymbosi in northern highbush blueberries G D bb h (1) A S hild (2) Field observations of ascospore discharge of Monilinia vaccinii-corymbosi in northern highbush blueberries G. Dabbah (1), A. Schilder (2) ( ), ( ) (1) CSU Monterey Bay, U.S.A.; (2) Michigan State University, Department of Plant, Soil and Microbial Sciences, U.S.A. Monilinia vaccinii-corymbosi (Mvc), is the causal agent of a disease known as mummy berry in highbush blueberries (Vaccinium corymbosum L.). The primary inoculum of Mvc is discharged from apothecia grown from mummified blueberry fruits that fall to the ground in previous seasons. In order to better predict the release of primary inoculum a study of ascospore discharge was conducted using aerial spore trapping and several weather variables. A Burkard spore trap was used to collect ascospores within infested blueberry fields in 2002, 2003, 2004, and 2009. A previously developed degree-day model was also assessed in order to compare the amount of chill-hours and degree-days required for southwest Michigan grown blueberries. Peak ascospore discharge was inversely related to daily relative humidity and the majority of ascospores were discharged between 67-107 growing degree- days and after 3000 chill-hours were attained. Additionally, peak ascospore discharge was reached when apothecial cup size was between 2-7mm in diameter which correlated with previous laboratory studies. The information in the study may be used to better understand the conditions needed to predict mummy berry apothecia emergence and ascospore discharge with the aim of improving future disease management practices. A leaf litter and fruit brown rot life style of Phytophthora syringae in California citrus W. HAO (1), H. Förster (1), T. Miles (2), F. Martin (3), G. Browne (4), J. Adaskaveg (1) (1) University of California, Riverside, Riverside, CA, U.S.A.; (2) California State University, Monterey Bay, Seaside, CA, U.S.A.; (3) USDA-ARS, Salinas, CA, U.S.A.; (4) University of California, Davis and USDA-ARS, Davis, CA, U.S.A. A leaf litter and fruit brown rot life style of Phytophthora syringae in California citrus W. HAO (1), H. Förster (1), T. Miles (2), F. Martin (3), G. Browne (4), J. Adaskaveg (1) (1) University of California, Riverside, Riverside, CA, U.S.A.; (2) California State University, Monterey Bay, Seaside, CA, U.S.A.; (3) USDA-ARS, Salinas, CA, U.S.A.; (4) University of California, Davis and USDA-ARS, Davis, CA, U.S.A. Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior In contrast, the high prevalence of avirulence genes Avrlm4, AvrLm6, AvrLm7, and AvrLm11 indicates their interacting resistance genes could be safely used. protection. In contrast, the high prevalence of avirulence genes Avrlm4, AvrLm6, AvrLm7, and AvrLm11 indicates their interacting resistance genes could be safely used. Transmission of Rhodococcus spp. causing pistachio bushy top syndrome in California E. FICHTNER (1), S. Dhaouadi (1), T. Kapaun (2), J. Cardwell (3) (1) University of California Division of Agriculture and Natural Resources, Tulare, CA, U.S.A.; (2) University of California Division of Agriculture and Natural Resources, Exeter, CA, U.S.A.; (3) Companion Animal Clinic, Visalia, CA, U.S.A. Pistachio bushy top syndrome (PBTS) is a new pistachio disease caused by two Rhodococcus spp., highly related to Rhodococcus fascians (Rf) and Rhodococcus corynebacteriodes (Rc). Determination of potential modes of pathogen transmission is necessary for protection of replants and asymptomatic trees in orchards, as well as prevention of similar future epidemics. The objectives of this study were to assess the potential for PBTS isolates to be transmitted to healthy plants via naturally-infested soil at replant sites, using pruning tools, or by rodents. Clonal UCB-1 (Pistacia atlantica x Pistacia integerrima) plants were planted in naturally-infested soil; positive and negative control treatments were established by placing root- inoculated and uninoculated plants in steam-disinfested soil. Pruning transmission studies were conducted by cutting into symptomatic tissue in advance of cutting healthy plants; positive and negative control treatments were established by artificial infestation and flame sterilization of pruners, respectively. Mice trapped in the greenhouse were evaluated for internal and external populations of Rf and Rc. The results demonstrate that naturally infested soil and infested pruning tools may transmit the bacteria to healthy plants. Mice were also found to carry Rhodococcus sp. on fur and in their digestive tract. The results underscore the need for vigilant sanitation, both in the orchard and in nursery propagation systems. Quantification of airborne inoculums of Magnaporthe oryzae by using TaqMan Real-time PCR X. CHEN (1), K. Yuan (1), S. Wang (2), F. Guo (2), B. Wu (2) (1) China Agricultural University, China; (2) China Agricultural University, China Rice blast caused by Magnaporthe oryzae is one of the most devastating diseases in China and worldwide. Accurate and timely quantification of airborne spores of the pathogen is important to disease forecast and management of the disease. Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior The experimental design was a randomized complete block with five pre-anthesis simulated rainfall treatments: 1) rainfall every day (Rain_1); 2) rainfall only on the first and last two days of the window (Rain_2); 3) rainfall only on the middle 3-4 days of the window (Rain_3); 4) rainfall every other day (Rain_4); and 5) check. Plots that received simulated rain had numerically higher mean FHB incidence (INC), index (IND), Fusarium damaged kernels (FDK), and DON than the check at all locations. Rain_1 consistently resulted in higher mean INC and IND than Rain_2, Rain_3 and Rain_4. However, Rain_2 and Rain_4 had comparable mean FDK and DON to Rain_1. Thus, dry-wet moisture cycles may be associated with disproportionately high DON for a given level of IND. Changes in frequency of Leptosphaeria maculans avirulence genes in North Dakota S. Mansouripour (1), K. Chittem (1), Z. Liu (1), L. del Rio Mendoza (1) (1) North Dakota State University, U.S.A. Blackleg, caused by Leptosphaeria maculans is resurging as a threat to North Dakota canola industry. A shift in the virulence profile of L. maculans populations is thought to be responsible for it. Estimating the prevalence of specific avirulence genes in L. maculans populations could be used to infer the relative value of its interacting resistance genes for a plant breeding program. The objective of this study was to determine the frequency of avirulence genes in L. maculans isolates from North Dakota. Single-spore cultures of L. maculans isolates collected from commercial grower’s fields in 2004, 2007 and 2014 were used. Presence of avirulence genes AvrLm1, AvrLm6, AvrLm4-7 and AvrLm11 was determined using PCR assays. The results indicated that the frequency of AvrLm1 increased from 3% among isolates collected in 2004 to 45% among isolates collected in 2014 while the frequencies of AvrLm6 and AvrLm11 remained unchanged (91 to 100% and 91 to 95%, respectively) between the same two periods. AvrLm4 and AvrLm7 alleles were present in 90 and 100% of the isolates, respectively in 2014. Determination of frequencies of AvrLm4 and AvrLm7 in 2004 and 2007 is underway. The higher frequency of isolates carrying avrLm1 in 2014 suggests its corresponding resistance gene, Rlm1, can no longer provide effective S4.131 protection. In contrast, the high prevalence of avirulence genes Avrlm4, AvrLm6, AvrLm7, and AvrLm11 indicates their interacting resistance genes could be safely used protection. Potential species distributions of Armillaria solidipes and Pseudotsuga menziesii under contemporary and changing climates in the interior Understanding the seasonal dynamics of these two species in leaf litter can provide knowledge to control CBS inoculum in Floridian groves. 1000 leaves were collected biweekly from 40 arbitrarily selected trees in a grove in south Florida since May 2014. The number of leaves with fructifications, the number of Phyllosticta spores collected from the leaf litter, and the average leaf degradation level from each sample were recorded. The quantity of P. citricarpa and P. capitalensis DNA in leaf litter and spore suspensions was determined via qPCR. The results showed that the fructification abundance is correlated with leaf degradation level (pseudothecium: P = 0.0008, pycnidium: P = 0.0333), conidia were always present, and ascospores were occasionally absent. The quantity of P. capitalensis DNA was constant while the quantity of P. citricarpa DNA increased throughout the two-year sample collection period. Field observations of ascospore discharge of Monilinia vaccinii-corymbosi in northern highbush blueberries G. Dabbah (1), A. Schilder (2) In recent years, Phytophthora syringae has become a major pathogen causing brown rot of citrus fruit in California during the winter season. This is a serious concern for the citrus industry because P. syringae is a quarantine pathogen in some export countries. The source of P. syringae inoculum infecting citrus fruit has never been clearly established. Roots and rhizosphere soil from trees where brown rot was found to be caused by P. syringae were sampled in three orchards. Samples were plated onto a Phytophthora-selective medium and TaqMan qPCR and Droplet Digital PCR were performed. P. citrophthora and P. parasitica were consistently detected by isolation in roots and soil and by PCR in roots. P. syringae was not found causing root rot and was not detected in the soil using these methods. Leaf litter on the ground and soil were sampled from the same locations and immersed in water. Mature green pears were used as baits and incubated at 12°C for 1 to 2 wk. Brown lesions that developed on pears at the water line were plated onto selective medium. P. syringae was detected in most leaf litter samples but only in two soil samples. Thus, leaf litter is a major inoculum source for P. syringae and changes our understanding of the disease cycle of this important pathogen and separates it from other Phytophthora species that cause citrus root and brown rots. Recognition of the distinct epidemiology of the disease opens the opportunity for new cultural management practices. S4.132 Specific TaqMan qPCR and a PvABC qPCR assays for monitoring airborne sporangia of Plasmopara viticola f. sp. riparia, aestivalis and vinifera O. CARISSE (1), M. Tremblay (2), A. Lefebvre (3) (1) Agriculture and AgriFood Canada, Canada; (2) Agriculture and Agroalimentaire Canada, Canada; (3) Agriculture and AgriFood Canada, Canada d a PvABC qPCR assays for monitoring airborne sporangia of Plasmopara viticola f. sp. riparia, aestivalis and vinifera ay (2), A. Lefebvre (3) Grape downy mildew (Plasmopara viticola) is among the most important grapevine diseases in eastern Canada. Recently, five new cryptic formae speciales of P. viticola from wild and cultivated Vitis species were described. In Eastern Canada, only P. viticola f.sp. riparia (clade A) and P. viticola f.sp. aestivalis (clade B) have been detected. Field observations of ascospore discharge of Monilinia vaccinii-corymbosi in northern highbush blueberries G. Dabbah (1), A. Schilder (2) As part of a biovigilance network aiming at tracking new pathogens and new genotypes, molecular assays for simultaneous quantification of sporangia of clade A and B and detection of clade C (P. viticola f. sp. vinifera) from air samples were developed. The assays are a specific triplex TaqMan qPCR and a PvABC qPCR developed from sequence alignments of internal transcribed spacer 1 and 2 (ITS1-2) DNA regions for P. viticola clade A, B, C and other closely related species. These essays were used to study the temporal and spatial occurrence of these genotypes in two vineyards. At one site, almost exclusively (99%) clade A was found while at the other site it was mostly (83%) clade B. Hence the genotype A was found at both sites, however, it was observed for the first time in early July at one site and in early June at the other site. The impact of these genotypes on grape downy mildew development is under study. Using these new tools, it will be possible to acquire knowledge on aerobiology and spatio-temporal distribution of genotypes A and B and to monitor clade C not present in Eastern Canada, but detected in north-east of USA. First report of the Asian citrus psyllid, vector of Candidatus Liberibacter asiaticus in Tanzania and their potential further spread in Africa M. SHIMWELA (1), H. Narouei-Khandan (1), S. Halbert (2), M. Keremane (3), G. Minsavage (1), S. Timilsina (1), D. Massawe (4), J. Jones (1), A. van Bruggen (1) (1) U i i f Fl id G i ill FL U S A (2) Di i i f Pl I d G i ill FL U S A (3) USDA Ri id CA U S A (4) (1) University of Florida, Gainesville, FL, U.S.A.; (2) Division of Plant Industry, Gainesville, FL, U.S.A.; (3) USDA, Rivers Sokoine University of Agriculture, Morogoro, Tanzania (1) University of Florida, Gainesville, FL, U.S.A.; (2) Division of Plant Industry, Gainesville, FL, U.S.A.; (3) USDA, Riverside, CA, U.S.A.; (4) Sokoine University of Agriculture, Morogoro, Tanzania Citrus surveys were conducted at high (>700m), medium (300-600m) and low (<200m) altitudes in Tanzania. Adults and nymphs of Trioza erytreae were abundant in the highlands and less abundant at medium altitudes. Unexpectedly, adults and nymphs of Diaphorina citri, the Asian citrus psyllid, were found at medium altitudes, around Morogoro. No psyllids were observed at low altitudes. Field observations of ascospore discharge of Monilinia vaccinii-corymbosi in northern highbush blueberries G. Dabbah (1), A. Schilder (2) Severe huanglongbing symptoms and tree decline were evident at high altitudes, while mild symptoms were observed at intermediate and low altitudes. DNA was extracted from leaf and psyllid samples and subjected to conventional PCR with seven different primer sets and qPCR with two primer sets for Candidatus Liberibacter spp. PCR products were sequenced and subjected to phylogenetic analysis. C. Liberibacter africanus (Laf) was detected in leaf and T. erytreae samples from high and medium altitudes by all methods. Sequences of C. Liberibacter DNA from leaves and psyllids were similar to those of Laf from South Africa. Even though Las primers gave a positive reaction in qPCR with medium altitude leaf samples, cPCR and sequencing results were negative for Las, suggesting cross reactivity of Las primers with Laf. Neither Laf nor Las were detected in D. citri samples. This is the first report of D. citri in Africa. Further surveys are needed to detect Las. The potential distribution of D. citri and Las in Africa was predicted using the correlative models MAXENT and Multi-Model Framework. Genetic diversity of pathogenic Clavibacter michiganensis subsp. michiganensis strains from Central Chile M. VALENZUELA (1), S. Fuentes (2), F. Claverias (2), X. Besoain (3), M. Seeger (4) Genetic diversity of pathogenic Clavibacter michiganensis subsp. michiganensis strains from Central Chile M. VALENZUELA (1), S. Fuentes (2), F. Claverias (2), X. Besoain (3), M. Seeger (4) (1) C d i l í A i id d i d i S i l i Chil (2) C d i l í A Genetic diversity of pathogenic Clavibacter michiganensis subsp. michiganensis strains from Central Chile M. VALENZUELA (1), S. Fuentes (2), F. Claverias (2), X. Besoain (3), M. Seeger (4) (1) Centro de Biotecnología DAL, Universidad Tecnica Federico Santa Maria, Valparaiso, Chile; (2) Centro de Biotecnología DAL, Universidad T (1) Centro de Biotecnología DAL, Universidad Tecnica Federico Santa Maria, Valparaiso, Chile; (2) Centro de Biotecnología DAL, Universidad Técnica Federico Santa María, Valparaíso, Chile; (3) Escuela de Agronomía, Pontificia Universidad Católica de Valparaíso, Valparaíso, Chile; (4) Laboratorio de Microbiología Molecular y Biotecnología Ambiental, Universidad Técnica Federico Santa María, Valparaíso, Chile The bacterial canker of tomato caused by Clavibacter michiganensis subsp. michiganensis (Cmm) is the most important bacterial disease in tomato. The genetic diversity of Cmm strains isolated during 20 years from tomato plants in different Chilean tomato regions was studied. Spatiotemporal analysis of wheat blast epidemiology (Magnaporthe oryzae Triticum pathotype) under natural field conditions K. MILLS (1), L. Madden (1), P. Paul (1), D. Salgado (1) (1) The Ohio State University, U.S.A. Spatiotemporal analysis of wheat blast epidemiology (Magnaporthe oryzae Triticum pathotype) under natural field conditions K. MILLS (1), L. Madden (1), P. Paul (1), D. Salgado (1) (1) The Ohio State University, U.S.A. Wheat blast is an emerging fungal disease of wheat caused by the Triticum pathotype of Magnaporthe oryzae. In years when weather is conducive for disease development, growers can suffer total crop losses. In order to understand the epidemiology of wheat blast, it is important to characterize the pattern of disease spread within a field. This pattern provides clues about the characteristics of the pathogen that affect the spatiotemporal dynamics of the disease. In this study, inoculum build-up and temporal and spatial spread from naturally occurring in-field disease foci (hotspots) were investigated in six 27 × 27 m2 plots at three locations in Bolivia during the 2015 growing season. Plots were assessed for spike blast incidence and severity every 3 days. Assessments were made on a grid, with twenty spikes rated every 2.7 m. Hotspots increased in size over time and were associated with the highest disease ratings. Initially, plants adjacent to the hotspots had higher disease levels than plants located farther from the hotspots. As the disease progressed the steepness of the disease gradient decreased away from hotspots, and high levels of incidence and severity were observed throughout the plots. This trend was observed across the three locations. Preliminary spatial analyses suggest that hotspots are important in-field sources of inoculum. Field observations of ascospore discharge of Monilinia vaccinii-corymbosi in northern highbush blueberries G. Dabbah (1), A. Schilder (2) Twenty five isolates were characterized by growth on different media, Gram staining and enzymatic activities. For bacterial identification PSA8/R PCR amplification and 16S rRNA gene sequencing were used. In addition, the housekeeping gyrB and kdpA genes and the pathogenicity tomA and chpC genes were sequenced. All genes were polymorphic in these isolates. The kdpA gene showed the highest variability. Except for the kdpA gene, Chilean isolates genes showed high identity with genes from reference strain NCPPB382. Phylogenetic trees of gyrB and pathogenicity genes showed close relationship between Chilean stains and those from New York and Serbia. Analyses of concatenated kdpA-gyrB genes yield 7 Chilean haplotypes. Geographical distribution of the isolates was analyzed. Most of the Chilean isolates clustered separately from the Serbian and New Yorker strains, suggesting that Chilean bacteria have a different origin. Additional housekeeping genes and a higher number of isolates will be studied. A Snapshot of Citrus Black Spot in Southwest Florida: Spatial Distribution in Two ‘Valencia’ Citrus Groves K. HENDRICKS (1), P. Roberts (1) (1) University of Florida - IFAS A Snapshot of Citrus Black Spot in Southwest Florida: Spatial Distribution in Two ‘Valencia’ Citrus Groves K. HENDRICKS (1), P. Roberts (1) 1) University of Florida - IFAS Citrus black spot (CBS) an emerging disease of citrus in Florida caused by the fungus Guignardia citricarpa Kiely (anamorph Phyllosticta citricarpa (McAlpine) van der Aa) was identified in March 2010. The source of the introduction into the United States is still unknown. Disease inoculum, conidia and ascospores are dispersed by water and wind respectively. Due to quarantine restrictions and the potential for severe climatic events – hurricanes, it is imperative that the spatial distribution of the disease be monitored within the state and within infected groves. This study investigates the spatial distribution in two commercial ‘Valencia’ citrus groves located within the quarantine zones mandated by the USDA. Trees were mapped for the presence or absence of CBS in groves. Positive trees were defined as having fruit with at least a single hard or shot-hold spot lesion. The hard or shot-hole spot lesion was chosen due to its ease of recognition as a symptom of CBS. Preliminary spatial analysis of disease intensity and clustering were analyzed in R using the binomial dispersion index (D), and Ripley’s K function (K). The incidence of CBS in Grove 1 and 2 were 7.1% and 2.6% respectively in the 2013-2014 citrus season. The binomial dispersion index (D), revealed clustering of symptomatic trees for both groves in all quadrant sizes tested. Monte Carlo simulations for the L function confirmed CBS aggregation in both groves. Monitoring is ongoing. S4.133 Sensitivity survey of Alternaria species to SDHI fungicides reveals regional and temporal resistance changes in pistachio orchards of California P. LICHTEMBERG (1), R. Puckett (1), C. Cunningham (2), V. Leschenne (3), T. Michailides (1) (1) University of California - Davis, U.S.A.; (2) Fresno State University, U.S.A.; (3) Swiss Federal Institute of Technology, Switzerland In California, resistance of Alternaria spp. to succinate dehydrogenase inhibitor (SDHI) fungicides is common in pistachio orchards and results in difficulties in managing Alternaria late blight (ALB). The objective of this study was to monitor the development and spread of SDHI resistant Alternaria phenotypes and evaluate the cross-resistance among fluopyram (Fp), penthiopyrad (Pe), fluxapyroxad (Fd) and boscalid (Bo). In total, 798 isolates of Alternaria spp. were collected from 35 pistachio orchards where SDHI were used. A Snapshot of Citrus Black Spot in Southwest Florida: Spatial Distribution in Two ‘Valencia’ Citrus Groves K. HENDRICKS (1), P. Roberts (1) (1) University of Florida - IFAS Tomato chlorotic spot virus (TCSV), Groundnut ringspot virus (GRSV) and Tomato spotted wilt virus (TSWV), are three tospoviruses currently known to infect tomato in this area. These tospoviruses can cause losses in tomato ranging from a reduction in fruit quality to a complete destruction of the crop. TCSV was first detected from tomato plants in Homestead, FL in 2012, and it has caused significant losses to commercial tomato growers since the fall of 2014. During 2015-2016 growing season, a survey on tospoviruses infecting tomato in Homestead, Florida was conducted to determine the disease incidence and distribution of the tospoviruses in commercial tomato production fields. The percentage of tomato plants showing tospovirus-like symptoms in Homestead tomato fields ranged from 4 - 80% with an average of 35%. Two hundreds and seventy two tomato samples showing the symptoms were collected from over forty tomato fields. RNAs were extracted from the samples and tested by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) using specific primers against TCSV, GRSV and TSWV to determine the incidence of both single and mixed infection of these viruses. RT-PCR results confirmed the infection of tomato plants with tospoviruses in Homestead. The results of the RT-PCR tests will be presented and more details will be discussed. Spatial dynamics of a begomovirus disease in processing tomato in central Brazil A. BERGAMIN FILHO (1), A. Bergamin Filho (1) (1) University of Sao Paulo, Brazil The begomovirus disease is one of the most important viral diseases in the tomato crop in Brazil. The objective of this work was to study the spatial aggregation of begomovirus disease in two central-pivot irrigated areas for processing tomatoes in central Brazil. A total of 24 plots (15 × 15 plants) was weekly evaluated by visual inspection for identification of begomovirus symptomatic plants (BSP). The spatial distribution of the disease was analyzed by: binominal index of dispersion, Taylor’s power law, and ordinary run. Tomato severe rugose virus was the predominant begomovirus species in tomato. It was also detected in a few non-tomato plants. The apatial pattern of sumptomatic plants was slightly aggregated. A relevant difference was observed between plots located at the center (PC) and at the edge (PE) of the pivots. The distribution of BSP in PC was more aggregated than in PE. It is most likely that at PE the primary spread is the major component of virus epidemics in the field. A Snapshot of Citrus Black Spot in Southwest Florida: Spatial Distribution in Two ‘Valencia’ Citrus Groves K. HENDRICKS (1), P. Roberts (1) (1) University of Florida - IFAS Classification and regression tree analysis indicated that host species was the most important segregating factor based on severity, were E. tereticornis plantations showed a significantly lower severity than E. camaldulensis and their hybrids. KLB is widespread and serious on susceptible Eucalyptus spp., posing a threat to commercial plantation forestry in Uruguay. Future studies will concentrate on understanding the genetic diversity of a large collection of isolates that have emerged from this study and on selecting planting stock resistant to KLB. The occurrence pattern of plant viruses and their vectors in pepper fields in Korea S. KWON (1), S. Kwon (1), I. Cho (1), S. Choi (1), J. Yoon (1), G. Choi (1) (1) National Institute of Horticultural and Herbal Science, South Korea Pepper viruses usually cause severe symptoms on pepper resulting in a significant loss of the yield. While many viruses have been reported to infect pepper, some of them, including Cucumber mosaic virus (CMV), Broad bean wilt virus 2 (BBWV2), Pepper mottle virus (PepMoV), Pepper mild mottle virus, Tomato spotted wilt virus (TSWV), Beet western yellows virus (BWYV), and Potato virus Y, have already caused epidemics in pepper in Korea. In this study, we performed nationwide surveys from May to October 2015 to examine the occurrence of pepper viruses. Interestingly, the incidence of TSWV was highest in May but decreased dramatically month by month, whereas the occurrence of BWYV and PepMoV was detected from July and increased gradually until October. In addition, CMV and BBWV2 are the most prevalent in July and August and their incidences were maintained at high levels until October. In our surveys, we also investigated the incidences of thrips and aphids. Interestingly, the rate of population of thrips was greatest in May but decreased dramatically from July. On the other hand, the population of aphids gradually increased from May to August and the rate of alate aphids in the population was increased in September in pepper fields. Taken together, our survey results on the occurrence of viruses and their insect vectors suggested that the incidence of the pepper viruses is highly correlated with the population dynamic and life-cycle of their insect vectors. Field Survey of Tospoviruses Infecting Tomato in South Florida O. Abdalla (1), S. Zhang (1) (1) University of Florida, U.S.A. Several viruses are known to infect tomato plants in south Florida, leading to serious losses to growers. A Snapshot of Citrus Black Spot in Southwest Florida: Spatial Distribution in Two ‘Valencia’ Citrus Groves K. HENDRICKS (1), P. Roberts (1) (1) University of Florida - IFAS In vitro assays were used to identify the frequency of SDHI resistant phenotypes and to compare the sensitivity distribution of isolates from previous years. The results revealed 85.8, 83, and 80% phenotypes in the current population resistant to Bo, Fd, and Pe, respectively. The Fp showed the smallest EC50 value 16.74 µg/ml and the greatest frequency of sensitive phenotypes, at 45.8%. For the first time, highly resistant phenotypes for Fp were observed in California pistachio orchards, showing a moderate cross- resistance with Bo, Fd and Pe. Finally, the current population of Alternaria isolates (2015) showed sensitivity shifts toward resistance for Fd and Fp in comparison to those of the 2010 survey. Regional and temporal changes in the resistance of Alternaria spp. population will be discussed as well as the impact of future studies in order to delay SDHI resistance in California. Teratosphaeria pseudoeucalypti: An emerging pathogen with uncertain impact on Eucalyptus plantations C. PEREZ (1), G. Balmelli (2), S. Simeto (2), E. a (3), M. Codina (3), R. Garcia (3), N. Ramirez (3), O. Bentancur (3), M. Wingfield (4) (1) EEMAC, Universidad de la Republica, Paysandu, Uruguay; (2) INIA Tacuarembo, Uruguay; (3) EEMAC, Universidad de la Republica, Uruguay; (4) FABI, University of Pretoria, Uruguay Teratosphaeria pseudoeucalypti: An emerging pathogen with uncertain impact on Eucalyptus plantations C. PEREZ (1), G. Balmelli (2), S. Simeto (2), E. a (3), M. Codina (3), R. Garcia (3), N. Ramirez (3), O. Bentancur (3), M. Wingfield (4) (1) EEMAC, Universidad de la Republica, Paysandu, Uruguay; (2) INIA Tacuarembo, Uruguay; (3) EEMAC, Universidad de la Republica, Uruguay; (4) FABI, University of Pretoria, Uruguay Teratosphaeria pseudoeucalypti was first discovered in Queensland, Australia in 2010 causing a serious leaf blight disease known as Kirramyces Leaf Blight (KLB) on Eucalyptus commercial plantations. The pathogen was subsequently reported in 2014 in Argentina, Brazil and Uruguay, representing the first occurrence outside Australia. During 2015, a survey was conducted to assess prevalence, incidence and severity of KLB on Eucalyptus camaldulensis and E. tereticornis in Uruguay. A total of 80 plantations were visited, including these species and their hybrids. In each plantation, one transect of 20 trees in a row was scored for crown damage, based on percentage of defoliation and overall disease severity. The disease was found in all regions of the country. There was no clear pattern of distribution or severity of the disease with a distribution appearing to be generalized. Composition of leaf fibrous ring mediates Diaphorina citri preference of Citrus sinensis over Citrus aurantium H. SHUGART (1), M. Rogers (1) (1) University of Florida- Citrus Research & Education Center, U.S.A. 6SrIII-L phytoplasma by leafhoppers (Scaphytopius marginelineatus) to cassava (Manihot esculenta Crantz) in Colom BETANCOURTH (1) As much as 30% of cassava production in Colombia, is affected by cassava frogskin disease (CFSD). The disease is associated with a 16SrIII-L phytoplasma that is transmitted between plants through vegetative propagation or by sap-sucking insect vectors. We collected 250 insect samples in Palmira and Santander de Quilichao; analyzed them for the presence of phytoplasmas during 2009 and 2012–2014; and detected a 16SrIII-L phytoplasma. Greenhouse experiments with symptomatic plants showed that potential insect vectors for cassava were hemipterans, particularly the Cicadellidae and Cixiidae families, and include the cicadellid leafhopper Scaphytopius marginelineatus. Colonies of this leafhopper were mass-reared on cassava (‘SM 909-25’) and bean (‘ICA Pijao’) plants. To determine the acquisition period, 50 individual insects were selected and exposed to phytoplasma-infected cassava plants for 2, 4, 6, and 8 days. The insects then underwent an additional 2 weeks’ incubation period, after which surviving insects were allowed to infest healthy cassava plants over a 5-to-7-day inoculation period. Molecular studies of plant tissues, based on PCR amplification and DNA sequencing, confirmed both the acquisition (6 days after inoculation) and transmission (3 weeks) of a phytoplasma. Sequences KP75989, and KP759898-KP759902 were reported to GenBank. Previous studies indicated that potential infestations of cassava crops by leafhoppers can be controlled by organic neem oil spray. Cladosporium interactions with the introduced insect spotted wing drosophila (Drosophila suzukii) may alter raspberry fruit rot epidemiology C. SWETT (1), C. Carignan (1), K. Hamby (1), E. Koivunen (1) (1) University of Maryland, U.S.A. In fruit crops, the interaction between frugivorous insects and fungi can alter pest and disease epidemiology, reducing marketable crop yields. In 2015 field surveys of fall red raspberries in Maryland, Cladosporium was recovered from fruit-rot affected berries at three farms, and 30% of Cladosporium- infested berries were co-infested with SWD larvae. Analyses of larval frass microbes recovered Cladosporium from 45% of larvae, with 3.3 colony forming units/ larvae on average. Sequence analysis identified both berry and larval frass isolates as either Cladosporium cladosporioides or C. pseudocladosporioides. All three tested isolates of each species were pathogenic on raspberries in detached berry assays, and one tested isolate could infect both ripe and unripe fruit in attached-berry assays. Disease incidence was significantly enhanced (P < 0.05) both when inoculated detached berries were mechanically wounded and when naturally infested attached berries were exposed to lab-reared SWD adults (ovipositor wounds), compared tounwounded controls. First report of laurel wilt on sassafras in Arkansas R. OLATINWO (1), C. Barton (2), J. Hwang (3), W. Johnson (3) (1) USDA Forest Service, Southern Research Station, U.S.A.; (2) Arkansas Forestry Commision, U.S.A.; (3) USDA Forest Service, Forest Health Protection, U.S.A. A Snapshot of Citrus Black Spot in Southwest Florida: Spatial Distribution in Two ‘Valencia’ Citrus Groves K. HENDRICKS (1), P. Roberts (1) (1) University of Florida - IFAS These results suggest that the distribution of BSP in PC and PE is ruled out by at least three different dissemination mechanisms: a random (primary spread), and two aggregation components, a real secondary spread, and a “false” secondary spread. However, it is clear that the primary spread is the most important component in begomovirus epidemics. Hence, management of begomovirus diseases focusing only on the target fields is certainly not enough for an efficient control. S4.134 Candidatus Liberibacter asiaticus, causal agent of citrus greening disease, is transmitted by Diaphorina citri, and together they pose the greatest risk to Citrus production worldwide. Currently research into host plant resistance to the vector and bacterial pathogen is ongoing, with the aim of developing resistant Citrus selections. Our objective was to test the influence of leaf anatomy on D. citri probing behaviors that regulate bacterial transmission. Leaf morphometrics might inhibit bacterial transmission, and thus could be candidates for resistance breeding. We performed an electropenetrographic study and histological examination of leaves of two Citrus species differently preferred by the psyllid, C. sinensis (var. Valencia) and C. aurantium (var. Sour orange). We quantified durations, frequencies, and order of probing behaviors associated with bacterial transmission, and correlated those behaviors with leaf anatomy. Young Sour Orange leaves were less preferred and exhibited a thicker and more complete fibrous ring than did same-aged Valencia leaves. This more developed barrier impacted probing behaviors such that D. citri took longer to reach the phloem and ingested from it for shorter durations. Delay and reduction of phloem associated behaviors influences the success of bacterial transmission by D. citri. Future resistant varieties could use fibrous ring morphometrics as an anatomical marker for resistance to pathogen transmission. 6SrIII-L phytoplasma by leafhoppers (Scaphytopius marginelineatus) to cassava (Manihot esculenta Crantz) in Colom BETANCOURTH (1) Many endophytic Cladosporium species are entomopathogens, so species causing fruit rot may be deleterious to SWD fitness; alternately, this may represent a commensal or mutually beneficial interaction. Overall, these results are among the first to suggest that SWD may interact with Cladosporium to significantly alter pre- and post-harvest fruit rot epidemiology. The ability of Olpidium brassicae to vector blueberry mosaic virus in southern highbush blueberries T MILES (1) A Shands (1) The ability of Olpidium brassicae to vector blueberry mosaic virus in southern highbush blueberries T. MILES (1), A. Shands (1) (1) CSU Monterey Bay, U.S.A. The ability of Olpidium brassicae to vector blueberry mosaic virus in southern highbush blueberries T. MILES (1), A. Shands (1) (1) CSU Monterey Bay, U.S.A. Blueberry mosaic associated Virus (BlMaV) is a recently described ophiovirus that is of concern in blueberry propagation systems. Members of the fungal genus Olpidium commonly found in plant roots, have the ability to move in these production systems using flagella, and possess the ability to vector Ophioviruses. In 2015, BlMaV affected blueberry plants were identified in a propagation nursery and a series of trap plants were utilized to determine potential vectors. Trap plant experiments were conducted with varying soil treatments (n=5), and hosts (carrot, lettuce, broccoli, cucumber) to determine the presence of Olpidium spp. Olpidium was observed in only lettuce trap plants (average of 6 resting spores/cm of root) and absent in the remaining trap plants. The ability of Olpidium species to vector BlMaV was tested through a series of concurrent inoculation experiments consisting of inoculated groups and control groups, treated with lettuce roots containing Olpidium resting spores or water, respectively. Each series of inoculation experiments varied in exposure time (10, 8, and 6 months) to root inoculum containing Olpidium resting spores and water, respectively. Transmission experiments showed Olpidium to be readily observed in the roots of inoculated southern highbush blueberry. By determining the ability of Olpidium spp. to vector BlMaV will aid in the management of blueberry propagation and increase our understanding of the basic biology of BlMaV. Olive Escudete, caused by Botryosphaeria dothidea, as Result of the Interaction fly-mosquito-fungus Olive Escudete, caused by Botryosphaeria dothidea, as Result of the Interaction fly-mosquito-fungus J. MORAL (1), I. Eldesouki-Arafat (2), F. López-Escudero (3), E. Vargas-Osuna (2), T. Antonio (4), H. Aldebis (2) (1) University of California, Davis, Kearney Agricultural Research and Extension Center, Parlier, CA, U.S.A.; (2) Dpto. Ciencias y Recursos Agrícolas y Forestales, Universidad de Córdoba. Campus de Rabanales, Edifico C4, 14071, Cordoba, Spain; (3) Dpto. Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C4, 14071, Cordoba, Spain; (4) Dpto. Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C4, 14071, Cordoba, Saint Kitts Nevis J. MORAL (1), I. Eldesouki-Arafat (2), F. López-Escudero (3), E. Vargas-Osuna (2), T. Antonio (4), H. Aldebis (2) (1) University of California, Davis, Kearney Agricultural Research and Extension Center, Parlier, CA, U.S.A.; (2) Dpto. Ciencias y Recursos Agrícolas y Forestales, Universidad de Córdoba. The ability of Olpidium brassicae to vector blueberry mosaic virus in southern highbush blueberries T. MILES (1), A. Shands (1) (1) CSU Monterey Bay, U.S.A. Campus de Rabanales, Edifico C4, 14071, Cordoba, Spain; (3) Dpto. Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C4, 14071, Cordoba, Spain; (4) Dpto. Agronomía, ETSIAM, Universidad de Córdoba, Campus de Rabanales, Edif. C4, 14071, Cordoba, Saint Kitts Nevis The Dalmatian disease or escudete, caused by Botryosphaeria dothidea, is the most important disease of table olive fruit. The typical escudete (small shield) symptom is a sunken, necrotic, and circular (≈8mm) lesion that appears in green fruits. The olive fly (Bactrocera oleae) and mosquito (Prolasioptera berlesiana) whose larvae prey on fly eggs, are disputed as vectors of the pathogen. Here, we study the interaction among these three agents: fly-mosquito-fungus. In the field, we periodically sampled for the presence of the mosquito in fruits, which were naturally punctured by the olive fly or artificially by a needle for three seasons. Under lab conditions, the presence of the fungus in the mosquito and the fly was studied by microscopy, isolation on media, and DNA sequencing. According to our results, the mosquito is attracted by ovipositor punctures on the olive surface made by the olive fly. When the mosquito deposits its own egg adjacent to the fly egg, it also inoculates the fungus in the puncture. This allows the mosquito larvae to feed on B. dothidea mycelium when the fly egg is absent; a need that arises due to frequent false punctures (no oviposition) made by the fly to avoid predation by the mosquito larvae. Microscopic studies show that the mosquito female has a special structure (mycangia) to carry B. dothidea spores in the last abdominal segments close to the ovipositor, which suggests a mutualistic association between B. dothidea and P. berlesiana. First report of laurel wilt on sassafras in Arkansas R. OLATINWO (1), C. Barton (2), J. Hwang (3), W. Johnson (3) (1) USDA Forest Service, Southern Research Station, U.S.A.; (2) Arkansas Forestry Commision, U.S.A.; (3) USDA Forest Service, Forest Health Protection, U.S.A. S4.135 Laurel wilt, caused by the fungus Raffaelea lauricola T.C. Harrin., Aghayeva & Fraedrich, is a lethal disease of redbay (Persea borbonia (L.) Spreng.), sassafras (Sassafras albidum (Nutt.) Nees), and other species in the laurel family (Lauraceae). The fungus is transmitted by an exotic ambrosia beetle (Xyleborus glabratus Eichh.) native to Asia introduced into the United States in 2002. It was first reported at Port Wentworth, Georgia and rapidly spread across the south causing extensive redbay mortality. ctor population trends, species composition, and the role they play in the barley yellow dwarf complex in Northeaster The recombinant isolate H12 was chosen because it does not elicit symptoms in turnips; the aphid’s preference of H12-infected plants over healthy turnips indicated that aphids were not attracted to plants based on differences in color or a pattern associated with disease. We are now focused on identifying differences in volatile profiles between healthy, W260- and NY8153-infected turnips that might influence the preference of an aphid for a specific host. These results suggest that CaMV infection affects the aphid’s choice of host and this in turn may have implications for the spread of different strains of CaMV. Aphid-mediated potyvirus transmission: Host plant chemistry, vector biology and component interactions K. GADHAVE (1), B. Dutta (2), J. Schmidt (2), R. Srinivasan (2) (1) University of Georgia U S A ; (2) University of Georgia U S A ; (3) University of Georgia U S A Aphid-mediated potyvirus transmission: Host plant chemistry, vector biology and component interactions K. GADHAVE (1), B. Dutta (2), J. Schmidt (2), R. Srinivasan (2) (1) University of Georgia, U.S.A.; (2) University of Georgia, U.S.A.; (3) University of Georgia, U.S.A. Potyviruses pose a significant threat to cucurbit production in Georgia and in the Southeastern United States. Aphids transmit these viruses in a non- circulative manner, causing extensive yield losses worth millions of dollars annually. The present virus and aphid management strategy involves the use of insecticides and transgenic cucurbit cultivars. However, none of these approaches have proven to be sustainable, especially due to the development of vector resistance to insecticides, and the narrow spectrum of virus resistance in transgenic cultivars. It is therefore critical to explore the novel avenues for reducing vector populations and viral incidences. To attain this, we have undertaken a holistic approach that explores the fundamental mechanisms underlying aphid-potyvirus interactions in cucurbit systems. Through an array of lab and field experiments, we have been studying the virus transmission, component interactions in the pathosystem and their impacts on vector behavior, fitness and transcriptome. The impact of these results in the context of virus epidemics and management will be discussed. Transovarial and Sexual Transmission of Tomato yellow leaf curl virus in Whiteflies W. MARCHANT (1), R. Srinivasan (1) (1) University of Georgia, U.S.A. Tomato yellow leaf curl virus (TYLCV) is whitefly-transmitted Geminivirus that causes yield losses in tomato crops. The virus is persistently transmitted by the sweetpotato whitefly, Bemisia tabaci. ctor population trends, species composition, and the role they play in the barley yellow dwarf complex in Northeaster Determination of vector population trends, species composition, and the role they play in the barley yellow dwarf com Determination of vector population trends, species composition, and the role they play in the barley yellow dwarf complex in Northeastern Kansas M. RAMOS (1), D. Rotenberg (1), A. Laney (1), A. Kieffaber (1), A. Whitfield (1) (1) Kansas State University, U.S.A. M. RAMOS (1), D. Rotenberg (1), A. Laney (1), A. Kieffaber (1), A. Whitfield (1) 1) Kansas State University, U.S.A. Barley yellow dwarf is a complex disease that includes grass hosts, aphid vectors, and yellow dwarf viruses (YDVs) in the family Luteoviridae. BYD is responsible for crop losses worldwide, and is an especially important disease of KS wheat. The goals of this project were to characterize aphid vector species composition in native grasses and crops, and to document associated YDVs in NE KS. Aphid populations were monitored during fall, spring, and summer using yellow sticky cards that were placed in fields of sorghum, corn, wheat, and big bluestem, and aphids were identified via morphological features. Rhopalosiphum padi was found to be the most prevalent aphid species. Other species of importance were Sitobion avenae, R. rufiabdominalis, and Schizaphis graminum. Aphid species overlapped between crops and native grasses, supporting the hypothesis that perennial grasses can serve as reservoirs for YDVs. Multiplex RT-PCR was used for virus detection in plants and aphids, and both tested positive for several YDVs. We found barley yellow dwarf virus (BYDV)-PAS to be the virus species most common in both aphids and plants, followed by BYDV-PAV and BYDV-RPV. Aphids and plants were either singly- or co-infected with YDV species. Ultimately, this will lead to determination of the most efficient vectors of YDVs, their association with YDV-species composition in croplands, and the temporal influx of viruliferous aphid populations across agroecosystems in KS. Variation in transmission of Tomato spotted wilt virus (TSWV) from Thrips tabaci is not solely influenced by virus titer within individual thrips J. BROWN (1), A. Jacobson (2), G. Kennedy (1), S. Lommel (1), T. Sit (1) (1) North Carolina State University, U.S.A.; (2) Auburn University, U.S.A. Thrips tabaci, a vector of TSWV, has been shown to vary greatly in the ability to transmit different isolates of TSWV. The objective of this study is to determine if the virus titer in individual T. tabaci influences the variation in transmission of TSWV from established T. ctor population trends, species composition, and the role they play in the barley yellow dwarf complex in Northeaster tabaci isofemale lines collected from several locations in North Carolina. The isofemale lines were previously shown to differ in their ability to transmit various TSWV isolates collected from the same series of locations as the thrips. The virus-thrips line combinations were grouped in sympatric and allopatric pairings and the groups classified as low, moderate, or high transmitters based on the percent transmission of TSWV to Emilia sonchifolia leaf discs. Quantitative real-time PCR was done on T. tabaci from 12 selected virus-thrips line combinations to quantify virus titer in transmitting and nontransmitting individuals to determine if the variation in transmission was correlated with virus titer. The results show that variation in transmission efficiency of the different virus-thrips pairings are not explained by differences in virus titer within the thrips. This is in contrast to findings with other TSWV thrips vectors where virus titer was directly correlated with transmission efficiency. This suggests that the differences in transmission among these thrips-virus isolate pairings may be determined by the genetics of both the virus and the thrips, and their interaction. Turnip aphid preference for turnip plants infected with different strains of Cauliflower mosaic virus M. ADHAB (1), J. Schoelz (1), D. Finke (1) (1) University of Missouri - Columbia, MO, U.S.A. Turnip aphid preference for turnip plants infected with different strains of Cauliflower mosaic virus M. ADHAB (1), J. Schoelz (1), D. Finke (1) (1) University of Missouri - Columbia, MO, U.S.A. (1) University of Missouri - Columbia, MO, U.S.A. Cauliflower mosaic virus (CaMV) is transmitted in a semi-persistent manner by several species of aphids. In this study, we investigated whether the turnip aphid (Lipaphis erysimi) exhibits a preference for healthy turnip plants (Brassica rapa L. var. rapa) or plants infected with CaMV. Two strains of CaMV, W260 and NY8153, as well as one recombinant isolate, H12, were used in this study. Preference assays were conducted with detached leaves in petri dishes or with whole plants. When given a choice between infected or healthy plants, aphids chose W260-infected and H12-infected plants significantly more often, regardless of the test. Interestingly, aphids did not exhibit a preference for turnips infected with CaMV strain NY8153 over healthy plants. The ability of Olpidium brassicae to vector blueberry mosaic virus in southern highbush blueberries T. MILES (1), A. Shands (1) (1) CSU Monterey Bay, U.S.A. On December 2, 2015, visit to a landowner in Bradley County near Warren, Arkansas (33.64511°N, 92.05134°W) led to the first reported mortality of sassafras caused by laurel wilt in Arkansas. Moderate degrees of staining and beetle galleries were found from symptomatic trees at this location. Suspect beetles collected from sapwood of affected trees were later identified as X. glabratus. The presence of R. lauricola was confirmed by morphological characterization and molecular identification of cultures obtained from stained sapwood tissues. This is the first report of laurel wilt on sassafras or any other Lauraceae species in Arkansas. In 2014, laurel wilt was reported on sassafras in northern Louisiana (83 miles southwest). This new report in Arkansas may indicate northward movement of the disease into natural habitat of sassafras. However, the lack of redbay (a more attractive host) in this region may slow the northward spread of laurel wilt in Arkansas. The genetic structure, virulence, and fungicide sensitivity of Fusarium fujikuroi in Taiwan The genetic structure, virulence, and fungicide sensitivity of Fusarium fujikuroi in Taiwan The genetic structure, virulence, and fungicide sensitivity of Fusarium fujikuroi in Taiwan C. CHUNG (1), Y. Chen (2), M. Lai (3), C. Wu (2), A. Cheng (2), T. Lin (3), A. Cheng (4), C. Yang (2), H. Wu (5), S. Chu (6), C. Kuo (7), Y. Wu (4), G. Lin (4), M. Tseng (8), Y. Tsai (9), C. Lin (10), C. Chen (11), J. Huang (11), H. Lin (2) (1) National Taiwan University, Taipei City, Taiwan; (2) National Taiwan University, Taiwan; (3) Taiwan Agricultural Research Institute, Taiwan; (4) T i Di t i t A i lt l R h d E t i St ti T i (5) T Di t i t A i lt l R h d E t i St ti T i (6) C. CHUNG (1), Y. Chen (2), M. Lai (3), C. Wu (2), A. Cheng (2), T. Lin (3), A. Cheng (4), C. Yang (2), H. Wu (5), S. Chu (6), C. Kuo (7), Y. Wu (4), G. Lin (4), M. Tseng (8), Y. Tsai (9), C. Lin (10), C. Chen (11), J. Huang (11), H. Lin (2) (1) National Taiwan University, Taipei City, Taiwan; (2) National Taiwan University, Taiwan; (3) Taiwan Agricultural Research Institute, Taiwan; (4) Tainan District Agricultural Research and Extension Station, Taiwan; (5) Taoyuan District Agricultural Research and Extension Station, Taiwan; (6) Miaoli District Agricultural Research and Extension Station, Taiwan; (7) Taichung District Agricultural Research and Extension Station, Taiwan; (8) Kaohsiung District Agricultural Research and Extension Station, Taiwan; (9) Hualien District Agricultural Research and Extension Station, Taiwan; (10) Taitung District Agricultural Research and Extension Station, Taiwan; (11) National Chung Hsing University, Taiwan Fusarium fujikuroi is the causal agent of bakanae disease, a common rice disease causing abnormal growth of seedlings and poorly filled grains. In Taiwan, the disease has long been well managed, but a disease outbreak was found in eastern Taiwan in 2009. To better understand the population genetics of F. fujikuroi, 16 polymorphic simple sequence repeat markers were newly developed and used to analyze 637 F. fujikuroi isolates collected in 14 rice-growing areas around Taiwan from 1996 to 2013. The isolates were classified into four highly differentiated clusters by Bayesian clustering analysis. The highly diversified vegetative compatibility groups and good numbers of MAT1-1 and MAT1-2 isolates sampled from all around Taiwan supported sexual reproduction. ctor population trends, species composition, and the role they play in the barley yellow dwarf complex in Northeaster The virus is considered non-propagative and non-transmissible transovarially or through mating. Feeding on infected plant tissue is the only method of acquisition according to most of the literature. However, a few recent studies from Israel demonstrate transovarial and sexual transmission of TYLCV as well as propagation within the whitefly. We have tested to see if transovarial and sexual transmission occur with the whiteflies and TYLCV present in Georgia using both molecular methods and plant transmission experiments. We found a very low rate of transovarial and sexual transmission suggesting negligible impact on virus epidemics. COI sequences from our local Bemisia tabaci and full-length TYLCV S4.136 genomes have been compared with sequences from Israel available on GenBank. Our B. tabaci COI haplotype is present in Israel, but differences in the TYLCV genome were found at the amino acid level. These differences could potentially explain the differences seen in insect-virus interactions. genomes have been compared with sequences from Israel available on GenBank. Our B. tabaci COI haplotype is present in Israel, but differences in the TYLCV genome were found at the amino acid level. These differences could potentially explain the differences seen in insect-virus interactions. Molecular interactions between citrus tristeza virus and its aphid vector, Toxoptera citricida N. KILLINY (1), S. Harper (2), C. C. El Mohtar (2), B. Dawson (2) (1) University of Florida, IFAS, CREC, Department of Plant Pathology, U.S.A.; (2) University of Florida, Depart Molecular interactions between citrus tristeza virus and its aphid vector, Toxoptera citricida N. KILLINY (1), S. Harper (2), C. C. El Mohtar (2), B. Dawson (2) (1) University of Florida, IFAS, CREC, Department of Plant Pathology, U.S.A.; (2) University of Florida, Department of Plant Pathology, U.S.A. AS, CREC, Department of Plant Pathology, U.S.A.; (2) University of Florida, Department of Plant Pathology, U.S.A. Citrus tristeza virus is transmitted by several aphid species, including Aphis gossypii, Aphis spiraecola, Toxoptera aurantii, and Toxoptera citricida. Considerable variations in the efficacy of transmission both among different vector species, and among CTV isolates are recorded. T. citricida, the brown citrus aphid, is the most efficient and specialized vector. It has a host range limited primarily to rutaceous species and transmits most CTV isolates at higher rates than other aphids. We demonstrated that CTV binds specifically to the lining of the cibarium of the aphid using fluorescently-labelled virions. ctor population trends, species composition, and the role they play in the barley yellow dwarf complex in Northeaster In vitro competitive-binding assays between fluorescent virions and free viral proteins determined that the minor coat protein and two heat- shock-like proteins (p61 and p65) are involved in vector interaction. Treating the dissected mouthparts with proteases prior to the binding assay did not affect the binding of CTV virions while, chitinase treatment reduced the CTV binding to foregut. These findings suggest that CTV binds to sugar moieties of the cuticular surface of the aphid cibarium and not to embedded proteins. Additionally, the specific binding involves the concerted activity of three virally-encoded proteins. A strategy to study population variation of Xanthomonas axonopodis pv. manihotis using a new set of multiple-locus variable number tand repeats A strategy to study population variation of Xanthomonas axonopodis pv. manihotis using a new set of multiple-locus variable number tandem repeats A strategy to study population variation of Xanthomonas axonopodis pv. manihotis using a new set of multiple-locus v repeats o study population variation of Xanthomonas axonopodis pv. manihotis using a new set of multiple-locus variable num p L. RACHE CARDENAL (1), C. Flores (2), C. López (3), B. Szurek (2), C. Vernièr (2), R. Koebnik (2), S. Restrepo (4), A. Bernal (4) (1) Universidad de los Andes, BOGOTA, Colombia; (2) UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France, France; (3) Universidad Nacional de Colombia, Colombia; (4) Universidad de los Andes, Colombia L. RACHE CARDENAL (1), C. Flores (2), C. López (3), B. Szurek (2), C. Vernièr (2), R. Koebnik (2), S. Restrepo (4), A. Bernal (4) (1) Universidad de los Andes, BOGOTA, Colombia; (2) UMR Interactions Plantes Microorganismes Environnement, IRD-Cirad-UM, Montpellier, France, France; (3) Universidad Nacional de Colombia, Colombia; (4) Universidad de los Andes, Colombia Multiple-Locus Variable Number Tandem Repeat Analysis (MLVA) is being used to analyze genetic diversity in phytopathogenic microorganisms. One of the most important pathogens affecting Cassava crops is Xanthomonas axonopodis pv. manihotis (Xam) as its infection relates to high loses in crop productivity. Several studies have reported that Xam populations change over time. It is important to assess population diversity in this pathogen as one of the basis to develop cassava resistant varieties. Studies to typify Xam strains performed by our group showed that using Variable Number of Tandem Repeats (VNTRs) have advantages over Amplified Fragment Length Polymorphisms (AFLPs), such as low costs and high discriminatory power using small sets of markers. ctor population trends, species composition, and the role they play in the barley yellow dwarf complex in Northeaster Consequently, in this study we standardized and tested a new scheme of VNTR loci to analyze Xam populations. First, by analyzing 12 of 65 previously sequenced genomes of Xam, which represent the worldwide variability, we selected 16 VNTR loci. We standardized four multiplex PCR to amplify four microsatellite loci in each multiplex. Finally, the 16-VNTR scheme was used to assess population diversity of Xam strains collected in four Colombian Caribbean-region localities from 2008 to 2010, 2013 and 2014. Our results show that most of Xam genetic diversity is found within populations, and that the 16-VNTR scheme is a useful tool to discriminate Xam populations up to a local scale. q y y y p p y analyzing 12 of 65 previously sequenced genomes of Xam, which represent the worldwide variability, we selected 16 VNTR loci. We standardized four multiplex PCR to amplify four microsatellite loci in each multiplex. Finally, the 16-VNTR scheme was used to assess population diversity of Xam strains collected in four Colombian Caribbean-region localities from 2008 to 2010, 2013 and 2014. Our results show that most of Xam genetic diversity is found within populations, and that the 16-VNTR scheme is a useful tool to discriminate Xam populations up to a local scale. Diversity of Aspergillus flavus on important agricultural crops produced in Bangladesh M. ISLAM (1), P. Cotty (2) Diversity of Aspergillus flavus on important agricultural crops produced in Bangladesh M. ISLAM (1), P. Cotty (2) Diversity of Aspergillus flavus on important agricultural crops produced in Bangladesh M. ISLAM (1), P. Cotty (2) (1) USDA ARS, U.S.A.; (2) USDA-ARS, School of Plant Sciences, the University of Arizona, U.S.A. Diversity of Aspergillus flavus on important agricultural crops produced in Bangladesh M. ISLAM (1), P. Cotty (2) (1) USDA ARS, U.S.A.; (2) USDA-ARS, School of Plant Sciences, the University of Arizona, U.S.A. M. ISLAM (1), P. Cotty (2) (1) USDA ARS, U.S.A.; (2) USDA-ARS, School of Plant Sciences, the University of Arizona, U.S.A. . S ( ), . Co y ( ) 1) USDA ARS, U.S.A.; (2) USDA-ARS, School of Plant Sciences, the University of Arizona, U.S.A. ( ), y ( ) DA ARS, U.S.A.; (2) USDA-ARS, School of Plant Sciences, th The hot, humid climate of Bangladesh is ideal for aflatoxin producing fungi. As a result, rice, maize, peanut, wheat and other crops grown in Bangladesh may become infected by these fungi and contaminated with dangerous concentrations of aflatoxins. Aflatoxins are toxic fungal metabolites produced by A. flavus, A. parasiticus, and closely related species. Aflatoxins cause increased risk of liver cancer, stunting, immune suppression, and in cases of acute exposure, liver necrosis and death. However, frequencies of aflatoxins and diversity of aflatoxin-producing fungi in crops of Bangladesh are poorly understood. To assess the magnitude of aflatoxin contamination and diversity of A. flavus in crops produced in Bangladesh, maize, rice, wheat and peanut samples were collected from 12 districts covering the major agro-ecological regions of Bangladesh. Aflatoxins were quantified in crop samples using immunochromatographic assays. Variable levels of aflatoxins were detected across the samples with a maximum concentration of 122.8 µg/kg total aflatoxins. Fungi associated with the maize and belonging to the L-strain morphotype were genotyped using 16 SSR markers. Genetic diversity of the L morphotype was high with 5 to 24 alleles per SSR locus, and 46 haplotypes among 50 isolates. Atoxigenic strains of A. flavus of potential value in aflatoxin prevention and native to Bangladesh were characterized. The genetic structure, virulence, and fungicide sensitivity of Fusarium fujikuroi in Taiwan pseudophaseolina clades with a bootstrap support value of 100%. Population structure of the wheat pathogen Zymoseptoria tritici in France at different scales A. SIAH (1), L. EL CHARTOUNI (2), B. TISSERANT (2), P. HALAMA (1), P. REIGNAULT (3) (1) Institut Charles Viollette (EA 7394), Institut Supérieur d’Agriculture, Lille, France; (2) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d’Opale, Calais, France; (3) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d’Opale, Lille, France Population structure of the wheat pathogen Zymoseptoria tritici in France at different scales A. SIAH (1), L. EL CHARTOUNI (2), B. TISSERANT (2), P. HALAMA (1), P. REIGNAULT (3) (1) Institut Charles Viollette (EA 7394), Institut Supérieur d’Agriculture, Lille, France; (2) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d’Opale, Calais, France; (3) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d’Opale, Lille, France . S ( ), . C OUN ( ), . SS N ( ), . ( ), . GN U (3) (1) Institut Charles Viollette (EA 7394), Institut Supérieur d’Agriculture, Lille, France; (2) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d’Opale, Calais, France; (3) Unité de Chimie Environnementale et Interactions sur le Vivant (EA 4492), Université du Littoral Côte d’Opale, Lille, France Zymoseptoria tritici is currently the most damaging disease on wheat crops in Europe, especially in France, where it causes each year 1.7 t/ha yield loss in average. In order to better understand its genetic features, we characterized several Z. tritici populations (more than 1500 isolates), isolated in France over a ten-year period (2005-2015) and at different spatial scales (whole country, Nord-Pas de Calais region, field, 30 cm2 square, plant, leaf and lesion) using SSR markers and mating type idiomorphs. Results revealed high and similar levels of genic and genotypic diversities at the country, region and field scales, but the diversity values gradually decreased from the field to finer scales such as plant, leaf and lesion. Bayesian and non-Bayesian statistical analyses revealed significant structuration of the global French population into three genetic clusters, and the Nord-Pas de Calais population into three sub-clusters, all distributed according to their geographical origin. However, no differentiation was found at finer scales. Preliminary screening of genetic markers for population analyses of Magnaporthe oryzae Triticum pathotype J. STACK (1), C. Cruz (1), S. Dobham (1) Wood (4) (1) USDA/ARS SE Fruit & Tree Nut Research Laboratory, 21 Dunbar Rd., Byron, GA, U.S.A.; (2) USDA-ARS SE Fruit and Tree Nut Research Laboratory, Byron, GA, U.S.A.; (3) Department of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.; (4) USDA/ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A. Scab (Fusicladium effusum) is the most destructive disease of pecan in the Southeast US. Infection is thought to occur solely through asexually produced conidia. To explore the population structure and genetic diversity of F. effusum, populations were hierarchically sampled from 11 orchards in Alabama, Florida, Georgia, Illinois, Kansas, Louisiana, Mississippi, Oklahoma and Texas. A total of 734 isolates was collected from up to 20 leaves from each of 3 to 6 trees in each orchard. Isolates were screened against 30 microsatellite markers. Polymorphic loci per population were ≥96.7%. Populations were genetically diverse (Nei’s measure of genetic diversity = 0.505-0.692). An analysis of molecular variance showed 81% of variation occurred in the populations of F. effusum within trees, 3% in populations between trees within orchards, and 16% between orchards. Population differentiation was low (GST = 0.17) and gene flow relatively high (Nm=2.5). However, there was some association between population genetic and log physical distance (r=0.471, P=0.0003). Ten of the 11 populations showed evidence of gametic disequilibrium (r?d = 0.024-0.057), which might be explained by a predominance of within-season asexual reproduction, or possibly by founder effects or migration rates. The results show that F. effusum is a genetically diverse pathogen, with the degree and distribution of genetic diversity often associated with sexual recombination. Spatial and temporal structure of Sclerotinia sclerotiorum in New York State A. DUNN (1), J. Kikkert (2), S. Pethybridge (3) (1) Hobart and William Smith Colleges, U.S.A.; (2) Cornell Cooperative Extension, Cornell Vegetable Program, U.S.A.; (3) Cornell University, U.S.A. Spatial and temporal structure of Sclerotinia sclerotiorum in New York State A. DUNN (1), J. Kikkert (2), S. Pethybridge (3) ( ), ( ), y g ( ) (1) Hobart and William Smith Colleges, U.S.A.; (2) Cornell Cooperative Extension, Cornell Vegetable Program, U.S.A.; (3) Cornell University, U.S.A. 1) Hobart and William Smith Colleges, U.S.A.; (2) Cornell Cooperative Extension, Cornell Vegetable Program, U.S.A.; (3) Sclerotinia sclerotiorum causes white mold on many vegetable and field crops in New York State, but the population structure has not been thoroughly studied. To assess spatial and temporal variation of S. Preliminary screening of genetic markers for population analyses of Magnaporthe oryzae Triticum pathotype J. STACK (1), C. Cruz (1), S. Dobham (1) Preliminary screening of genetic markers for population analyses of Magnaporthe or J. STACK (1), C. Cruz (1), S. Dobham (1) (1) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A. ( ), ( ), ( ) (1) Department of Plant Pathology, Kansas State University, Manhattan, KS, U.S.A. ( ) ( ) nt Pathology, Kansas State University, Manhattan, KS, U.S.A. Wheat blast, caused by Magnaporthe oryzae Triticum pathotype (MoT), is a devastating disease of wheat in South America; primarily in Brazil, Bolivia and Paraguay, and recently in a small area of northeast Argentina. It is a potential threat to wheat production worldwide with implications for food security. Very little is known about the population biology of the MoT. Therefore, understanding the population biology of wheat blast is important. Screening of current populations from Bolivia, Brazil, and Paraguay was performed using 30 inter-simple sequence repeat (ISSR) and retrotransposon microsatellite amplified polymorphism (REMAP) primers. Genes for MLST were also screened using primer sets targeting conserved genes and genes involved in different cellular functions. Combined phylogenetic analyses using ISSR and REMAP loci was able to differentiate populations from the three different regions based on 151 total loci, including 119 polymorphic and 32 monomorphic loci; showed 78.80% of percent polymorphism. The MLST based on genes involved in cellular function was found to be informative for the population genetics study. The screening of more markers for the population study is underway. This study will provide valuable information about genetic variation within and among current MoT populations of value for disease management and for the development of reliable and accurate detection tools for this emerging disease. Population structure and genetic diversity of Fusicladium effusum in the USA C. BOCK (1), M. Hotchkiss (2), K. Stevenson (3), B. Wood (4) (1) USDA/ARS SE Fruit & Tree Nut Research Laboratory, 21 Dunbar Rd., Byron, GA, U.S.A.; (2) USDA-ARS SE Fruit and Tree Nut Research Laboratory, Byron, GA, U.S.A.; (3) Department of Plant Pathology, University of Georgia, Tifton, GA, U.S.A.; (4) USDA/ARS SE Fruit & Tree Nut Research Laboratory, Byron, GA, U.S.A. C. BOCK (1), M. Hotchkiss (2), K. Stevenson (3), B. epidemics of wheat and barley and races of Puccinia striiformis identified in the United States in 2015 ) A W (2) Stripe rust epidemics of wheat and barley and races of Puccinia striiformis identified in the United States in 2015 X. CHEN (1), A. Wan (2) Stripe rust epidemics of wheat and barley and races of Puccinia striiformis identified in the Un X. CHEN (1), A. Wan (2) (1) USDA-ARS, U.S.A.; (2) Department of Plant Pathologist, Washington State University, U.S.A. ( ), ( ) 1) USDA-ARS, U.S.A.; (2) Department of Plant Pathologist, Washington State University, U.S.A. ( ), ( ) (1) USDA-ARS, U.S.A.; (2) Department of Plant Pathologist, Washington State University, U.S.A. In 2015, stripe rust of wheat, caused by Puccinia striiformis f. sp. tritici (Pst), occurred throughout the United States and caused severe yield losses in the Great Plains. Stripe rust of barley, caused by P. striiformis f. sp. hordei (Psh), occurred in California, Montana, Oregon, Utah, and Washington. For monitoring virulence changes of the pathogens, stripe rust samples collected from 21 states were tested on 18 wheat and 12 barley differentials to identify Pst and Psh races, respectively. From 32 Psh isolates, three previously existing races were detected, of which PSH-48 (virulent only on Topper among the 12 barley differentials) and PSH-33 (virulent on differentials Topper and Abed Binder 12) were the major races with frequencies of 53% and 54%, respectively, similar to 2014. From 310 Pst isolates, 32 races were detected including 5 new races (PSTv-140, PSTv-141, PSTv-142, PSTv-143, and PSTv-144). The top five frequent Pst races were PSTv-37 (36%), PSTv-52 (33%), PSTv-18 (5%), PSTv-53 (4%), and PSTv-48 (3%). PSTv-37 and PSTv-52 were detected in every epidemiological regions, while PSTv-18, PSTv-53, and PSTv-48 were detected only in the western U.S. High virulence frequencies (>50%) were detected for Yr6, Yr7, Yr8, Yr9, Yr17, Yr27, Yr43, Yr44, and YrExp2; moderate (10-50%) for Yr1, YrTr1, and YrTye; low (<10%) for Yr10, Yr24, Yr32, and YrSP; and none for Yr5 and Yr15. The information is useful for breeding for resistance and management of stripe rust. The genetic structure, virulence, and fungicide sensitivity of Fusarium fujikuroi in Taiwan However, the biased mating type ratios, reduced effective population numbers, and linkage disequilibrium suggest nonrandom mating between individuals. Analysis of isolation by distance revealed a significant relationship between spatial distance and genetic similarity, which implies a geographical restricted gene flow and low dissemination ability of F. fujikuroi. Evaluation of 24 representative isolates on 8 rice varieties revealed differential levels of virulence, however no clear pattern of specific variety x isolate interaction was observed. Investigations of 60 representative isolates from early and recent years indicate that F. fujikuroi in Taiwan has developed increased resistance to prochloraz. Exploring genetic diversity in Macrophomina phaseolina, the causal agent of charcoal rot on soybean V. ORTIZ LONDONO (1), M. Chilvers (1), K. Wise (2) (1) Michigan State University, U.S.A.; (2) Purdue University, U.S.A. Macrophomina phaseolina is a soil and seed-borne fungal pathogen infecting more than 500 plant species and causing charcoal rot disease on soybean (Glycine max). Recently, a set of Macrophomina isolates collected from three plant species in Senegal, morphologically similar and phylogenetically closely related to M. phaseolina, were described as a new species, M. pseudophaseolina. However, occurrence of M. pseudophaseolina is unknown outside of Senegal. In this study, 36 putative M. phaseolina isolates, collected from soybean grown in the northern and the southern US were identified to the species level. ITS, EF-1α and ACT loci were sequenced and analyzed. Additional sequences were obtained from GenBank. The Incongruence Length Difference (ILD) test was not significant (p=0.95) and supported a decision to combine the sequences. Phylogenetic relationships of the isolates were explored using Maximum Parsimony (MP), Neighbor-Joining (NJ), Maximum Likelihood (ML) and Bayesian Inference (BI). The MP, ML, NJ and BI S4.137 phylogenies resulted in two separate clades corresponding to M. phaseolina and M. pseudophaseolina and placed all 36 isolates from soybeans in the U.S. in M. phaseolina. The Shimodaira-Hasegawa test indicates the ML tree as the best, but there were not significant differences among any of them. The ML tree reconstructed the M. phaseolina and the M. pseudophaseolina clades with a bootstrap support value of 100%. phylogenies resulted in two separate clades corresponding to M. phaseolina and M. pseudophaseolina and placed all 36 isolates from soybeans in the U.S. in M. phaseolina. The Shimodaira-Hasegawa test indicates the ML tree as the best, but there were not significant differences among any of them. The ML tree reconstructed the M. phaseolina and the M. The genetic structure, virulence, and fungicide sensitivity of Fusarium fujikuroi in Taiwan Both mating types were found at equal proportions at the country, region and field scales and co-occurred at all other investigated scales. This study highlighted for Z. tritici in France a high potential for sexual reproduction and genetic diversification, as well as a structuration of its populations at largest scales, agreeing with its high fitness degree and its ability of adaptation to local agro-climatic conditions. Molecular characterization and genetic diversity of Mycosphaerella fijiensis in Costa Rica using sequence based nuclear markers J. RISTAINO (1), A. Saville (1), M. Charles (1), M. Wyatt (1), S. Chavan (1) (1) North Carolina State University, U.S.A. Mycosphaerella fijiensis is the causal agent of black sigatoka, a devastating disease of banana that can cause 20-80% yield loss in the absence of fungicides. M. fijiensis first appeared in Honduras in 1972 and spread to the rest of the western hemisphere, including major banana production regions such as Costa Rica. In order to characterize the genetic diversity of M. fijiensis in Costa Rica, one hundred and eighteen isolates, collected from Costa Rica and Honduras between 2010 and 2014, were examined using multilocus genotyping of six loci and compared to a previously published global dataset. A total of 13 haplotypes were identified from concatenated sequences of Honduran and Costa Rican populations, six of which were unique. Outside of the Americas, Costa Rican and Honduran populations shared haplotype diversity with populations in both Oceania and Southeast Asia for all loci, in particular Australia, Futuna, Tonga, and Papua New Guinea, the latter of which has been proposed as the center of origin for both banana and M. fijiensis. The genetic similarity to both regions suggests M. fijiensis may have been introduced into Honduras and Costa Rica multiple times. Given a relatively recent introduction, subsequent admixture events are expected to increase diversity of Costa Rican populations over time. Genetic and phenotypic diversity of Puccinia kuehnii of sugarcane from Brazil T. MISTURA (1), A. Urashima (1), L. Porto (1), C. Sakuno (2), R. Arias (3) (1) Federal University of Sao Carlos, Brazil; (2) Syngenta Crop Protection, Brazil; (3) USDA, ARS, South Atlantic Area Nat Peanut Res Lab, U.S.A. Genetic and phenotypic diversity of Puccinia kuehnii of sugarcane from Brazil T. MISTURA (1), A. Urashima (1), L. Porto (1), C. Sakuno (2), R. Arias (3) (1) Federal University of Sao Carlos, Brazil; (2) Syngenta Crop Protection, Brazil; (3) Resistant genotypes are the most employed method to control orange rust of sugarcane (Puccinia kuehnii). This approach has been adopted in Brazil; however, recent outbreaks on previously unreported varieties show that the efficacy of this method is dependent and requires better knowledge of pathogen diversity. This can be achieved by applying genetic and phenotypic tools. Therefore, the present work aimed to examine the genetic distance and pathotype variability among isolates of P. kuehnii from São Paulo, the most important growing state of the Country. Molecular characterization and genetic diversity of Mycosphaerella fijiensis in Costa Rica using sequence based nuclear markers J. RISTAINO (1), A. Saville (1), M. Charles (1), M. Wyatt (1), S. Chavan (1) (1) North Carolina State University, U.S.A. Thirty six rust isolates were genotyped by eight SSR markers and their genetic distances verified by the UPGMA clustering analysis using the software NTSYSpc v.2.2. Phenotypic data employed 22 isolates inoculated on five sugarcane varieties and evaluated at 21 days by examining percent diseased leaf area with Assess 2.0 and rating resistant/susceptible based on Santos et al. (2004). Moreover, an assay of disease progress over time was carried out with three isolates against five sugarcane cultivars examining disease score (Tai et al., 1981) at days 21, 28, 35 and 42. Our results showed two genetically distinct subpopulations of the pathogen with 79% of diversity, phenotypic data identified nine physiological races. In addition, disease progress over time showed that one cultivar considered resistant at day 21 became susceptible at day 42. Part of MSc dissertation by the second author at PPGPVBA, CAPES Scholarship. Short-term Host Selection Pressure Has Little Effect on the Evolution of Verticillium dahliae K. PURI (1), S. Gurung (1), D. Short (2), G. Sandoya (3), R. Hayes (4), K. Subbarao (1) (1) University of California, Salinas, CA, U.S.A.; (2) Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, U.S.A.; (3) UC Davis Genome Center, Davis, CA, U.S.A.; (4) United States Department of Agriculture, Agricultural Research Service, Salinas, CA, U.S.A. Short-term Host Selection Pressure Has Little Effect on the Evolution of Verticillium dahliae K. PURI (1), S. Gurung (1), D. Short (2), G. Sandoya (3), R. Hayes (4), K. Subbarao (1) (1) University of California, Salinas, CA, U.S.A.; (2) Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, U.S.A.; (3) UC Davis Genome Center, Davis, CA, U.S.A.; (4) United States Department of Agriculture, Agricultural Research Service, Salinas, CA, U.S.A. ( ), g ( ), ( ), y ( ), y ( ), ( ) (1) University of California, Salinas, CA, U.S.A.; (2) Division of Plant and Soil Sciences, West Virginia University, Morgantown, WV, U.S.A.; (3) UC Davis Genome Center, Davis, CA, U.S.A.; (4) United States Department of Agriculture, Agricultural Research Service, Salinas, CA, U.S.A. Understanding pathogen evolution over time is vital for breeding and deployment of host resistance. To study this in the context of a soilborne pathogen, within-field population structure from three major hosts (spinach, tomato and lettuce) and the potential of host-directed evolution of V. dahliae race 1 were determined. Molecular characterization and genetic diversity of Mycosphaerella fijiensis in Costa Rica using sequence based nuclear markers J. RISTAINO (1), A. Saville (1), M. Charles (1), M. Wyatt (1), S. Chavan (1) (1) North Carolina State University, U.S.A. A total of 408 representing 192, 44, and 172 isolates from spinach, tomato and lettuce, respectively, were collected from WA and CA. An additional 411 isolates were recovered over five years from an experimental screening plot infested with a race 1 strain in Salinas, CA. Isolates were genotyped using 13 microsatellite loci, race and mating type-specific primers. Results indicated no significant variation among the within-field populations from the three hosts. Isolates recovered from the screening plot over five years revealed no demonstrable changes in the race, species composition and mating type structure, except for six SSR locus variants. Replicated virulence assays of these six variants with the wild type race 1 strain on susceptible and resistant lettuce differentials suggested no significant virulence differences between them. This suggests that the deployed host resistance will remain stable against the native V. dahliae population, unless novel pathogen genotypes are introduced into the system. Population structure of Sclerotinia subarctica in England, Scotland and Norway J. CLARKSON (1), R. Warmington (2), B. Nordskog (3) (1) Warwick Crop Centre, University of Warwick, United Kingdom; (2) The Eden Project, United Kingdom; (3) Norwegian Institute of Bioeconomy Research, Norway Population structure of Sclerotinia subarctica in England, Scotland and Norway J. CLARKSON (1), R. Warmington (2), B. Nordskog (3) (1) Warwick Crop Centre, University of Warwick, United Kingdom; (2) The Eden Project, United Kingdom; (3) Norwegian Institute of Bioeconomy Research, Norway g ( ) g ( ) e, University of Warwick, United Kingdom; (2) The Eden Project, United Kingdom; (3) Norwegian Institute of Bioeconomy Sclerotinia subarctica is closely related to S. sclerotiorum which is a major pathogen of a wide range of dicotyledonous crop plants in many countries across the world. In contrast to S. sclerotiorum, S. subarctica is little studied as it was only identified relatively recently. However, preliminary evidence suggests that it is usually found sympatrically with S. sclerotiorum on the same host plants, but may be confined geographically to more northern latitudes. Using molecular approaches, we established that S. subarctica was rarely found in crop plants or wild buttercup in England (4.7% of Sclerotinia sp. isolates) and was more prevalent in Scotland (18.3%) and Norway (48.0%). Microsatellite data analysis of 157 S. subarctica isolates revealed a multiclonal population structure (75 haplotypes) with one or a few haplotypes more common than the rest. Preliminary screening of genetic markers for population analyses of Magnaporthe oryzae Triticum pathotype J. STACK (1), C. Cruz (1), S. Dobham (1) sclerotiorum in New York State, 200 isolates were sampled from 10 bean fields in 2014, and 36 isolates were obtained from a historical collection (sampled from various hosts between 1982 and 2008). Isolates were genotyped using eight microsatellite loci. Mycelial compatibility group (MCG), in vitro growth rate, and aggressiveness on snap bean plants were also assessed for 22 selected isolates. Twenty-eight unique multilocus genotypes (MLGs) were identified (24 in 2014). One MLG represented 44 and 47 percent of isolates sampled in 2014 and prior to 2014, respectively. In 2014, genotypic diversity was low (3 to 8 MLGs per field) and Nei’s genetic diversity varied among fields (0.42 to 0.90). Analysis of molecular variance indicated no significant spatial structure within or between fields, and the index of association differed significantly from zero in all fields. Isolates of the same MCG almost always shared the same MLG. Growth rate and aggressiveness varied slightly but S4.138 significantly among some isolates, but no significant association was found between the two measurements. Thus, the S. sclerotiorum population in New York State is highly clonal, and a small number of representative isolates would likely be sufficient to test new disease management strategies. significantly among some isolates, but no significant association was found between the two measurements. Thus, the S. sclerotiorum population in New York State is highly clonal, and a small number of representative isolates would likely be sufficient to test new disease management strategies. Sub-lethal fungicides induce microsatellite mutation in Sclerotinia sclerotiorum B. Amaradasa (1), S. Everhart (1) (1) University of Nebraska, U.S.A. Stress-induced mutations from sub-lethal fungicide exposure may be an important source of genetic variation for clonal plant pathogens. We used Sclerotinia sclerotiorum to assess the role of sub-lethal fungicide exposure in mutation at microsatellite loci. Nine sensitive isolates were exposed to long-term, sub-lethal doses of fungicides with different modes of action: boscalid, iprodione, thiophanate methyl, azoxystrobin and pyraclostrobin. Fungicides were plated in a logarithmic gradient and each isolate sub-cultured from the 50-100% inhibition zone for 12 consecutive generations and experiment repeated. Isolates at the first and twelfth generation from exposed and non-exposed lines were genotyped at six microsatellite loci. Control isolates showed no difference after 12 generations. Five of nine and two of nine fungicide-exposed isolates showed microsatellite mutations in both experiments. Preliminary screening of genetic markers for population analyses of Magnaporthe oryzae Triticum pathotype J. STACK (1), C. Cruz (1), S. Dobham (1) One locus did not mutate; all others range from 1 to 34 mutations per locus, with an average of 4.58 stepwise mutations. Most mutations were less than 20 bp, with one 80 bp mutation. The average mutation rate per locus among fungicide-exposed isolates ranged from 4-29 fold greater than the expected mutation rate of 1.3 × 10–5 estimated for microsatellites in Saccharomyces. All fungicides resulted in mutations, with iprodione- and azoxystrobin-exposed isolates showing the highest frequency. Results suggest long-term sub-lethal fungicide exposure increase mutation rates. Molecular characterization and genetic diversity of Mycosphaerella fijiensis in Costa Rica using sequence based nuclear markers J. RISTAINO (1), A. Saville (1), M. Charles (1), M. Wyatt (1), S. Chavan (1) (1) North Carolina State University, U.S.A. Molecular characterization and genetic diversity of Mycosphaerella fijiensis in Costa Rica using sequence based nucle J. RISTAINO (1), A. Saville (1), M. Charles (1), M. Wyatt (1), S. Chavan (1) 1) North Carolina State University, U.S.A. Is host specificity influencing the population structure of Phytophthora spp. collected from potato and tree tomato in central and southern Colombia? C. Chaves (1), M. Mideros (1), G. Danies (1), S. Restrepo (1) lactucae population over the past 33 years and an increase in the B1 mating type. The Californian population has predominately been the B2 mating type and reproduces asexually. The occurrence of the B1 mating type indicates the potential for sexual reproduction with prevalent B2 isolates. Sexual progeny between recent B1 and B2 isolates are fit and highly variable and may explain the recent rise in novel virulence phenotypes. This finding necessitates a change in strategy to achieve more durable disease resistance. We have also sequenced the genomes of representative isolates collected throughout the three decades as a tool for understanding variation of B. lactucae in California and the world. Population structure of Pseudoperonospora cubensis isolates in Michigan and Ontario, Canada J. BELLO (1), Y. Guo (1), M. Hausbeck (1) (1) Michigan State University, U.S.A. Pseudoperonospora cubensis is a highly destructive obligate oomycete pathogen that incites downy mildew disease in cucurbits worldwide. The pathogen populations in Michigan and Ontario are primarily affected by the movement of sporangia annually from the southern U.S. and Mexico or northern greenhouses where the pathogen can overwinter. The objective of our research was to monitor the population structure change over time in Michigan and Ontario, Canada. Sporangia were collected from cucurbit downy mildew (CMD) lesions using leaves of field grown cucurbits from 2007 to 2010, and sporangia collected in 2015. Population structure and genetic diversity were characterized using three marker types: (1) Selected genomic regions from three mitochondrial and nine nuclear genes previously used in oomycete genetic diversity and phylogenetic studies; (2) Selection from 942 simple sequencing repeat (SSR) markers derived from P. cubensis reference genome that were inside or partially overlapping with the annotated transcripts; (3) Genomic regions containing higher frequency of host and/or species specific single nucleotide polymorphisms (SNPs) adapted from previous studies. This research provides insight into changes of CMD populations in Michigan and Ontario over time which may contribute to overall management strategies. A shift in the population of Phytophthora infestans on Egyptian potato crops S. EL-GANAINY (1), Y. Ahmed (1), M. Soliman (1), A. Ismail (1), A. Tohamy (1), E. Randall (2), D. Cooke (2) (1) Plant Pathology Research Institute, Egypt; (2) The James Hutton Institute, Scotland Potato is an economically important crop in Egypt that is cultivated over three growing seasons, summer, fall (Nili) and winter. Is host specificity influencing the population structure of Phytophthora spp. collected from potato and tree tomato in central and southern Colombia? C. Chaves (1), M. Mideros (1), G. Danies (1), S. Restrepo (1) Late blight, caused by Phytophthora infestans, has been a major threat to global food security. In Colombia, late blight is considered one of the most limiting diseases on potato and tomato production. Additionally to potato and tomato crops, the pathogen has been associated with outbreaks on semi- domesticated plants such as tree tomato (Solanum betaceum) and other Solanaceous plants. Recently, a new Phytophthora species has been described infecting tree tomato crops in Southern Colombia (State of Nariño). Nonetheless, the distribution range of this new emerging pathogen in the country is unknown. Thus, the aim of this study was to investigate: i) if populations of Phytophthora associated to tree tomato in the central region of Colombia (States of Antioquia and Cundinamarca), the largest potato producing regions in the country, are genetically different from populations found on potato crops in this region; ii) if host preference exists in populations of Phytophthora associated to tree tomato and those associated to potato crops in this region; and iii) if populations of Phytophthora associated to tree tomato in the central region of Colombia are genetically distinct from those found in the southern region of the country. Preliminary results indicate that all isolates are of the A1 mating type and that host preference seems to exist between populations of Phytophthora associated to tree tomato and those associated to potato crops in the central regions of Colombia. Thirty-three years of lettuce downy mildew and counting: A phenotypic and genotypic narrative C. TSUCHIDA (1), F. Martin (2), R. Gil (1), O. Ochoa (3), I. Simon (3), R. Michelmore (3) (1) Department of Plant Pathology, University of California, Davis, U.S.A.; (2) USDA-ARS, Salinas, U.S.A.; (3) Genome Center, University of California, Davis, U.S.A. Downy mildew, caused by Bremia lactucae, is the number one foliar lettuce disease in California and worldwide. This heterothallic oomycete pathogen is highly variable and can rapidly change to overcome resistant cultivars. We are characterizing the pathogen population to determine which sources of wild germplasm will provide effective resistance in California that minimizes dependency on chemical protectants and aids organic farmers. We have been monitoring and archiving isolates of B. lactucae in California since 1982, mostly on an opportunistic basis. In December 2013, we began monthly surveys to provide a more comprehensive data set on pathogen variability. We report the patterns in virulence phenotypes observed in the Californian B. Is host specificity influencing the population structure of Phytophthora spp. collected from potato and tree tomato in central and southern Colombia? C. Chaves (1), M. Mideros (1), G. Danies (1), S. Restrepo (1) The winter crop that is grown from September to the end of February is particularly prone to late blight caused by Phytophthora infestans due to weather conditions that are conducive from the start of November and many crops suffer severe yield losses. We obtained 145 purified P. infestans isolates from the 2013 and 2014 winter crops which were genotyped using 12 SSR markers to generate data that would inform disease management. In contrast to previous work (3-year period 2010-12) in which the 13_A2 lineage was found, all isolates belonged to the 23_A1 lineage. There was no evidence for the existence of the A2 mating type or 13_A2 lineage even in the destroyed field crops of some cultivars (Cara, Bellini and Valor) that had been reported as resistant to 23_A1. This suggests that the previously reported virulence profile of 23_A1 has changed. Unlike 13_A2 the 23_A1 genotype is reported as sensitive to intermediate in response to metalaxyl. Polymorphisms in mainly 3 loci (SSR3, G11 and D13) discriminated 14 sub-clonal lineages of 23_A1 in Egypt suggesting the population has been present in Egypt for several years. The data have entered into the Euroblight database to allow temporal and spatial genetic diversity to be examined in comparison with other regional P. infestans populations. Molecular characterization and genetic diversity of Mycosphaerella fijiensis in Costa Rica using sequence based nuclear markers J. RISTAINO (1), A. Saville (1), M. Charles (1), M. Wyatt (1), S. Chavan (1) (1) North Carolina State University, U.S.A. Eight haplotypes were shared between populations from Scotland and Norway while none were shared with England. S. subarctica isolates from England were also less diverse and confined to one location suggesting geographic isolation. Preliminary data on the effect of temperature on mycelial growth and sclerotial germination S. subarctica is discussed in relation to the pathogen’s distribution. Tools for analysis of clonal population genetic data in R Z. KAMVAR (1), J. Brooks (2), N. Grünwald (3) (1) Oregon State University, U.S.A.; (2) Oregon State University, U.S.A.; (3) USDA-ARS, U.S.A. Tools for analysis of clonal population genetic data in R Z. KAMVAR (1), J. Brooks (2), N. Grünwald (3) (1) Oregon State University, U.S.A.; (2) Oregon State University, U.S.A.; (3) USDA-ARS, U.S.A. S4.139 S4.139 Knowledge of the population dynamics and evolution of plant pathogens allows inferences on evolutionary processes involved in their adaptation to hosts, pesticides, and other environmental pressures. These microbial pathogens often require different tools for analysis due to the fact that they can be clonal or partially clonal. With the advent of high-throughput sequencing technologies, obtaining genome-wide data has become easier and cheaper than ever before with techniques such as genotyping-by-sequencing. In 2013, we created the widely used R package poppr for analysis of clonal populations. In 2015, we published several additional extensions to poppr for use with genomic data including the ability to define clones based on a genetic distance threshold, minimum spanning networks with reticulation, and sliding window analysis of the index of association. We present here an overview of poppr as it pertains to traditional and high-throughput population genetic data with select applications. Is host specificity influencing the population structure of Phytophthora spp. collected from potato and tree tomato in central and southern Colombia? Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. Floodwaters can harbor human and plant pathogens and present a major risk of contamination to fresh fruit and vegetable production in Louisiana. This study evaluated the effect of flooding on the microbial quality and safety of cantaloupe in an agricultural setting. Fields (raised or flat bed) containing mature cantaloupe fruit were flooded with surface water spiked with generic Escherichia coli ATCC strains 23716, 25922, and 11775. Generic E. coli levels on the surface of cantaloupe were enumerated at 24, 48 and 72 hr after flooding. Incidence of Sclerotium rolfsii and Phytophthora capsici was measured weekly beginning 7 days prior to flooding. A significant reduction of generic E. coli was observed on the surface of cantaloupe 24, 48 and 72 S4.140 hr post flooding on fruit harvested from flat beds but only after 72 hr on the fruit from raised beds. Coliform levels remained the same over the 72 hr on fruit from both bed types. After 72 hr E. coli populations were reduced by an average of 1.8 and 2.5log MPN on fruit harvested from raised beds and flat beds respectively. No differences in S. rolfsii or P. capsci incidence on fruit from flat compared to raise beds were observed. However, disease incidence was significantly higher in flooded plots compared to non-flooded plots. These results provide science-based data that can be used to develop good agricultural practices programs to minimize food safety and plant disease hazards in flood stricken production regions. Weather patterns associated with Gibberella Ear Rot of Corn in Ohio F. DALLA LANA (1), R. Minyo (1), P. Thimison (1), L. V. Madden (1), P. A. Paul (1) (1) The Ohio State University, U.S.A. Gibberella ear rot (GER), caused by Fusarium graminearum is an important disease of corn. Since outbreaks of GER are sporadic, understanding the weather patterns associated with this disease is fundamental for GER risk assessment. The objective of this study was to determinate the association between weather conditions and GER intensity. During 2015, 15 hybrids were tested at 10 locations in Ohio. Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. At the R1 growth stage, ten arbitrarily- selected primary ears per hybrid were inoculated in the silk channel with 2 ml of a spore suspension containing 2.5 × 105 macroconidia of F. graminearum per mL. Air temperature, relative humidity (RH), surface wetness, and rainfall data were collected at 15-min intervals using local weather stations. At R6 all inoculated ears plus five non-inoculated ears were hand-harvested to estimate the incidence and severity of GER. Symptomatic infection was not observed on non-inoculated ears of any hybrid at any location, indicating that natural infection likely did not occur. Two locations had relatively high GER incidence (18 to 25%), four had low incidence (0.7 to 1.4%), while incidence was zero at the other locations. A preliminary descriptive analysis of the data indicated that GER was associated with 3 to 5 rainy days during the 7 days before R1, moderate temperatures (approximately 20?C) during R1, and RH above 80% during the week after R1. Evaluating soybean production in fields infested with Heterodera glycines and Macrophomina phaseolina with spatial regression analyses H. LOPEZ-NICORA (1), J. Carr (1), A. Dorrance (2), T. Niblack (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A. Evaluating soybean production in fields infested with Heterodera glycines and Macrophomina phaseolina with spatial regression analyses H. LOPEZ-NICORA (1), J. Carr (1), A. Dorrance (2), T. Niblack (1) (1) The Ohio State University, U.S.A.; (2) The Ohio State University, U.S.A. Heterodera glycines, the soybean cyst nematode, and Macrophomina phaseolina, causal agent of charcoal rot, are economically important soybean pathogens. The spatial distribution of both pathogens was previously described as aggregated. The objective of this study was to use and compare ordinary least squares (OLS) and spatial regression models to evaluate soybean production in fields co-infested with these pathogens and to assess their role in soybean performance. Six trials were conducted in fields naturally infested with H. glycines and M. phaseolina. Within 30 days after planting, a 10 × 10 grid of contiguous quadrats (7.62 × 7.62 m) was marked in each field. Fifteen to 20 soil cores were collected at each grid for initial (Pi) and final (Pf) H. glycines egg and M. phaseolina colony-forming unit (CFU) counts, respectively. Twenty-five plants were collected and hand-threshed from every grid for yield analysis. Moran’s I test indicated that yields from five fields were spatially correlated (P < 0.05). Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. Ancona (1) (1) Texas A&M Kingsville Citrus Center, U.S.A.; (2) Texas A&M Kingsville Citrus Center, Weslaco, TX, U.S.A.; (3) Texas A&M AgriLife Research and Extension Center, U.S.A.; (4) Texas A&M Kingsville Citrus Center, TX, U.S.A. Phytophthora foot and root rot disease, caused primarily by P. nicotianae, is present in Texas citrus orchards but there is a lack of information on the incidence and severity of the disease in the State. In addition, Texas citrus growers lack information on relative levels of P. nicotianae inoculum which are needed to make treatment decisions based on established thresholds. To address these knowledge gaps, a survey was conducted from February to June of 2015 across the three-county of Rio Grande Valley region where commercial citrus orchards in Texas are located. Twenty-four grapefruit, two tangerines and four sweet orange orchards were surveyed. 20 trees per orchard were rated for foot rot and gummosis lesion severity on a scale of 0 (no lesion) to 5 (entire trunk covered with lesion and gummosis). The survey revealed that 96% of Texas citrus groves are affected by foot rot and gummosis with the disease severity index ranging from 1 to 57%. Further analysis revealed that 20% of the surveyed citrus orchards had more than 50% disease incidence with an average disease severity of 23.6%. Phytophthora propagule counts in the soil ranged from less than, five to 118 cfu/cm3 with 46% of orchards having more than the reported damaging levels of 20 cfu/ cm3. The data will be useful to growers for making treatment decisions and in predicting yield losses in the region due to foot rot and gummosis. Potential global distribution of blueberry twig blight (Diaporthe vaccinii) predicted by two species distribution modeling approaches A. VAN BRUGGEN (1), H. Narouei-Khandan (1), C. Harmon (1), P. Harmon (1), C. Gardi (2), I. Koufakis (2), W. van der Werf (3), J. West (4) (1) University of Florida, Gainesville, FL, U.S.A.; (2) European Food Safety Authority, Parma, Italy; (3) Wageningen University, Wageningen, Netherlands; (4) Rothamsted Research, Harpenden, United Kingdom Blueberry twig blight (Diaporthe vaccinii) is an endemic disease in parts of the USA and Canada. It has been detected in limited locations in Belarus, Chile, China, Europe and Russia, but was reported to be eradicated in Chile and most of Europe, where it is a quarantine organism. Models on the potential spread of D. vaccinii do not exist. Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. The disease can occur prior to flag leaf emergence but the impact of onset time on yield has not been determined. Using 390 disease cases collected across 11 counties in North Carolina from 2012 to 2014, the effect of SNB onset on yield was analyzed to identify a disease onset threshold. A model was then developed to predict the time of SNB onset using pre-planting factors and cumulative daily infection values (cDIV) starting 1 to 3 weeks prior to the disease onset threshold. Time of disease onset explained 32% of the variation in yield (P < 0.0001), and day of year (DOY) 102 was identified as the disease onset threshold. Below-average yield occurred in 87% of the disease cases when onset occurred before DOY 102, but only in 28% of those cases where onset occurred on or after DOY 102. A binary logistic regression model indicated that cDIV l t d til t k i t DOY 102 d h t id i ifi t (P < 0 0001) di t f SNB t Th d l h d t year (DOY) 102 was identified as the disease onset threshold. Below-average yield occurred in 87% of the disease cases when onset occurred before DOY 102, but only in 28% of those cases where onset occurred on or after DOY 102. A binary logistic regression model indicated that cDIV accumulated until two weeks prior to DOY 102 and wheat residue were significant (P < 0.0001) predictors of SNB onset. The model had a correct classification rate of 0.94, and specificity and sensitivity rates were ≥ 0.91. Internal model validation based on two cross-validation techniques was also high. The model could serve as a useful decision support tool for fungicide application for SNB management in wheat. , y y g g accumulated until two weeks prior to DOY 102 and wheat residue were significant (P < 0.0001) predictors of SNB onset. The model had a correct classification rate of 0.94, and specificity and sensitivity rates were ≥ 0.91. Internal model validation based on two cross-validation techniques was also high. The model could serve as a useful decision support tool for fungicide application for SNB management in wheat. Incidence and severity of Phytophthora disease and assessment of inoculum levels in Texas citrus orchards S. CHAUDHARY (1), M. Setamou (1), O. Alabi (3), J. daGraca (1), M. Kunta (1), V. Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. In these fields, spatial error and spatial lag regression models were fitted and compared with the OLS model, which does not account for spatial correlation. Spatial regression analyses revealed a significant interaction effect between H. glycines and M. phaseolina on soybean yield for fields with high initial Pi of both pathogens. Results from this study will aid in long-term disease management and evaluation of pathogen interaction in reducing crop production. A model for predicting onset of Stagonospora nodorum blotch in winter wheat based on pre-planting and weather factors P. OJIAMBO (1), L. Mehra (1), C. Cowger (1) (1) North Carolina State University, U.S.A. A model for predicting onset of Stagonospora nodorum blotch in winter wheat based on pre-planting and weather factors P. OJIAMBO (1), L. Mehra (1), C. Cowger (1) (1) North Carolina State University, U.S.A. Stagonospora nodorum blotch (SNB), caused by Parastagonospora nodorum, is a serious disease of wheat. Its management relies mainly on fungicide application after flag leaf emergence. The disease can occur prior to flag leaf emergence but the impact of onset time on yield has not been determined. Using 390 disease cases collected across 11 counties in North Carolina from 2012 to 2014, the effect of SNB onset on yield was analyzed to identify a disease onset threshold. A model was then developed to predict the time of SNB onset using pre-planting factors and cumulative daily infection values (cDIV) starting 1 to 3 weeks prior to the disease onset threshold. Time of disease onset explained 32% of the variation in yield (P < 0.0001), and day of year (DOY) 102 was identified as the disease onset threshold. Below-average yield occurred in 87% of the disease cases when onset occurred before DOY 102, but only in 28% of those cases where onset occurred on or after DOY 102. A binary logistic regression model indicated that cDIV accumulated until two weeks prior to DOY 102 and wheat residue were significant (P < 0.0001) predictors of SNB onset. The model had a correct classification rate of 0.94, and specificity and sensitivity rates were ≥ 0.91. Internal model validation based on two cross-validation techniques was also high. The model could serve as a useful decision support tool for fungicide application for SNB management in wheat. Stagonospora nodorum blotch (SNB), caused by Parastagonospora nodorum, is a serious disease of wheat. Its management relies mainly on fungicide application after flag leaf emergence. Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. Published data on its worldwide occurrence were inventoried and supplemented with data from the National Plant Diagnostic Network. Occurrence and long-term climate data were entered in the species distribution models MaxEnt and Multi-Model Framework. The models correctly predicted that the climate in central-eastern USA, the US and Canadian west coasts, Latvia, Belarus and western Russia would be conducive to blueberry twig blight. Large areas in Europe, Eastern Australia, New Zealand, and smaller areas in South America and East Asia also were predicted to be conducive. Precipitation in the driest quarter contributed most to the prediction, followed by mean annual temperature, with an optimum of 10°C. The potential impact of D. vaccinii in Europe and its potential spread to native Vaccinium species is unknown. S4.141 The pathogen can be spread by natural and human-assisted means, and the risk of introduction, establishment and spread of D. vaccinii along different pathways, as well as potential impacts and the efficacy of risk reduction measures, will be assessed by the Plant Health Panel of the European Food Safety Authority. Symptomatology and epidemiology of Exobasidium leaf and fruit spot of blueberry R. INGRAM (1), H. Scherm (1), R. Allen (1) (1) University of Georgia, U.S.A. Over the past decade, Georgia has become one of the largest producers of cultivated blueberries in the United States. As production has expanded, several new blueberry diseases have emerged, with Exobasidium leaf and fruit spot caused by the fungal pathogen Exobasidium maculosum being the most recent. The disease causes early-season leaf spotting and green spots on fruit. The pathogen produces both basidiospores and yeast cells, but its life cycle, including its overwintering biology and primary and secondary inocula, is unknown. Epidemiological field studies initiated in 2014 included the use of trap plants, a leaf spot demography study, spore trapping, and epiphytic population enumeration. Monitoring of surface populations showed that E. maculosum is capable of overwintering epiphytically on all blueberry tissues tested during dormancy. Demography and trap plant data collected in 2015 suggested that dispersal of primary inoculum occurs shortly after leaf/flower emergence, and that infection of primarily young and tender tissue is favored by prolonged rainy periods (>3 days/week). Disease progress in 2015 indicated that the disease is active from March through late May, and that initial fruit and leaf infection occur simultaneously. Additional observations revealed a novel symptom, lesions on emerging shoots that cause girdling and blighting. Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. Although initial results suggest that the disease is monocyclic, further confirmatory studies are needed. Effect of soybean canopy closure on Sclerotinia sclerotiorum apothecia production, ascospore release, and primary plant infection of soybean M. FALL (1), M. Chilvers (1), A. Byrne (1), J. Wilbur (2), D. Smith (2) (1) Michigan States University, U.S.A.; (2) University of Wisconsin-Madison, U.S.A. Effect of soybean canopy closure on Sclerotinia sclerotiorum apothecia production, ascospore release, and primary plant infection of soybean M. FALL (1), M. Chilvers (1), A. Byrne (1), J. Wilbur (2), D. Smith (2) (1) Michigan States University, U.S.A.; (2) University of Wisconsin-Madison, U.S.A. Identifying the best timing for fungicide applications to get the optimum white mold control poses a major challenge to soybean producers. This study investigated the impact of canopy closure on apothecia development and ascospore release, to optimize fungicide application timing. In 2015, 0.355 m and 0.762 m row spacing replicated soybean plots were established at the MSU Montcalm Research Farm. The foliar fungicide Endura (boscalid) was applied to treated plots at the beginning of flowering, R1. The number of apothecia, ascospores, and the disease severity were monitored two times/week. Apothecia were first observed in the 0.355 m plots at full flower, R2, 8 d before they were observed in the 0.762 m plots, at beginning of pod development, R3. The number of apothecia was 50 times higher in the 0.355 m plots than in the 0.762 m plots. Incubated plants were symptomatic up to 13 and 18 days prior to symptom development in the 0.355 m and 0.762 m plots, respectively. There were no significant differences between non-treated and Endura treated plots in terms of final disease intensity and yield. However, there was a significant delay in disease severity (P<0.000) between treated and non-treated fungicide plots for the first 5 and 10 d after fungicide application in the 0.355 m and 0.762 m plots, respectively. There was a quantitative relationship between the number of apothecia and the soil temperature and between the number of apothecia and canopy closure. Modeling regulatory decisions in response to exotic pest detections: Pest case studies for decision makers L. BROWN (1), L. Brown (1) Modeling regulatory decisions in response to exotic pest detections: Pest case studies for decision makers L. BROWN (1), L. Brown (1) (1) APHIS Plant Protection & Quarantine, Science & Technology, U.S.A. Detections of exotic pest require responses from decision makers. An impact network analysis for management of Xanthomonas wilt of banana: Adoption thresholds for regional success of single diseased stem removal K. GARRETT (1), C. Ocimati (2), J. Ntamwira (3), G. Blomme (4) GARRETT (1), C. Ocimati (2), J. Ntamwira (3), G. Blomme (4) University of Florida, U.S.A.; (2) Bioversity, Uganda; (3) Bioversity, DR Congo, DR Congo; (4) Bioversity, Ethiopia Xanthomonas wilt of banana (BXW) is a major challenge to production for smallholder farmers of Central and Eastern Africa. Lack of resistant varieties and the often high rate of disease spread necessitate timely management. Single diseased stem removal (SDSR; removal at soil level of all visibly infected plants) is a promising approach to management requiring lower labor input compared to the previously advocated whole mat removal. We evaluate SDSR in a regional context using a new framework, Impact Network Analysis (INA). An INA is an evaluation of how technologies can have system-level impact through linked socioeconomic and biophysical networks. Estimates of short-term success rates for on-farm application of SDSR are available for several locations. INA includes projection of long-term regional outcomes based on rates of SDSR adoption. The risk of spread by garden tools is a given, but successful BXW management strategies vary by region. In most highland areas of eastern DR Congo, there is lower insect transmission risk associated with AAA type highland cooking/beer bananas. There, a regional strategy for community-level management does not depend on collective action; individual farmers can effectively manage BXW using SDSR, even if their neighbors do not manage effectively. By contrast, in most lowland areas, collective action is necessary for management, especially in zones planted to ABB type cooking/beer bananas highly susceptible to insect transmission. mpact network analysis for management of Xanthomonas wilt of banana: Adoption thresholds for regional success o l An impact network analysis for management of Xanthomonas wilt of banana: Adoption thresholds for regional success of single diseased stem removal K. GARRETT (1), C. Ocimati (2), J. Ntamwira (3), G. Blomme (4) (1) University of Florida, U.S.A.; (2) Bioversity, Uganda; (3) Bioversity, DR Congo, DR Congo; (4) Bioversity, Ethiopia Evaluation of the microbial quality of cantaloupe fruit produced on raised or flat beds following a flooding event I. KIKWAY (1), M. Lewis Ivey (1), I. Kikway (1) (1) Louisiana State University, U.S.A. Response options include management actions such as: eradication, containment, localized control, or the decision to not implement a program. When uncertainty is high, situational context can drive decision-making, leading to responses that are counter intuitive to epidemiological principles of infectivity, symptom expression, and pathogen spread. Spatially explicit models like Webidemics or spatially implicit models like ExPAT are used to model outcomes of decisions, reducing the reliance on highly uncertain information. These tools are applied based on the situation presented by the particular case study. Based on multiple case studies of all pathogen groups, we will present a matrix highlighting for decision-makers which tools are best suited for different exotic pest detections. In the future, the selection and parameterization of proper tools will supply decision makers with quantitative data to evaluate exotic pest detections. Seedborne inoculum thresholds of Pseudomonas syringae pv. aptata, causal agent of bacterial leaf spot, in ‘baby leaf’ Swiss chard crops L. DU TOIT (1), M. Derie (1), B. Holmes (1), I. Safni (2), C. Bull (2) (1) Washington State University U S A ; (2) Pennsylvania State University U S A Ramirez (1) (1) Universidad de Antioquia, Colombia; (2) Reading University, England; (3) Universidad Eafit, Colombia Ralstonia solanacearum is a soilborne plant pathogen that causes bacterial wilt on a wide range of hosts of economic importance. This disease is particularly devastating in Musa spp., where it is known as Moko disease. A major challenge in controlling this disease is to understand the diversity of the pathogen and how it adapts over time. With such knowledge, it may be possible to identify drivers of adaptation and inform changes in management practices. We therefore sought to characterize the genotypic and phenotypic diversity of R. solanacearum isolates obtained from plantations of Musa spp. in Colombia, and infer their phylogenetic relationship. Sixty-six isolates were collected. The genetic diversity and phylogenetic relationship among all the isolates were assessed by multiplex PCR and sequence analyses (mutS, rplB, and egl). Multiplex PCR assigned all the strains to phylotype II/sequevars 4 and 6. Similarly, the phylogenetic analysis of the gene sequences confirmed the classification of the strains into phylotype II; but, in addition, identified three separate clusters: IIA/6 (banana), IIB/4 (banana), and IIB/4 (plantain). Pathogenicity tests showed symptoms of wilt on banana plants after one month, but only strains isolated from plantain infected tomato. This study shows that plantain and banana plantations in Colombia harbor genetically diverse strains of R. solanacearum, belonging to at least three separate clades, and with differences in pathogenicity. Phylogenomic analysis supports polyphyly in Pythium sensu lato E. GOSS (1), M. Ascunce (2), J. Huguet-Tapia (2), E. Braun (2), A. Ortiz-Urquiza (2), N. Keyhani (2) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Pythium is a large and morphologically diverse genus that includes economically costly root pathogens. Molecular phylogenies support the morphological clustering of Pythium into three monophyletic groups: Clades A-D are characterized by filamentous sporangia, clades E-J by globose sporangia, and clade K has been reassigned to the new genus Phytopythium. However, different markers have supported different relationships among these clades and to the genus Phytophthora. The absence of a robust phylogeny has prevented advances in Pythium systematics and evolutionary genomics. In the last six years, whole genome sequences have been generated for seven Pythium species and ten other oomycetes. We conducted a phylogenomic analysis using the nucleotide sequences of 277 orthologous genes found in the 17 analyzed oomycete genomes to examine the evolutionary relationships among the major clades of Pythium. Seedborne inoculum thresholds of Pseudomonas syringae pv. aptata, causal agent of bacterial leaf spot, in ‘baby leaf’ Swiss chard crops L. DU TOIT (1), M. Derie (1), B. Holmes (1), I. Safni (2), C. Bull (2) (1) Washington State University U S A ; (2) Pennsylvania State University U S A Koike (5), V. Stockwell (6), C. Bull (2) (1) Department of Agroecotechnology, Faculty of Agriculture, University of Sumatera Utara, Utara, Indonesia; (2) Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park, PA, U.S.A.; (3) USDA/ARS, Salinas, CA, U.S.A.; (4) Washington State University, Mount Vernon, WA, U.S.A.; (5) Univ of California Cooperative Extension, Salinas, CA, U.S.A.; (6) USDA/ARS, Corvallis, OR, U.S.A. Genetic diversity of Pseudomonas syringae causing bacterial leaf spot on table beet (Beta vulgaris) and Swiss chard (Beta vulgaris subsp. cicla) I. SAFNI (1), L. Ramos-Sepulveda (2), P. Goldman (3), M. Derie (4), L. du Toit (4), S. Koike (5), V. Stockwell (6), C. Bull (2) (1) Department of Agroecotechnology, Faculty of Agriculture, University of Sumatera Utara, Utara, Indonesia; (2) Department of Plant Pathology and Environmental Microbiology, Penn State University, University Park, PA, U.S.A.; (3) USDA/ARS, Salinas, CA, U.S.A.; (4) Washington State University, Mount Vernon, WA, U.S.A.; (5) Univ of California Cooperative Extension, Salinas, CA, U.S.A.; (6) USDA/ARS, Corvallis, OR, U.S.A. Table beet and Swiss chard are grown throughout the US, with a significant proportion of baby leaf production in California and 90% of US seed production in Oregon and Washington. Bacterial leaf spot on beet and Swiss chard, caused by Pseudomonas syringae pv. aptata (Psa), has been reported from California and Oregon, but not yet from Washington. Since 2001, pseudomonads were isolated from surface-disinfested, symptomatic beet and chard plants grown in the western US, including Washington. Strains were variable for fluorescence on KMB agar medium, indicating populations may be diverse. Genetic diversity of the 99 strains was evaluated by repetitive extragenic palindromic PCR (rep-PCR) assay using the BOX AIR primer. Strains from a single Washington outbreak from a chard crop planted with infested seed had DNA fragment-banding patterns identical to the pathotype strain of Psa. The majority of the other isolates were identical, with a pattern similar to but distinct from the pattern of the pathotype. Three additional DNA fragment-banding patterns were observed. Strains from single outbreaks were represented in each of the alternative banding patterns. Preliminary multilocus sequence analysis and pathogenicity tests suggest a majority of bacterial leaf spot outbreaks on chard and table beet in California, Oregon, and Washington are caused by haplotypes distinct from Psa. Diversity and phylogeny of moko disease-causing Ralstonia solanacearum strains in Colombia M. RAMIREZ (1), R. Jackson (2), V. Villegas-Escobar (3), J. Correa-Álvarez (3), C. Seedborne inoculum thresholds of Pseudomonas syringae pv. aptata, causal agent of bacterial leaf spot, in ‘baby leaf’ Swiss chard crops L. DU TOIT (1), M. Derie (1), B. Holmes (1), I. Safni (2), C. Bull (2) (1) Washington State University U S A ; (2) Pennsylvania State University U S A Garrett (5) (1) University of Florida, Gainesville, FL, U.S.A.; (2) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Lima, Peru; (3) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Beijing, China; (4) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Quito, Ecuador; (5) Plant Pathology Department, Emerging Pathogens Institute and Institute for Sustainable Food Systems, University of Florida, Gainesville, FL, U.S.A. ( ), ( ), ( ), ( ), ( ), ( ), ( ) (1) University of Florida, Gainesville, FL, U.S.A.; (2) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Lima, Peru; (3) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Beijing, China; (4) International Potato Center, CGIAR Research Program on Roots Tubers and Bananas (RTB), Quito, Ecuador; (5) Plant Pathology Department, Emerging Pathogens Institute and Institute for Sustainable Food Systems, University of Florida, Gainesville, FL, U.S.A. The potato seed system is a multilayer network that includes social, economic, and agroecological components. Producers, breeding institutions, markets, technical support, and diseases are components of this complex system that interact and modify each other. We studied the structure of the networks of seed sources, markets, and IPM information sources, to understand impacts on the potato seed system of the Consortium of Small Potato Producers (CONPAPA) in Tungurahua, Ecuador. Using information about the structure of the seed system, we can consider how resilient the system is likely to be to stressors such as the introduction of a new pathogen. Which types of farmers may be more vulnerable to losses to such new stressors? One development goal is for there to be good access to quality seed and quality management recommendations, regardless of gender or farm size. Female farmers reported fewer diseases causing seed degeneration than male farmers, and fewer sources of IPM recommendations among the sources less likely to have commercial biases. Identification of key nodes that influence the success of seed systems (e.g., farmers, information, and seed sources) supports enhancement of the system, for improvements such as maximizing the distribution of good varieties, managing outbreaks, and targeted improvement of communication and infrastructure. Genetic diversity of Pseudomonas syringae causing bacterial leaf spot on table beet (Beta vulgaris) and Swiss chard (Beta vulgaris subsp. cicla) I. SAFNI (1), L. Ramos-Sepulveda (2), P. Goldman (3), M. Derie (4), L. du Toit (4), S. Seedborne inoculum thresholds of Pseudomonas syringae pv. aptata, causal agent of bacterial leaf spot, in ‘baby leaf’ Swiss chard crops L. DU TOIT (1), M. Derie (1), B. Holmes (1), I. Safni (2), C. Bull (2) (1) Washington State University U S A ; (2) Pennsylvania State University U S A Seedborne inoculum thresholds of Pseudomonas syringae pv. aptata, causal agent of bacterial leaf spot, in ‘baby leaf’ Swiss chard crops L. DU TOIT (1), M. Derie (1), B. Holmes (1), I. Safni (2), C. Bull (2) (1) Washington State University, U.S.A.; (2) Pennsylvania State University, U.S.A. Most US table beet and Swiss chard seed production (~90%) occurs in western Washington and Oregon. Pseudomonas syringae pv. aptata (Psa), the cause of bacterial leaf spot, has been identified in beet and chard seed from this region, but seedborne inoculum thresholds have not been established. Chard seed naturally infested with Psa at 8.6 × 103 CFU/g was mixed with seed of the same cultivar infested at <10 CFU/g to generate a log10 series of infestation. Five replicate plots/infestation level were planted in a field in Mount Vernon, WA at ~10.7 × 106 seeds/ha in May 2015 in a randomized complete block design. Bacterial leaf spot incidence was 52 ± 18% in plots planted with seed infested at 8.6 × 103 CFU/g vs. 15 to 21% in plots sown with seed infested at <103 CFU/g. Seed infestation level did not affect plant stand or amount of Psa recovered from leaves onto KBC medium. The trial was repeated in the fall, when moist, cool conditions resulted in a leaf spot incidence of 93 ± 2% across all plots, and Psa was recovered from every plot (9.97 × 1011 ± 1.82 × 1011 CFU/g dry leaf tissue), with no differences among seed infestation levels. Plots sown with seed infested at 8.6 × 103 CFU/g had 33% fewer plants, 29% less leaf dry weight, and 126% more severe leaf spot than plots planted with seed infested at <103 CFU/g. Psa was transmitted from infested chard seed, even at <10 CFU/g. Environmental conditions in the two trials affected seed transmission and leaf spot severity. S4.142 S4.142 Multilayer Networks Supporting Healthy Seed Systems for Smallholder Potato Producers in Tungurahua, Ecuador tilayer Networks Supporting Healthy Seed Systems for Smallholder Potato Producers in Tungurahua, Ecuador ERNANDEZ NOPSA (1), J. Andrade-Piedra (2), G. Forbes (3), P. Kromann (4), S. Lei (5), J. Brisbane (5), K. Garrett (5) Multilayer Networks Supporting Healthy Seed Systems for Smallholder Potato Producers in Tungurahua, Ecuador J. HERNANDEZ NOPSA (1), J. Andrade-Piedra (2), G. Forbes (3), P. Kromann (4), S. Lei (5), J. Brisbane (5), K. Seedborne inoculum thresholds of Pseudomonas syringae pv. aptata, causal agent of bacterial leaf spot, in ‘baby leaf’ Swiss chard crops L. DU TOIT (1), M. Derie (1), B. Holmes (1), I. Safni (2), C. Bull (2) (1) Washington State University U S A ; (2) Pennsylvania State University U S A Our results supported the position of Phytopythium vexans as a sister clade to Phytophthora. As expected, the remaining Pythium taxa formed two monophyletic groups. One group was composed of three taxa that correspond to Pythium clades A, B and C, and the other group contained taxa representing clades F, G and I, which was consistent with previous Pythium phylogenies. However, the group containing Pythium clades F, G and I formed a sister group to the Phytophthora-Phytopythium clade and thus Pythium clades F-J are not monophyletic. hemical changes in the wheat cell-wall resulting from stripe rust infection in compatible and incompatible near isogen R. LAHLALI (1), G. Brar (2), D. Qutob (3), C. Karunakaran (1), H. Kutcher (2) (1) Canadian Light Source Inc., Saskatoon, SK, Canada; (2) Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon, Canada; (3) Canadian National Research Council- Plant Biotechnology Institute, Saskatoon, SK, Canada; (4) Department of Plant Sciences/Crop Development Centre, University of Saskatchewan, Saskatoon, SK, Canada Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most devastating diseases in North America. The biotrophic fungus produces uredia, consisting asexual urediospores, on wheat leaves along the veins and obtains nutrition from host through haustoria. In a compatible interaction, the fungus damages the cell-wall in a susceptible cultivar, however in a resistant cultivar the incompatible interaction occurs as a result of hypersensitive cell death in the host. The purpose of this study was to understand the biomolecular changes in cell-wall compounds in each of the interactions. Two Avocet lines: one carrying Yr10 and other susceptible (no resistance allele) were inoculated with two isolates (one virulent on both and the other virulent only on Yr10). Infected and freeze dried leaves collected at 1 day and 15 days post inoculation were assessed by Fourier transform infrared (IR) spectroscopy. A significant difference in disease severity was observed between the two wheat lines. Principal component analysis of the IR spectra indicated substantial differences in the cell-wall components of the two wheat lines before and after pathogenic infection. There was a significant difference in the biomolecular changes between compatible and incompatible interactions. The most important IR peaks in the leaf cell wall that contributed to such differences were assigned to phenolic and aromatic rings, and proteins. Small RNA processing in Sclerotinia sclerotiorum S. MARZANO (1), L. Domier (2) ( ), ( ) h Dakota State University, U.S.A.; (2) USDA/ARS, University of Illinois, Urbana-Champaign, U.S.A. ( ) ( ) (1) South Dakota State University, U.S.A.; (2) USDA/ARS, University of Illinois, Urbana-Champaign, U.S.A. RNA silencing pathways are under-studied in plant pathogenic fungi. Once elucidated, they can be utilized to improve plant health by more effectively silencing fungal genes from host plants or viral vectors. In this study, we characterized changes in gene expression and small RNA accumulation in Sclerotinia sclerotiorum, a damaging and widely distributed fungal plant pathogen, in response to hypovirus infection. Our results indicated that the fungus produced small RNAs of predominantly 22 nt. The S. cation of essential protein machinery involved in the plant disease resistance-signaling node in Arabidopsis, NDR1 RION (1), B. Day (2) A. CORRION (1), B. Day (2) (1) Michigan State University, East Lansing, MI, U.S.A.; (2) Michigan State University, U.S.A. ( ), y ( ) (1) Michigan State University, East Lansing, MI, U.S.A.; (2) Michigan State University, U.S.A. Plant disease epidemics result in an estimated $200 billion in crop losses globally each year. To understand how plants and pathogens interact, and moreover, to develop a fundamental understanding of the mechanisms that drive pathogen virulence and adaptation, research in our group is focused on the molecular-genetic processes underpinning host resistance. To do this, we use the Arabidopsis thaliana – Pseudomonas syringae pv. tomato (Pst DC3000) pathosystem to study the role of non-host disease resistance-1 (NDR1), a key regulator of the of the disease signaling network of CC-NB-LRR (coiled-coil nucleotide binding leucine rich repeat) resistance (R) proteins. In the current study, we present a comprehensive analysis, utilizing a combination of genetic-, biochemical- and cell biology-based approaches to identify and define the NDR1 interactome. In brief, we utilized immunoprecipitation methods, coupled with liquid chromatography mass spectroscopy, to identify a list of candidate interacting proteins. Through this approach, we have identified a family of proteins that interact with NDR1 directly in planta, and have begun to characterize the role of these interactions in the NDR1-dependent resistance-signaling pathway. Data will be presented that provides support for a model that posits NDR1’s association with key, Rho-related GTPases from plants (ROPs), plasma membrane-localized signaling proteins involved in the resistance to Pst DC3000. S4.143 Examining putative motility genes in Clavibacter michiganensis subsp. michiganensis C. PERITORE (1), M. Tancos (2), C. Smart (2) (1) Cornell University, U.S.A.; (2) Cornell University, U.S.A. The Gram-positive bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) is the causative agent of bacterial canker of tomato (Solanum lycopersicum). Cmm historically has been characterized as a non-motile pathogen, however, it is known to move acropetally and basipetally in the xylem of infected plants. Under artificial xylem conditions in microfluidic chambers, non-flagellated Cmm actively moved against the media flow at ~6 µm/minute; these speeds were similar to those observed in planta. We have hypothesized that Cmm uses a type IV pilus (Tfp) mediated motility system similar to that of Xylella fastidiosa. Tfp are diverse in that biosynthesis can be mediated by pil, com, and/or tad operons in both Gram-negative and positive bacteria. of Fusarium thapsinum on host nitric oxide (NO) synthesis among stalk rot resistant and susceptible sorghum lines C Li l (1) Differential effects of Fusarium thapsinum on host nitric oxide (NO) synthesis among stalk rot resistant and susceptible sorghum lines A. BANDARA (1), C. Little (1) (1) Kansas State University, U.S.A. Fusarium thapsinum (FT) is a hemibiotrophic fungus causing stalk rot in sorghum (Sorghum bicolor (L.) Moench). NO is a powerful oxidant involved in programed cell death and FT-mediated NO synthesis was investigated in host stalk tissues. Two greenhouse-grown stalk rot resistant (SC599, SC35) and susceptible (Tx7000, BTx3042) sorghum lines were inoculated with FT and phosphate-buffered saline (control). At 7 and 30 d post-inoculation (d.p.i.), FT- and mock-inoculated stem cross sections were treated with 4-amino-5-methylamino-2?,7?-difluorofluorescein diacetate, and observed under a confocal microscope (excitation 488; emission 515 nm). The fluorescence generated by controls was used as the baseline. At 7 d.p.i, strong green fluorescence was observed in FT-inoculated resistant lines, confirming the presence of NO, while no (or weak) signal was evident at 30 d.p.i. The susceptible lines revealed the opposite phenomenon, except for the signal detected in BTx3042 at 7 d.p.i. These patterns suggested that FT switches from biotrophy to necrotrophy by 30 d.p.i. While the presence of NO in SC599 and SC35 at 7 d.p.i contributes to resistance against the biotrophic phase, its absence at 30 d.p.i may decrease vulnerability to the necrotrophic phase. Tx7000 showed the opposite response and is susceptible to both phases of the pathogen’s lifestyle. Based on NO response, BTx3042 appeared to be moderately resistant to the biotrophic phase but prone to the necrotrophic phase of FT. Stalk sugar content and charcoal rot disease reaction in grain sorghum A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1) (1) Kansas State University, U.S.A. Charcoal rot, caused by Macrophomina phaseolina (MP), is an important fungal disease in grain sorghum (Sorghum bicolor (L.) Moench). An RNA-Seq experiment was conducted to explore the molecular basis of the sorghum-MP interaction. Stalk RNA extracted from MP- and mock-inoculated resistant (SC599) and susceptible (Tx7000) sorghum lines at 2, 7, and 30 d post-inoculation (d.p.i.) was subjected to RNA-Seq. Genes involved in UDP-glucose conversion, sucrose degradation to ethanol and lactate, and fructose degradation to pyruvate and lactate metabolism were significantly up-regulated in Tx7000 at 7 d.p.i., while those of SC599 were not differentially expressed. of Fusarium thapsinum on host nitric oxide (NO) synthesis among stalk rot resistant and susceptible sorghum lines C Li l (1) To confirm the results, stalk juice was extracted at 7 d.p.i from two MP- and mock-inoculated resistant (SC599, SC35) and susceptible (Tx7000, BTx3042) lines and subjected to HPLC. Concentrations (mg/mL) of sucrose, glucose, and fructose in stalk juice of SC599 (95.3, 23.9, 20.7, respectively) and SC35 (66.2, 31.2, 23.4) were significantly greater than those of Tx7000 (2.7, 6.5, 4.1) and BTx3042 (9.1, 10.9, 6.9) across MP- and mock-inoculations (P < 0.0001). Moreover, the reduction of sucrose, glucose, and fructose in MP-inoculated susceptible lines was 86.4, 26.5, and 19.8%, respectively, while those of the resistant lines were 17.0, 21.5, and 7.3%. Therefore, sorghum genotypes with higher stalk sugar content along with lesser sugar reduction under MP infection appeared to be more resistant to charcoal rot disease. cation of essential protein machinery involved in the plant disease resistance-signaling node in Arabidopsis, NDR1 RION (1), B. Day (2) Two tight adhesion (tad) operons have been identified through sequence similarity in the annotated genome of Cmm, and literature suggests they may play a crucial role in biosynthesis of Tfp. Cmm transformants were generated by disrupting the tad operon promoter. Microfluidic chambers will be utilized to mimic in planta conditions of xylem flow to assay and compare motility between mutants and wildtype. Genetic and microscopic studies will be complemented with in planta virulence assays to investigate the role of tad genes in the Cmm-tomato pathosystem. Understanding how the pathogen infects and moves in the plant will strengthen our biological knowledge of Cmm, possibly leading to novel disease control strategies. hemical changes in the wheat cell-wall resulting from stripe rust infection in compatible and incompatible near isogen sclerotiorum genome was predicted to contain 59 families of microRNA-like (milRNA) genes, which similar to other recently characterized fungal species, were poorly conserved with other eukaryotes. In degradome analyses, 39 of the milRNAs sequences were predicted to mediate cleavage of 47 S. sclerotiorum mRNAs. Consistent with the role of RNA silencing in defense against mobile and infectious genetic elements, most of the small RNA sequences in virus-free S. sclerotiorum were derived from retrotransposons. Similarly, large amounts of predominantly 22 nt sequences were detected from the hypovirus genome, indicating that it also was targeted for degradation by host RNA-mediated antiviral defenses. These findings suggest that virus-induced hypovirulence could result from disruption of RNA silencing and that a robust RNA silencing pathway exists in S. sclerotiorum that can be exploited for RNA-based pathogen resistance strategies. S4.144 Investigation on the role of molecular composition and lignification of canola plant cell wall in resistance mechanisms Rcr1 Investigation on the role of molecular composition and lignification of canola plant cell wall in resistance mechanisms associated with CR gene Rcr1 R. LAHLALI (1), T. Song (2), M. Chu (2), F. Yu (3), L. McGregor (2), B. Gossen (2), S. Kumar (2), C. Karunakaran (1), G. Peng (2) (1) Canadian Light Source Inc., Saskatoon, SK, Canada; (2) Agriculture and Agri-Food Canada, Saskatoon, SK, Canada; (3) Agriculture and Agri-Food Canada, Saskatoon, Canada Rcr1 R. LAHLALI (1), T. Song (2), M. Chu (2), F. Yu (3), L. McGregor (2), B. Gossen (2), S. Kumar (2), C. Karunakaran (1), G. Peng (2) (1) Canadian Light Source Inc., Saskatoon, SK, Canada; (2) Agriculture and Agri-Food Canada, Saskatoon, SK, Canada; (3) Agriculture and Agri-Food Canada, Saskatoon, Canada Clubroot disease, caused by Plasmodiophora brassicae Woronin, is a serious soil-borne disease of crucifer crops worldwide. It poses a serious threat to canola production in western Canada. Rcr1 is a new clubroot resistance gene identified in Brassica rapa. A multifaceted approach was employed to understand the molecular changes in canola plant cell wall associated with Rcr1-mediated clubroot resistance and the expression of defense-related genes involved in the lignin pathway. Fourier Transform mid Infrared (FTIR) spectral analysis showed that non-inoculated canola roots from both resistant (R) and susceptible (S) populations were grouped together and distinctly separated from those inoculated with P. brassicae. A sensitive method for quantifying cercosporin, a fungal-derived secondary metabolite, in plant tissue J. SMITH (1), J. Lay (2), B. Bluhm (2) (1) University of Arkansas, U.S.A.; (2) University of Arkansas, U.S.A. A sensitive method for quantifying cercosporin, a fungal-derived secondary metabolite, in plant tissue J. SMITH (1), J. Lay (2), B. Bluhm (2) (1) University of Arkansas U S A ; (2) University of Arkansas U S A Cercospora species infect a wide range of host plants, including soybean and maize. Many species of Cercospora produce the phytotoxic secondary metabolite cercosporin. The role of cercosporin as a virulence factor has been demonstrated in several Cercospora species. However, it is not clear whether cercosporin is important for virulence in all species of Cercospora. To gain a more complete understanding of the role of cercosporin in plant pathogenesis, sensitive methods for its detection and quantification are required. Therefore, the objective of this research was to develop a mass spectrometry based method for quantification of cercosporin from plant tissues with high sensitivity. To this end, extraction, sample cleanup, chromatographic separation, and mass spectrometry based detection methods were developed and optimized. The use of liquid chromatography coupled with a triple quadrupole mass spectrometer allowed detection of cercosporin at concentrations as low as 1 ng/ml. Currently, the feasibility of using this method for quantifying cercosporin in soybean leaves and seed infected with C. kikuchii and maize leaves infected with C. zeae-maydis is being examined. A sensitive method for detecting and quantifying cercosporin will provide a valuable tool for determining the role of this compound in virulence among Cercospora species. Role of Cryphonectria parasitica Cdc48-like gene, CpCdc48 in regulation of growth & cell division D. KIM (1) Role of Cryphonectria parasitica Cdc48-like gene, CpCdc48 in regulation of growth & cell division D. KIM (1) (1) Chonbuk National University, South Korea Functional analysis of a cell division cycle 48 (CDC48) ortholog, CpCdc48, from Cryphonectria parasitica was performed via construction of a CpCdc48-null mutant. Genotype analysis revealed that the putative CpCdc48-null mutant was a heterokaryotic transformant containing two different types of nuclei (i.e., one with the wild-type CpCdc48 allele and the other with the CpCdc48-null mutant allele). Although stable mycelial growth of the heterokaryotic transformant was observed on media containing hygromycin B, neither germination nor growth of the resulting spores was observed on selection media, suggesting that the CpCdc48 gene is essential. Microscopic analysis of germinated conidia from the heterokaryon demonstrated that although there were normal germinating spores due to the wild-type conidia, there were many residual conidia that did not germinate. However, with prolonged incubation, non-germinating conidia began to swell into gigantic globose spores. DAPI staining and FACS analysis of the gigantic spores revealed the presence of multiple nuclei. These gigantic conidia did not show any signs of polarized growth and underwent autolysis with further incubation. These findings indicate that the CpCdc48 gene is responsible for delayed cell cycle during spore germination, resulting in some karyokinesis, but not following spore cytokinesis. Thus, CpCdc48 is essential for cell division and polarized growth. hemical changes in the wheat cell-wall resulting from stripe rust infection in compatible and incompatible near isogen R and S plants responded differentially to the infection; it appeared that the cell-wall components of roots were changed for plants carrying Rcr1, including lipids, proteins, lignin, aromatic and phenolics ring, polysaccharides, and carbohydrates. qPCR identified the upregulation of nine defense-related genes involved in the lignin pathway, as well as in chitinase and xyloglucan endo-transglycosylate activities in roots carrying Rcr1 relative to those of non-inoculated, but the level of increase was more pronounced with plants carrying Rcr1. The changes cell-wall components may reflect differential regulation of genes involved in host defense responses, in particular, the lignin pathways and chitinase synthesis mediated by Rcr1 during clubroot resistance. Wheat streak mosaic virus-encoded NIa-Pro and coat protein are involved in virus superinfection exclusion S. TATINENI (1) (1) USDA-ARS, U.S.A. Cross protection or superinfection exclusion (SE) is defined as the phenomenon whereby initial infection by one virus prevents subsequent infection by closely related viruses. The mechanisms of SE are just beginning to be understood. Wheat streak mosaic virus (WSMV; genus: Tritimovirus; family: Potyviridae) is an economically important wheat virus. Proteins encoded by WSMV were individually expressed in wheat using an unrelated Triticum mosaic virus (TriMV) gene expression vector, followed by challenge inoculation with GFP-tagged WSMV (WSMV-GFP). Wheat plants infected with TriMV expressing WSMV P1, HC-Pro, P3, 6K1, CI, 6K2, NIa-VPg or NIb cistrons did not prevent superinfection by WSMV-GFP. However, expression of WSMV NIa-Pro or CP greatly reduced superinfection by WSMV-GFP, suggesting that NIa-Pro and CP are involved in SE. Expression of WSMV NIa-Pro or CP cistron with frameshift mutations did not exhibit SE against WSMV-GFP, indicating that the NIa-Pro and CP proteins but not the RNA sequences are involved in SE. A series of deletions comprising 60 amino acid (aa) sets covering the entire NIa-Pro cistron revealed that the complete NIa-Pro protein is required for SE activity. In contrast, WSMV CP aa 3-100 and 301-349 were dispensable for SE but not aa 101-300. Expression of WSMV CP aa 101 to 300 in TriMV exhibited the same SE activity as full-length CP. In reciprocal experiments, wheat infected with WSMV expressing TriMV CP prevented superinfection by TriMV-GFP. Overexpression of a modified plant thionin enhances disease resistance to citrus canker and Huanglongbing (HLB, citrus greening) G. HAO (1), E. Stover (1) (1) USDA/ARS/HRL U S A Overexpression of a modified plant thionin enhances disease resistance to citrus canker and Huanglongbing (HLB, citrus greening) G. HAO (1), E. Stover (1) (1) USDA/ARS/HRL, U.S.A. Huanglongbing (HLB or citrus greening disease) caused by Candidatus Liberibacter asiaticus (Las) is a great threat to the US citrus industry. Citrus canker is also an economically important disease associated with a bacterial pathogen (Xanthomonas citri). In this study, we characterized endogenous citrus thionins and investigated their expression in different citrus tissues. We aim to develop citrus resistant to both HLB and citrus canker through expression of modified plant thionin. In order to improve effective for disease resistance, we modified and synthesized the sequence encoding plant thionin and cloned into binary vector pBinPlus/ARS. The construct was introduced into Agrobacterium strain EHA105 and used for citrus transformation. Transgenic Carrizo plants expressing the modified plant thionin were successfully generated through Agrobacterium-mediated transformation. Transgenic Carrizo plants expressing the modified thionin gene were challenged with X. citri 3213 at various concentrations, and showed a significant reduction in canker symptoms and a decrease in bacterial growth compared to nontransgenic plants. Furthermore the transgenic citrus plants were challenged with HLB via graft inoculation. The results showed significant decrease in the Las titer in transgenic Carrizo compared with nontransgenic plants. These data, thus, provide a promising approach to engineering citrus disease resistance against HLB and canker. Transgenic Carrizo expressing ‘Candidatus Liberibacter asiaticus’ effector P235 displays Huanglongbing-like symptom and alters some gene expression G. HAO (1) (1) USDA/ARS/HRL, U.S.A. Transgenic Carrizo expressing ‘Candidatus Liberibacter asiaticus’ effector P235 displays Huanglongbing-like symptom and alters some gene expression G HAO (1) ‘Candidatus Liberibacter asiaticus’ (Las) is the agent causing destructive citrus huanglongbing worldwide. A putative protein (P235) was identified in Las genome prophage region, which encodes a 123 amino-acid protein with a molecular weight of 14.19 kD. Green fluorescence protein (GFP)-fused with P235 appeared to accumulate in Nicotiana benthamiana cell nuclei by Agrobacterium-mediated transient expression, however, P235-GFP fusion protein in transgenic citrus targets the citrus chloroplast. P235 alone was constructed into a binary vector for citrus transformation. Over 40 independent transgenic Carrizo plants were obtained. Gene integration and expression were confirmed by molecular analysis. We observed that some P235- expressing Carrizo plants displayed HLB-like symptoms with leaf chlorosis and plant growth retardation. P235 expression levels, determined by RT- qPCR, correlated with HLB-like symptoms. Overexpression of a modified plant thionin enhances disease resistance to citrus canker and Huanglongbing (HLB, citrus greening) G. HAO (1), E. Stover (1) (1) USDA/ARS/HRL U S A Transcriptome analysis revealed that carbohydrate metabolism and biotic stress pathways were significantly affected in transgenic citrus expressing p235 effector. Higher transcript levels were determined for several stress-related genes involved in hormone metabolism and zinc ion binding. This is the first report that ‘Candidatus Liberibacter asiaticus’ encodes a protein that targets the chloroplast of plant cells and alters plant-host phenotypes. Bioluminescent imaging as a sensitive tool for evaluation of Ralstonia solanacearum infection dynamics in resistant and susceptible pepper lines H. DU (1), B. Chen (1), X. Zhang (1), S. Miller (2), G. Rajashekara (3), X. Xu (1), S. Geng (1) (1) Beijing Vegetable Research Center (BVRC) of BAAFS, China; (2) Department of Plant Pathology, Ohio Agricultural Research Development Center, Ohio State University, Wooster, U.S.A.; (3) Food Animal Health Research Program, Ohio Agricultural Research Development Center, Ohio State University, Wooster, U.S.A.; (4) Beijing Vegetable Research Center (BVRC) of BAAFS, Bejing, China Bacterial wilt, caused by Ralstonia solanacearum, is a major disease affecting pepper (Capsicum annuum) production worldwide. In this study, we evaluated the resistance of 100 pepper lines using R. solanacearum strain Rs-SY1 (phylotype I, isolated from sweet pepper in South China) and identified one resistant pepper line, BVRC 1. In order to study the bacterial spatial-temporal distribution in planta in real time, we generated a strongly bioluminescent R. solanacearum strain (BL-Rs7) using pXX3 carrying luxCDABE genes. BL-Rs7 was similar to its parent strain Rs-SY1 in morphology and pathogenicity. Furthermore, luminescence intensity of BL-Rs7 strongly correlated with bacterial population in planta (R2 = 0.88). The utility of bioluminescence assay was validated by real-time in vivo monitoring of R. solanacearum infection dynamics in resistant and susceptible pepper lines BVRC 1 and BVRC 25, respectively, following root inoculation. The luminescence signals were detected in susceptible pepper plants from 3 days post inoculation (dpi), while little signal was detected in roots or stem of the resistant pepper line over 10 dpi and only a very low titer of bacteria was detected in the root. The results suggested that pepper line BVRC 1 exhibits resistance by interfering with translocation and multiplication of R. solanacearum in the stem. approaches for the genetic improvement of cowpea (Vigna unguiculata(L.)Walp) and soybean (Glycine max(L.) Merr HANDRAN (1) Transgenic approaches for the genetic improvement of cowpea (Vigna unguiculata(L.)Walp) and soybean (Glycine max(L.) Merrill) V. RAMACHANDRAN (1) (1) BHARAT UNIVERSITY, India Herbicide-resistant transgenic improvement of Cotyledone explants excised from 3 day old in vitro of Vigna unguiculata and Glycine max were infected with Agrobacerium tumefaciens (LBA4404) harboring chitinase (chi II) and bar gene. Co- cultivated cotyledon explants were inoculated on MS medium containing B5 vitamins BA, TDZ, L-glutamine, adenine sulphate, basta and cefotaxime. Shoots formed at the proximal end of cotyledons were transferred to MS medium containing B5 vitamins, GA3, basta and cefotaxime for elongation. Elongated shoots rooted in hormone free half strenth MS medium. Rooted plants were subsequently established in soil. Transformed plants were conformed by herbicide resistance and PCR analysis. These ready systems enable us to improve seed qualities and agronomic characteristics by transgenic approaches. In addition, with the accumulation of cowpea and soybean genomic resources, convenient or promising approaches will be requisite for the determination and use of gene function in cowpea and soybean. In this article, we describe recent advances in and problems of cowpea and soybean transformation, and survey the current transgenic approaches for applied and basic research in India. Displaying foreign proteins and peptides on the surface of Pepino mosaic virus virions C. LI (1), S. Shi (1), N. Yu (1), Z. Xiong (1) (1) University of Arizona, U.S.A. In planta interaction between Tobacco mosaic virus 126 kDa protein and a host membrane protein (SNARE) and its i accumulation In planta interaction between Tobacco mosaic virus 126 kDa protein and a host membrane protein (SNARE) and its influence on virus RNA accumulation R. NELSON (1), A. Ibrahim (1), J. Schoelz (2) (1) Samuel Roberts Noble Foundation, Inc., U.S.A.; (2) University of Missouri, U.S.A. Tobacco mosaic virus (TMV) 126 kDa protein aids virus accumulation and is necessary for virus intercellular movement. This protein also co-localizes or associates with the endoplasmic reticulum and vacuole membrane (reviewed in Liu et al. 2013 Front. Plant Sci. 4:12. doi: 10.3389/fpls.2013.00012). The importance of the 126 kDa protein interaction with different host membranes as it relates to virus accumulation is not understood. As shown through yeast two hybrid analysis the 126 kDa protein interacts with two host proteins: one putatively involved in membrane fusion with vacuoles and the second with intra-vacuolar protease activity. Here we will show the co-localization of the membrane fusion protein, a plant SNARE (soluble N-ethylmaleimide sensitive attachment protein receptor) protein, in planta with the 126 kDa protein. Additionally, we are analyzing the influence of this and other SNARE proteins on tobamovirus [TMV and Turnip vein clearing virus (TVCV)] RNA accumulation using Arabidopsis T-DNA mutants. TVCV is being studied because of the different characteristics of its 126 kDa protein homolog, the 125 kDa protein (e.g. different aggregation states displayed during their ectopic expression). The importance of this SNARE protein for subcellular localization of the TMV 126 kDa protein and virus RNA accumulation will be summarized. S4.145 Co-expression of proteins by two virus vectors in the same cells of infected plants M. Mendoza (1), H. Scholthof (1) (1) Texas A&M University, U.S.A. Co-expression of proteins by two virus vectors in the same cells of infected plants M. Mendoza (1), H. Scholthof (1) (1) Texas A&M University, U.S.A. Plant viral vectors represent a technology that allows expression of valuable proteins at high levels in a relatively short time. For many purposes it may be desirable to express more then one protein in a single cell but that is often not feasible when using a single virus vector. Such a co-expression strategy requires the simultaneous delivery by two non-competitive viruses to express proteins. Here we report on the use of two agro-launchable virus vector systems based on Tomato bushy stunt virus (TBSV) and Tobacco mosaic virus (TMV) that both express suppressors to minimize the effects of Argonaute2-mediated silencing. To co-express proteins we used TBSV and TMV each expressing green fluorescent protein (GFP) of different sizes, in addition to co-expressing TBSV-GFP with TMV expressing red fluorescent protein (RFP). The results in Nicotiana benthamiana and tomato demonstrated that these two vectors accumulated in the same plant, same leaves, and in the same cells. Therefore, co-expression by these two vectors provides a biotechnological platform for fast and high level expression of potentially valuable proteins that need to form oligomers for activity. Displaying foreign proteins and peptides on the surface of Pepino mosaic virus virions C. LI (1), S. Shi (1), N. Yu (1), Z. Xiong (1) (1) University of Arizona, U.S.A. S4.146 S4.146 A single nucleotide polymorphism at the right terminal region of Mexican papita viroid is a virulent determinant factor on tomato K. LING (1), R. Li (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. Novel transcription activator like (TAL) effectors and repeat variable domains (RVD) were discovered. Predicted TAL effector target sequences were identified in the available L. perrieri genome. Rice susceptibility gene homologs and unique TAL effector targets were also uncovered. Pathogenicity screening on L. hexandra and diverse rice cultivars confirmed that Xcl can colonize rice and produces weak not progressive symptoms. Overall, based on average nucleotide identity, effector repertoires and phenotype, we propose to rename Xcl to X. oryzae pv. leersiae and use this parallel system to better understand the evolution of pathogenicity in grasses. Phytopathogenic interaction between Herbaspirillum rubrisubalbicas and different genotypes of Sorghum T. TULESKI (1), T. Tuleski (1), C. Espinoza (2), F. Plucani do Amaral (2), T. Pereira (2), R. Monteiro (4), E. Maltempi de Souza (4), F. Pedrosa (4), G. Stacey (2) (1) University of Missouri Columbia MO U S A ; (2) University of Missouri U S A ; (3) University of Missouri columbia U S A ; (4) Federal Phytopathogenic interaction between Herbaspirillum rubrisubalbicas and different genotypes of Sorghum T. TULESKI (1), T. Tuleski (1), C. Espinoza (2), F. Plucani do Amaral (2), T. Pereira (2), R. Monteiro (4), E. Maltempi de Souza (4), F. Pedrosa (4), G. Stacey (2) Phytopathogenic interaction between Herbaspirillum rubrisubalbicas and different genotypes of Sorghum T. TULESKI (1), T. Tuleski (1), C. Espinoza (2), F. Plucani do Amaral (2), T. Pereira (2), R. Monteiro (4), E. Maltempi de Souza (4), F. Pedrosa (4), G. Stacey (2) (1) University of Missouri, Columbia, MO, U.S.A.; (2) University of Missouri, U.S.A.; (3) University of Missouri, columbia, U.S.A.; (4) Federal University of Parana, Brazil y of Missouri, Columbia, MO, U.S.A.; (2) University of Missouri, U.S.A.; (3) University of Missouri, columbia, U.S.A.; (4) f Parana, Brazil Missouri, Columbia, MO, U.S.A.; (2) University of Missouri, U.S.A.; (3) University of Missouri, columbia, U.S.A.; (4) Fed ana Brazil Herbaspirillum rubrisubalbicans is a nitrogen fixing and plant growth promoting bacterium found in association with important grasses, such as maize and wheat. In contrast, this bacterium is a pathogen on some genotypes of Sorghum bicolor, being responsible for red stripe disease. The pathogen infects sorghum leaves leading to tissue necrosis, a reduction in photosynthesis and impaired leaf development. Twenty five genotypes of sorghum were inoculated with H. rubrisubalbicans with the great majority showing symptoms after 7 days. A single nucleotide polymorphism at the right terminal region of Mexican papita viroid is a virulent determinant factor on tomato K. LING (1), R. Li (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. Bacteria were recovered from portions of the leaf increasingly distal from the site of inoculation. The results showed the spread of both bacteria and symptoms along the expanding leaf. Plants have the ability to mount a defense response to invading pathogens due to recognition of conserved motifs, termed microbe-associated molecular patterns (MAMPs). We tested the ability of MAMP-triggered immunity to protect sorghum plants from H. rubrisubalbicans infection. Sorghum leaves were pre- treated with chitin and flg22 (a conserved 22 amino acid peptide derived from bacterial flagellin) and then challenged with the pathogen. Evaluation of disease symptoms 5 days after inoculation showed strong plant protection in those leaves pretreated with chitin and flg22. The data suggest that H. rubrisubalbicans will be a useful pathogen in which to study the innate immunity response of sorghum, an important grain, forage and bioenergy crop. Expression of FLS2s from ‘Nagami’ kumquat and ‘Sun Chu Sha’ mandarin enhance citrus canker resistance in ‘Duncan’ grapefruit V. FEBRES (1), Q. Shi (1), G. Moore (1) (1) University of Florida, U.S.A. Expression of FLS2s from ‘Nagami’ kumquat and ‘Sun Chu Sha’ mandarin enhance citrus canker resistance in ‘Duncan’ grapefruit V. FEBRES (1), Q. Shi (1), G. Moore (1) (1) University of Florida, U.S.A. Expression of FLS2s from ‘Nagami’ kumquat and ‘Sun Chu Sha’ mandarin enhance citrus canker resistance in ‘Duncan’ grapefruit V. FEBRES (1), Q. Shi (1), G. Moore (1) In citrus, flagellin 22 (flg22), a pathogen-associated molecular pattern (PAMP) from Xanthomonas citri ssp. citri (Xflg22), triggers stronger defense responses in canker resistant genotypes than susceptible ones (Shi et al. Mol. Plant Pathol. 2025, 16:507). We have now identified the citrus genes encoding putative bacterial flagellin/flg22 receptors (FLS2). We isolated two genes each from ‘Sun Chu Sha’ mandarin (Citrus reticulata, CrFLS2-1 and CrFLS2-2) and ‘Duncan’ grapefruit (C. paradisi, CpFLS2-1 and CpFLS2-2), a canker resistant and a highly susceptible citrus species, respectively. Only one FLS2 gene was obtained from the highly canker-resistant ‘Nagami’ kumquat (Fortunella margarita, FmFLS2-1). Flanking sequences suggest a rearrangement event resulted in the deletion of FLS2-2 from the genome of kumquat. Transcriptional analysis indicated that the FLS2-2s reached higher expression levels in response to Xflg22 than the FLS2-1s, and the induction levels positively correlated with the level of canker resistance. Kumquat showed the highest FLS2-1 pre-treatment expression level among the citrus species tested. We also transiently expressed FmFLS2-1, CrFLS2-2 and CpFLS2-2 in leaves of susceptible grapefruit. A single nucleotide polymorphism at the right terminal region of Mexican papita viroid is a virulent determinant factor on tomato K. LING (1), R. Li (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. Mexican papita viroid (MPVd), a pospiviroid, causes serious disease outbreaks on greenhouse tomato in North America. Two dominant genotypes (MPV-S and MPV-M), sharing 93.8% sequence identity, incited striking different symptom expression (severe and mild) on tomato ‘Rutgers’. To determine genetic factor that is responsible for differential pathogenicity, using synthetic DNA approach, a series of chimeric infectious clones were generated through progressive replacement of each secondary structural domain in MPVd-M with their corresponding domain of MPVd-S. In plant inoculation assays, a chimera containing the Terminal Right domain of MPVd-S incited severe symptom on tomato similar to that induced by MPVd-S. The other three chimeras incited only mild symptoms similar to that of MPVd-M. A single nucleotide polymorphism in the Terminal Right region between MPVd-M (176U:A185) and MPVd-S (174G:C183) was identified as the virulent determinant factor for MPVd. This result supports variable pathogenicity functions in different pospiviroids, which may lead us to design a better strategy in managing this emerging viroid disease. Two complete genome sequences of a new pathovar of Xanthomonas oryzae infecting wild grasses provide insight into evolution of pathogenicity J. LANG (1), R. Koebnik (2), A. Pérez-Quintero (2), E. DuCharme (1), B. Szurek (2), J. Leach (1), V. Verdier (2) (1) Colorado State University, U.S.A.; (2) Institut de recherche pour le développement (IRD), France Two complete genome sequences of a new pathovar of Xanthomonas oryzae infecting wild grasses provide insight into evolution of pathogenicity J. LANG (1), R. Koebnik (2), A. Pérez-Quintero (2), E. DuCharme (1), B. Szurek (2), J. Leach (1), V. Verdier (2) (1) Colorado State University, U.S.A.; (2) Institut de recherche pour le développement (IRD), France Xanthomonas oryzae (Xo) is a group of plant pathogenic bacteria composed of weak and highly virulent members that are genetically distinct from one another. Strains belonging to different virulent lineages from Africa and Asia and less virulent strains from the US have been used for comparative studies of pathogenicity and establishing effector repertoires. A close relative of Xo, X. campestris pv. leersiae (Xcl) was first described in 1957, and causes bacterial streak on the perennial grass, Leersia hexandra. L. hexandra is closely related to rice, globally ubiquitous around rice paddies, and is a reservoir of pathogenic Xo. We used draft and long read, single molecule, real time (SMRT) genome sequences of two strains of Xcl from China and Burkina Faso to determine the genetic relatedness of this organism with Xo. S4.146 Pepino mosaic virus (PepMV), a member of Potexvirus genus, infects tomatoes and causes only mild symptoms under high intensity light conditions. However, its infection is highly productive and yields a large amount of virions in tomatoes. The flexuous PepMV virions (510 nm x ~13 nm in dimensions) are made of 1290 identical copies of a 25 kDa capsid protein (CP) with exposed N-terminus at the virion surface. Therefore, PepMV virions are ideal nanoparticles to display useful antigens and antibodies fused at the N-terminus of the CP. Using a highly infectious cDNA clone as the backbone, we have developed a virus-based expression system that is capable of displaying foreign proteins and peptides on the surface of PepMV virions. Two unique restrictions sites have been engineered immediately downstream the AUG translation initiation codon of PepMV CP to allow convenient and directional cloning of any foreign proteins and peptides into the expression vector. Several 2A self-cleavage peptides from picornaviruses have also been inserted between the restriction sites and PepMV CP to modulate variable ratios of CP fusion proteins and mature CP. m7 G-capped in vitro transcripts synthesized from the vectors are highly infectious, yielding high level expressions of foreign proteins and peptides in inoculated plants. The PepMV-based protein display system is expected to have a wide range of applications in pharmaceuticals and diagnosis. Homologs of CsLOB1 can function as disease susceptibility genes in citrus canker J. ZHANG (1), J. Tapia (1), Y. Hu (2), J. Jones (1), N. Wang (1), S. Liu (3), W. Frank (1) (1) University of Florida, Gainesville, FL, U.S.A.; (2) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China, Gainesville, FL, U.S.A.; (3) Kansas State University, Gainesville, FL, U.S.A. J. ZHANG (1), J. Tapia (1), Y. Hu (2), J. Jones (1), N. Wang (1), S. Liu (3), W. Frank (1) (1) University of Florida, Gainesville, FL, U.S.A.; (2) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China, Gainesville, FL, U.S.A.; (3) Kansas State University, Gainesville, FL, U.S.A. The Type II-dependent secretion of virulence factors is necessary for systemic colonization by the xylem-limited pathogen, Xylella fastidiosa B. INGEL (1), P. Wang (2) (1) University of California Riverside U S A ; (2) University of California Riverside U S A The Type II-dependent secretion of virulence factors is necessary for systemic colonization by the xylem-limited pathogen, Xylella fastidiosa B. INGEL (1), P. Wang (2) (1) University of California Riverside, U.S.A.; (2) University of California Riverside, U.S.A. ( ) g ( ) (1) University of California Riverside, U.S.A.; (2) University of California Riverside, U.S.A. Xylella fastidiosa (Xf) is a xylem-limited, gram-negative bacterium that causes Pierce’s disease (PD) in grapevines. PD symptom progression is highly correlated with systemic colonization of the xylem, which is achieved by bacterial movement via the degradation of pit membranes separating adjacent xylem vessels. Pit membranes are readily dismantled by Xf cell wall-degrading enzymes (CWDEs), three of which have been implicated as virulence factors: a polygalacturonase (PG), and two endoglucanases (EngXCA1 and EngXCA2). As pathogenicity was nullified in a PG-deficient mutant, the PG has been a focal point for Xf inhibition studies. However, PG expression has proved to be difficult, and we have sought alternative CWDE inhibition targets. A knockout mutation of the Type II secretion system (T2SS) ATPase gene (ΔxpsE) resulted in a complete loss of pathogenicity similar to that displayed by the PG-deficient mutant. As Xf lacks a Type III secretion system, it is predicted that virulence factors such as CWDEs are secreted via the T2SS. Secretome analyses of both the Xf wild-type and ΔxpsE mutant strains had distinct differences in protein composition, and an antibody targeting EngXCA2 revealed that secretion of this endoglucanase is Type II-dependent. These results provide clear evidence that Xf relies on the T2SS for secretion of the CWDEs necessary for systemic colonization, and inhibition of this secretion system will significantly reduce Xf pathogenicity in grapevines. The chemotaxis regulator pilG of Xylella fastidiosa is required for virulence in Vitis vinifera grapevines H. LIN (1), X. Shi (2) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. The chemotaxis regulator pilG of Xylella fastidiosa is required for virulence in Vitis vinifera grapevines H. LIN (1), X. Shi (2) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. Xylella fastidiosa is a Gram-negative, xylem-limited pathogenic bacterium that causes Pierce’s disease of grapevines. Type IV pili of X. fastidiosa are regulated by pilG, a chemotaxis regulator in Pil-Chp operon involving signal transduction pathways. To elucidate the role of pilG in twitching motility and pathogenicity, phenotypes of wild type, pilG-mutant and a complementary strain were characterized. While in vitro studies showed that all tested strains had similar growth curves, mutant XfΔpliG exhibited a twitching defective phenotype and significant reduction in biofilm formation and cell-cell aggregation. Greenhouse in planta experiment further confirmed that grapevines infected with XfΔpilG showed very mild PD symptoms whereas grapevines inoculated with X. fastidiosa wild type and XfΔpilG-C developed typical PD symptoms. These results demonstrate that pilG of X. fastidiosa is required for twitching motility and full virulence. Biological functional Analysis of tatB in Acidovorax citrulli Biological functional Analysis of tatB in Acidovorax citrulli ( ) (1) Chinese academy of agricultrual sciences, institute of plant protection, China Acidovorax citrulli causes bacterial fruit blotch of cucubits world wide. In order to explore the relationship between Twin-arginine Translocation system (Tat) and pathogenicity of A. citrulli, a tatB disruption mutant (?tatB) and its complement strains were constructed by homologous recombination. The virulence, motility, growth ability, extracellular polysaccharide and cellulase producing ability and biofilm formation of the wild type, mutant and the complement strains were tested to illustrate the influence of Tat system on A. citrulli. By comparing the expression of trbC, hrcN, Aave_1810, Aave_0034 and hrpE gene with real-time fluorescent quantitative PCR, we explored the relationship between tatB genes associated with type III secretion system, Tat system related genes and cell toxicity related genes. The results showed the deficiency of tatB weakened the virulence, motility and growth ability of A. citrulli, while had no effect on the production of extracellular polysaccharide (EPS)?extracellular cellulase and biofilm formation. Real-time fluorescent quantitative analysis showed that the deficiency of tatB made the expressions of trbC and hrcN genes in mutant strain significantly increase, while the expressions of other genes notably decreased. Consequently, the deficiency of functional gene tatB in Tat affects some biological characteristics of A. citrulli, and ultimately weakens pathogenic ability. Secretome annotation in the citrus greening bacterial pathogen L. Cano (1), M. Pitino (2), Y. Duan (2) (1) University of Florida, U.S.A.; (2) US Horticultural Research Laboratory, U.S. ( ) ( ) ( ) (1) University of Florida, U.S.A.; (2) US Horticultural Research Laboratory, U.S.A. Citrus greening or also known as Huanglongbing (HLB) is one of most deadly citrus disease in Florida and in the world. The causal agent is the bacterium Candidatus liberibacter asiaticus (CLas). Plant pathogens secrete molecules that manipulate host physiology to achieve colonization. The main goal of our study was to identify and make available to the citrus community a set of candidate pathogen secreted proteins from CLas using genome sequences and in planta gene expression. Identified secreted protein candidates can be employed in high throughput functional genomics screens, commonly referred to as effectoromics, to test citrus germplasm for specific pathogen recognition by resistance R proteins. Our findings highlight about 10 candidate secreted proteins for pathogen-assisted breeding in citrus for resistance to HLB. The bifunctional catalase/peroxidase KatG is required for pathogenicity of Xanthomonas albilineans on sugarcane M. JAIN (1), L. Fleites (2), S. Ca. Liberibacter asiaticus carries a highly conserved, chromosomal CI repressor that binds an early gene promoter in its prophage L. FLEITES (1), M. Jain (1), S. Zhang (1), D. Gabriel (1) (1) University of Florida, Gainesville, FL, U.S.A. Ca. Liberibacter asiaticus carries a highly conserved, chromosomal CI repressor that binds an early gene promoter in its prophage L. FLEITES (1), M. Jain (1), S. Zhang (1), D. Gabriel (1) (1) University of Florida, Gainesville, FL, U.S.A. Candidatus Liberibacter asiaticus (Las) is one of three species of bacteria associated with Huanglongbing, arguably the most devastating disease of citrus. The majority of Las strains harbor bacteriophage that are maintained as stable lysogens in psyllids, but begin to enter a lytic cycle in planta. Las carries a CI phage repressor (LCI) with internal direct repeats that readily recombine and can create truncated translation products. LCI has been used as an epidemiological marker to follow the movement of Las populations; however, LCI has not been functionally characterized. Las strain UF506 has been maintained in isolation in a containment facility and transmitted from citrus to citrus and to periwinkle via dodder. All five LCI sequences isolated from curated Las strain UF506 in infected citrus encoded a full-length protein. By contrast, all seven UF506 LCI sequences derived from artificially infected periwinkle encoded a truncated version. Mobility shift assays demonstrated that LCI binds its own promoter and an early gene promoter on SC1, indicating potential involvement in lytic cycle activation. Promoter reporters are being evaluated to quantify repression in the presence of the full-length and truncated proteins, and additional potential binding sites are being investigated. If the LCI does function as a phage repressor, it would become a promising target for HLB control, since interfering with this function could trigger the lytic cycle in Las. The Type II-dependent secretion of virulence factors is necessary for systemic colonization by the xylem-limited pathogen, Xylella fastidiosa B. INGEL (1), P. Wang (2) (1) University of California Riverside U S A ; (2) University of California Riverside U S A The Type II-dependent secretion of virulence factors is necessary for systemic colonization by the xylem-limited path B. INGEL (1), P. Wang (2) ( ) i i f lif i i id ( ) i i f lif i i id A single nucleotide polymorphism at the right terminal region of Mexican papita viroid is a virulent determinant factor on tomato K. LING (1), R. Li (1) (1) USDA-ARS, U.S. Vegetable Laboratory, U.S.A. FmFLS2-1 and CrFLS2-2 conferred stronger responses to Xflg22 and reduced canker symptom development when compared to CpFLS2-2. We conclude that these two genes are valuable for engineering enhanced canker resistance and possibly other bacterial pathogens. Homologs of CsLOB1 can function as disease susceptibility genes in citrus canker J. ZHANG (1), J. Tapia (1), Y. Hu (2), J. Jones (1), N. Wang (1), S. Liu (3), W. Frank (1) (1) University of Florida, Gainesville, FL, U.S.A.; (2) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China, Gainesville, FL, U.S.A.; (3) Kansas State University, Gainesville, FL, U.S.A. Homologs of CsLOB1 can function as disease susceptibility genes in citrus canker J. ZHANG (1), J. Tapia (1), Y. Hu (2), J. Jones (1), N. Wang (1), S. Liu (3), W. Frank (1) (1) University of Florida, Gainesville, FL, U.S.A.; (2) Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, P. R. China, Gainesville, FL, U.S.A.; (3) Kansas State University, Gainesville, FL, U.S.A. The lateral organ boundary domain (LBD) genes is a plant-specific family of proteins that function as transcription factors for plant growth and development. Citrus sinensis lateral organ boundary 1 (CsLOB1) functions as a disease susceptibility (S) gene in citrus bacterial canker (CBC). The Citrus sinensis genome contains thirty-four LBD members. We assessed the potential for additional LBD genes in citrus to function as surrogates for S4.147 CsLOB1 and compared host gene expression upon induction of different genes. Using custom designed TAL effectors (dTALes), CsLOB2 and CsLOB3 functioned similarly to CsLOB1 in CBC. Quantitative RT-PCR of mRNA during infection revealed a set of cell wall metabolic genes that are associated with CsLOB1, CsLOB2 and CsLOB3 expression. The genes may represent downstream genes involved in CBC. Biological functional Analysis of tatB in Acidovorax citrulli Loria (1) (1) Department of Plant Pathology, University of Florida, Gainesville, FL, U.S.A.; (2) Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada; (3) Department of Medicinal Chemistry, University of Florida, Gainesville, FL, U.S.A.; (4) College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, China Approximately ten Streptomyces species including the most widely distributed Streptomyces scabiei cause the potato scab disease. The main pathogenicity determinant of scab-causing Streptomyces species is the phytotoxin thaxtomin A (ThxA). In S. turgidiscabies, ThxA biosynthetic genes are localized on a mobile pathogenicity island (PAI). However, the PAI mobilization in other Streptomyces species remains uncharacterized. We investigated the mobilization of the PAI of S. scabiei 87-22 both in vivo and in vitro. We sequenced genomes of ten pathogenic Streptomyces isolates from different locations, inferred the evolutionary relationships of pathogenic Streptomyces species, and identified a novel pathogenic species (S. sp. 96- 12) that was isolated from Egypt. The PAI in S. sp. 96-12 is identical to the PAI in S. scabiei 87-22 despite differences in their whole genome sequences. This strongly suggested in vivo mobilization of this PAI in these two strains. To test whether the PAI in S. scabiei 87-22 could indeed be mobilized, S. scabiei 87-22 deletion mutants with antibiotic resistance markers in the PAI, were mated with S. diastatochromogenes, a nonpathogenic species. The PAI of S. scabiei was found to be site-specifically inserted into the aviX1 gene of S. diastatochromogenes and conferred pathogenicity. Our results demonstrated that the mobilization of the S. scabiei PAI could be responsible for the emergence of novel pathogenic species. Biological functional Analysis of tatB in Acidovorax citrulli Zhang (2), D. Gabriel (2) (1) Department of Plant Pathology, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A. The bifunctional catalase/peroxidase KatG is required for pathogenicity of Xanthomonas albilineans on sugarcane M. JAIN (1), L. Fleites (2), S. Zhang (2), D. Gabriel (2) (1) Department of Plant Pathology, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A. Survival and multiplication of phytopathogenic bacteria, and disease progression are essentially contingent upon the pathogen’s ability to detoxify reactive oxygen species (primarily H2O2) generated either endogenously through aerobic respiration or by the oxidative burst of plant cells during plant- pathogen interactions. Xanthomonas albilineans (Xal) is a gram-negative, xylem-invading pathogen that causes sugarcane leaf scald disease. Two S4.148 putative catalase genes XALc_0265 (srpA) and XALc_2991 (katE), and a bifunctional catalase/peroxidase XALc_0959 (katG) have been annotated in silico in the genome of Xal FL07-1. Xal katG was replaced by an enhanced Green Florescent Protein marker gene (gfp) via splice overlap PCR. Challenge inoculations of decapitated sugarcane plants by Xal ÄkatG::gfp exhibited a significant reduction in pathogenic symptoms on the newly emerged leaf blades. In addition bacterial viability in planta was significantly reduced compared to the wild type Xal FL07-1. Full-length katG cloned in the broad host range vector pUFR047 complemented the ÄkatG::gfp mutant strain for both pathogenicity and endophytic survival of the bacterium. Given that Xal katG is predicted to have a non-classical secretion potential, its role is also being investigated in dampening the apoplastic, non-specific low amplitude systemic wave of H2O2 elicited by the host cell that is essential for establishment of innate plant immunity. putative catalase genes XALc_0265 (srpA) and XALc_2991 (katE), and a bifunctional catalase/peroxidase XALc_0959 (katG) have been annotated in silico in the genome of Xal FL07-1. Xal katG was replaced by an enhanced Green Florescent Protein marker gene (gfp) via splice overlap PCR. Challenge inoculations of decapitated sugarcane plants by Xal ÄkatG::gfp exhibited a significant reduction in pathogenic symptoms on the newly emerged leaf blades. In addition bacterial viability in planta was significantly reduced compared to the wild type Xal FL07-1. Full-length katG cloned in the broad host range vector pUFR047 complemented the ÄkatG::gfp mutant strain for both pathogenicity and endophytic survival of the bacterium. Biological functional Analysis of tatB in Acidovorax citrulli ( ), ( ), ( ), ( ) (1) Department of Plant Sciences, University of California, Davis, U.S.A.; (2) Department of Microbiology and Molecular Genetics, University of California, Irvine, U.S.A. (1) Department of Plant Sciences, University of California, Davis, U.S.A.; (2) Department of Microbiology and Molecular Genetics, University of California, Irvine, U.S.A. Pathogens that cause foodborne illness pose a challenge to food safety and security, as crops that are vectors for these human diseases may appear healthy, resulting in their integration to marketplaces throughout the world. Although some human pathogens that are introduced to the phyllosphere elicit an immune response within the plant, Salmonella enterica serovar Typhimurium strain SL1344 has been shown to disrupt plant innate immune signaling (including stomatal immunity) and survive for long periods of time inside the leaf. We have determined that, like SL1344, S. enterica serovar Typhimurium strain 14028 (14028) also disrupts stomatal immunity after 4 hours post-inoculation (hpi). We set up a genetic screen to elucidate the mechanism(s) by which 14028 modulates stomatal movement using a mutant library with high-coverage and multi-gene deletions created with lambda- red mediated gene-replacement. Leaf sections of lettuce (Lactuca sativa cultivar Salinas) were floated on bacterial inoculum (108 CFU/mL) or a water control under light and stomatal aperture widths were measured via microscopy at 4 hpi. A total of 535 mutants were analyzed for their inability to stimulate stomatal opening at 4 hpi and compared to the wild type and water control. We observed that mutant strains lacking operons involved in secretion could not open stomata. Further characterization of identified mutants will aim to determine the individual genes that are pertinent for stomatal opening. In vivo and in vitro investigation of the mobilization of pathogenicity islands of Streptomyces Y. ZHANG (1), D. Bignell (2), R. Zuo (3), Q. Fan (4), J. Huguet-Tapia (1), Y. Ding (3), R. Loria (1) (1) Department of Plant Pathology, University of Florida, Gainesville, FL, U.S.A.; (2) Department of Biology, Memorial University of Newfoundland, St. John’s, Newfoundland and Labrador, Canada; (3) Department of Medicinal Chemistry, University of Florida, Gainesville, FL, U.S.A.; (4) College of Plant Protection, Fujian Agriculture and Forestry University, Fuzhou, China In vivo and in vitro investigation of the mobilization of pathogenicity islands of Streptomyces Y. ZHANG (1), D. Bignell (2), R. Zuo (3), Q. Fan (4), J. Huguet-Tapia (1), Y. Ding (3), R. Biological functional Analysis of tatB in Acidovorax citrulli Given that Xal katG is predicted to have a non-classical secretion potential, its role is also being investigated in dampening the apoplastic, non-specific low amplitude systemic wave of H2O2 elicited by the host cell that is essential for establishment of innate plant immunity. The role of the tomato genotype in susceptibility to Salmonella enterica M. TEPLITSKI (1), M. Teplitski (1), M. Marvasi (2), J. Giovannoni (3) (1) University of Florida, U.S.A.; (2) Middlesex University, United Kingdom; (3) Boyce Thompson Institute, Cornell University, U.S.A. The role of the tomato genotype in susceptibility to Salmonella enterica M. TEPLITSKI (1), M. Teplitski (1), M. Marvasi (2), J. Giovannoni (3) (1) University of Florida, U.S.A.; (2) Middlesex University, United Kingdom Tomatoes were linked to multiple outbreaks of salmonellosis. A hypothesis that some tomato genotypes are less conducive to Salmonella growth was tested. A screen of 30 cultivars revealed 10-1,000 fold differences in the ability of this human pathogen to multiply within fruits. Expression of Salmonella genes involved in the interactions was tomato cultivar- and ripening-dependent. Salmonella reached lower numbers within immature fruit, however, pigment accumulation per se did not have an effect on its growth within tomatoes. Salmonella reached similar population densities in tomatoes carrying green flesh or Hyper pigmented mutations. Growth of Salmonella was reduced in the tomato genotypes with defects in ethylene synthesis, perception and signal transduction. While mutation in the ripening-related ethylene receptor Nr resulted only in a modest reduction in Salmonella numbers within tomatoes, strong reduction of the Salmonella growth was observed in rin and nor tomato mutants. A commercial cultivar Sebring heterozygous for rin was less susceptible to Salmonella under the greenhouse conditions than other large-fruited varieties. No significant differences in growth of Salmonella within green and red tomatoes of cv. Sebring were observed in the greenhouse experiments, consistent with the hypothesis that ethylene signaling is responsible for the phenotype of green tomatoes. However, in the field, the phenotype of the cv. Sebring was masked by environmental conditions. Determining the genetic basis for stomatal aperture modulation by Salmonella enterica serovar Typhimurium strain 14028 J. MONTANO (1), S. Porwollik (2), M. McClelland (2), M. Melotto (1) (1) Department of Plant Sciences, University of California, Davis, U.S.A.; (2) Department of Microbiology and Molecular Genetics, University of California, Irvine, U.S.A. Characterization of the T6SS secretion system of Ralstonia solanacearum and its role in virulence at low temperatures A. BOCSANCZY (1), J. Yuen (1), A. Mangravita-Novo (1), D. Norman (1) (1) University of Florida MREC, U.S.A. Characterization of the T6SS secretion system of Ralstonia solanacearum and its role in virulence at low temperatures A. BOCSANCZY (1), J. Yuen (1), A. Mangravita-Novo (1), D. Norman (1) (1) University of Florida MREC, U.S.A. Ralstonia solanacearum is the causal agent of Bacterial Wilt, the worldwide devastating disease of hundreds of plant species. R. solanacearum is a ‘complex’ species due to its genetic diversity and wide host range; however all strains possess conserved secretion systems. The type VI secretion system (T6SS) has been associated with virulence, symbiosis and bacterial competition, however its function is not known in R. solanacearum. Previously, we discovered a functional T6SS in several strains of R. solanacearum, through a proteomic comparison. We created a T6SS related (Vip-B like) gene defective mutant in strain P673. In this work we confirmed that this mutant renders the T6SS non-functional. We show that the T6SS contributes to P673 virulence at low temperatures, only when soil inoculated. We hypothesized that the T6SS contributes indirectly to virulence, either by competition with other bacterial species, or with populations of the same species. To test this hypothesis we performed bacterial competition assays with other bacterial species commonly found in soil, however we did not find a difference. Currently, we are developing intra-species competition assays at 30°C and 18°C in order to determine the T6SS function and its dependency on temperature. We also present a comparative genomics of the T6SS system for all the strains sequenced to date. Characterization of the T6SS system in R. solanacearum will help elucidate its role in virulence at low temperatures. Expression of the FST1 from Fusarium verticillioides in a yeast strain lacking a major myo-inositol transporter gene C. NIU (1), G. Payne (2), C. Woloshuk (3) (1) Purdue University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) Purdue University, U.S.A. ( ), g ( ), ( ), ( ), ( ) (1) Texas A&M University, Plant Pathology & Microbiology, Texas A&M University, College Station, TX, 77843, College Station, TX, U.S.A.; (2) Texas A&M University, Plant Pathology & Microbiology, Texas A&M University, College Station, TX, 77843, College Station, TX, U.S.A.; (3) Texas A&M University, U.S.A.; (4) Texas A&M University Electrical & Computer Engineering, Texas A&M University, College Station, TX, 77843, U.S.A.; (5) Texas A&M University Electrical & Computer Engineering, Texas A&M University, College Station, TX, 77843, TX, U.S.A. Symptoms of autumn decline include black root rotting with stunted and yellowish rice foliage starting as early as two weeks following permanent flood establishment. The problem is often most severe where cold well water first enters a rice field and may spread throughout the field, except on levees. The phenomenon was reported in Arkansas in a limited number of fields in 2004. However, there were several reports of autumn decline across the state of Arkansas in 2012 to 2015. Observations have shown fields having a clay loam soil texture as more prone to the autumn decline phenomenon than others commonly cropped to rice. The root rotting symptoms often start a few weeks after flood establishment and become progressively worse throughout the season if unmitigated. In situations where root rotting is severe, fungi grow into the crown which limits function of the whole root system and prevents translocation of water and nutrients from the soil to the plant. In moderate to severe cases, tillers break off easily and plant death may occur rapidly leading to significant yield losses. Twenty commercial rice varieties were evaluated in the field in 2015 and based on their reactions a rating scale was developed. The nature of autumn decline problem, reactions of the varieties and the rating scale will be presented. Global Analysis of Horizontal Gene Transfer in Fusarium verticillioides S. GAO (1), J. Wisecaver (2), Y. Zhang (3), L. Ma (3), A. Rokas (2), S. Gold (4), A. Glenn (4) (1) Department of Plant Pathology, University of Georgia, Athens, GA, U.S.A.; (2) Department of Biological Sciences, Vanderbilt University, Nashville, TN, U.S.A.; (3) Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst, MA, U.S.A.; (4) USDA ARS, Richard B. Russell Research Center, Toxicology & Mycotoxin Research Unit, Athens, GA, U.S.A. Expression of the FST1 from Fusarium verticillioides in a yeast strain lacking a major myo-inositol transporter gene C. NIU (1), G. Payne (2), C. Woloshuk (3) (1) Purdue University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) Purdue University, U.S.A. The co-occurrence of microbes within plants and other specialized niches may facilitate horizontal gene transfer (HGT) affecting host-pathogen interactions. We recently identified fungal-to-fungal HGTs involving metabolic gene clusters. For a global analysis of HGTs in the maize pathogen Fusarium verticillioides (Fv), we used a phylogenomic pipeline to identify potential candidates. From an initial output of 1801 genes, manual curation revealed 117 strong HGT candidates acquired mostly from bacteria. Preliminary evaluation suggested several enriched metabolic pathways (e.g. lysine biosynthesis and nitrogen metabolism) compared to the frequency of such genes within the Fv genome. Interestingly, among the five candidates that were categorized as being involved in lysine biosynthesis, FVEG_09873 appears only in Fv and has no orthologs in other fungi. This gene was acquired from plant-associated Proteobacteria and putatively encodes a diaminopimelate epimerase in the diaminopimelic acid pathway employed by most bacteria but not commonly found in fungi. Another promising HGT candidate acquired from bacteria is FVEG_10494 that encodes a putative class-III aminotransferase, functioning in nitrogen metabolism. It is highly expressed when Fv is exposed to nitric oxide. Additional analyses will focus on other HGT candidates having limited fungal distribution. Functional characterization is underway to determine the significance of select HGT candidates in fungal adaptation. A Battle in a Kernel: Molecular Exploration of Antagonisms between Two Maize Endophytes, Fusarium verticillioides and Acremonium zeae M. GAO (1), A. Glenn (2), S. Gold (2) (1) The University of Georgia, U.S.A.; (2) United States Department of Agriculture Toxicology and Mycotoxin Research Unit, U.S.A. A Battle in a Kernel: Molecular Exploration of Antagonisms between Two Maize Endophytes, Fusarium verticillioides and Acremo M. GAO (1), A. Glenn (2), S. Gold (2) (1) The University of Georgia, U.S.A.; (2) United States Department of Agriculture Toxicology and Mycotoxin Research Unit, U.S.A. M. GAO (1), A. Glenn (2), S. Gold (2) (1) The University of Georgia, U.S.A.; (2) United States Department of Agriculture Toxicology and Mycotoxin Research Unit, U.S.A. ( ), ( ), ( ) (1) The University of Georgia, U.S.A.; (2) United States Department of Agriculture Toxicology and Mycotoxin Research Unit, U.S.A. Fusarium verticillioides (Fv) is a prevalent seed-borne maize endophyte capable of causing severe kernel rot and fumonisin mycotoxin contamination. Within maize kernels, Fv is primarily confined to the pedicel, while another seed-borne fungal endophyte, Acremonium zeae (Az), is observed in embryos. Global regulatory changes allow for the increased growth of Salmonella in soft rotted tomatoes A. GEORGE (1), A. George (2) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Global regulatory changes allow for the increased growth of Salmonella in soft rotted tomatoes A GEORGE (1) A George (2) Global regulatory changes allow for the increased growth of Salmonella in soft rotted tomatoes A. GEORGE (1), A. George (2) (1) University of Florida U S A ; (2) University of Florida U S A Global regulatory changes allow for the increased growth of Salmonella in soft rotted tomatoes A GEORGE (1) A George (2) While little is known about the mechanisms that allow Salmonella to multiply in tomatoes, even less is understood about how interactions of this human pathogen with phytobacteria contribute to its survival in alternate hosts such as plants. It is now known that bacterial soft rot contributes to an increased fitness of Salmonella enterica sv. Typhimurium in tomato fruits. The goal of this research is to understand the underlying mechanisms involved in this S4.149 phenomenon. The hypothesis is that a change in the tomato environment caused by Pectobacterium provides an overall benefit for Salmonella. In order to determine the full complement of functions that may be vital for the success of Salmonella in soft rots, we employed high throughput transposon sequencing (TN-seq). Libraries of mutants made via transposon mutagenesis were seeded into tomatoes inoculated with fully virulent Pectobaterium, less virulent Pectobacterium, and no Pectobacterium. As a result, pathways involved in nitrogen metabolism, motility, and other regulatory pathways have been identified as potentially playing a role in these interactions. Deletion mutants of genes identified by the TN-seq were then constructed via Wanner-Datsenko mutagenesis for individual testing in tomatoes. These individual mutations have confirmed the results of the TN-seq. We conclude that a variety of metabolic changes do in fact contribute to the increased growth of Salmonella in tomatoes. phenomenon. The hypothesis is that a change in the tomato environment caused by Pectobacterium provides an overall benefit for Salmonella. In order to determine the full complement of functions that may be vital for the success of Salmonella in soft rots, we employed high throughput transposon sequencing (TN-seq). Libraries of mutants made via transposon mutagenesis were seeded into tomatoes inoculated with fully virulent Pectobaterium, less virulent Pectobacterium, and no Pectobacterium. As a result, pathways involved in nitrogen metabolism, motility, and other regulatory pathways have been identified as potentially playing a role in these interactions. Global regulatory changes allow for the increased growth of Salmonella in soft rotted tomatoes A. GEORGE (1), A. George (2) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Deletion mutants of genes identified by the TN-seq were then constructed via Wanner-Datsenko mutagenesis for individual testing in tomatoes. These individual mutations have confirmed the results of the TN-seq. We conclude that a variety of metabolic changes do in fact contribute to the increased growth of Salmonella in tomatoes. Expression of the FST1 from Fusarium verticillioides in a yeast strain lacking a major myo-inositol transporter gene C. NIU (1), G. Payne (2), C. Woloshuk (3) (1) Purdue University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) Purdue University, U.S.A. Expression of the FST1 from Fusarium verticillioides in a yeast strain lacking a major myo-inositol transporter gene C. NIU (1), G. Payne (2), C. Woloshuk (3) (1) Purdue University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) Purdue University, U.S.A. Expression of the FST1 from Fusarium verticillioides in a yeast strain lacking a major myo-inositol transporter gene C. NIU (1), G. Payne (2), C. Woloshuk (3) Fusarium verticillioides causes an important seed disease on maize and produces fumonisin B1 (FB1), a mycotoxin that is detrimental to human and animal health. Previous studies discovered that expression of FST1 is required for FB1 production. In addition, strains lacking a functional FST1 are less virulence on maize seeds and more inhibited by hydrogen peroxide than the wild-type strain. The mutation also causes reduced hydrophobicity of hyphae and macroconidia production. FST1 encodes a putative protein with 12 transmembrane domains with sequence similarity to hexose transporters. However previous studies have failed to prove its ability to transport glucose, fructose or mannose. In this research, we used an Saccharomyces cerevisiae strain lacking a functional myo-inositol transporter (ITR1). This strain grows poorly in myo-inositol medium and is not inhibited by increasing concentrations of FB1. Expression of FST1 in the itr1 mutant restored growth on myo-inositol medium and the inhibitory effects of FB1 to levels observed in the wild-type yeast strain. These results indicate that FST1 can function as a myo-inositol transporter. The evidence also suggests that FST1 can transport FB1 in to the fungal cell. H. YAN (1), H. Zhang (2), M. Kim (3), B. Yoon (4), W. Shim (4) ( ), g ( ), ( ), ( ), ( ) (1) Texas A&M University, Plant Pathology & Microbiology, Texas A&M University, College Station, TX, 77843, College Station, TX, U.S.A.; (2) Texas A&M University, Plant Pathology & Microbiology, Texas A&M University, College Station, TX, 77843, College Station, TX, U.S.A.; (3) Texas A&M University, U.S.A.; (4) Texas A&M University Electrical & Computer Engineering, Texas A&M University, College Station, TX, 77843, U.S.A.; (5) Texas A&M University Electrical & Computer Engineering, Texas A&M University, College Station, TX, 77843, TX, U.S.A. Expression of the FST1 from Fusarium verticillioides in a yeast strain lacking a major myo-inositol transporter gene C. NIU (1), G. Payne (2), C. Woloshuk (3) (1) Purdue University, U.S.A.; (2) North Carolina State University, U.S.A.; (3) Purdue University, U.S.A. Az produces two lactam-containing antibiotics, named pyrrocidine A (PA) and B (PB), with PA exhibiting greater inhibitory activity against Fv and other fungi. PA differs from PB only by having a double bond in the lactam ring, suggesting the importance of the lactam moiety for toxicity. Preliminary experiments indicated the fungus is able to metabolize PB. In the Fv genome, 38 out of the 46 identified lactamase domain-containing proteins are predicted to carry out lactam hydrolysis. We recently characterized one lactamase gene (FVEG_08291) essential for the hydrolysis of 2- benzoxazolinone, a maize phytoanticipin, that significantly induced the expression of this lactamase and adjacent clustered genes. Likewise, we predict that pyrrocidines will induce one or more lactamase gene(s) and that the encoded enzyme(s) will function to hydrolyze these Az metabolites. RNA-seq experiments are underway to assess the Fv transcriptional response to pyrrocidines and to Az co-inoculation. RNAseq data should allow us to identify the interacting lactamase gene(s) and help us better understand the molecular mechanisms underlying the metabolic antagonism between these endophytes. A dynamic regulatory network model underlying stomatal infection in Cercospora zeae-maydis J. RIDENOUR (1), B. Bluhm (1) (1) University of Arkansas, U.S.A. Macrophomina phaseolina induces stalk senescence in charcoal rot susceptible sorghum genotypes through up-regula biosynthesis Macrophomina phaseolina induces stalk senescence in charcoal rot susceptible sorghum genotypes through up-regulated host chlorophyllase-2 biosynthesis A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1) (1) Kansas State University, U.S.A. A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1) (1) Kansas State University, U.S.A. Charcoal rot (Macrophomina phaseolina, MP) and Fusarium stalk rot (Fusarium thapsinum, FT) are important sorghum diseases worldwide. An RNA- Seq experiment was conducted to explore the interaction of MP and FT with known resistant and susceptible sorghum genotypes. SC599 (R) and TX7000 (S) sorghum lines were grown in the greenhouse and inoculated with FT, MP, and phosphate-buffered saline (control). RNA extracted from stem tissues at 2, 7, and 30 days post-inoculation (d.p.i.) was subjected to RNA-Seq. At 7 d.p.i., a gene that encodes chlorophyllase-2 (Sb02g012300) showed two and 200-fold up-regulation in FT- and MP-inoculated Tx7000, respectively. SC599 did not show a significant differential expression. To confirm the effect of up-regulated chlorophyllase-2, stalk chlorophyll was extracted with acetone from two pathogen- and mock-inoculated resistant (SC599, SC35) and susceptible (Tx7000, BTx3042) sorghum lines at 7 d.p.i. Total chlorophyll content (mg/g stalk) was computed using spectrophotometer readings. Compared to control, MP significantly reduced total chlorophyll of Tx7000 (control, 1.56; MP, 0.40) and BTx3042 (control, 1.45; MP, 0.63) (P < 0.016). No such effect was evident with the two resistant genotypes. FT did not significantly affect the chlorophyll content of any genotype. Results revealed the ability of MP to induce stalk senescence through increased chlorophyll degradation, which contributes to the enhanced vulnerability to this necrotrophic fungus. ( ) p ( ) g p p y ( g g ) p g spectrophotometer readings. Compared to control, MP significantly reduced total chlorophyll of Tx7000 (control, 1.56; MP, 0.40) and BTx3042 (control, 1.45; MP, 0.63) (P < 0.016). No such effect was evident with the two resistant genotypes. FT did not significantly affect the chlorophyll content of any genotype. Results revealed the ability of MP to induce stalk senescence through increased chlorophyll degradation, which contributes to the enhanced vulnerability to this necrotrophic fungus. Genome-wide study on cultivar-specific and stripe rust responsive miRNAs in Triticum aestivum S. RAMACHANDRAN (1), N. Mueth (1), P. Zheng (1), S. Hulbert (1) (1) Washington State University, U.S.A. Wheat represents approximately 30% of the world’s grain production with more than 220 million cultivated hectares. Macrophomina phaseolina promotes charcoal rot susceptibility in sorghum through induced host nitric oxide (NO) production A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1) (1) Kansas State University, U.S.A. Macrophomina phaseolina promotes charcoal rot susceptibility in sorghum through induced host nitric oxide (NO) production A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1) (1) Kansas State University, U.S.A. Macrophomina phaseolina (MP) is an important necrotrophic pathogen causing charcoal rot disease in sorghum (Sorghum bicolor (L.) Moench). Exploring the host-pathogen interaction is a key to understanding the molecular basis of resistance. An RNA-Seq experiment was conducted to discover differentially expressed genes (DEG) in response to MP infection. SC599 (resistant) and Tx7000 (susceptible) sorghum lines were grown in the greenhouse and inoculated with MP and phosphate-buffered saline (mock-inoculated control). RNA was extracted from three biological replicates at 2, 7, and 30 d post-inoculation (d.p.i.) from stem tissues adjacent to the inoculation point and subjected to RNA-Seq. Among the DEG, three genes (Sb08g011530, Sb05g000680, and Sb04g027860) involved in NO biosynthesis were significantly upregulated in Tx7000 at 7 d.p.i., while those of SC599 were not differentially expressed. To further confirm the findings, confocal microscopy was used. At 7 d.p.i, in situ NO production in MP- and mock- inoculated stem cross sections of two resistant (SC599, SC35) and two susceptible (Tx7000, BTx3042) lines was analyzed using 4-amino-5- methylamino-2?,7?-difluorofluorescein diacetate (DAF-FM DA). Strong green fluorescence was observed in both MP-inoculated susceptible lines confirming the presence of NO while no signal was detected in the resistant lines. We believe that NO-mediated cell death contributes to the rapid proliferation of MP within susceptible hosts. A dynamic regulatory network model underlying stomatal infection in Cercospora zeae-maydis J. RIDENOUR (1), B. Bluhm (1) (1) University of Arkansas, U.S.A. Andargie (2) (1) South China Botanical Garden, Chinese Academy of Sciences, China; (2) South China Botanical Garden, Chinese Academy of Scicnecs, China ) al Garden, Chinese Academy of Sciences, China; (2) South China Botanical Garden, Chinese Academy of Scicnecs, China g ( ) ina Botanical Garden, Chinese Academy of Sciences, China; (2) South China Botanical Garden, Chinese Academy of Scicn Rice false smut fungus which is a biotrophic fungal pathogen causes an important rice disease and brings a severe damage where rice is cultivated. We established a new fungal-plant pathosystem where Ustilaginoidea virens was able to interact compatibly with the model plant Arabidopsis thaliana. Disease symptoms were apparent on the leaves of the plants after 6 days of post inoculation in the form of chlorosis. Cytological studies showed that U. virens caused a heavy infestation inside the cells of the chlorotic tissues. Development and colonization of aerial mycelia in association with floral organ, particularly on anther and stigma of the flowers after 3 weeks of post inoculation was evident which finally caused infection on the developing seeds and pod tissues. The fungus adopts a uniquely biotrophic infection strategy in roots and spreads without causing a loss of host cell viability. We have also demonstrated that U. virens isolates infect Arabidopsis and the plant subsequently activates different defense response mechanisms which are witnessed by the expression of pathogenesis-related genes, PR-1, PR-2, PR-5, PDF1.1 and PDF1.2. The established A. thaliana–U. virens pathosystem will now permit various follow-up molecular genetics and gene expression experiments to be performed to identify the defense signals and responses that restrict fungal hyphae colonization in planta and also provide initial evidence for tissue-adapted fungal infection strategies. Induced rhamnose biosynthesis in sorghum by Macrophomina phaseolina contributes to charcoal rot susceptibility A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1) (1) Kansas State University, U.S.A. Induced rhamnose biosynthesis in sorghum by Macrophomina phaseolina contributes to charcoal rot susceptibility A. BANDARA (1), D. Weerasooriya (1), S. Liu (1), C. Little (1) (1) Kansas State University, U.S.A. Macrophomina phaseolina (MP) is a necrotrophic pathogen that causes charcoal rot disease in sorghum (Sorghum bicolor (L.) Moench). To detect differentially expressed genes (DEG) in response to MP infection, an RNA-Seq experiment was conducted. SC599 (resistant) and Tx7000 (susceptible) sorghum lines were stalk-inoculated with MP hyphal fragments and phosphate-buffered saline (control). Stalk RNA extracted at 2, 7, and 30 d post- inoculation (d.p.i.) was subjected to RNA-Seq. Among the DEG, 7 genes involved in the rhamnose biosynthetic pathway (Sb01g038050, Sb01g039220, Sb01g043370, Sb02g029130, Sb08g022850, Sb09g018070, and Sb10g024490) were significantly upregulated in Tx7000 at 7 d.p.i., while those of SC599 showed no differential expression. To confirm the results, stem juice extracted from two MP- and mock-inoculated resistant (SC599, SC35) and susceptible (Tx7000, Btx3042) sorghum lines were subjected to HPLC. Compared to control, the MP-inoculated charcoal rot susceptible lines had significantly higher (P < 0.005) stalk rhamnose concentrations (mg/mL) (Tx7000, 7.2; BTx3042, 5.8), while no such effect was evident in the resistant lines (SC599, 1.6; SC35, 1.4). To test the effect of rhamnose on MP growth, two isolates were grown on minimal medium supplemented with rhamnose at 0, 25, 50, 75, 100, and 125 mM concentrations. MP grew on rhamnose in a concentration-independent manner. Results suggested the potential role of host rhamnose as a virulence factor for MP. A dynamic regulatory network model underlying stomatal infection in Cercospora zeae-maydis J. RIDENOUR (1), B. Bluhm (1) (1) University of Arkansas, U.S.A. Cercospora zeae-maydis causes gray leaf spot of maize, which is considered to be one of the most important foliar diseases of maize worldwide. Intriguingly, C. zeae-maydis has evolved an infection strategy that exploits host stomata. Prior to entering the host, the fungus undergoes a series of defined morphogenetic transitions collectively termed pre-infectious development. Despite the importance of pre-infectious development, the underlying genetics are poorly understood. Genetic regulatory network (GRN) modeling offers a novel approach to understand the complexity underlying plant- S4.150 pathogen interactions. The overarching goal of this project is to utilize GRN modeling to identify central regulatory hubs that control pre-infectious development in C. zeae-maydis. To this end, we first developed a methodology to collect fungal tissue from inoculated maize during pre-infectious development and extract RNA of sufficient quality for RNA sequencing (RNA-seq). RNA-seq data is being generated via the Ion Torrent platform, and will be used to generate a GRN model. Preliminary transcriptome analysis has revealed that genes involved in transcriptional regulation, signaling, and stress and defense are enriched during pre-infectious development. Defining pre-infectious development at the systems level will provide mechanistic insight into the developmental program employed by C. zeae-maydis and augment efforts to identify antifungal targets and improve disease resistance. pathogen interactions. The overarching goal of this project is to utilize GRN modeling to identify central regulatory hubs that control pre-infectious development in C. zeae-maydis. To this end, we first developed a methodology to collect fungal tissue from inoculated maize during pre-infectious development and extract RNA of sufficient quality for RNA sequencing (RNA-seq). RNA-seq data is being generated via the Ion Torrent platform, and will be used to generate a GRN model. Preliminary transcriptome analysis has revealed that genes involved in transcriptional regulation, signaling, and stress and defense are enriched during pre-infectious development. Defining pre-infectious development at the systems level will provide mechanistic insight into the developmental program employed by C. zeae-maydis and augment efforts to identify antifungal targets and improve disease resistance. Arabidopsis thaliana: A model host plant to study plant-pathogen interaction using rice false smut isolates of Ustilaginoidea virens J. LI (1), M. Andargie (2) (1) South China Botanical Garden, Chinese Academy of Sciences, China; (2) South China Botanical Garden, Chinese Academy of Scicnecs, China J. LI (1), M. Macrophomina phaseolina induces stalk senescence in charcoal rot susceptible sorghum genotypes through up-regula biosynthesis Productivity at harvest is mainly governed by genetic composition and environmental factors influencing growth and development. Understanding the genetic factors that regulate these processes will help in developing varieties with better yield potential and disease resistance. MiRNAs are regulators of gene expression in eukaryotes that control myriad processes from development to abiotic and biotic stress responses. Using 12 small RNA libraries prepared from two wheat cultivars infected with stripe rust fungus (Puccinia striiformis), we identified 43 previously-known miRNAs, 50 novel variants of known miRNAs and 93 S4.151 candidate novel miRNAs. Digital gene expression revealed 20 cultivar-specific miRNAs, 6 miRNAs differentially expressed between the two cultivars and 4 miRNAs responsive to stripe rust infection. These results were validated using RT-PCR and qRT-PCR. Using different target prediction algorithms, 69 wheat miRNAs were found to target fungal genes - a majority of which coded for small, secreted, cellular proteins. These results suggest a trans-kingdom regulation of gene expression potentially involved in the cultivar-specific resistance to stripe rust. Overall, this study contributes to the current repository of wheat miRNAs and provides novel information on the as yet uncharacterized roles for miRNAs in wheat host-pathogen interactions. candidate novel miRNAs. Digital gene expression revealed 20 cultivar-specific miRNAs, 6 miRNAs differentially expressed between the two cultivars and 4 miRNAs responsive to stripe rust infection. These results were validated using RT-PCR and qRT-PCR. Using different target prediction algorithms, 69 wheat miRNAs were found to target fungal genes - a majority of which coded for small, secreted, cellular proteins. These results suggest a trans-kingdom regulation of gene expression potentially involved in the cultivar-specific resistance to stripe rust. Overall, this study contributes to the current repository of wheat miRNAs and provides novel information on the as yet uncharacterized roles for miRNAs in wheat host-pathogen interactions. Genotype and chemotype profiles of endophytes associated with wild barley Genotype and chemotype profiles of endophytes associated with wild barley M. YI (1), J. Kaste (2), N. Charlton (1), W. Hendricks (1), N. Krom (1), P. Nagabhyru (3), D. Panaccione (4), C. Young (1) (1) The Samuel Roberts Noble Foundation, Ardmore, OK, U.S.A.; (2) Cornell University, Ithaca, NY, U.S.A.; (3) University of Kentucky, Lexington, KY, U.S.A.; (4) West Virginia University, Morgantown, WV, U.S.A. The genetic and chemotypic diversity of Epichloë species (Clavicipitaceae, Ascomycota) isolated from wild barley (Hordeum spp.) was explored to provide potential valuable resources for forage improvement. Exploring the Monilinia vaccinii-corymbosi-blueberry pathosystem for the discovery of genes governing host-specificity K. BANSAL (1), J. Rollins (1) (1) University of Florida, U.S.A. Exploring the Monilinia vaccinii-corymbosi-blueberry pathosystem for the discovery of genes governing host-specificity K. BANSAL (1), J. Rollins (1) (1) University of Florida, U.S.A. Monilinia vaccinii-corymbosi, a Sclerotiniaceae member, causes mummy berry disease on blueberries. Primary infection of vegetative tissues is caused by ascospores and secondary infection of flowers is caused by conidia. Fruit infection is highly specific as conidial germ tubes enter the host through flowers. Conidia germinate on stigma, traverse the stylar canal to colonize developing ovules, converting fruit into a mummy. To understand the underlying genetics of this specificity, we are developing standardized tools for generating inoculum, visualizing the course of infection, and collecting infected materials for transcriptomic analysis. A comparison of culture media and temperature conditions demonstrated that four day old cultures of M. vaccinii-corymbosi isolate RL1 (Mvc-RL1) grown on oatmeal agar medium, in the dark, at 22°C followed by a low temperature shift (15°C) induced sporulation most abundantly after 24 hours. For characterization of the gynoecium infection process, a protoplast-mediated genetic transformation system was utilized to produce constitutive GFP-expressing Mvc-RL1 strains. Floral inoculation of Southern High Bush variety Legacy with conidia of GFP-Mvc-RL1 documented germinating conidia and the course of hyphae traversing the stylar canal. Using these tools, floral infections will be utilized for comparison with uninoculated flowers for transcriptomic analysis and identification of genes underlying host specificity in M. vaccinii-corymbosi. Fungal transcriptome analysis of the Sclerotinia sclerotiorum and Pisum sativum interaction M. CHILVERS (1), J. Rojas (1), P. Santos (2), J. Wang (1) (1) Michigan State University, U.S.A.; (2) University of Nevada, U.S.A. Sclerotinia sclerotiorum is a broad host range necrotrophic pathogen that causes white mold on pea (Pisum sativum). Gene expression profiling during infection has been characterized for S. sclerotiorum on several model plants. However, the S. sclerotiorum-P. sativum interaction is still poorly understood. In this study, fungal transcriptomic data was collected at three time points: 12 h, 24 h, and 48 h post inoculation (hpi), from susceptible and partially resistant pea cultivars inoculated with S. sclerotiorum. As a control, transcriptomic data was also collected from mycelia grown on PDA medium used in the inoculations. Four gene clusters were identified using expression profile analysis, one cluster was exclusively up-regulated on PDA, while the remaining three clusters were either associated with the host or sampling time. Aspartyl protease mediated cleavage of AtBAG6 triggers autophagy and fungal resistance in plants Y. LI (1), M. Kabbage (2), M. Dickman (1) (1) Texas A&M University, U.S.A.; (2) University of Wisconsin-Madison, U.S.A. Aspartyl protease mediated cleavage of AtBAG6 triggers autophagy and fungal resistance in plants Y. LI (1), M. Kabbage (2), M. Dickman (1) (1) Texas A&M University, U.S.A.; (2) University of Wisconsin-Madison, U.S.A. The Bcl-2-associated-athanogene (BAG) family is an evolutionarily conserved group of co-chaperones that modulate numerous cellular processes. Previously we found that Arabidopsis BAG6 (AtBAG6) is required for basal immunity against the fungal phytopathogen Botrytis cinerea. However, the mechanism(s) by which AtBAG6 controls immunity are obscure. Here, we address this important question by determining the molecular mechanisms responsible for AtBAG6 mediated basal resistance. We show that AtBAG6 is cleaved in vivo in a caspase-1 like dependent manner, and via a combination of pull-downs, mass spectrometry, yeast-two-hybrid and chemical genomics, we demonstrate that AtBAG6 interacts with a C2-GRAM domain protein (AtBAGP1) and an aspartyl protease (AtAPCB1), both of which are required for AtBAG6 processing. Furthermore, fluorescence and transmission electron microscopy established that AtBAG6 cleavage triggers autophagy in the host that coincides with disease resistance. Targeted inactivation of AtBAGP1 or AtAPCB1 results in the blocking of AtBAG6 processing and loss of resistance. Mutation of the cleavage site blocks cleavage, inhibits autophagy and disease resistance is compromised. Taken together, these results couple an aspartyl protease with a molecular co- chaperone to trigger autophagy and plant defense, providing a key link between fungal recognition and the induction of cell death and resistance. The MAP kinase kinase kinase gene, CpBck1, regulates sectorization and pathogenicity of the phytopathogenic fungu D KIM (1) The MAP kinase kinase kinase gene, CpBck1, regulates sectorization and pathogenicity of the phytopathogenic fu D. KIM (1) ( ) h b k i l i i The MAP kinase kinase kinase gene, CpBck1, regulates sectorization and pathogenicity of the phytopathogenic fungus Cryphonectria parasitica D. KIM (1) (1) Chonbuk National University, Korea ( ) (1) Chonbuk National University, Korea CpBck1, an ortholog of the cell-wall integrity mitogen-activated protein kinase kinase kinase of Saccharomyces cerevisiae, was cloned and characterized from the chestnut blight fungus Cryphonectria parasitica. The CpBck1-null mutant displayed cell wall integrity–related phenotypic changes such as abnormal cell morphology and wall formation and hypersensitivity to cell wall-disrupting agents. In addition, the mutant showed severely retarded growth without any sign of normal development, such as hyphal differentiation, conidiation, or pigmentation. As the culture proceeded, the mutant colony showed sporadic sectorization. Once sectored, the sectored phenotype of robust mycelial growth without differentiation was stably inherited. Exploring the Monilinia vaccinii-corymbosi-blueberry pathosystem for the discovery of genes governing host-specificity K. BANSAL (1), J. Rollins (1) (1) University of Florida, U.S.A. Gene ontology enrichment analysis demonstrated that 83% of the up-regulated cluster 1 genes were categorized as oxidoreductases at 12 hpi. Whereas, 43% of the up-regulated cluster 2 genes were involved in hydrolysis and transport functions at 24 and 48 hpi. The temporal gene expression profile change may indicate the lifestyle transition of the pathogen from biotroph to necrotroph. In addition, three out of 486 predicted effector candidate genes were up-regulated in planta at 24 and 48 hpi. The identification of key genes involved in the S. sclerotiorum-P. sativum interaction will facilitate effector assisted breeding for disease management. Macrophomina phaseolina induces stalk senescence in charcoal rot susceptible sorghum genotypes through up-regula biosynthesis We screened 56 wild barley PI lines from the US Department of Agriculture’s National Plant Germplasm System (USDA-NPGS), and identified 16 PI lines with endophyte incidence. Five distinct endophyte genotypes representing three nonhybrid and two hybrid Epichloë species were identified among the isolated endophytes from nine viable PI lines. Genotypes were identified based on the presence of alkaloid biosynthesis and mating type genes. Molecular phylogenetic analyses indicated that all nonhybrid genotypes are E. bromicola, whereas the two hybrid genotypes are E. bromicola by either E. typhina or an ancestor of E. amarillans or E. baconii. One of the hybrid genotypes was identified as a new taxonomic group currently referred to as HboTG-3. Alkaloid profiles were predicted from the genotyping data and the chemical profiles of distinct isolates were confirmed by HPLS and mass spectrometry with endophyte-infected plants, which confirmed the predicted chemotypes. Whole genome sequencing of four isolates provided insights for the complete alkaloid biosynthesis clusters in the wild barley endophytes and the unique features of the Epichloë species genomes. The beneficial effects each endophyte may provide the host will be tested under various biotic and abiotic stresses. Exploring the Monilinia vaccinii-corymbosi-blueberry pathosystem for the discovery of genes governing host-specificity K. BANSAL (1), J. Rollins (1) (1) University of Florida, U.S.A. A band of misfits: Role of unexpected proteins in the plant symbiotic signaling pathway A band of misfits: Role of unexpected proteins in the plant symbiotic signaling pathway M. VENKATESHWARAN (1), A. Wiley-Kalil (2), D. Jayaraman (3), M. Banba (4), A. Binder (5), S. Bernard (6), J. Maeda (6), M. Otegui (7), H. Imaizumi-Anraku (8), M. Parniske (5), J. Ané (9) (1) University of Wisconsin-Platteville, Platteville, WI, U.S.A.; (2) NDSU Williston Research Extension Center, U.S.A.; (3) University of Wisconsin- Madison, U.S.A.; (4) Division of Plant Sciences, National Institute of Agrobiological Sciences, Japan; (5) Ludwig-Maximilians-Universität München, Germany; (6) Department of Agronomy, University of Wisconsin, Madison, U.S.A.; (7) Department of Botany, University of Wisconsin-Madison, U.S.A.; (8) Division of Plant Sciences, National Institute of Agrobiological Sciences, U.S.A.; (9) Departments of Bacteriology and Agronomy, University of Wisconsin, Madison, Madison, WI, U.S.A. ( ), ( ), ( ) (1) University of Wisconsin-Platteville, Platteville, WI, U.S.A.; (2) NDSU Williston Research Extension Center, U.S.A.; (3) University of Wisconsin- Madison, U.S.A.; (4) Division of Plant Sciences, National Institute of Agrobiological Sciences, Japan; (5) Ludwig-Maximilians-Universität München, Germany; (6) Department of Agronomy, University of Wisconsin, Madison, U.S.A.; (7) Department of Botany, University of Wisconsin-Madison, U.S.A.; (8) Division of Plant Sciences, National Institute of Agrobiological Sciences, U.S.A.; (9) Departments of Bacteriology and Agronomy, University of Wisconsin, Madison, Madison, WI, U.S.A. Rhizobia and arbuscular mycorrhizal fungi produce lipo-chito-oligosaccharidic signals that are perceived by legume hosts at the plasma membrane level. These signals are then transduced to the nucleus where they trigger oscillations of the nuclear calcium concentration (calcium spiking) that, in turn, regulates symbiotic gene expression. Genetic and protein interaction studies identified some relatively expected components in such a signaling pathway: receptor-like kinases at the plasma membrane that perceive the microbial signals, ion channels and ion pumps on the nuclear envelope that allow calcium spiking, and nuclear transcription factors that regulate gene expression. We will present new data on more unexpected members of this signaling pathway. (1) An HMGR (3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase) that interacts with and is regulated by the symbiotic receptor kinases. In particular, we will show that the activity of this HMGR is not only necessary but also sufficient to trigger calcium spiking. (2) Nucleoporins of the NUP107-160 sub-complex that have been identified through forward genetic approaches. We will show that these nucleoporins are required for the proper localization of symbiotic ion channels to the inner nuclear membrane. A band of misfits: Role of unexpected proteins in the plant symbiotic signaling pathway Finally, we will compare results between two legumes, Medicago truncatula and Lotus japonicus, and show the value of comparing carefully these two genetic systems. Functional analysis of root-knot nematode (Meloidogyne javanica) virulence genes in rice y gy j g D. FERNANDEZ (1), D. Fernandez (2), M. Grossi de Sá (3), I. Mezzalira (4), M. Beneventi (4), M. Lisei de Sá (5), D. Am Petitot (7), J. de Almeida Engler (8), É. Saliba Albuquerque (4), M. Grossi de Sá (4) D. FERNANDEZ (1), D. Fernandez (2), M. Grossi de Sá (3), I. Mezzalira (4), M. Beneventi (4), M. Lisei de Sá (5), D Petitot (7), J. de Almeida Engler (8), É. Saliba Albuquerque (4), M. Grossi de Sá (4) g (1) IRD - French Research Institute for Development, Montpellier cedex 5, France; (2) IRD- French Research Institute for Development, Montpellier, France; (3) IRD, Montpellier, France; (4) EMBRAPA CENARGEN, Brazil; (5) Epamig and Embrapa Cenargen, Brazil; (6) Parakou University, Benin; (7) IRD- French Research Institute for Development, France; (8) INRA, France Root-knot nematodes are endo-parasites with a wide host range, encompassing mono- and dicotyledonous plant crops. Meloidogyne javanica is responsible for rice (Oryza sativa) production losses in Brazil and Africa (Benin). Successful infection is likely achieved by effector proteins produced in the nematode esophageal gland cells and released in the host plant cells. The aim of this study was to assess the functional role of three esophageal gland cell proteins of M. javanica in rice - nematode interactions. RT-qPCR assays showed that Mj-SP2, Mj-SP18 and Mj-SP19 genes are over-expressed all along the infection cycle in rice roots. Immunodetection experiments using specific antibodies raised against synthetic peptides showed that the nematode proteins are localized in circular granules structures within the nematode body. Transgenic rice (O. sativa Nipponbare) plants expressing the candidate proteins or artificial micro-RNAs (amiRNAs) able to silence the cognate genes in the nematode were produced. Assessment of nematode reproduction on homozygous transgenic lines allowed the identification of rice lineages with altered susceptibility, indicating that these proteins may be involved in M. javanica virulence. Data obtained significantly widen our knowledge of molecular players contributing to nematode pathogenicity, and open new avenues for nematode control strategies in rice and other crops of interest. Aspartyl protease mediated cleavage of AtBAG6 triggers autophagy and fungal resistance in plants Y. LI (1), M. Kabbage (2), M. Dickman (1) (1) Texas A&M University, U.S.A.; (2) University of Wisconsin-Madison, U.S.A. Compared with the wild type, both the parental CpBck1-null mutant and the sectored progeny exhibited marked impaired virulence. The present study S4.152 revealed that a mutation in a signaling pathway component related to cell-wall integrity resulted in sporadic sectorization and these sectored phenotypes were stably inherited, suggesting that this signal transduction pathway is implicated in adaptive genetic changes for sectorization. revealed that a mutation in a signaling pathway component related to cell-wall integrity resulted in sporadic sectorization and these sectored phenotypes were stably inherited, suggesting that this signal transduction pathway is implicated in adaptive genetic changes for sectorization. Molecular dissection of resistance signaling in watermelon fruit through transcriptomic approach M. MANDAL (1), J. Ikerd (2), A. Soorni (3), C. Kousik (2) M. MANDAL (1), J. Ikerd (2), A. Soorni (3), C. Kousik (2) (1) ORISE Participant sponsored by the U.S. Vegetable Laboratory, USDA, ARS., Charleston, SC, U.S.A.; (2) U.S. department of Agriculture, Agricultural Research Service, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (3) Department of Horticulture, Virginia Tech, Blacksburg, WV, U.S.A. Plant defense signaling involves a complex array of events initiated by direct or indirect interactions between pathogen effectors and host resistance proteins. Our study involves developing watermelon (Citrullus lanatus) lines resistant to various pests and pathogens through conventional breeding techniques. One of the most common and widely spread diseases in watermelon is Phytophthora fruit rot caused by Phytophthora capsici, which is especially severe in the southeastern US due to prevalence of favorable weather conditions. In order to elucidate the molecular mechanism of signaling events that occur during watermelon - Phytophthora interactions we are utilizing the available watermelon and P. capsici genome sequence data along with our RNA-seq data to identify the differentially regulated transcriptome between resistant and susceptible watermelon lines. Our preliminary data indicates that most sample sequence reads matched to watermelon genome (70-90%) and the pathogen transcript reads were higher in susceptible cultivar ‘Sugar Baby’ (5-30%) when compared to resistant germplasm line ‘USVL020-PFR’ (0.5-1%). The genotypic data correlates well with the phenotypic data. The overall goal is to identify plant defense markers contributing to host resistance against P. capsici that will prove useful in watermelon breeding programs. Identification of silencing suppressor proteins in Maize chlorotic mottle virus N. BACHELLER (1), H. Garcia-Ruiz (1) (1) University of Nebraska-Lincoln, U.S.A. MetacodeR: An R package for comparative analysis and visualization of microbial communities and evaluation of metabarcoding primer specificity Z FOSTER (1) N G ld (1) MetacodeR: An R package for comparative analysis and visualization of microbial communities and evaluation of metabarcoding primer specificity Z. FOSTER (1), N. Grunwald (1) Metabarcoding, the sequencing of a locus amplified from environmental DNA, is revealing the unculturable majority of microbes for the first time, but new methods present new challenges. Complex hierarchical data are difficult to plot and the commonly used stacked bar charts are ineffective when plotting multiple taxonomic ranks or treatments. In addition, the choices of loci and barcode primers are a source of underexplored bias. MetacodeR is a new tool kit for metabarcoding and community ecology, focusing on visualization of taxonomic data and in silico primer/barcode evaluation. It provides tools to integrate quantitative data from thousands of taxa and multiple communities into intuitive tree-based plots. Data from one or more communities are mapped to tree element size or color, as in a heat map. This allows for rapid exploration of data and information-rich, publication-quality graphics. In addition, metacodeR provides critical tools for evaluating potential primers and barcode loci. In silico PCR evaluates primer specificity and barcode gap analysis estimates error rates and resolution. MetacodeR is currently being applied to evaluating the diversity of fungi and oomycetes in the rhizosphere of nursery-grown rhododendron. The ability to easily test new primers/barcodes will accelerate the adoption of metabarcoding to emerging or understudied pathogen groups, such as oomycetes, and intuitive plotting will make subtle patterns in complex data more apparent. Molecular dissection of resistance signaling in watermelon fruit through transcriptomic approach M. MANDAL (1), J. Ikerd (2), A. Soorni (3), C. Kousik (2) Resistance of cultivated soybean to Clover yellow vein virus apparently originated during domestication from wild soybean K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2) (1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A. Resistance of cultivated soybean to Clover yellow vein virus apparently originated during domestication from wild soybean K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2) (1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A. Clover yellow vein virus (ClYVV) (genus Potyvirus, family Potyviridae), is pathogenic to legume plants, including broad bean (Vicia faba) and pea (Pisum sativum). However, inoculation of dozen cultivated soybean (Glycine max) genotypes with ClYVV strain no30 (ClYVV-no30) did not result in systemic infection in any of the inoculated plants. In contrast, inoculation of 24 accession lines of wild soybean (G. soja) with the green fluorescent protein (GFP)-tagged ClYVV-no30 (ClYVV-no30-GFP) resulted in systemic infection in all, except one line. This observation led us to hypothesize that resistance to ClYVV in cultivated soybean originated from wild soybean during domestication. It seems likely that during soybean domestication, parental plants with natural resistance to ClYVV were selected. To test this hypothesis, and to elucidate the genetic basis of the resistance, recombinant inbred lines (RILs) were generated from a cross between cultivated soybean TK780 and wild soybean line B01167. Analysis of responses of 64 derivative RILs inoculated with ClYVV-no30-GFP resulted in identification of one major and one minor quantitative trait loci (QTL). The role of each of these loci in conferring resistance to ClYVV in cultivated soybean is being investigated. Examination of the mechanism of superinfection exclusion by Citrus tristeza virus S. FOLIMONOVA (1), O. Atallah (1), S. Kang (2) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Superinfection exclusion (SIE), referred to as cross-protection when used as an agricultural practice, is a phenomenon in which a primary virus infection prevents a secondary infection with the same or closely related virus. Earlier we demonstrated that SIE by Citrus tristeza virus requires viral p33 protein, yet p33 alone is not sufficient for virus exclusion. In this study we show that a 5’-proximal genomic region encoding leader proteases L1 and L2 is involved in SIE. Substitution of this region with the cognate sequences from a heterologous strain affected virus ability to exclude the parental virus. As the next step, we are interested to assess whether SIE is conferred by the L1/L2 proteins or by their coding sequences. Resistance of cultivated soybean to Clover yellow vein virus apparently originated during domestication from wild soybean K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2) (1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A. We created four mutant viruses having alterations in the RNA sequences of the L1 and L2 N-terminal (NTD) and protease domains by changing the wobble base of each possible codon to avoid changing the amino acid sequences of these proteins. All mutant viruses were able to replicate in Nicotiana benthamiana at the levels comparable to the wild type virus, and three of them except L1 NTD mutant were able to establish systemic infection in N. benthamiana and citrus plants. Additional constructs containing mutations within the L1 NTD domain are being made with the goal to produce virus variants capable of systemic infection of both plant hosts. The effect of the introduced mutations in the L1-L2 domains on SIE ability will be assessed. Exchange of HC-Pro cistron between Soybean mosaic virus and Clover yellow vein virus: Impact on pathogenicity M.R. HAJIMORAD (1), Y. Wang (1,2), J. Abe (3), K. Nakahara (3) (1) University of Tennessee, Knoxville, TN, U.S.A., (2) Jilin Academy of Agricultural Sciences, Jilin, China, (3) Hokkaido University, Japan Soybean mosaic virus (SMV)-N and Clover yellow vein virus (ClYVV)-no30, members of Potyvirus genus within Potyviridae family, have narrow host range. SMV-N is well adapted to cultivated soybean (Glycine max) while infects systemically wild-type soybean (G. Soja) as well. However, SMV-N is incapable of systemic infection in broad bean (Vicia faba). In contrast, ClYVV-no30 is capable of systemic infection in both broad bean and wild-type soybean; however, incapable of systemic movement in cultivated soybean. Because SMV-N and ClYVV-no30 both establish local infection in all these plants, we hypothesized that lack of systemic movement by SMV-N in broad bean and by ClYVV-no30 in cultivated soybean is due to incompatibility of respective HC-Pro, a key long-distance movement potyviral protein, in these two hosts. To test this hypothesis, chimeras were constructed with precise exchanges of HC-Pro cistrons between SMV-N and ClYVV-no30 and inoculated onto cultivated soybean “Williams”, wild-type soybean “B01167” and broad bean “Windsor”. Analyses showed that pathogenicity of recombinant viruses did not differ from those of the parental viruses. This observation suggests that i) HC-Pro of SMV-N and ClYVV-no30 are functionally equivalent, and ii) the determinant(s) for systemic infection in cultivated soybean by SMV-N and by ClYVV-no30 in broad bean reside elsewhere. 3K unigene-based Nicotiana benthamiana microarray to study host-pathogen interactions at the transcriptome level 1), P. Thammarat (1), T. Sit (2), S. Identification of silencing suppressor proteins in Maize chlorotic mottle virus N. BACHELLER (1), H. Garcia-Ruiz (1) (1) University of Nebraska-Lincoln, U.S.A. Maize Lethal Necrosis (MLN) is a devastating disease of maize caused by a synergistic interaction between Maize chlorotic mottle virus (MCMV) and Sugarcane mosaic virus (SCMV), Wheat streak mosaic virus (WSMV) or Triticum mosaic virus (TriMV). RNA silencing is a prominent antiviral S4.153 defense system in plants. In single and double infections, MCMV and SCMV activate maize antiviral RNA silencing machinery, resulting in the accumulation of virus-derived small RNAs. Most plant viruses encode silencing suppressor proteins to inactivate RNA silencing and overcome the host defense system. No silencing suppressor has been identified in MCMV. To identify silencing suppressors in MCMV, individual proteins were cloned into binary vectors for transient expression in Nicotiana benthamiana. Proteins with silencing suppression activity were identified by the ability to complement the pathogenicity defect in two different suppressor-deficient viruses. Identification and characterization of silencing in MCMV and SCMV establishes the foundation to further study the molecular mechanisms involved in Maize Lethal Necrosis Disease. defense system in plants. In single and double infections, MCMV and SCMV activate maize antiviral RNA silencing machinery, resulting in the accumulation of virus-derived small RNAs. Most plant viruses encode silencing suppressor proteins to inactivate RNA silencing and overcome the host defense system. No silencing suppressor has been identified in MCMV. To identify silencing suppressors in MCMV, individual proteins were cloned into binary vectors for transient expression in Nicotiana benthamiana. Proteins with silencing suppression activity were identified by the ability to complement the pathogenicity defect in two different suppressor-deficient viruses. Identification and characterization of silencing in MCMV and SCMV establishes the foundation to further study the molecular mechanisms involved in Maize Lethal Necrosis Disease. Resistance of cultivated soybean to Clover yellow vein virus apparently originated during domestication from wild soybean K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2) (1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A. Lommel (2) Devising a custom 13K unigene-based Nicotiana benthamiana microarray to study host-pathogen interactions at the transcriptome level P. THAMMARAT (1), P. Thammarat (1), T. Sit (2), S. Lommel (2) (1) Functional Genomics Program, North Carolina State University, U.S.A.; (2) Department of Entomology and Plant Pathology, North Carolina State University, U.S.A. ( ), ( ), ( ), ( ) (1) Functional Genomics Program, North Carolina State University, U.S.A.; (2) Department of Entomology and Plant Pathology, North Carolina State University, U.S.A. (1) Functional Genomics Program, North Carolina State University, U.S.A.; (2) Department of Entomology and Plant Pathology, North Carolina State University, U.S.A. Selecting a model plant to study host/pathogen interactions at the transcriptome level is a crucial part of experimental design. Although Arabidopsis thaliana is a preferred model based on the availability of comprehensive genomic information, it is only susceptible to a restricted number of pathogens. Due to this limitation, exploring alternative plant models is necessary. Nicotiana benthamiana (Nb) is highly susceptible to a wide range of pathogens. In order to enable the use of Nb in high-throughput studies, we created a genomic tool for this plant. We developed the first Nb array from 13,014 unigenes (average 752 bp) which were originally derived from 40,000 expressed sequence tags (ESTs; ~70% of publicly available Nb ESTs representing ~38% of the transcriptome). Homology searches against GenBank, KEGG, and GO databases (BLASTX, E-value < 1 × 10–5) indicated that 66% of the unigenes have > one hit, with the most frequent hit to Solanaceae plants. Our unigene-based array has broad coverage of plant pathways: metabolism, transportation, transcription, translation, signal transduction and defense. 261 unigenes were involved in transcription with the most abundant ones belonging to the AP2/ERF, WRKY and GRAS families. The Nb array consists of 9 probes per unigene. We conducted an array performance test to study interactions between Nb and Red clover necrotic mosaic virus. The array successfully yielded 100% hybridization capacity to Nb mock and infected samples. transportation, transcription, translation, signal transduction and defense. 261 unigenes were involved in transcription with the most abundant ones belonging to the AP2/ERF, WRKY and GRAS families. The Nb array consists of 9 probes per unigene. We conducted an array performance test to study interactions between Nb and Red clover necrotic mosaic virus. The array successfully yielded 100% hybridization capacity to Nb mock and infected samples. Resistance of cultivated soybean to Clover yellow vein virus apparently originated during domestication from wild soybean K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2) (1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A. Xiong (4) (1) Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (2) Institute of Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (3) Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (4) University of Arizona, Tucson, AZ, U.S.A. ( ), ( ), g ( ), g ( ), g ( ), ( ), g ( ) (1) Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (2) Institute of Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (3) Chinese Academy of Tropical Agricultural Sciences, Haikou, China; (4) University of Arizona, Tucson, AZ, U.S.A. Pepper veinal mottle virus (PVMV) causes a major disease on the Yellow Lantern chili peppers (Capsicum chinense), an economically important specialty crop on Hainan Island, China. PVMV is a Potyvirus with a genome-linked protein (VPg) attached at the 5’ end of the (+)-strand RNA genome. Interaction between PVMV VPg and pepper eIF4E is necessary for the PVMV to infect peppers. As there is no natural resistance against PVMV in the Yellow Lantern chili pepper, disruption of the VPg-eIF4E interaction by genome editing appears to be a feasible approach to engineer PVMV resistance. Four eIF4E genes were identified from bioinformatic analysis of pepper genomes, cloned by RT-PCR with specific primers, and analyzed by sequencing. The pepper eIF4E family consists of two eIF4E genes (Cc-eIF4E and Cc-eIF4E-Xm), one eIF(iso)4E gene (Cc-eIF(iso)4E), and one novel cap binding protein gene (Cc-nCPB9090). Only Cc-eIF4E and Cc-eIF(iso)4E interacted with PVMV VPg in the LexA yeast two-hybrid system. The specific interaction between these two pepper eIF4E proteins and PVMV VPg was further studied in vivo with bimolecular fluorescence complementation (BiFC) tests in infiltrated tobacco cells. Cc-eIF4E showed a strong and consistent interaction while Cc-eIF(iso)4E displayed a weak and inconsistent interaction with PVMV VPg by BiFC. These two eIF4E genes will be further evaluated as potential targets for genome editing in our quest for PVMV resistance in the chili pepper. QTL Analysis of flg22-triggered Basal Resistance in Maize X. ZHANG (1), P. Balint-Kurti (1), . Stacey (2), Y. Cao (2) (1) North Carolina State University, U.S.A.; (2) University of Missouri, U.S.A. Plants are constantly under attack by different pathogens. Resistance of cultivated soybean to Clover yellow vein virus apparently originated during domestication from wild soybean K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2) (1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A. Molecules released or generated during microbial entry can be perceived by the plant and produce activate defense, including early responses such as the oxidative burst. An important component of this defense system is based upon the recognition of microbe-associated molecular patterns (MAMPs also known as pathogen-associated molecular patterns or PAMPs). Once MAMPs are detected by the plant, a defense response, termed MAMP-triggered immunity (MTI, also known as PAMP-triggered immunity or PTI) happens. In a few cases, a direct link has been established between MTI and speci?c quantitative trait loci (QTLs) for partial resistance. The MTI response has been widely studied, mainly in the model plant Arabidopsis and a few crops, such as Brassica and soybean. Few studies have examined MTI in maize. There is signi?cant potential in harnessing maize MTI to improve cultivars to withstand a variety of pathogens. In this study, we evaluated the MTI responses in 26 different maize genotypes by challenging with one well known MAMP (flg22), and then assessed a recombinant inbred line mapping population (CML228×B73) derived from parent lines with differential flg22 responses. We identified one major QTL on chromosome 2 associated with variation in the flg22 MAMP response. We will describe our progress in characterizing this QTL and in identifying QTL associated with other MAMP responses. Physiological and Molecular-Genetic Characterization of Basal Resistance in Sorghum J. KIMBALL (1), D. Chen (2), G. Stacey (2), P. Balint-Kurti (1) (1) North Carolina State University, U.S.A.; (2) University of Missouri, U.S.A. Early detection of pathogens is a critical component of plant immunity. Basal resistance, aka innate immunity, is the first line of defense to protect plants against a range of pathogens. Triggered by the recognition of microbe-associated molecular patterns (MAMPs) by pathogen recognition receptors (PRRs), the induction of basal resistance has been shown to vary quantitatively within and across species. In sorghum (Sorghum bicolor (L.) Moench), preliminary results supports this hypothesis. The goal of this project is to investigate the genetic architecture and transcriptional response associated with the basal defense response in sorghum. Specific objectives in this project are to 1. Develop robust assays to measure disease resistance and the MAMP response in sorghum, and screen a set of diverse sorghum germplasm for variation in these traits, 2. Identify genes differentially regulated during the MAMP response in high and low responding sorghum genotypes, and 3. Resistance of cultivated soybean to Clover yellow vein virus apparently originated during domestication from wild soybean K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2) (1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A. Reimagining the Cauliflower mosaic virus genome as an interactome between host and virus proteins J. SCHOELZ (1), R. Nelson (2), S. Leisner (3), C. Angel (4) Reimagining the Cauliflower mosaic virus genome as an interactome between host and virus proteins J. SCHOELZ (1), R. Nelson (2), S. Leisner (3), C. Angel (4) (1) University of Missouri, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.; (3) University of Tole Reimagining the Cauliflower mosaic virus genome as an interactome between host and virus proteins J. SCHOELZ (1), R. Nelson (2), S. Leisner (3), C. Angel (4) (1) University of Missouri, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.; (3) University of Tol ( ), ( ), ( ), g ( ) (1) University of Missouri, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.; (3) University of Toledo, U.S.A.; (4) Cenicafe, Colombia Missouri, U.S.A.; (2) The Samuel Roberts Noble Foundation, U.S.A.; (3) University of Toledo, U.S.A.; (4) Cenicafe, Colom The genome of Cauliflower mosaic virus (CaMV) was the first plant-associated organism to be completely sequenced, in 1980. The CaMV nucleotide sequence revealed that its genome was composed of seven substantial open reading frames (ORFs), and six proteins were subsequently matched up with their respective ORFs; the seventh putative protein has never been found in CaMV-infected plants. Since the initial CaMV sequence was finalized, numerous studies have been completed to ascertain the functions of the six CaMV proteins, as well as their subcellular localization. Furthermore, many of the CaMV proteins have been the subject of yeast two-hybrid and co-immunoprecipitation screens to characterize the interactions of host and virus proteins. For example, the P6 protein of CaMV is a multifunctional protein with distinct roles in translation, intracellular movement, elicitation of defenses in resistant hosts or symptoms in susceptible hosts, and modulation of plant defenses. The P6 protein has been shown to interact with at least 14 S4.154 host and virus proteins. This presentation will illustrate how interactome maps coupled with subcellular localization studies can be used to describe the CaMV disease cycle. host and virus proteins. This presentation will illustrate how interactome maps coupled with subcellular localization studies can be used to describe the CaMV disease cycle. Two eIF4E proteins in Yellow Lantern chili pepper interact with Pepper veinal mottle virus VPg Z. ZHANG (1), N. Yu (1), X. Zhang (2), Y. Zhang (1), J. Wang (1), Z. Liu (3), Z. Resistance of cultivated soybean to Clover yellow vein virus apparently originated during domestication from wild soybean K. NAKAHARA (1), J. Abe (1), T. Yamada, (1) Y. Wang, (2), M. Hajimorad (2) (1) Hokkaido University, Japan, (2) University of Tennessee, Knoxville, TN, U.S.A. Examine the activity of fungal extracts of sorghum pathogens in inducing the MAMP response, 4. Measure the effect of the MAMP response on disease resistance in controlled assays, and 5. Determine whether control of variation in the MAMP response and variation in disease resistance is under shared genetic control. The Role of Ethylene in PAMP-Triggered Immunity in Fusarium Crown Rot (FCR) Disease Resistance in Wheat S. ALLEN (1), X. Xiao (1), K. Bhide (1), S. Scofield (2) (1) Purdue University, U.S.A.; (2) USDA-ARS, Purdue University, U.S.A. The Role of Ethylene in PAMP-Triggered Immunity in Fusarium Crown Rot (FCR) Disease Resistance in Wheat S. ALLEN (1), X. Xiao (1), K. Bhide (1), S. Scofield (2) (1) Purdue University, U.S.A.; (2) USDA-ARS, Purdue University, U.S.A. Fusarium Crown Rot (FCR) is a devastating cereal crop disease cause by necrotrophic fungal pathogens Fusarium graminearum and Fusarium pseudograminearum. Plants detect conserved pathogen-associated molecular patterns (PAMPs) through pattern-recognition receptors. Then, a signal cascade is triggered resulting in PAMP-Triggered Immunity (PTI) response such as expression of defense response genes and production of plant hormones jasmonic acid, salicylic acid, and ethylene. A model proposed by Zipfel in 2013 suggests that ethylene functions to prime additional signaling pathways for sustained PTI response. Our laboratory has shown that disease resistance to FCR can be enhanced in susceptible wheat cultivars by stimulating the ethylene-signaling pathway with ethylene biosynthetic precursor 1-aminocyclopropene-1carboxylic acid (ACC). Additionally, resistant cultivars become more susceptible when ethylene-signaling is inhibited by treatment with the competitive inhibitor 1-methylcylopropene (1-MCP). RNA-Seq analysis of FCR-susceptible wheat cv ‘Kennedy’ in response to ACC or 1-MCP application and F. graminearum inoculation demonstrates significant difference in Differentially Expressed Genes (DEGs) within several disease-response categories such as peroxidases, pathogenesis-related proteins, and WRKY transcription factors. Currently, we are examining the transcriptome of FCR-resistant cv ‘Bobwhite’ to identify additional candidate genes involved in FCR resistance. Agroinfiltration-based screening to discover new potato germplasm with resistance against Globodera nematode pests S. Chen (1), R. Cui (1), X. Wang (2) (1) Cornell University U S A ; (2) USDA ARS U S A Potato cyst nematodes (PCN) (Globodera rostochiensis and G. pallida) are of worldwide regulatory concern and considered to the most economically important pests of potato, causing > 80% yield loss. The U.S. potato industry has been adversely affected due to the emergence of G. pallida in Idaho and a virulent G. rostochiensis pathotype in New York and the lack of resistant potato cultivars. Using host resistance is the most effective and sustainable means for nematode control. Like other plant pathogens, nematodes secrete effector proteins into root cells to suppress host plant defenses, leading to successful infection. However, specific effectors can be recognized by host resistance proteins and trigger plant defense, which often results in a hypersensitive response (HR) believed to halt pathogen infection. “Effectoromics” is a high-throughput, functional genomics approach that uses effectors to potentially detect R genes in plants. Wild potato species offer an extremely rich source of resistance against Globodera nematodes. We have established an agroinfiltration assay in wild potato species. Our initial screening of forty-two wild potato accessions with a group of PCN-secreted effectors showed the occurrence of an HR phenotype when specific effectors were transiently expressed in potato leaves. The results indicate that this “Effectoromics” approach may be a valuable tool that helps to accelerate resistance breeding against Globodera nematode pests. Transcriptome analysis of resistance against Phytophthora cinnamomi and the role of ß-cinnamomin elicitins in pathogenicity M. ISLAM (1), H. Hussain (1), J. Rookes (2), A. Chambery (3), A. Schallmey (4), W. Oßwald (5), D. Cahill (2) (1) Deakin University, Waurn Ponds, Australia; (2) Deakin University, Waurn Ponds, Australia; (3) Università degli Studi di Napoli, Italy; (4) RWTH Aachen University, Aachen, Germany; (5) Technische Universität München, Freising, Germany Transcriptome analysis of resistance against Phytophthora cinnamomi and the role of ß-cinnamomin elicitins in pathogenicity M. ISLAM (1), H. Hussain (1), J. Rookes (2), A. Chambery (3), A. Schallmey (4), W. Oßwald (5), D. Cahill (2) (1) Deakin University, Waurn Ponds, Australia; (2) Deakin University, Waurn Ponds, Australia; (3) Università degli Studi di Napoli, Italy; (4) RWTH Aachen University, Aachen, Germany; (5) Technische Universität München, Freising, Germany Resistance to the destructive oomycete pathogen Phytophthora cinnamomi is rare. We have explored resistance in roots, following infection, of the Australian native monocot Lomandra longifolia. Inoculated plants developed restricted lesions and analysis of lignin, callose and hydrogen peroxide indicated their involvement in resistance. Development of HRM markers tightly linked to extreme resistance to Potato virus Y using next-generation sequencing X. NIE (1), D. De Koeyer (1), A. Murphy (1), V. Dickison (1), M. Singh (2) (1) Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Canada; (2) Agricultural Certification Services, Use of synchrotron Fourier transform spectroscopy to shed light on the chemical composition of cell wall appositions papillae on winter wheat leaf R. LAHLALI (1), T. Song (2), C. Karunakaran (1), G. Peng (2), Y. Wei (3) (1) Canadian Light Source Inc., Canada; (2) Agriculture and Agri-Food Canada, Canada; (3) Department of Biology, University of Saskatchewan, Canada leaf R. LAHLALI (1), T. Song (2), C. Karunakaran (1), G. Peng (2), Y. Wei (3) (1) Canadian Light Source Inc., Canada; (2) Agriculture and Agri-Food Canada, Canada; (3) Department of Biology, University of Saskatchewan, Canada Papillae formation is known as one of the most important plant immunity mechanisms. Its major function is to prevent the entry of fungal pathogens into the host plant cells. In this study, a synchrotron based Fourier transform spectroscopy (sFTIR) was used to elucidate the chemical composition of the cell wall of forming papillae and non-forming papillae in response to powdery mildew infection. Spectroscopic results indicated an increase in peaks intensity of asymmetric and symmetric stretching vibrations of CH3 and CH2 that correspond to fatty acid groups, phenolics groups, lignification, cellulose, hemicellulose, and glucans following pathogenic infection. Principal Component Analysis (PCA) applied to sFTIR spectra confirmed these findings and distinguished two different groups with significant differences in cell wall composition including lignin, phenolics, polysaccharides and carbohydrates. This study highlighted, for the first time, the potential application of synchrotron-based infrared technique to study the in situ composition of lignified papillae and plant cell in the plant disease resistance, which remained to be a challenge with biochemical approaches. Subsequently, further S4.155 studies with more cultivars, as well as other synchrotron imaging techniques will be undertaken to confirm these preliminary findings and explore other defense response mechanisms. Agroinfiltration-based screening to discover new potato germplasm with resistance against Globodera nematode pests S. Chen (1), R. Cui (1), X. Wang (2) (1) Cornell University U S A ; (2) USDA ARS U S A We then used a transcriptomic approach to further examine resistance in this system. As the genome of L. longifolia is not sequenced, we performed RNA-seq analysis and the data showed the elevated expression of a range of resistance-related genes including those involved in jasmonic acid synthesis, lignin biosynthesis and in disease resistance signalling. Blast2GO analysis of contigs revealed the presence of elicitin and elicitin-like proteins. We then investigated the role of elicitins in pathogenicity of P. cinnamomi. Elicitins were isolated from liquid culture and different isoforms were identified by isoelectric focusing analysis. A β-cinnamomin elicitin was purified, an antibody produced and its specificity was shown by Western blot analysis. β-cinnamomin production at different life stages of the pathogen was confirmed by confocal microscopy. Elicitins were also detected in inoculated roots of the susceptible plant species, Lupinus angustifolius. Elucidation of resistance-related mechanisms and pathogenicity factors provides opportunities for modification of susceptible plants and identification of targets to prevent infection. A synergistic effect of two plant antimicrobial peptides from defensin and lipid-transfer protein families towards Phytophthora infestans E. ROGOZHIN (1), D. Zaytsev (2) (1) Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry Russian Academy of Sciences, Russia; (2) Timiryazev Russian State Agrarian University, Russia Late blight caused by Phytophthora infestans (Mont.) de Bary is one of the most important diseases afflicting potatoes and tomatoes worldwide. It results in economically meaningful yield losses and decreases in the quality of harvested produce. In the last two decades, researchers have focused on developing chemical and biological pesticides to treat against the diseases following plant tissue necrotization and dying off. Antimicrobial peptides are one of the primary components of a plant innate immunity to biotic stress factors involving diseases and pests. Most of these peptides can act directly against plant pathogenic microorganisms (fungi, oomycetes and bacteria) based on disruption of cell wall and plasma membrane integrity. In our study, we investigated two antimicrobial peptides (plant defensin NsD2 isolated from Nigella sativa seeds and lipid-transfer protein EcLTP purified from Echinochloa crusgalli kernels) belonging to different plant defense families. We showed that these peptides exhibited synergistic action against Phytophthora infestans on potato tuber disks assays when used in tandem. An application of plant antimicrobial peptides that demonstrates a diverse mode of action towards Phytophthora infestans would enable increases in the inhibition effect up to complete elimination of the pathogen. miR858 functions as a negative regulator of plant susceptibility to the beet cyst nematode Heterodera schachtii S. PIYA (1), T. Baum (2), T. Hewezi (1) (1) University of Tennessee, U.S.A.; (2) Iowa State University, U.S.A. miR858 functions as a negative regulator of plant susceptibility to the beet cyst nematode Heterodera schachtii S. PIYA (1), T. Baum (2), T. Hewezi (1) (1) University of Tennessee, U.S.A.; (2) Iowa State University, U.S.A. Recent studies have indicated that microRNA genes play key roles in shaping the compatibility of the interaction between plant-parasitic cyst nematodes and host plants. In this study, we report a novel regulatory role of the Arabidopsis miR858 and its MYB83 transcription factor during plant-cyst nematode interactions. Promoter–GUS fusion assays pointed to a role of miR858 in post-transcriptional regulation of MYB83 in the syncytium induced by the beet cyst nematode Heterodera schachtii. qPCR quantification further supported the negative association between miR858 and MYB83 expression levels at different time points post H. schachtii infection. Overexpression of miR858 in Arabidopsis resulted in significant decreases of plant susceptibility to infection by H. schachtii, whereas a mir858 mutant showed the opposite effect of increased susceptibility. In addition, we found that T- DNA insertional mutation of MYB83 was less susceptible to nematode infection. In contrast, overexpression of miR858–resistant myb83 produced hyper-susceptibility phenotypes. Taken together, these data revealed the importance of tight regulation of MYB83 transcription factor expression by miR858 in the syncytium, apparently to control downstream target genes that encode components that may be needed for syncytium formation/function. Identification of target genes that are directly or indirectly regulated by MYB83 is currently underway. Agroinfiltration-based screening to discover new potato germplasm with resistance against Globodera nematode pests S. Chen (1), R. Cui (1), X. Wang (2) (1) Cornell University, U.S.A.; (2) USDA-ARS, U.S.A. Wheat streak mosaic virus P1: Defining the minimal region required for the suppression of RNA silencin A. KASAMSETTY (1), G. Hein (2), S. Tatineni (1) (1) USDA-ARS and University of Nebraska-Lincoln, U.S.A.; (2) University of Nebraska-Lincoln, U.S.A. Wheat streak mosaic virus P1: Defining the minimal region required for the suppression of RNA silencing A. KASAMSETTY (1), G. Hein (2), S. Tatineni (1) (1) USDA-ARS and University of Nebraska-Lincoln, U.S.A.; (2) University of Nebraska-Lincoln, U.S.A. Wheat streak mosaic virus (WSMV) is the most economically important wheat virus in the Great Plains region of the USA. WSMV is the type species of the genus Tritimovirus, family Potyviridae, and is transmitted by the wheat curl mite, Aceria tosichella Keifer. Previously, we reported that WSMV P1 functions as the viral suppressor of RNA silencing (VSR) and pathogenicity enhancer. In this study, we examined the minimal region of P1 required for VSR function. Twenty one deletion mutants of WSMV P1 were generated by progressively deleting from the N- and C-terminal regions. VSR assays were conducted by agro-infiltration of deletion mutants in pCASS4 together with the 35S-GFP into Nicotiana benthamiana leaves. WSMV P1 with deletions comprising up to 23 amino acids at the N-terminal region exhibited VSR activity, while deletion of N-terminal 25 amino acids completely abolished the VSR activity. In contrast, even a single amino acid at the C-terminus is indispensable for VSR activity of P1, suggesting that the C- terminal region of WSMV P1 is crucial for VSR activity. Mechanistic studies of WSMV P1 in VSR activity will be discussed. Development of HRM markers tightly linked to extreme resistance to Potato virus Y using next-generation sequencing X. NIE (1), D. De Koeyer (1), A. Murphy (1), V. Dickison (1), M. Singh (2) (1) Fredericton Research and Development Centre, Agriculture and Agri-Food Canada, Canada; (2) Agricultural Certification Services, Canada S4.156 Tetraploid potato breeding clone F87084 possesses a resistance gene (Ry) that confers extreme resistance (ER) to Potato virus Y (PVY). However, markers reported to be highly linked to different Ry genes were not detected in F87084. Next generation sequencing of genomic DNA from F87084 (Rrrr), F02010 (rrrr) and the respective bulk DNA of resistant and susceptible progenies of F87084 x F02010 was carried out by Illumina HiSeq for identification of single nucleotide polymorphisms (SNP). Polymorphic positions matching the expected pattern in parents and progeny bulk samples were determined. The genomic regions flanking the target SNP were extracted and used to design primers and probes for high-resolution DNA melting (HRM) assays. The genome of the endophytic Curtobacterium strain ER 1.4/2 and its potential as a biocontrol of several plant diseases L. CURSINO (1), K. Pawlak (2), G. Li (2), R. Worobo (3) (1) Keuka College, U.S.A.; (2) Hobart and William Smith Colleges, U.S.A.; (3) Cornell University, U.S.A. Curtobacterium are Gram-positive bacteria that can live inside of plants as endophytes and also have the ability to cause disease such as wilting disease in dry beans. Curtobacterium sp. strain ER1.4/2 (ER1.4/2) was isolated from branches of healthy sweet-orange (Citrus sinensis). The genomic DNA of this bacterium was extracted and the whole DNA sequencing was performed using next-generation DNA sequencing by the Illumina Hiseq 2000 method. The genome annotation of Curtobacterium strain ER1.4/2 was initially performed using a RAST (Rapid Annotation using Subsystem Technology) server. The draft genome contains 3,692,034 bases with a GC content of 71.6%. Important genes such as a bacteriocin and siderophores were found in the genome. This bacteria showed antibiosis in vitro against several important phytopathogens such as Xylella fastidiosa CVC, Xanthomonas campestris pv. Glycines; Pseudomonas syringae pv. syringae and Agrobacterium tumefaciens. We are currently identifying ER1.4/2 at the species level by FAME to confirm its 16S rDNA identification. In addition, studies on its interaction with several cultivars of Phaseoulus vulgaris and Glycine max are underway. The ability of ER1.4/2 to colonize multiple plants, with its wide-spectrum antibiosis and non-pathogenicity to all plants tested make this bacterium an excellent biocontrol candidate against bacterial diseases. The dlt locus contributes to virulence in the necrotrophic pathogen Pectobacterium carotovorum I. RUBIO (1), C. Guerin (1), R. Schaub (1), A. Charkowski (1) (1) University of Wisconsin-Madison, U.S.A. In Gram-positive bacteria, the dlt locus contributes to antimicrobial peptide resistance through D-alanylation of cell wall teichoic acids. Homologs of dlt are present in the Gram-negative, soft rot pathogens Pectobacterium and Dickeya. Gram-negative bacteria lack teichoic acids, so the function of the dlt operon is unclear. We mutated the Pectobacterium carotovorum dltB and dltD genes and assayed for virulence attenuation in Solanum tuberosum (potato) stems and tubers. Red Norland plant stems were stab-inoculated and evaluated with a rating scale for wilt and stem maceration symptoms. Stem colonization by the different strains was determined by slicing and plating 1cm stem sections. Russet tubers were inoculated with serial dilutions of bacteria, and evaluated for the presence or absence of maceration. None of the mutants assayed were reduced in ability to cause symptoms on stems. However, ΔdltD was reduced in ability to spread inside stems. In tubers, both mutants were reduced in virulence compared to the wild type strain. Therefore, the dlt appears to contribute to P. carotovorum virulence in tubers and spread in stems. Wheat streak mosaic virus P1: Defining the minimal region required for the suppression of RNA silencin A. KASAMSETTY (1), G. Hein (2), S. Tatineni (1) (1) USDA-ARS and University of Nebraska-Lincoln, U.S.A.; (2) University of Nebraska-Lincoln, U.S.A. Of the 10 primer/probe sets designed and tested, five produced amplicons that exhibit different HRM profiles between the resistant parent/bulk and the susceptible parent/bulk. Further analysis of the HRM primer/probe sets on a segregating population comprised of 300 progenies revealed near-perfect matches between the HRM assays and the phenotyping results, demonstrating high linkage between these newly developed HRM markers and Ry in F87084. The efficacy of the HRM markers for marker-assisted selection of ER to PVY is validated with different populations involving F87084. The virulence function and regulation of the metalloprotease gene prtA in the bacterial plant pathogen, Burkholderia glumae T. DE PAULA LELIS (1), J. Peng (1), S. Osti (1), J. Hyun Ham (1) (1) Louisiana State University, U.S.A. The virulence function and regulation of the metalloprotease gene prtA in the bacterial plant pathogen, Burkholderia glumae T. DE PAULA LELIS (1), J. Peng (1), S. Osti (1), J. Hyun Ham (1) (1) Louisiana State University, U.S.A. Bacterial panicle blight caused by Burkholderia glumae is a major bacterial disease of rice in the United States. Our preliminary RNA-seq study showed that a serine metalloprotease gene, named prtA, is positively regulated by the TofI/TofR-mediated quorum sensing system like other genes for producing important virulence factors of B. glumae, such as toxoflavin. To study the function of prtA in B. glumae 336rg-1, a null mutant of prtA was generated through an insertional mutagenesis approach. Extracellular protease activity was not detected when the prtA mutant was grown on LB-agar plates amended with 1% skim milk, indicating that prtA is solely responsible for the extracellular protease activity of B. glumae 336gr-1. In addition, inoculation of rice panicles with the prtA mutant resulted in reduction of disease severity compared to the wild type strain, showing the requirement of ptrA for full virulence. This study also revealed that the regulation pattern of extracellular protease activity is similar to that of toxoflavin production. Particularly, it was newly found that qsmR, known as the quorum-sensing master regulator gene, is essential for both extracellular protease activity and toxoflavin production by B. glumae 336gr-1. Furthermore, additional genetic elements that control extracellular protease activity were newly identified through screening of random mini-Tn5 mutants, which provide useful information to understand the mechanism of prtA regulation. The genome of the endophytic Curtobacterium strain ER 1.4/2 and its potential as a biocontrol of several plant diseases L. CURSINO (1), K. Pawlak (2), G. Li (2), R. Worobo (3) (1) Keuka College, U.S.A.; (2) Hobart and William Smith Colleges, U.S.A.; (3) Cornell University, U.S.A. The genome of the endophytic Curtobacterium strain ER 1.4/2 and its potential as a biocontrol of several plant diseases L. CURSINO (1), K. Pawlak (2), G. Li (2), R. Worobo (3) (1) Keuka College, U.S.A.; (2) Hobart and William Smith Colleges, U.S.A.; (3) Cornell University, U.S.A. The genome of the endophytic Curtobacterium strain ER 1.4/2 and its potential as a biocontrol of several plant diseases L. CURSINO (1), K. Pawlak (2), G. Li (2), R. Worobo (3) (1) Keuka College, U.S.A.; (2) Hobart and William Smith Colleges, U.S.A.; (3) Cornell University, U.S.A. Investigating possible targets of gene products using HPLC and biolog analysis are on-going and will provide insight into dlt function in P. carotovorum. ematic computational network-based analysis to predict subnetwork modules associated with pathogenicity and fumo illi id Systematic computational network-based analysis to predict subnetwork modules associated with pathogenicity and fumonisins in Fusarium verticillioides M. KIM (1), B. Yoon (1), W. Shim (1) (1) Texas A&M Univ., U.S.A. Fusarium verticillioides is a notorious pathogen of maize causing ear rot and fumonisin contamination. In order to develop innovative preharvest control strategies, such as new biocontrol agents and resistant hybrids, to minimize the entry of fumonisins into our food supply, there is a need to identify new molecular targets for disrupting F. verticillioides pathogenicity. In this study, we performed a systematic computational network-based comparative analysis of two distinct F. verticillioides – maize kernel RNA-seq datasets (one on moderately resistant inbred and the other on susceptible hybrid). For the systematic analysis of the pathogenicity mechanism, we first inferred F. verticillioides co-expression networks. Subsequently, we identified functional subnetwork modules on the co-expression networks consisting of interacting genes that display harmoniously coordinated behavior in the respective datasets. A computationally efficient branch-out technique applied with an adopted probabilistic pathway activity inference method was used to identify functional subnetwork modules likely involved in F. verticillioides pathogenicity. Here we exhibit key potential subnetwork modules, where the modules contain several enriched GO terms as well as potential pathogenicity genes from other pathogenic fungi. Putative hub genes in each subnetwork will be functionally characterized to test their candidacy as new targets for ear rot and fumonisin control strategies. Transcriptomic analysis identifies specificity in Fusarium verticillioides metabolic response to Bacillus mojavensis lipop A. BLACUTT (1), S. Gold (2) (1) University of Georgia, U.S.A.; (2) USDA-ARS TMRU, U.S.A. Fusarium verticillioides is a mycotoxigenic fungus capable of both pathogenic and asymptomatic endophytic lifestyles in maize; such intimate association renders efficient chemical control cost-prohibitive. Bacillus mojavensis RRC101 is a maize endophyte demonstrating both in vitro antagonism of F. verticillioides and in planta reductions of disease and mycotoxin accumulation, the former attributed to production of lipopeptide antibiotics. Although both RRC101 surfactins and fengycins induce increase mycotoxin accumulation in plate assays, only fengycins are antagonistic and induce hyphal distortion, pigment accumulation indicative of a stress response, and violent lysis. Preliminary analysis of RNA sequencing data has identified common functional groups in transcripts enriched in F. verticillioides under fengycin antagonism, particularly structural proteins and hydrolytic enzymes. Genes responsible for secondary metabolism, specifically antibiotic production, are also upregulated under lipopeptide challenge. These data suggest that differential lipopeptide responses previously observed in F. verticillioides reflect an underlying cross-kingdom “conversation” of secondary metabolites between maize endophytes. Temporal succession of an Aspergillus flavus biocontrol agent in commercial cornfields in Texas M. SEXTON (1), I. Carbone (1), J. White (1), T. Isakeit (2) (1) Center for Integrated Fungal Research, North Carolina State University, U.S.A.; (2) Texas A&M Uni Temporal succession of an Aspergillus flavus biocontrol agent in commercial cornfields in Texas M. SEXTON (1), I. Carbone (1), J. White (1), T. Isakeit (2) Temporal succession of an Aspergillus flavus biocontrol agent in commercial cornfields in Texas M. SEXTON (1), I. Carbone (1), J. White (1), T. Isakeit (2) (1) Center for Integrated Fungal Research, North Carolina State University, U.S.A.; (2) Texas A&M University, U.S (1) Center for Integrated Fungal Research, North Carolina State University, U.S.A.; (2) Texas A&M University, U.S.A. (1) Center for Integrated Fungal Research, North Carolina State University, U.S.A.; (2) Texas A&M University, U.S.A. Aspergillus flavus contaminates corn by producing carcinogenic aflatoxins (AF). This results in devastating economic losses worldwide and poses serious health risks. The most effective control strategy for reducing AF contamination is the application of non-aflatoxigenic strains of A. flavus, such as NRRL 21882 (=Afla-Guard®) or NRRL 18543 (=AF36). However, we have a limited understanding of the succession of biocontrol agents in cornfields. From a genomic perspective, we are examining the influence of NRRL 21882 on the populations of A. flavus from Texas commercial cornfields. In 2014, A. flavus strains were isolated from kernels from fields that were not treated or treated with NRRL 21882 in 2011. functional analyses of MADS-box transcription factor-mediated regulation of fumonisin biosynthesis in Fusarium Computational and functional analyses of MADS-box transcription factor-mediated regulation of fumonisin biosy verticillioides verticillioides A. HILTON (1), A. Hilton (1), M. Kim (1), C. Ortiz (2), C. Woloshuk (3), B. Yoon (2), W. Shim (2) (1) Texas A&M University, U.S.A.; (2) Texas A&M University, U.S.A.; (3) Purdue University, U.S.A. A. HILTON (1), A. Hilton (1), M. Kim (1), C. Ortiz (2), C. Woloshuk (3), B. Yoon (2), W. Shim (2) (1) Texas A&M University, U.S.A.; (2) Texas A&M University, U.S.A.; (3) Purdue University, U.S.A. Fusarium verticillioides is an important maize pathogen, leading to Fumonisin B1 (FB1) in infested kernels. MADS-box transcription factors (TF) were found to modulate polyketide synthase (PKS) gene expression and FB1 production in F. verticillioides. With next-generation sequencing of F. verticillioides wild type and MADS-box TF mutant cultures, we used a suite of computational network-based tools, e.g. partial correlation, log-likelihood S4.157 ratio matrix and seed-and-extend approach, to perform a system-oriented transcriptome analysis to predict downstream genetic subnetwork modules associated with FB1 production. Our aim was to identify and characterize system-level changes across correlated genes rather than simply focusing on individual gene expression. The resulting subnetwork modules are predicted to contain hub genes, which likely play a crucial regulatory role within functional modules. Two predicted subnetwork modules were analyzed in silico, and five putative hub genes were subjected to functional characterization. Deletion of RAS GTPase (FvRSR1) and methyltransferase (FvEFM3) led to a significant reduction in FB1 levels. A comparative qPCR was performed to investigate the impact of FvRsr1 and FvEfm3 on the expression of neighboring genes in two subnetworks, which did show significant changes. Collectively, these results support our prediction that FvRSR1 and FvEFM3 serve as a key hub gene in each subnetwork and regulate FB1 biosynthesis. ratio matrix and seed-and-extend approach, to perform a system-oriented transcriptome analysis to predict downstream genetic subnetwork modules associated with FB1 production. Our aim was to identify and characterize system-level changes across correlated genes rather than simply focusing on individual gene expression. The resulting subnetwork modules are predicted to contain hub genes, which likely play a crucial regulatory role within functional modules. Two predicted subnetwork modules were analyzed in silico, and five putative hub genes were subjected to functional characterization. Deletion of RAS GTPase (FvRSR1) and methyltransferase (FvEFM3) led to a significant reduction in FB1 levels. Transcriptomic analysis identifies specificity in Fusarium verticillioides metabolic response to Bacillus mojavensis lipop A. BLACUTT (1), S. Gold (2) (1) University of Georgia, U.S.A.; (2) USDA-ARS TMRU, U.S.A. To obtain fine resolution of genetic variation, we conducted genome-wide sequencing, which allowed us to detect changes in the genomes of NRRL 21882-derived strains and introgression of NRRL 21882 into native strains over a three year period. From an analysis of 168 A. flavus strains, 32 strains are missing their entire AF gene cluster, a marker of NRRL 21882; 14 of these were in untreated fields. Eight of the strains missing the cluster are putative recombinants, phylogenetically grouping with full cluster strains. Ongoing sexual reproduction is indicated by approximately equal distributions of both mating types in treated and untreated fields. g , ; g g p , phylogenetically grouping with full cluster strains. Ongoing sexual reproduction is indicated by approximately equal distributions of both mating types in treated and untreated fields. th full cluster strains. Ongoing sexual reproduction is indicated by approximately equal distributions of both mating types in Comparative genomics of S and L morphotypes of Aspergillus flavus M. OHKURA (1), M. Ohkura (1), P. Cotty (2), M. Orbach (1) School of Plant Sciences, University of Arizona, U.S.A.; (2) Agricultural Research Services USDA, University of Arizona, U Strains of Aspergillus flavus vary greatly in their level of aflatoxin production and atoxigenic strains are not uncommon in nature despite the fungus’ notoriety for producing the carcinogenic mycotoxin. Aspergillus flavus strains are of two morphotypes; the S morphotype that produces numerous small sclerotia and limited conidiospores and the L morphotype that produces relatively fewer large sclerotia and abundant conidiospores. S strains are consistently toxigenic, whereas L strains vary greatly in toxin production ranging from atoxigenic to highly toxigenic. This suggests the two morphotypes have adapted to different niches where selection pressure to maintain aflatoxin production is high in the environment the S strains inhabit, and low in which the L strains inhabit. To develop hypotheses on differential niche adaption, we compared the genomes of three S strains and three L strains to identify genes unique to each morphotype. A notable difference in secondary metabolite genes was observed and the results from the comparative genomics analyses will be presented. functional analyses of MADS-box transcription factor-mediated regulation of fumonisin biosynthesis in Fusarium A comparative qPCR was performed to investigate the impact of FvRsr1 and FvEfm3 on the expression of neighboring genes in two subnetworks, which did show significant changes. Collectively, these results support our prediction that FvRSR1 and FvEFM3 serve as a key hub gene in each subnetwork and regulate FB1 biosynthesis. Independent amplification of a housekeeping gene and its evolutionary significance in the Dothideomycetes B. DHILLON (1), G. Kema (2), S. Goodwin (3), B. Bluhm (1) (1) University of Arkansas, U.S.A.; (2) Plant Research International, Wageningen University and Research Centre, Netherlands; (3) USDA/ARS Purdue University, U.S.A. Transposable elements (TEs) are key drivers of genome evolution, but the exact mechanisms of how they modulate gene and genome function are largely unknown. We identified an event where a housekeeping gene (histone H3) was captured and amplified by a hAT DNA transposon in the genome of the banana pathogen Pseudocercospora fijiensis. Comparative genome analysis in related Capnodiales revealed a similar event occurred in the corn pathogens Cercospora zeae-maydis and C. zeina, but was lacking in the soybean pathogens C. sojina and C. flagellaris. Histone H3 amplification had been described previously in Pleosporales (Pyrenophora tritici-repentis). Our analysis showed key differences in the amplification events between the Pleosporales and Capnodiales, namely, 1) extent of histone H3 amplification, 2) difference in H3 nucleotide composition and 3) number of functional H3 genes present. In the Capnodiales, all amplified H3 copies were inactivated by RIP, which suggests the original H3 gene was under strong negative selection pressure. The interplay of TEs and RIP can result in various outcomes for gene and genome evolution. For instance, when TEs act as breeding grounds for pathogenicity genes, such interactions can influence gene function and/or regulation, and ultimately affect host-pathogen interactions. Transcriptomic analysis identifies specificity in Fusarium verticillioides metabolic response to Bacillus mojavensis lipopeptides A. BLACUTT (1), S. Gold (2) (1) University of Georgia, U.S.A.; (2) USDA-ARS TMRU, U.S.A. Transcriptomic analysis identifies specificity in Fusarium verticillioides metabolic response to Bacillus mojavensis lipopeptides A. BLACUTT (1), S. Gold (2) A metabolomic approach to investigate the putative role of drought stress in Esca disease of grapevine M. LIMA (1), A. Machado (1), W. Gubler (1) (1) University of California Davis, U.S.A. Esca is a destructive disease of grapevine known to be associated with several xylem inhabiting fungi, namely Phaeomoniella chlamysdospora (Pch) and Phaeoacremonium alephilum (Pal). However, the disease mechanism(s) remains unclear. These fungi seem capable of living for several years as endophytes without apparently causing the disease. It is unknown why these fungi become pathogenic, but symptom appearance has been associated with abiotic factors, particularly water stress. We hypothesize that changes induced by drought on the components of xylem sap contribute to symptom appearance. H1-Nuclear Magnetic Resonance spectroscopy coupled with multivariate statistical techniques was used to investigate the interaction of drought stress and Esca-associated pathogen infection. Young, greenhouse-grown, Vitis vinifera cv. Chardonnay cuttings were inoculated with Pch or Pal six weeks before inducing drought stress. Xylem sap was collected thirteen weeks after inoculation. Drought stress induced major changes in several compound classes, e.g. amino acids, carbohydrates and aromatic compounds. The modulation of these metabolic changes by fungal inoculation is being considered. This “omic” approach allowed to gain better insight into the biotic-abiotic interaction, and suggested candidates that could be tested in the control of esca-associated fungi, thus leading Science closer to Practice. PiCS, a predicted protein-protein interactome of the frog eye leaf spot pathogen Cercospora sojina A. SUBEDI (1), B. Musungu (1), M. Geisler (2), B. Bluhm (3), A. Fakhoury (1) (1) Southern Illinois University Carbondale, U.S.A.; (2) Southern Illinois University Carbondale, U.S.A.; (3) University of Arkansas Fayetteville, U.S.A. PiCS, a predicted protein-protein interactome of the frog eye leaf spot pathogen Cercospora sojina A. SUBEDI (1), B. Musungu (1), M. Geisler (2), B. Bluhm (3), A. Fakhoury (1) (1) Southern Illinois University Carbondale, U.S.A.; (2) Southern Illinois University Carbondale, U.S.A.; (3 Frog eye leaf spot (FLS) is a foliar disease of soybean caused by the fungus Cercospora sojina Hara. FLS is a severe problem for soybean production, particularly in the mid-southern United States causing millions of dollars in losses every year. A predicted protein-protein interaction (PPI) map provides an overall snapshot of all the protein interactions occurring in an organism. Therefore, the development of PiCS, a predicted protein-protein interactome of C. sojina, can decipher many of the primary metabolic processes, biological processes and developmental processes in the fungus. This will help further our understanding of the mechanisms governing pathogenesis in the C. sojina – soybean pathosystem. ctional characterization of candidate effector proteins identified from the wheat scab fungus Fusarium graminearum u (1), M. Edwards (1), S. Lu (1) Functional characterization of candidate effector proteins identified from the wheat scab fungus Fusarium graminearum S. Lu (1), M. Edwards (1), S. Lu (1) (1) USDA-ARS Cereal Crops Research Unit, U.S.A. Fungal pathogens often produce certain small secreted cysteine-rich proteins (SSCPs) during pathogenesis that may function in triggering resistance or susceptibility in specific host plants. We have recently identified a total of 190 SSCPs encoded in the genome of the wheat scab fungus Fusarium graminearum and found at least 23 SSCPs to be true extracellular proteins that are expressed in planta. To facilitate functional characterization, we compared the expression patterns between compatible and incompatible interactions by transcriptional analysis. A majority of SSCPs examined were found to be expressed in identical or similar patterns when the fungus attacked susceptible wheat cultivars such as Grandin and Wheaton. However, the expression patterns of at least 13 SSCPs were altered when the same pathogen infected the resistant cultivar Sumai 3: the expression of 11 SSCPs appeared to be suppressed or “down-regulated” at certain stages of infection while two others were apparently “up-regulated” at 4-8 days post inoculation. Gene knock-out experiments are underway with the priority given to these differentially expressed SSCPs. The resultant deletion mutants will be subjected to pathogenicity assays on both susceptible and resistant wheat cultivars to determine the roles of these candidate effector proteins in F. S4.158 graminearum-wheat interactions. This study may provide new molecular insights into Fusarium head scab, a devastating disease of wheat crops worldwide. graminearum-wheat interactions. This study may provide new molecular insights into Fusarium head scab, a devastating disease of wheat crops worldwide. Analysis of a ketide synthase from fungal endophytes of toxic locoweeds A. NOOR (1), D. Baucom (2), D. Cook (3), R. Creamer (2) (1) Molecular Biology, New Mexico State University, U.S.A.; (2) New Mexico State University, U.S.A.; (3) USDA, ARS, U.S.A.; (4) New Mexico State University, U.S.A. Analysis of a ketide synthase from fungal endophytes of toxic locoweeds A. NOOR (1), D. Baucom (2), D. Cook (3), R. Creamer (2) (1) Molecular Biology, New Mexico State University, U.S.A.; (2) New Mexico State University, U.S.A.; (3) USDA, ARS, U.S.A.; (4) New Mexico State University, U.S.A. y, New Mexico State University, U.S.A.; (2) New Mexico State University, U.S.A.; (3) USDA, ARS, U.S.A.; (4) New Mex A Toxic Astragalus and Oxtropis locoweeds are legumes in which toxicity is due to fungal endophytes of Alternaria section Undifilum sp. (A. A metabolomic approach to investigate the putative role of drought stress in Esca disease of grapevine M. LIMA (1), A. Machado (1), W. Gubler (1) (1) University of California Davis, U.S.A. PPI maps have been generated for some model organisms such as human, fruit fly, and arabidopsis using high throughput experimental methods. In this study, we used PPI data from 11 model organisms to predict the PPI map in C. sojina via the interlog method using the INPARANOID and ENSEMBL algorithms. PiCS is comprised of 4,092 proteins (nodes) and 144,488 interactions (edges). It covers 33% of the total C. sojina proteome. Subnetworks were also built for secreted proteins in C. sojina as well as for putative pathogenicity related genes. This is a first attempt to build a PPI map for a member of the Cercospora genus. It is a new tool that could be used to understand the interaction between soybean and C. sojina. Comparative analysis of predicted proteomes from forma speciales of Fusarium oxysporum causing palm wilt S. PONUKUMATI (1), S. Ponukumati (2), J. Huguet (2), J. Rollins (2), M. Elliott (1) (1) University of Florida, U.S.A.; (2) University of Florida, U.S.A. Fusarium oxysporum f. sp. palmarum [FOP] and Fusarium oxysporum f. sp. canariensis [FOC] cause lethal fusarium wilt of palms in Florida. While FOC and FOP cause similar diseases, FOP has a wider host range and appears to be more aggressive than FOC. The focus of the current study is to compare the predicted FOC and FOP proteomes with various formae speciales of F. oxysporum to identify common and unique gene families. To date, the FOP and FOC genomes were sequenced, assembled and annotated using multiple bioinformatic approaches. Genome sizes of FOP and FOC are 46.9 Mbp and 46 Mbp, respectively, and the predicted proteomes consist of 15528 (FOC) and 15974 (FOP) proteins. CAZy analysis indicate FOP and FOC share similar copies of sugar transporters, cellulase, and secretory lipase, whereas FOP has additional copies of necrosis inducing proteins, cysteine rich secretory proteins and cell degrading enzymes, such as cutinase and pectate lyase. OrthoMCL was used to cluster related proteins among Fusarium genomes, resulting in 396 (FOC) and 511 (FOP) unique clusters. Secondary metabolite genes were present in 22 OrthoMCL clusters. Two gene clusters related to nonribosomal peptide synthases have increased copy number in FOP, indicating the possible role of toxins in pathogenicity. Further annotation is underway to identify putative functions of unique and common genes and to develop hypotheses concerning differences in pathogenic specificity. ctional characterization of candidate effector proteins identified from the wheat scab fungus Fusarium graminearum u (1), M. Edwards (1), S. Lu (1) sec. U. sp.). The fungi produce swainsonine, an alkaloid α-mannosidse inhibitor that causes locosim disease when consumed by grazing animals. Polyketide synthase (PKSs) is a key enzyme in swainsonine biosynthesis. KS is the most highly conserved and informative of the multi-domain enzymes for predicting pathway associations. The objective of this study was to analyze the KS sequences from A. sec. U. sp. isolated from locoweed plants of Astragalus lentiginosus var. diphysus, lentiginosus, araneosus, and wahweapensis, and A. mollissimus var. biglovii, earleii, wootoni and mollissimus that were collected from the western USA. The KS from these endophytic fungi were compared to A. sec. U. oxytropis isolated from Oxytropis lamberteii and O. sericea using PCR. A 741 bp amplicon was sequenced and analyzed to produce maximum parsimony trees. Fungi isolated from different varieties of locoweed and locations had different morphologies and colors in culture. The KS sequences were highly conserved across A. sec. U. sp., but differed slightly between A. sec. U. oxytropis and A. sec. U. fulvum and A. sec. U. cinereum. Comparing the KS from additional locoweed endophytes and A. sec. U. sp. will allow the further examination of phylogenetic relationships among these fungi. A metabolomic approach to investigate the putative role of drought stress in Esca disease of grapevine M. LIMA (1), A. Machado (1), W. Gubler (1) (1) University of California Davis, U.S.A. Fungal endophyte community analysis of green coffee beans: A comparison across growing regions and qualities R. CAPOUYA (1), T. Mitchell (1), V. Devi Ganeshan (1) (1) The Ohio State University, U.S.A. Fungal endophyte community analysis of green coffee beans: A comparison across growing regions and qualities R. CAPOUYA (1), T. Mitchell (1), V. Devi Ganeshan (1) (1) The Ohio State University, U.S.A. Coffee is considered a global dietary staple due to its distinct flavor, longstanding cultural significance, and stimulating caffeine content. During the production process, defective green coffee beans are sorted out so the batch contains below a certain percentage of them, as a non-homogenous batch can negatively impact the roasting process. A reduction of this percentage of defects would result in higher yields and better quality coffee. We hypothesize that there are notable differences in the endophyte communities between good and defective green coffee beans, and one or more fungal taxa can be associated with bean health or with particular economically significant defects. Through next-generation amplicon sequencing, we analyzed the endophytes present within beans from six defect categories across three coffee growing regions and made comparisons to identify the fungal taxa associated with these categories. We identified a variety of fungal communities present within the samples and were able to make statistical associations between both healthy and defective beans and certain endophytic taxa. Particularly, our studies indicate that multiple species from the Pichia genus may be responsible for promoting healthy bean development. Our findings will open the door to new research regarding the manipulation of endophytic fungi to improve overall coffee production and quality. S4.159 Transcriptomic response of Fusarium verticillioides associated with nitrogen availability and development of sugarcane pokahh boeng disease M. ZHANG (1), Z. Lin (2), J. Wang (3), Y. Bao (3), Q. Guo (3), C. Powell (1), S. Xu (3), B. Chen (3) (1) IRREC-IFAS, University of Florida, U.S.A.; (2) Guangxi University, China; (3) Guangxi University, China Pokahh boeng, caused by Fusarium verticillioides, is a serious disease in sugarcane industry. The impact of nitrogen source (ammonium sulfate, urea or sodium nitrate) on sugarcane pokahh boeng disease and its pathogen was investigated in planta and fungal growth and sporulation production was measured in vitro. The results showed that ammonium and nitrate were beneficial to fungal mycelium growth, cell densities and sporulation and enhanced the disease symptomology of sugarcane pokahh boeng in comparison to urea fertilization. Total 1,779 transcripts out of 13,999 annotated genes identified from global transcriptomic analysis by RNA sequencing were differentially expressed in F. verticillioides CNO-1 grown in the different sources of nitrogen. Apple replant disease and the –omics: Interaction of apple rootstock metabolome and the soil microbiome R. Leisso (1), R. Leisso (2), M. Mazzola (2) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. Apple replant disease and the –omics: Interaction of apple rootstock metabolome and the soil microbiome R. Leisso (1), R. Leisso (2), M. Mazzola (2) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. Apple replant disease (ARD) negatively impacts tree health and reduces crop yield in new orchard plantings. Use of tolerant rootstock cultivars can diminish the growth limiting effects of ARD; however specific rootstock attributes enabling ARD tolerance are not understood. Systems biology tools were used to contrast root exudate biochemical profiles and corresponding soil microbial ecology between ARD tolerant and susceptible rootstock genotypes. A metabolomic approach utilizing LC-MS/MS QTOF was used to characterize water-soluble root exudate metabolites collected periodically from water percolated through rootstock roots planted in pasteurized quartz sand over 12 weeks. Soil microbial community profiling was conducted by terminal restriction fragment length polymorphism (TRFLP) or next-generation sequencing (NGS) on samples collected concurrently from soil with a history of ARD treated daily with the flow through exudates from rootstocks cultivated in quartz sand. Results indicate rootstock cultivars differ in both root exudate composition and quantity, and correspondingly soil microbial communities are altered in a rootstock genotype-dependent manner. Differences in exudate metabolome associated with ARD cultivar tolerance, and temporal dynamics of root exudate production and microbial populations during early stages of rootstock growth following dormancy, may offer insight into ARD tolerance and the early stages of disease development. Differential metabolome analysis of field-grown maize kernels in response to drought stress L. YANG (1), J. Fountain (1), X. Ni (2), R. Lee (1), S. Chen (3), B. Scully (2), R. Kemerait (1), B. Guo (2) (1) University of Georgia, U.S.A.; (2) USDA-ARS, U.S.A.; (3) University of Florida, U.S.A. Drought stress constrains maize kernel development and can exacerbate aflatoxin contamination. In order to identify drought responsive metabolites and explore pathways involved in kernel responses, a metabolomics analysis was conducted on kernels from a drought tolerant line, Lo964, and a sensitive line, B73, with and without drought stress treatment for 7 and 14 days after drought induction beginning 7 days after pollination. Using ultra- performance liquid chromatography coupled with MS/MS, we profiled 445 metabolites covering 42 pathways. Drought stress induced higher accumulation of glycerolipid and phospholipid metabolites, and decreased content of galactolipid, amines and polyamines, amino sugar, and nucleotide sugar metabolites in both lines. However, B73 was found to accumulate fewer metabolites involved in dipeptide and pyrimidine metabolism under drought stress than Lo964. Apple replant disease and the –omics: Interaction of apple rootstock metabolome and the soil microbiome R. Leisso (1), R. Leisso (2), M. Mazzola (2) (1) USDA-ARS, U.S.A.; (2) USDA-ARS, U.S.A. Lo964 also exhibited increases in sphingolipid, sterol, and purine metabolism in comparison to B73. Collectively, higher sugar accumulation, a lower rate of energy metabolism, and elevated lipid and nucleic acid turnover was observed in B73. Conversely, higher amino acid and dipeptide accumulation, a lower TCA cycle rate, and elevated lipid and nucleic acid turnover was observed in Lo964 in response to drought stress. These results suggest that drought sensitivity is associated with differential metabolite accumulation, and diverse metabolic pathways in maize kernels. Metagenomic analysis of oomycete communities in rhizosphere soil from field pea on the Canadian prairies using Illumina Miseq B. GOSSEN (1), S. Chatterton (2), N. Foroud (3), A. Esmaeili Taheri (3), D. McLaren (3) (1) AAFC, Canada; (2) AAFC, Canada; (3) AAFC, Canada Oomycetes include several important soil-borne pathogens that cause damping-off and root rot in many crops including pea. Samples of soil from the rhizosphere of pea plants were collected from patches of healthy or diseased plants (127 samples from 26 commercial fields) on the Canadian prairies in 2013 and 2014. Oomycete communities were characterized using metagenomic analysis of the ITS1 region on Illumina Miseq. Roughly 4 million high quality sequences were obtained. Clustering took place at 85% coverage and 97% similarity. A representative sequence of each operational taxonomic unit (OTU) was compared to NCBI sequences. Species were identified at (minimum) 90% coverage and 97% similarity, and genera at 80% coverage and similarity. Clusters below 80% coverage or identity were labelled as unidentified. From 102 identified OTUs, 43 species and 16 oomycete genera were identified. Pythium was the dominant genus and P. heterothalicum was the most prevalent species. Aphanomyces euteiches, a very important pea root rot pathogen, was detected in 71 sites, but in very low abundance (≈ 0.2%). Multivariate analysis revealed difference in oomycete communities of healthy and diseased sites, and among years and provinces. This study shows that deep amplicon sequencing on Miseq can reveal unknown diversity of oomycete communities in agricultural soil, and that primer optimization for higher detection power of economically important oomycetes such as A. euteiches is warranted. Fungal endophyte community analysis of green coffee beans: A comparison across growing regions and qualities R. CAPOUYA (1), T. Mitchell (1), V. Devi Ganeshan (1) (1) The Ohio State University, U.S.A. A subset of these genes were associated with organic nitrogen assimilation and reproductive processes of F. verticillioides. The identified DEGs suggested the modes of nitrogen uptake and assimilation for F. verticillioides during different nitrogen regimes. Our results further demonstrated that nitrogen availability might play an important role in disease development by increasing fungal cell growth as well as influencing the expression of genes required for successful pathogenesis. These genes included those associated with phenotypic alteration, reproduction, nitrogen and carbon metabolism as well as several transcription factors responsible for these metabolisms. Metagenomic analysis of oomycete communities in rhizosphere soil from field pea on the Canadian prairies using Illumina Miseq B. GOSSEN (1), S. Chatterton (2), N. Foroud (3), A. Esmaeili Taheri (3), D. McLaren (3) (1) AAFC, Canada; (2) AAFC, Canada; (3) AAFC, Canada Identification of defense-related genes associated with tomato Sw-7 line against Tomato spotted wilt virus in tomato through transcriptome analysis Transcriptome profiling to discover defense-related genes associated with resistance line ty-5 against Tomato yellow leaf curl virus in tomato C. PADMABHAN (1), Y. Zheng (2), R. Shekaste-band (3), K. Stewart (1), D. Hasegawa (1), J. Scott (3), Z. Fei (4), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Boyce Thompson Institute for Plant Research, Ithaca, NY, U.S.A.; (3) University of Florida, IFAS, Gulf Coast Research and Education Center, Wimauma, FL, U.S.A.; (4) zf25@cornell.edu, Ithaca, NY, U.S.A. Tomato yellow leaf curl virus (TYLCV), a whitefly-transmitted begomovirus, has caused serious economic losses to tomato crops in the U.S. and around the world. The most effective management would be the use of a TYLCV-resistant tomato cultivar. Several sources of TYLCV resistance genes have been identified and incorporated in tomato breeding. The ty-5 gene recently discovered in ‘Tyking’ was a recessive gene. Although its function has not been well-characterized, near-isogenic breeding materials with TYLCV resistance have been generated. In the present study, after feeding by viruliferous whiteflies (Bemisia tabaci, MEAM1) at 0, 4, 7, 14 and 21 days post inoculation (dpi), leaf materials were collected on the test tomato plants for RNA- seq. Differentially expressed (DE) genes based on genome wide transcriptome profiling were analyzed and categorized based on their functions. Those defense-related genes included pathogenesis related 1 (PR1) and 4b (PR4b), CC-NBS-LRR, defensin protein, and heat shock protein were differentially expressed in the ty-5 line. Furthermore, RNA silencing and transcriptional silencing pathway genes, such as RdRp family protein, DNA (Cytosine-5)- methyltransferase 3 and microRNA target genes were also identified. To conclude, the genome wide transcriptome analysis allows us to have a global view on the differential gene expression, which may lead us to an effective management of TYLCV through breeding for durable disease resistance cultivars. National Seed Health System: Standardizing seed health practices in the United States for seed exports N. GONZALEZ (1), T. Bruns (1), G. Munkvold (2) (1) Iowa State University, U.S.A.; (2) Iowa State University, U.S.A. The U.S. National Seed Health System (NSHS) is a program authorized by USDA-APHIS and administered by the Iowa State University Seed Science Center in Ames, Iowa. NSHS provides accreditation to non-government entities to perform certain activities needed to support phytosanitary certification for the international movement of seed, and conducts peer review of protocols for approval as standard phytosanitary seed health testing methods. Comparison of Fusaruim graminearum transcription factors affecting Fusaruim graminearum virus 1 accumulation based on phenome analysis J. Yu (1), Y. Lee (1), K. Kim (1) (1) Seoul National University Korea BRRV was detected by PCR in 100% and 25% of the ‘Emerald’ and V. arboreum samples, respectively. Since sequences of species in the Caulimoviridae are known to be present as integrants in their hosts, back to back primers were designed to detect the episomal form of BRRV genome. The episomal form of BRRV was detected in root tissue of ‘Emerald’ but not V. arboreum, which indicated that the BRRV sequences in V. arboreum are most likely those of integrants. In addition, BLASTx analysis of 16 blueberry libraries revealed contigs that match sequences in 20 virus genera and 10 families. Overall, this study demonstrates the feasibility of using available host transcriptome data for mining viral sequences. Replication and transcription are independently modulated for each Banana bunchy top virus DNA component N. YU (1), H. Xie (1), J. Wang (1), X. Zhang (1), Z. Liu (1), Z. Xiong (2) (1) Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, China; (2) U Replication and transcription are independently modulated for each Banana bunchy top virus DNA component N. YU (1), H. Xie (1), J. Wang (1), X. Zhang (1), Z. Liu (1), Z. Xiong (2) (1) Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, China; (2) University of Arizona, U.S.A. N. YU (1), H. Xie (1), J. Wang (1), X. Zhang (1), Z. Liu (1), Z. Xiong (2) (1) Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, China; (2) University of Arizona, U.S.A. g ( ) g ( ) ( ) g ( ) cience and Biotechnology, Chinese Academy of Tropical Agricultural Sciences, China; (2) University of Arizona, U.S.A. Banana bunchy top virus (BBTV) causes a severe disease in banana world-wide. BBTV is a single-stranded DNA virus with a genome consisting of six circular molecules of ~1kb each. Some BBTV isolates also carry satellite DNA molecules that are not essential for BBTV infection. To understand the coordinated actions of the multiple genomic components during BBTV infection, we measured the relative and absolute amounts of each DNA component of isolate B2 and its transcript levels by real-time PCR (qPCR) and reverse transcription (RT)-qPCR, respectively. Significant differences in the absolute amounts and expression levels were found among BBTV genomic components. The most abundant DNA molecules are those of DNA-U3, DNA-M, and DNA-N, 4 to 6 times more than those of DNA-R. Comparison of Fusaruim graminearum transcription factors affecting Fusaruim graminearum virus 1 accumulation based on phenome analysis J. Yu (1), Y. Lee (1), K. Kim (1) (1) Seoul National University Korea (1) Seoul National University, Korea The Fusarium graminearum virus 1 (FgV1) causes hypovirulent phenotypic changes in Fusarium graminearum. It has been reported that the numerous F. graminearum genes were differentially expressed upon FgV1 infection including genes encoding regulatory factors. Using gene-deletion mutant library of 657 putative transcription factors (TFs) in F. graminearum, we transferred FgV1 by hyphal anastomosis to screen transcription factors that might be associated with viral replication or symptom induction. FgV1-infected TF mutants were divided into three groups according to the mycelial growth phenotype compare to the wild-type FgV1-infected strain (WT-VI). The FgV1-infected TF mutants in Group 1 exhibited slow or weak mycelial growth compare to that of WT-VI on CM media at 5-dpi. In contrast, Group 3 consists of virus-infected TF mutants showing faster mycelial growth and mild symptom compared to that of WT-VI. The hyphal growth of FgV1-infected TF mutants in Group 2 was not significantly different from that of WT- VI. These growth differences among the FgV1-infected TF mutant groups might be related with FgV1 RNA accumulations in infected host fungi. To confirm this, we tested FgV1 RNA accumulation of some of FgV1-infected TF mutants in each group. Taken together, our analysis will help us identifying regulator(s) of FgV1-triggered signaling and antiviral responses and thus expand our understanding on complex regulatory networks between FgV1 and F. graminearum interaction. S4.160 Root transcriptome analysis reveals viral diversity in two species of blueberry N. SAAD (2), J. Olmstead (1), R. Alcalá-Briseño (2), A. Varsani (3), J. Polston (2) (1) Horticultural Sciences Department, University of Florida, U.S.A.; (2) Department of Plant Pathology, University of Florida, U.S.A.; (3) School of Biological Sciences, University of Canterbury, Christchurch, New Zealand A growing number of metagenomics based approaches have been used for the elucidation of viral diversity in insects, cultivated plants, and water in agricultural production systems. However, while the generation of plant transcriptomes has greatly increased over the last few years, their use for data mining viral sequences has been largely unexplored. In this study, blueberry root transcriptomes (8 libraries each of Vaccinium arboreum and V. corymbosum x V. darrowi ‘Emerald’) were analyzed using bioinformatic analyses for plant viral sequences. De novo assembly of ‘Emerald’ libraries yielded a complete viral genome of the Blueberry red ringspot virus (BRRV), genus Soymovirus, family Caulimoviridae, a plant virus with a dsDNA genome. Identification of defense-related genes associated with tomato Sw-7 line against Tomato spotted wilt virus in tomato through transcriptome analysis y C. PADMABHAN (1), Y. Zheng (2), R. Shekaste-band (3), K. Stewart (1), J. Scott (3), Z. Fei (2), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Boyce Thompson Institute for Plant Research, Ithaca, NY, U.S.A.; (3) University of Florida, IFAS, Gulf Coast Research and Education Center, Wimauma, FL, U.S.A. C. PADMABHAN (1), Y. Zheng (2), R. Shekaste-band (3), K. Stewart (1), J. Scott (3), Z. Fei (2), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Boyce Thompson Institute for Plant Research, Ithaca, NY, U.S.A.; (3) University of Florida, IFAS, Gulf Coast Research and Education Center, Wimauma, FL, U.S.A. Tomato spotted wilt virus (TSWV) is a highly infectious tospovirus, and one of the most damaging plant viruses infecting tomatoes worldwide. Developing a tomato cultivar with TSWV-resistance would be the most effective approach for disease management. Comparative analysis of differential expression of genes in plant functional pathways between highly isogenic tomato breeding lines with striking difference in resistance and susceptibility to TSWV may lead to understanding of the mechanism of disease resistance. In the present study, RNA-seq was performed on samples from a near- isogenic breeding line containing Sw-7 and its recurrent TSWV-susceptible parent at 0, 4, 7, 14, 21 and 35 days post inoculation (dpi). Comparative transcriptome analysis revealed a large number of genes that are differentially expressed (DE) by TSWV infection over time, including pathogenesis related 1 (PR1) and 5 (PR5) (Osmotic-like protein), Pto-like, Serine/threonine kinase protein, N-methyltransferase-like protein, auxin, glycine/proline rich protein, MADS box transcription factors, histidine kinase, histone proteins, as well as the RNA silencing pathway genes, argonaute, DCL3 and PolIV and microRNA target genes. Genome-wide transcriptome analysis offers us an opportunity to understand the mechanism of disease resistance, which may lead us to an effective management of TSWV in tomato and other crops. Transcriptome profiling to discover defense-related genes associated with resistance line ty-5 against Tomato yellow leaf curl virus in tomato C. PADMABHAN (1), Y. Zheng (2), R. Shekaste-band (3), K. Stewart (1), D. Hasegawa (1), J. Scott (3), Z. Fei (4), K. Ling (1) (1) USDA-ARS, U.S. Vegetable Laboratory, Charleston, SC, U.S.A.; (2) Boyce Thompson Institute for Plant Research, Ithaca, NY, U.S.A.; (3) University of Florida, IFAS, Gulf Coast Research and Education Center, Wimauma, FL, U.S.A.; (4) zf25@cornell.edu, Ithaca, NY, U.S.A. Comparison of Fusaruim graminearum transcription factors affecting Fusaruim graminearum virus 1 accumulation based on phenome analysis J. Yu (1), Y. Lee (1), K. Kim (1) (1) Seoul National University Korea The least abundant DNA molecules are those of DNA-C, 12 times less than that of DNA- U3. Interestingly, two satellite DNA components are also present at high levels, with amounts similar to that of DNA-U3. Transcript levels determined by RT-qPCR did not correspond to the absolute amount of each DNA component. The most abundant transcripts came from DNA-N, followed by DNA- U3, and DNA-S, and the least abundant transcripts came from DNA-C, 110-fold less than those from DNA-N. The ratios of transcript copies over DNA copies ranged from 0.15 to 2.63. These data suggest that both replication and expression levels are independently modulated for each DNA component, possibly by cis-acting DNA sequence elements. Identification of defense-related genes associated with tomato Sw-7 line against Tomato spotted wilt virus in toma analysis eFile and the USDA Animal Plant Health Inspection Service’s (APHIS) PPQ Pest Permitting Process J. ABAD (1) (1) USDA-APHIS, U.S.A. Londo (6) (1) The Ohio State University, Department of Plant Pathology, Columbus, OH, U.S.A.; (2) The Ohio State University, Ohio Agricultural Research and Development Center, Department of Plant Pathology, Wooster, OH, U.S.A.; (3) The Ohio State University, Ohio Agricultural Research and Development Center, Department of Entomology, Wooster, OH, U.S.A.; (4) Ohio State University Extension, Xenia, OH, U.S.A.; (5) Ohio State University Extension, Cleveland, OH, U.S.A.; (6) Ohio State University Extension, Columbus, OH, U.S.A.; (7) Ohio State University Extension, Urbana, OH, U.S.A. (1) The Ohio State University, Department of Plant Pathology, Columbus, OH, U.S.A.; (2) The Ohio State University, Ohio Agricultural Research and Development Center, Department of Plant Pathology, Wooster, OH, U.S.A.; (3) The Ohio State University, Ohio Agricultural Research and Development Center, Department of Entomology, Wooster, OH, U.S.A.; (4) Ohio State University Extension, Xenia, OH, U.S.A.; (5) Ohio State University Extension, Cleveland, OH, U.S.A.; (6) Ohio State University Extension, Columbus, OH, U.S.A.; (7) Ohio State University Extension, Urbana, OH, U.S.A. In response to the growing demand for qualified professionals to address emerging trends and challenges in plant health, the Master in Plant Health Management (MPHM) was established by the Department of Plant Pathology and the Department of Entomology at The Ohio State University. The MPHM program, which is affiliated with the Professional Science Master’s Association, is distinguished by an interdisciplinary curriculum that integrates agricultural science coursework with professional development and workforce skills in the College of Food, Agricultural, and Environmental Sciences. An online option was added in 2014. The program has attracted a diverse population of students, many of whom are working professionals and non-traditional students. A new graduate associateship program was established with OSU Extension to recruit, educate and support future Extension educators to work in the field of agriculture and natural resources. Students work directly with Extension teams and mentors on projects that include agronomic field trials, ornamental pathology, and citizen science programs. The MPHM program’s placement rate is very strong, with graduates working in Extension, industry and research. Virtual disease management using Blightpro DSS M. MCKELLAR (1), I. Small (1), W. Fry (1) (1) Cornell University, U.S.A. Experiential learning activities can support student understanding of material in an introductory plant pathology course where student base knowledge related to content can be limited. Unfortunately not all course content can be successfully translated into authentic hands-on activities. Computer-based activities such as simulations can be successful in meeting this challenge. eFile and the USDA Animal Plant Health Inspection Service’s (APHIS) PPQ Pest Permitting Process J. ABAD (1) (1) USDA-APHIS, U.S.A. USDA APHIS safeguards U.S. agriculture against the entry, establishment, and spread of economically and environmentally significant pests. APHIS PPQ uses a permit type called the “PPQ 526 permit” to regulate the interstate movement and importation of plant pathogens (such as bacteria, fungi), pests (such as insects and nematodes), and biocontrols. We currently issue permits through ePermits, a web-based system that allows users to submit and track applications. PPQ receives over 6,000 of these applications per year. The average processing time is 80 days. Depending on the risk of the organism(s) requested, we may require that the work to be done in a USDA APHIS- inspected containment facility to adequately contain the organisms. This may increase processing time as PPQ determines if the facility is adequate. In most cases, this applies for the receipt of foreign organisms, federal quarantine pests, and diagnostic facilities. APHIS is developing a new permitting system known as “eFile” which we will deploy soon. We expect that this new system could substantially reduce the processing time for certain permits. National Clean Plant Network for Roses K. ONG (1), D. Byrne (2), D. Golino (3), N. Anderson (2), S. Sim (3), B. Pemberton (4) (1) Texas Plant Disease Diagnostic Lab, Texas A&M AgriLife Extension Service, College Station, TX, U.S.A.; (2) Department of Horticultural Sciences, Texas A&M University, College Station, TX, U.S.A.; (3) Foundation Plant Services, University of California-Davis, Davis, CA, U.S.A.; (4) Texas A&M AgriLife Research, Overton, TX, U.S.A. K. ONG (1), D. Byrne (2), D. Golino (3), N. Anderson (2), S. Sim (3), B. Pemberton (4) (1) Texas Plant Disease Diagnostic Lab, Texas A&M AgriLife Extension Service, College Station, TX, U.S.A.; (2) Department of Horticultural Sciences, Texas A&M University, College Station, TX, U.S.A.; (3) Foundation Plant Services, University of California-Davis, Davis, CA, U.S.A.; (4) Texas A&M AgriLife Research, Overton, TX, U.S.A. At the urging of the grape and fruit tree industries, the National Clean Plant Network (NCPN) was established to provide high quality asexually propagated plant material free of targeted plant pathogens and pests for growers in the United States. In 2008, the NCPN was initiated with grapes and fruit tree crops. This was followed by the addition of citrus, berries and hops in 2010 and roses and sweet potatoes in 2015. Each specialty crop has its own commodity network composed of representatives from industry, academia, extension, state government and other interested experts. Seed treatment with multiple rhizobacterial strains promotes soybean growth and suppresses reproduction of soybean cyst nematode in the field L. CHEN (1), Y. Zhou (2), Y. Wang (2), X. Zhu (2), R. Liu (2), P. Xiang (3), J. Chen (4), X. Liu (2), Y. Duan (2) Identification of defense-related genes associated with tomato Sw-7 line against Tomato spotted wilt virus in tomato through transcriptome analysis NSHS is governed by a Policy and Procedures Advisory Board in collaboration with the USDA-APHIS Seed Health Accreditation Manager and the National Plant Board Council/PPQ Strategy Team. Currently, there are 19 entities accredited by NSHS to perform field inspections, seed sampling and/or the seed health testing necessary to obtain phytosanitary certificates for exporting seeds. These organizations include private seed companies, state crop improvement associations and private testing labs. NSHS has been operating since 2001 and has approved 86 methods as standards for testing seeds for 66 different pathogens within a range of vegetable and field crops. NSHS works closely with international organizations to harmonize seed health testing methods that are accepted around the globe for phytosanitary certification. The standardization of seed health methods and practices increases confidence in the health status of US seed exports and facilitates safe and efficient international movement of seed for research and commercial purposes. S4.161 Implementation of a Quality Management System in an ISO 17025 Accredited Plant Pathogen Diagnostic Laboratory D. PICTON (1), A. Barth (1), K. Burch (2), P. Shiel (2) (1) USDA-APHIS-PPQ-S&T-CPHST, U.S.A.; (2) USDA-APHIS-PPQ-S&T-CPHST, U.S.A. eFile and the USDA Animal Plant Health Inspection Service’s (APHIS) PPQ Pest Permitting Process J. ABAD (1) (1) USDA-APHIS, U.S.A. This program supports translational research, education and extension initiatives to maintain the network’s high quality collections and strengthen its services. Since being established in 2015, the National Clean Plant Network for Roses (NCPNR) has been working to augment, renovate and improve the current rose collection at the Foundation Plant Services (Davis, CA), to assess the frequency of viruses in roses in the USA, to develop educational materials, and to establish the best protocols for determining the pathogen status of a rose. Master in Plant Health Management: Education and training to meet 21st century trends and challenges in Extension and industry M. LEWANDOWSKI (1), A. Dorrance (2), L. Canas (3), B. Kleinke (4), E. Roche (5), J. Schoenhals (6), S. Williams (1), F. Peduto Hand (1), J. Jasinksi (7), M. Gardiner (3), P. Paul (2), A. Londo (6) (1) The Ohio State University, Department of Plant Pathology, Columbus, OH, U.S.A.; (2) The Ohio State University, Ohio Agricultural Research and Development Center Department of Plant Pathology Wooster OH U S A ; (3) The Ohio State University Ohio Agricultural Research and Master in Plant Health Management: Education and training to meet 21st century trends and challenges in Extension and industry M. LEWANDOWSKI (1), A. Dorrance (2), L. Canas (3), B. Kleinke (4), E. Roche (5), J. Schoenhals (6), S. Williams (1), F. Peduto Hand (1), J. Jasinksi (7), M. Gardiner (3), P. Paul (2), A. Londo (6) Master in Plant Health Management: Education and training to meet 21st century trends and challenges in Extension and industry M. LEWANDOWSKI (1), A. Dorrance (2), L. Canas (3), B. Kleinke (4), E. Roche (5), J. Schoenhals (6), S. Williams (1), F. Peduto Hand (1), J. Jasinksi (7), M. Gardiner (3), P. Paul (2), A. Londo (6) Master in Plant Health Management: Education and training to meet 21st century trends and challenges in Extension and industry M. LEWANDOWSKI (1), A. Dorrance (2), L. Canas (3), B. Kleinke (4), E. Roche (5), J. Schoenhals (6), S. Williams (1), F. Peduto Hand (1), J. Jasinksi (7), M. Gardiner (3), P. Paul (2), A. Implementation of a Quality Management System in an ISO 17025 Accredited Plant Pathogen Diagnostic Laboratory D. PICTON (1), A. Barth (1), K. Burch (2), P. Shiel (2) (1) USDA-APHIS-PPQ-S&T-CPHST, U.S.A.; (2) USDA-APHIS-PPQ-S&T-CPHST, U.S.A. In 2015, the USDA-APHIS-PPQ-S&T Beltsville lab (Bldg 580) became the first ISO 17025:2005 accredited regulatory plant pathogen diagnostic lab in the United States. The ISO/IEC 17025 Standard is used world-wide by testing labs in the human clinical realm to the Federal Bureau of Investigation criminal analysis labs to ensure accuracy and reliability of test results. As a result of incorporating a system for technical and quality management, the diagnostics and results generated by the lab are internationally recognized to be technically competent, traceable, repeatable and statistically valid. The implementation of a Quality Management System (QMS) required the establishment of a systematic approach to control all laboratory processes as well as well-defined work instructions that were developed using statistically sound validation and verification data. The newly implemented Laboratory Information Management System (LIMS) has been integrated into the Laboratory QMS to provide complete sample, equipment, reagent and report tracking. This provides an added level of confidence that all data is sufficiently documented, reproducible, and traceable. An annual assessment by an accreditation body ensures that our laboratory continues to meet the requirements of technical competence. Continual improvements to the lab and its commitment to stakeholders through use of the QMS are now an operational component of the USDA-APHIS-PPQ-S&T Beltsville Lab. eFile and the USDA Animal Plant Health Inspection Service’s (APHIS) PPQ Pest Permitting Process J. ABAD (1) (1) USDA-APHIS, U.S.A. We developed a laboratory exercise using a practice version of BlightPro DSS (Decision Support System), which models the progress and control of potato late blight epidemics. The major learning outcome for students is to synthesize and apply their knowledge of disease management to the “real-time” management of late blight in relation to weather, cultivar resistance, and fungicide application. Working in small groups, students developed fungicide schedules to protect their potato crop from late blight while minimizing economic costs and environmental impact. A class discussion on the outcome of student group data concluded the exercise. Students demonstrated a strong cognitive presence throughout the exercise, thinking critically about plant disease management. They sought information from instructors and electronic resources during the exercise, beyond what was required for completion. Student collaboration in small groups promoted cooperative learning opportunities with students sharing previous experiences and knowledge applicable to the exercise outside of content presented in lecture. S4.162 (1) Shenyang Agricultural University, Ithaca, NY, China; (2) Shenyang Agricultural University, Shenyang, China; (3) Heihe Branch, Heilongjiang Academy of Agricultural Sciences, Heihe, China; (4) Daqing Branch, Heilongjiang Academy of Agricultural Sciences, Daqing, China (1) Shenyang Agricultural University, Ithaca, NY, China; (2) Shenyang Agricultural University, Shenyang, China; (3) Heihe Branch, Heilongjiang Academy of Agricultural Sciences, Heihe, China; (4) Daqing Branch, Heilongjiang Academy of Agricultural Sciences, Daqing, China The soybean cyst nematode (Heterodera glycines, SCN) is the most damaging pathogen of soybean (Glycine max) worldwide. Seed coating with nematicides or biological control agents represent a valuable method for controlling nematode pests. Here, we presented the study that evaluated the effects of combining three rhizobacterial strains, including Sinarhizobium fredii, Bacillus megaterium, and Bacillus simplex, as a seed treatment on SCN control and soybean yield enhancement under greenhouse and field conditions in northeast China. An optimal formulation consisting of different proportion of the above three rhizobacterial strains was determined based on the results of the germination vigor of the coated soybean seeds and subsequently designated as SN101. SN101 coated soybean seeds were then used in greenhouse studies and field trials conducted in two field locations for a period of three years. After each growing season, SCN reproduction was determined by nematode egg and cyst numbers and soybean growth and yield were also evaluated. Rapid differentiation of Claviceps species occurring in Oregon and Washington using high resolution melting curve analysis N. KAUR (1), R. Cating (1) (1) Oregon State University, Hermiston, OR, U.S.A. Rapid differentiation of Claviceps species occurring in Oregon and Washington using high resolution melting curve analysis N. KAUR (1), R. Cating (1) (1) Oregon State University, Hermiston, OR, U.S.A. Ergot disease of grass seed crops can be caused by Claviceps purpurea and C. humidiphila, a species recently reported to occur in certain regions of OR and WA. Routine identification of Claviceps species involves conventional PCR amplification and sequencing of ITS, β-tubulin or EF-1α genes. In this study, a high resolution melting (HRM) assay was developed to rapidly identify and differentiate C. purpurea and C. humidiphila based on the melting temperatures (Tm) of partial β-tubulin amplicons. PCR primers were designed to target and amplify a 110 bp region of the β-tubulin gene containing multiple single nucleotide polymorphisms generating amplicons with distinct melting profiles for each species. Forty-six fungal cultures, previously classified based on RAPD profiles, and ITS, β-tubulin, and EF-1α gene sequences, were used to validate the HRM assay. The Tm for C. purpurea and C. humidiphila ranged between 79.8 to 80.3 and 78.9 to 79.3, respectively. Classification of Claviceps species based on Tm using linear contrasts (P < 0.05) correctly grouped 96% (N=46) of samples previously classified using sequence data. In two cases, C. purpurea was misclassified as a mixture of the two Claviceps species. Therefore HRM analysis allows rapid detection and differentiation of these two Claviceps species and will be useful to study their distributions within OR and WA cool-season grass seed growing regions. eFile and the USDA Animal Plant Health Inspection Service’s (APHIS) PPQ Pest Permitting Process J. ABAD (1) (1) USDA-APHIS, U.S.A. Our results showed that SN101 treatment greatly inhibited SCN reproduction and significantly promoted plant growth and yield production in both greenhouse and field trials, suggesting that SN101 is a promising seed-coating agent that may be used as an alternative tool for controlling SCN in soybean fields. Effect of root pathogens Bipolaris sorokiniana and Fusarium graminearum on germination and seedling blight in wheat in South Dakota N. Kaur (1), S. Ali (1), S. Sehgal (1), K. Glover (1), S. Kumar (1) (1) South Dakota State Universty, Brookings, SD, U.S.A. Effect of root pathogens Bipolaris sorokiniana and Fusarium graminearum on germination and seedling blight in wheat in South Dakota N. Kaur (1), S. Ali (1), S. Sehgal (1), K. Glover (1), S. Kumar (1) (1) South Dakota State Universty, Brookings, SD, U.S.A. Bipolaris sorokiniana (Bs) and Fusarium graminearum (Fg) are important wheat root pathogens that effect seed germination and seedlings establishment thus ultimately impact the crop productivity. The objective of this study was to explore the effect of Bs and Fg infested seed on germination and seedlings establishment (blight) of 7 wheat cultivars, Advance, Briggs, Forefront, Oxen, Russ, Prevail, and SD4215 which were planted at Volga, SD in 2015. The treatments included, uninfested + untreated, uninfested + treated with fungicide, infested (Bs) + treated, infested (Bs) + untreated, infested (Fg) + treated, infested (Fg) + untreated. Seed germination and seedling blight data was recorded after the germination for 3 consecutive weeks. Wheat cultivars varied in seed germination and seedling blight to both pathogens; low seed germination (ranging from 35-71%) and high seedling blight (30- 65%) was observed in Fg infested seed, whereas, in Bs infested seed the germination ranged 58-100% and seedling blight ranged 0-42%. The cultivars Oxen and Prevail had the lowest (33%) and the highest (71%) seed germination respectively. Also the treatments including the fungicide significantly increased the germination (ranging from 5-44%) and the seedling blight got reduced in almost all the cultivars. Frequent isolation of Colletotrichum species complex form citrus tissues infected with leaf and fruit spot disease in Ethiopia A. D. Moges (1,3), D. Belew (2), B. Admassu (1), M. Yesuf (1), S. Maina (3), S. R. GHIMIRE (3) (1) Ethiopian Institute of Agricultural Research, Ethiopia; (2) Jimma University, Ethiopia; (3) Biosciences eastern and central Africa Hub-International Livestock Research Institute (BecA-ILRI) Hub, Box 30709, Nairobi 00100, Kenya; Email: s.ghimire@cgiar.org Frequent isolation of Colletotrichum species complex form citrus tissues infected with leaf and fruit spot disease in Ethiopia A. D. Moges (1,3), D. Belew (2), B. Admassu (1), M. Yesuf (1), S. Maina (3), S. R. GHIMIRE (3) (1) Ethiopian Institute of Agricultural Research, Ethiopia; (2) Jimma University, Ethiopia; (3) Biosciences eastern and central Africa Hub-International Livestock Research Institute (BecA-ILRI) Hub, Box 30709, Nairobi 00100, Kenya; Email: s.ghimire@cgiar.org Frequent isolation of Colletotrichum species complex form citrus tissues infected with leaf and fruit spot disease in Ethiopia A. D. Moges (1,3), D. Belew (2), B. Admassu (1), M. Yesuf (1), S. Maina (3), S. R. GHIMIRE (3) (1) Ethiopian Institute of Agricultural Research, Ethiopia; (2) Jimma University, Ethiopia; (3) Biosciences eastern and central Africa Hub-International Livestock Research Institute (BecA-ILRI) Hub, Box 30709, Nairobi 00100, Kenya; Email: s.ghimire@cgiar.org Citrus are economically important fruit crops in Ethiopia with estimated acreage of 7,040 hectares and annual production of 72,459 tons. The citrus leaf and fruit spot is among the major diseases contributing to a low productivity (10.3 t/ha) of citrus in Ethiopia. Therefore, a study was conducted to study diversity and relationships of the pathogens associated with leaf and fruit spot disease in major citrus growing regions of the country. Citrus leaves and fruits samples with distinct leaf and fruit spot disease symptoms were collected from 16 districts of Ethiopia and a total of 167 fungal isolates were recovered and monosporic or hyphal tip cultures were developed for each isolates. The genomic DNAs of all 167 isolates were amplified and sequenced for internal transcribed spacers (ITS), long subunit (LSU) and actin (ACT) genes. The sequences obtained were compared with the GenBank database sequences. The multilocus sequences based phylogenetic analysis revealed that 167 isolates were from either C. gloeosporioides or C. boninense species complexes. Surprisingly, this study did not recover any Pseudocercospora angolensis isolate, the causal agent reported for citrus leaf and fruit spot diseases. The detached leaf assay revealed the ability of both C. gloeosporioides and C. boninense isolates to cause foliar symptoms on multiple citrus species. S4.163
https://openalex.org/W2320017656	http://www.odermatol.com/odermatology/42013/21.SeroepidemiologyI-AlsamaraiAM.pdf	English	null	Seroepidemiology of Toxoplasma, Rubella, Cytomegalovirus and Herpes Simplex Virus -2 in Women with Bad Obstetric History. PART I: Toxoplasma and Rubella infections	Nasza Dermatologia Online	2,013	cc-by	13,758	Review Article Review Article DOI: 10.7241/ourd.20134.135 Introduction primary infection is likely to have a more important effect on fetus than recurrent infection and may cause congenital anomalies, spontaneous abortion, intrauterine fetal death, intrauterine growth retardation, prematurity, stillbirth, and live born infants with the evidence of disease [6]. Bad obstetric history (BOH) implies previous unfavorable fetal outcome in terms of two or more consecutive spontaneous abortions, history of intrauterine fetal death, intrauterine growth retardation, stillbirth, early neonatal death, and/or congenital anomalies [1]. The causes of BOH may be genetic, hormonal, abnormal maternal immune response, and maternal infection [2,3]. Most of the TORCH infections cause mild maternal morbidity but have serious fetal consequences [7]. The ability of the fetus to resist infectious organisms is limited and the fetal immune system is unable to prevent the dissemination of infectious organisms to various tissues [8]. Abstract Abstract Bad obstetric history (BOH) is associated with social and psychological impacts on society worldwide. The causes of BOH may be genetic, hormonal, abnormal maternal immune response, and maternal infection. In women with bad obstetric history (BOH), Toxoplasma (T) IgG high rate has been reported for Nepal (55.2%), while high (42.5%) and lowest (6.97%) active toxoplasma infections has been reported for India. In Arab countries, IgG and IgM higher and lowest seroprevalence rates were for Iraq. The higher susceptibility rates for Rubella in Arab countries excluding Iraq were reported in Morocco (83.4%), Sudan (34.7%), Qatar (25.1%), and Tunisia (20.3%). The lowest susceptibility was reported for Saudi Arabia (6.7%). In Iraq, studies indicate a high susceptibility rates in Thi Qar (98.05%), Kirkuk (91%), Baghdad (79%), and Waset (45.7%). The lowest susceptibility rates were reported for Diyala (0%) in women with previous abortion, and 3.9% in pregnant women without history of BOH. Key words: TORCH; Toxoplasma; Rubella; CMV; Cytomegalovirus; HSV Cite this article: Abdulghani Mohamed Alsamarai, Zainab Khalil Mohamed Aljumaili: Seroepidemiology of Toxoplasma, Rubella, Cytomegalovirus and Herpes Simplex Virus -2 in Women with Bad Obstetric History. PART I: Toxoplasma and Rubella infections. Our Dermatol Online. 2013; 4(4): 522-535. SEROEPIDEMIOLOGY OF TOXOPLASMA, RUBELLA, CYTOMEGALOVIRUS AND HERPES SIMPLEX VIRUS -2 IN WOMEN WITH BAD OBSTETRIC HISTORY. PART I: TOXOPLASMA AND RUBELLA INFECTIONS Abdulghani Mohamed Alsamarai1, Zainab Khalil Mohamed Aljumaili2 1Departments of Medicine, Tikrit University College of Medicine, Tikrit, Iraq 2Departments of Microbiology, Tikrit University College of Medicine, Tikrit, Iraq 1Departments of Medicine, Tikrit University College of Medicine, Tikrit, Iraq 2Departments of Microbiology, Tikrit University College of Medicine, Tikrit, Iraq Source of Support: Nil Competing Interests: None Source of Support: Nil Competing Interests: None Corresponding author: Prof. Abdulghani Mohamed Alsamarai Date of submission: 18.07.2013 / acceptance: 04.10.2013 Our Dermatol Online. 2013; 4(4): 522-535 TORCH Complex: In Maracaibo, Venezuela the overall prevalence of toxoplasmosis was 33%, while 18.2% were positive IgM [34]. In Qatar among 823 women of childbearing age, the T. gondii IgG and IgM was 35.1% and 5.2% respectively [35]. i Although congenital toxoplasmosis is not a nationally reportable disease in Iraq, it represents a health care problem. Reported studies indicated an estimated 400 to 4,000 cases occur in the U.S. each year [11,15,16]. The overall prevalence and incidence varies in different communities and contributes significantly to heavy morbidity [10]. Congenital toxoplasmosis mainly results from a primary infection acquired during pregnancy [17], but not from the reactivation of a latent infection in immunocompetent pregnant women [18]. However, it is believed that latent toxoplasmosis could reactivate and cause a congenital transmission of the parasite to infants who then become infected in utero [19]. Sixty-five studies [3,33,36-97] characterizing the prevalence of maternal infections with T. gondii in developing and developed countries and fifty-nine [35,98-155] studies in Arab countries (30 studies reported for Iraq) were identified. The features and results of these studies are summarized in Tables I and II.The majority of studies had small sample sizes, between 0– 4112 subjects. Most of these studies were conducted in antenatal clinics, hospitals, health care facilities or prenatal clinics. The remaining studies (3.3%) were community-based and the study setting was not specified in 7.4% of the studies. The most commonly used test was ELISA, which is the gold standard for T. gondii analysis. The median of IgG Toxoplasma prevalence was 38.5% [64] for Bangladesh. IgG high rate of detection was reported for Brazil [50] (75%, 832 pregnant women), while the lowest rate was for Thailand [38] (5.3%, 831 pregnant women). IgM lowest rate reported for China [49] (0%, 235 pregnant women) and Vietnam [59] (0%, 300 pregnant women), while the highest rate reported for Ghana [87] (76.1%, 159 pregnant women). Countries with high disease prevalence have instituted successful secondary prevention programs via widespread maternal serologic screening [20], but universal maternal serologic screening for toxoplasmosis is not currently recommended in most of countries [21-24]. Instead, current practice suggests maternal serological screening when abnormal fetal findings or presence of infertility problem indicate possible infection [22]. ELISA methods is commonly performed in many countries to detect anti-toxoplasma antibodies [25]. TORCH Complex: TORCH infections in the mother are transmissible to fetus in the womb or during the birth process and cause a cluster of symptomatic birth defects. Many sensitive and specific tests are available for serological diagnosis of TORCH complex [9]; however, ELISA test is more routinely used for its sensitivity. An attempt is being made to find out the correlation of TORCH infection during pregnancy in the Iraqi population. Toxoplasma gondii is an obligate intracellular protozoan parasite, which is linked to one of the most prevalent chronic infections affecting one third of the world’s human population [10]. The TORCH infections can lead to severe fetal anomalies or even fetal loss. They are a group of viral, bacterial, and protozoan infections that gain access to the fetal bloodstream transplacentally via the chorionic villi. Hematogenous transmission may occur at any time during gestation or occasionally at the time of delivery via maternal-to-fetal transfusion [4]. Primary infections caused by TORCH-Toxoplasma gondii, Rubella virus, cytomegalovirus (CMV), and herpes simplex virus (HSV)-are the major causes of BOH [5]. These infections usually occur before the woman realizes that she is pregnant or seeks medical attention. The 522 © Our Dermatol Online 4.2013 www.odermatol.com The infection is characterized by non-specific symptoms with the consequent formation of cysts that may remain in latent form in many organs [11]. Primary infection is usually subclinical but the infection hazard is its occurrence during pregnancy. There are four groups of individuals in whom the diagnosis of toxoplasmosis is most critical: a) pregnant women who acquire their infection during gestation, b) fetuses, c) newborns who are congenitally infected, immunocompromised patients, and d) those with chorioretinitis [12-14]. toxoplasmosis reference laboratory, and followed by serial titers at least 3 weeks apart [12,28,32]. The infection is characterized by non-specific symptoms with the consequent formation of cysts that may remain in latent form in many organs [11]. Primary infection is usually subclinical but the infection hazard is its occurrence during pregnancy. There are four groups of individuals in whom the diagnosis of toxoplasmosis is most critical: a) pregnant women who acquire their infection during gestation, b) fetuses, c) newborns who are congenitally infected, immunocompromised patients, and d) those with chorioretinitis [12-14]. There are different Toxoplasma seropositivity reports from all over the world. The population of Turkish childbearing age women has the seropositivity of T. gondii as 1.34% for IgM and 24.6% for IgG [33]. TORCH Complex: ELISA results are generally well accepted by clinicians because of their excellent sensitivities and specificities, the rapid availability of results, and the relatively low costs of the tests. It is important to understand that a single serologic test is not enough for the diagnosis of toxoplasmosis [26]. In worldwide, commercial test kits for Toxoplasma-specific IgG and IgM antibodies are readily available. The presence of IgM antibodies is not always an indication of a recent infection since IgM maybe present for many months [27,28]. Misdiagnosis of recent infections may be as a result of the presence of specific T. gondii IgM antibodies in the chronic stage of an infection, or false-positive IgM positivity [17,29]. IgM test results are difficult to interpret and the reliability of test kits is largely dependent upon other factors. A negative IgM with a positive IgG result can indicate infection at least 1 year before. A positive IgM result may indicate more recent infection or may also be a false positive reaction [25]. In women with bad obstetric history (BOH), IgG high rate was reported for Nepal [62] (55.2%, 345 BOH) and the lowest one was that reported by Natu et al [74] (19.44%, 499 BOH). IgM in BOH high rate was reported for India [36] (42.5%, 200 BOH), while the highest one for India also [91] (6.97%, 86 BOH). In Arab countries, the median of IgG prevalence was 41.9% which was reported for Sudan [144]. IgG highest rate of detection reported for Iraq [132] (94%, 54 pregnant women) Bahrain [137] (15.8%, 146 Pregnant women), while the corresponding values for IgM were 55.5% (Iraq, 180 pregnant women) [129] and 2.8% (Egypt, 323 pregnant women) [153] respectively. Concerning BOH, IgG ranges between 77.1% (Iraq,122 BOH) [114] and 6.84% (Iraq, 190 BOH) [130], while the range of IgM was between 58% (Iraq, 50 BOH) [127] and 0.97% (Iraq, 310 BOH) [104]. Rubella virus Currently worldwide, there is no systematic screening of pregnant women to detect seroconversion during gestation and most clinicians make decisions depending on result of single serum sample. This approach is not effective to detect toxoplasma infections during pregnancy, thus monthly serological screening for pregnant women is the recommended approach [30]. The presence of elevated levels of Toxoplasma specific IgG antibodies indicates infection has occurred at some point, but does not distinguish between an infection acquired recently and one acquired in the distant past. In acute infection, IgG and IgM antibodies generally rise within 1 to 2 weeks of infection [31]. Given the potential for false-positive results, the true value of IgM testing is in ruling out the presence of acute infection. In other words, negative IgM results are reassuring, whereas positive results should be interpreted carefully, confirmed in a Rubella is a contagious viral disease caused by a togavirus and usually goes unnoticed. However, maternal infection during pregnancy may result in fetal loss or in congenital rubella syndrome (CRS) [156,157]. Infection in the first eight to ten weeks of pregnancy results in damage in up to 90% of surviving infantswhere multiple defects are common. The risk of damage declines to about 10 to 20% with infection occurring between 11 and 16 weeks gestation [158]. Fetal damage is rare with infection after 16 weeks of pregnancy, with only deafness being reported following infections up to 20 weeks of pregnancy. Some infected infants may appear normal at birth but perceptive deafness may be detected later [157,158]. Before the introduction of Rubella immunisation, Rubella was commonly prevalent in children, and more than 80% of adults had evidence of previous rubella infection [159]. Rubella virus © Our Dermatol Online 4.2013 523 Article Location, setting of study Type, Duration Population Results Wanachiwanawin et al [38] Thailand, antenatal clinic Cross sectional , 2 years 831 Pregnant women 5.3% IgG, IgM positive in 4.5% of IgG positive Lopes et al [39] Brazil, antenatal clinic Cross sectional, 7 months 492 Pregnant women 49.2% IgG, IgM 1.2% of IgG positive Varella et al [40] Brazil, Hospital Cross-sectional, 7 years 41112 Pregnant women 0.48% seroprevalence Khurana et al [41] India, antenatal clinic Cross sectional, No information 300 Pregnant women 15.3% IgG, 3% IgM Vaz et al [42] Brazil, No information Cross-sectional, 15 months 20389 Pregnant women 53.3% IgG, 3.26% IgM Alvarado-Esquivel et al [43] Mexico, Rural Community based, 439 Pregnant women 8.2% IgG, 2.3% IgM Sakikawa M et al [44] Japan, antenatal clinic All cases screening, 7.5 years 4466 Pregnant women 10.3% seroprevalence ,0.25% primary infection Maggi et al [45] Albania, General outpatient centre Screening, 6 months 498 Pregnant women 48.6% IgG, IgM 1.3% of IgG positive Sen MR et al [46] India, Hospital Descriptive case control, 2 years 380 pregnant women 19.4% IgM Sarkar et al [47] India, antenatal clinic Descriptive case control, 10 months 105 Pregnant women 49.52% IgG, 21.9% IgM Barbosa et al [48] Brazil, Maternity hospital Cross-sectional, 10 months 190 Pregnant women 66.3% IgG, 0.53% IgM Liu et al [49] China, antenatal clinic Cross-sectional 235 Pregnant women 10.6% IgG, 0% IgM Ribeiro et al [50] Brazil, PHC Cross sectional, 3.5 years 832 Pregnant women 75.1% IgG, 2% IgM Rosso et al [37] Colombia, Healthcare facility Cross-sectional, 5 months 955 Pregnant women 45.8% IgG, 2.8% IgM Abdi et al [51] Iran, Cross-sectional, 553 Pregnant women 44.8% IgG Mostavi N [52] Iran, Survey Cross-sectional, 1 year 217 Child bearing age 47.5% seroprevalence (IgG) Hajsoleimani [53] Iran, PHC Cross-sectional, 500 Pregnant women 37.2% IgG, 1.4% IgM Ndiaye et al [54] Senegal, Hospital Cross-sectional, 1 year 109 Pregnant Women 22% IgG, 3% IgM Spalding et al [55] Brazil, PHC Cross-sectional, 18 months 2128 Pregnant women 71.5% IgG, 3.6% IgM Castilho-Pelloso et al [56] Brazil, Public health care Observational Retrospective, 3 years 290 Pregnant women 1.07% IgM Sharifi-Mood et al [57] Iran, Hospital Cross sectional, 200 Pregnant women 27% serpositive Ndir et al [58] Senegal, Health centre Case control, 6 months 70 Pregnant & 70 Abortion cases 37.1% in pregnant, 40% in abortion Buchy et al [59] Vietnam, Cross-sectional, 300 Pregnant women 11.2% IgG, 0% IgM Akoijam et al [60] India, Antenatal clinic Cross-sectional, 1 year 503 Pregnant women 41.75% seroprevalence Mahdi et al [61] Iran, Antenatal clinic Cross-sectional, 245 Pregnant women 49.2% seroprevalence Rai et al [62] Nepal, Antenatal clinic Cross-sectional, 2 years 345 BOH 55.2% seroprevalence Chintana et al [63] Thailand, Antenatal Clinic Cross-sectional, 6 months 1200 Pregnant women 13.2% IgG Ashrafunnessa et al [64] Bangladesh, Antenatal clinic Cross-sectional 286 Pregnant women 38.5% IgG Zhang et al [65] China Cross-sectional 1250 Pregnant women 7.28% seroprevalence Sroka et al [66] Brazil, Hospital Cohort, 10 weeks 963 Pregnant women 68.6% IgG, 0.5% IgM Zhang et al [67] China, antenatal clinic Cross-sectional 4126 Pregnant women 3.38% IgM Gonzalez-Morales et al [68] Cuba, Health centres Cross-sectional, 2 years 3913 Pregnant women 70.9% seroprevalence Galvan Ramirez et al [69] Mexico, Hospital Case control 350 High risk pregnancy 34.9% IgG, 20.7% IgM Lelong et al [70] Madagascar, 599 Pregnant women 83.5% seroprevalence Sun et al [71] China, hospital Cross sectional 1211 Pregnant women 39.14% IgG, 4.21% IgM Martinez Sanchez et al [72] Cuba, Community survey Cross sectional, 6 months 362 Pregnant women 71% seroprevalence Bari et al [73] India, antenatal clinic Cross sectional 302 Pregnant women 46% IgG, 27.7% IgM Natu et al [74] Case control 499 BOH 19.44% seroprevalence Bittencourt [75] Brazil, Public health services Cross sectional, 16 months 4022 Pregnant women 59.8% IgG, 1.1% IgM Shanmugam et al [76] India, antenatal Cross sectional 225 Pregnant women 23.6% Seropositive Reis et al [77] Brazil, Hospital Cross sectional, 6 years 10468 61.1% Seroprevalence Harma et al [78] Turkey, Prenatal clinic Cross-sectional, 1149 Pregnant women 60.4% IgG, 3% IgM Hou et al [79] China, Hospital Cross-sectional 347 Pregnant and post partum women 5.5% seroprevalence. Rubella virus Doehring et al [80] Tanzania, Hospital Cross-sectional 849 Pregnant women 35% Seropositive Soto et al [81] Venezuela, Hospital Cross sectional 7969 Pregnant women 53.91% Seroprevalence 524 © Our Dermatol Online 4.2013 524 © Our Dermatol Online 4.2013 Article Location, setting of study Type, Duration Population Results Khurana et al [82] India, Antenatal clinic Cross-sectional 300 Pregnant women 15.33% IgG, 3% IgM Ouermi et al [83] Burkina Faso, Healthcare facility Cross-sectional 6 months 276 Pregnant women 27.2% IgG, 4.7% IgM Zemene et al [84] Ethiopia, Community based Cross-sectional, 2 months 201 Pregnant women 81.1% IgG, 2.5% IgM Flatt A & Shetty N [85] UK, Antenatal clinic Cohort, 2 years 5000 Pregnant women 17.32 % IgG Surpam et al [86] India, Antenatal clinic Case control, 150 BOH 14.66% IgM Ayi et al [87] Ghana, Antenatal clinic Cross-sectional, 4 months 159 Pregnant women 73.6% IgG, 76.1 IgM Cvetkovic D et al [88] Macedonia, Retrospective, 2 years 235 Pregnant women 20.4% overall seroprevalence Karabulut A et al [89] Turkey, Antenatal clinic Case control, 1 year 1102 Pregnant women 37% IgG, 1.4% IgM Kumari N et al [1] Nepal, Hospital Case control, 4 months 12 BOH 50% seropositive Nabi SN et al [90] Bangladesh, Hospital Case control, 10 months 111 Pregnant women 23.42% IgG, 0.9% IgM Sadik MS et al [91] India, Hospital Case control, 2 years 86 BOH 20.93% IgG, 6.97% IgM Akyar I [33] Turkey, Hospital Cross sectional, 7.5 years 17751 Child bearing age 24.6% IgG, 1.34% IgM Frischknecht F et al [92] Switzerland, Hospital Cross sectional, 1 yr 723 Pregnant women 44.11% serpositive Inagaki ADM, et al [93] Brazil, Antenatal clinic Cross sectional, 1 year 9559 Pregnant women 69.3% IgG, 0.4% IgM Turbadkar D, et al [3] India, Antenatal clinic Case control, 1 year 380 BOH 42.1% IgG, 10.52% IgM Linguissi LS et al [94] Burkia Faso, Cross sectional, 3 years Pregnant women 20.37% IgG Chopra S et al [36] India, Antenatal clinic Case control, 1 year 200 BOH 42.5% IgM Koksaldi-Motor et al [95] Turkey, Hospital Cross sectional, 1 year 1103 Childbearing age 59.9% IgG Vilibik-Cavlek T, et al [96] Croatia, Hospital Cross sectional, 5 years Pregnant & non pregnant women 29.1% IgG, 0.25% IgM Goncalves MA, et al, [97] Brazil, Hospital Retrospective, 2 years 574 Pregnant women 62% IgG, 3.4% IgM Table I. Characteristics and results of studies reporting prevalence of maternal Toxoplasma infection (continued). Rubella virus Article Location, setting of study Type, duration of study Population Results Al-Ani RT [103] Iraq, Al- Anbar, Hospital Cross sectional, 6 months 50 Pregnant women 50% IgM Razzak et al [104] Iraq, Duhok, Hospital Case control, 18 months 310 Women with BOH 0.97% IgM El Mansouri et al [105] Morocco, Institute National Hygiene Cross-sectional 2456 Pregnant women 50.6% seroprevalence Elnahas et al [106] Sudan, Antenatal clinic 7 months 487 Pregnant women 34.1% IgG, 14.3% IgM Abdel-Hafez et al [107] Jordan, Case control, 1 year 55 Aborted women 46 Pregnant women 58.2% Aborted women, 26.1% Pre gnant women Hammouda et al [108] Egypt, Hospital Case control, 100 BOH 65% seroprevalence Abdulmohaymen N [99] Iraq (Baghdad), Hospital Case control, 9 months 119 Aborted women 24.2% IgM recurrent spontaneous abortion 14.7% IgM non recurrent spontaneous abortion. 8.1% IgG recurrent spontaneous abortion 5.9% IgG non recurrent spontaneous abortion Salih HA [109] Iraq, Najaf, Hospital Case control 260 Aborted women 30.76% IgG, 11.92% IgM Al-Mohammad et al [110] Saudi Arabia, Maternity Hospital Cross-sectional, 1 year 554 Pregnant women 51.4% IgG, 8.8 IgM Jasim et al [100] Iraq, Waset, Hospital Case control, 1 year 162 Aborted women 53.9% IgG, 54.8% IgM Al- Taie et al [101] Iraq, Mosul, Private laboratory Case control, 1 year 100 BOH 43% IgM Al Seadawy MAH [111] Iraq, Al Muthana, Hospital Case control, 3 months 81 Aborted women 44.5% IgM Mousa DA [112] Libya, Hospital Case control, 6 months 143 BOH 44.8% IgG, 8.4% IgM Mahmood SH et al [113] Iraq, Baghdad, Public Health Central Laboratory Case control, 8 months 120 Aborted women 39.16% IgG, 17.79% IgM Aziz & Drueish[114] Iraq, Baghdad, Hospital Case control 122 Aborted women 77.1% IgG, 58.1% IgM Al-Hamdani & Mahdi [115] Iraq, Basrah, PHC Case control, 8 months 81 Habitual abortion 18.5% seropositive Al-Sodany & Saleh [116] Iraq, Basrah, Hospital Case control, 8 months 81 Habitual abortion 81.5% seropositive Table II. Characteristics and results of studies in Arab countries reporting prevalence of maternal Toxoplasmosis infection. Rubella virus © Our Dermatol Online 4.2013 525 Article Location, setting of study Type, duration of study Population Results Majeed AK [117] Iraq, Baghdad, Case control, 3 years 260 Aborted women for IgG 259 Aborted women for IgM 21.2% IgG35.1% IgM Alsaeed et al [118] Iraq, Al-Hila, Hospital Case control, 6 months 120 Aborted women 41.66% seropositive Almishhadani & Aljanabi [119] Iraq, Al- Anbar, Medical Laboratory Case control study, 3 years 230 Aborted women 58.3% IgG, 8.3% IgM Khudair M K [120] Iraq, Diala, Hospital Case control, 5 months 50 Aborted women 54% seropositive Hasan SF [121] Iraq, Karbala, Immunology Centre Cross sectional, 3 months 82 Childbearing age women 18.3% IgG Ali AA [122] Iraq, Al- Tameem, Hospital Cross sectional, 1 year 100 Pregnant women 97 BOH 61% Seroprevalence 74.22% BOH non pregnant Kadir MA et al [123] Iraq, Kirkuk, Hospital & PHC Cross sectional, 7 months 319 Pregnant women 121 Aborted women 36.6% seroprevalence LAT, 16.92 IgM ELISA52% LAT, 25.61% IgM ELISA Alkulabi R [124] Iraq, Najaf, Hospital Cross sectional study 137 Pregnant women 60.5% IgG, 43.7% IgM Yousif JJ et al [125] Iraq, Najaf, PHC Cross sectional, 3 months 120 Pregnant women 120 Non pregnant 40% IgG29.2% IgG Al-khafaji & Mohsen [126] Iraq, Thi Qar, Hospital Case control, 10 months 74 Habitual abortion 23% IgG, 31.1% IgM Alkhashab FMBA, et al [127] Iraq, Mosul, Hospital Case control, 16 months 50 Aborted women, 100 Pregnant women 34% IgG, 58% IgM20% IgG, 41% IgM Alaa Z [128] Iraq, Tikrit, Hospital Case control, 15 months 226 BOH 26.1% IgG, 3.1% IgM Rashid KN [102] Iraq, Tikrit, Private laboratory ????? Rubella virus 100 Women 15 -45 years age 46% IgG, 32% IgM of IgG positive cases, Al-Marzoqi AHM, et al [129] Iraq, Babylon, Hospital Cross sectional, 6 months 180 Pregnant women 62.2% IgG, 55.5% IgM Hadi NJ [130] Iraq, Thi Qar, Hospital Case control 190 Aborted women 6.84% IgG, 12.63% IgM Salman YG [131] Iraq, Kirkuk, Hospital Case control, 11 months 184 BOH 4.84% Seropositive, 17% IgM Mossa HAL [132] Iraq, Baghdad, Hospital Retrospective, 2 years 54 Pregnant women 94% IgG, 33% IgM Al- Shimmery MN [133] Iraq, Diwanya, Hospital Case control, 5 months 125 Aborted women 45.6% IgG, 29.6% IgM Bouratbine A, et al [134] Tunisia, Hospital Cross sectional 1421 community sample 70% seroprevalence at age of 30 years Barkat A et al [135] Morocco, Hospital Cross sectional, 1 year 368 Pregnant women 44.3% IgG Bouhamdan SF et al [136] Lebanon, Hospital & Private laboratories Retrospective, 1year 3516 Female for IgG 3426 Female for IgM 62.2% IgG6.8% IgM Tabbara & Saleh [137] Bahrain, Hospital Cross-sectional, 46 months 146 Delivering women 15.8% IgG Ibrahim HM et al [138] Egypt, Private Clinical Laboratory Cross sectional, 101 Pregnant women 51.49% seroprevalence Al-Hindi & Lubbad [139] Palestine, Hospital Case control, 6 months 312 Aborted women 17.9% IgG, 12.8% IgM Abu- Madi MA, et al [35] Qatar, Hospital Cross sectional, 3 years 847 Women > 20 yr age 38.2% IgG, 5.1% IgM Gashout A, et al [140] Libya, Hospital Case control, 5 years 692 Aborted women 45% IgG, 17.6% IgM Al-Qahtani & Hassan [141] Saudi Arabia, Hospital Cross sectional, 5 months 75 Adult female 44% seroprevalence Al-Harithi SA et al [142] Saudi Arabia, Hospital Cross sectional, 6 months 197 Pregnant women 29.4% IgG, 5.6% IgM Elamin MH, et al [143] Sudan, Hospital Case control, 94 Pregnant Aborted during study 94 Pregnant with normal outcome 35.1% IgG, 15.2% IgM, 39.4% IgG, 16.2% IgM, Overall 37.2% IgG, 5.9% IgM Khalil KM, et al [144] Sudan, Hospital Case control, 245 Pregnant women 209 Aborted women 35.9% seroprevalence58.3% Seroprevalence Mohamad K, et al [145] Sudan, Hospital Cross sectional, 253 Childbearing age women 73.1% IgG Al- Nahari AM, et al [146] Yemen, Central Laboratory Cross sectional, 2 years 463 Pregnant women 41.9% IgG, 11.88% IgM Ghazi HO, et al [147] Saudi Arabia, Hospital Cross sectional 926 Pregnant women 35.6% IgG Sellami H, et al [148] Tunisia, Hospital Cross sectional, 13 years 40 566 Pregnant women 39.3% seroprevalence, 1.3% acute infection during pre gnancy. Rubella virus Rubella infection of a pregnant woman may have devastating effects on the developing fetus and once congenital infection occurred there is no availability of treatment for the foetus. Thus the mainstay of prevention is the universal immunization of all infants and identification and immunization of women at risk [156]. [ ] Fetal infection is acquired hematogenously, and the rate of transmission varies with the gestational age at which maternal infection occurs, with higher frequency in first trimester [160]. Periconceptual maternal infection does not seem to increase the risk of CRS [160]. Maternal immunity, either after vaccination or naturally derived, is generally protective against intrauterine rubella infection [162,163]. However, there have been cases of CRS after maternal reinfection [163]. Therefore, CRS should always be considered in a fetus or neonate with a clinical picture suggestive of congenital infection [162]. It should be noted that no case of CRS has been reported when maternal reinfection occurred after 12 weeks of pregnancy [164]. The higher susceptibility rates for Arab countries excluding Iraq were reported [35,216,220,221] in Morocco (83.4%), Sudan (34.7%), Qatar (25.1%), and Tunisia (20.3%). The lowest susceptibility was reported [217] for Saudi Arabia (6.7%). In Iraq, reports indicate a high susceptibility rates in Thi Qar (98.05%), Kirkuk (91%), Baghdad (79%), and Waset (45.7%). While the lowest susceptibility rates were reported for Diyala (0%) in women with previous abortion, and 3.9% in pregnant women without history of BOH [215]. The same figures was reported later by another research group in Babylon [213]. The higher susceptibility rates for Arab countries excluding Iraq were reported [35,216,220,221] in Morocco (83.4%), Sudan (34.7%), Qatar (25.1%), and Tunisia (20.3%). The lowest susceptibility was reported [217] for Saudi Arabia (6.7%). In Iraq, reports indicate a high susceptibility rates in Thi Qar (98.05%), Kirkuk (91%), Baghdad (79%), and Waset (45.7%). While the lowest susceptibility rates were reported for Diyala (0%) in women with previous abortion, and 3.9% in pregnant women without history of BOH [215]. The same figures was reported later by another research group in Babylon [213]. In Iraq, reports indicate a high susceptibility rates in Thi Qar (98.05%), Kirkuk (91%), Baghdad (79%), and Waset (45.7%). While the lowest susceptibility rates were reported for Diyala (0%) in women with previous abortion, and 3.9% in pregnant women without history of BOH [215]. The same figures was reported later by another research group in Babylon [213]. Fifty- nine studies (Tabl. Rubella virus Almogren A [149] Saudi Arabia, Hospital Retrospective, 1 year 2176 Pregnant women 38% IgG, 0% IgM Al- Hindi A, et al [150] Palestine, IVF centre Retrospective, 6 years 1954 Women with infertility or abortion 7.9% IgM Table II. Characteristics and results of studies in Arab countries reporting prevalence of maternal Toxoplasmosis infection (continued). Table II. Characteristics and results of studies in Arab countries reporting prevalence of maternal Toxoplasmosis infection (continued). 526 © Our Dermatol Online 4.2013 Article Location, setting of study Type, duration of study Population Results El-Gozamy BR, et al (151) Egypt, Hospital Cross sectional, 17 months Rural 57.6% seroprevalence, 46.5% Urban Hussein AH, et al (152) Egypt, Hospital Case control, 152 randomly selected individuals, 31 full term pregnant, 38 BOH IgG- 57.9%, 58.1%, 44.7%IgM – 10.5%, 6.5%, 23.7% El- Deeb HK, et al (153) Egypt, Hospital Cross sectional 323 Pregnant women 67.5% IgG, 2.8% IgM El- Ridi AM, et al, (154) Egypt, Hospital Case control 72 BOH 27.8 % Seropositive Jumaian NF (98) Jordan, Antenatal Cross sectional,17 months 280 Pregnant women 47.1 seropositive, Mohammed TK (155) Iraq, Baghdad, Hospital Cross sectional, 6 months 212 Pregnant women 28.77% IgG, 23.8% IgM Table II. Characteristics and results of studies in Arab countries reporting prevalence of maternal Toxoplasmosis infection (continued). and of the total 13 (22.4%) studies deals with women with bad obstetric history (BOH). These studies detected the presence of maternal anti-rubella IgG as a marker of past infection or immunization and mothers who did not possess these antibodies were susceptible to Rubella infection. Maternal IgM was detected in some studies as a marker of recent or current infection, which is associated with an increased risk of vertical transmission. The range of maternal susceptibility to Rubella was 2.1% to 43% in pregnant women [186,189] and 21.1% - 71.04% in women with BOH [91,190]. Higher susceptibility rates were reported [1,91,93,178,209,210] in Nigeria (84.8%), India (71%), Nepal (50%), Brazil (28.4%), Iran (25%), and Sri Lanka (24%). Rubella infection of a pregnant woman may have devastating effects on the developing fetus and once congenital infection occurred there is no availability of treatment for the foetus. Thus the mainstay of prevention is the universal immunization of all infants and identification and immunization of women at risk [156]. Rubella virus III) characterizing the epidemiology of maternal rubella were identified mostly for low and middle income countries [1,3,36,89-97,165-211] and 19 studies (Tabl. IV) for Arab countries [35,100,101,129-131,140,150,212-221]. Seven studies were with a retrospective (12.1%) study design Article Location, setting of study Type, duration of study Population Results Lin et al, [166] Taiwan, Hospital Cross-sectional, 7 yrs 10,089 pregnant women Seronegativity was 14% Tamer et al, [167] Turkey, Antenatal clinic Cross-sectional, 1972 pregnant women Anti-rubella IgG 96.1% Anti-rubella IgM 0.2% Ai & Ee, [168] Malaysia, Antenatal & hospital Cross-sectional 500 pregnant women Seronegativity 11.4% Majlessi et al, [169] Iran, PHC Cross-sectional, 2 yrs 965 Pregnant women Seronegativity 8.9% Das et al, [170] India, hospital Case control 1115 pregnant BOH Seropositivity 3.6% Ocak et al, [171] Turkey, Antenatal Retrospective, 23 months 1652 Pregnant women Anti-rubella IgG 95% Anti-rubella IgM 0.54% Pehlivan et al, [172] Turkey, Community based Cross-sectional, 7 months 824 Women Anti-rubella IgG 93.8% Anti-rubella IgM 0.6% Negative 5.6% Tseng et al, [173] Taiwan, Hospital Retrospective observational, 4 yrs 5007 pregnant women 13.4% susceptible Table III. Characteristics and results of studies reporting prevalence of maternal rubella infection. Rubella virus © Our Dermatol Online 4.2013 527 Article Location, setting of study Type, duration of study Population Results Bareto et al, [174] Mozambique, antenatal Cross-sectional, 3 months 974 pregnant women Anti-rubella IgG 95.3% Corcoran & Hardie, [175] South Africa, Antenatal clinic Cross-sectional 1200 serum sample 96.5% immune Desinor et al, [176] Haiti, hospital Cross-sectional, 4 months 495 pregnant women 95.2% seropositive Weerasekera et al,[177] Sri Lanka, antenatal Cross-sectional, 2 yrs 500 pregnant women 82% positive for rubella IgG Palihawadana et al, [178] Sri Lanka, antenatal Cross-sectional, 620 pregnant women 76% seropositive Ashrafunnessa Khatun, et al [179] Bangladesh, hospital Cross-sectional, 11 months 609 pregnant women 14.1% seronegative Dos Santos et al, [180] Brazil, antenatal Cross-sectional, 8 months 1024 pregnant women 77.4% seropositive Surpam et al [181] India, Antenatal clinic Case control, 150 BOH 4.66% IgM Uyar Y et al [182] Turkey, Hospital Cross sectional, 1 year 600 Pregnant women 94.3% IgG, 1.7% IgM Karabulut A et al [89] Turkey, Antenatal clinic Cross sectional, 1 year 1268 Pregnant women 95.1% IgG, 0% IgM Kumari N et al [1] Nepal, Hospital Case control, 4 months 12 BOH 50% Seropositive Nabi SN et al [90] Bangladesh, Hospital Cross sectional, 10 months 111 Pregnant women 81.08% IgG, 6.3% IgM Sadik MS et al [91] India, Hospital Case control, 2 years 86 BOH 29.06% IgG, 4.65% IgM Fomda BA [183] Kashmire, Hospital Case control, 892 Pregnant with BOH1028 Pregnant with previous normal pregnancy 26.12% IgM8.96% IgM Bamgboye AE et al [184] Nigeria, Antenatal clinic Cross sectional, 159 Pregnant women 68.5% IgG Linguissi LS et al [94] Burkina Faso, Cross sectional, 3 years Pregnant women 77% IgG Jubaida N, et al [185] Bangladesh, Outpatient clinic Cross sections, 6 months 134 Pregnant women 84.33% IgG, 0.75% IgM Amina MD et al [186] Nigeria, Antenatal clinic Cross sectional, 10 months 430 Pregnant women 97.9% IgG Chopra S et al [36] India, Antenatal clinic Case control, 1 year 200 BOH 17.5% IgM Ogbounnaya EC [187] Nigeria, Hospital Cross sectional, 1 year 190 Pregnant women 6.84% IgM Koksaldi-Motor et al [95] Turkey, Hospital Cross sectional, 1 year 1103 women childbearing age 93.6% IgG Langiano E et al [188] Italy, Hospital Cross sectional, 23 months 1242 Child bearing age 77.9% IgG Onakewhor & Chiwuzie [189] Nigeria, Hospital Cross sectional, 270 Pregnant women 57% IgG, 91.3% IgM Raveendran V et al [190] India, Hospital Case control, 1 year 182 BOH 78.9% IgG, 31.58% IgM Fokunang et al [191] Cameroon, Hospital Cross sectional, 4 months 211 Pregnant women 88.6% IgG Calimeri S et al [192] Italy, Hospital Cross sectional, 18 months 500 Pregnant women 85.8% IgG Corcoran & Hardie [193] South Africa, Hospital Cross sectional, 1 year 1200 Pregnant women 95.3% - 98 % IgG Mora- Garcia GJ et al [194] Colombia, Hospital Cross sectional, 1 year 1528 female 10-49 yrs 93% IgG Uysal A et al [195] Turkey, Hospital Cross sectional, 8 years 5959 Pregnant women 97.8% IgG, 0.37% IgM Combich JJ et al [196] Kenya, Hospital Cross sectional, 7 months 470 Pregnant women 92.9% IgG Kearns MJ et al [197] Canada, Provincial Public Health Laboratory Retrospective Observational study, 3.5 years 140 473 Pregnant women 91.2% IgG Jahromi AS et al [198] Iran, Hospital Case control, 8 months 220 Aborted women 91.2% IgG, 10.8% IgM Ramana BV et al [199] India, Hospital Case control, 150 BOH 12.67% IgM Cheong & Khoo [200] Malaysia, Hospital Cross sectional, 500 Pregnant women 11.4% susceptibility Honarvar B, et al [201] Iran, Hospital Cross sectional, 3 months 138 Pregnant women 96% IgG Nwanegbo et al [202] USA, Prenatal care clinic Retrospective Cross sectional, 1 year 642 Pregnant women 6.9% Non rubella immune Eslamian L [203] Iran, Hospital Cross sectional, 10 months 500 Pregnant women 76% IgG Ozdemir M et al [204] Turkey, Hospital Cross sectional, 6 months 249 Pregnant women 95.9% IgG, 0.4% IgM Adesina OA [205] Nigeria, Hospital Cross sectional, 230 Childbearing age women 93.5% IgG Frischknecht F et al [92] Switzerland, Hospital Cross sectional, 1 yr 723 Pregnant women in labor 93.08% seropositive Ang LW et al [206] Singapore, Retrospective Epidemiological data 1991- 2007 84.2% Immune to rubella Upreti SR et al [207] Nepal, Retrospective 2004-2009 2224 Childbearing age 90.8% IgG Table III. Rubella virus Characteristics and results of studies reporting prevalence of maternal rubella infection (continued). p g p ( ) Article Location, setting of study Type, duration of study Population Results Abdulmohaymen N [99] Iraq (Baghdad), Hospital Case control, 9 months 119 Aborted women 4.8% IgM recurrent spontaneous abortion 2.9% IgM non recurrent spontaneous abortion. Rubella virus 6.5% IgG recurrent spontaneous abortion 20.6% IgG non recurrent spontaneous abortion Jasim et al [100] Iraq, Waset, Hospital Case control, 1 year 162 Women with spontaneous abortion 54.3% IgG, 62.3% IgM Al- Taie et al [101] Iraq, Mosul, Private laboratory Case control, 1 year 100 BOH 16% IgM Hadi NJ [130] Iraq, Thi Qar, Hospital Case control 190 Aborted women 1.05% IgG, 4.21% IgM Salman YG [131] Iraq, Kirkuk, Hospital Case control, 11 months 75 BOH 8.88% Seropositive, 6.75% IgM Abdul-kareem ET, et al [212] Iraq, Baghdad, Hospital Case control, 8 months 79 Aborted women 34.2% seropositive Al-rubaii B, et al [214] Iraq, Babylon, Hospital Cross sectional , 14 months 250 Childbearing age women 78.33% Pregnant, 75.71% non- pregnant Hasan ARS, et al [215] Iraq, Diyala, PHC Case control 46 Pregnant - BOH, 52 Pregnant - Non BOH 47 Non pregnant Without Abortion 39 Non pregnant with Abortion IgG- 76% IgG- 96.1 IgG – 85.1% IgG- 100% Hammod AM, et al [213] Iraq, Babylon, Hospital Case control, 20.5 m0nths 46 Pregnant - BOH, 52 Pregnant - Non BOH 47 Non pregnant Without Abortion 39 Non pregnant with Abortion IgG- 76% IgG- 96.1 IgG – 85.1% IgG- 100% Hamdan HZ, et al [216] Sudan, Hospital Cross sectional, 2 months 231 Pregnant women 65.3% IgG, 3.4% IgM Ghazi HO, et al [217] Saudi Arabia, Hospital Cross sectional 926 Pregnant women 93.3% IgG Al-Marzoqi AHM, et al [129] Iraq, Babylon, Hospital Cross sectional, 6 months 180 Pregnant women 73.9% IgG, 53.9% IgM Gashout A, et al [140] Libya, Hospital Case control, 5 years 692 Aborted women 89% IgG, 4.3% IgM Abu- Madi MA, et al [35] Qatar, Hospital Cross sectional, 3 years 847 Women > 20 yr age 74.9% IgG Barah & Chehada [219] Syria, University Laboratory Cross sectional, 3 months 90 university female students 85.6% IgG Caidi H, et al [220] Morocco, Hospital Cross sectional, 1 year 967 childbearing age 16.6% IgG Article Location, setting of study Type, duration of study Population Results Odland JO, et al [208] Russia, Hospital Case control, 4 months 182 Pregnant & 127 Aborted women 77.5% versus 59.8% seroprevalence Goncalves MA, et al, [97] Brazil, Hospital Retrospective, 2 years 574 Pregnant women 93.1% IgG, 0.6% IgM Turbadkar D, et al [3] India, Antenatal clinic Case control, 1 year 380 BOH 61.3% IgG, 26.8% IgM Inagaki ADM, et al [93] Brazil, Antenatal clinic Cross sectional, 1 year 9559 Pregnant women 71.6% IgG, 0.1% IgM Agbede OO, et al [209] Nigeria, Antenatal clinic Cross sectional, 3 months 92 Pregnant women 15.2% IgG, 3.3% IgM Ebadi p, et al [210] Iran, Hospital Case control, 3 years 120 Aborted women 75% seropositive Malarvizhi et al [211] India, Private hospital Cross sectional, 2 years 232 Pregnant women 50.9% IgG, 3.4% IgM Vilibik-Cavlek T, et al [96] Croatia, Hospital Cross sectional, 5 years Pregnant & non pregnant women 94.6% IgG, 0% IgM Ballal M et al [165] India, Hospital Case control, 334 BOH 4.49% IgM Table III. Rubella virus Characteristics and results of studies reporting prevalence of maternal rubella infection (continued). 528 © Our Dermatol Online 4.2013 Article Location, setting of study Type, duration of study Population Results Odland JO, et al [208] Russia, Hospital Case control, 4 months 182 Pregnant & 127 Aborted women 77.5% versus 59.8% seroprevalence Goncalves MA, et al, [97] Brazil, Hospital Retrospective, 2 years 574 Pregnant women 93.1% IgG, 0.6% IgM Turbadkar D, et al [3] India, Antenatal clinic Case control, 1 year 380 BOH 61.3% IgG, 26.8% IgM Inagaki ADM, et al [93] Brazil, Antenatal clinic Cross sectional, 1 year 9559 Pregnant women 71.6% IgG, 0.1% IgM Agbede OO, et al [209] Nigeria, Antenatal clinic Cross sectional, 3 months 92 Pregnant women 15.2% IgG, 3.3% IgM Ebadi p, et al [210] Iran, Hospital Case control, 3 years 120 Aborted women 75% seropositive Malarvizhi et al [211] India, Private hospital Cross sectional, 2 years 232 Pregnant women 50.9% IgG, 3.4% IgM Vilibik-Cavlek T, et al [96] Croatia, Hospital Cross sectional, 5 years Pregnant & non pregnant women 94.6% IgG, 0% IgM Ballal M et al [165] India, Hospital Case control, 334 BOH 4.49% IgM Table III. 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Rubella virus 6.5% IgG recurrent spontaneous abortion 20.6% IgG non recurrent spontaneous abortion Jasim et al [100] Iraq, Waset, Hospital Case control, 1 year 162 Women with spontaneous abortion 54.3% IgG, 62.3% IgM Al- Taie et al [101] Iraq, Mosul, Private laboratory Case control, 1 year 100 BOH 16% IgM Hadi NJ [130] Iraq, Thi Qar, Hospital Case control 190 Aborted women 1.05% IgG, 4.21% IgM Salman YG [131] Iraq, Kirkuk, Hospital Case control, 11 months 75 BOH 8.88% Seropositive, 6.75% IgM Abdul-kareem ET, et al [212] Iraq, Baghdad, Hospital Case control, 8 months 79 Aborted women 34.2% seropositive Al-rubaii B, et al [214] Iraq, Babylon, Hospital Cross sectional , 14 months 250 Childbearing age women 78.33% Pregnant, 75.71% non- pregnant Hasan ARS, et al [215] Iraq, Diyala, PHC Case control 46 Pregnant - BOH, 52 Pregnant - Non BOH 47 Non pregnant Without Abortion 39 Non pregnant with Abortion IgG- 76% IgG- 96.1 IgG – 85.1% IgG- 100% Hammod AM, et al [213] Iraq, Babylon, Hospital Case control, 20.5 m0nths 46 Pregnant - BOH, 52 Pregnant - Non BOH 47 Non pregnant Without Abortion 39 Non pregnant with Abortion IgG- 76% IgG- 96.1 IgG – 85.1% IgG- 100% Hamdan HZ, et al [216] Sudan, Hospital Cross sectional, 2 months 231 Pregnant women 65.3% IgG, 3.4% IgM Ghazi HO, et al [217] Saudi Arabia, Hospital Cross sectional 926 Pregnant women 93.3% IgG Al-Marzoqi AHM, et al [129] Iraq, Babylon, Hospital Cross sectional, 6 months 180 Pregnant women 73.9% IgG, 53.9% IgM Gashout A, et al [140] Libya, Hospital Case control, 5 years 692 Aborted women 89% IgG, 4.3% IgM Abu- Madi MA, et al [35] Qatar, Hospital Cross sectional, 3 years 847 Women > 20 yr age 74.9% IgG Barah & Chehada [219] Syria, University Laboratory Cross sectional, 3 months 90 university female students 85.6% IgG Caidi H, et al [220] Morocco, Hospital Cross sectional, 1 year 967 childbearing age women 15-39 yrs 16.6% IgG Hannachi N, et al [221] Tunisia, Hospital Cross sectional, 404 Pregnant women 79.7% seroprevalence Al- Hindi A, et al [150] Palestine, IVF centre Retrospective, 6 years 1954 Women with infertility or abortion 7% IgM Nama J et al [218] Iraq, Najaf, Hospital Case control, 10 months 300 Aborted women 77% IgG, 4.66% IgM Table IV. 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https://openalex.org/W3113043049	https://ukralmanac.univ.kiev.ua/index.php/ua/article/download/371/356	Ukrainian	null	THE PROBLEM OF FINITENESS OF HUMAN EXISTENCE IN UKRAINIAN FOLKLORE	Ukraïnoznavčij alʹmanah	2,020	cc-by-sa	3,199	Володимир Ятченко доктор філософських наук, професор, професор кафедри економіки підприємств та соціальних технологій, Державний університет телекомунікацій, м. Київ Володимир Ятченко доктор філософських наук, професор, професор кафедри економіки підприємств та соціальних технологій, Державний університет телекомунікацій, м. Київ ORCID: 0000-0003-0533-8580 Email: yatchych@ukr.net ORCID: 0000-0003-0533-8580 Email: yatchych@ukr.net Оксана Олійник кандидат соціологічних наук, доцент, доцент кафедри методологій та методів соціологічних досліджень Київський національний університет імені Тараса Шевченка, м. Київ Оксана Олійник кандидат соціологічних наук, доцент, доцент кафедри методологій та методів соціологічних досліджень Київський національний університет імені Тараса Шевченка, м. Київ ORCID: 0000-0003-0568-7940 Email o_oliinyk@ukr.net ORCID: 0000-0003-0568-7940 Email o_oliinyk@ukr.net Українознавчий альманах. Випуск 26 victim behavior]. Nizhyn: Vydavets P.P. Lysenko. 29 р. (in Ukranian). 13. Цюрупа М. Военные теоретики Европы о войне, мире и национальной безопасности. Киев: КГВИ, 1996. 250 с. 8. Skyba, V., Horbatenko, V., Turenko, V. (1996). Vstup do politolohii. Ekskurs v istoriiu pravnycho- politychnoi dumky [Introduction into political science. Excursus on the history of the legal and political thinking]. K.: Osnovy. 538 р. (in Ukranian). References 1. Baikov, L. (1918). Pidhotovka narodu ta viiska do zakhystu Batkivshchyny [Preparing the people and the army for the motherland defense]. Kyiv. 1-12 p. ( in Ukranian). 9. Skoropadskyi, P. (2016). 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Випуск 26 DOI: https:// doi.org/10.17721/2520-2626/2020.26.15 УДК 616.001: 101: 316 DOI: https:// doi.org/10.17721/2520-2626/2020.26.15 УДК 616.001: 101: 316 ПРОБЛЕМА СКІНЧЕННОСТІ ЛЮДСЬКОГО ІСНУВАННЯ В УКРАЇНСЬКОМУ ФОЛЬКЛОРІ Анотація. В статті піддаються аналізові екзистенційні проблеми життя, смерті й безсмертя в українському фольклорі (на матеріалі українських казок). В корпусі українських народних казок широко побутують теми, які в європейській філософії й літературі отримали назву «трагічні основи людського буття», – усвідомлення неуникності смерті в земному бутті людини, пошук форм здобуття індивідуального безсмертя. В українських казках спостерігається двоїсте ставлення особистості до неминучості власної смерті. З одного боку тут наявний мотив замирення з фатумом людської долі, а задля пом’якшення болючого відчуття власної скінченності педалюється настанова про вищий сенс існування смерті. Смерть виправдовується, бо вона постає як запобігання безглуздості нескінченного 100 Київський національний університет імені Тараса Шевченка людського існування або як перепона розгнузданості примх і небезпечних забаганок індивіда, або ж врешті- решт як покарання людей за здійснені порушення заповідей Вищої духовної істоти. Іншими словами – смерть постає в низці казок як втілення вищої світової справедливості. При цьому смерть здебільшого постає в казках як опредмечене язичницьке уявлення про Смерть як конкретну живу істоту зі своїми примхами, симпатіями й слабкостями. Проблема ж пошуків шляхів досягнення безсмертя прослідковується в українських казках теж двояким чином. Найчастіше цей пошук розгортається в площині перемоги героя казки над смертю або через ув’язнення смерті, або через одруження героя з божественною істотою. Це дуже поширений мотив у казках народів світу. Рідше зустрічається мотив досягнення безсмертя через моральне самовдосконалення героя, дотримання ним моральних заповідей Бога. Це вже відображення в казках впливу християнства на духовний світ предків сучасних українців. Ключові слова: український фольклор, українська казка, екзистенційні виміри українського життя, смерть і безсмертя в українській казці. Volodymyr Yatchenko Doctor of Sciences in Philosophy, Professor, Departament of Enterprise Economics and Social Technologies, State University of Telekommunications of Kyiv Oksana Oliinyk PHD. in Sociology, Associate professor, Departament of Metodology and Methods of Sociological Researches Taras Shevchenko National University of Kyiv THE PROBLEM OF FINITENESS OF HUMAN EXISTENCE IN UKRAINIAN FOLKLORE Abstract. The article analyzes the existential problems of life, death and immortality in Ukrainian folklore (based on Ukrainian fairy tales). In the corpus of Ukrainian folk tales there are widely used topics, which in European philosophy and literature are called "tragic foundations of human existence" - awareness of the inevitability of death in the earthly existence of man, the search for forms of individual immortality. In Ukrainian fairy tales there is a dual attitude of the individual to the inevitability of his own death. On the one hand, there is the motive of reconciliation with the fate of human destiny, and in order to relieve the painful feeling of one's own finitude, the instruction on the higher meaning of the existence of death is forced. Death is justified because it appears as the prevention of the absurdity of infinite human existence or as an obstacle to the debauchery of the whims and dangerous wishes of the individual, or ultimately as the punishment of people for violating the commandments of the Supreme Spiritual Creature. In other words, death appears in a number of fairy tales as the expression of the highest world justice. At the same time, death mostly appears in fairy tales as an objectified pagan idea of Death as a concrete living creature with its whims, sympathies and weaknesses. The problem of finding ways to achieve immortality is traced in Ukrainian fairy tales in two ways. Most often, this search unfolds in the plane of the victory of the hero of the fairy tale over death, or through the imprisonment of death, or through the marriage of the hero to a divine being. This is a very common motive in the tales around the world. Less common is the motive of achieving immortality through the moral self-improvement of the hero, his compliance to the moral commandments of God. This is already a reflection in fairy tales of the influence of Christianity on the spiritual world of the ancestors of modern Ukrainians. Keywords: Ukrainian folklore, Ukrainian fairy tales, existential dimension of Ukrainian folklore, life, death and immortality in Ukrainian fairy tales. p krainian folklore, Ukrainian fairy tales, existential dimension of Ukrainian folklore, life, death and krainian fairy tales. Серед 1907-1986) [5;], старозаповітних текстів, міфів культур різних континентів Дж. Кемпбеллом (Campbell, 1904- 1987) [див. 6], міфів і звичаїв південноафриканських племен Дж. Фрезером (Frazer, 1854-1941) [див. 12] і багатьох інших недвозначно засвідчили умоглядність європоцентричного сприйняття не лише духовності раніше зневажуваних «неісторичних» народів, але й обмеженість традиційних, усталених поглядів на духовний світ людини докласових, дорелігійних культур загалом. Перегляд цих застарілих уявлень потребував, окрім збагачення новим фактологічним матеріалом, ще й нових узагальнень в європейській філософії, фольклористиці в царині екзистенційного досвіду, екзистенційних переживань людини. Українознавчий альманах. Випуск 26 нескінченно триваючого) життя (а придатніше в цьому випадкові – існування) людини. Добре ілюструють цю настановленість казки «Бідний чоловік і Смерть» або «Чоловік і Смерть», записані відповідно В. Лесевичем на Полтавщині й М. Зінчуком на Буковині. Сюжет першої казки вістує про чоловіка, який зумів так улестити Смерть, що вона дарує йому пів сотні літ життя, а далі й зовсім відмовляється від нього: «Як узяв жить, і сини вже померли, і внуки оженились, а він став уже негодящий нікуди, і ніхто його не вважає ні за віщо, він, як головешка качається, ніхто його не доглядає, усі від нього одцурались і смерть від нього відцуралася». За сюжетом другої казки на знак помсти за своє ув’язнення Смерть карає чоловіка безсмертям «… не вмреш ти, а будеш гнити, поки не зогниєш», і погроза ця у фіналі казки здійснюється. Екзистенційний аспект таких сюжетів полягає в тім, що життя, яке проживається без вищого сенсу, є безблагодатним і не заслуговує на безсмертя [14, с.53]. Аналіз досліджень. Проблеми скінченності людського існування досліджувались в літературі в багатьох контекстах. Гроно дослідників ще з ХІХ ст. обирали об’єктом розгляду культурні контексти явища смерті в філософських та поетичних творах – Ф. Ар’єc [див.1]; в етичних вимірах співвідношення добра і зла – Р. Каюа [див.7]; в міфах первісних народів різних континентів – Ю. Берьозкін [див.2]. Українські дослідники в основному зосереджуються на розгляді питань скінченності людського буття у весільних чи поховальних обрядах, через систему символів: в космогонічному вимірі – В. Телеуця [див.10], як символу зародження нового життя, переходу у вічність, обрядових дійств [див.9] . Однак поза межами аналізу текстів українського фольклору (особливо казок і демонологічних оповідок) залишається потужний пласт наявного в них загальнолюдського екзистенційного досвіду, латентних спроб розв’язати «вічні» проблеми смерті й безсмертя в індивідуальному бутті. Досить поширеними в корпусі українських казок є сюжети про усвідомлення непозбутності смерті як перепони розгнузданості примх і норовів людської істоти і в цьому плані як вияву вищої справедливості. Так в казці «Не вмирають тільки грішні люди» (записана В. Кравченком) святі Петро й Павло запитують в Бога, чому в одній з багатодітних сімей вже протягом трьох поколінь жодна людина не померла. І тут з уст Бога звучить твердження, що це все грішні люди, тому вони всі живі. Тобто вони не сподобились зустрітись з Богом після своєї смерті. Ця ж моральна настанова, але вже в іншій проєкції розгортається в бойківській казці «Пригоди діда- гуцула». Герой казки (Солдат) ув’язнює Смерть в торбі. Українознавчий альманах. Випуск 26 Як наслідок «Люди Бога не бояться, бо ніхто не вмирає, Люди бавляться, п’ють, ріжуться, вмирати ніхто не вмирає, бо нема смерті». Нарешті Бог через янгола примушує героя випустити Смерть на волю. Отже причина існування й необхідності смерті в цій казці закорінена в самій природі людини. Мета написання статті. В статті здійснено спробу проаналізувати на матеріалах українського фольклору ставлення людини до феноменів смерті й безсмертя з погляду наявності в них екзистенційних проривів і виходу на метафізичні аспекти буття. Виклад основного матеріалу. Європейська філософська й теологічна думка протягом століть розробила дві базові позиції ставлення людини до факту неуникності смерті в індивідуальному бутті людини як спроби подолання природного страху перед необхідністю уривання всього життєвого шляху й життєвих виявів. Суть першої полягає в доведенні доцільності й вищого сенсу смерті людини в космічному вимірі буття. Тому прижиттєве надзавдання індивіда полягає в формуванні почуття замиреності з фактом скінченності свого земного перебування й підготовці до іншої – вже вічної – форми існування в потойбічному світі. Друга позиція відбиває непотайно негативне ставлення до смерті як до несправедливого, нічим не виправданого явища й прагне знайти шляхи перебороти смерть та здійснити прорив людини на поле безсмертя. Світоглядну настановленість прихильників цієї позиції можна окреслити так: Серед українських казок зустрічаються надзвичайно цікаві тексти з погляду витлумачення смерті як детермінатора соціальної активності, самореалізації й самоствердження людини. Ось в гуцульській казці «Музикант і Смерть» до Музиканта приходить Смерть й дорікає йому за пустопорожньо пройдений життєвий шлях: «Яку ти славу по собі залишиш? Дітей не маєш, господарства не маєш, грати нікого не навчив. Ніхто за тобою не заплаче!» [11; ХІІІ; с.371]. Тут бачимо виображення не тільки суто індивідуальної драми конкретного індивіда, але – що важливіше – осмислення трансцендентальних вимірів життя й безсмертя, демаскування безплідності голого заперечення розуміння смерті як безглуздого явища. а) первинний і «істинний» стан людини – безсмертя; б) смертність – вияв спотворення справжнього ходу речей; в) смертність – трагічний збій у стосунках людини з вищими силами [14; с.14]. Український фольклор багатий на приклади виображення обох цих позицій, на утримання в них потенцій життєстверджуючих сил та екзистенційних очікувань людини. Не можна, нарешті, оминути ще один екзистенційного характеру аспект ставлення людини до феномена смерті, а саме примирення людини зі своєю долею – долею бути смертною істотою. Але це суперечить самій природі людини. Постановка проблеми. Постановка проблеми. Серед набору мисленевих штампів, які побутують і серед широкого загалу, і навіть в середовищі інтелектуалів либонь ще від часів Просвітництва, неабиякої популярності набуло переконання, ніби розмисли й розгорнуті рефлексії щодо скінченності людського існування (щодо життя й смерті, щодо безсмертя й причин існування смерті й т. под.) були зовсім не притаманними людям архаїчних культур. Між тим аналіз первісних міфів етнографами, релігієзнавцями, психоантропологами на матеріалах давньогрецьких, скандинавських, близькосхідних міфів [див. 3], польові дослідження серед меланезійських племен Б. Маліновського (Malinowski, 1984-1942) [див.8], племен Північної Австралії М. Еліаде (Eliade, 101 Українознавчий альманах. Випуск 26 References 1.Aries R. (1989). Chelovek pered licom smerti [A Man in the Face of Death]. M., 438 p.(in Russian). 1.Aries R. (1989). Chelovek pered licom smerti [A Man in the Face of Death]. M., 438 p.(in Russian). Давніша й примітивніша форма здобуття безсмертя в українських казках (як і в казках багатьох інших народів) – нейтралізація Смерті, її ув’язнення за допомогою хитрощів та винахідливості героя. Казок з таким сюжетом дуже багато. Вони доносять до нас первісні уявлення далеких предків сучасних українців про добрих і злих духів, добрих і злих божеств і богів, про співпрацю людей і богів у створенні й упорядкуванні світу й організації суспільного життя та об’єднання в боротьбі зі злими вищими силами. 2.Bereskin Yu. (2007). Proishozhdenie smerti – drevnejshiy mif [The Origin of Death is an Ancient Myth]. Еtnograficheskoje Obozrienie. № 1. P. 70-89 (in Russian). 2.Bereskin Yu. (2007). Proishozhdenie smerti – drevnejshiy mif [The Origin of Death is an Ancient Myth]. Еtnograficheskoje Obozrienie. № 1. P. 70-89 (in Russian). 2.Bereskin Yu. (2007). Proishozhdenie smerti – drevnejshiy mif [The Origin of Death is an Ancient Myth]. Еtnograficheskoje Obozrienie. № 1. P. 70-89 (in Russian). 3.James E. (2006). Tajny jazycheskih bogov [Secrets of the Pagan Gods]. M., 329 p. 3.James E. (2006). Tajny jazycheskih bogov [Secrets of the Pagan Gods]. M., 329 p. 4.Dmytrenko M., Ivannikova L., Lozko H., Muzychenko Ja., Shalak O. (1994). Ukrayinski symvoly [Ukrainian Symbols]. K., 140 (in Ukrainian). З іншого боку в українських казках присутній і інший шлях досягнення особистого безсмертя. Це, наприклад, язичницький мотив одруження героя з божественною істотою або прилучення до сонму божественних істот через вірність тотемам свого роду. Але наявний в казках й інший мотив – зміна, трансформація людської природи через самовдосконалення. Це вже вплив християнського світогляду. 5.Eliade M. (2002). Simvolika smerti v obriadie posviashchieniia. Tajnyie obshchestva. Obriady iniciacii i posviashchieniia [Symbolism of Death in the Initiation Rite // Eliade M. Secret societies. Initiation and Initiation Rites]. M. P.46-49 (in Russian). Rite // Eliade M. 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Найпоширеніша така «прокладка» – турбота про батьків, належне виховання дітей, передача їм у спадок нажитого добра. Це проглядається в казках усіх регіонів і всіх етнічних груп українців і через такого роду сюжети набувається й культивується вищий метафізичний сенс життєвої програми та усвідомлення сенсу життя особистості [див.14; с.52]. 4. Дмитренко М., Іваннікова Л., Лозко Г., Музиченко Я., Шалак О. Українські символи. К., 1994. 140 с. 5. Элиаде М. Символика смерти в обряде посвящения. Тайные общества. Обряды инициации и посвящения. М., 2002. С. 46-49. 6. Каюа Р. Людина та сакральне. К., 2003. 238 с. 7. Кэмпбелл Дж. Мифический образ. М., 2004. 686 с. 8. Малиновский Б. Магия, наука и релігія. Магический кристалл. М., 1992. С. 17-59. 9. Словник символів культури України. К., 2002. 257 с. 10. Телеуця В. Символи космогонічного ряду в художній системі фольклору Подунав’я . Література. Фольклор, Проблеми поетики: Зб. наук. праць. Вип. 28. Ч. 1. К., 2007. С. 286-299. 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https://openalex.org/W2027820788	https://escholarship.org/content/qt8d95r5b4/qt8d95r5b4.pdf?t=qaek0l	English	null	Increased Peripheral Nerve Excitability and Local Nav1.8 RNA Up-Regulation in Painful Neuropathy	Molecular pain	2,009	cc-by	10,682	UCLA UCLA Previously Published Works Title Increased Peripheral Nerve Excitability and Local Nav1.8 RNA Up-Regulation in Painful Neuropathy Permalink https://escholarship.org/uc/item/8d95r5b4 Authors Thakor, Devang Kashyap Lin, Audrey Matsuka, Yoshizo et al. Publication Date 2009 DOI 10.1186/1744-8069-5-14 Peer reviewed UCLA UCLA Previously Published Works Title Increased Peripheral Nerve Excitability and Local Nav1.8 RNA Up-Regulation in Painful Neuropathy Permalink https://escholarship.org/uc/item/8d95r5b4 Authors Thakor, Devang Kashyap Lin, Audrey Matsuka, Yoshizo et al. Publication Date 2009 DOI 10.1186/1744-8069-5-14 Peer reviewed UCLA UCLA Previously Published Works Title Increased Peripheral Nerve Excitability and Local Nav1.8 RNA Up-Regulation in Painful Neuropathy Permalink https://escholarship.org/uc/item/8d95r5b4 Authors Thakor, Devang Kashyap Lin, Audrey Matsuka, Yoshizo et al. Publication Date 2009 DOI 10.1186/1744-8069-5-14 Peer reviewed UCLA UCLA Previously Published W Title Increased Peripheral Nerve Excitability a Neuropathy Permalink https://escholarship.org/uc/item/8d95r5b Authors Thakor, Devang Kashyap Lin, Audrey Matsuka, Yoshizo et al. Publication Date 2009 DOI 10.1186/1744-8069-5-14 Peer reviewed Powered by the California Digital Library University of California eScholarship.org BioMed Central BioMed Central Increased peripheral nerve excitability and local NaV1.8 mRNA up-regulation in painful neuropathy Address: 1Division of Oral Biology and Medicine, School of Dentistry, University of California, Los Angeles, 10833 Le Conte Avenue Los Angeles, CA 90095-1668, USA, 2Jane and Jerry Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, Biomaterials, and Hospital Dentistry, School of Dentistry, University of California, Los Angeles, 10833 Le Conte Avenue, Los Angeles, CA 90095-1668, USA, 3Neuroengineering Training Program, Biomedical Engineering Interdepartmental Program, Henry Samueli School of Engineering and Applied Science, University of California, Los Angeles, CA 90095, USA and 4Departments of Neurosurgery and Physical Medicine & Rehabilitation, Harvard Medical School, Boston, MA, USA Email: Devang Kashyap Thakor - dthakor@ucla.edu; Audrey Lin - audlin23@yahoo.com; Yoshizo Matsuka - matsuka@md.okayama-u.ac.jp; Edward M Meyer - hybradoma@hotmail.com; Supanigar Ruangsri - tulip2772@yahoo.com; Ichiro Nishimura - inishimura@dentistry.ucla.edu; Igor Spigelman* - igor@ucla.edu * Corresponding author Published: 25 March 2009 Molecular Pain 2009, 5:14 doi:10.1186/1744-8069-5-14 Received: 26 November 2008 Accepted: 25 March 2009 This article is available from: http://www.molecularpain.com/content/5/1/14 © 2009 Thakor et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 26 November 2008 Accepted: 25 March 2009 This article is available from: http://www.molecularpain.com/content/5/1/14 © 2009 Thakor et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Molecular Pain Open Access Background g Neuropathic pain is a chronic disorder defined as pain ini- tiated or caused by a primary lesion or dysfunction in the nervous system [1]; it affects 1.4% of the U.S. population and remains extremely difficult to treat, due to poorly understood etiology and a lack of well-defined molecular targets [2]. Several peripheral nerve injury-based rat mod- els have been developed that exhibit various behavioral characteristics representative of neuropathic symptoma- tology, including hindpaw hypersensitivity to mechanical and thermal stimuli [3]. Aberrant hyperexcitability and ectopic burst discharge of primary sensory neurons in these models is widely considered to be the major contrib- utor to neuropathic pain symptomatology. Functional studies have implicated ion channels, including sodium channels, in this pathology. In particular, loss-of-function studies using antisense oligodeoxynucleotides and siRNA have highlighted the importance of the NaV1.8 tetrodo- toxin resistant (TTX-r) sodium channel in the neurophys- iological and behavioral effects observed in spinal nerve ligation (SNL) and chronic constriction injury (CCI) models of peripheral neuropathy [4-7]. Among TTX-r sodium channels, Nav1.8 has been shown to contribute substantially to action potential electrogenesis in dissoci- ated DRG neurons [8,9] and is expressed in both C- and A-fiber populations [10,11]. NaV1.8 has a higher inactiva- tion threshold, slower inactivation kinetics, and faster recovery from inactivation than TTX-sensitive Na+ chan- nels [12,13], suggesting that its local upregulation could facilitate signaling during neuropathic pain. Additionally, it has been found that NaV1.8-null transgenic mice do not exhibit abnormal neurophysiology in injured neuroma tissue [14]. To address the mechanisms of NaV1.8 translocation and its potential relationship to hyperexcitability, we studied sciatic nerve excitability and NaV1.8 expression in a rat model of painful neuropathy induced by sciatic nerve entrapment (SNE). SNE is a variation on CCI in which the loose ligatures placed around the sciatic nerve are replaced by chemically inert fixed-diameter polyethylene cuffs, resulting in decreased variability across animals [22]. We found increased sciatic nerve axonal excitability and TTX- resistance proximal to the injury. This was concommitant with increased NaV1.8 immunoreactivity in proximal axons and decreased immunoreactivity in the cell bodies of DRG neurons on the side of the injury, analogous to previous findings after CCI [11]. Importantly, we observed selective and large increases in sciatic nerve NaV1.8 mRNA in the absence of changes in NaV1.8 mRNA within DRG or changes in NaV1.8 polyadenyla- tion. Results Sciatic nerve excitability and TTX-resistance after SNE Sciatic nerve excitability and TTX-resistance after SNE The CAP response amplitudes from contralateral, naïve control nerves, and ipsilateral sham nerves were similar and therefore grouped together. 2 weeks after SNE, the amplitudes of both A- and C-fiber CAPs were significantly increased in ipsilateral sciatic nerves when compared to control, sham or contralateral nerves (Fig. 1). The increases were observed in the plateau responses to maxi- mal stimulation (Fig. 1A, B) Although leftward shifts in the stimulus-response relationsips were also detected for the A-fiber and to a lesser extent the C-fiber CAP after nor- malizing responses to maximal amplitudes, these shifts were not statistically significant (Fig. 1A and 1B insets). The conduction velocity (CV) of the peak A-fiber CAP was not different between nerves ipsilateral to SNE (32.4 ± 1.5 m/s) and control, sham or contralateral nerves (29.9 ± 1.7 m/s). The CV of the ipsilateral C-fiber CAP 1.2 ± 0.1 m/s) was also similar to that of control, sham and contralateral nerves (1.1 ± 0.1 m/s). These and other studies have suggested that peripheral NaV1.8 increases in sensory neuron axons contribute to peripheral hypersensitivity. For example, in human patients with various neuropathies such as causalgia and brachial plexus injury, NaV1.8 has been shown to accu- mulate in peripheral axons near the injury site [15-17] and in a causalgic finger [18]. Increases in NaV1.8-like immunoreactivity in axons of the rat sciatic nerve were demonstrated after CCI [11]. Also, after SNL injury sciatic nerve NaV1.8-like immunoreactivity increased selectively in 'uninjured' axons; this was accompanied by increased TTX-resistance of the C-fiber compound action potential, suggesting a functional peripheral upregulation of NaV1.8 [5]. However, discrepancies between these findings and the observed reduction of NaV1.8 protein and mRNA expression in the cell bodies of injured sensory neurons [19], as well as the normal development of neuropathic pain behavior in NaV1.8–null mice [20,21] continue to raise the question of what role, if any, is played by NaV1.8 in the development of neuropathic pain. Also, the mech- Concentration-dependent decreases in CAP amplitude were detected within 30 sec after TTX (0.1–1 μM) was applied in the recording chamber (p < 0.05, two-way ANOVA with Tukey post-hoc) and measurements were obtained after 20–25 min to ensure a full drug response. The C-and A-fiber CAPs in both injured and uninjured nerves were blocked completely in the presence of 1 μM TTX (not shown). Abstract Background: Neuropathic pain caused by peripheral nerve injury is a chronic disorder that represents a significant clinical challenge because the pathological mechanisms have not been fully elucidated. Several studies have suggested the involvement of various sodium channels, including tetrodotoxin-resistant NaV1.8, in affected dorsal root ganglion (DRG) neurons. We have hypothesized that altered local expression of NaV1.8 in the peripheral axons of DRG neurons could facilitate nociceptive signal generation and propagation after neuropathic injury. Results: After unilateral sciatic nerve entrapment injury in rats, compound action potential amplitudes were increased in both myelinated and unmyelinated fibers of the ipsilateral sciatic nerve. Tetrodotoxin resistance of both fiber populations and sciatic nerve NaV1.8 immunoreactivity were also increased. Further analysis of NaV1.8 distribution revealed that immunoreactivity and mRNA levels were decreased and unaffected, respectively, in the ipsilateral L4 and L5 DRG; however sciatic nerve NaV1.8 mRNA showed nearly an 11-fold ipsilateral increase. Nav1.8 mRNA observed in the sciatic nerve was likely of axonal origin since it was not detected in non-neuronal cells cultured from nerve tissue. Absence of changes in NaV1.8 mRNA polyadenylation suggests that increased mRNA stability was not responsible for the selective peripheral mRNA increase. Furthermore, mRNA levels of NaV1.3, NaV1.5, NaV1.6, NaV1.7, and NaV1.9 were not significantly different between ipsilateral and contralateral nerves. We therefore propose that selective NaV1.8 mRNA axonal transport and local up-regulation could contribute to the hyperexcitability of peripheral nerves in some neuropathic pain states. Conclusion: Cuff entrapment injury resulted in significantly elevated axonal excitability and increased NaV1.8 immunoreactivity in rat sciatic nerves. The concomitant axonal accumulation of NaV1.8 mRNA may play a role in the pathogenesis of this model of neuropathic pain. Page 1 of 12 (page number not for citation purposes) Page 1 of 12 (page number not for citation purposes) Molecular Pain 2009, 5:14 http://www.molecularpain.com/content/5/1/14 anism by which peripheral translocation of NaV1.8 occurs in the CCI model is unclear [11]. Background Our data collectively introduce the possibility that NaV1.8 mRNA could be peripherally transported from the neuronal cell bodies to the sciatic nerve and locally trans- lated, thereby contributing to axonal hyperexcitability and neuropathy symptoms. Page 2 of 12 (page number not for citation purposes) Detection of NaV1.8 mRNA in sciatic nerves Inset (bottom right) shows normalized data with error bars removed for clarity. Stimulus durations were 0.1 ms and 0.5 ms for A- and C-fiber CAP, respectively throughout. Tissue was harvested 2 weeks after SNE or sham surgery. Increased excitability of ipsilateral sciatic nerve after SNE Figure 1 Increased excitability of ipsilateral sciatic nerve after SNE. A: Stimulus-output plot of A-fiber CAP peak for sciatic nerve ipsilateral to SNE (n = 8) compared to preparations from contralateral, naïve or sham control rats (n = 8). Inset (left) shows examples of ipsilateral and contralateral A-fiber CAP with peaks aligned for clarity (0.1 mA stimulus). , p < 0.05 (two-way ANOVA, Tukey post-hoc). Inset (bottom right) shows normalized data with error bars removed for clarity. Note the leftward shift of data from ipsilateral nerves. B: Stimulus-output plot of C-fiber CAP peak for sciatic nerve ipsilateral to SNE compared to preparations from contralat- eral, naïve or control rats. Inset (left) shows examples of ipsi- lateral and contralateral C-fiber CAP (10 mA stimulus). Inset (bottom right) shows normalized data with error bars removed for clarity. Stimulus durations were 0.1 ms and 0.5 ms for A- and C-fiber CAP, respectively throughout. Tissue was harvested 2 weeks after SNE or sham surgery. Increased T Figure 2 Increased TTX resistance of the sciatic nerve after SNE Figure 2 Increased TTX-resistance of the sciatic nerve after SNE. A: TTX-resistance of the A-fiber sciatic nerve com- pound action potential (CAP) ipsilateral to SNE (n = 8) com- pared to contralateral, naïve and sham surgery nerves (n = 8). CAP response is expressed as a % of recorded amplitude before TTX administration. A-fiber CAP stimulus was 0.1 mA, 0.1 ms throughout. B: Changes in TTX-resistance of the C-fiber sciatic nerve CAP after SNE. C-fiber CAP stimulus was 10 mA, 0.5 ms throughout. indicates significant differ- ence from control nerve sensitivity to TTX (p < 0.05, two- way ANOVA, Tukey post-hoc). Tissue was harvested 2 weeks after SNE or sham surgery. sed TTX-resistance of the sciatic nerve after Increased TTX-resistance of the sciatic nerve after SNE. A: TTX-resistance of the A-fiber sciatic nerve com- pound action potential (CAP) ipsilateral to SNE (n = 8) com- pared to contralateral, naïve and sham surgery nerves (n = 8). CAP response is expressed as a % of recorded amplitude before TTX administration. A-fiber CAP stimulus was 0.1 mA, 0.1 ms throughout. Detection of NaV1.8 mRNA in sciatic nerves Detection of NaV1.8 mRNA in sciatic nerves Real-time PCR detected the presence of NaV1.8 mRNA in the sciatic nerve and revealed a significant up-regulation in the ipsilateral nerve as compared to the contralateral and uninjured nerve (p < 0.05, one-way ANOVA with Tukey post-hoc; Fig. 4A). In contrast, ipsilateral, contralat- eral, and uninjured DRG showed no significant differ- ences in NaV1.8 mRNA levels (Fig. 4B). To confirm that the NaV1.8 PCR signals represent mRNA and not genomic DNA contamination, 3'RACE PCR was performed. South- ern blot hybridization analysis showed a positive band at the predicted size of 830 bp in both contralateral and ipsi- lateral sciatic nerve samples (Fig. 4C). After agarose gel separation and cloning of amplicons, DNA sequencing positively identified the 3' end of the coding region and Increased TTX-resistance of the sciatic nerve after SNE Figure 2 Increased TTX-resistance of the sciatic nerve after SNE. A: TTX-resistance of the A-fiber sciatic nerve com- pound action potential (CAP) ipsilateral to SNE (n = 8) com- pared to contralateral, naïve and sham surgery nerves (n = 8). CAP response is expressed as a % of recorded amplitude before TTX administration. A-fiber CAP stimulus was 0.1 mA, 0.1 ms throughout. B: Changes in TTX-resistance of the C-fiber sciatic nerve CAP after SNE. C-fiber CAP stimulus was 10 mA, 0.5 ms throughout. * indicates significant differ- ence from control nerve sensitivity to TTX (p < 0.05, two- way ANOVA, Tukey post-hoc). Tissue was harvested 2 weeks after SNE or sham surgery. Increased TTX-resistance of the sciatic nerve after SNE Figure 2 Increased Figure 1 Increased excitability of ipsilateral sciatic nerve after SNE Figure 1 Increased excitability of ipsilateral sciatic nerve after SNE. A: Stimulus-output plot of A-fiber CAP peak for sciatic nerve ipsilateral to SNE (n = 8) compared to preparations from contralateral, naïve or sham control rats (n = 8). Inset (left) shows examples of ipsilateral and contralateral A-fiber CAP with peaks aligned for clarity (0.1 mA stimulus). , p < 0.05 (two-way ANOVA, Tukey post-hoc). Inset (bottom right) shows normalized data with error bars removed for clarity. Note the leftward shift of data from ipsilateral nerves. B: Stimulus-output plot of C-fiber CAP peak for sciatic nerve ipsilateral to SNE compared to preparations from contralat- eral, naïve or control rats. Inset (left) shows examples of ipsi- lateral and contralateral C-fiber CAP (10 mA stimulus). Results At lower concentrations, the TTX-resist- Page 2 of 12 (page number not for citation purposes) Page 2 of 12 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/14 Molecular Pain 2009, 5:14 Increased excitability of ipsilateral sciatic nerve after SNE Figure 1 Increased excitability of ipsilateral sciatic nerve after SNE. A: Stimulus-output plot of A-fiber CAP peak for sciatic nerve ipsilateral to SNE (n = 8) compared to preparations from contralateral, naïve or sham control rats (n = 8). Inset (left) shows examples of ipsilateral and contralateral A-fiber CAP with peaks aligned for clarity (0.1 mA stimulus). , p < 0 05 (two-way ANOVA Tukey post-hoc) Inset (bottom ance of both the A-fiber and C-fiber CAPs was signifi- cantly increased in nerves ipsilateral to SNE (p < 0.05, two-way ANOVA with Tukey post-hoc) (Fig. 2A, B). ance of both the A-fiber and C-fiber CAPs was signifi- cantly increased in nerves ipsilateral to SNE (p < 0.05, two-way ANOVA with Tukey post-hoc) (Fig. 2A, B). Peripheral nerve NaV1.8 immunoreactivity after SNE Peripheral nerve NaV1.8 immunoreactivity after SNE At two weeks after SNE injury, NaV1.8-like immunoreac- tivity in the ipsilateral L4 and L5 dorsal root ganglia (DRG) was reduced (Fig. 3A, B). Specifically, the staining intensities of NaV1.8+ small-, medium-, and large diame- ter neurons were all significantly decreased (p < 0.05, one- way ANOVA with Tukey post-hoc; Fig. 3B). In marked contrast to the DRG, NaV1.8-like immunoreactivity increased more than two-fold in ipsilateral nerves (p < 0.05, t-test; Fig. 3C, D). Detection of NaV1.8 mRNA in sciatic nerves B: Changes in TTX-resistance of the C-fiber sciatic nerve CAP after SNE. C-fiber CAP stimulus was 10 mA, 0.5 ms throughout. * indicates significant differ- ence from control nerve sensitivity to TTX (p < 0.05, two- way ANOVA, Tukey post-hoc). Tissue was harvested 2 weeks after SNE or sham surgery. Page 3 of 12 (page number not for citation purposes) Page 3 of 12 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/14 Molecular Pain 2009, 5:14 NaV1.8 protein is down-regulated in the DRG and up-regu- lated in the nerve after SNE Figure 3 NaV1.8 protein is down-regulated in the DRG and up-regulated in the nerve after SNE. A: Nav1.8-ir in representative DRG sections after SNE. B: Quantification of fluorescence intensity in DRG neurons. Neurons are classi- fied by cell area. h: contralateral. ▪: ipsilateral. DRG measure- ments were from 3 contralateral and 6 ipsilateral sections (n = 3. ; p < 0.05, one-way ANOVA, Tukey post-hoc). C: NaV1.8-ir in representative sections of sciatic nerve after SNE. D: Quantification of fluorescence intensity in sciatic nerve. Measurements were made from 8 contralateral and 8 ipsilateral sections. (n = 3, ; p < 0.05, t-test). Scale bar: 100 μm for A and C. Tissue was harvested 2 weeks after SNE. For both DRG and nerve sections, average intensity was measured using ImageJ software. Output intensity values were on a 0–255 scale based on the 255 shades of grey present in the image, with 0 representing black and 255 rep- resenting white. The output numerical intensity value was defined as "relative fluorescence." Discussion The NaV1.3, NaV1.8, and NaV1.9 sodium channels are differentially regulated after nerve injury [24], and the TTX-resistant NaV1.8 and NaV1.9 are selectively distrib- uted in peripheral sensory neurons [24]; these properties have identified them as potential molecular targets for neuropathic pain. Antisense oligodeoxynucleotide and siRNA studies suggest that sensory neuron NaV1.8 expres- sion and spinal cord NaV1.3 expression are important for behavioral phenotypes and aberrant neurophysiology in rats with peripheral nerve injuries [4-7,25]. Rat loss-of- function studies have not yet been performed for NaV1.9, but studies of NaV1.9-null transgenic mice suggest involvement in inflammatory rather than neuropathic pain [26,27]. the 3' untranslated region of rat Nav1.8 as well as an addi- tional poly A tail (data not shown). Selectivity of peripheral nerve NaV1.8 mRNA up- regulation after SNE After confirmation of the presence of NaV1.8 mRNA in the sciatic nerve, we compared steady-state levels of mRNA for NaV1.3, NaV1.5, NaV1.6, NaV1.7, NaV1.8, and NaV1.9 between ipsilateral and contralateral segments of the sciatic nerve immediately proximal to the injury site (Fig. 6A). Ipsilateral NaV1.8 mRNA was significantly up- regulated by 10.8-fold (p < 0.05, Mann-Whitney). Ipsilat- eral NaV1.6 mRNA expression was increased by 3.48-fold, but this difference did not reach statistical significance (p > 0.05, Mann-Whitney). None of the other sodium chan- nels showed significant up-regulation in the sciatic nerve (p > 0.05, Mann-Whitney). We further examined mRNA expression of the same sodium channels in the DRG at the same time point. Ipsilateral NaV1.5, NaV1.6, NaV1.7, and NaV1.9 mRNA levels decreased by 28.6%, 39.8%, 54.1%, and 54.9%, respectively, however only the decrease was only statistically significant for NaV1.7 (p < 0.05, Mann- Whitney; Fig. 6B). N V1 8 t i i d l t d i th DRG d l t d i th ft SNE Fi 3 NaV1.8 pr lated in th Figure 3 NaV1.8 protein is down regulated in the DRG and up regu lated in the nerve after SNE Figure 3 NaV1.8 protein is down-regulated in the DRG and up-regulated in the nerve after SNE. A: Nav1.8-ir in representative DRG sections after SNE. B: Quantification of fluorescence intensity in DRG neurons. Neurons are classi- fied by cell area. h: contralateral. ▪: ipsilateral. DRG measure- ments were from 3 contralateral and 6 ipsilateral sections (n = 3. ; p < 0.05, one-way ANOVA, Tukey post-hoc). C: NaV1.8-ir in representative sections of sciatic nerve after SNE. D: Quantification of fluorescence intensity in sciatic nerve. Measurements were made from 8 contralateral and 8 ipsilateral sections. (n = 3, ; p < 0.05, t-test). Scale bar: 100 μm for A and C. Tissue was harvested 2 weeks after SNE. For both DRG and nerve sections, average intensity was measured using ImageJ software. Output intensity values were on a 0–255 scale based on the 255 shades of grey present in the image, with 0 representing black and 255 rep- resenting white. The output numerical intensity value was defined as "relative fluorescence." Polyadenylation of peripheral NaV1.8 mRNA To investigate potential inhibition of peripheral NaV1.8 mRNA degradation by polyadenylation, we conducted the polyA tail length assay as originally described by Salles and Strickland [23]. NaV1.v8 mRNA isolated from both injured nerve and uninjured DRG primarily exhibited a discrete polyA tail of about 50 bases, with additional poly- adenylation occurring out to 200 bases (Fig. 7). However, there were no apparent differences in the maximum polyA tail length or smearing pattern. Detection Figure 4 g Detection of NaV1.8 mRNA in the sciatic nerve. A. NaV1.8 mRNA levels were significantly increased in the sciatic nerve ipsilateral to the injury site as compared to contralateral and uninjured DRG. (n = 3; p < 0.05, one-way ANOVA with Tukey post-hoc). B. NaV1.8 mRNA levels in SNE-injured DRG were not significantly different from those of uninjured DRG (n = 4 for uninjured DRG and n = 3 for all other samples). C: Southern blot of the Nav1.8 3' RACE product from contralateral and ipsi- lateral sciatic nerve. Note the presence of a distinct band representing the expected 3'RACE product of 830 bp. Sequencing of the RACE product confirmed the presence of the 3' end of the NaV1.8 coding region as well as the NaV1.8 3' un-translated region and polyA tail. Tissue was harvested 2 weeks after SNE. plateau (Fig. 1). Because increased CAP amplitude is typi- cally interpreted as a recruitment of more axons, a stimu- lus-output plateau suggests that all available axons have been recruited. A further CAP increase could potentially arise from a change in the total number of axons; how- ever, after SNE the numbers of large- and small-diameter afferents do not change proximal to the injury site, where the recorded tissues were sampled [22]. It is therefore more likely that the observed CAP increase represents peripherally increased Na+ conductance in individual axons. It is further likely that such an increase in conduct- ance would be at least partially mediated by TTX-resistant channels, as CAP TTX resistance increased concomitantly with CAP amplitude for both C- and A-fibers after SNE injury (Fig. 2). An alternative explanation for these obser- vations is that increased expression of TTX-sensitive Na+ channels could increase both the CAP amplitudes and the safety factor for axonal action potential conduction dur- ing TTX application. Another possibility we considered was the upregulation of a third TTX-R channel, Nav1.5, which is normally expressed in sensory neurons during development but declines to very low levels at birth and adulthood [36]. Nav1.5 exhibits faster kinetics than Nav1.8 and is significantly more sensitive to TTX (IC50 ~2 μM) than Nav1.8 or Nav1.9 [36]; thus upregulation of Nav1.5 could potentially account for the observed increases in TTX-resistance, including the blockade of A and C-fiber CAP conduction by 1 μM TTX after SNE. Evidence for neuronal origin of NaV1.8 mRNA in sciatic nerve We hypothesized that if sciatic nerve NaV1.8 mRNA was of non-neuronal origin, it would be detectable in cultures of dissociated sciatic nerves where there are no neuronal somata and neuronal axons have degenerated. No real- time PCR signal was detected for NaV1.8 in sciatic nerve cultures (Fig 5A, C), whereas robust NaV1.8 expression was observed in DRG cultures that served as a positive control (Fig. 5B, C). However, similar signals for 18s rRNA were detected in the sciatic nerve and DRG cultures (Fig. 5D), suggesting similar concentrations of template cDNA. Additionally, we were able to detect strong expression of s100B, a Schwann cell marker, in both sciatic nerve and DRG cultures, with Ct values ranging from 26–30. Inter- estingly, although s100B expression was strong in nerve cultures, it was still stronger in DRG cultures (Fig. 5E). The requirement of NaV1.8 and NaV1.3 for neuropathic pain remains controversial, however, as neuropathic pain behavior develops normally in NaV1.8- and NaV1.3-null mice [20,21,28], as well as in mice treated with diptheria toxin to selectively destroy all postmitotic sensory neu- rons expressing NaV1.8 [29]. It is possible that the dis- crepancy between these findings and those of rat loss-of- Page 4 of 12 (page number not for citation purposes) Page 4 of 12 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/14 Molecular Pain 2009, 5:14 Detection of NaV1 8 mRNA in the sciatic nerve Figure 4 Detection of NaV1.8 mRNA in the sciatic nerve Figure 4 Detection of NaV1.8 mRNA in the sciatic nerve. A. NaV1.8 mRNA levels were significantly increased in the sciatic nerve ipsilateral to the injury site as compared to contralateral and uninjured DRG. (n = 3; p < 0.05, one-way ANOVA with Tukey post-hoc). B. NaV1.8 mRNA levels in SNE-injured DRG were not significantly different from those of uninjured DRG (n = 4 for uninjured DRG and n = 3 for all other samples). C: Southern blot of the Nav1.8 3' RACE product from contralateral and ipsi- lateral sciatic nerve. Note the presence of a distinct band representing the expected 3'RACE product of 830 bp. Sequencing of the RACE product confirmed the presence of the 3' end of the NaV1.8 coding region as well as the NaV1.8 3' un-translated region and polyA tail. Tissue was harvested 2 weeks after SNE. Axonal or Figure 5 Axonal or Figure 5 g g Axonal origin of sciatic nerve NaV1.8 mRNA. A, B: Axonal origin of sciatic nerve NaV1.8 mRNA. A, B: Representative images of cells cultured from the sciatic nerve and dorsal root ganglia. Cells with Schwann- and fibroblast- like morphology were present in both nerve and DRG cul- tures, whereas neurons were only present in the DRG cul- ture. C: Expression of 18s rRNA in nerve and DRG cultures (n = 3). Expression is given in terms of the absolute cycle count where signal was first detected. D. Expression of NaV1.8 mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the DRG sam- ple with the lowest NaV1.8 expression and internally against 18s expression rRNA expression. E. Expression of s100B mRNA in nerve and DRG cultures (n = 3). Relative expres- sion was normalized externally against the nerve sample with the lowest s100B expression and internally against 18s expression rRNA expression. Strong expression was observed in both cultures with Ct values of 26–30. However, relative expression was still higher in DRG cultures. Axonal origin of sciatic nerve NaV1.8 mRNA. A, B: Representative images of cells cultured from the sciatic nerve and dorsal root ganglia. Cells with Schwann- and fibroblast- like morphology were present in both nerve and DRG cul- tures, whereas neurons were only present in the DRG cul- ture. C: Expression of 18s rRNA in nerve and DRG cultures (n = 3). Expression is given in terms of the absolute cycle count where signal was first detected. D. Expression of NaV1.8 mRNA in nerve and DRG cultures (n = 3). Relative expression was normalized externally against the DRG sam- ple with the lowest NaV1.8 expression and internally against 18s expression rRNA expression. E. Expression of s100B mRNA in nerve and DRG cultures (n = 3). Relative expres- sion was normalized externally against the nerve sample with the lowest s100B expression and internally against 18s expression rRNA expression. Strong expression was observed in both cultures with Ct values of 26–30. However, relative expression was still higher in DRG cultures. To address this hypothesis, we first determined the pres- ence of NaV1.8 mRNA in the sciatic nerve using RT-PCR (Fig. 4 &5; also see [44]. The 3'RACE sequencing identi- fied a polyA tail in NaV1.8 PCR amplicons, suggesting that our PCR data did not reflect genomic DNA contami- nation. Detection Figure 4 How- ever, we did not observe increased NaV1.5 mRNA expres- sion in the DRG or the sciatic nerve after SNE. function studies could arise from compensation for NaV1.8 or NaV1.3 deletion, as well as species differences, although it has also been shown that intrathecal antisense knockdown of NaV1.3 did not reverse pain symptoms in a rat spared nerve injury (SNI) model [30]. We hypothesized that SNE injury would produce sensory neuron hyperexcitability which would be related to altered local expression of NaV1.8. We found that SNE injury in rats induced a unique electrophysiological phe- notype characterized by increased sciatic nerve CAP amplitude in both C- and A-fiber populations (Fig. 1). The C-fiber increase likely contributed to the thermal hyper- sensitivity we have previously observed in this model, while Aδ-fiber excitability would be expected to contrib- ute to the observed mechanical hypersensitivity [31]. However, the overall magnitude of the A-fiber CAP increase and lack of change in conduction velocity suggest increased excitability of Aβ-fibers instead of or in addition to Aδ-fibers. Aβ-fibers are not normally involved in nocic- eption [32] with the exception of a small subpopulation [33], but phenotypic changes in Aβ-fibers have been reported after nerve injuries including SNE [34,35] that result in nociceptive afterdischarge in spinal wide dynamic range neurons [35]. It is possible that such a phe- notypic switch might have been involved in the observed A-fiber CAP enhancement and concomitant mechanical or thermal hypersensitivity. It is notable that increased A- and C-fiber CAP amplitudes persisted even after the stimulus-output plot reached a Page 5 of 12 (page number not for citation purposes) Page 5 of 12 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/14 Molecular Pain 2009, 5:14 Axonal origin of sciatic nerve NaV1.8 mRNA Figure 5 Axonal origin of sciatic nerve NaV1.8 mRNA. A, B: Representative images of cells cultured from the sciatic nerve and dorsal root ganglia. Cells with Schwann- and fibroblast- like morphology were present in both nerve and DRG cul- tures, whereas neurons were only present in the DRG cul- ture. C: Expression of 18s rRNA in nerve and DRG cultures (n = 3). Expression is given in terms of the absolute cycle count where signal was first detected. D. Expression of NaV1.8 mRNA in nerve and DRG cultures (n = 3). Detection Figure 4 Relative expression was normalized externally against the DRG sam- ple with the lowest NaV1.8 expression and internally against 18s expression rRNA expression. E. Expression of s100B mRNA in nerve and DRG cultures (n = 3). Relative expres- sion was normalized externally against the nerve sample with the lowest s100B expression and internally against 18s expression rRNA expression. Strong expression was observed in both cultures with Ct values of 26–30. However, relative expression was still higher in DRG cultures. reported for the CCI model [11], underscoring simmilari- ties with SNE. This peripheral translocation of Nav1.8 together with increased TTX-resistance suggests involve- ment of peripherally accumulated NaV1.8 in sciatic nerve hyperexcitability after SNE injury. Several mechanisms may underly the peripheral translo- cation of NaV1.8 protein observed in multiple injury models. In the present study, we detected ipsilaterally decreased DRG NaV1.8 immunoreactivity (Fig. 3), but NaV1.8 mRNA levels in the DRG were not affected by SNE injury (Figs. 4 &5). Similar post-CCI immunohistochemi- cal redistribution of NaV1.8 to sciatic nerve axons without increased DRG mRNA expression previously led Novako- vic et al. to suggest peripheral trafficking of existing NaV1.8 protein with increased local insertion [11]. Decreased NaV1.8 mRNA levels and attenuated NaV1.8 currents that could be compatible with such a mechanism were also observed in injured small-diameter DRG somata cultured from CCI-injured rats [39]; NaV1.8 pro- tein levels in injured neurons within the DRG were also shown to decrease in rats after SNL, CCI, SNI, and axot- omy injuries [24]. However, while the majority of de novo protein synthesis is believed to occur in DRG somata, an increasing number of studies suggest that axons contain mRNAs such as β-actin, cofilin and RhoA that are locally translated in response to guidance cues during nervous system development [40-42]. Recently, intra-axonal CREB mRNA translation was shown to be essential for neuronal survival in response to nerve growth factor application [43]. We thus hypothesized that axonal NaV1.8 mRNA accumulation might contribute to the increased immuno- reactivity in the nerve proximal to SNE injury. A l i i f i ti N V1 8 Fi 5 RNA A l i i f i Fi 5 Axonal or Figure 5 In addition, we determined the cellular origin of NaV1.8 transcripts because sciatic nerve contains non- neuronal cells such as Schwann cells and fibroblasts in addition to neuronal axons. Cells cultured from both naïve DRG and sciatic nerve showed expression of s100B, confirming the presence of Schwann and/or satellite 'glial' cells. However, NaV1.8 expression was not detected in the nerve cultures (Fig. 6), suggesting that NaV1.8 transcripts detected in intact nerve tissue were likely derived from axons. Much larger increases in CAP TTX-resistance, and only in C-fibers (resistant to block by 100 μM TTX), were reported for the SNL model [5], underscoring major differences between the SNE and SNL models. SNL is thought to cause extensive Wallerian degeneration of injured axons from L5 and L6 DRG, leaving only uninjured L4 axons in the sciatic nerve [5]. In contrast, CCI and SNE cause selec- tive degeneration of Aβ-fibers distal to the constriction site with some remyelination occurring 2–4 weeks after injury [37,38]; this partial, distal degeneration leaves proximal injured and uninjured axons structurally intact. Significant up-regulation of NaV1.8 mRNA was demon- strated in the ipsilateral sciatic nerve when compared to the contralateral or uninjured nerve (Fig. 4). To explore the mechanism of the peripheral NaV1.8 mRNA increase, we examined the polyadenylation state. There were no dif- We also found that sciatic nerve NaV1.8 immunoreactiv- ity was increased after SNE injury, while immunoreactiv- ity within cell bodies of DRG sensory neurons ipsilateral to SNE was reduced. Similar findings were previously Page 6 of 12 (page number not for citation purposes) Page 6 of 12 (page number not for citation purposes) Molecular Pain 2009, 5:14 http://www.molecularpain.com/content/5/1/14 Selective peripheral translocation of NaV1.8 mRNA after SNE injury Figure 6 Selective peripheral translocation of NaV1.8 mRNA after SNE injury. A: Sodium channel mRNA levels in naive, con- tralateral, and ipsilateral sciatic nerve. Black columns: ipsilateral side. White columns: contralateral side. B: Sodium channel mRNA levels in contralateral and ipsilateral L4 and L5 DRG. Black columns: ipsilateral side. Grey columns: contralateral side. Note that NaV1.8 showed a significant 10.8-fold increase in the ipsilateral sciatic nerve (; p < 0.05, Mann-Whitney) and that this increase did not occur in the DRG. NaV1.6 showed a 3.48-fold increase in the nerve, but this did not reach statistical sig- nificance (p = 0.19, Mann-Whitney). Selective p Figure 6 Therefore, intra-axonal polyadenylation resulting in reduced turnover and increased steady-state mRNA levels [45] was ruled out as a mechanism of increased NaV1.8 mRNA accumulation in SNE axons. Strikingly, simultaneous analysis of multiple sodium channels further demonstrated that after SNE injury, steady-state NaV1.8 mRNA was significantly increased in the ipsilateral nerve by nearly 11-fold when compared to the contralateral nerve. In contrast, we were unable to observe any changes in ipsilateral mRNA levels of NaV1.3, NaV1.6, NaV1.7, or NaV1.9 (Fig. 5). It is unlikely that the selective NaV1.8 mRNA increase was the indirect result of ultrastructural changes, as both the fiber number and composition remain unaltered at the sampling location proximal to the injury site [37]. These findings thus sug- gest a novel mechanism of selective NaV1.8 mRNA accu- mulation in injured axons. Selective p Figure 6 Selective peripheral translocation of NaV1.8 mRNA after SNE injury Figure 6 Selective peripheral translocation of NaV1.8 mRNA after SNE injury. A: Sodium channel mRNA levels in naive, con- tralateral, and ipsilateral sciatic nerve. Black columns: ipsilateral side. White columns: contralateral side. B: Sodium channel mRNA levels in contralateral and ipsilateral L4 and L5 DRG. Black columns: ipsilateral side. Grey columns: contralateral side. Note that NaV1.8 showed a significant 10.8-fold increase in the ipsilateral sciatic nerve (; p < 0.05, Mann-Whitney) and that this increase did not occur in the DRG. NaV1.6 showed a 3.48-fold increase in the nerve, but this did not reach statistical sig- nificance (p = 0.19, Mann-Whitney). NaV1.5 showed an 80.2% decrease in the ipsilateral nerve, but this also did not reach sta- tistical significance (p = 0.079, Mann-Whitney) Ipsilateral NaV1.5, NaV1.6, NaV1.7, and NaV1.9 mRNA levels decreased by 28.6%, 39.8%, 54.1%, and 54.9%, respectively, but the downregulation only reached statistical significance for NaV1.7 (p < 0.05, Mann-Whitney). All samples were normalized externally against the mean contralateral expression and internally against 18s rRNA expression (n = 3–7). Means and standard errors are shown for convenience; parametric analyses of non-parametric data returned similar results. proteins, although no changes were observed in the steady-state protein levels of NaV1.8, NaV1.3, or the α 2δ calcium channel [46]. It has also been shown that axon- ally transported importin β and vanilloid receptor mRNAs can be locally translated in sciatic nerve axons after nerve injury and dorsal horn synaptic terminals during inflam- mation, repsectively [47,48]. Various mRNAs coding cytoskeletal and metabolic proteins have been found to be axonally transported and locally translated in a cul- tured axonal preparation [49]. Thus, we propose here that enhanced and selective peripheral transport is the most likely mechanism for axonal NaV1.8 mRNA up-regula- tion, and that local mRNA accumulation could contribute to the observed increase in sciatic nerve NaV1.8 immuno- reactivity. Our findings also support the involvement of peripheral NaV1.8 protein in sciatic nerve hyperexcitabil- ity after neuropathic injury. ferences in maximum NaV1.8 polyA tail length or smear- ing pattern between injured nerve and uninjured DRG samples (Fig. 7). Therefore, intra-axonal polyadenylation resulting in reduced turnover and increased steady-state mRNA levels [45] was ruled out as a mechanism of increased NaV1.8 mRNA accumulation in SNE axons. ferences in maximum NaV1.8 polyA tail length or smear- ing pattern between injured nerve and uninjured DRG samples (Fig. 7). Axonal or Figure 5 NaV1.5 showed an 80.2% decrease in the ipsilateral nerve, but this also did not reach sta- tistical significance (p = 0.079, Mann-Whitney) Ipsilateral NaV1.5, NaV1.6, NaV1.7, and NaV1.9 mRNA levels decreased by 28.6%, 39.8%, 54.1%, and 54.9%, respectively, but the downregulation only reached statistical significance for NaV1.7 (p < 0.05, Mann-Whitney). All samples were normalized externally against the mean contralateral expression and internally against 18s rRNA expression (n = 3–7). Means and standard errors are shown for convenience; parametric analyses of non-parametric data returned similar results. Conclusion While our experimental design did not permit the direct discrimination of somatically- and axonally-derived pro- tein, our findings collectively introduce the possibility that sodium channel mRNA could be peripherally trans- A recent study of rat saphenous nerve neuromas revealed increased local translation of structural and metabolic Page 7 of 12 (page number not for citation purposes) Page 7 of 12 (page number not for citation purposes) http://www.molecularpain.com/content/5/1/14 Molecular Pain 2009, 5:14 NaV1.8 mRNA polyadenylation does not increase in the injured sciatic nerve Figure 7 NaV1.8 mRNA polyadenylation does not increase in the injured sciatic nerve. A: Schematic representation of PolyA Tail (PAT) assay. 1. Reverse transcription with oligo dT-Adaptor primer. 2. NaV1.8-specific PCR amplification with Adaptor primer. Amplicon length depends on polyA tail length. 3. Southern blot detection with digoxygenin (DIG)-labeled NaV1.8-spe- cific probe after gel electrophoresis. Multiple polyA tail lengths result in smearing and multiple bands. B: Southern blot of PAT amplicons. : NaV1.8 mRNA from both ipsilateral nerve and uninjured DRG tissue predominantly exhibits a discrete polyA tail < 50 bases long, with additional polyadenylation occurring out to 200 bases. Tissue was harvested 2 weeks after SNE. NaV1.8 mRNA polyadenylation does not increase in the injured sciatic nerve Figure 7 NaV1.8 mRNA polyadenylation does not increase in the injured sciatic nerve. A: Schematic representation of PolyA Tail (PAT) assay. 1. Reverse transcription with oligo dT-Adaptor primer. 2. NaV1.8-specific PCR amplification with Adaptor primer. Amplicon length depends on polyA tail length. 3. Southern blot detection with digoxygenin (DIG)-labeled NaV1.8-spe- cific probe after gel electrophoresis. Multiple polyA tail lengths result in smearing and multiple bands. B: Southern blot of PAT amplicons. : NaV1.8 mRNA from both ipsilateral nerve and uninjured DRG tissue predominantly exhibits a discrete polyA tail < 50 bases long, with additional polyadenylation occurring out to 200 bases. Tissue was harvested 2 weeks after SNE. NGS and 0.3% Triton X-100). The NaV1.8 antibody, which was previously extensively characterized, 15 was generously provided by Dr. John Wood, London, UK. Sec- tions were then incubated in a FITC-labeled goat anti-rab- bit secondary antibody (Molecular Probes, Carlsbad, CA) at 1:500 dilution. The sections were mounted in an anti- fade medium (ProLong, Molecular Probes, Carlsbad, CA). To minimize variability between specimens, ipsilateral and contralateral tissues were processed simultaneously and representative sections grouped on the same slides. ported from the DRG to the sciatic nerve and locally trans- lated. Confocal Microscopy Confocal fluorescence images were taken at 20× magnifi- cation on a Leica TCS-SP confocal fixed-stage upright microscope (Heidelberg, Germany) equipped with argon (488 nm excitation: JDS Uniphase) and DPSS diode (561 nm excitation: Melles-Griot) lasers. All laser excitation and detection parameters were kept constant across all samples. Image stacks were averaged and given identical high and low intensity thresholds using LCS lite software (Leica, Heidelberg, Germany). For DRG images, neuronal area was measured by outlining NaV1.8 immunoreactive somata and then calculating the area with ImageJ software http://rsb.info.nih.gov/ij/. Fluorescence intensity was measured by outlining the cytosolic region of NaV1.8 immunoreactive somata; outlines were in the shape of an annulus, to exclude the darker nuclei. Average cytosolic fluorescence intensity was then measured using ImageJ software. When overlapping NaV1.8-immunoreactive somata were detected, the overlapping regions were Sciatic nerve entrapment (SNE) Surgery was performed as described previously [22] using adult male Sprague-Dawley rats weighing 200–250 g. Briefly, three Tygon® cuffs (length = 1 mm, outer diameter = 2.28 mm, inner diameter = 0.76 mm) were placed around the exposed sciatic nerve proximal to the trifurca- tion. Conclusion If this is the case, the local up-regulation of NaV1.8 mRNA shown here after injury may at least partially con- tribute to the observed peripheral increases in NaV1.8 protein levels and nerve excitability, which in turn may play a role in the aberrant nociception that characterizes neuropathic pain. Page 8 of 12 (page number not for citation purposes) Cell Culture S i i Sciatic nerve sections and lumbar DRG were extracted as above and cut into small pieces in ice-cold Hank's bal- anced salt solution (HBSS) with 20% fetal bovine serum (FBS). The tissue was incubated for 75 min at 37°C in HBSS with 0.125% collagenase P (Roche, Indianapolis, IN), and 0.05% DNAse. 0.25% trypsin was added for 5 min, after which the tissue was centrifuged for 5 min at 2000 rpm. After washing with 20% FBS in HBSS, the cell pellet was resuspended in HBSS containing 0.295% MgSO4 and 0.05% DNAse. The tissue was then triturated 10 times with a fire-polished siliconized Pasteur pipette and centrifuged for 5 min at 2000 rpm. The resulting cell pellet was resuspended in 1 ml of F12 nutrient mixture with 10% FBS, and 300 μl were plated into each of 3 wells of a 12-well cell culture plate. The plate was incubated at 37°C for 1 hr, and then another 700 μl of medium was added. The medium was then changed every 2 days. After 4 days, culture plates were washed in PBS, snap-frozen in liquid nitrogen, and then stored at -80°C. For RNA extrac- tion, culture plates were thawed, and 350 μl of buffer RLT from the RNEasy Plus Mini Kit (Qiagen, Valencia, CA) were immediately added to each well. Wells were incu- bated for 15 minutes at room temperatue and scraped with pipette tips. The buffer RLT containing scraped cells from each well was then placed into separate RNAse-free 1.5 mL microcentrifuge tubes. The RNEasy Plus protocol with gDNA eliminator columns was then followed for RNA extraction. Surgery, tissue collection and preparation for electrophysiology At 2 weeks after injury, rats were anesthetized with sodium pentobarbital (50 mg/kg) and L4 and L5 DRG were excised with their dorsal roots, the ventral roots, the spinal nerves and a length of attached sciatic nerves cut just proximal to the site of entrapment. The preparations were further trimmed at 0–4°C in the low-Na+ artificial cerebrospinal fluid (ACSF) composed of (in mM): NaCl, 62; KCl, 3.5; NaH2PO4, 1.25; CaCl2, 2; MgCl2, 2; NaHCO3, 26; glucose, 10 and sucrose, 124. Isolated DRG and sciatic nerve sections were then placed in cryogenic vials, frozen in liquid nitrogen and stered at -80°C. Alter- natively, sections of sciatic nerve were then transferred to normal ACSF (20–23°C) composed of (in mM): NaCl, 124; KCl, 3.5; NaH2PO4, 1.25; CaCl2, 2; MgCl2, 2; NaHCO3, 26 and glucose, 10. The ACSF was continuously bubbled with a 95/5% mixture of O2/CO2. For recording, preparations were transferred to a custom recording chamber (0.3 ml volume) that was perfused (2.7 ml/min) with oxygenated ACSF at 34.5°C. TTX (0.1–1 μM, Sigma) was dissolved and applied in ACSF. recording. All data are presented as mean ± standard error of the mean (SEM). recording. All data are presented as mean ± standard error of the mean (SEM). excluded from intensity analysis. For sciatic nerve analy- ses, 1–2 111 × 320 pixel rectangles were drawn on each image such they did not cover any gap, break, or artifact. The average intensity within each rectangle was measured using ImageJ software. For both DRG and nerve sections, output intensity values were on a 0–255 scale based on the 255 shades of grey present in the image, with 0 repre- senting black and 255 representing white. The output numerical intensity value was defined as "relative fluores- cence." Immunohistochemistry At 2 weeks after injury, rats were anesthetized with pento- barbital (100 mg/kg, i.p.) and fixed by intracardiac per- fusion of 500 ml ice-cold 4% paraformaldehyde and 0.14% picric acid in 0.1 M phosphate buffer (PB), pH 7.4. Ipsilateral and contralateral L4 and L5 DRG were har- vested along with sciatic nerve segments. Tissue was post- fixed for 24 hrs and cryoprotected overnight in 30% sucrose in 0.1 M PB. Samples were mounted in Tissue-Tek (Sakura Finetek USA, Inc., Torrance, CA), and cryosec- tioned at 25 μm (DRG) or 20 μm (sciatic nerve). Free- floating sections were incubated overnight with an anti- NaV1.8 antibody (1:2000 dilution, in PBS containing 5% Page 8 of 12 (page number not for citation purposes) Page 8 of 12 (page number not for citation purposes) Molecular Pain 2009, 5:14 http://www.molecularpain.com/content/5/1/14 3'Rapid amplification of cDNA ends (3'RACE) p p f f ( ) At 2 weeks after injury, total RNA (800 ng) extracted sep- arately from ipsilateral and contralateral sciatic nerve was converted to first strand cDNA and then subjected to 3'RACE PCR using the SMART RACE cDNA Amplification Kit (Clontech, Mountain View, CA). The anchoring primer for the 3'RACE PCR reaction was 5'-GTTTAT- GGCGACAATCTCTCCAAAG-3' (Tm = 65°C), located approximately 830 bp upstream of the NaV1.8 mRNA polyA tail. Approximately 18 ng of first strand cDNA tem- plate was consistently used for all samples. Samples were denatured at 94°C for 30 sec, annealed at 68°C for 30 sec, and elongated at 72°C for 3 min. First strand cDNA was amplified for 35 cycles with the anchoring primer and oligo d(T) primer. Southern blot hybridization analysis was used to identify positive PCR amplicons containing NaV1.8 3'end sequence. The DIG-labeled DNA probe against the NaV1.8 3' end coding sequence was synthe- sized using the PCR DIG Probe Synthesis Kit (Roche, Indi- anapolis, IN) with forward primer 5'- GGAAGACCTCTCAGCCACAG-3' and reverse primer 5'- CAGGGTGTTGGAGAGTGTCA-3'. After hybridization, the membrane was washed, blocked, incubated with anti- DIG antibody, and detected according to the manufac- turer's instructions. Chemiluminescence was detected by a LAS-3000 Luminescent Image Analyzer (Fujifilm, Tokyo, Japan). 18S ribosomal RNA was amplified from the con- tralateral and ipsilateral sciatic nerve RNA samples for use as an internal control during PCR. Statistical analysis Ipsilateral and contralateral data sets were checked for normality using Shapiro-Wilkes and Liliefors tests, with p < 0.05 accepted as a non-normal distribution. Compari- sons of two groups with normal distributions were made using Student's t-test. Comparisons of a non-normally distributed group with any other group were made using the Mann-Whitney U test. Comparisons of more than two groups, all of which were normally distributed, were made using 1-way ANOVA with Tukey post-hoc tests for pairwise comparisons. Two-way ANOVA with Tukey post- hoc tests was used for behavioral and electrophysiological data where a second variable (i.e. stimulus strength or time point) was analyzed simultaneously with laterality (i.e. ipsilateral vs. contralateral). PolyA tail length assay At 2 weeks after injury, total RNA was extracted as described above. Reverse transcription was performed using 5'-GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT-3' as the primer. Reaction mixtures were prepared on a block heated to 94°C and then subjected to 35 cycles of PCR amplification using high fidelity Ex Taq polymerase (Takara, Otsu, Japan). Each cycle consisted of 30 sec dena- turation, 1 min annealing, and 2 min extension. The primers were 5'-ACTCCTCCATTCTGACGTCCCTTCC-3' and 5'-GCGAGCTCCGCGGCCGCGTTTTTTTTTTTT-3,' and the annealing temperature was 68°C. Authors' contributions DKT, IN, and IS conceived of, designed, and coordinated the study. DKT, IN, and IS interpreted the results. DKT drafted the manuscript and performed: immunohisto- chemistry, confocal microscopy, image analysis, cell cul- ture, real-time PCR, and polyA tail assay experiments as well as statistical analyses. AL performed real-time PCR and 3' RACE experiments as well as statistical analyses. YM and IS prepared SNE animals. YM performed electrophys- iology experiments and statistical analyses. TO performed behavioral testing. EM performed immunohistochemis- try. SR performed real-time PCR and statistical analyses. IS and IN edited and helped to draft the manuscript. All authors read and approved the final manuscript. Acknowledgements Support was contributed by NIH grants DE14573, DA023163 and NS049137, NSF IGERT DGE9972802, the Takanawakai, the Clinical Implant Society of Japan, and the Japan Society for the Promotion of Sci- ence. This investigation was conducted in part in a UCLA facility con- structed with support from Research Facilities Improvement Program Grant Number C06 RR014529 from NCRR/NIH, and in the Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan with kind sup- port of Dr. Yasuhiko Tabata. DKT is currently supported by a US Depart- ment of Veterans Affairs Laboratory Research and Development Grant (PI: YD Teng) and would like to thank Dr. Teng for his support. Digoxigenin-labeled probes were generated by PCR using a standard kit and protocol (Roche). The primers were 5'- TTACCGGCTGAGTGTCCATAACAGG-3' and 5'-CATTCT- GACGTCCCTTCCGAGTTCC-3', and the annealing tem- perature was 66°C. PCR amplicons from the PolyA tail length assay were run on a 1.5% agarose gel, transferred and cross-linked to a positively charged nylon membrane by UV light. After probe hybridization, the membrane was washed and blocked with a standard kit (Roche, Indiana- polis, IN), and 1:10000 Anti-Digoxigenin-AP conjugate was applied. The membrane was washed again and devel- The authors declare that they have no competing interests. The authors declare that they have no competing interests. Compound action potential recording Compound action potentials (CAPs) were recorded from the sciatic nerves using a suction recording electrode [50,51]. Signals from the recording electrode and the arti- fact suppression electrode were fed into a differential amplifier (Dam 50, WPI, Sarasota, FL) in AC mode. A cal- ibration pulse (± 2 V across a 100 MΩ series resistor) was always included in the CAP recordings to estimate the resistance at the nerve/recording electrode junction [50,51]. Signals from CAP recordings were further ampli- fied (Brownlee Precision Instruments, model 440, Santa Clara, CA). Amplified signals were digitized at 10–20 kHz via the Digidata 1200B interface (Molecular Devices, Union City, CA) and displayed on a computer screen using the pCLAMP 8 software package (Molecular Devices). Data were analyzed offline using the pCLAMP 8 software package (Molecular Devices). Since CAP ampli- tude/area varies linearly with changes in this resistance [50,51], the CAP signal was adjusted based on the calibra- tion pulse amplitude changes during analysis of any given At 2 weeks after injury, rats were anesthetized (50 mg/kg pentobarbital, i.p.), and ipsilateral L4 and L5 DRG were harvested together with sciatic nerve segments. RNA was extracted using a combination of phenol/chloroform extraction and glass fiber purification (Trizol, Invitrogen, Carlsbad, CA, and RNAqueous, Ambion, Austin, TX), DNAse treated (Ambion, Austin, TX), and reverse-tran- scribed (Superscript III, Invitrogen, Carlsbad, CA) using random hexamer primers. Samples were subjected in trip- licate to real-time PCR under default cDNA conditions (Applied Biosystems, Foster City, CA) using a ready-made 18s rRNA primer and probe set (Applied Biosystems, Fos- ter City, CA) as an internal control. Ready-made Taqman expression assay sets (Applied Biosystems, Foster City, CA) were used for PCR amplification of various sodium channel cDNAs; the expression assay IDs were: NaV1.3, Rn00565438_m1; NaV1.5, Rn00565502_m1; NaV.16, Rn00570506_m1; NaV1.7, Rn00581647_m1; NaV1.8, Rn_00568393; NaV1.9, Rn00570487_m1. Page 9 of 12 (page number not for citation purposes) Molecular Pain 2009, 5:14 http://www.molecularpain.com/content/5/1/14 oped with CDP-Star (Roche, Indianapolis, IN). The mem- brane was exposed for 0.5–15 min and developed. oped with CDP-Star (Roche, Indianapolis, IN). The mem- brane was exposed for 0.5–15 min and developed. 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https://openalex.org/W4214768287	https://www.ssoar.info/ssoar/bitstream/document/77799/1/ssoar-griot-2022-1-brandao-Dilectio_resignacao_e_injustica_possibilidade.pdf	Portuguese	null	Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho	Griot	2,022	cc-by	14,628	www.ssoar.info Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho Brandão, Ricardo Evangelista Empfohlene Zitierung / Suggested Citation: Brandão, R. E. (2022). Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot: Revista de Filosofia, 22(1), 251-265. https:// doi.org/10.31977/grirfi.v22i1.2622 Empfohlene Zitierung / Suggested Citation: Brandão, R. E. (2022). Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot: Revista de Filosofia, 22(1), 251-265. https:// doi.org/10.31977/grirfi.v22i1.2622 Nutzungsbedingungen: Dieser Text wird unter einer CC BY Lizenz (Namensnennung) zur Verfügung gestellt. Nähere Auskünfte zu den CC-Lizenzen finden Sie hier: https://creativecommons.org/licenses/by/4.0/deed.de ISSN 2178-1036 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 https://doi.org/10.31977/grirfi.v22i1.2622 Recebido: 03/09/2021 \| Aprovado: 12/02/2022 Received: 09/03/2021 \| Approved: 02/12/2022 RESUMO: No “Comentário da primeira Epístola de São João”, em específico fragmento do texto em que o foco é a controvérsia donatista, Agostinho afirma que a despeito do fato de que o amor é norma incontornável à verdadeira vida cristã, no caso de testemunhar uma ação injusta, o cristão não pode aceitar passivamente a tal ato. Todavia, nesse mesmo texto, em outro contexto, é defendida uma espécie de resignação como prova de que o cristão de fato está vivendo uma prática cristã eivada pelo amor de Deus. Na De civitate Dei Livro XIX, no “Sobre os costumes da igreja católica e dos maniqueus”, e em outras obras, o mesmo paradoxo persiste: se por um lado o filósofo cristão reconhece que mesmo vivendo em intensa dor e sofrimento é possível se conformar e encontrar a paz, uma paz possível ante a situação, por outro lado, defende que não é justo um homem se submeter a outro visto que somos ontologicamente iguais, logo, aparentemente não incentivando a eupatia nessa injusta situação. Assim, é nossa meta nessa comunicação: investigar como podemos compreender a relação do amor com a resignação, e se ambos os conceitos podem ser aplicados às situações de flagrante injustiça vividas ou testemunhadas. PALAVRAS – CHAVE: Dilectio; Injustiça; Resignação. BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. DILECTIO, RESIGNAÇÃO E INJUSTIÇA: POSSIBILIDADE DE INTERPRETAR O AMOR COMO RESIGNAÇÃO E CONFORMAÇÃO COM A INJUSTIÇA, À LUZ DE SANTO AGOSTINHO Ricardo Evangelista Brandão1 Instituto Federal de Pernambuco (IFPE) https://orcid.org/0000-0003-2217-4661 E-mail: ricardobrand75@gmail.com Ricardo Evangelista Brandão1 Instituto Federal de Pernambuco (IFPE) https://orcid.org/0000-0003-2217-4661 E-mail: ricardobrand75@gmail.com 1 Doutor(a) em Filosofia pela Universidade Federal de Pernambuco (UFPE), Recife – PE, Brasil. Professor(a) do Instituto Federal de Pernambuco (IFPE), Caruaru – PE, Brasil. Introdução Os termos latinos dilectio, amor e caritas significam amor, e embora Agostinho em algumas obras utilize os termos de forma livre, com significados intercambiáveis e até como sinônimos como no De civitate Dei, em outras obras como In epistolam Ioannis ad Parthos (Comentário da primeira epístola de São João) no próprio texto esclarece a distinção que faz entre os termos amor e dilectio, sendo o primeiro um termo autorizado a ser usado para objetos inferiores, como quando se diz que se ama determinada refeição, todavia igualmente pode ser utilizado para objetos superiores. Já dilectio é utilizado para objetos mais elevados como para expressar a devoção a Deus (Cf. AGOSTINHO, In ep. Joan. ad Parthos VIII, 5)2. Dessa forma, levando em consideração a eloquência no uso das palavras e expressões da pena do filósofo, que o faz atribuir sentidos diversos ao sabor das circunstâncias e contextos, na prática o que determina o significado de dilectio, amor e caritas nas obras do filósofo é o contexto em que as palavras são usadas. Esse amor com sentido mais elevado do termo dilectio presente no “Comentário da primeira epístola de São João”, independente de quais dos três termos latinos o filósofo utiliza, é decorrente de uma conversão ao amor de Deus, e, por conseguinte por uma metafísica imersão no amor divino, habilitando assim o cristão a amar a Deus e ao próximo com o amor de Deus. Isto posto, doravante nesse artigo trabalharemos os termos dilectio, amor e caritas com o sentido de amor a objetos mais elevados, como a dilectio utilizada no “Comentário da primeira epístola de São João”, claro que sempre respeitando a intenção do autor ditada pelo contexto de uso dos termos. Assim, amor, sempre que o contexto permitir, será interpretado como um sentimento que predispõe uma inclinação ao objeto amado, tornando esse objeto um pondus que direciona a vida, decisões e ações do amante em direção do objeto amado, objeto esse de ordem superior como Deus e o próprio homem. Após essas breves mas necessárias observações acerca de como significar a dilectio, nos cabe doravante compreender se podemos interpretar a dilectio como uma resignação3 ao sofrimento de si mesmo e do próximo, ou pelo contrário, se o verdadeiro amor impossibilita esse conformar-se com os tormentos mencionados. 252 2 Ver também AGOSTINHO, De ver. rel. XLVII, 91. 3 O termo resignação nesse texto detém o seguinte significado: se conformar com uma situação de dor e sofrimento, sendo, portanto, uma forma de sujeição tranquila aos infortúnios, dificuldades, penúrias, fatalidades e até injustiças da vida (Cf. FERREIRA, 1986, p. 452, 1493). BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. ABSTRACT: In the “Commentary on the First Epistle of St. John”, in a specific fragment of the text in which the focus is the Donatist controversy, Augustine states that despite the fact that love is an unavoidable norm for the true Christian life, in case of witnessing an action unjust, the Christian cannot passively accept such an act. However, in that same text, in another context, a kind of resignation is defended as proof that the Christian is in fact living a Christian practice permeated by the love of God. In De civitate Dei Livro XIX, in “On the customs of the Catholic Church and the Manicheans”, and in other works, the same paradox persists: if on the one hand the Christian philosopher recognizes that even living in intense pain and suffering it is possible to conform and finding peace, a possible peace before the situation, on the other hand, defends that it is not fair for a man to submit to another since we are ontologically equal, therefore, apparently not encouraging eupathy in this unfair situation. Thus, our goal in this communication is: to investigate how we can understand the relationship between love and resignation, and whether both concepts can be applied to situations of flagrant injustice experienced or witnessed. KEYWORDS: Dilectio; Iustitia; Resignation. KEYWORDS: Dilectio; Iustitia; Resignation. 1 Doutor(a) em Filosofia pela Universidade Federal de Pernambuco (UFPE), Recife – PE, Brasil. Professor(a) do Instituto Federal de Pernambuco (IFPE), Caruaru – PE, Brasil. Artigo publicado em acesso aberto sob a licença Creative Commons Attribution 4.0 International License Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum Começando por um dos primeiros escritos entre os mencionados, o “A verdadeira religião” escrito entre 389-3914, período esse entre a tentativa de ócio teológico-filosófico e a ordenação sacerdotal de Agostinho, escrito na intenção de apresentar a verdadeira religião aos amigos que outrora o viram apregoar o maniqueísmo, como o seu benfeitor Romaniano. Dessa forma temos uma obra em que Agostinho contava com 36 anos, e com aproximadamente três anos de conversão à fé cristã quando começou a redigir o livro, se tratando assim de um Agostinho jovem, e embora sem longa vivência na fé e com poucas responsabilidades no cristianismo, não podemos considerá-lo como um neófito nesse período ante ao teor do texto em análise. Destarte, em certo trecho da “A verdadeira religião” o pensador descreve o que em sua compreensão seria o homem justo que foi inundado pelo amor de Deus, e ao descrever essas características estabelece que esse homem dispõe de uma capacidade para suportar tribulações diversas, devido ao fato de ele saber que seu real repouso e felicidade não está nessa esfera terrena, mas em uma vida mais plena e vindoura com a parousia de Cristo. Esse justo aproveitaria as farturas com moderação, aprenderia com os sofrimentos e até amadureceria espiritualmente com eles, ou seja, o homem justificado pelo amor de Deus é inabalável, pois é cônscio de que nada nessa terra lhe pertence, de forma que esse desapego o capacita a suportar as maiores tormentas sem se entristecer “Para tal homem é pouco não se entristecer com as tribulações. Ele gloria-se delas [...]” (AGOSTINHO, De ver. rel. XLVII, 92)5. Esse estranho regozijo (gaudeat) oriundo da tribulação acontece por dois motivos: por ter condições de se aperfeiçoar interiormente com o sofrimento, e pelo desapego que esse justo sente pelas coisas materiais que nos podem dar conforto e prazer nessa vida terrena. Assim, a pessoa que lutou muito para conquistar muitos recursos materiais, pelo esforço que teve acaba se apegando ao que conquistou, e embora aos olhos de todos possa parecer vencedora, na medida em que perde esses recursos por alguma possível adversidade, o apego a tais bens lhes causará sofrimento, esse supostamente bem sucedido na verdade pode ser considerado fracassado. 5 “Huic viro in tribulationibus parum est non contristari, nisi etiam gaudeat [...]” (AGOSTINHO, De ver. rel. XLVII, 92 – PL 34). 6 Essa reflexão de Agostinho lembra muito, nas devidas proporções, as elucubrações do segundo estoicismo, pois, Epíteto nos revela como o grande princípio da sabedoria e consequente felicidade, distinguir entre o que podemos alterar e o que não podemos alterar, e é óbvio que essa distinção por si só não vai adiantar muito na vida se não for seguida pela ação. Dessa forma, depois da ciência que ressaltamos, o homem verdadeiramente sábio deve escolher (proaireses) viver segundo essa distinção, ou seja, se preocupar com o que realmente ele pode alterar, e não desperdiçar suas energias com aquilo que não se pode alterar. “Das coisas existentes, umas estão em nosso poder e outras não estão em nosso poder. Em nosso poder certamente a suposição, o impulso, o desejo, o desvio e numa palavra todas as nossas ações; não estão em nosso poder o corpo, as opiniões, os reinos e numa palavra quantas não são as nossas ações” (EPÍTETO, Manual, I, 1). Quando o homem escolhe viver levando em consideração a sábia distinção que apontamos, não será conduzido à inação, muito pelo contrário aperfeiçoará as suas ações para o que realmente importa na vida, amadurecendo no que deve amadurecer e aproveitando melhor cada instante da vida. Mas se pelo contrário, quando no viver não se considera como norteador das ações essa distinção, se desperdiçará energias com o que não vala à pena, de forma que viverá amaldiçoando a vida e todos ao redor, pois nunca conseguirá tirar o melhor proveito do viver (Cf. EPÍTETO, Manual, I, 3; II, 1, 2). Introdução Para esclarecer e refletirmos sobre o problema posto à luz do pensamento de Agostinho, elencamos como objeto de análise alguns fragmentos de quatro obras do pensador que lançam importante luz sobre a questão, notadamente: De vera religione, De moribus Ecclesiae Catholicae et de moribus manichaeorum, In Epistolam Ioannis ad Parthos tratactus decem, De civitate Dei. No primeiro capítulo examinaremos as duas primeiras obras, que inclusive foram escritas em períodos muito próximos, notadamente entre 387 – 391, revelando as reflexões de Agostinho sobre o assunto pouco tempo depois de seu batismo, período marcado pelo batismo, tentativa de ócio filosófico e o começo da ordenação sacerdotal. E no segundo capítulo abordaremos a temática por meio da análise de fragmentos das duas últimas obras listadas, escritas em 416 e 413-426 respectivamente, fase intelectual de um Agostinho bispo, maduro na fé que adotou, e com muitas responsabilidades civis e eclesiásticas. 252 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 ISSN 2178-1036 BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. 4 A datação das obras que analisaremos nesse texto, terá como base a cronologia das obras de Agostinho presente no primeiro volume da coleção “Obras Completas de San Agustin” da Biblioteca de Autores Cristianos (BAC) organizada por Victorino Capanaga (Cf. CAPANAGA, 1994, v. 1, p. 384-387). Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum De forma que o verdadeiro vencedor é o homem que não se apega ao que não depende dele para a manutenção, mas o que se apega às coisas que o perder e o manter dependem absolutamente dele6 (Cf. AGOSTINHO, De ver. rel. XLVII, 92). 253 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 ISSN 2178-1036 No mesmo contexto do fragmento citado, Agostinho afirma que o homem que ama Deus de todo o coração se torna invencível, pois a sua principal atenção estará voltada a Deus, e esse Deus lhe trará uma paz e contentamento que independe de circunstâncias exteriores. Além disso, poderíamos questionar, e se o sofrimento for com os outros? Pois uma coisa é ter a capacidade interior para absolver os sofrimentos da vida e resignadamente viver em paz, na medida em que se sabe que como cristão a sua real felicidade não está nesse mundo, e outra coisa bem diferente é perceber a dor e o sofrimento do próximo e ficar em paz com isso. A questão é essa, na medida em que essa resignação do cristão provém justamente do amor a Deus, visto que esse amor a Deus nos exige um amor da mesma qualidade para com o nosso próximo, o sofrimento do outro seria o limite dessa resignação? Vale à pena transcrever o que pensa Agostinho sobre o problema posto: Quem ama a Deus de todo o coração não se aflige, pois, com a morte de ninguém. Sabe bem que não perece para ele, quem não perece para Deus, pois Deus é o senhor dos vivos e dos mortos. Não fica desesperado pela miséria de ninguém, como também não se sente justificado pela justiça dos outros (AGOSTINHO, De ver. rel. XLVII, 91)7. A morte não deve causar sofrimento, devido à consciência de que a vida continua após a morte (Deus autem dominus est et vivorum et mortuorum – Deus é senhor dos vivos e dos mortos), de forma que a morte nada mais é que uma passagem para outra etapa de nossas vidas, é uma referência apenas humana da transitoriedade corpórea. 254 7 “Non ergo iste affligitur morte cuiusquam, quoniam qui toto animo Deum diligit, novit nec sibi perire quod Deo non perit. Deus autem dominus est et vivorum et mortuorum. Non cuiusquam miseria miser est, quia nec cuiusquam iniustitia fit iniustus. Et ut nemo illi iustitiam et Deum, sic nemo aufert beatitudinem” (AGOSTINHO, De ver. rel. XLVII, 91 – PL 34). 8 “Et si quando forte alicuius periculo, vel errore, vel dolore commovetur; usque ad illius auxilium, aut correctionem, aut consolationem, non usque ad suam subversionem valere patitur” (AGOSTINHO, De ver. rel. XLVII, 91 – PL 34). BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum Obviamente Agostinho está fiado na tese da imortalidade da alma, visto que Deus continua sendo o Senhor dessa alma imortal, que em algum momento (parousia) receberá corpo (corpo transformado) novamente por meio da ressurreição, pois esse fragmento faz referência ao texto bíblico de Rom., 14, 9, que defende o senhorio universal de Cristo e que todos acertarão as contas com Deus na parousia, no grande tribunal de Deus. Com essa base teológica, certa justificação existe na relação do homem com a morte, todavia, o não sentir empatia com o sofrimento e com as alegrias do próximo fica mais difícil de justificar. Estamos diante de uma forma de apatheia cristã, em que o cristão eivado pela certeza de uma relação futura com Deus se resigna com as mais severas dores, sofridas por si ou pelo próximo. De fato estamos ante algo muito semelhante à imperturbabilidade estoica, em formato cristão é óbvio, pois essa ampla resignação provém da imersão no amor de Deus, e esse que a alcançou é de fato um sábio na prática da fé. Semelhantemente, como no estoicismo existem diversos níveis de sabedoria, pois o próprio Epíteto entendia não ser ele um modelo de sabedoria estoica, remetendo esse modelo a Diógenes (Cf. REALE, 1994, vol. IV, p. 95). O pensador de Hipona menciona que alguns com nível menor de resignação, não conseguirão não sofrer com as dores do próximo: “Se alguma vez é atingido por demais, pelos perigos ou extravios e dores de outrem, ele aceita o impacto dessa emoção no sentido de ir socorrer, corrigir e consolar o outro. E não ficar ele mesmo transtornado por essas coisas” (AGOSTINHO, De ver. rel. XLVII, 91)8. Essa invencibilidade decorrente da tranquila resignação é possível ser rompida, no caso exemplificado no texto quando diante do sofrimento alheio. Essa exceção apontada é bastante coerente, pois, na medida em que o cristão ama o próximo com um amor de qualidade superior, 254 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 ISSN 2178-1036 semelhante ao despendido a Deus9, seria dissonante esse se conformar. Do capítulo 45 até o 48, Agostinho se concentra em refletir acerca da transformação realizada pelo amor na vida do fiel, e no fragmento citado essa imersão no amor de Deus, visto que o cristão passa a amar com esse amor, não lhe permite a indiferença. BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. 9 Nosso filósofo se refere ao amor a Deus, expressando uma devoção elevada e desinteressada, todavia, sem embargo aplica essa qualidade de amor igualmente ao nosso próximo. Ou seja, após a conversão espiritual por meio da aceitação do sacrifício de Cristo na cruz, o agora cristão deve amar ao próximo com esse amor superior, o qual conceituamos na introdução como dilectio, ou simplificando ainda mais, o cristão deve amar ao próximo com o amor de Deus. 10 A despeito de ter mencionado a semelhança das intuições de Agostinho com as reflexões do segundo estoicismo no corpo do texto e em nota de rodapé, é relevante ressaltar que existiu certa influência no movimento monástico cristão (monaquismo cristão) de teorias e modo de vida estoico. De certa forma os ensinamentos estoicos relacionados à aphatheia, em que os afetos externos empanam a verdadeira sabedoria de forma que o sábio se dedicaria ao aperfeiçoamento da alma e do controle das paixões tanto corpóreas como das inquietações advindas do mundo, estão presentes nas orientações monásticas (Cf. GONZALEZ, 1995, v. 2, p. 61). Dessa forma, é prudente lembrar que Agostinho pouco antes de sua adesão ao cristianismo, foi fortemente impactado pela história do monge anacoreta Antônio do Egito, conhecido como Santo Antão, relatado por ele mesmo no oitavo livro das Confissões. Agostinho narra que enquanto ele estava imerso nas consequências de uma possível adesão ao cristianismo, pois teria que abandonar prazeres, honras, cargos e mulheres, é visitado por um dignitário do império romano de naturalidade africana chamado Ponticiano. Este ao ver as cartas de Paulo sobre a mesa começa a relatar a história de vida de Antônio do Egito (Cf. AGOSTINHO, Confissões, VIII, 6, 14), que este teve contato pela leitura do livro “Vida e Conduta de Santo Antão” escrito pelo bispo católico Atanásio. Pelo testemunho escrito de Atanásio, Antônio do Egito viveu entre 251-269, nasceu rico, mas nunca se interessou em demasia em aproveitar a riqueza do pai. Quando ainda muito jovem, entre 18 a 20 anos, os pais morreram, e ao entrar em uma igreja escutou a leitura do Evangelho de Mateus 19: 21, que em síntese exorta a vender tudo e seguir a Cristo, o que antão faz reservando uma pequena parte da fortuna para cuidar da irmã menor. Todavia volta a entrar na igreja em outro momento e ouve a leitura de Mateus 6:34, passagem do sermão do monte em que Jesus orienta a não se preocupar com o dia de amanhã. Conta-nos Atanásio que depois dessa segunda experiência Antão distribuiu o restante dos bens, deixou sua irmã aos cuidados de uma casa de virgens para ser cuidada e educada, e começou a se preparar para a vida monástica (Cf. ATANÁSIO, Vida e Conduta de Santo Antão, I, 3). Segundo a narrativa de Atanásio Antão na intenção de aprender sobre a prática da vida monástica com outros monges mais experientes, principia no monaquismo cenobítico, e quando se sentiu preparado iniciou no monaquismo anacoreta (Cf. ATANÁSIO, Vida e Conduta de Santo Antão, I, 4 – I, 15). A vida desses monges era extremamente simples como nos descreve Justo Gonzalez: “A vida dessas pessoas era extremamente simples. Alguns cultivavam hortas, mas a maioria se sustentava tecendo cestas e esteiras que vendiam em troca de um pouco de azeite. [...] A dieta da maioria dos monges consistia em pão e, às vezes, frutas, legumes e azeite. Suas posses não excediam a vestimenta necessária e uma esteira para dormir” (GONZALEZ, 1995, v. 2, p. 67). Com relação ao desapego de bens materiais do monge Antão, conta-nos Atanásio: “Comia só uma vez por dia, depois do pôr-do-sol, e acontecia por vezes de tomar alimento apenas de dois em dois dias, muitas vezes até de quatro em quatro. Ora, seu alimento era pão e sal; a bebida água pura. [...] Para dormir, contentava-se com uma esteira, e na maior parte do tempo dormia na terra nua (ATANÁSIO, Vida e Conduta de Santo Antão, I, 7). Ao relatar o primeiro contato de Antão com as pessoas após vinte anos solitário no deserto, nos revela a aphatheia ante a quaisquer afetos: “[...] nele nem riso nem tristeza; a multidão não o perturbava, as muitas pessoas que o saudavam não lhe davam alegria excessiva: sempre igual a si mesmo governado pela razão [...] (ATANÁSIO, Vida e Conduta de Santo Antão, I, 14). Com uma leitura um pouco mais atenta das Confissões VIII, é perceptível o tremendo impacto da exposição de Ponticiano sobre Antão na compreensão de Agostinho sobre a vida cristã, e apesar de nos textos analisados e que analisaremos no corpo do texto não existir clara sinalização, entendemos ser perfeitamente possível que essa aparência de aphatheia estoica nos textos discutidos seja na verdade decorrente do forte efeito do testemunho de autoridades espirituais do quilate do Monge Antão, na medida em que é possível encontrar nos enfrentamentos das paixões e no desapego absoluto com relação aos bens materiais, certo ponto de encontro entre as máximas estoicas e as práticas dos monges anacoretas. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum Com relação ao desapego de bens materiais do monge Antão, conta-nos Atanásio: “Comia só uma vez por dia, depois do pôr-do-sol, e acontecia por vezes de tomar alimento apenas de dois em dois dias, muitas vezes até de quatro em quatro. Ora, seu alimento era pão e sal; a bebida água pura. [...] Para dormir, contentava-se com uma esteira, e na maior parte do tempo dormia na terra nua (ATANÁSIO, Vida e Conduta de Santo Antão, I, 7). Ao relatar o primeiro contato de Antão com as pessoas após vinte anos solitário no deserto, nos revela a aphatheia ante a quaisquer afetos: “[...] nele nem riso nem tristeza; a multidão não o perturbava, as muitas pessoas que o saudavam não lhe davam alegria excessiva: sempre igual a si mesmo governado pela razão [...] (ATANÁSIO, Vida e Conduta de Santo Antão, I, 14). Com uma leitura um pouco mais atenta das Confissões VIII, é perceptível o tremendo impacto da exposição de Ponticiano sobre Antão na compreensão de Agostinho sobre a vida cristã, e apesar de nos textos analisados e que analisaremos no corpo do texto não existir clara sinalização, entendemos ser perfeitamente possível que essa aparência de aphatheia estoica nos textos discutidos seja na verdade decorrente do forte efeito do testemunho de autoridades espirituais do quilate do Monge Antão, na medida em que é possível encontrar nos enfrentamentos das paixões e no desapego absoluto com relação aos bens materiais, certo ponto de encontro entre as máximas estoicas e as práticas dos monges anacoretas. 9 Nosso filósofo se refere ao amor a Deus, expressando uma devoção elevada e desinteressada, todavia, sem embargo aplica essa qualidade de amor igualmente ao nosso próximo. Ou seja, após a conversão espiritual por meio da aceitação do sacrifício de Cristo na cruz, o agora cristão deve amar ao próximo com esse amor superior, o qual conceituamos na introdução como dilectio, ou simplificando ainda mais, o cristão deve amar ao próximo com o amor de Deus. 10 A despeito de ter mencionado a semelhança das intuições de Agostinho com as reflexões do segundo estoicismo no corpo do texto e em nota de rodapé, é relevante ressaltar que existiu certa influência no movimento monástico cristão (monaquismo cristão) de teorias e modo de vida estoico. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum De certa forma os ensinamentos estoicos relacionados à aphatheia, em que os afetos externos empanam a verdadeira sabedoria de forma que o sábio se dedicaria ao aperfeiçoamento da alma e do controle das paixões tanto corpóreas como das inquietações advindas do mundo, estão presentes nas orientações monásticas (Cf. GONZALEZ, 1995, v. 2, p. 61). Dessa forma, é prudente lembrar que Agostinho pouco antes de sua adesão ao cristianismo, foi fortemente impactado pela história do monge anacoreta Antônio do Egito, conhecido como Santo Antão, relatado por ele mesmo no oitavo livro das Confissões. Agostinho narra que enquanto ele estava imerso nas consequências de uma possível adesão ao cristianismo, pois teria que abandonar prazeres, honras, cargos e mulheres, é visitado por um dignitário do império romano de naturalidade africana chamado Ponticiano. Este ao ver as cartas de Paulo sobre a mesa começa a relatar a história de vida de Antônio do Egito (Cf. AGOSTINHO, Confissões, VIII, 6, 14), que este teve contato pela leitura do livro “Vida e Conduta de Santo Antão” escrito pelo bispo católico Atanásio. Pelo testemunho escrito de Atanásio, Antônio do Egito viveu entre 251-269, nasceu rico, mas nunca se interessou em demasia em aproveitar a riqueza do pai. Quando ainda muito jovem, entre 18 a 20 anos, os pais morreram, e ao entrar em uma igreja escutou a leitura do Evangelho de Mateus 19: 21, que em síntese exorta a vender tudo e seguir a Cristo, o que antão faz reservando uma pequena parte da fortuna para cuidar da irmã menor. Todavia volta a entrar na igreja em outro momento e ouve a leitura de Mateus 6:34, passagem do sermão do monte em que Jesus orienta a não se preocupar com o dia de amanhã. Conta-nos Atanásio que depois dessa segunda experiência Antão distribuiu o restante dos bens, deixou sua irmã aos cuidados de uma casa de virgens para ser cuidada e educada, e começou a se preparar para a vida monástica (Cf. ATANÁSIO, Vida e Conduta de Santo Antão, I, 3). Segundo a narrativa de Atanásio Antão na intenção de aprender sobre a prática da vida monástica com outros monges mais experientes, principia no monaquismo cenobítico, e quando se sentiu preparado iniciou no monaquismo anacoreta (Cf. ATANÁSIO, Vida e Conduta de Santo Antão, I, 4 – I, 15). A vida desses monges era extremamente simples como nos descreve Justo Gonzalez: “A vida dessas pessoas era extremamente simples. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum Dessa forma, essa aphatheia cristã10 teorizada pelo hiponense não pode ser confundida com frieza ante as dores do próximo. Contudo, ao deixar de se conformar com a mencionada situação, não deve permitir que esse sentimento de inconformação se transforme em lamúria, mas deve-se canalizar esse se afetar em direção ao socorro do outro. A despeito da sensibilidade do filósofo no fragmento analisado, o que nos causa certa estranheza não é a exceção apontada, mas a possibilidade de imperturbabilidade mesmo ante o sofrimento alheio, aliás, pela forma como foi construída a argumentação agostiniana, evidencia- se a impressão que o se afetar com essas dores do outro seria o resultado de um menor amadurecimento espiritual, uma vez que até mesmo as vitórias dos outros não nos devem gerar regozijo. O texto não deixa dúvidas quanto a isso, é dito Et si quando (E se alguma vez), quer dizer, a irresignação para com os infortúnios de outrem se alguma vez te afetar se devem tomar 9 Nosso filósofo se refere ao amor a Deus, expressando uma devoção elevada e desinteressada, todavia, sem embargo aplica essa qualidade de amor igualmente ao nosso próximo. Ou seja, após a conversão espiritual por meio da aceitação do sacrifício de Cristo na cruz, o agora cristão deve amar ao próximo com esse amor superior, o qual conceituamos na introdução como dilectio, ou simplificando ainda mais, o cristão deve amar ao próximo com o amor de Deus. 10 A despeito de ter mencionado a semelhança das intuições de Agostinho com as reflexões do segundo estoicismo no corpo do texto e em nota de rodapé, é relevante ressaltar que existiu certa influência no movimento monástico cristão (monaquismo cristão) de teorias e modo de vida estoico. De certa forma os ensinamentos estoicos relacionados à aphatheia, em que os afetos externos empanam a verdadeira sabedoria de forma que o sábio se dedicaria ao aperfeiçoamento da alma e do controle das paixões tanto corpóreas como das inquietações advindas do mundo, estão presentes nas orientações monásticas (Cf. GONZALEZ, 1995, v. 2, p. 61). Dessa forma, é prudente lembrar que Agostinho pouco antes de sua adesão ao cristianismo, foi fortemente impactado pela história do monge anacoreta Antônio do Egito, conhecido como Santo Antão, relatado por ele mesmo no oitavo livro das Confissões. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum Agostinho narra que enquanto ele estava imerso nas consequências de uma possível adesão ao cristianismo, pois teria que abandonar prazeres, honras, cargos e mulheres, é visitado por um dignitário do império romano de naturalidade africana chamado Ponticiano. Este ao ver as cartas de Paulo sobre a mesa começa a relatar a história de vida de Antônio do Egito (Cf. AGOSTINHO, Confissões, VIII, 6, 14), que este teve contato pela leitura do livro “Vida e Conduta de Santo Antão” escrito pelo bispo católico Atanásio. Pelo testemunho escrito de Atanásio, Antônio do Egito viveu entre 251-269, nasceu rico, mas nunca se interessou em demasia em aproveitar a riqueza do pai. Quando ainda muito jovem, entre 18 a 20 anos, os pais morreram, e ao entrar em uma igreja escutou a leitura do Evangelho de Mateus 19: 21, que em síntese exorta a vender tudo e seguir a Cristo, o que antão faz reservando uma pequena parte da fortuna para cuidar da irmã menor. Todavia volta a entrar na igreja em outro momento e ouve a leitura de Mateus 6:34, passagem do sermão do monte em que Jesus orienta a não se preocupar com o dia de amanhã. Conta-nos Atanásio que depois dessa segunda experiência Antão distribuiu o restante dos bens, deixou sua irmã aos cuidados de uma casa de virgens para ser cuidada e educada, e começou a se preparar para a vida monástica (Cf. ATANÁSIO, Vida e Conduta de Santo Antão, I, 3). Segundo a narrativa de Atanásio Antão na intenção de aprender sobre a prática da vida monástica com outros monges mais experientes, principia no monaquismo cenobítico, e quando se sentiu preparado iniciou no monaquismo anacoreta (Cf. ATANÁSIO, Vida e Conduta de Santo Antão, I, 4 – I, 15). A vida desses monges era extremamente simples como nos descreve Justo Gonzalez: “A vida dessas pessoas era extremamente simples. Alguns cultivavam hortas, mas a maioria se sustentava tecendo cestas e esteiras que vendiam em troca de um pouco de azeite. [...] A dieta da maioria dos monges consistia em pão e, às vezes, frutas, legumes e azeite. Suas posses não excediam a vestimenta necessária e uma esteira para dormir” (GONZALEZ, 1995, v. 2, p. 67). 9 Nosso filósofo se refere ao amor a Deus, expressando uma devoção elevada e desinteressada, todavia, sem embargo aplica essa qualidade de amor igualmente ao nosso próximo. Ou seja, após a conversão espiritual por meio da aceitação do sacrifício de Cristo na cruz, o agora cristão deve amar ao próximo com esse amor superior, o qual conceituamos na introdução como dilectio, ou simplificando ainda mais, o cristão deve amar ao próximo com o amor de Deus. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum rel., 46, 87), pois como defender essa imperturbabilidade perante o sofrimento do outro e mesmo assim assumir o amor ao próximo como valor incondicionalmente oriundo da relação com o Sagrado? Uma forma de interpretar o fragmento preservando-o de possíveis incongruências seria interpretar que essa passividade que é regra, se refere exclusivamente aos sentimentos para não se perder a paz interior, e não a ação do cristão no socorro ao combalido. Embora tal compreensão não estaria fora de remotas possibilidades, o texto não é claro quanto a isso, de forma que tal interpretação poderia ser uma exegese criativa não autorizada pelo texto na intenção de preservar o autor de incoerências. Dessa forma, para evitar o risco de eisegese, se faz necessário o estudo de outros textos do filósofo em análise escritos sobre o assunto. tais medidas, nos dando a informação que nesse texto o hiponense entende que esse sentimento de inconformação é uma exceção, pois a regra é a resignação. Sem dúvida a perícope analisada não parece coerente com o amor bidimensional (a Deus e ao próximo) dos evangelhos sinóticos assumidos por Agostinho (Cf. AGOSTINHO, De civ. Dei, XIX, 23, 5; AGOSTINHO, In ep. Joan. ad Parthos, V, 3; AGOSTINHO, De ver. rel., 46, 87), pois como defender essa imperturbabilidade perante o sofrimento do outro e mesmo assim assumir o amor ao próximo como valor incondicionalmente oriundo da relação com o Sagrado? Uma forma de interpretar o fragmento preservando-o de possíveis incongruências seria interpretar que essa passividade que é regra, se refere exclusivamente aos sentimentos para não se perder a paz interior, e não a ação do cristão no socorro ao combalido. Embora tal compreensão não estaria fora de remotas possibilidades, o texto não é claro quanto a isso, de forma que tal interpretação poderia ser uma exegese criativa não autorizada pelo texto na intenção de preservar o autor de incoerências. Dessa forma, para evitar o risco de eisegese, se faz necessário o estudo de outros textos do filósofo em análise escritos sobre o assunto. 11 O De moribus Ecclesiae Catholicae et de moribus manichaeorum, foi escrito entre 387 - 388, logo, pouco mais de um ano após a sua adesão ao cristianismo. Obra dividida em duas partes, uma sobre os costumes da igreja e outro sobre os costumes dos maniqueus. O próprio Agostinho esclarece a motivação da obre em seu livro “Retratações” o qual transcrevemos: “Estando e Roma já batizado, e não podendo tolerar a jactância dos maniqueus a respeito de sua falsa e falaz continência ou abstinência com a qual, para enganar os ignorantes, colocavam-se superiores aos verdadeiros cristãos, aos quais não podem ser comparados, escrevi dois livros: um Sobre os costumes da Igreja Católica, o outro Sobre os costumes dos maniqueus” (AGOSTINHO, Retract. I, 7, 1). “Iam baptizatus autem cum Romae essem, nec ferre tacitus possem Manichaeorum iactantiam de falsa et fallaci continentia vel abstinentia, qua se ad imperitos decipiendos, veris Christianis, quibus comparandi non sunt, insuper praeferunt, scripsi duos libros, unum De moribus Ecclesiae catholicae, et alterum De moribus Manichaeorum” (AGOSTINHO, Retract. I, 7, 1 – PL 32). Dessa forma, trata-se de uma obra de cunho apologética, visto que ele pretende demonstrar que a santidade propagada pelos ensinos dos discípulos de Mani além de ser exterior, é muito inferior à santidade dos autênticos discípulos de Cristo (Cf. PRIETO, 2011, p. 208). É importante ressaltar que o hiponense viveu por nove anos na categoria de ouvinte na seita maniqueísta, de maneira que a conhecia com propriedade, de forma que sentiu a necessidade de esclarecer apologeticamente a falta de fundamento da moralidade maniqueia e os fundamentos da moralidade cristã. Ao fazer essa comparação entre as duas doutrinas morais, Agostinho acaba ensinando bastante sobre a filosofia cristã, e de forma clara e aprofundada nos explica o papel do amor e da relação desse amor com o socorro ao próximo na vivência cristã. BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum Alguns cultivavam hortas, mas a maioria se sustentava tecendo cestas e esteiras que vendiam em troca de um pouco de azeite. [...] A dieta da maioria dos monges consistia em pão e, às vezes, frutas, legumes e azeite. Suas posses não excediam a vestimenta necessária e uma esteira para dormir” (GONZALEZ, 1995, v. 2, p. 67). Com relação ao desapego de bens materiais do monge Antão, conta-nos Atanásio: “Comia só uma vez por dia, depois do pôr-do-sol, e acontecia por vezes de tomar alimento apenas de dois em dois dias, muitas vezes até de quatro em quatro. Ora, seu alimento era pão e sal; a bebida água pura. [...] Para dormir, contentava-se com uma esteira, e na maior parte do tempo dormia na terra nua (ATANÁSIO, Vida e Conduta de Santo Antão, I, 7). Ao relatar o primeiro contato de Antão com as pessoas após vinte anos solitário no deserto, nos revela a aphatheia ante a quaisquer afetos: “[...] nele nem riso nem tristeza; a multidão não o perturbava, as muitas pessoas que o saudavam não lhe davam alegria excessiva: sempre igual a si mesmo governado pela razão [...] (ATANÁSIO, Vida e Conduta de Santo Antão, I, 14). Com uma leitura um pouco mais atenta das Confissões VIII, é perceptível o tremendo impacto da exposição de Ponticiano sobre Antão na compreensão de Agostinho sobre a vida cristã, e apesar de nos textos analisados e que analisaremos no corpo do texto não existir clara sinalização, entendemos ser perfeitamente possível que essa aparência de aphatheia estoica nos textos discutidos seja na verdade decorrente do forte efeito do testemunho de autoridades espirituais do quilate do Monge Antão, na medida em que é possível encontrar nos enfrentamentos das paixões e no desapego absoluto com relação aos bens materiais, certo ponto de encontro entre as máximas estoicas e as práticas dos monges anacoretas. 255 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 tais medidas, nos dando a informação que nesse texto o hiponense entende que esse sentimento de inconformação é uma exceção, pois a regra é a resignação. Sem dúvida a perícope analisada não parece coerente com o amor bidimensional (a Deus e ao próximo) dos evangelhos sinóticos assumidos por Agostinho (Cf. AGOSTINHO, De civ. Dei, XIX, 23, 5; AGOSTINHO, In ep. Joan. ad Parthos, V, 3; AGOSTINHO, De ver. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum Um texto que lançará importante luz sobre o tema, escrito aproximadamente dois anos antes do texto outrora analisado, é o “Sobre os costumes da Igreja Católica e dos maniqueus”11, livro interessante pois construído em contexto de vida semelhante ao livro anterior, quando Agostinho após abandonar a categoria de ouvinte maniqueu, se converte ao cristianismo, decidindo confrontar a superioridade da nova fé com as falhas da fé apostatada. Rito contínuo, no capítulo vinte e sete do primeiro livro, o que trata dos costumes da igreja, Aurélio Agostinho na intenção de expandir a extensão do amor ao próximo, afirma que podemos dizer que amamos o próximo em duas dimensões: a corpórea e a da alma, pois o homem é esse composto de corpo e alma, logo, para amar o homem é necessário amá-lo integralmente e não delimitar a dimensão que lhe é mais conveniente (Cf. AGOSTINHO, De mor. Eccl. cath et mor. Man. I, 27, 52,53). Pela atenção que ele dá ao amor ao corpo, nas entrelinhas possivelmente está implícito a insistência de alguns cristãos que dizem amar o próximo se dedicando exclusivamente a alma do mesmo, ou seja, ensinando, exortando, aconselhando, orando pela salvação, etc. O problema não é o cuidado com a alma do outro, mas fazê-lo em detrimento do cuidado com a dimensão corpórea desse outro. Cuidar do corpo é: alimentar, saciar a sede, cuidar das feridas, vestir, dá abrigo, etc. Ou seja, o amor verdadeiro ao próximo, envolve toda essa preocupação e cuidado com a alma, mas também ação de cuidar do corpo do outro quando houver necessidade. Nesse contexto, a questão deixada em aberto na análise do De vera religione é melhor esclarecida no texto atual objeto de nossa análise. Assim, em exortação acerca da importância 256 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 do cuidado com as necessidades corpóreas do outro como evidência do amor ao próximo, afirma o filósofo cristão: São misericordiosos quem por cortesia e humanidade oferecem o que é necessário para resistir a estes males e dores, ainda que alcance tal nível de sabedoria, que sua alma não se afete com dor ou tribulação alguma. Quem não sabe que a palavra misericórdia significa tornar miserável ou infeliz o coração que se aflige como mal dos outros? (AGOSTINHO, De mor. Eccl. cath et mor. Man. I, 27, 53)12. 257 12 Quare illa omnia, quibus huiuscemodi malis incommodisve resistitur, qui officiose atque humiliter praebent, misericordes vocantur, etiamsi sapientes usque adeo sint, ut iam nullo animi dolore turbentur; nam quis ignoret ex eo appellatam esse misericordiam, quod miserum cor faciat condolescentis alieno malo? (AGOSTINHO, De mor. Eccl. cath et mor. Man. I, 27, 53 – PL 32). 13 O cisma donatista surge no início de século IV, liderado por Donato, em consequência da interpretação do comportamento que a igreja deveria ter em relação às pessoas que apostataram a fé, na época da última grande perseguição romana ao cristianismo desenvolvida pelo imperador Diocleciano. Os denominados donatistas entendiam que as pessoas que apostataram a fé entregando as escrituras às autoridades romanas devido ao decreto imperial, não deveriam ser mais aceitos como autoridades eclesiástica, e seus atos eclesiais não deveriam ser autenticados pela igreja. Assim, no ano 311 um grupo de cristãos liderados por Donato solicitaram a deposição do recém escolhido bispo de Cartago Ceciliano, afirmando que a sua ordenação não era legítima por ter sido realizada pelo bispo Félix que o grupo acusava de apostatar da fé na perseguição mencionada (Cf. CAIRNS, 1995, p. Resignação e o amor ao próximo à luz das obras: De vera religione e De moribus ecclesiae Catholicae et de moribus manichaeorum Está presente no texto a ideia de que o sábio dispõe de total controle de suas emoções, de forma que possui controle racional de qualquer afecção, e, portanto uma forma de resignação gerada pela sabedoria. Contudo, o sofrimento alheio, quando é intenso, de forma que outrem se encontre em estado de penúria, existe uma natural tendência de afecção, que no fragmento citado igualmente está no controle do sábio. Entretanto, esse controle resignado com relação à inópia alheia, não significa inação. Agostinho reconhece que o termo misericordia significa compadecer-se pelo sofrimento dos miseráveis, todavia entende que existem pessoas que alcançaram tal nível de sabedoria que socorrem os miseráveis sem ser movidos por empatia, mas pelo simples dever de agir com bondade, uma forma de deontologia cristã. Contudo, mesmo nesse caso Agostinho entende que esses sábios por causa de não se permitir a inação, podem ser chamados de misericordiosos. Se existiam algumas interrogações suspensas, entendemos que elas foram deveras respondidas, pois, a despeito da existência do ideal do sábio imperturbável em qualquer situação, inclusive ante o sofrimento alheio, há uma clara exortação de que isso não pode conduzir à inércia. Isso fica patente no contexto do fragmento citado, no qual o hiponense critica severamente os homens que se afastam dos miseráveis para não serem afetados com tal estado de sofrimento, e ao não serem abalados agem como se tal situação de dor alheia não existisse. Esses não agem com serenidade provinda da sabedoria, mas como uma frieza decorrente de falta de humanidade (Cf. AGOSTINHO, De mor. Eccl. cath et mor. Man. I, 27, 54). Na prática o pensador ressignifica o termo misericordia, como agir para afastar o sofrimento do próximo ao invés de sentir as estocadas das dores do próximo, de forma que apenas se abalar e nada fazer não seria de fato uma ação misericordiosa, mas apenas emocionalismo. BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos No belíssimo texto “Comentário à Primeira Epístola de São João” (In Epistolam Ioannis ad Parthos tratactus decem) existe uma importante contribuição acerca do assunto proposto. O texto foi escrito em 416, período em que Agostinho já exercia o cargo de bispo efetivo de Hipona há aproximadamente 19 anos, período esse deveras conturbado no episcopado do filósofo, pois estava administrando animosidades decorrentes do cisma donatista13. Esse problema cismático 257 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 não era nada modesto em Hipona, pois a maioria da população da cidade era donatista, e na medida em que a sede episcopal defendia a perspectiva católica, a tensão e animosidade na igreja em Hipona tornaram desafiador ser titular dessa sede. A hostilidade era patente de ambos os lados, chegando em alguns casos mais radicais a ser violenta com os circunceliões do lado donatista, e com a força coercitiva do Estado pelo lado dos católicos (Cf. BROWN, 2005, p. 285- 287). O fato é que Agostinho estava no centro desse rio de animosidades, e no texto em questão defende a tese de que o amor genuíno, fruto de uma real conversão deve ultrapassar todas as barreiras, inclusive a da inimizade consequente do cisma presente no contexto. Todos devem ser incondicionalmente amados, sejam eles católicos ou donatistas14. No sétimo tratado Agostinho faz uma exaltada exortação acerca do amor, afirmando a princípio que a própria expiação na cruz foi um ato de puro amor, pois Jesus não tinha a menor obrigação de ser cravado na cruz, e sendo filho de Deus teria o poder para evitar tal sofrimento, mas ele quis expiar a culpa de outros por esse terrível sofrimento, e ele quis por um ato de puro amor (Cf. AGOSTINHO, In ep. Joan. ad Parthos, VII, 2). Ato contínuo, o filósofo defende a resignação como uma forma de amor, ou melhor, como evidência do amor ao outro. Pois, Cristo, que sofreu todas as agonias da cruz sem ter feito nada para merecê-las, teria todos os motivos do mundo para se irar contra seus algozes, mas ao contrário disso os amou a todos, pedindo ao seu Pai que perdoasse seus ofensores. Ante essa forte colocação, questiona: “Se aquele que tinha o poder soberano não fez nenhuma ameaça de vingança, por que hás de ameaçar tu, que estás colocado sob o poder de outros?” (AGOSTINHO, In ep. Joan. 84). Como a solicitação não foi aceita, elegeram outro bispo, o Majorino, e a igreja de Cartago ficou com dois bispos, um donatista e outro católico, de forma que depois da morte de Marjorino, Donato o sucedeu na sede cismática (Cf. MARKUS, in: FITZGERALD, 2006, p. 440-444). BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. 14 Embora bem mais possa ser dito acerca da ocasião da obra, e do estilo de texto do comentário, as informações colocadas até aqui sobre o contexto são o suficiente para o que se pretende em nossa análise, não cabendo aqui fazer uma análise acurada acerca de todas as minúcias envolvidas nas possíveis intenções da obra. 15 “Si autem non minabatur qui potestatem habebat; tu quid minaris, quid sufflas in potestate aliena constitutus?” (AGOSTINHO, In ep. Joan. ad Parthos, VII, 3 – PL 35). 84). Como a solicitação não foi aceita, elegeram outro bispo, o Majorino, e a igreja de Cartago ficou com dois bispos, um donatista e outro católico, de forma que depois da morte de Marjorino, Donato o sucedeu na sede cismática (Cf. MARKUS, in: FITZGERALD, 2006, p. 440-444). 14 Embora bem mais possa ser dito acerca da ocasião da obra, e do estilo de texto do comentário, as informações colocadas até aqui sobre o contexto são o suficiente para o que se pretende em nossa análise, não cabendo aqui fazer uma análise acurada acerca de todas as minúcias envolvidas nas possíveis intenções da obra. 15 “Si autem non minabatur qui potestatem habebat; tu quid minaris, quid sufflas in potestate aliena constitutus?” (AGOSTINHO, In Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos Mesmo não sendo o motivo principal, esse argumento da submissão ao poder dos outros para justificar a resignação causa certo incômodo, pois, permanece um espaço para se interpretar que o subalterno deveria se sujeitar às injustiças cometidas apenas pelo fato de sua condição de subalternidade. Dessa forma, a submissão do homem a outros é usados nos textos não como o fator principal da resignação, pois esse é o amor, mas como um argumento a mais para incentivar a resignação pelo exemplo da conformação de Cristo. Mesmo não sendo o motivo principal, esse argumento da submissão ao poder dos outros para justificar a resignação causa certo incômodo, pois, permanece um espaço para se interpretar que o subalterno deveria se sujeitar às injustiças cometidas apenas pelo fato de sua condição de subalternidade. Essa possibilidade de entendermos se de fato Agostinho defendeu no analisado comentário a resignação frente aos poderosos mesmo ante as injustiças, é parcialmente aprofundada no oitavo tratado. Pois, no tratado em questão interpretando vários versículos da Primeira Epístola de São João que admoestam que o amor ao próximo não pode ser apenas teórico, mas deve conduzir o cristão à prática benevolente, afirma que até mesmo essa benevolência deve ser pensada. Visto que embora seja um eco da dilectio auxiliar ao próximo que necessita, não devemos desejar a existência de miseráveis para ter oportunidade de exibir a nossa benevolência16, logo, deve-se desejar a igualdade para que o que ajuda não se sinta superior ao outro, nem para que o auxiliado se sinta inferior: “Ele necessita, tu o ajudas; mas por ter ajudado, tu te imaginas superior àquele a quem ajudas. Deseja ser igual, para que ambos estejam sujeitos Àquele a quem nada se pode dar” (AGOSTINHO, In ep. Joan. ad Parthos, VIII, 5)17. O que seria esse: Opta aequalem - Deseja ser igual? Agostinho chega a mencionar a Nova Jerusalém onde não existirá miséria de nenhum tipo, mas levando em consideração que essa igualdade da Nova Jerusalém é para a eternidade, e a igualdade almejada refere-se no contexto às necessidades da vida terrena, questionamos: seria um comportamento que expressa a dilectio apenas o querer ser igual ao outro? Ou está implícito a ação para tentar de alguma forma agir para concretizar esse desejo? 16 Em diversos trechos de seu “Comentário da primeira epístola de João”, que é uma dos mais belos e profundos tributos ao amor entre as obras do mestre de Hipona, é dito que esse amor inevitavelmente envolve benevolência, de forma que não consiste em apenas um bom sentimento romanticamente verbalizado, mas algo que envolve ação de quem ama em prol do objeto amado. No capítulo cinco do sétimo tratado, de forma dura, contudo coerente insiste que se deve ser benevolente para com os que necessitam, todavia, jamais se deve desejar a miséria alheia para exercitar a benevolência, pois isso é qualquer outra coisa menos dilectio (Cf. AGOSTINHO, In ep. Joan. ad Parthos VIII, 5). Essa forma elevada de amor, a despeito de ser concretizada na ajuda ao próximo, não deve ter como fim nos fazer sentir melhores pessoas, mas o cuidado desinteressado para com o outro. Não que o cristão não deva se sentir bem em socorrer quem precisa, pois em certa medida demonstra que foi realizada por uma pessoa que se compraz com a melhora do outro, mas isso não pode ser o sentimento protagonista na ação, mas o bem em si mesmo ao próximo, pois se assim não for, essa ação benevolente pode se tornar um egoísmo disfarçado de altruísmo. 17 “Ille indiguit, tu impertitus es; quasi maior videris quia tu praestitisti, quam ille cui praestitum est. Opta aequalem, ut ambo sub uno sitis cui nihil praestari potest” (AGOSTINHO, In ep. Joan. ad Parthos, VIII, 5 – PL 35 ). 18 Eis o trecho da Primeira carta de São João que exorta sair do discurso para a prática do amor cristão, que sem dúvida impactou o hiponense em seu comentário: “Se alguém, possuindo bens deste mundo, vê seu irmão na necessidade e lhe fecha o coração, como permanecerá nele o amor de Deus? Filhinhos, não amemos com palavras nem com a língua, mas com ações e em verdade” (I João, III, 17, 18). BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos ad Parthos, VII, 3)15. p ( p ) É nítida no texto citado a marca da tensão vivida na época, pois ambos os partidos estavam com os sentimentos à flor da pele, chegando a imprecar ameaças aos cristãos do outro partido. A despeito dessa conjuntura, entendemos que essa compreensão, embora escrita movida pela situação, enquanto compreensão de Agostinho de como lidar com os infortúnios gerados por outros, não está restrito ao contexto. Assim, se exorta uma resignação pelo exemplo do filho de Deus, e pela constatação da pequenez humana. Pois, se o próprio Cristo nos horríveis sofrimentos na cruz não proferiu ameaças para quem o torturava, levando em consideração que sendo a segunda pessoa da santíssima Trindade é onipotente, quem somos nós para querer reparação ou vingança quando sofremos com a ação, mesmo que injusta dos outros. O texto faz questão de ressaltar que um dos motivos para a resignação seria a própria condição humana de estar sobre o poder de outros. Sabemos que no entorno do texto está presente a comparação com Deus que possui pleno poder, e mesmo assim se conformou com a humilhação imposta pela morte por tortura na cruz, todavia, ao mencionar a falta de poder dos homens reportando o fato de estarem sob o poder dos outros, poderia indicar apologia discreta a um resignar-se por sua posição hierárquica. Ou seja, com cuidado para não forçar demais os limites exegéticos do texto, poderíamos afirmar nas entrelinhas que um dos motivos para que o homem se conforme com os danos causados pelos outros seja o fato de estar submetido ao poder de outros. Na verdade esse não é o motivo principal presente no contexto, pois, o que levou Cristo a se entregar ao sacrifício expiatório não foi seu poder, mas seu amor incondicional, seu poder é utilizado para ressaltar seu amor, pois, ele teria poder para não aceitar a injusta situação, mas aceitou apenas por amor. 258 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 ISSN 2178-1036 Dessa forma, a submissão do homem a outros é usados nos textos não como o fator principal da resignação, pois esse é o amor, mas como um argumento a mais para incentivar a resignação pelo exemplo da conformação de Cristo. 16 Em diversos trechos de seu “Comentário da primeira epístola de João”, que é uma dos mais belos e profundos tributos ao amor entre as obras do mestre de Hipona, é dito que esse amor inevitavelmente envolve benevolência, de forma que não consiste em apenas um bom sentimento romanticamente verbalizado, mas algo que envolve ação de quem ama em prol do objeto amado. No capítulo cinco do sétimo tratado, de forma dura, contudo coerente insiste que se deve ser benevolente para com os que necessitam, todavia, jamais se deve desejar a miséria alheia para exercitar a benevolência, pois isso é qualquer outra coisa menos dilectio (Cf. AGOSTINHO, In ep. Joan. ad Parthos VIII, 5). Essa forma elevada de amor, a despeito de ser concretizada na ajuda ao próximo, não deve ter como fim nos fazer sentir melhores pessoas, mas o cuidado desinteressado para com o outro. Não que o cristão não deva se sentir bem em socorrer quem precisa, pois em certa medida demonstra que foi realizada por uma pessoa que se compraz com a melhora do outro, mas isso não pode ser o sentimento protagonista na ação, mas o bem em si mesmo ao próximo, pois se assim não for, essa ação benevolente pode se tornar um egoísmo disfarçado de altruísmo. p g it, tu impertitus es; quasi maior videris quia tu praestitisti, quam ille cui praestitum est. Opta aequalem, ut ambo s ihil praestari potest” (AGOSTINHO, In ep. Joan. ad Parthos, VIII, 5 – PL 35 ). Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos No contexto da perícope em análise, o filósofo de Hipona destaca diversos exemplos de que o cristão deve afastar a tentação de sentir-se superior ao outro ante a dependência dele à sua ajuda, utilizando até mesmo o eloquente exemplo da relação entre o mestre e o discípulo. Pois, o mestre deve trabalhar para que seu discípulo adquira autonomia para pensar por si mesmo, e nunca permitir que essa relação mantenha o discípulo sempre na dependência do mestre para o saber, alimentando assim uma forma de submissão na sabotagem, mesmo que não consciente, da autonomia do discípulo (Cf. AGOSTINHO, In ep. Joan. ad Parthos, VIII, 8). Entendemos que essa não aceitação do sentimento de superioridade do discípulo de Cristo frente aos auxiliados por ele, está devidamente demonstrada nos textos supra analisados, todavia surge uma questão relacionada: poderíamos estender essa não aceitação aos que não estão sendo frutos de nossos atos de misericórdia? Ou seja, até que ponto poderíamos aplicar essa não resignação às relações de submissões sofridas em outras relações que não estamos envolvidos diretamente, ou até mesmo naquelas em que nós estamos em submissão a outros? Embora possamos querer uma visão mais progressista de Agostinho, com uma defesa para que o cristão não aceite a submissão, entendemos que na situação retratada, o cristão, visto que possui pela fé certeza da vida eterna, deve se resignar por essa certeza. Temos fortes indícios disso no tratado VIII, 11, onde no contexto de apaziguar as desavenças entre católicos e donatistas, afirma que se deve perdoar qualquer mal praticado contra si, fiado na esperança da vida eterna, e na convicção de que se Deus tudo pode, e poderia evitar determinado dano gerado a você e decide permitir, a Sua permissão deve ter algum propósito que desconhecemos (Cf. AGOSTINHO, In ep. Joan. ad Parthos, VIII, 11). A convicção mencionada supra, não deve conduzir o cristão à passividade ante ao erro, de forma que em fragmento que traz os traços do cisma donatista, Agostinho afirma que o amor incondicional recebido após a real conversão não significa compactuar com erro, mas justamente repreender por amor: “Se quiseres conservar a caridade, irmãos guardai-vos, sobretudo de pensar que ela seja sem iniciativa, sem atividade e que para conservá-la basta certa mansidão – que digo – não mansidão, mas antes indolência e moleza (AGOSTINHO, In ep. Joan. ad Parthos, VII, 11)19. Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos Entendemos que embora o filósofo não expresse claramente, pelo contexto do fragmento, a ação para tornar concreto o desejo de igualdade é necessária, pois, permanecer apenas no desejo de igualdade poderia equivaler ao amar só no discurso e não na prática, criticado nessa carta de São João por ele comentada18. Destarte, ninguém deve estar acima do outro, pois, todos foram criados à imagem e semelhança de Deus, ou acaso o Criador ao ordenar a criação disse que os homens deveriam dominar outros homens? (Cf. AGOSTINHO, In ep. Joan. ad Parthos, VIII, 6). Sabemos que embora esse não tenha sido o plano original do Ordenador supremo, a desigualdade adentrou na ordem pelo pecado, de forma que os homens não se conformam em dominar a criação não humana, e se comprazem ao se pensarem superiores aos outros. Ciente de que essa desigualdade apenas penetrou na ordem humana por meio do mal moral, não seria obrigação do homem cristão não se resignar com essa marca do pecado, e ao não se conformar se dispor a ser agente p p ( p ) 18 Eis o trecho da Primeira carta de São João que exorta sair do discurso para a prática do amor cristão, que sem dúvida impactou o hiponense em seu comentário: “Se alguém, possuindo bens deste mundo, vê seu irmão na necessidade e lhe fecha o coração, como permanecerá nele o amor de Deus? Filhinhos, não amemos com palavras nem com a língua, mas com ações e em verdade” (I João, III, 17, 18). 259 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 ISSN 2178-1036 que propicie a igualdade? Levando em consideração que ao falar opta aequalem Agostinho estava justamente criticando certa forma de benevolência, a possibilidade da igualdade não ser apenas por atos benevolentes é grande. Se a nossa leitura estiver correta, temos indícios de uma defesa de uma ação social voltada para a autonomia do indivíduo, para que ele deixe de necessitar do outro e alcance a dignidade de não ser inferior a outrem. Assim, mesmo ante a ideia teológica do desarranjo da ordem que gerou a submissão de um homem para com outro, o texto em análise defende opta aequalem na tentativa de retornar à ordem divinamente orientada, onde todos os homens são iguais sendo inferiores apenas ao Criador, que por conceito nada necessita, sendo o único necessário. 19 “Si qui forte vultis servare caritatem, fratres, ante omnia ne putetis abiectam et desidiosam; nec quadam mansuetudine, imo non mansuetudine, sed remissione et neglegentia servari caritatem” (AGOSTINHO, In ep. Joan. ad Parthos, VII, 11 – PL 35 ). 20 “[...] non est ista caritas, sed languor. Ferveat caritas ad corrigendum, ad emendandum” (AGOSTINHO, In ep. Joan. ad Parthos, VII, 11 – PL 35). BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos E após alguns exemplos de situações que em síntese apresentam que amar de forma verdadeira não prescinde a repreensão, arremata: “[...] Isso não é caridade, é tibieza. Que a caridade seja ardente para corrigir, para repreender (AGOSTINHO, In ep. Joan. ad Parthos, VII, 11)20. Dessa forma, no que tange à correção ao próximo, o amor não gera a resignação, mas antes a correção, pois o amor não pode ser confundido com a aceitação irrestrita de qualquer situação. Essa eloquente perícope coloca um limite: o erro do próximo, pois, a indulgência com o erro alheio não pode ser conceituado como amor, mas como languor (frouxidão, moleza, tepidez). 260 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 Nos exemplos elencados por Agostinho em sua argumentação temos a correção: ao servo, filho e ao vizinho, todos realizando atos incorretos que devido ao amor deveriam ser repreendidos. A correção ao filho e ao servo está na esfera íntima da família, e em relação que se tem certa autoridade natural para a repreensão, todavia, ao repreender o vizinho a situação muda completamente de figura. Já que é proposto nesse fragmento a não aceitação de erros praticados por vizinhos, é possível sem forçar muito os limites exegéticos do texto, estender essa não aquiescência a atos injustos em geral dos outros. Logo, não seria uma consequência natural do amor se resignar com as injustiças provocadas aos outros, pois tais atos injustos igualmente se constituem em erros passíveis de serem repreendidos21, de maneira que ser resignado ante um ato de injustiça praticado equivaleria ao languor. Como essa interpretação supra não foi distintamente apresentada pelo pensador de Hipona, é possível que seja apenas isso de fato, uma interpretação. Assim, convém nas linhas que nos restam, iluminar o assunto em fragmentos da última obra a ser analisada nessa investigação. Para concluir as nossas reflexões acerca do assunto em estudo, analisaremos alguns fragmentos do livro XIX do De civitate Dei22 que sem dúvida nos auxiliará a pensar na perspectiva de um Agostinho no ápice de sua maturidade intelectual, maturidade essa que lhe possibilitou escrever um verdadeiro texto de filosofia da história, costurando uma impressionante gama de assuntos sob a perspectiva da filosofia cristã. A partir do capítulo treze do livro XIX, o filósofo na intenção de exortar acerca da paz eterna, elabora interessante reflexão acerca da paz que toca lateralmente no objeto investigado. 21 Na obra “Dos Deveres”, Cícero classifica a injustiça em dois tipos, a que se pratica com a intenção deliberada de cometer atos injustos, e a que se pratica por inação, ou seja, quando se tem o poder para evitar um ato injusto, mas nada faz (Cf. CÍCERO, Dos Deveres, I, 6). Na perspectiva de Agostinho igualmente a inação quando se possuía a potência para algo fazer, é algo indigno do amor cristão, logo, um ato de injustiça. É como se por inércia, o cristão que possui o dever de amar com o amor de Deus, se tornasse cúmplice do ato injusto. 22 A De civitate Dei, foi escrito entre os anos 413 a 426, portanto, três anos após a queda de Roma por Alarico. A obra foi escrita com o incentivo de seu amigo e irmão de fé Marcelino, e a intenção fica absolutamente clara já nos primeiros capítulos do primeiro livro (Cf. AGOSTINHO, De civ. Dei, I, 1-10). Dessa forma, ao sair em defesa do cristianismo, desenvolve uma interpretação da história na perspectiva da fé cristã, escrevendo a primeira teologia e filosofia da história (Cf. LEÃO, In. AGOSTINHO, 2000, p. 17). BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. 261 p ) 23 Segundo Agostinho a paz é uma tendência natural de tudo o que existe, pois de alguma forma tudo tende para o seu lugar natural na ordem, essa tendência permanece mesmo depois da queda. Logo, nem sempre a paz buscada é aquela presente na ordem original, mas uma deformada ou adaptada à nova ordem estabelecida por consequência do pecado. Portanto, todos indistintamente buscam a paz, até mesmo quem propõe uma guerra não o faz pela guerra em si mesma, mas para alcançar a paz, paz essa segundo seus propósitos. No corpo, a paz se manifesta com o funcionamento harmônico de suas partes em relação com a alma, e na família a paz acontece quando harmonicamente a hierarquia de obediência e funções é respeitada. A mesma coisa Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos A mesma coisa 261 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 ISSN 2178-1036 Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 Sabemos que a inteligência suprema do Criador ao tudo criar, pensou e estabeleceu uma ordem natural para cada criatura em particular, e para a totalidade da criação em suas relações e inter-relações holísticas, todavia, devido à queda, principalmente, mas não exclusivamente na esfera humana, essa ordem foi alterada para encaixar as consequências do pecado, o que tornou mais difícil ocupar seu lugar na ordem e, por conseguinte alcançar a paz. Mesmo com as consequências da queda, que gerou entre outras coisas o sofrimento, a miséria, as desigualdades sociais, etc., o ser humano permanece, conscientemente ou não, buscando a paz, ou melhor, se adaptando resignadamente à nova ordem. Como nos esclarece emblematicamente o pensador cristão refletindo acerca da paz na miséria e sofrimento: Portanto, como os miseráveis, enquanto tais, não estão em paz, não gozam da traquilidade da ordem, isenta de turbações, mas, porque merecida e justamente miseráveis, mesmo na miséria não podem estar fora da ordem [...]. Neles há, portanto, certa tranquilidade na respectiva ordem e, por conseguinte, certa paz. São miseráveis, porque, embora estejam onde devem estar, não estão onde não se veriam precisados de sofrer. São mais miseráveis, se não estão em paz com a lei que rege a ordem natural (AGOSTINHO, De civ. Dei, XIX, 13, 1)24. Dessa forma, na medida em que a paz é consequência de cada coisa estar em seu devido lugar, com o advento do pecado, dor, miséria e sofrimento passaram a ser partícipes da ordem, tendo assim seu devido lugar na vida humana adaptada à nova ordem. Agostinho percebe a paz na miséria com certa dubiedade, pois, se por um lado a miséria gera falta de tranquilidade por não fazer parte da ordem original, na medida em que foi incorporada na ordem, é possível ter paz mesmo em sofrimento. Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos Aristóteles em sua Ética a Nicômaco já dizia que todas as coisas possuem seu próprio bem, na medida em que tudo o que existe enquanto existe possui seu telos natural, e esse telos é o seu bem (Cf. ARISTÓTELES, Ética a Nicômaco, I, 1, 1094a). Em toada semelhante, Agostinho pensa que igualmente existe uma tendência em toda a natureza, como se fosse um pondus natural de tudo o que existe, a paz. A felicidade sem dúvida é objeto de busca de todos os homens, mas até que ponto seria possível a felicidade sem a paz? Além disso, podemos afirmar que a paz é ainda mais universal que a própria felicidade, pois essa última é objeto de busca consciente, enquanto a paz além de não se restringir apenas a busca humana, pois, é uma tendência comum aos homens, animais e plantas, nem sempre é busca consciente, além do fato de que o homem a consegue encontrar mesmo nas situações mais inóspitas. A despeito de estarmos habituados a conceituar a paz como certa tranquilidade oriunda da ausência de conflitos, o pensador de Hipona acrescenta que essa ausência de conflitos de diversas ordens acontece por certo ordenamento, ou seja, cada coisa encontra a sua específica paz ao ocupar seu devido lugar natural23. 261 23 Segundo Agostinho a paz é uma tendência natural de tudo o que existe, pois de alguma forma tudo tende para o seu lugar natural na ordem, essa tendência permanece mesmo depois da queda. Logo, nem sempre a paz buscada é aquela presente na ordem original, mas uma deformada ou adaptada à nova ordem estabelecida por consequência do pecado. Portanto, todos indistintamente buscam a paz, até mesmo quem propõe uma guerra não o faz pela guerra em si mesma, mas para alcançar a paz, paz essa segundo seus propósitos. No corpo, a paz se manifesta com o funcionamento harmônico de suas partes em relação com a alma, e na família a paz acontece quando harmonicamente a hierarquia de obediência e funções é respeitada. Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos De fato é perceptível a perspicácia do hiponense na observação do comportamento humano, dado que de fato ninguém fica satisfeito no estado de miséria, e sem dúvida essa inconformação gera falta de paz, contudo, a permanência prolongada nessa condição faz com que a pessoa encontre, por sobrevivência, espaço em sua vida para encontrar certa paz na resignação tranquila de uma consciência de que tal situação é inalterável ou aparentemente inalterável25. acontece na relação entre patrão e empregado, quando a cada um faz a sua parte respeitando a hierarquia de obediência, temos paz nessa relação, rito contínuo a paz na cidade se dá pela concórdia entre governantes e governados (Cf. AGOSTINHO, De civ. Dei, XIX, 12 -13). E suma essa busca, que é mais uma tendência, é anseio presente em toda a criação, como arremata Agostinho: “A paz de todas as coisas é a tranquilidade da ordem. A ordem é a disposição dos seres iguais e desiguais que distribui a cada um os seus lugares” (AGOSTINHO, De civ. Dei, XIX, 13, 1). 24 “Proinde miseri, quia, in quantum miseri sunt, utique in pace non sunt, tranquillitate quidem ordinis carent, ubi perturbatio nulla est; verumtamen quia merito iusteque sunt miseri, in ea quoque ipsa miseria sua praeter ordinem esse non possunt; [...] ac per hoc inest eis ordinis nonnulla tranquillitas, inest ergo nonnulla pax. Verum ideo miseri sunt, quia, etsi in aliqua securitate non dolent, non tamen ibi sunt, ubi securi esse ac dolere non debeant; miseriores autem, si pax eis cum ipsa lege non est, qua naturalis ordo administratur” (AGOSTINHO, De civ. Dei, XIX, 13, 1 – PL 41). 24 “Proinde miseri, quia, in quantum miseri sunt, utique in pace non sunt, tranquillitate quidem ordinis carent, ubi perturbatio nulla est; verumtamen quia merito iusteque sunt miseri, in ea quoque ipsa miseria sua praeter ordinem esse non possunt; [...] ac per hoc inest eis ordinis nonnulla tranquillitas, inest ergo nonnulla pax. Verum ideo miseri sunt, quia, etsi in aliqua securitate non dolent, non tamen ibi sunt, ubi securi esse ac dolere non debeant; miseriores autem, si pax eis cum ipsa lege non est, qua naturalis ordo administratur” (AGOSTINHO, De civ. Dei, XIX, 13, 1 – PL 41). BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. acontece na relação entre patrão e empregado, quando a cada um faz a sua parte respeitando a hierarquia de obediência, temos paz nessa relação, rito contínuo a paz na cidade se dá pela concórdia entre governantes e governados (Cf. AGOSTINHO, De civ. Dei, XIX, 12 -13). E suma essa busca, que é mais uma tendência, é anseio presente em toda a criação, como arremata Agostinho: “A paz de todas as coisas é a tranquilidade da ordem. A ordem é a disposição dos seres iguais e desiguais que distribui a cada um os seus lugares” (AGOSTINHO, De civ. Dei, XIX, 13, 1). Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos 25 O filósofo e economista contemporâneo Amartya Sen em seu magnífico texto “Desigualdade Reexaminada”, adverte que um aparente estado de satisfação e prazer não pode ser indício real de bem estar, pois as pessoas expostas por tempo prolongado a um estado de penúria, não se lastima por grande período de tempo, na medida em que ante a inalterabilidade da situação buscam limitar suas aspirações, encontrando satisfação e prazer em situações tão miseráveis que inviabilizam um real bem-estar: “O problema é particularmente grave no contexto das desigualdades e privações arraigadas. Uma pessoa totalmente desprovida, levando uma vida bastante limitada, poderia não parecer pobre em termos de uma métrica mental do desejo e sua satisfação, se a miséria for aceita com silenciosa resignação. Em situação de privação duradoura, as vítimas não continuam lamentosas e pesarosas todo o tempo, e muito frequentemente fazem grandes esforços para tirar prazer de pequenas dádivas e reduzir desejos pessoais a modestas – “realistas” – proporções” (SEN, 2001, p. 96). Na mesma toada, a filósofa Adela Cortina em seu texto “Aporofobia, a aversão ao pobre”, afirma que os pobres ao se resignarem com a miséria em que vivem, ficam satisfeitos por quaisquer pequenas ajudas ou melhoras, de forma que muitas vezes ficam por meios de pacífica resignação pouco conscientes de sua situação de injustiça social, tendo assim que ser conscientizados da indignidade da situação em que estão expostos (Cf. CORTINA, 2020, p. 48, 49). 262 BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. Griot : Revista de Filosofia, Amargosa - BA, v.22, n.1, p.251-265, fevereiro, 2022 ISSN 2178-1036 ISSN 2178-1036 Segundo a perícope analisada, alguma forma de paz é possível mesmo em meio a mais agonizante miséria, ao tentar na conformação e aceitação desse estado banir expectativas de iminentes mudanças para possuir certa tranquilidade. A paz nesse formato supra descrita é exatamente correspondente ao conceito de nosso objeto de pesquisa, a resignação. Pois, essa paz acontecerá exatamente quando por meio da aquiescência da inalterabilidade de tal situação, se aceita de forma tranquila a situação de miséria. Resignação e o amor ao próximo à luz das obras: In Epistolam Ioannis ad Parthos e De civitate Dei contra paganos Assim, segundo Agostinho, a resignação não só é possível em estado de extrema adversidade, como seria uma condição humana se resignar em algum momento com tais circunstâncias para ter alguma paz, pois, se isso não acontecer, a miséria seria superlativada, na medida em que se sofreria pelo estado de miséria e pela constante inconformação ante certa estabilidade desse cenário. A despeito de no analisado fragmento não se fazer referência a uma possível relação entre resignação e o amor, achamos por bem analisá- lo, porquanto, fica evidenciado que a resignação em situações extremas acaba se tornando o único meio possível para a paz, e como já mencionamos sem paz a felicidade é impossível. Além disso, apesar do fato de que embora o amor não seja textualmente mencionado, não podemos descartar a sua presença nas entrelinhas, posto que o texto também discorre sobre a situação de miséria mesmo para os cristãos, que ao serem cristãos foram inundados com o amor de Deus, irradiando esse superior amor em todas as situações de sua vida. BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. BRANDÃO, Ricardo Evangelista. Dilectio, resignação e injustiça: possibilidade de interpretar o amor como resignação e conformação com a injustiça, à luz de Santo Agostinho. Griot : Revista de Filosofia, Amargosa – BA, v.22 n.1, p.251-265, fevereiro, 2022. Considerações finais É perceptível em nosso pensador, quando se trata de injustiças geradas a si, certa resignação gerada pela imersão no amor, por vários motivos declarados e não declarados. Entre os declarados poderíamos elencar: a certeza na vida eterna em que existirá a verdadeira justiça, e que o próprio Deus lhe consolará enxugando as suas lágrimas, como retrata a bela imagem no livro do Apocalipse (Cf. Apocalipse, XXI, 4); a imitação do perfeito amor de Cristo, que não se enfureceu mesmo no calvário em que era o único justo sofrendo por uma sentença injusta, tendo poder para desfazer o sofrimento e castigar seus algozes; a resignação trazida pela própria conversão no verdadeiro amor, que o faz ter um temperamento diferente ante ao sofrimento, percebendo um propósito divino por trás do sofrimento. Entre os motivos não declarados, e que por tanto interpretamos no silêncio de possíveis contextos, e que por esse motivo pedimos vênia máxima caso estejamos forçando os limites exegéticos do assunto, pensamos que Agostinho tinha enorme responsabilidade quando falava e escrevia ao povo. Pois, ele estava em uma comunidade de extrema desigualdade social, com pessoas em estado de profunda miséria vivendo literalmente da misericórdia e migalhas dos ricos e da igreja. Nessa situação, levando em conta a força das palavras de um bispo na região, a responsabilidade se redobra, pois um sermão mal interpretado poderia gerar consequências desastrosas incentivando o embate dos pobres para com os ricos, que naquele contexto, poderia ser muito pior para os pobres. Naquelas circunstâncias a resignação e o apelo ao auxílio do outro, foi a alternativa mais prudente. Todavia, quando as injustiças em questão são as executadas contra o nosso próximo, não se exorta uma resignação, aliás, o amor gera uma irresignação para com a dor do outro, levando esse que verdadeiramente foi imerso no amor divino à ação. É óbvio que em hipótese alguma estamos discorrendo sobre alguma ação revolucionária contra as instituições que geraram o estado de injustiça, mas em se comover com a dor do próximo, partindo assim para ações misericordiosas. Em suma, evidenciou-se nessa pesquisa que as misérias, dores, desigualdades sociais, injustiças, etc., são oriundas da desordem gerada pela introdução do mal no mundo. Referências AGUSTÍN, San. De las costumbres de la Iglesia catolica y de las costumbres de los Maniqueos. In: Obras completas de San Agustín. ed. bilíngüe. Trad., introd. y notas de Teófilo Prieto. Madrid: La Editorial Católica / BAC, 2011. tomo IV, p. 207-388. AGUSTÍN, San. De las costumbres de la Iglesia catolica y de las costumbres de los Maniqueos. 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Trad., introd. y notas de Teófilo Prieto. Madrid: La Editorial Católica / BAC, 2011. tomo IV, p. 204-227. p REALE, Gilvanni. História da filosofia antiga. 2. ed. Trad. de Marcelo Perine; Henrique Cláudio de Lima Vaz. São Paulo: Loyola, 1994. vol. IV, 608 p. SEN, Armatya Kaumar. Desigualdade Reexaminada. Trad. de Ricardo Doninelli Mendes. Rio de Janeiro: Record, 2001. _______________________________________________________________________________________ Autor(a) para correspondência / Corresponding author: Ricardo Evangelista Brandão. ricardobrand75@gmail.com 265
https://openalex.org/W2808497360	https://njg.geologi.no/images/NJG_articles/NJG_Vol98_Nr1_Art9_Henriksen_et_al.pdf	English	null	Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides: a result of long-term staging followed by catastrophic release of sediments	Norwegian journal of geology	2,018	cc-by	15,185	E-mail corresponding author (Sverre Henriksen): shen@statoil.com June 2018 to inferred Early Silurian, low-grade metasedimentary rocks (Bergström, 1979, 1997; Bruton & Bockelie, 1980; Ryan et al., 1980; Neuman & Bruton, 1989; Neuman et al., 1997). These magmato-sedimentary associations constitute a part of the Upper Allochthon of Caledonide tectonostratigraphy. These are interpreted to represent NORWEGIAN JOURNAL OF GEOLOGY Vol 98 Nr. 1 https://dx.doi.org/10.17850/njg98-1-09 E-mail corresponding author (Sverre Henriksen): shen@statoil.com E-mail corresponding author (Sverre Henriksen): shen@statoil.com The Ordovician metasedimentary rocks in the Caledonian nappes in Mid Norway were deposited in a back-arc setting, following ophiolite obduction, uplift and erosion, related to continental uplift and changing directions of subduction during this period. Deep-water conditions were established in the basin and this accommodated a thick succession of variously coarse-grained, conglomeratic and argillaceous sediments. The conglomerates typically display the well-rounded clast material of variable igneous, volcanic, sedimentary and carbonate origin and alternate between being clast-supported and matrix-supported with interbedded sandstone beds. The rounded clasts suggest significant reworking and long-term storage of sediments in the source area before transport into the deeper part of the basin. The basal surface of conglomerates always shows an erosive contact with underlying shales or coarsening-upward sandstone successions. The basal surfaces probably represent sedimentary fairways, where several conglomerate units separated by erosional contacts are stacked on top of each other. The conglomerate successions likely result from episodic release of large quantities of sediments. Between each burst of release, sediments accumulated in alluvial fans, fan deltas and fluvial delta systems along the basin shoreline and carbonate reefs established on the outer shelf. Laterally accreting channel sandstone beds were probably important in setting up sedimentary fairways for the succeeding conglomerates. In the inferred deepest part of the basin, smaller channellised features and thin-bedded turbidites seem to dominate. Channel features and hummocky cross-stratification in shelf to upper slope sandstone beds are alternatively interpreted to result from unidirectional flows and constitute an integral part of the turbidite system. By including all the observed facies within the turbidite system we invoke a depositional model which does not require a eustacy-driven sequence-stratigraphic model for explanation. We rather suggest a tectonic model involving an elevated source area, either volcanic or continental, where erosional products are stored and reworked in the alluvial accommodation zone. These sediments are built up to a threshold level and flushed to the sea by a sudden availability of large amounts of water. The actual cause of this sudden availability of water remains uncertain, but the consequent mixture of water and sediments resulted in a super-concentrated high-energy density current. Keywords: Ordovician, back arc basin, conglomerates, catastrophic floods, turbidites Received 20. September 2017 / Accepted 12. April 2018 / Published online10. Keywords: Ordovician, back arc basin, conglomerates, catastrophic floods, turbidites Henriksen, S., Roberts, D. & Pedersen, P.Å. 2018: Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides: a result of long-term staging followed by catastrophic release of sediments. Norwegian Journal of Geology 98, 141–164. https://dx.doi.org/10.17850/njg98-1-09. Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides: a result of long-term staging followed by catastrophic release of sediments Sverre Henriksen1, David Roberts2 & Per-Åge Pedersen3 1 Statoil Research Center, Rotvoll, Arkitekt Ebbels veg 10, 7005 Trondheim, Norway 2 Geological Survey of Norway, Postbox 6315 Torgarden, 7491 Trondheim, Norway 3 Statoil ASA, Sandsli, Sandsliveien 90, 5254 Sandsli, Norway 1 Statoil Research Center, Rotvoll, Arkitekt Ebbels veg 10, 7005 Trondheim, Norway 2 Geological Survey of Norway, Postbox 6315 Torgarden, 7491 Trondheim, Norway 3 Statoil ASA, Sandsli, Sandsliveien 90, 5254 Sandsli, Norway py g This work is licensed under a Creative Commons Attribution 4.0 International License. © Copyright the authors. Introduction The Caledonides of central Norway are especially well known for their fragmented ophiolite assemblages and, in some areas, the overlying, richly fossiliferous, Ordovician Henriksen, S., Roberts, D. & Pedersen, P.Å. 2018: Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides: a result of long-term staging followed by catastrophic release of sediments. Norwegian Journal of Geology 98, 141–164. https://dx.doi.org/10.17850/njg98-1-09. © Copyright the authors. This work is licensed under a Creative Commons Attribution 4.0 International License. Copyright the authors. This work is licensed under a Creative Commons Attribution 4.0 International License. 141 S. Henriksen et al. 142 S. Henriksen et al. 142 go back to two, unpublished, Master’s theses (Pedersen, 1981; Strømmen, 1983). Both before and since that time, detailed 1:50,000-scale mapping had revealed local features of sedimentology and ichnology that pointed to a deep-marine, depositional environment during the accumulation of these thick successions (Gale & Roberts, 1974; Roberts, 1969, 1972, 1984). This interpretation was later verified from a more detailed study of trace fossils by Uchman et al. (2005). Subsequent international excursions to the area and supplementary fieldwork over the last thirty years have revealed additional sedimentary features which lend further support to the earlier interpretations. At the same time, these new observations have allowed corrections and additions to be made to the preliminary 1:50,000 map-sheet ‘Frosta’ (Roberts, 1985), which will be prepared for publication during the coming months. the exotic oceanic terranes of the Iapetus Ocean deriving from far outside the Baltoscandian margin of Baltica, Laurentia, or even an intervening microcontinent (Roberts & Gee, 1985; Stephens & Gee, 1989; Roberts et al., 2002; Lippard, & Roberts, 2010; Hollocher et al., 2012; Nilsson & Roberts, 2014). Although well known for their paleontology, the Ordovician basinal successions in this part of Norway have received comparatively little attention with regard to their sedimentology and overall development, with a few exceptions (Siedlecka, 1967; Roberts, 1972; Buller & Johnsen, 1982). As the region has a quite detailed map coverage at 1:50,000 scale, it clearly holds the potential to provide new insight into our current knowledge of deep-marine deposits, basin formation and basin-filling processes. the exotic oceanic terranes of the Iapetus Ocean deriving from far outside the Baltoscandian margin of Baltica, Laurentia, or even an intervening microcontinent (Roberts & Gee, 1985; Stephens & Gee, 1989; Roberts et al., 2002; Lippard, & Roberts, 2010; Hollocher et al., 2012; Nilsson & Roberts, 2014). Earlier research on turbidite systems Our general understanding of sedimentary structures and facies models for turbidite successions was quite advanced and for a large part well established by the 1980s (e.g., Kuenen & Migliorini, 1950; Kuenen & Menard, 1952; Sloss, 1963; Mutti & Ricci-Lucchi, 1975; Payton, 1977; Walker, 1978). The further development of sequence-stratigraphic concepts and the placement of the turbidite system within this framework accelerated during the 1980s and 1990s and major advancements were summed up in significant contributions by Wilgus et al. (1988) and Weimer & Posamentier (1994). In parallel with this, a growing body of publications on deep-marine turbidite deposits emerged in the scientific community (e.g., Weimer & Link, 1991; Mutti, 1992; Weimer et al., 1994; Plink-Börklund, et al., 2001). The increased focus on deep-marine deposits and processes was largely driven by the oil industry and the identification of these deposits as exploration targets. Our understanding of deep-water depositional systems has since advanced significantly mainly due to the advent of high-quality 3D seismic data from continental margins worldwide (Posamentier & Kolla, 2003; Mayall et al., 2006; Janocko et al., 2013; Gong et al., 2015). If a hyper-concentrated/hyperpycnal river plunges directly into a newly formed slump scar and submarine canyon, the sediment delivery to the deep-marine domain may be efficient as long as the river stays hyperpycnal (Porebski & Steel, 2006). In the case of a collapse of an over-steepened shelf edge, this will invariably trigger the initial phase of the turbidity current. Several studies (e.g., Hiscott et al., 1997; Mulder et al., 2001; Henriksen et al., 2011) have suggested hyperpycnal flows as an important mechanism for formation and maintenance of submarine canyons in areas of high sediment supply, either as frequent slide-induced surges or by sustained hyperpycnal flows (Mulder et al., 2003). In this scenario, the hyperpycnal flows confined within the incised shelf will set up a sediment fairway that will adjust its gradient towards the lowest point (basin floor). This will imply a stratigraphic base level different from mean sea level. Such a model will only be meaningful if there is a sustained flow from source to sink in the basin (Mutti et al., 2006; Mutti et al., 2009). Whereas early models of deep-marine systems were largely derived from outcrop studies in active tectonic settings, most of the recent models and interpretations originate in industry-based seismic studies from passive continental margins. Introduction Although well known for their paleontology, the Ordovician basinal successions in this part of Norway have received comparatively little attention with regard to their sedimentology and overall development, with a few exceptions (Siedlecka, 1967; Roberts, 1972; Buller & Johnsen, 1982). As the region has a quite detailed map coverage at 1:50,000 scale, it clearly holds the potential to provide new insight into our current knowledge of deep-marine deposits, basin formation and basin-filling processes. To partly redress this imbalance, in this contribution we describe the sedimentology and sedimentary development of part of the post-ophiolite, Ordovician to inferred Early Silurian, lithostratigraphical succession of the Åsenfjord district, located 25–40 km northeast of Trondheim (Fig. 1). The seeds of this present study Our study of the turbidites in the Åsenfjord area has led us to repudiate the traditional eustacy-driven sequence-stratigraphic model for deposition of such sediments. Instead, we favour a model involving long- term storage of erosional products in an alluvial to Figure 1. Location of the study area in the inner part of Trondheimsfjorden, Mid Norway. Block diagram with legend shows the lithostratigraphy and also illustrates the main, large-scale, Scandian fold structures in the area. The localities are all on 1:50,000 map-sheet 'Frosta' 1622-2. In some cases, the given localities extend along strike over several tens of metres. All the localities are in UTM zone 32V NR. UTM coordinates for the localities 1–9 are: Loc. 1. 59310–705425; Loc. 2. 59600–705430; Loc. 3. 59420–705290; Loc. 4. 59230–704970; Loc. 5. 59265–704985; Loc. 6. 59490–704940; Loc.7. 59540–704900; Loc. 8. 59290–704765. Figure 1. Location of the study area in the inner part of Trondheimsfjorden, Mid Norway. Block diagram with legend shows the lithostratigraphy and also illustrates the main, large-scale, Scandian fold structures in the area. The localities are all on 1:50,000 map-sheet 'Frosta' 1622-2. In some cases, the given localities extend along strike over several tens of metres. All the localities are in UTM zone 32V NR. UTM coordinates for the localities 1–9 are: Loc. 1. 59310–705425; Loc. 2. 59600–705430; Loc. 3. 59420–705290; Loc. 4. 59230–704970; Loc. 5. 59265–704985; Loc. 6. 59490–704940; Loc.7. 59540–704900; Loc. 8. 59290–704765. 143 Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides NORWEGIAN JOURNAL OF GEOLOGY to create incision of the highstand shoreface (e.g., Miall, 1991; Posamentier et al., 1992; Schumm, 1993; Bullimore et al., 2005). Introduction It is also well known that rivers may flood and become supersaturated with sediments independent of sea level, and deliver sediments directly to deep water if they are close to the shelf edge and can feed directly into the slope. In a basin where super-concentrated/ hyperpycnal flows occur from time to time, the sediment delivery to the shelf and shelf edge may be very high. This may result in accumulation of thick sediment successions at the shelf edge and over-steepening of the slope. These sediments are prone to collapse, and in the case of a hyperpycnal delivery system, the sediment discharge and current velocity may be sufficient to incise the shelf, even during a highstand situation. Indeed, Mulder et al. (2003) documented that a single flood may cause a river to incise 3 m as far as 2 km inland of the river mouth without implying sea level fall at all. deltaic accommodation zone, wherein such sediments are eventually and catastrophically flushed into the sea by a sudden availability of large amounts of water, thus generating a hyper-concentrated, high-density current. The sequence-stratigraphic line of thinking, and its predefined terminology, has been found unsuitable to describe and interpret the stratigraphy in the study area. Therefore, we have chosen a descriptive and neutral terminology in the following text, where beds and sets of beds constitute genetically linked depositional units. Stacking of units constitute successions of strata that may, or may not, be genetically linked. General The Trondheim Region of central Norway is dominated by a series of nappes or thrust sheets that were transported southeastwards onto the Baltoscandian margin of the Fennoscandian Shield during the Siluro– Devonian Scandian orogeny. This has been interpreted to result in the formation and migration of an oceanic island arc above a subduction zone influenced by continentally derived sediments (Slagstad et al., 2014). The Upper Allochthon here comprises three major nappes—from east to west the Meråker, Gula and Støren nappes—that together constitute the Trondheim Nappe Complex (TNC), which in turn occupies a deep depression in the Precambrian crystalline basement (Wolff, 1984; Gee et al., 1985; Hurich et al., 1988). Below the TNC in some areas there are diverse nappes of the Middle Allochthon, most of which have various local names (Gee et al., 1985; Roberts & Stephens, 2000). The Støren Nappe is particularly well known for its fragmented ophiolites and rich assemblage of Ordovician body fossils. The classical work of Vogt (1945) in the Hølonda–Horg area southwest of Trondheim (Fig. 1) led to the establishment of four major groups, in ascending order the Støren, Lower Hovin, Upper Hovin and Horg groups, ranging in age from Late Cambrian to Late Ordovician and possibly Early Silurian. The ophiolite at the base of the Støren lies with tectonic contact above a generally higher-grade, heterogeneous unit, the Gula Complex of the Gula Nappe (Fig. 1). Overlying the ophiolite are two main groups of sedimentary rocks, locally with a volcanic component, which Wolff (1976) named the Lower and Upper Hovin groups, following Vogt’s (1945) and Kiær’s (1932) nomenclature. On the preliminary 1:50,000 map-sheet ’Frosta’, Roberts (1985) retained the term Lower Hovin Group but used the name Ekne Group for the higher unit, following Törnebohm (1896). During ongoing revision of this map-sheet, the term Lower Hovin Group has been replaced by the local name Vuddudalen Group (Figs. 1, 2). The basal formation of the Vuddudalen Group is the Stokkvola conglomerate (Roberts, 1975), a polymict lithology lying unconformably upon the ophiolite and replete with material eroded from the latter. The clasts are dominated by basaltic greenstone, but there are also pebbles and cobbles (up to 40 cm across) and subangular blocks of jasper, tuffs, quartz-keratophyre, siltstone, sandstone, carbonate and pelitic rocks, quartzite, chert, trondhjemite and minor gabbro. Earlier research on turbidite systems really means in the evolution of a geological system, and how to recognise such an event in the rock record, has been a subject of discussion (Mutti et al., 1996; Mulder & Chapron, 2011). However, catastrophic events do occur and, as suggested in this and other studies, these events appear to be cyclic and affect the equilibrium profile of the depositional system (e.g., Mutti et al., 1996). In a larger perspective, the catastrophic events may be a natural part of the normal evolution of a sedimentary basin, especially where triggered by earthquakes in volcanically active regions. The Åsenfjord study area The greenschist-facies sedimentary succession of the Åsenfjord area and adjacent Frosta peninsula forms part of the Støren Nappe (Fig. 1). Although the succession has been deformed during three main phases of Mid Silurian– Early Devonian, Scandian folding (Roberts, 1969), the low-grade rocks display surprisingly well preserved sedimentary structures. The oldest rocks in the area are those of the Forbordfjell ophiolite, comprising mainly metabasaltic greenstones with minor trondhjemite, felsic extrusive rocks and gabbro. Zircons from two separate felsic rocks considered to be broadly coeval with the mafic volcanism have yielded U–Pb crystallisation ages of circa 485.6 and 488 Ma (Gromet & Roberts, 2015). Earlier research on turbidite systems The scale of the passive continental margin systems is generally several orders of magnitude larger than any exposed deep-water system onshore. The actual relevance of the outcrop studies from active tectonic settings to the large passive continental-margin basins and vice-versa is thus also a question to bear in mind (e.g., Lien et al., 2003; Higgs, 2015; Henstra et al., 2016). The tectonically active and episodically high supply margin discussed in this paper may provide excellent conditions for the formation of well-defined unconformities with only modest changes in relative sea level. The processes involved in transporting and depositing the sediments, however, are universal and in this study, we aim at utilising the sedimentological and stratigraphic data from a volcanic arc-adjacent basin and propose a depositional model that we believe may have general validity for understanding deep-marine deposits in similar basins. In this, we infer a linked depositional system from alluvial and deltaic to deep marine and the significance of sudden, catastrophic event release of large quantities of sediments stored in the alluvial and near- shore environments. Most workers, however, have related incision of the shelf and further emplacement of turbidites to the deep- marine environment to a lowstand situation, and indeed, even for the most extreme sedimentation rates this is most likely to happen during a fall in relative sea level. It is however, increasingly recognised that even during a relative sea-level fall, a difference in gradient is necessary These sudden releases of large volumes of sediments may be termed a catastrophic event. What a catastrophic event S. Henriksen et al. 144 excised in some areas. This is also the case in the Åsenfjord area, where it has been necessary to adopt a more local lithostratigraphic nomenclature. really means in the evolution of a geological system, and how to recognise such an event in the rock record, has been a subject of discussion (Mutti et al., 1996; Mulder & Chapron, 2011). However, catastrophic events do occur and, as suggested in this and other studies, these events appear to be cyclic and affect the equilibrium profile of the depositional system (e.g., Mutti et al., 1996). In a larger perspective, the catastrophic events may be a natural part of the normal evolution of a sedimentary basin, especially where triggered by earthquakes in volcanically active regions. General The matrix is a green to greenish-grey siltstone, generally coarser near the base of the formation and becoming increasingly finer grained and phyllitic towards the top (Fig. 2B). In the Fættenfjord area, the top of the conglomerate defines a major, basin-scale event in terms of both sedimentology and tectonics. In other areas, shallow- marine green sandstones and shales generally follow equivalents of the Stokkvola conglomerate (e.g., Buller & Johnsen, 1982), but in our study area a ’fold-fault’ or tectonic slide (sensu Fleuty, 1964) is present at the top of the conglomerate and has excised most of the shallow- marine strata. The lowermost lithologies (an unnamed formation) above the slide in our area are sheared grey shales, fan-fringe sandstone beds and a chaotic debris- flow unit (Fig. 2C). These are followed by fine-grained phyllitic claystones and siltstones, informally named the Fætta formation (Fig. 2). The highest unnamed unit in the Vuddudalen Group is a series of turbiditic greywacke-sandstone beds and intercalated phyllitic In a compilation of the 1:250,000 map-sheet ‘Trondheim’, Wolff (1976) adopted and extended Vogt’s nomenclature over the entire map area, and this fourfold lithostratigraphy was also incorporated into many of the 1:50,000 preliminary (black and white) map-sheets from the region compiled in the 1970s and 80s. Since that time, however, further research and mapping have shown that although the basic subdivisions are still largely acceptable, there are significant and, in places, rapid lithofacies changes along strike from area to area. In addition, parts of successions have been tectonically NORWEGIAN JOURNAL OF GEOLOGY NORWEGIAN JOURNAL OF GEOLOGY Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides 145 Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides 145 Figure 2. Logged section through the Fættenfjord profile. This is the most complete record of the lithostratigraphy in the study area. T to the right illustrates the field expression of some of the sandstones and conglomerates. See text for discussion and Fig. 1 for location Figure 2. Logged section through the Fættenfjord profile. This is the most complete record of the lithostratigraphy in the study area. The column to the right illustrates the field expression of some of the sandstones and conglomerates. See text for discussion and Fig. 1 for location of profile. formation, post-date the Tremadocian or older Forbordfjell ophiolite. Body fossils are rare in the study area and are not particularly definitive in terms of age. General Gastropods, cephelopods stromatoporoids and corals of wide-ranging Mid to Late Ordovician age have been reported from the Tautra limestone (Spjeldnæs, 1985), whilst conodonts from a nearby limestone on Frosta peninsula (Tolmacheva & Roberts, 2007) are Katian in age. Fragments of gastropods and corals also occur in the limestones in the Åsen–Hopla district. shales, some beds of which contain the deep-marine, Nereites trace fossils (Roberts, 1969; Uchman et al., 2005). This unit occurs directly below the base of the Ekne Group, represented by the Hopla conglomerate, which is excellently exposed on the promontory Blekkpynten. About 4–5 km farther north, between Åsen and Hopla, black pyritous shales and some limestones occur directly beneath the Hopla conglomerate (Roberts, 1985). The age of the Vuddudalen Group is predominantly Ordovician, as the basal parts, including the Stokkvola S. Henriksen et al. 146 In the several localities marked in Fig. 1, the Hopla conglomerate contains well-rounded clasts of granite, diorite, gneiss, trondhjemite, greenstone, quartz keratophyre, chert, sandstone, siltstone, quartzite and minor gabbro, but the percentages of the different rock- types vary from locality to locality and along strike (details are given in Pedersen, 1981). In addition, in some places, notably near Hopla and on Langøya, there are large fragments of sandstones and shales, and angular blocks of limestone between one and two metres across which are clearly short-transported. Although the Hopla conglomerate is indicated as one unit on maps at 1:50,000 scale, the formation is actually composed of twenty or more conglomerate beds separated by thin sandstone beds and phyllitic shales. Sections through the formation have been logged in detail at Hopla, Langøya, Digerberget, Gullgruva, Blekkpynten and Vikane (Fig. 3) for thickness, clast/matrix ratios, fining directions and sequence analysis (Pedersen, 1981). Due to the post- depositional tectonism and the varying appearance of conglomerates in the study area, any correlation between locations remains tentative, at best. conglomerate and pebbly greywacke beds. Higher up there is a series of thick-bedded, tuffaceous sandstone beds with shaly and rare tuffitic interbeds, reaching several hundred metres in thickness. The total thickness of the Ekne Group is estimated to be more than 2800 m, based on careful calculation of dips of bedding in the upward-grading succession. Taking average rates of deposition of arenaceous sediments into account, this would suggest that the youngest sedimentary strata in the group could conceivably be of Llandovery age. General Preliminary results of a provenance study (work in progress) are, in fact, indicating a Silurian age, and even possibly for the Hopla conglomerate. Facies associations/ depositional sub-environments section is a composite log starting at Paradisbukta (locality 7), through the Vuddudalen Group and ending at the uppermost exposures at Blekkpynten (locality 6) (Fig. 1). The subsequent discussion of basin evolution, stratigraphy, sedimentary processes and the final depositional model is largely based on detailed observations made in this area. Observations and interpretations from several other localities in the study area have provided vital information which strongly support our interpretations and help to rule out other possible scenarios. Facies associations/ depositional sub-environments The Fættenfjord profile represents the most complete record of deposition in the Lower Hovin Group from the top of the Stokkvola conglomerate, through successions of deep-water shale- and turbiditic sandstone beds and farther into the Hopla conglomerate and associated sandstone beds of the Ekne Group (Fig. 2A). The logged Above the Hopla formation, the Ekne Group comprises alternating units of shales, siltstones and grey-green turbiditic sandstone beds with several polymict Figure 3. Logged sections of the Hopla conglomerate from selected localities in the study area. Tentative correlation datum at the base of the Hopla conglomerate. See Fig. 1 for location of logs Figure 3. Logged sections of the Hopla conglomerate from selected localities in the study area. Tentative correlation datum at the base of the Hopla conglomerate. See Fig. 1 for location of logs NORWEGIAN JOURNAL OF GEOLOGY WEGIAN JOURNAL OF GEOLOGY Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides 147 147 Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides 147 The thin-bedded sandstone succession is overlain by a poorly exposed succession of shale and another sandstone unit (Fig. 2E). These sandstone beds show a coarsening and fining-up trend, where the lower and upper parts have facies similar to the thin-bedded sandstone unit below. This unit shows good lateral continuity and is traceable for several hundred metres along strike. Within the middle and coarser part of the unit, the individual sandstone beds thin and thicken laterally (Fig. 2E). Beds attain a maximum thickness of 1 m and pinch out to just a few centimetres laterally. Within the lenses there are imbricated beds suggesting lateral accretion in smaller channel features (Fig. 2E). There is little or no shale separating the individual sandstone beds and sand-on-sand is the rule rather than the exception. Internal sedimentary structures are not evident, giving the beds a massive structureless appearance (Fig. 2E). section is a composite log starting at Paradisbukta (locality 7), through the Vuddudalen Group and ending at the uppermost exposures at Blekkpynten (locality 6) (Fig. 1). The subsequent discussion of basin evolution, stratigraphy, sedimentary processes and the final depositional model is largely based on detailed observations made in this area. Observations and interpretations from several other localities in the study area have provided vital information which strongly support our interpretations and help to rule out other possible scenarios. Interpretation The apparent incorporation and rotation of sandstone slabs into the finer matrix is quite similar to what is seen in mass-transport complexes worldwide and interpreted as debris flows or slumps (e.g., Martinsen et al., 2003; Odonne et al., 2011). Although outcrop with good stratigraphic control is limited to this location, we interpret the deformed unit as a debris-flow unit possibly representing the first sandy input into the basin after establishment of comparatively deep-marine conditions. It is possible that this could represent a regional mass- transport complex transported in from a long distance. However, the clast material in this unit consists of fine- grained sandstone beds incorporated in a mud-rich silty matrix. The fine-grained nature of both clasts and matrix suggests that these sediments originally were deposited in a lower basin-slope to basin-floor environment and thus probably represent a slumped-in unit of local origin. The lenses and lateral accretion surfaces are suggestive of deposition in a channel in a deep-water fan setting, possibly related to a channel-mouth environment. Because of the close association with distal, basin-floor, fan environments below and above, we interpret this unit as a basinward progradation or lateral shift of a sandy basin-floor fan relative to the underlying beds. Description p The Stokkvola conglomerate is overlain by a shaly succession followed by a deformed unit with fragments of shale, siltstone and sandstone beds which have apparently rotated into the main body of strata (Fig. 2B, C). Neither the shale below nor the sandstones above show signs of syndepositional deformation. Interpretation The pinch-and-swell bedding of sandstones in fine- grained background sediment and planar-parallel lamination is an association typical for distal deep- marine settings. This would indicate that we are dealing with either a distal or quite possibly a lateral fan-fringe environment. The laterally continuous sandstone beds show coarsening and fining-up trends. These sandstones beds may represent a slightly more proximal part of the deep-water fan system. The lack of sedimentary structures makes it difficult to see if these sandstone lenses represent erosive features later filled by sediments or ‘depositional channels’ representing sandy ‘fingers’ spreading out from an up-dip source. Description p Directly above the lowermost deformed unit is a succession of thin (1–3 cm) sandstone beds alternating with laminae of siltstone and shale, and showing relatively good lateral continuity (Fig. 2D). Primary structures are difficult to identify, and bedding generally coincides with the main schistosity, but there are many internal features which are similar to small-scale, ripple- drift, cross-lamination structures. The beds also show examples of pinch-and-swell bedding of sandstone beds in fine-grained background sediment (Fig. 2D). Apart from these features, planar-parallel lamination dominates. This association is typical for distal deep- marine settings, and a distal or possibly lateral fan-fringe environment is suggested. Description p Successions of shale are present between sandstone units and represent the background sedimentation in the basin. The shales are generally dark grey, slightly phyllitic and commonly contain small cubes of pyrite. The dark colour and the presence of pyrite are probably due to anoxic conditions in the deeper parts of the basin. Although dominantly fine grained, the shaly intervals contain an alternation between beds of clay and sand in variable amounts throughout the section from 0 to 700 metres (Fig. 2A). These sandstone beds have been divided into eight classes based on the identification of diverse sedimentary structures and the development of graded bedding (Table 1). S. Henriksen et al. 148 S. Henriksen et al. 148 148 Class Thickness Grain size Bed boundaries Internal features Additional observations/Comments I Two beds: 31 and 76 cm Coarser fraction consists of medium to coarse sst. Undulating upper and lower boundaries Traceable 10- 20m laterally Planar parallel lamination and small current ripples. Constitute a complete Bouma sequence. In cross-bedding the height of each ripple is about 1, 5 cm. The wave- length of ripples from trough to trough is 1,3cm. II 3,5-125 cm Coarser fraction of the beds ranges between fine and very coarse sandstone. Even surfaces at the base and undulating tops. Traceable > 20 m laterally. Planar parallel lamination and small-scale cross- bedding. planar parallel lamination in lower/ middle part of the beds, Small -scale cross bedding in the upper parts of the beds. III 1-22,5 cm Fine and fine- medium sst. Lower boundary of the beds may be even or undulating. Traceable > 20 m laterally. Small-scale cross- bedding may be developed throughout the entire bed Completely even beds are not observed, but this may be due to the intense phases of folding post deposition IV 1-6 cm Very fine- medium sst. Dominating grain size is very fine- fine. “Pinch and swell” bedding. Traceable > 20 m laterally. Ripples in the thickest part of the beds. In places, almost isolated lenses of sandstone, but variable degree of undulation. V ~10 cm Fine - medium sst. Under- and overlying surfaces are generally even Graded bedding in their upper parts, as well as climbing ripple successions Laminations 2-3 cm thick probably caused by variable grain size. Some climbing ripples show sandy stoss sides while the lee side and trough consist of clay. VI 2-100cm. Description Stacked successions of thin-bedded turbidite beds are found in several locations within the study area, and most notably on the Blekkpynten location (Figs. 2F, 5A). These appear as alternations of thin-bedded turbidites and shale layers (Figs. 2F, 5A). The sandy deposits thin out from a few decimetres (maximum 50 cm) to a few centimetres over a lateral distance of 100 m. Over the same distance the sandstone beds also become finer grained and grade from medium-grained sandstone to a very fine-grained sandstone and siltstone. Levees/overbank (thin-bedded sandstone successions) Levees/overbank (thin-bedded sandstone successions) Interpretation Th f p The unconformable lower boundary and overall geometry points to a channellised deposit incising into the finer-grained substratum. The presence of coarse- grained deposits at the base of sandstone beds probably represents lag deposits indicative of sediment bypass and throughgoing turbidity currents. The horizontal and vertical stacking of sandstone beds strongly suggests lateral migration of sandy channel belts in a deep-marine environment (e.g., Beaboef, 2004, Mayall et al., 2006; Wynn et al., 2007). The outcrops do not allow for plan Description Fine - medium sandstone Even boundaries Traceable > 20 m laterally. Planar parallel lamination is 4 -20 mm thick, largely limited to the lower parts of the bed. Where the normal grading is more evident, the thicker the bed is. VII 1-109 cm Very fine - very coarse sandstone Generally medium sst Generally extensive and even surfaces. Sole marks (flute casts) at the base of some beds Massive structureless sst. a) Sole mark with a deeper part which widens and thins at one end, resulting in a cone shape feature. b) Erosional furrows with thin parallel stripes but with varying relief between the furrows. c) Flute cast similar to 1), but with the thickest part in the wider end. VIII 5-288 cm Very coarse sandstone and gravel Thin beds show even surfaces and thickness. Thicker beds, show lateral variations Massive structureless sst. Small-scale cross- bedding near the top of one bed One layer shows a, local, weak trend of upward coarsening (reverse/inverse grading). Table 1. Schematic illustration of the sandstone classes as identified in the study area. Table 1. Schematic illustration of the sandstone classes as identified in the study area. 149 NORWEGIAN JOURNAL OF GEOLOGY Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides NORWEGIAN JOURNAL OF GEOLOGY Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides Description Description Successions of sandstone beds typically occur below major conglomerate units and appear as 4–5, laterally accreting bodies of sandstone beds (Fig. 4). The lower boundary of any one sandstone bed shows a sharp and erosive contact with underlying shales and to some degree also with adjoining sand bodies. The sandstone beds constitute larger units that stack and amalgamate horizontally and to some degree also vertically to form a succession of sand bodies. Individual sandstone beds in successions defined by horizontal boundaries at their top and base display a series of inclined surfaces (Fig. 4A). The inclined surfaces are not following the structural grain and appear as primary bedding surfaces and thereby suggest lateral accretion during deposition (Fig. 4A). It is generally difficult to identify any grain-size separation within and between accreting surfaces, but in a few places coarser-grained sandstone or even conglomeratic sediments are found near the base of individual sandstone beds (Fig. 4B). Moreover, distinctly deformed units within otherwise well-stratified beds appear at certain positions within the sandstone successions (Fig. 4C). The deformed units are composed of the same material as the adjoining sandstone beds and parts of the primary bedding are preserved within these units. The appearance of the deformed units thus probably reflects a local origin and short distance of transport. Interpretation Interpretation view of the sandstone geometries, but the associations have a strong resemblance to sinuous channel systems (e.g., Janocko et al., 2013; Li et al., 2016) (Fig. 4D). p The trace fossils identified are found within thin-bedded turbidites and all belong to the Nereites ichnofacies which represents an opportunistic style of colonisation of the seabed by organisms adapted to fluctuations in food supply introduced by turbidity currents (Bromley, 1996). The eight sandstone classes within the overall fine-grained interval suggest that the turbidite current activity never really shut down totally in the basin. Bottom conditions were thus probably oxygenated in certain areas or in the entire basin from time to time (e.g., Torske, 1965; Roberts, 1969, 1984; Uchman et al., 2005). The alternation between sand and clay probably reflects an episodic supply of sediments to the basin, either due to sediment shut down or avulsion of feeder systems up-dip. Channels A variety of sedimentary structures are observed within this succession. Most common are planar lamination ripples and climbing ripple successions and one bed even displays a complete Bouma sequence (Fig. 5A). Examples of penecontemporaneous deformation of individual sand- stone beds also occur within this succession (Fig. 5B), where both the overlying and the underlying beds seem to be virtually unaffected by this slump-type deforma- tion. The deformed beds display contortions and unidirec- tional folds of the sandstone and shale, which in places are also broken up and rotated, suggesting that the beds must have been consolidated to some degree at the moment of deformation (Fig. 5A, B). The massive sandstone of the overlying bed appears to fill in the sea bottom relief caused by the slump-deformation process. It is thus likely that this is a local feature, and most probably a cohesive slump or debris flow released on an inclined surface of a slope or channel margin/levee. Evidence for current activ- ity is given by the presence of the above-mentioned sedi- mentary structures. In addition, there are also some nice examples of flute casts at the bases of some beds that docu- ment current activity during deposition (Fig. 5). In the more fine-grained parts of the succession there are beds containing the deep-marine, Nereites ichnofacies (Roberts, 1969; Uchman et al., 2005) (Fig. 5D). Small disconformi- ties within the otherwise more or less planar-bedded sand- stone succession occur as local erosive features (Fig. 5A). These features display a sharp erosive boundary and are filled with well stratified thin-bedded sandstone (Fig. 6A). Apart from these subordinate erosional features the suc- cession is dominated by planar-stratified beds, quite a few of them exposing successions of climbing ripples (Fig. 6B) which also display signs of synsedimentary deformation within individual beds (Fig. 5B). Interpretation The abundant evidence of current activity points to a close association with a sediment conduit or turbidite channel on the continental/basin slope. Both the S. Henriksen et al. 150 S. Henriksen et al. 150 Figure 4. Laterally accreting bodies of sandstone (A) in a shaly background environment at the Lofjorden locality. (B) Coarse-grained channel lag deposits with a fining-up trend via coarse sand into medium to fine sandstone. (C) Deformed sandstone embedded between well stratified sandstone beds. (D) Simplistic model for the occurrence of sedimentary features in laterally accreting sandstone units in sinuous channels. See Fig. 1 for location. Figure 4. Laterally accreting bodies of sandstone (A) in a shaly background environment at the Lofjorden locality. (B) Coarse-grained channel lag deposits with a fining-up trend via coarse sand into medium to fine sandstone. (C) Deformed sandstone embedded between well stratified sandstone beds. (D) Simplistic model for the occurrence of sedimentary features in laterally accreting sandstone units in sinuous channels. See Fig. 1 for location. Figure 4. Laterally accreting bodies of sandstone (A) in a shaly background environment at the Lofjorden locality. (B) Coarse-grained channel lag deposits with a fining-up trend via coarse sand into medium to fine sandstone. (C) Deformed sandstone embedded between well stratified sandstone beds. (D) Simplistic model for the occurrence of sedimentary features in laterally accreting sandstone units in sinuous channels. See Fig. 1 for location. beds or laminae and thin out both laterally and texturally away from the channel. The climbing ripple successions result from sediment fallout and net deposition and suggest that sediments contained in channel overflows were deposited relatively rapidly after leaving the main conduit (Fig. 6). The overflow from channels is most stratigraphic and the lithological thinning of sandstone beds seen in these facies are features characteristic of levees bounding deep-water channel systems (e.g., Janocko et al., 2013). Whilst the coarser-grained part of the turbidity current is kept within the channel, sand and clay particles will spill over channel margins as very thin NORWEGIAN JOURNAL OF GEOLOGY 151 NORWEGIAN JOURNAL OF GEOLOGY Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides 151 Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides 151 Figure 5. Features of the sandstone succession at the Blekkpynten locality in the Fættenfjord profile. (A) Alternating thin-bedded turbidite sandstone and shale. Interpretation Sandstone beds increase in thickness and content of sand upwards, and deformed sandstones with slump folds are found beneath thicker beds. (B) Detail of (A) displaying the undisturbed lower surface of the deformed sandstone and the well-structured sandstone above, filling in the relief on top of the inferred mass-movement deposit. (C) Flute casts on the bottom surface of a sandstone bed. (D) Trace fossils of the Nereites (Megagrapton) ichnofacies on the bottom surface of a sandstone bed found along the track leading to the Blekkpynten location. Figure 5. Features of the sandstone succession at the Blekkpynten locality in the Fættenfjord profile. (A) Alternating thin-bedded turbidite sandstone and shale. Sandstone beds increase in thickness and content of sand upwards, and deformed sandstones with slump folds are found beneath thicker beds. (B) Detail of (A) displaying the undisturbed lower surface of the deformed sandstone and the well-structured sandstone above, filling in the relief on top of the inferred mass-movement deposit. (C) Flute casts on the bottom surface of a sandstone bed. (D) Trace fossils of the Nereites (Megagrapton) ichnofacies on the bottom surface of a sandstone bed found along the track leading to the Blekkpynten location. Figure 6. (A) Crevasse splay channel in inferred levee deposit at the Blekkpynten location. Stratigraphic up is towards the left in the photo. See text for discussion. (B) Enlarged section of (A) showing thin-bedded turbidites with climbing ripples. Note also small-scale deformation structures in some of the beds. Figure 6. (A) Crevasse splay channel in inferred levee deposit at the Blekkpynten location. Stratigraphic up is towards the left in the photo. See text for discussion. (B) Enlarged section of (A) showing thin-bedded turbidites with climbing ripples. Note also small-scale deformation structures in some of the beds. and create crevasse channels and eventually splays on the levee surface (Lopez, 2001). Channel levee breaks may thus be a viable explanation to the erosive features within the thin-bedded sandstone succession. likely not a continuous process and some consolidation of beds may occur between each event. This may account for the deformation of inferred semi-consolidated beds on levee slopes. From time to time the levee will break S. Henriksen et al. 152 Conglomerate successions feature of the Hopla conglomerate is the well-rounded clast material (Fig. 7A). Another characteristic feature of the conglomerate successions is the alternation between clast-supported and matrix-supported conglomerates (Fig. 7A, Table 2). Notably the conglomerate beds show trends of coarsening up, fining up and coarsening-fining up-, as well as massive bedding (Table 2). Description Description The Hopla conglomerate contains pebbles and cobbles of a variety of rock types, mainly quartzite, grey granite, fine-grained gneiss, porphyritic andesitic greenstone and green-grey sandstone, with subordinate felsic volcanites and gabbro (Törnebohm, 1896; Vogt, 1945). A principal Figure 7. Conglomerates in the study area. (A) The Hopla conglomerate at Blekkpynten in the Fættenfjord succession. Note the well-rounded conglomerate clasts and the alternation between clast-supported and matrix-supported conglomerate. (B) Shale clasts and an angular clast of limestone embedded in coarse-grained immature sandstone beds in alternation with structured sandstones from the Langøya location in Lofjorden. (C) Sandstone in alternation with fine-grained thin-bedded sediments showing a sharp transition towards the overlying Hopla conglomerate. (D) Example of shale separating conglomerate successions at the Gullgruva location. (E) Conglomerate succession deposited directly on underlying fine-grained/shaly turbidite deposits, Location Langøya. Note the folding and shearing of beds below the conglomerate and an angular sandstone clast (1 m across) 'floating' on top of the conglomerate unit. (F) Conglomerate succession deposited directly on underlying fine-grained/shaly turbidite deposits, at Langøya. Note the folding and shearing of beds below the conglomerate. See Fig. 1 for location of photos. Figure 7. Conglomerates in the study area. (A) The Hopla conglomerate at Blekkpynten in the Fættenfjord succession. Note the well-rounded conglomerate clasts and the alternation between clast-supported and matrix-supported conglomerate. (B) Shale clasts and an angular clast of limestone embedded in coarse-grained immature sandstone beds in alternation with structured sandstones from the Langøya location in Lofjorden. (C) Sandstone in alternation with fine-grained thin-bedded sediments showing a sharp transition towards the overlying Hopla conglomerate. (D) Example of shale separating conglomerate successions at the Gullgruva location. (E) Conglomerate succession deposited directly on underlying fine-grained/shaly turbidite deposits, Location Langøya. Note the folding and shearing of beds below the conglomerate and an angular sandstone clast (1 m across) 'floating' on top of the conglomerate unit. (F) Conglomerate succession deposited directly on underlying fine-grained/shaly turbidite deposits, at Langøya. Note the folding and shearing of beds below the conglomerate. See Fig. 1 for location of photos. Figure 7. Conglomerates in the study area. (A) The Hopla conglomerate at Blekkpynten in the Fættenfjord succession. Note the well-rounded conglomerate clasts and the alternation between clast-supported and matrix-supported conglomerate. (B) Shale clasts and an angular clast of limestone embedded in coarse-grained immature sandstone beds in alternation with structured sandstones from the Langøya location in Lofjorden. Description (C) Sandstone in alternation with fine-grained thin-bedded sediments showing a sharp transition towards the overlying Hopla conglomerate. (D) Example of shale separating conglomerate successions at the Gullgruva location. (E) Conglomerate succession deposited directly on underlying fine-grained/shaly turbidite deposits, Location Langøya. Note the folding and shearing of beds below the conglomerate and an angular sandstone clast (1 m across) 'floating' on top of the conglomerate unit. (F) Conglomerate succession deposited directly on underlying fine-grained/shaly turbidite deposits, at Langøya. Note the folding and shearing of beds below the conglomerate. See Fig. 1 for location of photos. 153 NORWEGIAN JOURNAL OF GEOLOGY Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides Table 2. Summary table of conglomerates in the study area. Both the matrix-supported and the clast-supported conglomerates can be separated into four classes: massive, inverse graded, inverse to normal graded and normal graded. Thickness ranges, average thicknesses and mean clast size are indicated. Average measurements were carried out on representative beds (those that occur most commonly). Outliers are not included in the calculations Class Relative abundance % Thickness Range/average cm Mean Particle Size cm Bed boundaries Internal features Additional observations Matrix supported conglomerates 68,5% of total conglomerates in study area Massive 38,3 10.0 -211.0 Average: 70 2.5 - 29.0 Mean: 10.3 Conformable Random distribution of clasts of all sizes. One location, show clasts of very fine- grained sandstone in alternation with millimetre-thick laminae of clay or silt. Inverse 10,4 7.0 – 181.0 Average: 55.8 7.5 - 28.5 Mean: 15.4 Erosive on sst. Sharp on conglomerate Increasing matrix content and clast size upwards Constant grain size in matrix Inverse - normal 17,4 23.0 -212.0 Average: 77.5 6.0 - 58.0 Mean: 20.3 cm Mostly erosive Less matrix in inverse part of bed In places, the inverse grading first appears as ungraded beds with only a few larger clasts concentrated in distinct layers Normal 33,9 7.0 – 190.0 Average: 46.4 2.5 – 30.0 Mean: 13.8 Erosional base. Gradational top Upward increase in matrix content In some rare cases this matrix shows a gradual transition into overlying sandstone beds. Clast supported conglomerates 31,5% of total conglomerates in study area Massive 30,2 21.0 -143.0 Average: 66,7 3,0 – 41,0 Mean: 12.8 Even and concordant Random distribution of clasts of all sizes. Description Beds up to 175 cm observed; likely amalgamation Inverse 15,1 20.0 – 215.0 Average: 117.0 6,5 - 33,0 Mean: 20,9 Sharp, but no obvious erosion Matrix increase 10-15% upwards: the opposite occurs in one bed Inverse - normal 13,2 24 - 128 Average: 52. 3 9.0 – 30,0 Mean: 19.1 Erosive channel- geometry or planar and evenly levelled surfaces inversely graded part 17.2-37.3% of total bed thickness One bed 360cm thick, may be a result of amalgamation Inverse 41,5 15-180 Average: 69 5,5 - 22,5 Mean 13.4 Even, non-erosive General increase in matrix upwards. 40%r reduction occurs Matrix grain size overall constant, fine-medium sandstone. calculations. Inverse S. Henriksen et al. 154 In addition, there are clasts of shale up to a metre in length, as well as angular blocks of sandstone and limestone, up to two metres across, which are clearly short-transported and derived from subjacent depositional units (Fig. 7E). The conglomerate also contains intercalated beds of immature sandstone, some of which are continuous within any one outcrop (Figs. 7A, 8). Some beds within these sandstone intercalations show large-scale cross-bedding with set thicknesses of up to 50 cm (Fig. 8C, D, F). These cross-bed sets extend laterally over several metres with variable degrees of erosion at their bases. They also show a basal section of thinly bedded strata displaying sets of climbing-ripple laminations, succeeded by more massive sandstone beds with discrete accretion surfaces building a dune or bar form (Fig. 8B, D). Notably, these dunes seem to have an element of aggradation which suggests that they are ‘climbing dunes’ in the sense of Mutti et al. (1996). Near the top of the same bar form, the thin-bedded strata display sets of climbing ripples (Fig. 8B). The massive sandstone beds are fine to medium grained, whereas the climbing-ripple successions consist of fine- to very fine- grained sandstone. The lower boundary of the conglomerates commonly represents an unconformity and shows clear evidence of erosion into the underlying sandstone- or claystone beds. There are also several erosional features within the conglomerates and between thicker successions of conglomerates. Smaller and larger erosional features Figure 8. (A) Sandstone dominated by evenly spaced, thin-bedded turbidites. Inserted photo is a close up of the same outcrop and shows an example of the climbing-ripple succession within the unit, Location Langøya. (B) Climbing ripples in sandy beds between conglomerate beds, Location Blekkpynten. Description (C) Coarser-grained channel with foresets within sandy section between conglomerates (pencil for scale), Location Blekkpynten. (D) Larger cross beds or foresets with climbing-ripple succession below and above, Location Langøya. (E) Alternating sandstone and conglomerate succession at Langøya, Lofjorden. Stratigraphic upwards is towards the right in the photo. (F) Enlarged section (red square in E) illustrating bed boundaries, scouring and fill relations between sandstones and conglomerates. Figure 8. (A) Sandstone dominated by evenly spaced, thin-bedded turbidites. Inserted photo is a close up of the same outcrop and shows an example of the climbing-ripple succession within the unit, Location Langøya. (B) Climbing ripples in sandy beds between conglomerate beds, Location Blekkpynten. (C) Coarser-grained channel with foresets within sandy section between conglomerates (pencil for scale), Location Blekkpynten. (D) Larger cross beds or foresets with climbing-ripple succession below and above, Location Langøya. (E) Alternating sandstone and conglomerate succession at Langøya, Lofjorden. Stratigraphic upwards is towards the right in the photo. (F) Enlarged section (red square in E) illustrating bed boundaries, scouring and fill relations between sandstones and conglomerates. 155 NORWEGIAN JOURNAL OF GEOLOGY urbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides 155 Figure 9. Vertical logs with horizontal correlations at the Blekkpynten location. Note that sands may appear as lenses that wedge out within a short distance. Deformation structures result from both synsedimentary and post-sedimentary processes (See Fig. 1 for location and text for discussion). Figure 9. Vertical logs with horizontal correlations at the Blekkpynten location. Note that sands may appear as lenses that wedge out within a short distance. Deformation structures result from both synsedimentary and post-sedimentary processes (See Fig. 1 for location and text for discussion). the slope, they are assumed to have the character of debris flows in the upper reaches of the fairway. Farther downslope they will naturally incorporate more water, and with that lose the capability of carrying larger clasts. The conglomerate material will be transported along the base of the fairway and the kinetic sieving and/or dispersive pressure of the throughgoing current will thus result in inverse grading. When the gradient flattens out, the inversely graded conglomerates will be deposited relatively quickly, while the remaining part of the current will bypass. The currents will then deposit inverse/normal-graded beds, and normally graded to massive beds will develop farther downslope. Interpretation p The lower boundaries of both sandstone and conglomerate beds appear to erode into the underlying shale successions which commonly contain the deep- marine Nereites ichnofacies (Fig. 5). This implies that even though the conglomerates are transported and possibly also deposited in a channellised environment, the overall setting must have been deep marine and close to the basin floor. The climbing ripples are indicative of a waning flow and net deposition, whereas the massive, slightly coarser- grained sandstone beds are suggestive of waxing flow and an element of bypass. Building a larger bar or dune form in deep-marine settings will generally occur gradually over some time and will require a sustained flow lasting for hours or days, or even longer (Mutti et al., 1996; Zavala et al., 2011). Description It is thus still possible that the sandy successions and thin-bedded turbidites beneath conglomerates may represent parts of the same mass flow, but they must then be explained as lateral or distal equivalents of the massive, coarse- grained input. Whereas the sandy units are local features, most of the basin fill comprises thin-bedded fine-grained sandstones within mainly shaly intervals. at the base of the conglomerates and within the conglomerate successions are filled in with conglomerate beds of variable thickness and extent. Many of these beds show an even thickness over 2–4 m and then wedge out rapidly to zero within 0.5 m (Fig. 9). The sediments underlying conglomerates commonly display syn-depositional deformation structures and examples of the underlying deposits having been detached and incorporated into the conglomerates as large angular clasts (Fig. 7). Description h k p Thick successions of very fine to fine-grained greenish sandstone beds and shales are found in the western parts of the study area (Fig. 10). These facies comprise thin-bedded graded sandstone beds with very few sedimentary structures preserved. Ripples and cross- bedding structures are preserved in a few places, but it is often quite difficult to distinguish primary planar structures from the regional schistosity in parts of the study area (Fig. 10). However, a repetitive pattern of thin- bedded normal-graded sandstone beds characterises the unit (Fig. 10). Larger bedforms such as lenticular beds with a size and geometries similar to hummocky cross-bedding do occur (Fig. 10). Larger erosive features The conglomerates may be regarded as hyper concentrated gravity flows (sensu Mutti, 1992) that experienced several phases of flow transition on their way to the basin sink. The variable fining up, coarsening up, coarsening up-fining up and massive trends for the conglomerate beds suggest highly variable current conditions. If the conglomerates were triggered by one specific event, and initially move en masse down S. Henriksen et al. 156 Figure 10. (A) Inclined surfaces in sandstone succession interpreted to represent hummocky cross stratification or large-crossbedding. (B) Channel geometry within thin-bedded turbidites in the western and inferred most proximal part of the study area. (C) Ripple structures and faint graded bedding. (D) Apparent (false) ripple structures between bedding due to schistosity induced by later tectonic phases. Figure 10. (A) Inclined surfaces in sandstone succession interpreted to represent hummocky cross stratification or large-crossbedding. (B) Channel geometry within thin-bedded turbidites in the western and inferred most proximal part of the study area. (C) Ripple structures and faint graded bedding. (D) Apparent (false) ripple structures between bedding due to schistosity induced by later tectonic phases. Discussion occur in the form of a channel geometry incising into the underlying strata. The infill of these channel features is little different from the underlying lithology (Fig. 10). The post-depositional erosion of the sedimentary succession and subsequent Scandian deformation inhibits a full source to sink analysis of the basin. Whereas the deep-water part of the depositional system is well preserved in the study area, only a few examples of inferred hummocky cross-stratification in close association with a possible channel feature are found in the western part of the area (Fig. 10). Although hummocky cross-bedding has traditionally been interpreted to result from storm wave action on the continental shelf, it has been found to originate from several different processes and can easily fit into a model involving high sedimentation rates and unidirectional flows (Southard et al., 1990; Mutti et al., 2007; Quin, 2011). The interpretation remains speculative, but the repeated pattern of graded, thin-bedded sandstones, hummocky cross-stratification and channellisation may represent an integral part of the turbidity system. Interpretation This accounts for the deformed beds seen in the channel successions (Fig. 4C). Since the turbidity current is driven by gravity, most sediment movement is naturally downslope. However, the current also interacts with the flow separation along channel margins and local flow cells will develop, similar to eddies and back-logs in fluvial systems. This flow pattern is likely to contribute to lateral movement of sediments and the creation of bar forms and accretion surfaces in the channels. The few observations of channels in the study area do not justify our proposing a detailed model for channel evolution, but nicely documents channellised flows and allows for a general model to explain the observed stacking pattern (Fig. 4D). The interchangeable sandstone and conglomerate beds and the incorporation of coarse-grained material in the sandstone beds as well as sands within the matrix- supported conglomerates suggest that these sediment types are closely associated and parts of the same event. The sandstone beds associated with conglomerates generally show well preserved sedimentary features with relatively thick units of evenly spaced, thin-bedded turbidites dominated by rippledrift and climbing-ripple structures (Fig. 8A, B). Sandy beds may settle out from the ‘tail’ of the turbidity current or possibly also from separate events of current flow. If sandstone beds are deposited as separate events, they may accumulate in proximal parts of the system. Bypassing currents will subsequently erode and entrain the channel sands and deposit these as part of the normally graded beds at the distal end of the system. In this model, winnowing of sands in the proximal parts will make it more likely to find inversely graded, clast-supported conglomerates in the upper reaches of the channel fairway, a transitional zone with relatively thin beds of inverse/normal-graded beds, and thicker, normally graded, matrix-supported conglomerates in the more distal setting. This depositional model should be regarded with some caution since even hyper-concentrated flows will vary in velocity and concentration. Consequently, individual currents will deposit proximal and distal facies at different locations, and the distal–proximal criterion for the classification of deep-marine conglomerates becomes difficult to apply. The proximal-distal model for explaining the matrix content and bedding style may be redundant in any event, since even the slightest sinuosity of the channel will result in a lateral sorting of material (e.g., Jobe et al., 2010). Interpretation p Thick successions of normal-graded, thin-bedded, fine- grained sandstone point to a relatively high sediment supply with repeated input of fine-grained sediments. The pale greenish colour of the sediment points to an oxygenated depositional environment, quite unlike the dark-coloured shale successions in the inferred deep-marine parts of the basin. Considering a very sandy sediment with a narrow grain-size distribution, deposition offset, but not too far from a main feeder system is suggested. The exact location of the sediment in a source to sink perspective is difficult to pinpoint due to the quality of the outcrop, but the hummocky cross- bedding points to a shallow-marine shelf environment. The channel feature is not diagnostic in itself, but the lack of coarse-grained lag suggests low gradients with little sediment bypass before the infill of sediments that are quite similar to the substrate. Based on the observations and overall appearance, we interpret these thin-bedded sandstone and shale beds to represent either shelf or upper slope deposits. Evident from the trace fossil assemblage, facies associations and sedimentary structures, the sandstone channels and levee facies were deposited in a deep- marine slope environment. Moreover, these facies NORWEGIAN JOURNAL OF GEOLOGY 157 Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides associations are commonly found beneath the conglomerate successions and it is plausible that they occupied the same sedimentary fairway on the slope. The lateral migration of sandstone facies within and among inferred channel geometries suggests some degree of sinuosity of the channel systems. Sinuous and laterally accreting channels tend to form as a result of sediment accumulation at inner and outer channel bends, unlike fluvial systems that consistently erode at outer bends and deposit sediments on inner channel bends (Posamentier & Kolla, 2003; Kane et al., 2008; Janocko et al., 2013). The variable degree of deposition and bypass in submarine systems allows for more stable channel geometries than in the fluvial counterpart. There is, however, general consensus that the axial part of the channel will be a zone dominated by coarse-grained sediment bypass lags (Fig. 4B), and that lateral migration will still occur (e.g., Abreu et al., 2003; Posamentier & Kolla, 2003). Because of the asymmetry in channel cross-section created by the variable flows, instability and slumping is likely to occur at channel margins when the deepest part of the channel is shifted towards either of the channel margins. Interpretation The active channel will be dominated by conglomerates, while sandy material will be deposited along channel margins both as clean sandstone beds and, as a result of channel ‘overflow’, also as matrix between the clast material. Flow velocities will vary within a sinuous channel system, and flow separation will result in the development of lateral accretion surfaces and bar forms at channel bends (e.g., Posamentier & Kolla, 2003; Mayall et al., 2006; Janocko et al., 2013). Flows may erode the matrix-rich deposits in fast-flowing parts of the channel and deposit its material where the flow slows down at the next channel bend. This process will result in winnowing of finer-grained material and deposition of clast-supported material along the channel thalweg. An important prerequisite for this model to work is that there is very little time between erosion and deposition, and that there is not time enough to properly incorporate all the fine-grained material in the suspension cloud of the turbidity current. In this way, clast-supported conglomerates may be deposited in the channel thalweg (or areas of fastest flow) and the matrix-supported conglomerates deposited where flows are slower. Clast- supported and matrix-supported conglomerates may Channellisation within and at the base of conglomerate successions suggests that conglomerates were transported into the basin along the same sedimentary fairways as the sandy channel deposits. Whether the sandstone and conglomerate beds represent separate events or just grain-size variations within the same event of sediment release, remains uncertain. What is readily observed is that several events of conglomerate deposition are stacked on top of each other within these fairways, and thus suggest repeated events of deposition (Figs. 7D, 8, 9). The occurrence of conglomerates in deep- water settings will, in general, be limited to channels or larger conduits on the basin slope. Such channels may attain significant dimensions and some have been recorded up to 25–30 km in width and 200 m in depth (e.g., Hickson & Lowe, 2002). The actual distribution of conglomerates in the study area is difficult to perceive due to the complex structuring of the area. The suggested datum for correlation (Fig. 3) may therefore represent several conglomerate-filled channels, offset in both time and space. S. Henriksen et al. 158 S. Henriksen et al. 158 thus occur side by side without implying a proximal- distal concept. Interpretation energy needed to overcome the barrier represented by the ‘energy littoral fence’ set up by the longshore currents is quite considerable (sensu Mazullo & Erlich, 1980). In reality, only flood-generated, supersaturated flows have sufficient energy to escape marine diffusion processes and transport coarse-grained material away from the shore zone (Allen, 1970; Mutti et al., 1996). Although reworking of carbonate material is evident, there are also examples of large, angular blocks or clasts of carbonate, sandstone and shale that must be locally derived and transported over only short distances. The abundant occurrence of carbonate clasts, some of significant size, provides evidence for periods of carbonate reef growth along the basin margin. The presence of carbonate reefs suggests limited siliciclastic sediment supply to the shelf for long periods of time. This implies equally long periods of accumulation, storage and reworking of coarser-grained sediments in near-shore and onshore areas. The initial rounding of clasts may have occurred in a fluvial environment, but the presence of well-rounded carbonate clasts suggests that some of the abrasion of clasts results from reworking by marine processes in the littoral zone. The The coarse-grained sediments stored in the near- shore and shelfal areas may well have been triggered by slumping and submarine sliding on the basin margin. gure 11. Depositional model with suggested areal distribution of facies in the Åsenfjord mixed conglomerate-turbidite system. N diments deposited on the continental shelf are interpreted to result from the same catastrophic event as the conglomerates in the d ccession and are regarded as an integral part of the turbidite system. No scale intended. Figure 11. Depositional model with suggested areal distribution of facies in the Åsenfjord mixed conglomerate-turbidite system. Note that the sediments deposited on the continental shelf are interpreted to result from the same catastrophic event as the conglomerates in the deep-marine succession and are regarded as an integral part of the turbidite system. No scale intended. NORWEGIAN JOURNAL OF GEOLOGY Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides 159 Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides NORWEGIAN JOURNAL OF GEOLOGY exotic explanation is that the sediments could have been made available to marine processes by tsunamis, triggered by seismic activity, submarine slides or giant rock falls on land; truly a catastrophic event, but this remains difficult to prove for the above-stated reasons. Interpretation The actual triggering mechanism may never be known, even though seismogenic events are the most reasonable option, and we therefore limit the discussion to the stratigraphic signature left by the sudden release of large amounts of sediment and water. In any event, the ‘catastrophic model’ may largely explain the presence of thick successions of conglomerates in deep-marine environments. Depending on the amount of water available, the floods will probably last for a while and give rise to relatively long-duration, sustained, supersaturated flows to the basin. After a while the system will experience a subsequent decrease in water and sediment supply. The flood will eventually recede and this will cause a gradual back-stepping of the system until the water discharge becomes so small that the bulk of sediments will be stored in a landward If released by slumping, this implies that the slumps must have reached far enough into the shelf to reach the conglomerate material, and consequently also must have been of significant size. In such a case, shelf-crossing canyons may have acted as conduits for sediment transport. This situation represents ‘drainage’ of the source area from the seaward side of the basin, and does adequately explain the presence of both well-rounded clasts and the large angular blocks found in the deep- marine successions. Given the island-arc setting, frequent earthquakes capable of triggering the release of large quantities of clastic material are to be expected (e.g., Gutierrez-Pasto et al., 2009). However, the fluvial catchment area cannot have been very large, but rather favours a region of steep topography with many small rivers and a narrow continental shelf. The tectonic activity may have been combined with climatic cyclicity involving periods of heavy rainfall or the collapse of natural dams. A more Figure 12. A schematic back-stripped profile with four stages of evolution illustrates the basin-filling successions of the Åsenfjord turbidite system. The four stages are likely to be repeated in a cyclic manner. Note that a direct link between the alluvial and turbidite system is inferred. (A) Denudation of the landscape will result in accumulation of sediments in alluvial and fan-delta settings. Very little sediments are transported beyond the littoral zone during this stage and carbonate sedimentation prevails on the outer shelf. (B) Uplift of the source significantly increases the availability and accumulation of sediments in the alluvial system. Interpretation (C) After reaching a threshold value, and possibly/likely triggered by a seismogenic/tectonic pulse, vast amounts of clastic material are flushed to the marine part of the basin during a catastrophic event. (D) When the alluvial system is evacuated for sediments, the system gradually attains normal conditions and carbonate sedimentation eventually resumes on the outer continental shelf. Figure 12. A schematic back-stripped profile with four stages of evolution illustrates the basin-filling successions of the Åsenfjord turbidite system. The four stages are likely to be repeated in a cyclic manner. Note that a direct link between the alluvial and turbidite system is inferred. (A) Denudation of the landscape will result in accumulation of sediments in alluvial and fan-delta settings. Very little sediments are transported beyond the littoral zone during this stage and carbonate sedimentation prevails on the outer shelf. (B) Uplift of the source significantly increases the availability and accumulation of sediments in the alluvial system. (C) After reaching a threshold value, and possibly/likely triggered by a seismogenic/tectonic pulse, vast amounts of clastic material are flushed to the marine part of the basin during a catastrophic event. (D) When the alluvial system is evacuated for sediments, the system gradually attains normal conditions and carbonate sedimentation eventually resumes on the outer continental shelf. S. Henriksen et al. 160 S. Henr 160 160 transport complexes has recently been suggested by Ortiz-Karpf et al. (2018). position again. In sequence-stratigraphic terms, this will be equivalent to a highstand situation and only mud and silt will then be carried to the deeper parts of the basin. When siliciclastic sediment delivery to the basin is at its minimum, carbonate sedimentation and reef growth will resume on the shelf. position again. In sequence-stratigraphic terms, this will be equivalent to a highstand situation and only mud and silt will then be carried to the deeper parts of the basin. When siliciclastic sediment delivery to the basin is at its minimum, carbonate sedimentation and reef growth will resume on the shelf. Due to the post-depositional polyphase structuring of the area, measurement of paleocurrent flow directions may appear virtually meaningless. The exact basin physiography will therefore be difficult to reconstruct, but a scenario with both lateral sediment sources as well as an axial feeder system for turbidites and conglomerates is quite possible, as described by Pedersen (1981) (Fig. 13). Interpretation The conglomerates represent relatively proximal parts of the depositional system, likely residing in canyons or larger sediment fairways fed from the basin margins. The thin-bedded turbidites and inferred distal facies, however, could very well have been transported in an axial system with contributions from far-distant sediment sources as well as from shorter-transported volcanogenic sediments from the island arc in the ‘west’ and the uplifted continental mainland to the ‘east’ (Fig. 13). The vertical stacking of several conglomerate units suggests that the deposition was cyclic in nature. The classical sequence-stratigraphic principles and relative sea level causative explanation for cyclicity are, however, less likely to have worked in this active tectonic setting. Instead, we propose a four-stage model where the stratigraphic signature is explained in terms of repetitive phases of uplift and erosion of the drainage area and where the ‘shallow marine’ features are included in the turbidite system via a transfer zone (Figs. 11, 12). This transfer zone may to some degree have spread out laterally on the shelf and accounts for the hummocky cross-beds, ‘precursor beds’ and dune forms signalling a sustained flow of sediments to the basin (Fig. 11). Summary and conclusions If not released by slumping and canyon formation on the shelf, the release of sediments by catastrophic floods would have likely occurred around the time of maximum elevation and subsidence, which provided the source and accommodation for vast amounts of alluvium in the drainage area. The tectonic phases will naturally be long-term and provide the sediments available through time, whereas the triggering of individual conglomerate- turbidite successions records a higher frequency signal (Fig. 12). Similar tectonically induced release of mass • The Ordovician back-arc basin formation in Mid Norway is linked to changing subduction polarity in the Iapetus Ocean following Early Ordovician ophiolite obduction and gave rise to the gross distribution of land areas and uplift centres (Torsvik, 1996; Grenne & Roberts, 1998; Hollocher et al., 2012; Hollocher et al., 2016). The short-term tectonics and proximity to a volcanic arc were important features Figure 13. Simplified reconstruction of basin physiography and possible sediment routing during deposition. Both basin-axial and basin- transverse depositional systems are possible in explaining the observed facies and facies associations. Figure 13. Simplified reconstruction of basin physiography and possible sediment routing during deposition. Both basin-axial and basin- transverse depositional systems are possible in explaining the observed facies and facies associations. 161 JOURNAL OF GEOLOGY Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides Turbidite and conglomerate successions in an Ordovician back-arc basin, Mid-Norwegian Caledonides NORWEGIAN JOURNAL OF GEOLOGY Acknowledgements. Anna Pontén and Nils Erik Janbu at Statoil Rese- arch Center in Trondheim read and edited an earlier version of this manuscript and provided many useful comments and suggestions. Lars Reistad did a tremendous job in drafting of the figures. Deta Gasser and Gjis Henstra reviewed the manuscript thoroughly and their insightful comments significantly contributed to improvement of both text and figures. To all these persons, we offer our sincere thanks. in the staging of sediments in near-shore and shelfal areas and their sudden release and transport to the receptor basin. in the staging of sediments in near-shore and shelfal areas and their sudden release and transport to the receptor basin. • The well-rounded clasts in the Hopla conglomerate are taken as evidence for long-term storage and reworking of coarse-grained sediments. The presence of carbonate clasts in the conglomerates suggests build-up of reef structures on the shelf and a very limited sediment supply to the shelf during periods of carbonate reef build up. https://doi.org/10.1007/978-1-4899-2875-7 • By incorporating the thin-bedded turbidite successions and hummocky cross-bedded strata into the turbidite system, a holistic view on the turbidite system is introduced. These strata are interpreted to represent dispersive flows on the outer shelf and upper continental slope. Bruton, D.L. & Bockelie, J.F. 1980: Geology and paleontology of the Hølonda area, western Norway—A fragment of North America? In Wones, D.R. (ed.): The Caledonides in the USA, Virginia Polytechnic Institute and State University, Department of Geological Sciences Memoir 2, pp. 41–47. Buller, A.T. & Johnsen, S.O. 1982: Storm-influenced marine sandstones in the Ordovician Lower Hovin Group, Nord-Trøndelag. Norwegian Journal of Geology 62, 211–217. • The conglomerate beds have incised into the substrate and into each other and were thus probably confined to larger conduits (channel complexes), variably occupied by sandy turbidity currents and supersaturated conglomeratic flows. Bullimore S., Henriksen, S., Liestøl, F.M. & Helland-Hansen, W. 2005: Clinoform stacking patterns, shelf-edge trajectories and facies associations in Tertiary coastal deltas, offshore Norway: Implications for the prediction of lithology in prograding systems. Norwegian Journal of Geology 85, 169–187. Fleuty, M.J. 1964: Tectonic slides. Geological Magazine 101, 452–456. https://doi.org/10.1017/S0016756800049840. • Laterally accreting sandstone units are interpreted as sinuous turbidite channels. Such channel sandstone beds are always found below major conglomerate successions and constitute an embryonic sediment fairway leading to the deep-marine environment. Gale, G.H. & Roberts, D. 1974: Trace element geochemistry of Norwegian Lower Palaeozoic basic volcanics and its tectonic implications. Earth and Planetary Science Letters 22, 380–390. https://doi.org/10.1016/0012-821X(74)90148-4. Gong, C., Wang, Y., Pyles, D.R., Steel, R.J., Xu, S., Xu, Q. & LI, D. 2015: Shelf-edge trajectories and stratal stacking patterns: Their sequence- stratigraphic significance and relation to styles of deep-water sedimentation and amount of deep-water sandstone. American Association of Petroleum Geologists Bulletin, Bulletin 99, 1211–1243. https://doi.org/10.1306/01311513229. • Sinuosity in the turbidite channels may have caused flow separation even for the coarsest grain sizes and may be an explanation for the interchangeable and juxtaposed matrix- and clast-supported conglomerates. Grenne, T. & Roberts, D. 1998: The Hølonda Porphyrites, Norwegian Caledonides: geochemistry and tectonic setting of Early-Mid Ordovician shoshonitic volcanism. Journal of the Geological Society of London 155, 131–142. https://doi.org/10.1144/gsjgs.155.1.0131. • The most distal turbidite units consist of sandstone beds and locally derived mass-transport deposits. These sediments probably dispersed freely on the sea floor and had no restriction on distribution other than the natural barriers set up by the relief of previous deposits. Gromet, L.P. 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https://openalex.org/W2023823730	https://digibug.ugr.es/bitstream/10481/64462/1/pone.0054856.pdf	English	null	Social Support, Socio-Economic Status, Health and Abuse among Older People in Seven European Countries	PloS one	2,013	cc-by	9,992	Abstract Competing Interests: The authors have declared that no competing interests exi Competing Interests: The authors have declared that no competing interests e * E-mail: g.melchiorre@inrca.it Maria Gabriella Melchiorre1, Carlos Chiatti2, Giovanni Lamura1, Francisco Torres-Gonzales3, Mindaugas Stankunas4,5, Jutta Lindert6, Elisabeth Ioannidi-Kapolou7, Henrique Barros8, Gloria Macassa9,10, Joaquim F. J. Soares9 Maria Gabriella Melchiorre1, Carlos Chiatti2, Giovanni Lamura1, Francisco Torres-Gonzales3, Mindaugas Stankunas4,5, Jutta Lindert6, Elisabeth Ioannidi-Kapolou7, Henrique Barros8, Gloria Macassa9,10, Joaquim F. J. Soares9 1 Centre of Socio-Economic Research on Ageing, Italian National Institute of Health and Science on Aging, I.N.R.C.A., Ancona, Italy, 2 Scientific Direction, Italian National Institute of Health and Science on Aging, I.N.R.C.A., Ancona, Italy, 3 Centro de Investigacio´n Biomedica en Red de Salud Mental (CIBERSAM), University of Granada, Granada, Spain, 4 School of Public Health, Griffith University, Gold Coast, Queensland, Australia, 5 Department of Health Management, Lithuanian University of Health Sciences, Kaunas, Lithuania, 6 Department of Public Health Science, Protestant University of Applied Sciences, Ludwigsburg, Germany, 7 Department of Sociology, National School of Public Health, Athens, Greece, 8 Department of Hygiene and Epidemiology, Medical School, University of Porto, Porto, Portugal, 9 Department of Public Health Sciences, Institution for Health Sciences, Mid Sweden University, Sundsvall, Sweden, 10 Division of Social Medicine, Department of Public Health Sciences, Karolinska Institute, Stockholm, Sweden January 2013 \| Volume 8 \| Issue 1 \| e54856 Abstract Background: Social support has a strong impact on individuals, not least on older individuals with health problems. A lack of support network and poor family or social relations may be crucial in later life, and represent risk factors for elder abuse. This study focused on the associations between social support, demographics/socio-economics, health variables and elder mistreatment. Methods: The cross-sectional data was collected by means of interviews or interviews/self-response during January-July 2009, among a sample of 4,467 not demented individuals aged 60–84 years living in seven European countries (Germany, Greece, Italy, Lithuania, Portugal, Spain, and Sweden). Results: Multivariate analyses showed that women and persons living in large households and with a spouse/partner or other persons were more likely to experience high levels of social support. Moreover, frequent use of health care services and low scores on depression or discomfort due to physical complaints were indicators of high social support. Low levels of social support were related to older age and abuse, particularly psychological abuse. Conclusions: High levels of social support may represent a protective factor in reducing both the vulnerability of older people and risk of elder mistreatment. On the basis of these results, policy makers, clinicians and researchers could act by developing intervention programmes that facilitate friendships and social activities in old age. Citation: Melchiorre MG, Chiatti C, Lamura G, Torres-Gonzales F, Stankunas M, et al. (2013) Social Support, Socio-Economic Status Older People in Seven European Countries. PLoS ONE 8(1): e54856. doi:10.1371/journal.pone.0054856 , Chiatti C, Lamura G, Torres-Gonzales F, Stankunas M, et al. (2013) Social Support, Socio-Economic Status, Health and Abuse among uropean Countries. PLoS ONE 8(1): e54856. doi:10.1371/journal.pone.0054856 Editor: Antony Bayer, Cardiff University, United Kingdom Received August 2, 2012; Accepted December 17, 2012; Published January 30, 2013 Copyright: 2013 Melchiorre et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This research was funded by grant support from the European Commission, through the Executive Agency for Health and Consumers (EAHC), (Public Health Programme 2008–2010), Grant Agreement n. 2007123, which made possible the realisation of the ABUEL Project, ‘‘Elder Abuse: A multinational prevalence survey’’. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Sources/Collection and Ethics Statement Sampling and administration procedures were carried out according to the national, ethical and legal requirements for this type of studies. The potential participants were informed about the study by means of a letter explaining aims and contents of the ABUEL project. Written informed consent from participants, regarding also their anonymity, rights and freedom to stop the interview at any moment, was obtained prior to data collection. Ethical permission/approval also was sought and received in each participating state from the national/university or regional ethics review boards. Greece was an exception. In detail, the full names of the ethics committees/institutional review boards were the following: Regional etisk kommittee vid Karolinska Institutet (Karolinska Institute, Regional Ethics Com- mittee) in Sweden; Ethikkommission des Landes Baden-Wuert- temberg (Ethics Committee of the State of Baden-Wuerttemberg) in Germany; Comitato di Bioetica INRCA, Istituto Nazionale di Riposo e Cura per Anziani, Ancona (National Institute of Health and Science on Aging, Bioethics Advisory Committee) in Italy; Kauno regioninio biomedicininiu tyrimu etikos komitetas (Kaunas Regional Research Ethics Committee) in Lithuania; Comite´ de E´tica do Hospital de Joa˜o, Porto (Ethics Committee of the John Hospital, Porto) in Portugal; Comite´ de Etica en Investigacio´n de la Universidad de Granada (Research Ethics Committee, Univer- sity of Granada) in Spain. In Greece the field work was carried out by the QED company which is member of ESOMAR that provides ethical guidance through global guidelines, and actively promotes self-regulation in partnership and researchers with a number of associations across the globe. The members, as well as their company contact details, are listed in the ESOMAR Members Directory. Members undersigned, and agreed to abide by the ICC/ESOMAR International Code on Market and Social Research, which has been jointly drafted by ESOMAR and the International Chamber of Commerce. Social isolation and a low level of social support may be crucial risk factors for elder abuse, besides older age, chronic health conditions and cognitive deficits. Also, if the old person is economically dependent this can add to the burden and stress experienced by family caregivers of older relatives and play a role in elder abuse. A systematic review of studies on the prevalence of elder abuse and neglect in various countries [24] has reported abuse rates ranging between 3.2–27.5% in the general population. Data Sources/Collection and Ethics Statement Concerning the elderly, social support may represent a main source of personal care and well-being [13], and the aspects already emphasized in the general context of social support become more critical and amplified by the various problems connected to an ageing population. Social vulnerability, which is a concept related to a low social support, is indeed higher among people with individual frailty, and it increases with age. Greater social vulnerability is associated with mortality in older adults [14]. Very old age is moreover associated with lower levels of income [15], and reduced social networks and social support are more frequent among older people with low socio-economic position [16]. Very old age is also associated with lower levels of health [17]. The positive influence of social support on the health of the elderly is well documented; in particular emotional support from offspring is positively associated with a higher degree of well-being, and less distress and cognitive impairments among older people without a spouse [18]. Conversely, loneliness in old age has been suggested to be a risk factor for morbidity and mortality [19]. In particular, an absence of informal support can have a serious impact on health and quality of life of low-income elderly women living alone, and this may also lead to premature institutionali- zation [20]. Further, recent findings emphasise the importance of family and friendship for healthy aging [21], and confirm that chronic stress and loss of functions in older people may be mitigated by informal and formal support [22]. Family solidarity, in its affective aspect, can indeed be considered a ‘‘robust concept’’ and a fundamental element for social integration in old age [23]. The present study is based on the data from the ABUEL survey (Elder Abuse: A multinational prevalence survey) carried out to investigate the prevalence and risk factors of violence against the elderly in seven urban centres within selected European countries: Ancona (Italy), Athens (Greece), Granada (Spain), Kaunas (Lithuania), Ludwigsburg (Germany), Porto (Portugal) and Stock- holm (Sweden). The data were collected cross-sectionally among community-dwelling elderly populations during January-July 2009 by face-to-face interview or interviews/self-response. All survey materials (e.g. the questionnaire) were translated into the relevant languages, back-translated, and culturally adapted. Interviewers in each country were carefully instructed about the administration of the questionnaire and ethical behaviour. Strong emphasis was put on voluntariness and confidentiality of participation. Introduction support is the subjective evaluation of the received help. Perceived support is a crucial resource when stress is experienced [6], and for individuals with limitations in daily living activities (ADLs, i.e. everyday routine activities generally involving functional mobility and personal care, including eating, bathing, dressing, toileting, walking and control of continence) [7]. Higher levels of interpersonal trust also appear to be positively correlated with good self-assessed physical health and mental well-being [8]. Social support is defined in terms of social network character- istics such as assistance from family, friends, neighbours and other community members. It involves ‘‘social transactions the aims of which are to assist individuals in coping with everyday life, and particularly in responses to critical situations’’ [1]. Hall & Wellman [2] proposed the concepts of social network and social support as mediating constructs towards well-being, to explain how various social exchanges among individuals, mainly in situations of need, may influence health outcomes. Further studies refer to the health benefits resulting from social support due to its capacity to reduce the risks for both physical and cognitive illnesses [3,4]. According to Krause [5], received support is the amount of tangible help provided by social network, whereas perceived The evidence suggests that social support depends on several demographic/socio-economic and geographical factors. In gener- al, the perception of available support is higher among younger and married persons, and persons with higher socio-economic and employment status [9]. Concerning gender, some studies find January 2013 \| Volume 8 \| Issue 1 \| e54856 1 PLOS ONE \| www.plosone.org Social Support and Elder Abuse in Europe major informal perceived support for women [10], whereas others report larger health benefits from social networks for men [11,12]. Materials and Methods Data Sources/Collection and Ethics Statement January 2013 \| Volume 8 \| Issue 1 \| e54856 Data Sources/Collection and Ethics Statement PLOS ONE \| www.plosone.org January 2013 \| Volume 8 \| Issue 1 \| e54856 2 Social Support and Elder Abuse in Europe number of diseases (e.g. cardio-vascular) from which the elderly were currently suffering. The questions were derived from the Stock- holm County Council health survey [37]. elderly (e.g. sheltered houses). The sample size calculation was based on municipal censuses in each participating city, and on an expected abuse prevalence of 13% derived from a recent systematic review [24]. Assuming this prevalence rate, with a precision of 2.6%, a sample size of 633 individuals in each city was required, but considering the infinite population assumption a maximum of 656 individuals was allowed. The sample size was adapted to each city according to the population of individuals aged 60–84 years (representative and proportional to sex and age). Mean response rate was 45.2% across countries. More detailed description of materials and methods, sampling strategy and data collection, target population, cooperation, completion and re- sponse rates by country, are reported in a separate paper [31]. Various demographic and socio-economic variables such as age, gender, marital status, living situation, habitation, education level, profession, financial support and financial strain were measured. Age was categorized into five-year groups (60–64, 65–69, 70–74, 75–79, and 80–84). Marital status was assessed as single, married/ cohabiting, divorced/separated and widow/er. Living situation (as type of relationship to the person living with the interviewee) was classified as alone, only with partners/spouse, with partner/ spouse/others (e.g. daughter), without partner/spouse and with others (e.g. daughter). Habitation was assessed as living in an own property, in a rented place, or other (e.g. housing for elderly). Education level was grouped into seven categories: cannot read/ write, without any degree, less than primary school, primary school/similar, secondary school/similar, university/similar, other (e.g. art school). Profession was grouped into six categories: managers/professionals/assistant professional, clerical support/ sales workers, skilled agricultural/forestry/fishery workers, assem- blers/elementary occupations, housewife/husband, and armed forces. Finally, the financial situation was assessed by means of financial support and financial strain. Financial support asked for the main source of income, and was categorised as work income, work pensions (e.g. age and early retirement pension), social/sick- leave/other pension benefits (e.g. sick-leave/unemployment/social support benefits, disability/sick pension), partner/spouse income (e.g. widower pension), and other (e.g. rentals from own capital, including no financial support). Statistical Analyses y The bivariate relation between social support and categorical variables (e.g. demographics/, socio-economics and abuse) was analysed with the Kruskall-Wallis test with a Bonferroni correc- tion, following a Shapiro-Wilks test to check for the normality of distributions. Associations between social support and numerical variables (household size, healthcare services use, depression, anxiety, somatic complaints, and number of events of abuse) were analysed with the Spearman correlation test. The analyses of the factors associated with abuse were expanded also with regard to each sub-categories of social support (from family, friends and significant other). Multivariate quantile linear regression models, based on median values, were used to examine the interrelations between social support and various variables (independent). The associations between social support and the independent variables were expressed in un-standardized Betas and their standard errors (SEs). Un-standardized Betas were used, despite difficulties in the interpretation of coefficients, because the main aim in the study was to establish associations among covariates and the dependent variable, not measuring them or comparing different coefficients. The choice of using the Beta coefficients and SEs, as outcome measures from the regression models, is also explained by the need to allow comparisons with other similar studies where this sort of data presentation is the most usual. The statistical packages SPSS 15.1 and STATA 11.1 were used to carry out the analyses. Somatic symptoms were measured with the short version of the Giessen Complaint List [35], consisting of 24 questions (graded 0– 4, no complaints-severely affected). The symptoms are organized according to four types, with six questions in each: exhaustion (e.g. tiredness); gastrointestinal (e.g. nausea); musculoskeletal (e.g. pains in joints or limbs); and heart distress (e.g. heavy, rapid or irregular heart-throbbing). The total score amounts to 96, and the sub-total score in each symptom category ranges from 0–24. The higher the scores, the more one is affected (sub-scales, total). For this study, the focus was on the total score. Depressive and anxiety symptoms were measured with the Hospital Anxiety and Depression Scale [36]. This consists of 14 questions (graded 0–3), with seven questions about depression (e.g. I feel as if I am slowed down) and seven about anxiety (e.g. I get sudden feelings of panic). The total score for depression and anxiety is 21 each. A score of 0–7 corresponds to no cases, 8–10 to possibly cases and 11–21 to probable cases. High scores correspond to high depression and anxiety levels. Measures The participants completed a standardized questionnaire with various validated instruments. Social support was measured with the Multidimensional Scale of Perceived Social Support [32]. It consists of 12 questions (graded 1–7), which can be divided into 3 sub-scales, i.e. support from family, significant other and friends. Each sub-scale has been calculated when all related 4 items have been answered. The possible range of each subtotal score is 4–28. Likewise, the total scale has been calculated when all 12 items have been answered. In this case, the possible range of total score (sum all responses) is 12–84. High scores correspond to high social support (sub-scales, total). For this study, the focus was on the total and sub-scales scores. Violence was assessed with 52 questions based on the UK study on elder abuse [33] and the CTS2 [34]. The participants were asked if during the past year they had been exposed to at least one single episode/event of: psychological (11 items), physical (17 items), sexual (8 items) and financial abuse (9 items), including injuries (7 items). The acts of abuse may have occurred once, twice, 3–5, 6– 10, 11–20 or .20 times during the past year, or did not occur the past year. In addition, we assessed neglect (e.g. not helped in routine housework) with 13 items where the participants were asked whether they needed help and received it, needed help but did not receive it or did not need help. Data concerning the perpetrator’s main characteristics were also gathered. For this study, the focus was on exposure to the above-mentioned abuse types, excluding neglect. Data Sources/Collection and Ethics Statement Self-reported financial strain (preoccupation with how to make ends meet) was investigated with the following question: ‘‘How often are you worried about the daily expenses? (e.g. for buying food)’’ It was measured in a ‘‘no/ sometimes/often/always’’ format. A participant was defined as having ‘‘financial strain’’ if she/he chose any response other than ‘‘no’’. The demographic and socio-economic variables were customised for each country, but similar in content. Data Sources/Collection and Ethics Statement In this context, social isolation may represent a crucial dimension of social insecurity and vulnerability affecting older people due to their minor (or lack of) role in society. When this is combined with a reduced independence in later life, it may expose older persons to mistreatment and/or to neglect. Isolation thus appears to be a risk factor for all forms of elder abuse (e.g. physical). Consistent correlations between different types of elder abuse and low social support [25], and also consistent positive associations between most subtypes of mistreatment and depression [26] have been found. In contrast, a high level of social support may represent one potential protective factor for elder mistreatment [27]. Greater levels of social support can modify and reduce depression in old age as a risk factor for elder abuse, mainly in women [28]. The final sample (sex and age-stratified) included 4,467 persons (2,559 women, 57.3%) randomly selected (registry/census based) from the general population, except for Greece and Portugal. In Greece a sampling by random route of the elderly was obtained, according to a walking scheme that allowed selecting elder persons in households, and in ‘Open Care Community Centres’ (KAPI). In Portugal a cluster sampling method was used, and subjects were recruited among the members of a cohort (EPIPorto) of urban dwellers previously selected using random digit dialling. The inclusion criteria across countries were: (a) women and men; (b) age 60–84 years; (c) not suffering from dementia, or other cognitive impairments, assessed by means of the Mini-Cog [30]; (d) having legal status (national citizens or documented migrants); (e) living in the community (own/rented houses) or homes for Scrutinizing this latter topic in Europe is very important as few studies have addressed elder abuse and its features from a comparison perspective, within multi-cultural and multi-national contexts [24,29]. The aim of this study was to determine whether social support (perceived help from family, friends and significant other) was related to various selected dimensions in old age in seven European countries, including the crucial aspect of elder abuse. We hypothesized that a high level of social support would be associated with increased health and well-being, and negatively linked with the risk of mistreatment. Lack of social support, in a context of dependency and vulnerability, may indeed represent a potential risk of exposure to abuse. January 2013 \| Volume 8 \| Issue 1 \| e54856 Social Support, Country and Demographic/Socio- economic Variables Social support by country and demographic/socio- economic variables. As shown in Table 2, participants from Lithuania reported higher mean scores on social support than those of the other countries, with Portugal showing the lowest (p,.001). Individuals under 70 years, in particular those aged 60– 64 years, scored higher on social support than older participants, with those aged 80–84 years reporting the lowest (p,.001). Participants who were male (p = .023), but also participants who were married/cohabiting, living only with spouse-partner, highly educated with university degree and owning their housing reported greater social support than their counterparts (p,.001). Those who had been managers-professionals and in the armed forces (p = .001), had their main financial support from a work pension and did not experience financial strain (p,.001) also scored high on social support. Drawing from the significant results of the multivariate analyses, we found that social support differed in relation to a wide range of associated factors. Respondents from Greece and Lithuania were more likely to report increased support, whereas the opposite emerged in Portugal and Italy. With regard to geographical differences in the level of perceived social support, some authors [38,39] observed cross-national/cultural variations. Other studies [40] reported a strong ‘‘familistic’’ cultural tradition in Mediter- ranean countries, and a greater support from non-family networks in the non-Mediterranean ones. In our study we partly found the abovementioned differences across countries. The positive relation with perceived social support, which is reported from Greeks, could thus be explained by a greater family support available in the Mediterranean Greece, whereas the high rate of perceived social support in Lithuania could be more related to non-family networks in the non-Mediterranean areas. Drawing from some national studies, the Lithuanian context could also be explained by the high level of education of elder participants from this country [41], which is linked in turn to a greater social involvement, comparing with the less educated ones [42]. In Italy and Portugal we found a negative association with the level of perceived social support. We can argue that these two typical Mediterranean contexts, with a strong ‘‘family-oriented’’ connotation, have been negatively influenced by recent and similar demographic/socio- economic changes (e.g. low fertility rates, smaller households, increasing presence of women in the labour market, urbanisation and increasing individualisation). Internal Reliability of Exposure Variables Reliability, considered as internal consistency of exposure variables across countries in the study, was assessed using the Cronbach’s a statistic. Cronbach a for total social support was.92, and for the three subscales of family, friends and significant other were.90,.94 and.87, respectively. Cronbach a for violence was: for psychological.85, for physical.80, for sexual.76, for financial.64, and for injuries.70. Finally Cronbach a was for somatic symptoms.92, for anxiety.81 and for depression.80. Results less marked than support from family) in the case of psychological and financial mistreatment. The support from significant other was perceived as much lower by respondents exposed to all kinds of violence, except for sexual abuse (no significant differences), and this result was more evident when injuries and physical violence were involved. Social Support, Country and Demographic/Socio- economic Variables These changes, according with some authors, could possibly account for a dramatic reduction of the traditional resilience of family networks, as primary welfare providers for older people in these areas [43,44]. Correlations between social support, household size and health variables. As shown in Table 3, household size was positively correlated with social support, indicating that the larger the household, the greater the social support received (r = 0.1276, p,.05). Conversely, depression(r = 20.2110, p,.05), anxiety (r = 20.2911, p,.05), and physical complaints (r = 20.2180, p,.05), were negatively correlated with social support, whereas no significant correlation was found with the frequency of health care contacts. Social support by abuse type and injuries. As shown in Table 4, elderly exposed to psychological, physical and financial abuse, and injury, reported significantly lower scores in total perceived social support than their counterparts. In particular, this was more evident among those who sustained injuries (56.3 vs. 67.5, p = .001), There were no significant differences concerning sexual abuse. Social support by abuse type and injuries. As shown in Table 4, elderly exposed to psychological, physical and financial abuse, and injury, reported significantly lower scores in total perceived social support than their counterparts. In particular, this was more evident among those who sustained injuries (56.3 vs. 67.5, p = .001), There were no significant differences concerning sexual abuse. Concerning subscales, family support was significantly perceived to be lower by respondents exposed to all kinds of violence, except for injuries (no significant differences), and the victims of sexual abuse had the lowest significant score (19.8 vs. 23.4, p = .005). With regard to perceived social support from friends, the differences between abused and non-abused respondents were significant (but Concerning subscales, family support was significantly perceived to be lower by respondents exposed to all kinds of violence, except for injuries (no significant differences), and the victims of sexual abuse had the lowest significant score (19.8 vs. 23.4, p = .005). With regard to perceived social support from friends, the differences between abused and non-abused respondents were significant (but Being aged 70–74 or 80–84 years was associated with low social support levels, which is in line with other findings from previous literature confirming that the oldest people are more isolated and less socially supported [45]. Multivariate Analyses y Factors associated with social support. As shown in Table 5, a higher level of social support was independently associated with being from Greece (ß = 4.91, p,.001) and Lithuania (ß = 5.56, p,.001), married/cohabitant (ß = 5.35, p,.01), divorced/separated (ß = 3.24, p,.01), widow/er (ß = 6.67, p,.001), living with spouse/partner (ß = 5.25, p,.01) or other persons e.g. daughters (ß = 3.26, p,.01), living in larger households (ß = 0.69, p,.05), frequent use of health care services (ß = 4.51, p,.001), and low scores in depression (ß = 27.21, p,.001) and physical complaints (ß = 20.06, p,.01). A lower level of social support was independently associated with being from Italy (ß = 22.15, p,.05) and Portugal (ß = 24.56, p,.001), older age (mainly those aged 80–84 years, ß = 22.28, p,.05) and male (ß = 21.63, p,.01), having a social/sick-leave/other pension as the main source of financial support (ß = 22.98, p,.01), and exposure to psychological mistreatment (ß = 22.51, p,.001). Descriptive Statistics Table 1 provides a full descriptive summary of the demograph- ics and socio-economic characteristics of the sample. The responses from 4,467 participants, besides 57.3% of women (as already highlighted), also put in evidence that 6.0% of them were single and 65% were married or cohabiting, and that 49.6% lived only with a partner/spouse and 24.2% alone. Further, 76% of the interviewees lived in an own habitation, 24.5% had a primary/ elementary education and 39.9% a secondary/intermediate one. With regard to the occupation, 27.6% of the sample were managers/professionals, 27.5% were clerical support/sale work- ers, and 14.9% were housewives. Finally, 65.9% lived on a work pension and 64% declared to experience financial strains. January 2013 \| Volume 8 \| Issue 1 \| e54856 Statistical Analyses For this study, the focus was on the total scores. Health care use was measured in form of the number of contacts with different types of health care staff (e.g. physician) and health care services (e.g. primary care). Additionally, we assessed the January 2013 \| Volume 8 \| Issue 1 \| e54856 January 2013 \| Volume 8 \| Issue 1 \| e54856 PLOS ONE \| www.plosone.org 3 Social Support and Elder Abuse in Europe Social Support, Country and Demographic/Socio- economic Variables The recent and actual demographic changes (different family patterns due to declining marriage, January 2013 \| Volume 8 \| Issue 1 \| e54856 PLOS ONE \| www.plosone.org 4 Social Support and Elder Abuse in Europe Table 1. Cont. Total (n = 4467) Variables N % Work 542 12.1 Work pensions 2939 65.9 Social/sick-leave/other pension benefitsd 243 5.4 Partner/spouse income 627 14.1 Othere 110 2.5 Financial strain No 1605 36.0 Yes 2857 64.0 a = e.g. daughter; b = e.g. housing for elderly; c = e.g. art school; d = e.g. sick pension; e = e.g. own capital. doi:10.1371/journal.pone.0054856.t001 Table 1. Cont. the sample. Total (n = 4467) Variables N % Country Germany 648 14.5 Greece 643 14.4 Italy 628 14.1 Lithuania 630 14.1 Portugal 656 14.7 Spain 636 14.2 Sweden 626 14.0 Age (group years) 60–64 1124 25.2 65–69 1088 24.4 70–74 961 21.5 75–79 749 16.8 80–84 545 12.2 Gender Female 2559 57.3 Male 1908 42.7 Marital status Single 270 6.0 Married/cohabiting 2903 65.0 Divorced/separated 343 7.7 Widow/er 950 21.3 Living situation Alone 1078 24.2 Only partner/spouse 2208 49.6 Partner/spouse//othersa 706 15.9 Without partner/spouse – with othersa 457 10.3 Habitation Own 3392 76.0 Rental 930 20.8 Otherb 143 3.2 Education Cannot read/write 136 3.0 Without any degree 187 4.2 Less than primary school 338 7.6 Primary school/similar 1092 24.5 Secondary school/similar 1782 39.9 University/similar 855 19.2 Otherc 73 1.6 Profession Managers/professionals/assistant profess. 1217 27.6 Clerical support/sale workers 1214 27.5 Skilled agricultural/forestry/fishery workers 707 16.0 Assemblers/elementary occupations 570 12.9 Housewife/husband 656 14.9 Armed forces 45 1.0 Financial support increasing divorce and cohabitation, reconstitutions of new family) indeed highlight significant family disruptions and a decreasing care role of the family. According to literature, this has crucial implications for the provision of social support to older persons, especially to the ‘‘oldest old’’ suffering from health problems and functional limitations [46] thus further affecting the reduction in social relations which takes place especially after retirement [47]. Males were less likely to receive social support than females, and consequently were at higher risk of not being adequately supported by the informal network. An explanation of these results is supported by the literature [48], showing that women have a more socially-oriented life-style, are more concerned about establishing social relations, and appear to receive support from multiple sources, whereas men tend to rely usually on the wife. January 2013 \| Volume 8 \| Issue 1 \| e54856 Social Support, Country and Demographic/Socio- economic Variables It is to be considered that in the multivariate analyses (Table 5), whose finding are discussed, men scored negatively (like those living on social sick leave and those aged 70–74 and 80–84 years), and this suggests that men, but not women, experience a lower social support. Conversely, in the bivariate analyses (Table 2) social support is higher for men. This may seem counter-intuitively but it is the result of the adjustment of other covariates in the regression analysis. Likewise, as mentioned in the introduction, the literature on gender and perceived social support is controversial. Some authors refer to major perceived support for women [10], whereas others report that men have larger social networks [12]. Social support from family, friends and significant other was more likely to be received by those who were not living alone but with partner/spouse or other persons. Additionally, being married/cohabitant, divorced/separated and widowed were asso- ciated with higher levels of social support. These findings suggest that living arrangements, as the effective presence of a spouse/ partner or other persons in the household, more than marital status (with the exception of single individuals) are predictors of stronger social support. Many studies have shown that married/ cohabiting persons can count on spousal/partner social support and that the spouse/partner is usually the principal figure providing help [49], whereas not having the assistance of a spouse/partner is a significant predictor of living alone [50]. Predictably, household size was found associated with increased social support levels. In this respect, previous studies have stressed that household size may have various implications for the well- being of older people. Some authors suggested that social support January 2013 \| Volume 8 \| Issue 1 \| e54856 January 2013 \| Volume 8 \| Issue 1 \| e54856 5 Social Support and Elder Abuse in Europe Table 2. Cont. Social supporta Variables n Mean SD Pb Work 532 67.8 14.3 Work pensions 2859 70.0 13.4 Social/sick-leave/other pension benefitsf 234 60.7 16.5 Partner/spouse income 622 66.4 15.7 Otherg 106 65.9 14.5 Financial strain ,.001 No 1563 68.7 14.1 Yes 2791 66.8 14.9 a = MSPSS, 12–84; b = Kruskall-Wallis test: P,.05; c = e.g. daughter; d = e.g. housing for elderly; e = e.g. art school; f = e.g. sick pension; g = e.g. own capital. doi:10.1371/journal.pone.0054856.t002 Table 2. Cont. Social Support, Country and Demographic/Socio- economic Variables Social supporta Variables n Mean SD Pb Country ,.001 Germany 580 68.0 14.8 Greece 642 67.8 15.7 Italy 610 67.2 12.6 Lithuania 629 70.4 12.7 Portugal 650 63.2 12.8 Spain 635 67.4 16.2 Sweden 612 67.6 16.4 Age (group years) ,.001 60–64 1098 69.1 13.5 65–69 1064 68.6 14.2 70–74 935 66.7 14.9 75–79 727 66.5 15.1 80–84 534 64.1 16.0 Gender = .023 Female 2493 66.9 15.2 Male 1865 68.3 13.7 Marital Status ,.001 Single 258 58.9 19.0 Married/cohabiting 2841 70.0 12.1 Divorced/separated 331 61.3 18.5 Widow/er 927 64.3 16.6 Living situation ,.001 Alone 1038 61.3 18.3 Only partners/spouse 2161 70.3 12.1 Partner/spouse/othersc 693 68.8 12.1 Without partner/spouse - with othersc 450 66.3 15.3 Habitation ,.001 Own 3332 68.5 13.9 Rental 887 64.1 16.3 Otherd 139 62.2 17.2 Education level ,.001 Cannot read/write 135 59.8 19.0 Without any degree 186 66.5 18.0 Less than primary school 335 64.3 16.2 Primary school/similar 1075 66.9 14.5 Secondary school/similar 1718 68.0 13.8 University/similar 835 69.8 13.4 Othere 71 66.9 14.3 Profession = .001 Managers/professionals/assistant profess. 1190 69.2 13.5 Clerical support/sale workers 1171 67.5 14.2 Skilled agricultural/forestry/fishery workers 694 66.6 14.5 Assemblers/elementary occupations 563 65.8 16.6 Housewife/husband 644 66.6 15.4 Armed forces 45 71.0 10.7 Financial support ,.001 itself can be seen as resulting from certain structural/quantitative characteristics of the social network, e.g. size and composition of one’s interpersonal ties [2,51]. Other studies found that larger households may improve intergenerational solidarity and support, and also reduce isolation in later life [52], thus buffering poor health and improving quality of life [53]. Social-sick-leave/other pension benefits as the main source of financial support was associated with low social support levels. This is in line with authors [54] indicating that respondents experiencing a poor economic situation and low occupational status, such as unskilled workers, report the poorest social support. This also confirms what previous studies have asserted, i.e.the perception of available support is higher among individuals with higher occupational status and higher income levels [55], whereas reduced social support is more frequent among socio-economically disadvantaged older people [16]. Table 3. Correlations between social support, household size and health variables. Social Support, Depression, Somatic Complaints and Health Care Services the health and well-being of older people, but due also to the lack of a theoretical framework useful for understanding this type of effect [63], the way it works is not clear [64], i.e. social isolation could also be a symptom of physical/mental health problems rather than a cause [65]. In any case, our findings are in line with above-mentioned research indicating that ‘‘deficiencies’’ in social support have a negative impact on health. In our study depressive symptoms were associated with decreased social support. Our results concerning depression confirm previous studies showing that strong social support may be associated with increased mental health, and it may act as coping resources in older age [47], whereas individuals who have more restricted networks are most likely to exhibit signs of depression [56]. Literature also shows that social support may exert a buffering effect reducing the impact of depression on functional status [57], and that support from marriage, in particular, protects against worse psychological health over time [58]. Our findings indicate also a positive relation between frequent use of health care services and increased social support levels. These results seem in line with studies indicating that frequent contacts with the social network and higher levels of social support are associated with greater use of general medical services [66], whereas conversely socially isolated older adults use fewer health services and seem to have small social networks resources [67]. Our findings seem thus to follow the ‘bridging’ mechanism [68] which indeed predicts a positive association between service use and social support (i.e. social networks bring individuals into contact with health services when needed). Therefore, our findings are not in line with Cantor’s ‘hierarchical-compensatory’ mech- anism [69], i.e. when social support is unavailable from the social network, the request for formal services increases. Further, somatic complaints were associated with low levels of social support. Our findings are consistent with those from recent studies emphasising a positive association between health and social support, and thus an overall protective effect of social networks on disability may be supposed. Literature on the topic highlights that this was especially true for older individuals with chronic disease, and for those suffering from stress associated with ADLs limitations [59], whereas social isolation may also increase the risk for coronary heart disease events and mortality [60]. Social Support, Country and Demographic/Socio- economic Variables Social supporta Variables Correlation coefficientsb Household sizec 0.1276* Health care services used 20.0244 Depressione 20.2110* Anxietye 20.2911* Physical complaintsf 20.2180* a = MSPSS, 12–84; b = Spearmann correlation: c = number of people in the household; d = number of health care contacts; e = HADS, 0–21; f = GBB 24, 0–96; P,.05. doi:10.1371/journal.pone.0054856.t003 January 2013 \| Volume 8 \| Issue 1 \| e54856 Table 3. Correlations between social support, household size and health variables. January 2013 \| Volume 8 \| Issue 1 \| e54856 PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org 6 Social Support and Elder Abuse in Europe Table 4. Social support by abuse type and injuries during the past 12 months. Abuse type and injuries Total Social supporta Social support from familyaa Social support from friendsaa Social support from significant otheraa n Mean SD Pb Mean SD Pb Mean SD Pb Mean SD Pb Psychologicalc ,.001 ,.001 = .001 = .001 No 3494 68.4 14.03 23.8 5.1 20.2 6.9 24.3 4.9 Yes 864 63.7 16.40 21.5 6.2 19.4 6.9 22.5 5.9 Physicald = .001 = .001 = .138 = .001 No 4246 67.6 14.50 23.4 5.4 20.1 6.9 24.0 5.1 Yes 112 61.5 18.15 21.1 6.3 19.0 7.3 21.6 6.3 Injuriese = .001 = .300 = .248 = .001 No 4329 67.5 14.56 23.4 5.4 20.0 6.9 23.9 5.1 Yes 29 56.3 21.04 18.6 7.3 18.3 7.8 18.5 7.1 Financialf = .001 = .001 = .013 = .001 No 4186 67.7 14.42 23.5 5.4 20.1 6.9 24.0 5.0 Yes 172 62.3 18.51 21.0 6.8 18.8 7.2 22.1 6.6 Sexualg = .318 = .005 = .281 = .385 No 4325 67.5 14.61 23.4 5.4 20.0 6.9 23.9 5.1 Yes 33 64.1 17.93 19.8 7.7 21.1 7.0 22.8 6.1 a = MSPSS, 12–84; aa = MSPSS, 4–28; b = Kruskall-Wallis test: P,.05; c = e.g. undermined or belittled what you do; d = e.g. kicked you; e = e.g. you passed out from being hit on the head; f = e.g. tried to make you give money, possessions or property; g = e.g. touched you in a sexual way against your will. doi:10.1371/journal.pone.0054856.t004 January 2013 \| Volume 8 \| Issue 1 \| e54856 Social Support, Depression, Somatic Complaints and Health Care Services Further authors showed that social support may also provide health benefits to older persons by facilitating recovery when ill or by protecting against illness through biological mechanisms [61]. Family members’ actions may indeed often support the recovery of their older relatives by providing intangible resources and emotional help [62]. The combination of the results of this section - frequent use of health care services was independently associated with increased social support, whereas depressive symptoms and somatic com- plaints were associated with decreased social support – indicates, on the whole, relations concerning the dimensions investigated. We are aware that, as the data are cross-sectional, they don’t allow the establishment of causal links between variables (but only hypothesis of relations), and thus no causal inference can be made in this regard. Anyway, our results seem to suggest that older Concerning physical and mental health on the whole, many studies have shown that social support has an important impact on PLOS ONE \| www.plosone.org January 2013 \| Volume 8 \| Issue 1 \| e54856 7 January 2013 \| Volume 8 \| Issue 1 \| e54856 Social Support and Elder Abuse in Europe Table 5. Multivariate quantile (median) linear regression analysis (un-standardized betas/standard error) of the association between social support, country, demographic, socio-economic, violence and other selected variables. Table 5. Multivariate quantile (median) linear regression analysis (un-standardized betas/standard error) of the association between social support, country, demographic, socio-economic, violence and other selected variables. Table 5. Multivariate quantile (median) linear regression analysis (un-standardized betas/standard error) of the association between social support, country, demographic, socio-economic, violence and other selected variables. Social Support, Depression, Somatic Complaints and Health Care Services Social supporta Independent variables Categories Beta Standard error Countryb, Germany+ Greece 4.91** 1.02 Italy 22.15* 0.92 Lithuania 5.56* 0.96 Portugal 24.56* 0.9 Spain 0.48 1.1 Sweden 0.3 0.88 Age bandsb, 60–64+ 65–69 years 20.47 0.66 70–74 years 21.69* 0.72 75–79 years 21.06 0.78 80–84 years 22.28* 0.89 Genderb, Female+ Male 21.63 0.53 Marital status b, Single+ Married/cohabiting 5.35 1.68 Divorced/separated 3.24 1.2 Widow/er 6.67* 1.07 With whom one livesb, Alone+ Only partner/spouse 5.25 1.57 Partner/spouse/othersd 2.61 1.76 Without partner/spouse - with othersd 3.26 1.01 Household sizec, e 0.69* 0.27 Main source of financial supportb, Work pensions+ Work 0.23 1.09 Social/sick-leave/other pensionf 22.98 1.05 Partner/spouse income 1.07 0.96 Otherg 22.09 1.48 Health care services usec, h 4.51* 1.08 Depressionc, i 27.21* 0.54 Physical complaintsc, j 20.06 0.02 Psychological abuseb, k, No+ Yes 22.51* 0.58 Constant 58.86* 2.98 individuals who report higher levels of social support, also use health care services, showing less physical complaints and low levels of depression,. It could also be hypothesized that the lack of social support exerts a negative influence on cognitive functioning and physical health in later life, especially when health care services are not, or very little, used. Greater levels of social support in old age might therefore be considered to reduce the risk for physical-functional diseases and mental illnesses, and mitigate depression. Acknowledgments The authors wish to acknowledge and express their appreciation to the staff of EAHC, Executive Agency for Health and Consumers, in particular to Dr. Guy Dargent for his precious assistance and to Sabrina Quattrini and Juan de La Luna for their work concerning data preparation and analyses. Moreover, the authors are especially grateful to the older people who participated in the study, for their kindness, efforts and answers. Limitations This study has some limitations. First, the generalizability of the findings can be questioned. Data are only from large urban centres in seven European countries and are based on self-reports by older participants, thus being subject to possible recall bias. Moreover, the study excluded elderly with cognitive impairment (not able to appropriately complete the survey). Second, the data are cross- sectional thus not allowing the establishment of causal links between variables. Third, the relatively low numbers of partici- pants who reported some types of abuse episodes (e.g. injury and sexual abuse) suggests caution in the interpretation of findings, and this doesn’t allow further inferences or generalizations. It is also very likely that this could be linked with a systematic under- reporting of abuses. Future research in this area will require a longitudinal design in order to test the correlation found between social support and other dimensions, elder abuse included. Despite these limitations, our study provides the following benefits: cross- national data on various aspects of elder abuse (e.g. social support); a workable definition of abuse (including injuries) and validated instruments to assess the phenomenon; findings and tools which could be used by policy makers, clinicians and researchers at the European and country levels for a range of activities (e.g. monitoring abuse, awareness campaigns). References 12. Thoits P (1995) Stress, coping, and social support processes: Where are we? What next? J Health Social Behav 37(Extra Issue): 53–79. 1. Pierce GR, Sarason BR, Sarason IG (1990) Integrating social support perspectives: Working models, personal relationships and situational factors. In Duck S, editor.Personal relationships and social support. London: Sage Publications. 173–215. 13. Litwin H, Landau R (2000) Social network type and social support among the old-old (Statistical Data Included). J Aging Stud 14: 213–228. 2. Hall A, Wellman B (1985) Social networks and social support. In Cohen S, Syme SL, editors. Social support and health. Orlando: Academic Press. 23–41. ( ) J g g 14. Andrew MK, Mitnitski AB, Rockwood K (2008) Social Vulnerability, Frailty and Mortality in Elderly People. PLoS ONE 3(5): e2232. and Mortality in Elderly People. PLoS ONE 3(5): e2232. 3. Seeman TE (2000) Health promoting effects of friends and family on health outcomes in older adults. Am J Health Promot 14: 362–370. 15. Berkman CS, Gurland BJ (1998) The relationship among income, other socioeconomic indicators, and functional level in older persons. J Aging Health 10: 81–98. outcomes in older adults. Am J Health Promot 14: 362–370. 4. Stroebe W (2000) Moderators of the stress-health relationship. In Stroebe W, editor. Social psychology and health. Philadelphia, PA: Open University Press. 236–273. 16. Weyers S, Dragano N, Mo¨bus S, Beck EM, Stang A, et al. (2008) Low socio- economic position is associated with poor social networks and social support: results from the Heinz Nixdorf Recall Study. Int J Equity Health 7: 13–19. 5. Krause N (2001) Social Support. In: Binstok RH, George LK, editors. Handbook of Aging and the Social Sciences. San Diego CA: Academic Press. 272–294. 17. Rundall TG (1992) Health services for an aging society. Med Care Res Rev 49: 3–18. 6. Gadalla TM (2010) The Role of Mastery and Social Support in the Association Between Life Stressors and Psychological Distress in Older Canadians. J Gerontol Soc Work 53: 512–530. 18. Okabayashi H, Liang J, Krause N, Akiyama H, Sugisawa H (2004) Mental health among older adults in Japan: do sources of social support and negative interaction make a difference? Soc Sci Med 59: 2259–2270. 7. Antonovsky A. (1979) Health, stress and coping: New Perspectives on Mental and Physical Well-Being. San Francisco: Jossey Bass. 255 p. 19. Social Support and Elder Abuse in Europe Social Support and Elder Abuse in Europe Social Support and Elder Abuse in Europe of social support involves such things as good intimate social relations (spouses/partners), feeling cared for, valued and being part of a network of relationships, with a resulting sense of well- being [4]. Psychological abuse, on the other hand, involves among other things harsh and insulting words, threats, silent ‘‘treatment’’ and being ignored [33,34], which seem to be the opposite of central components in social support. Thus, this may explain the relationship between psychological abuse and a low level of social support. It is worth noting that, according to many authors, social isolation, loneliness and low levels of social support seem to co- exist with elder abuse, but more as risk factors for violence than causes [25,71,72]. Apparently in our data low social support may be both a cause and effect of abuse, at least for psychological one. Low levels of social support may indeed create a potential violent emotional context, but psychological abuse itself could also lead to the perception of reduced social support. Conclusions Our study highlights the multiple connections between social support and various dimensions in later life. Variations and similarities between countries, concerning cultural attitude in relationships across Europe, were shown. Further, factors related to increased perceived social support were found, such as living in larger households and not alone, frequent use of health care services, low scores in depression and in physical complaints. A lower level of social support was conversely associated with older age, male gender, having a poor economic situation, and exposure to psychological mistreatment. High levels of social support may thus represent a key factor in reducing and preventing the vulnerability and isolation of older people, and the risk of elder mistreatment, at least emotional. On the basis of these results, policy makers and clinicians could act by developing adequate intervention programmes, which aim at promoting opportunities for older people to engage in social activities. Moreover, further more broadly and longitudinal research is needed to explore causality and direction of the relations found. In this respect, our findings confirm previous results and contribute with additional evidence, thus suggesting further directions and implications to investigate more in depth the impact of support systems on the life of frail elderly. Author Contributions Revised article critically for important intellectual content: FTG MS JL EIK HB GM JS. Final approval of the version to be published: MM CC GL FTG MS JL EIK HB GM JS. Conceived and designed the experiments: MM CC GL FTG MS JL EIK HB GM JS. Performed the experiments: MM CC GL FTG MS JL EIK HB GM JS. Analyzed the data: MM CC GL JS. Wrote the paper: MM CC GL JS. Social Support, Abuse Type and Injuries Only having been exposed to psychological abuse was independently associated with decreased social support. This finding indicate that greater levels of social support might exert a protective influence against the risk of psychological abuse in older age, a phase of life which is too often marked by a weakening of one’s social networks. These results are consistent with those of other studies revealing that generally emotional and affective support and solidarity from family might be more important than instrumental support for psychological symptoms [23,70]. Liter- ature in particular highlights that, on the one hand, the experience PLOS ONE \| www.plosone.org PLOS ONE \| www.plosone.org January 2013 \| Volume 8 \| Issue 1 \| e54856 January 2013 \| Volume 8 \| Issue 1 \| e54856 8 Social Support and Elder Abuse in Europe 47. Fiori KL, Antonucci TC, Cortina KS (2006) Social Network Typologies and Mental Health Among Older Adults. 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https://openalex.org/W3197239939	https://escholarship.umassmed.edu/cgi/viewcontent.cgi?article=3138&context=faculty_pubs	English	null	Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone	Communications biology	2,021	cc-by	17,501	1 Department of Biology and Biotechnology, Worcester Polytechnic Institute, Worcester, MA, USA. 2 Neural Signaling and Circuit Plasticity Group, Department of Biology, KU Leuven, Leuven, Belgium. 3 Neurobiology Department, University of Massachusetts Medical School, Worcester, MA, USA. 4 Department of Biology, University of Massachusetts Dartmouth, Dartmouth, MA, USA. 5Present address: Tufts University, Medford, MA, USA. 6Present address: AbbVie Foundational Neuroscience Center, Cambridge, MA, USA. 7Present address: Optum, Hartford, CT, USA. ✉email: jsrinivasan@wpi.edu https://doi.org/10.1038/s42003-021-02547-7 OPEN COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone Item Type Journal Article Authors Reilly, Douglas K.; McGlame, Emily J.; Vandewyer, Elke; Robidoux, Annalise N.; Muirhead, Caroline S.; Northcott, Haylea T.; Joyce, William; Alkema, Mark J; Gegear, Robert J.; Beets, Isabel; Srinivasan, Jagan Citation <p>Reilly DK, McGlame EJ, Vandewyer E, Robidoux AN, Muirhead CS, Northcott HT, Joyce W, Alkema MJ, Gegear RJ, Beets I, Srinivasan J. Distinct neuropeptide-receptor modules regulate a sex-specific behavioral response to a pheromone. Commun Biol. 2021 Aug 31;4(1):1018. doi: 10.1038/s42003-021-02547-7. PMID: 34465863; PMCID: PMC8408276. <a href="https://doi.org/10.1038/ s42003-021-02547-7">Link to article on publisher's site</a></p> DOI 10.1038/s42003-021-02547-7 Rights Copyright © The Author(s) 2021. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Download date 24/10/2024 04:49:12 Item License http://creativecommons.org/licenses/by/4.0/ Link to Item http://hdl.handle.net/20.500.14038/29914 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Pheromones are small-molecule signals between conspeciﬁcs that convey information on the sender’s current physiological state, and potentially life stage and developmental history6–8. How the nervous system responds to these stimuli is dependent on both the internal, physiological state of the animal9, and external, concurrently sensed stimuli10. We have previously shown that behavioral response to pheromones is directly dependent on the physiological state of the animal11, though the mechanisms which determine such responses remain enigmatic. Nematodes communicate through a large and growing class of pheromones termed ascarosides (ascr)6. These small molecules convey social as well as developmental information, and the assays used to understand the roles of these cues have varied4,6. There are multiple ascarosides found to communicate attractive behaviors, speciﬁcally in a sex-speciﬁc manner, including: ascr#1, ascr#2, ascr#3, ascr#4, and ascr#812. Unique among ascaroside structures is the presence of a p-aminobenzoate group—a folate precursor that C. elegans are unable to synthesize, yet obtain from bacterial food sources13—at the terminus of ascr#8. This pher- omone has previously been shown to act as an extremely potent male attractant, being sensed via a chemosensory pathway shared with ascr#3: the male-speciﬁc CEM neurons14. However, whereas ascr#3 is also sensed by over half a dozen chemosensory neurons14–17, ascr#8 has only been shown to be sensed by the male-speciﬁc CEM14. We identiﬁed two divergent FLP-3 receptors responsible for sensing the processed neuropeptides. Receptor activation studies elucidated that the previously identiﬁed ﬂp-3-sensing G protein- coupled receptor, NPR-1023, and the novel FRPR-16, are both activated by FLP-3 peptides at nanomolar afﬁnities. Additionally, loss-of-function mutations in either receptor result in behavioral defects that parallel those observed in ﬂp-3 mutants. ﬂ To more completely understand the role of ﬂp-3 in mediating the ascr#8 behavioral response, we adapted a peptide rescue-by- feeding protocol37. Using this method, we were able to rescue individual peptides in ﬂp-3 mutant animals and showed that a speciﬁc subset of FLP-3 peptides responsible for suppressing the avoidance differs from those responsible for driving male attraction to ascr#8. Here we show that individual neuropeptides encoded by the ﬂp-3 gene exhibit speciﬁc biological activity, by binding multiple receptors, to drive the behavioral valence to a cue in a sex-speciﬁc manner. p While the CEMs offer a sex-speciﬁc mode of chemosensation for ascr#8, neuromodulators and hormones are heavily implicated in all stages of the of sex-speciﬁc and pheromone-elicited C. elegans mating behaviors. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 S receptors. For instance, activation of NPR-4 by FLP-18 modulates reversal length26, while the sensation of the divergent FLP-4 by the same receptor contributes to food preference choice27. S ex-speciﬁc behaviors are unique aspects of survival throughout the animal kingdom from invertebrates to humans1–3. These behaviors include a wide range of coor- dinated and genetically preprogrammed social and sexual displays that ensure successful reproductive strategies, ultimately resulting in survival of the species in its natural environment2,3. The neural circuits regulating these behavioral responses are conserved, and often shared between sexes, but dependent on social experience and physiological state2. For example, the vomeronasal and main olfactory epithelium in mice are required for male aggression and mating, but in females they contribute towards receptivity and aggression3. Prominent among these stimuli are mating cues4; while the visual displays of higher order animals are among the most apparent of these, chemical mating cues are the most ubi- quitous, with entire sensory organs dedicated to pheromone sensation5. Here, we investigate the neuronal mechanisms governing the behavioral attractive response of male C. elegans to ascr#828. Males exhibit a unique behavioral tuning curve to ascr#8, pre- ferring concentrations in the 1 µM range, no longer being attracted to higher concentrations14. Given that multiple ﬂp NP/ NPR modules have been shown to play roles in setting physio- logical state29, as well as linking sensation to physiology and behavior30–32, we reasoned that peptidergic signaling is likely to play a role in the male ascr#8 behavioral response. p y p Previous studies of C. elegans behavioral responses to attractive social ascarosides employed a Spot Retention Assay (SRA)14,33. However, we found that the SRA contains several drawbacks, including male–male contact and the inability to track individual animals through the course of an assay. To address these issues, we have developed a single worm attraction assay (SWAA): a more robust assay that determines variables on a per-worm basis, and not solely at the population level. We utilized our novel SWAA to examine the responses of him-8 males defective in ﬂp neuropeptide genes expressed in male-speciﬁc neurons; ﬂp-3, ﬂp- 6, ﬂp-12, and ﬂp-1934. In doing so, we discovered that ﬂp-3 plays a role in determining the sex-speciﬁc behavioral valence: i.e., determining whether the response to ascr#8 is attractive or aversive16,35,36. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Prior to sensation of mating cues, the mate searching behavior of male C. elegans is modulated by the neuropeptide, PDF- 118,19. Interestingly, this neuropeptide also controls the sexual iden- tity of the ASJ chemosensory neurons20. The mating pheromone ascr#3 is modulated by insulin signaling21, while activation of ascr#3- sensing neurons also activates the NPR-1 receptor17. Finally, the physical act of male sexual turning during mating is mediated by multiple FMRFamide-like peptides22. This complex regulation of behaviors relies on speciﬁc neuropeptide–neuropeptide receptor (NP/NPR) modules. Distinct neuropeptide-receptor modules regulate a sex-speciﬁc behavioral response to a pheromone Douglas K. Reilly1,5, Emily J. McGlame 1,6, Elke Vandewyer2, Annalise N. Robidoux1, Caroline S. Muirhead1, Haylea T. Northcott1,7, William Joyce3, Mark J. Alkema3, Robert J. Gegear4, Isabel Beets 2 & Jagan Srinivasan 1✉ https://doi.org/10.1038/s42003-021-02547-7 OPEN Distinct neuropeptide-receptor modules regulate a sex-speciﬁc behavioral response to a pheromone Douglas K. Reilly1,5, Emily J. McGlame 1,6, Elke Vandewyer2, Annalise N. Robidoux1, Caroline S. Muirhead1, Haylea T. Northcott1,7, William Joyce3, Mark J. Alkema3, Robert J. Gegear4, Isabel Beets 2 & Jagan Srinivasan 1✉ Dioecious species are a hallmark of the animal kingdom, with opposing sexes responding differently to identical sensory cues. Here, we study the response of C. elegans to the small- molecule pheromone, ascr#8, which elicits opposing behavioral valences in each sex. We identify a novel neuropeptide-neuropeptide receptor (NP/NPR) module that is active in males, but not in hermaphrodites. Using a novel paradigm of neuropeptide rescue that we established, we leverage bacterial expression of individual peptides to rescue the sex-speciﬁc response to ascr#8. Concurrent biochemical studies conﬁrmed individual FLP-3 peptides differentially activate two divergent receptors, NPR-10 and FRPR-16. Interestingly, the two of the peptides that rescued behavior in our feeding paradigm are related through a conserved threonine, suggesting that a speciﬁc NP/NPR combination sets a male state, driving the correct behavioral valence of the ascr#8 response. Receptor expression within pre-motor neurons reveals novel coordination of male-speciﬁc and core locomotory circuitries. 1 Results Conversely, FMRFamide-like peptide (ﬂp) genes, many of which encode multiple peptides, signal through a complex network in which multiple receptors sense identical peptides, and multiple FLP peptides activate the same COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio 2 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 b c d e S V A S V A S V A S V A 0 100 200 300 400 500 * * ** * ++++ Mean Dwell Time (s) ** -1.0 -0.5 0.0 0.5 1.0 1.5 Log(fold-change) A/V N2 him-5 him-8 osm-3 00 600 ** * * ) -0.2 0.0 Q1 Q3 Q2 Q4 1 4 3 2 6 5 7 8 A S B V D N/A E F C A a N2 him-5 him-8 osm-3 + + Q1 Q3 Q2 Q4 1 4 3 2 6 5 7 8 A S B V D N/A E F C A a b c d e S V A S V A S V A S V A 0 100 200 300 400 500 * * ** * ++++ Mean Dwell Time (s) ** -1.0 -0.5 0.0 0.5 1.0 1.5 Log(fold-change) A/V N2 him-5 him-8 osm-3 N2 him-5 him-8 osm-3 S V A S V A S V A S V A 0 200 400 600 ** * * Mean Dwell Time (s) -0.8 -0.6 -0.4 -0.2 0.0 Log(fold-change) A/V Q1 Q3 Q2 Q4 1 4 3 2 6 5 7 8 A S B V D N/A E F C A a N2 him-5 him-8 osm-3 N2 him-5 him-8 osm-3 + + to ascr#8 is sex speciﬁc. a Design of the Single Worm Attraction Assay (SWAA). The outer 40 wells of a 48-well su with NGM agar and a thin lawn of OP50 E. coli. A random block design results in spatial control (light gray), vehicle purple) containing wells. Quadrants are recorded for 15 m. (Inset) The structure of ascr#8. b Raw dwell times of males ple), osm-3;him-5 (orange) in SWAA. c Transformed log(fold-change) of male dwell time data. d Raw dwell time of he g(fold-change) of SWAA data of hermaphrodites. Light gray denotes spatial controls (“S”) (when applicable), dark g olors denote ascr#8 values (“A”). ♂denotes male data, ⚥denotes hermaphrodite data. For all ﬁgures: Error bars d 0.01, *p < 0.001, *p < 0.0001, unless denoted otherwise. For 1b: ++++p < 0.0001, vehicle vs. spatial control. Results Spot retention assay vs. single worm assay. We adapted the spot retention assay (Supplementary Fig. 1)28,38 to allow for better characterization of individual worm behavior and robust inter- rogation of attraction to small molecules. We ﬁrst compared the attractiveness of 1 µM ascr#8 (Fig. 1a, inset) across multiple strains of C. elegans (the wild-type N2 strain, the high incidence of male him-5 and him-8 strains, and the chemosensory cilia defective osm-3), using our novel behavioral assay, the single worm attraction assay (SWAA) (Fig. 1a). In this assay, individual animals are placed directly into the spot of the ascaroside cue (A) while simultaneously removing any potential of male–male con- tact. A spatial control is included throughout the assay plate to allow us to investigate any innate differences in the number of visits to the well center, or the time spent therein, of which we have only found one strain to date with differences in male dwell time (Fig. 1a). Likewise, a vehicle control (V) is also included, to account of any changes in dwell time that are driven by the components of the vehicle. Unique NP/NPR modules are known to drive speciﬁc phy- siological and behavioral responses in C. elegans23. While DAF-2 propagates insulin-like peptide (ins) signaling, the speciﬁc peptide determines the effect. For example, ins-4 functions in learning, while ins-6 affects synapse formation24,25. Meanwhile, avoidance of ascr#3 by hermaphrodites is mediated in part by INS-18/DAF- 2 signaling—higher levels of ins-18 expression result in lower ascr#3 avoidance rates21. ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Male C. elegans exhibited a signiﬁcant increase in the amount of time spent within ascr#8 spot compared to the vehicle control, in all wild-type and him male strains tested (Fig. 1b, c). These results conﬁrm the attraction behavior of wild-type and him males observed with SRA (Supplementary Fig. 1). To measure the attraction behavior of the ascaroside, we then computed the normalized increase in dwell time, calculated as log(fold-change) [i.e., ascaroside dwell time over vehicle dwell time]. Using this log(fold-change) metric, we observed that the increase in attraction to ascr#8 is consistent across N2, him-5, and him-8 male strains (Fig. 1c). This metric enabled a direct comparison between strains and conditions while accounting for baseline variability in vehicle dwell times. avoid confounding variables, our criteria for selection stipulated that outside of male-speciﬁc neurons, expression proﬁles would be limited to a small number of neurons (ﬂp-5 was therefore excluded as it exhibits expression in the pharyngeal muscle; while ﬂp-21 and ﬂp-22 are expressed in a large number of neurons outside of the male-speciﬁc expression proﬁles). p p p We found that loss of ﬂp-3 strongly affected the ability of male C. elegans to respond to ascr#8, (Fig. 2a, b, Supplementary Fig. 3a, b). The log(fold-change) of ﬂp-3 is the only value signiﬁcantly different than that seen in the wild-type (Fig. 2b). Interestingly, there was defect seen neither in ﬂp-3 hermaphrodites, nor any other strain (Fig. 2d, e, Supplementary Fig. 3c, d). Because the defect in male response to ascr#8 was signiﬁcant, and the SWAA was designed to detect attractive behaviors, we sought to determine if ﬂp-3 loss-of-function (lof) animals were in fact avoiding ascr#8. Using a previously described drop avoidance assay11,41, we exposed forward moving animals to a drop of either vehicle control or ascr#8 and scored the avoidance index. Wild- type males did not avoid the cue, as expected for an attractive cue, while ﬂp-3 lof males strongly avoided the pheromone (Fig. 2c). The hermaphroditic behavior was unaffected by the loss of ﬂp-3. (Fig. 2f). Together, these results suggest that ﬂp-3 functions to control the behavioral valence of the ascr#8 response to be attractive in a sex-speciﬁc manner; serving in males to suppress a basal avoidance behavior observed in hermaphrodites. Given the setup of the SWAA assay, we can measure the number of visits per-worm under different genetic backgrounds and conditions (Supplementary Fig. 2a, c). ARTICLE This assay additionally helps calculate the percentage of attractive visits to the cue (Supplementary Fig. 2b). Our results suggest no difference in the attractive properties to ascr#8 between any of the wild-type strains (Supplementary Fig. 2b). Setting an “attractive visit” as any visit longer than two standard deviations above the mean vehicle dwell time, we show that males are indeed attracted to the cue itself, and not the male–male contact. Our results suggest that it is in fact a minority of animals (30–45%) that exhibit attractive visits to the cue (Supplementary Fig. 2b). This rate of behavioral attraction to ascr#8 is consistent with calcium imaging experi- ments wherein ascr#8 exposure elicits similar rates of calcium transients in the CEM neurons39. Rescue of ﬂp-3 under a 4-kb region of its endogenous promoter was able to restore the behavioral valence of males to wild-type levels (Fig. 2g–i, Supplementary Fig. 3e, f). While overexpression of neuropeptides can result in dominant negative phenotypes18, expression of the ﬂp-3 construct in wild-type animals did not alter wild-type behavioral response to ascr#8 (Fig. 2g–i, Supplementary Fig. 3e, f). Unlike the SRA, wherein hermaphrodites did not exhibit any difference in dwell time between vehicle and ascr#8 (Supplemen- tary Fig. 2d, e), the SWAA revealed that hermaphrodites from all strains consistently spent signiﬁcantly less time in ascr#8 than the vehicle, with no difference between the spatial and vehicle control dwell times (Fig. 2d, e). Hermaphrodites also visited the ascaroside cue less than they did vehicle or spatial control well centers and exhibited little-to-no attractive visits (Supplementary Fig. 2c, d). To rule out an allele speciﬁc effect of the ﬂp-3(pk361) mutation, which results in deletion of the entire coding sequence as well as 439 bp of upstream and 1493 bp of downstream genomic sequence42, we also assayed ﬂp-3(ok3265), an in-frame deletion of the coding sequence that retains expression of two peptides produced by the ﬂp-3 gene43 (FLP-3-1 and FLP-3-4) (Supple- mentary Fig. 4a). The ﬂp-3(pk361) and ﬂp-3(ok3265) mutant phenotypes were identical (Supplementary Fig. 4b–f), conﬁrming that the deletion in the pk361 allele did not cause any off-target effects, and that neither of the two peptides still encoded by the ok3625 allele (FLP-3-1 and FLP-3-4) are sufﬁcient to rescue the mutant phenotype. Whether these peptides contribute to the ascr#8 response in an another way remains to be elucidated. ARTICLE Together, these data validate the SWAA as a robust assay for the measurement of the attractiveness of a small-molecule cue on a single animal basis in both sexes. In addition, it provides data on visit count and the percent of attractive visits that was previously impossible utilizing the SRA. Interestingly, attractive visits to the ascr#8 cue were only observed in 30–45% of the time (Supplementary Fig. 2b), suggesting that the individual state of the animal plays a critical role in determining the behavioral response to the ascaroside, as seen in other ascaroside behavioral responses11,40. FLP-3 functions speciﬁcally to modulate the ascr#8 behavioral response. While ascr#8 is a potent male-attracting pheromone, previous studies have shown that ascr#2, ascr#3, ascr#4 also function synergistically in attracting males33. The CEM neurons that are required for ascr#8 sensation also function in ascr#3 sensation14. While ascr#3 signal propagation is processed through the hub-and-spoke circuit centered around RMG16,17,44, little is known about the mechanics of ascr#8 sensation outside of CEM involvement14. To determine if ﬂp-3 functions to regulate pheromone-mediated male attraction and avoidance in a general manner, or rather one speciﬁc to ascr#8, we assayed the response of wild-type and ﬂp-3 lof males to ascr#3, a cue for which behavioral valence has also recently been shown to be regulated in a sex-speciﬁc manner16. We found that ﬂp-3 lof males exhibited no defect in their attractive response to ascr#3 (Supplementary Fig. 5), suggesting that its role is indeed speciﬁc to that of ascr#8 sensation. Peptidergic signaling drives sex-speciﬁc ascr#8 behavioral response. Several neuropeptides of the FMRFamide-like-peptide (FLP) family have been implicated in the mechanosensory reg- ulation of male-mating behavior37. The genes encoding the neuropeptides ﬂp-8, ﬂp-10, ﬂp-12, and ﬂp-20 all suppress the number of turns around a hermaphrodite executed by a male prior to mating22. Despite this enrichment of ﬂp genes func- tioning in the mechanosensation of these male-speciﬁc behaviors22, there has been no neuropeptide found to regulate the chemosensation of mating ascarosides. We sought to under- stand why an attractive concentration of a mating pheromone does not result in consistent attraction (Supplementary Fig. 2b) by investigating potential peptidergic signaling pathways that function in the sensation of ascr#8. We focused our initial screening of neuropeptides on the FLP family. We generated him-8 lines of ﬂp genes expressed in male- speciﬁc neurons, speciﬁcally ﬂp-3, ﬂp-6, ﬂp-12, and ﬂp-1934. COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio Results a Q3 b c S V A S V A S V A S V A 0 100 200 300 400 500 * ** * ++++ Mean Dwell Time (s) ** -1.0 -0.5 0.0 0.5 1.0 1.5 Log(fold-change) A/V N2 him-5 him-8 osm-3 N2 him-5 him-8 osm-3 b c e N2 him-5 him-8 osm-3 -0.8 -0.6 -0.4 -0.2 0.0 Log(fold-change) A/V + d e N2 him-5 him-8 osm-3 S V A S V A S V A S V A 0 200 400 600 ** * * Mean Dwell Time (s) -0.8 -0.6 -0.4 -0.2 0.0 Log(fold-change) A/V N2 him-5 him-8 osm-3 + + d S V A S V A S V A S V A 0 200 400 600 * * Mean Dwell Time (s) N2 him-5 him-8 osm-3 + d e Fig. 1 Attraction to ascr#8 is sex speciﬁc. a Design of the Single Worm Attraction Assay (SWAA). The outer 40 wells of a 48-well suspension cell culture plate are seeded with NGM agar and a thin lawn of OP50 E. coli. A random block design results in spatial control (light gray), vehicle control (dark gray), and ascaroside (purple) containing wells. Quadrants are recorded for 15 m. (Inset) The structure of ascr#8. b Raw dwell times of males of N2 (blue), him-5 (red), him-8 (purple), osm-3;him-5 (orange) in SWAA. c Transformed log(fold-change) of male dwell time data. d Raw dwell time of hermaphrodites across strains and e log(fold-change) of SWAA data of hermaphrodites. Light gray denotes spatial controls (“S”) (when applicable), dark grey denotes vehicle controls (“V”), colors denote ascr#8 values (“A”). ♂denotes male data, ⚥denotes hermaphrodite data. For all ﬁgures: Error bars denote SEM. n ≥5. p < 0.05, p < 0.01, p < 0.001, *p < 0.0001, unless denoted otherwise. For 1b: ++++p < 0.0001, vehicle vs. spatial control. COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio 3 ARTICLE Transcriptional reporters have shown robust ﬂp-3 expression in the amphid IL1 neurons, as well as the sensory PQR and the male-speciﬁc interneuron, CP9, although our translational fusion exhibited no PQR or CP9 expression (Fig. 2j–l, Supplementary Fig. 6a–c). Previous studies employed 1–2 kb regions of promoter sequence driving GFP expression, while our construct employs a 4 kb region, thereb further regulatory elements that may restrict expr By including the full coding sequence in our trans we have also incorporated the regulatory element the introns of the ﬂp-3 gene46. ARTICLE To Expression analysis of a FLP-3 translational fusion (pﬂp-3::ﬂp- 3::mCherry) conﬁrmed previous expression analyses of the 4 within male-speciﬁc spicule neurons34,45 (Fig. 2j–l, Fig. 6a–c). Transcriptional reporters have shown while our construct employs a 4 kb region, thereby further regulatory elements that may restrict expre S V A S V A S V A S V A S V A 0 200 400 600 Mean Dwell Time (s) * * him-8 flp-3 flp-6 flp-12flp-19 him-8 him-8 flp-3 flp-3 him-8 flp-3 flp-6 flp-12 flp-19 0.0 0.5 1.0 a b - + - + 0.0 0.2 0.4 0.6 0.8 1.0 Avoidance Index c - + - + 0.0 0.2 0.4 0.6 0.8 1.0 Avoidance Index d e f g h i j l S V A S V A S V A S V A S V A 0 200 400 600 800 1000 Mean Dwell Time (s) ** * * * -1.0 -0.5 0.0 Log(fold-change) A/V S V A S V A S V A S V A 0 200 400 600 800 Mean Dwell Time (s) * 0.5 0.0 0.5 1.0 ◊◊ - + - + - + - + 0.0 0.2 0.4 0.6 0.8 Avoidance Index *** k * SPDL SPDR * SPDL SPDR * SPDL SPDR him-8 flp-3 flp-6 flp-12 flp-19 him-8 flp-3 flp-6 flp-12flp-19 him-8 him-8 flp-3 flp-3 pflp-3::flp-3 him-8 him-8 flp-3 flp-3 pflp-3::flp-3 him-8 him-8 flp-3 flp-3 pflp-3::flp-3 Log(fold-change) A/V -0.5 Log(fold-change) A/V - + + + 10 μm ONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 him-8 flp-3 - + - + 0.0 0.2 0.4 0.6 0.8 1.0 Avoidance Index **** c S V A S V A S V A S V A S V A 0 200 400 600 Mean Dwell Time (s) * * * him-8 flp-3 flp-6 flp-12flp-19 him-8 flp-3 flp-6 flp-12 flp-19 0.0 0.5 1.0 ** a b Log(fold-change) A/V -0.5 S V A S V A S V A S V A S V A 0 200 400 600 Mean Dwell Time (s) * * * him-8 flp-3 flp-6 flp-12flp-19 him-8 him-8 flp-3 flp-3 flp-6 flp-12 flp-19 0.0 0.5 1.0 a b - + - + 0.0 0.2 0.4 0.6 0.8 1.0 Avoidance Index c Log(fold-change) A/V -0.5 c a neuropeptide within male-speciﬁc spicule neurons34,45 (Fig. 2j–l, Supplementary Fig. 6a–c). ARTICLE Interestingly, we observed localization of m sensory cilia of male dorsal and ventral IL1 neur S V A S V A S V A S V A S V A 0 200 Mean him-8 flp-3 flp-6 flp-12flp-19 him-8 him-8 flp-3 flp-3 him-8 flp-3 flp-6 flp-12 flp-19 0.0 - + - + 0.0 0.2 Av - + - + 0.0 0.2 0.4 0.6 0.8 1.0 Avoidance Index d e f g h i j l S V A S V A S V A S V A S V A 0 200 400 600 800 1000 Mean Dwell Time (s) ** * * * -1.0 -0.5 0.0 Log(fold-change) A/V S V A S V A S V A S V A 0 200 400 600 800 Mean Dwell Time (s) * 0.5 0.0 0.5 1.0 ◊◊ - + - + - + - + 0.0 0.2 0.4 0.6 0.8 Avoidance Index *** k * SPDL SPDR * SPDL SPDR * SPDL SPDR him-8 flp-3 flp-6 flp-12 flp-19 him-8 flp-3 flp-6 flp-12flp-19 him-8 him-8 flp-3 flp-3 pflp-3::flp-3 him-8 him-8 flp-3 flp-3 pflp-3::flp-3 him-8 him-8 flp-3 flp-3 pflp-3::flp-3 Log(f -0.5 Log(fold-change) A/V - + + + 10 μm COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio him-8 flp-3 flp-6 flp-12flp-19 Avoidance Index d e f S V A S V A S V A S V A S V A 0 200 400 600 800 1000 Mean Dwell Time (s) * * * -1.0 -0.5 0.0 Log(fold-change) A/V him-8 flp-3 flp-6 flp-12 flp-19 him-8 flp-3 flp-6 flp-12flp-19 + + p p p p p him-8 flp-3 - + - + 0.0 0.2 0.4 0.6 0.8 1.0 Avoidance Index ** d e f S V A S V A S V A S V A S V A 0 200 400 600 800 1000 Mean Dwell Time (s) ** * * * -1.0 -0.5 0.0 Log(fold-change) A/V him-8 flp-3 flp-6 flp-12 flp-19 him-8 flp-3 flp-6 flp-12flp-19 + + + h 0.5 0.0 0.5 1.0 ◊◊ him-8 him-8 flp-3 flp-3 pflp-3::flp-3 Log(fold-change) A/V - him 8 flp 3 i - + - + - + - + 0.0 0.2 0.4 0.6 0.8 Avoidance Index * him-8 him-8 flp-3 flp-3 pflp-3::flp-3 i i g S V A S V A S V A S V A 0 200 400 600 800 Mean Dwell Time (s) * ** him-8 him-8 flp-3 flp-3 pflp-3::flp-3 h g j * SPDL SPDR p p p k * SPDL SPDR l * SPDL SPDR 10 μm l l neuropeptide within male-speciﬁc spicule neurons34,45 (Fig. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 and NPR-10 to be representative of one another due to their close phylogenetic relationship and separation by a recent gene duplication55,56. However, in our testing of these mutants using our SWAA, we found that npr-4 and npr-5 lof males respond similarly to him-8 males (Fig. 3b–d, Supplementary Fig. 9) while npr-10 lof animals exhibited a complete loss of attraction to the cue, as well as a partial avoidance phenotype matching that of ﬂp- 3 lof mutants (Fig. 3b–d, Supplementary Fig. 9). f g pp y g Transgenic rescue by an NPR-10::GFP translational fusion construct expressed under 1.6 kb of the endogenous promoter was able to restore wild-type levels off attraction in an npr-10 lof mutant background (Fig. 3c, Supplementary Fig. 9a, b). This construct was also able to suppress the avoidance phenotype of npr-10 (Fig. 3d, Supplementary Fig. 9c). Expression analysis of NPR-10::GFP revealed expression in both amphid and phasmid regions of the animals (Fig. 3e, f. Supplementary Fig. 9d, e). Among these head neurons are the inner labial IL2 neurons, as well as their respective socket cells (ILso). NPR-10::GFP ﬂuorescence was also observed in the ADL and ASG chemosen- sory neurons, cells which synapse onto AVD and AIA neurons, contributing to reversal control and turning circuitries, respec- tively. The localization of NPR-10 in the RMEL and RMEV neurons provides a direct input into neurons innervating muscle cells (Fig. 3e). Alongside expression in the interneuron AVK (Fig. 3e), which links the npr-10 circuitry to the backwards locomotion neuron AVE, AVF expression (Fig. 3e) links the circuit to the forward locomotion premotor interneuron, AVB. Because the spicule neurons are exposed to the environment50, we investigated whether they play a direct role in the sensation of ascr#8. To test this, we assayed ceh-30 lof males for their ability to avoid ascr#8. Male ceh-30 lof animals lack the male-speciﬁc CEM neurons responsible for ascr#8 sensation in the amphid region of the animal14,51. him-5 males did not avoid ascr#8 (Supplementary Fig. 7). Males lacking CEM neurons also did not avoid ascr#8 (Supplementary Fig. 7). However, with ﬂp-3 still present in these animals, it may be that they are still able to sense the cue, but do not avoid it due to the presence of the neuropeptide. We therefore generated a ceh-30;ﬂp-3 double mutant, and found that these animals still do not avoid the pheromone (Supplementary Fig. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 7), conﬁrming that the CEM neurons are the primary route of ascr#8 chemosensation which results in the male C. elegans behavioral response14. p , NPR-10 expression was also observed in the B-class ray neurons in the male tail (Fig. 3f). Given the tight localization of these cells, it may be that NPR-10 is also present in the HOB neuron, which is impossible to decipher without further colocalization studies. However, the RnB neurons that express NPR-10 to sense SPD-secreted FLP-3 peptides heavily innervate the male-speciﬁc interneuron, EF1, which travels from the tail to synapse onto neurons in the head of the animal, including the forward locomotion neuron, AVB. Interestingly, the hermaphro- dite tail exhibits expression in the dorso-rectal ganglion neurons DVA, DVB, DVC, and ALN (Supplementary Fig. 9e), which is not observed in the male tail. These data suggest that NPR-10 plays sex-speciﬁc roles based on sex-speciﬁc expression patterns. Using Chinese hamster ovarian (CHO) cell cultures stably expressing the promiscuous G protein, Gα16, and the calcium reporter, aequorin57, we found that both isoforms of NPR-10 are activated by seven of the ten FLP-3 peptides (Supplementary Fig. 10a, b), with half-maximal effective concentrations (EC50) in the nM range (Fig. 4a–i). Peptide FLP-3-6 (EDGNAPFGTMK- Famide) did not activate NPR-10 in our assay. This peptide contains an R-to-K mutation within the C-terminal motif, which may explain the lack of receptor activation. Likewise, peptide FLP-3-10 (STVDSSEPVIRDQ), which contains no sequence homology with any RFamide peptide (Fig. 4i) also failed to activate the receptor. Interestingly, FLP-3-8 (SADD- SAPFGTMRFamide) did not activate either NPR-10A or NPR- p NPR-10 expression was also observed in the B-class ray neurons in the male tail (Fig. 3f). Given the tight localization of these cells, it may be that NPR-10 is also present in the HOB neuron, which is impossible to decipher without further colocalization studies. However, the RnB neurons that express NPR-10 to sense SPD-secreted FLP-3 peptides heavily innervate the male-speciﬁc interneuron, EF1, which travels from the tail to synapse onto neurons in the head of the animal, including the forward locomotion neuron, AVB. Interestingly, the hermaphro- dite tail exhibits expression in the dorso-rectal ganglion neurons DVA, DVB, DVC, and ALN (Supplementary Fig. 9e), which is not observed in the male tail. These data suggest that NPR-10 plays sex-speciﬁc roles based on sex-speciﬁc expression patterns. FLP-3 regulates attractive behavior to ascr#8 by activation of two evolutionarily divergent G protein-coupled receptors. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Fig. 2 Peptidergic regulation of the behavioral response to ascr#8 is sex speciﬁc. Screen of FMRFamide-like peptide (ﬂp) defective mutants in response to ascr#8 in SWAA. a Male raw dwell time and b log(fold-change) values revealed speciﬁcity of ﬂp-3 in the ascr#8 behavioral response. c A drop avoidance assay was employed to reveal a change in behavioral valence, with ﬂp-3 males avoiding ascr#8 (See Methods for details). d Hermaphrodite raw dwell times and e log(fold-change) values in response to ascr#8 are consistent across ﬂp mutants. f ascr#8 avoidance in hermaphrodites is unaffected by the loss of ﬂp-3. g, i Attractive behavior in response to ascr#8 is restored in ﬂp-3 mutant males by expressing ﬂp-3 under its endogenous promoter. g raw dwell times in males and h log(fold-change) values), as well as i rescue of ﬂp-3 restores avoidance to ascr#8. j–l Expression pattern of pﬂp-3::ﬂp-3::mCherry in the male tail. Colocalization with gpa-1::GFP in the SPD spicule neurons suggests that ﬂp-3 is expressed in male-speciﬁc neurons in the tail (arrows). Asterisk denotes coelomocyte accumulation of GFP. j GFP, k mCherry, l merged image at ~×90 magniﬁcation. In panel h, ◊◊p < 0.01 for ﬂp-3 mutant versus transgenic rescue. Light gray denotes spatial controls (“S”) (when applicable), dark gray denotes vehicle controls (“V”), colors denote ascr#8 values (“A”) (him-8, purple; ﬂp-3, teal; ﬂp mutants, blue; transgenic rescues, light blue). ♂denotes male data, ⚥denotes hermaphrodite data. in puncta spanning their dendrites (Supplementary Fig. 6d, e), consistent with peptide packing into dense core vesicles47. Previous studies have observed dense core vesicles within, and being released from, dendritic arbors48, supporting our ﬁndings that FLP-3 is packaged within the dendrites of the IL1 neurons (Supplementary Fig. 6a–c). We found an identical expression pattern within the IL1 cilia and dendritic puncta of hermaphro- dites as well (Supplementary Fig. 6f). Recent single-cell RNA- sequencing of the adult nervous system has again found more proliﬁc expression of ﬂp-3 throughout the nervous system, including most of the VC neurons49. However, these studies were performed only in hermaphrodites, and were therefore unable to examine any male-speciﬁc changes in expression. In males, our ﬂp-3 construct also exhibited male-speciﬁc tail expression in the SPD spicule neurons (Fig. 2j–l, Supplementary Fig. 6a–c), and coupled with IL1 expression, completely rescued the attractive response to ascr#8 (Fig. 2g–i, Supplementary Fig. 3e, f), suggesting a physiologically relevant site-of-release from this small subset of neurons. ARTICLE ARTICLE ARTICLE 2j–l, Supplementary Fig. 6a–c). Transcriptional reporters have shown robust ﬂp-3 expression in the amphid IL1 neurons, as well as the sensory PQR and the male-speciﬁc interneuron, CP9, although our translational fusion exhibited no PQR or CP9 expression (Fig. 2j–l, Supplementary Fig. 6a–c). Previous studies employed 1–2 kb regions of promoter sequence driving GFP expression, while our construct employs a 4 kb region, thereby incorporating further regulatory elements that may restrict expression patterns. By including the full coding sequence in our translational fusion, we have also incorporated the regulatory elements found within the introns of the ﬂp-3 gene46. while our construct employs a 4 kb region, thereby incorporating further regulatory elements that may restrict expression patterns. By including the full coding sequence in our translational fusion, we have also incorporated the regulatory elements found within the introns of the ﬂp-3 gene46. Interestingly, we observed localization of mCherry within sensory cilia of male dorsal and ventral IL1 neurons, as well as COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio 5 5 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 The ﬂp-3 gene encodes multiple peptides23. Recent studies have uncovered a tenth peptide encoded by the gene; although this newest peptide does not contain the conserved GTMRFamide motif found in the remainder of ﬂp-3 peptides (Fig. 3a)52. We determined that the lysine-arginine sites ﬂanking the individual peptides are processed speciﬁcally by the proprotein convertase encoded by the egl-3 gene, and not by aex-5 or bli-422,53,54 (Supplementary Fig. 8), supporting previous studies that the egl-3 gene is the proprotein convertase involved in mating behaviors22. To better understand where the fully processed peptides act within the male-speciﬁc circuit, we assayed mutants for receptors that have previously shown activation upon FLP-3 peptide exposure. While activation of NPR-4 has been reported for only two peptides encoded by ﬂp-3, NPR-5 and NPR-10 have been shown to respond to four and six ﬂp-3 encoded peptides, respectively (Fig. 3a)23. Recent studies have considered NPR-4 Using Chinese hamster ovarian (CHO) cell cultures stably expressing the promiscuous G protein, Gα16, and the calcium reporter, aequorin57, we found that both isoforms of NPR-10 are activated by seven of the ten FLP-3 peptides (Supplementary Fig. 10a, b), with half-maximal effective concentrations (EC50) in the nM range (Fig. 4a–i). Peptide FLP-3-6 (EDGNAPFGTMK- Famide) did not activate NPR-10 in our assay. This peptide contains an R-to-K mutation within the C-terminal motif, which may explain the lack of receptor activation. Likewise, peptide FLP-3-10 (STVDSSEPVIRDQ), which contains no sequence homology with any RFamide peptide (Fig. 4i) also failed to activate the receptor. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Interestingly, FLP-3-8 (SADD- SAPFGTMRFamide) did not activate either NPR-10A or NPR- COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio 6 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Receptor FLP-3 Peptide C16D6.2 Y58G8A.4 C53C7.1 3-1 SPLGTMRFamide μM μM nM 3-2 TPLGTMRFamide 3-3 EAEEPLGTMRFamide nM 3-4 NPLGTMRFamide 3-5 ASEDALFGTMRFamide nM 3-6 EDGNAPFGTMKFamide μM nM 3-7 SAEPFGTMRFamide μM μM nM 3-8 SADDSAPFGTMRFamide μM nM 3-9 NPENDTPFGTMRFamide 3-10 STVDSSEPVIRDQ c d a b S V A S V A S V A S V A S V A S V A 0 200 400 600 800 Mean Dwell Time (s) ** ** * - + - + - + - + - + - + 0.0 0.2 0.4 0.6 0.8 1.0 Avoidance Index ** * -0.5 0.0 0.5 1.0 Log(fold -change) A/V ** * ◊◊ e A P V D AVF IL2 ADL ASG AVK RMEL RMEV ILso ILso f RnB him-8 flp-3 npr-4 npr-5 npr-10 pnpr-10:: npr-10 him-8 flp-3 npr-4 npr-5 npr-10 pnpr-10:: npr-10 him-8 flp-3 npr-4 npr-5 npr-10 pnpr-10:: npr-10 npr-4 npr-5 npr-10 10 μm 10 μm rotein-coupled receptor, NPR-10, is required for the male behavioral response to ascr#8. a FLP-3 peptide afﬁnities for Li and Kim, 201423. b, c Male raw dwell time and log(fold-change) values for npr receptor mutants and npr-10 rescue in npr-10 males exhibit increased avoidance to ascr#8 compared to the other receptors. Expression of npr-10 under its endo oidance phenotype. e Localization of pnpr-10::npr-10::GFP in the amphid region of the male head. Neurons expressing npr-10 IL2, and their respective socket cells (ILso), the interneurons RMEV and RMEL, the chemosensory neurons ADL and A AVF and AVK. f Expression of pnpr-10::npr-10::GFP in the mail tail. Localization is observed in the B-class Ray neurons. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 ARTICLE NPR-10 FRPR-16 FLP-3 Peptide EC50 95% CI 3-1 SPLGTMRFamide 262.1 nM 189.7 nM to 359.8 nM 7.4 nM 5.2 nM to 10.6 nM 3-2 TPLGTMRFamide 366.5 nM 270.4 nM to 491.3 nM 31.7 nM 19.0 nM to 51.2 nM 3-3 EAEEPLGTMRFamide 866.7 nM 706.6 nM to 1.1 μM 288.3 nM 203.7 nM to 402.3 nM 3-4 NPLGTMRFamide 427.1 nM 351.7 nM to 516.3 nM 10.7 nM 7.6 nM to 15.3 nM 3-5 ASEDALFGTMRFamide 1.1 μM 946.2 nM to 1.4 μM 72.1 nM 51.1 nM to 101.3 nM 3-7 SAEPFGTMRFamide 438.6 nM 351.3 nM to 530.1 nM 39.5 nM 28.5 nM to 54.0 nM 3-8 SADDSAPFGTMRFamide N/A N/A 1.6 μM 1.1 μM to 2.4 μM 3-9 NPENDTPFGTMRFamide 760.1 nM 624.1 nM to 923.3 nM 88.3 nM 65.2 nM to 119.6 nM i a b c d e f g h -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) ) % ( n oit a vitc A r o t p e c e R -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) Receptor Activation (%) -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) ) % ( n oit a vitc A r o t p e c e R -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) Receptor Activation (%) -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) ) % ( n oit a vitc A r o t p e c e R -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) Receptor Activation (%) -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) ) % ( n oit a vitc A r o t p e c e R -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) Receptor Activation (%) FLP-3-1 FLP-3-2 FLP-3-3 FLP-3-4 FLP-3-5 FLP-3-7 FLP-3-8 FLP-3-9 EC50 95% CI peptides activate two different G protein-coupled receptors, NPR-10 and FRPR-16, in vitro. Dose response curves of a FLP-3- P-3-4, e FLP-3-5, f FLP-3-7, g FLP-3-8, and h FLP-3-9 for activation of NPR-10B (blue circles) and FRPR-16 (red triangles). Pep did not activate either receptor. i EC50 values and 95% Conﬁdence Intervals for FLP-3 peptide activating NPR-10B and FRPR-16. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 ♂d TIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Receptor FLP-3 Peptide C16D6.2 Y58G8A.4 C53C7.1 3-1 SPLGTMRFamide μM μM nM 3-2 TPLGTMRFamide 3-3 EAEEPLGTMRFamide nM 3-4 NPLGTMRFamide 3-5 ASEDALFGTMRFamide nM 3-6 EDGNAPFGTMKFamide μM nM 3-7 SAEPFGTMRFamide μM μM nM 3-8 SADDSAPFGTMRFamide μM nM 3-9 NPENDTPFGTMRFamide 3-10 STVDSSEPVIRDQ a b S V A S V A S V A S V A S V A S V A 0 200 400 600 800 Mean Dwell Time (s) ** ** * him-8 flp-3 npr-4 npr-5 npr-10 pnpr-10:: npr-4 npr-5 npr-10 Receptor FLP-3 Peptide C16D6.2 Y58G8A.4 C53C7.1 3-1 SPLGTMRFamide μM μM nM 3-2 TPLGTMRFamide 3-3 EAEEPLGTMRFamide nM 3-4 NPLGTMRFamide 3-5 ASEDALFGTMRFamide nM 3-6 EDGNAPFGTMKFamide μM nM 3-7 SAEPFGTMRFamide μM μM nM 3-8 SADDSAPFGTMRFamide μM nM 3-9 NPENDTPFGTMRFamide 3-10 STVDSSEPVIRDQ a b npr-4 npr-5 npr-10 b b a a c -0.5 0.0 0.5 1.0 Log(fold -change) A/V ** * ◊◊ him-8 flp-3 npr-4 npr-5 npr-10 pnpr-10:: npr-10 d - + - + - + - + - + - + 0.0 0.2 0.4 0.6 0.8 1.0 Avoidance Index ** * him-8 flp-3 npr-4 npr-5 npr-10 pnpr-10:: npr-10 npr-10 d c e A P V D AVF IL2 ADL ASG AVK RMEL RMEV ILso ILso 10 μm e e f f RnB 10 μm RnB Fig. 3 The G protein-coupled receptor, NPR-10, is required for the male behavioral response to ascr#8. a FLP-3 peptide afﬁnities for known receptors, adapted from Li and Kim, 201423. b, c Male raw dwell time and log(fold-change) values for npr receptor mutants and npr-10 rescue in the SWAA, respectively. d npr-10 males exhibit increased avoidance to ascr#8 compared to the other receptors. Expression of npr-10 under its endogenous promoter rescues the avoidance phenotype. e Localization of pnpr-10::npr-10::GFP in the amphid region of the male head. Neurons expressing npr-10 include the inner labial neurons, IL2, and their respective socket cells (ILso), the interneurons RMEV and RMEL, the chemosensory neurons ADL and ASG, as well as the interneurons AVF and AVK. f Expression of pnpr-10::npr-10::GFP in the mail tail. Localization is observed in the B-class Ray neurons. ♂denotes male data. COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio 7 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 \| p // g/ / a -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) ) % ( n oit a vitc A r o t p e c e R FLP-3-1 b -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) Receptor Activation (%) FLP-3-2 c -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) ) % ( n oit a vitc A r o t p e c e R FLP-3-3 d -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) Receptor Activation (%) FLP-3-4 -10 -8 Log[peptide] (M) 10 8 Log[peptide] (M) f g[p p ] ( ) -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) Receptor Activation (%) FLP-3-7 e g[p p ] ( ) -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) ) % ( n oit a vitc A r o t p e c e R FLP-3-5 10 8 Log[peptide] (M) 10 8 Log[peptide] (M) g -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) ) % ( n oit a vitc A r o t p e c e R FLP-3-8 h -14 -12 -10 -8 -6 -4 0 50 100 Log[peptide] (M) Receptor Activation (%) FLP-3-9 NPR-10 FRPR-16 FLP-3 Peptide EC50 95% CI 3-1 SPLGTMRFamide 262.1 nM 189.7 nM to 359.8 nM 7.4 nM 5.2 nM to 10.6 nM 3-2 TPLGTMRFamide 366.5 nM 270.4 nM to 491.3 nM 31.7 nM 19.0 nM to 51.2 nM 3-3 EAEEPLGTMRFamide 866.7 nM 706.6 nM to 1.1 μM 288.3 nM 203.7 nM to 402.3 nM 3-4 NPLGTMRFamide 427.1 nM 351.7 nM to 516.3 nM 10.7 nM 7.6 nM to 15.3 nM 3-5 ASEDALFGTMRFamide 1.1 μM 946.2 nM to 1.4 μM 72.1 nM 51.1 nM to 101.3 nM 3-7 SAEPFGTMRFamide 438.6 nM 351.3 nM to 530.1 nM 39.5 nM 28.5 nM to 54.0 nM 3-8 SADDSAPFGTMRFamide N/A N/A 1.6 μM 1.1 μM to 2.4 μM 3-9 NPENDTPFGTMRFamide 760.1 nM 624.1 nM to 923.3 nM 88.3 nM 65.2 nM to 119.6 nM i EC50 95% CI i Fig. 4 FLP-3 peptides activate two different G protein-coupled receptors, NPR-10 and FRPR-16, in vitro. Dose response curves of a FLP-3-1, b FLP-3-2, c FLP-3-3, d FLP-3-4, e FLP-3-5, f FLP-3-7, g FLP-3-8, and h FLP-3-9 for activation of NPR-10B (blue circles) and FRPR-16 (red triangles). ARTICLE A full-gene deletion of frpr-16 was generated using CRISPR mutagenesis (Fig. 5a, b)58. We assayed frpr-16 lof males for their ability to respond to ascr#8. Males lacking frpr-16 exhibited a loss of attraction to ascr#8, as well as a partial avoidance phenotype, like that observed in npr-10 lof mutant animals (Fig. 5, Supplementary Fig. 11). Interestingly, a double mutant containing both npr-10 and frpr-16 null alleles not only did not result in an additive effect in the avoidance phenotype, but in fact also suppressed the avoidance phenotype, suggesting that these receptors are nonredundant in their functions (Fig. 5e, Supple- mentary Fig. 11c). To further validate the ability of this threonine (T) to suppress avoidance of ascr#8, we tested the ability of FLP-3-7 (SAEPFGTMR- Famide) to restore wild-type behavior (Fig. 6e–h). We observed that FLP-3-7 neither suppresses avoidance (Fig. 6h, Supplementary Fig. 13) nor drives attraction (Fig. 6f, g, Supplementary Fig. 13). However, when the ninth amino acid from the C-terminal, a glutamate (G), is replaced with a threonine, FLP-3-7T (Fig. 6e), we observed a suppression of the avoidance behavior (Fig. 6e, Supplementary Fig. 13), supporting our hypothesis that this threonine is critical for suppressing male-speciﬁc avoidance of ascr#8. Rescue of FRPR-16 under 1.9 kb of its endogenous promoter was able to restore wild-type attractive behavior (Fig. 5c, d, Supplementary Fig. 11a, b), as well suppress the avoidance phenotype (Fig. 5e, Supplementary Fig. 11c). Localization of the mCherry fusion reporter was observed in the premotor inter- neurons responsible for reverse locomotory control: AVA, AVE, and AVD (Fig. 5f). The ﬂuorescent protein was also seen anterior to the nerve ring in the gas-sensing BAG neuron (Fig. 5f). This expressing pattern was not sex speciﬁc, as a matching expression pattern was observed in hermaphrodites (Supplementary Fig. 11d). Together, these data show that NPR-10 and FRPR-16 function as receptors for FLP-3 peptides. While FLP-3-9, which contains this threonine, can drive attraction, we observed that FLP-3-7T, which contains an SA sequence upstream of the threonine in place for FLP-3-9’s NPEND sequence, is unable to drive attraction to ascr#8 (Fig. 6e–g, Supplementary Fig. 13). These results support our hypothesis that the NPEND sequence of FLP-3-9 is critical in regulating the attractive behavior. Together, these data argue that the threonine in the ninth position from the C-terminus is critical for suppression of the basal avoidance response (Fig. ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 10 or FRPR-16. The non-RFamide peptide was unable to rescue the avoidance phenotype (Fig. 6b, Supplementary Fig. 12a). 10B, despite its conserved terminal amino acid sequence (Supplementary Fig. 10a, b). 10 or FRPR-16. The non-RFamide peptide was unable to rescue the avoidance phenotype (Fig. 6b, Supplementary Fig. 12a). pp y g The lack of full avoidance phenotype observed in npr-10 lof mutants suggests that there are other FLP-3 receptors involved in regulating the ascr#8 avoidance behavior. Interestingly, while the human NPY/Drosophila NPF receptor NPR-10 is required for FLP-3 sensation, the Drosophila FR receptor homolog, FRPR-16, was also found to be reliably activated by FLP-3 peptides in vitro (Supplementary Fig. 10c). This evolutionarily divergent receptor exhibited potencies in the 10-nanomolar range for seven of the peptides, and submicromolar for an eighth peptide (Fig. 4). Again, FLP-3-6 and FLP-3-10 did not activate FRPR-16, supporting the notion that the terminal motif conserved in the remaining FLP-3 peptides is critical for receptor activation. Cells transfected with a control vector did not exhibit any activation following exposure to FLP-3 peptides, conﬁrming that the activation observed is speciﬁc to receptor-ligand interactions with NPR-10 and FRPR-16 (Supplementary Fig. 10d). FLP-3-1 and FLP-3-4 differ in sequence only in their N-terminal amino acid (Figs. 3a, 4i). Similarly, a single amino acid change is all that distinguishes either peptide from FLP-3-2 (TPLGTMRFamide), which exhibits the second highest afﬁnity for NPR-10 (Fig. 4b). We therefore tested FLP-3-2 for its ability to rescue the ﬂp-3 lof phenotype. Surprisingly, this peptide was able to abolish the avoidance phenotype observed in ﬂp-3 lof animals (Fig. 6b, Supplementary Fig. 12a), although it was not able to restore the animal’s ability to be attracted to ascr#8 (Fig. 6 c, d, Supplementary Fig. 12c). The only difference being the presence of a threonine in that position of the peptide, we hypothesized that this may be the required component to suppress the avoidance behavior. Peptide FLP-3-9 (NPENDTPFGTMRFamide) is a naturally occurring FLP-3 peptide that contains a threonine in the same location of the peptide. The N-terminus is capped with a NPEND sequence, and the lysine conserved in FLP-3-1, 3-2, and 3-4 is mutated to a phenylalanine. However, when ﬂp-3 lof animals were fed NPENDTPFGTMRFamide, they not only displayed lack of avoidance to ascr#8, but also a full rescue of their ability to be attracted to the cue (Fig. 6, Supplementary Fig. 12). ARTICLE 7a), as both FLP-3-2 and FLP-3-9, along with the modiﬁed FLP-3-7T peptide, were all capable of doing so, while FLP-3-1 and FLP-3-4 could not (Fig. 6, Supplementary Figs. 12, 13). Likewise, the NPEND sequence in FLP-3-9 may convey further speciﬁcity to the peptide, allowing it to drive attraction to the pheromone (Fig. 7a). It is not merely the presence of terminal amino acids that drives attraction, as the short sequence present in FLP-3-7T was unable to do so (Fig. 6). Rescue of individual FLP-3 peptides by feeding reveals a spe- ciﬁc subset of active peptides required for attractive behavior. To identify which FLP-3 peptides are required for male avoidance of ascr#8, we adopted a peptide feeding approach59, similar to RNAi feeding, as initially described previously37,59 (Fig. 6a). Using Gateway Cloning technology, we ﬁrst inserted FLP-3 peptide coding sequences into a bacterial expression vector. As a control, we ﬁrst tested the FLP-3 peptides that are still encoded in the ok3625 allele which are insufﬁcient to drive the proper ascr#8 response in vivo: FLP-3-1 (SPLGTMRFamide) and FLP-3-4 (NPLGTMRFamide) (Supplementary Fig. 4). We then reared ﬂp- 3 lof animals on lawns of bacteria expressing the rescue con- structs, and their progeny were assayed for avoidance (Fig. 6b–d, Supplementary Fig. 12). Neither FLP-3-1 nor FLP-3-4 were unable to rescue the avoidance phenotype on their own (Fig. 6b–d, Supplementary Fig. 12), supporting the ﬂp-3(ok3625) data and further suggesting that these two peptides are insufﬁ- cient to maintain wild-type behavior (Supplementary Fig. 4). This is surprising, as each peptide exhibits the highest afﬁnities for FRPR-16, and two of the three highest afﬁnities for NPR-10 (Fig. 4). COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Peptides FLP-3-6 and FLP-3-10 did not activate either receptor. i EC50 values and 95% Conﬁdence Intervals for FLP-3 peptide activating NPR-10B and FRPR-16. a–h Error bars denote SEM. n ≥6. Fig. 4 FLP-3 peptides activate two different G protein-coupled receptors, NPR-10 and FRPR-16, in vitro. Dose response curves of a FLP-3-1, b FLP-3-2, c FLP-3-3, d FLP-3-4, e FLP-3-5, f FLP-3-7, g FLP-3-8, and h FLP-3-9 for activation of NPR-10B (blue circles) and FRPR-16 (red triangles). Peptides FLP-3-6 and FLP-3-10 did not activate either receptor. i EC50 values and 95% Conﬁdence Intervals for FLP-3 peptide activating NPR-10B and FRPR-16. a–h Error bars denote SEM. n ≥6. COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio 8 Discussion f Expression pattern of pfrpr-16::frpr-16::SL2::mCherry in ans at ×20 magniﬁcation. Localization within the ventral cord denoted. f (inset), Amphid localization of pfrpr-16::frpr-16::SL2::mCherry at ~×120 everse locomotion command interneurons, AVA, AVE, and AVD, as well as the BAG neuron (anterior to the nerve ring). In panel d ◊◊p < 0.01 f mutant versus transgenic rescue. ♂denotes male data. LE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 d e c S V A S V A S V A S V A S V A 0 200 400 600 Mean Dwell Time (s) him-8 flp-3 frpr-16 frpr-16 pfrpr-16:: frpr-16 npr-10; frpr-16 * ** Avoidance Index - + - + - + - + - + 0.0 0.2 0.4 0.6 0.8 * -0.5 0.0 0.5 1.0 Log(fold-change) A/V * ◊◊ neoR prps-27 UTR UTR GFP pmyo-2 500 bp a b CRISPR Cut Site CRISPR Cut Site homology arm homology arm frpr-16 homology arm homology arm him-8 flp-3 frpr-16frpr-16 pfrpr-16:: frpr-16 npr-10; frpr-16 him-8 flp-3 frpr-16 frpr-16 pfrpr-16:: frpr-16 npr-10; frpr-16 f AVA AVD Ventral Cord Ventral Cord BAG AVE 50 μm 10 μm 50 μm Fig. 5 FRPR-16 is required for the male behavioral response to ascr#8. a, b Design of CRISPR/Cas9-mediated frpr-16 null mutation construct. a The wild- type gene, with CRISPR cut sites marked, along with 450 bp homology arm regions. b The mutant gene sequence, consisting of an inverted cassette driving loxP ﬂanked pmyo-2::GFP and prps-27::neoR expression. c, d frpr-16 lof male animals display loss of attraction to ascr#8 and rescue of frpr-16 under its endogenous promoter rescues the attraction phenotype. frpr-16;npr-10 double mutant animals do not display an additive phenotype in terms of attraction c Raw dwell time and d log(fold-change) values. e frpr-16 lof male animals display increased avoidance to ascr#8 and rescue of frpr-16 restores avoidance to wild-type male animals. frpr-16;npr-10 double mutant animals do not display avoidance to ascr#8. f Expression pattern of pfrpr-16::frpr-16::SL2::mCherry in male C. elegans at ×20 magniﬁcation. Localization within the ventral cord denoted. f (inset), Amphid localization of pfrpr-16::frpr-16::SL2::mCherry at ~×120 within the reverse locomotion command interneurons, AVA, AVE, and AVD, as well as the BAG neuron (anterior to the nerve ring). In panel d ◊◊p < 0.01 for frpr-16 lof mutant versus transgenic rescue. ♂denotes male data. attracted than their him-5 counterparts (Supplementary Fig. 1). Discussion Our results reveal a complex mechanism regulating the sex- speciﬁc behavioral response to a pheromone guided through the interaction of at least two peptides encoded by a single- neuropeptide precursor gene and two divergent GPCRs. We show that two distinct NP/NPR modules driven by the single- neuropeptide gene ﬂp-3 serve to drive sex-speciﬁc attraction to the male-attracting pheromone, ascr#814,28 (Fig. 2). We developed and validated our novel single worm behavioral assay (SWAA), which conﬁrmed that male C. elegans are indeed attracted to different pheromone cues (Fig. 1 and Supplementary Figs. 1, 2). Previous attraction assays, such as SRA (Supplemen- tary Fig. 1), resulted in attraction values that were skewed due to male–male contact. The SWAA overcomes those caveats as it We also tested FLP-3-10 (STVDSSEPVIRDQ), which exhibits a lack of consensus sequence and an inability to activate either NPR- COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio 9 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 ARTICLE d e c S V A S V A S V A S V A S V A 0 200 400 600 Mean Dwell Time (s) him-8 flp-3 frpr-16 frpr-16 pfrpr-16:: frpr-16 npr-10; frpr-16 * ** Avoidance Index - + - + - + - + - + 0.0 0.2 0.4 0.6 0.8 * -0.5 0.0 0.5 1.0 Log(fold-change) A/V * ◊◊ neoR prps-27 UTR UTR GFP pmyo-2 500 bp a b CRISPR Cut Site CRISPR Cut Site homology arm homology arm frpr-16 f homology arm homology arm him-8 flp-3 frpr-16frpr-16 pfrpr-16:: frpr-16 npr-10; frpr-16 him-8 flp-3 frpr-16 frpr-16 pfrpr-16:: frpr-16 npr-10; frpr-16 AVA AVD Ventral Cord Ventral Cord BAG AVE 50 μm 10 μm -16 is required for the male behavioral response to ascr#8. a, b Design of CRISPR/Cas9-mediated frpr-16 null mutation construct. a The wild- with CRISPR cut sites marked, along with 450 bp homology arm regions. b The mutant gene sequence, consisting of an inverted cassette driving pmyo-2::GFP and prps-27::neoR expression. c, d frpr-16 lof male animals display loss of attraction to ascr#8 and rescue of frpr-16 under its promoter rescues the attraction phenotype. frpr-16;npr-10 double mutant animals do not display an additive phenotype in terms of attraction c me and d log(fold-change) values. e frpr-16 lof male animals display increased avoidance to ascr#8 and rescue of frpr-16 restores avoidance to ale animals. frpr-16;npr-10 double mutant animals do not display avoidance to ascr#8. COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio Discussion f ﬂp-3 lof male animals raised on FLP-3-7T suppress avoidance to ascr#8 suggesting the importance of amino acid threonine in mediating avoidance. g, h ﬂp-3 animals raised on FLP-3-7 and FLP-3-7T display no rescue of attractive behavior to ascr#8. g Raw dwell time and h log(fold-change) of ﬂp-3 animals raised on FLP-3-7 and FLP-3-7T. ♂denotes male data. Previous studies have linked the entirety of the gene to a receptor based on binding studies and full transgenic rescue29,65,66. Here, we employ a rescue-by-feeding assay, following the design of RNAi feeding protocols, to rescue individual peptides37. While “feeding” of peptides through soaking is a valid approach, there are many constraints on such approaches, the most prominent being the ability to acquire puriﬁed peptides29,65. Using our rescue-by- feeding approach, we provide access to the peptide to the worms directly through their food source. This approach enables new avenues for characterizing the roles of the other neuropeptides in mediating diverse cellular and organismal processes. spatial control effects reminiscent of ‘edge effects’61—wherein animals spend more time along the edge rather than in the throughout the well—and instead spend more time the vehicle control more often (Fig. 1d). We observe that this dwell time is dramatically reduced in the presence of the pheromone, sup- porting the ﬁndings that hermaphrodites avoid ascr#8 (Fig. 2f). p g g p g Peptidergic modulation of neural circuits as long been hypo- thesized as complex62, and here we elucidate the recruitment of two neuropeptide/neuropeptide-receptor (NP/NPR) modules, FLP-3/NPR-10 and FLP-3/FRPR-16, that both serve to regulate the nervous system in sensing and respond to asr#8 in a sex- speciﬁc manner. Our results elucidate that not all peptides encoded by the FLP-3 propeptide are involved in the regulation of the sex-speciﬁc circuit, but rather a subset function through two unique NP/NPR modules to drive the behavioral response. This suggests that the complexity of the expansive class of FMRFamide-related peptide (FLP) genes in C. elegans, of which there are 31 genes encoding over 70 unique peptides42, function through an even greater number of NP/NPR modules to drive speciﬁc behavioral or physiological states. FLPs have been identiﬁed as regulators of a variety of behavioral and sensory mechanisms, including locomotion19, egg-laying42, gas sensing63, sleep29, and mating22,64. Here, we show that ﬂp-3 functions to coordinate ascr#8 sensation with attractive behavior. Discussion a Overview of rescue-by-feeding paradigm: (Top) A plasmid in generated which encodes the peptide of interest is ﬂanked by EGL-3 cleavage sites, with a 6x-His tag upstream. (Bottom) ﬂp- 3 lof animals are raised on bacteria expressing the FLP-3 peptide of interest for 72-96 h and are assayed as young adults. b Avoidance behavior of him-8 and ﬂp-3 animals raised on scramble peptide or speciﬁed FLP-3 peptides. him-8 and ﬂp-3 males fed SCRAMBLE display their characteristic avoidance to ascr#8. ﬂp-3 males fed peptides FLP-3-2 or FLP-3-9 restore avoidance to wild-type levels. c, d ﬂp-3 male animals fed FLP-3-9 peptides rescues attraction behavior to ascr#8 c Raw dwell time and d log(fold-change) values for him-8 and ﬂp-3 males fed peptides FLP-3-2 or FLP-3-9. e Mutational schematic of FLP-3-7 to generate FLP-3-7T: the glutamate (“E”) in position 9 was mutated to a threonine (“T”). f ﬂp-3 lof male animals raised on FLP-3-7T suppress avoidance to ascr#8 suggesting the importance of amino acid threonine in mediating avoidance. g, h ﬂp-3 animals raised on FLP-3-7 and FLP-3-7T display no rescue of attractive behavior to ascr#8. g Raw dwell time and h log(fold-change) of ﬂp-3 animals raised on FLP-3-7 and FLP-3-7T. ♂denotes male data. d-type behavior and reveals two active peptides within the FLP-3 precursor. a Overview of rescue-by-feeding thin the FLP-3 precursor. a Overview of rescue-by-feeding Fig. 6 Peptide feeding rescues wild-type behavior and reveals two active peptides within the FLP-3 precursor. a Overview of rescue-by-feeding paradigm: (Top) A plasmid in generated which encodes the peptide of interest is ﬂanked by EGL-3 cleavage sites, with a 6x-His tag upstream. (Bottom) ﬂp- 3 lof animals are raised on bacteria expressing the FLP-3 peptide of interest for 72-96 h and are assayed as young adults. b Avoidance behavior of him-8 and ﬂp-3 animals raised on scramble peptide or speciﬁed FLP-3 peptides. him-8 and ﬂp-3 males fed SCRAMBLE display their characteristic avoidance to ascr#8. ﬂp-3 males fed peptides FLP-3-2 or FLP-3-9 restore avoidance to wild-type levels. c, d ﬂp-3 male animals fed FLP-3-9 peptides rescues attraction behavior to ascr#8 c Raw dwell time and d log(fold-change) values for him-8 and ﬂp-3 males fed peptides FLP-3-2 or FLP-3-9. e Mutational schematic of FLP-3-7 to generate FLP-3-7T: the glutamate (“E”) in position 9 was mutated to a threonine (“T”). Discussion This may be due to a high degree of male–male contact within the ascr#8 spot, but not the vehicle spot in SRA (Supplementary Fig. 1). We additionally conﬁrmed that another previously described male attractant ascaroside, acsr#3, maintains its ability to attract males in the SWAA (Supplementary Fig. 5). We also demonstrate that hermaphrodites, which have previously been shown to leave food rarely in comparison to males60, overcome measures the attractive properties of individual animals in each spot (Fig. 1, Supplementary Fig. 2). It also helps calculate the percentage attractiveness of the cue adding another parameter to measure the robustness of the cue. Our SWAA suggests that both him-5 and him-8 males are equally attracted to ascr#8 (Fig. 1, Supplementary Fig. 2). This addresses one of the caveats of the SRA results, wherein him-5 and him-8 males responded differ- entially to ascr#8, with him-8 males being signiﬁcantly more 10 ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Fig. 6 Peptide feeding rescues wild-type behavior and reveals two active peptides within the FLP-3 precursor. a Overview of rescue-by-feeding paradigm: (Top) A plasmid in generated which encodes the peptide of interest is ﬂanked by EGL-3 cleavage sites, with a 6x-His tag upstream. (Bottom) ﬂp- 3 lof animals are raised on bacteria expressing the FLP-3 peptide of interest for 72-96 h and are assayed as young adults. b Avoidance behavior of him-8 and ﬂp-3 animals raised on scramble peptide or speciﬁed FLP-3 peptides. him-8 and ﬂp-3 males fed SCRAMBLE display their characteristic avoidance to ascr#8. ﬂp-3 males fed peptides FLP-3-2 or FLP-3-9 restore avoidance to wild-type levels. c, d ﬂp-3 male animals fed FLP-3-9 peptides rescues attraction behavior to ascr#8 c Raw dwell time and d log(fold-change) values for him-8 and ﬂp-3 males fed peptides FLP-3-2 or FLP-3-9. e Mutational schematic of FLP-3-7 to generate FLP-3-7T: the glutamate (“E”) in position 9 was mutated to a threonine (“T”). f ﬂp-3 lof male animals raised on FLP-3-7T suppress avoidance to ascr#8 suggesting the importance of amino acid threonine in mediating avoidance. g, h ﬂp-3 animals raised on FLP-3-7 and FLP-3-7T display no rescue of attractive behavior to ascr#8. g Raw dwell time and h log(fold-change) of ﬂp-3 animals raised on FLP-3-7 and FLP-3-7T. ♂denotes male data. Fig. 6 Peptide feeding rescues wild-type behavior and reveals two active peptides within the FLP-3 precursor. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 The NPEND sequence of FLP-3-9 drives male attraction to ascr#8. b Schematic of the neural circuit regulating male responses to ascr#8 pheromone. ascr#8 is sensed by the male-speciﬁc CEM neurons. The neuropeptide gene ﬂp-3 is expressed in the IL1 neurons in the head and the SPD spicule neurons in the tail (teal). Following release of processed FLP-3 peptides, FLP-3-2 and FLP-3-9 are sensed by NPR-10 and FRPR-16 expressing neurons (blue and red, respectively) to mediate dwelling in the mating pheromone by modulating forward and reverse locomotion. Green neurons are connections inferred from the male synaptic connectome73. Gold denotes the command interneuron AVB and the forward locomotory circuitry. Fig. 7 Two FLP-3 NP/NPR modules mediate the male behavioral response to ascr#8. a Sequence speciﬁcity of FLP-3 peptide function. FLP-3-1, FLP-3-4, and FLP-3-7 exhibit no sex-speciﬁc effects on ascr#8 behavioral response. The threonine in the 9th position of FLP-3-2 and FLP-3-9, and in the mutated FLP-3-7T (red) is required for suppressing the sex-speciﬁc avoidance of ascr#8. The NPEND sequence of FLP-3-9 drives male attraction to ascr#8. b Schematic of the neural circuit regulating male responses to ascr#8 pheromone. ascr#8 is sensed by the male-speciﬁc CEM neurons. The neuropeptide gene ﬂp-3 is expressed in the IL1 neurons in the head and the SPD spicule neurons in the tail (teal). Following release of processed FLP-3 peptides, FLP-3-2 and FLP-3-9 are sensed by NPR-10 and FRPR-16 expressing neurons (blue and red, respectively) to mediate dwelling in the mating pheromone by modulating forward and reverse locomotion. Green neurons are connections inferred from the male synaptic connectome73. Gold denotes the command interneuron AVB and the forward locomotory circuitry. 3-1, FLP-3-4, and FLP-3-10 are not. As such, multiple NP/NPR modules are implicated in the ascr#8 behavioral response. Further studies will allow for further separation of FLP-3-2 and FLP-3-9, and how they interact with NPR-10 and FRPR-16. 3-1, FLP-3-4, and FLP-3-10 are not. As such, multiple NP/NPR modules are implicated in the ascr#8 behavioral response. Further studies will allow for further separation of FLP-3-2 and FLP-3-9, and how they interact with NPR-10 and FRPR-16. The presence of FRPR-16 in the amphid premotor inter- neurons responsible for backwards locomotion (Fig. 5) suggests the FLP-3/FRPR-16 module serves to mediate reversals during ascr#8 sensation (Fig. 7b). Discussion g g p Combining biochemical receptor activation studies with behavioral rescue-by-feeding assays, we have been successful in elucidating discrete neuropeptide signaling modalities within the complex FLP-3 signaling system. The involvement of two evo- lutionarily divergent receptors in sensing speciﬁc FLP-3 neuro- peptides suggests that the sex-speciﬁc behavioral response to ascr#8 module is a result of the activity of two distinct NP/NPR modules that mediate both attractive and repulsive properties of the small molecule. Two GPCRs respond to FLP-3 peptides to function in the behavioral response to ascr#8: the previously identiﬁed NPR-10, and the novel FRPR-16 (Figs. 3–5). Both exhibit high potencies for multiple FLP-3 peptides, although our single-peptide rescues have shown that FLP-3-2 and FLP-3-9 are required for the wild-type response to ascr#8 (Fig. 6), while FLP- 11 COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 CEM flp-3 expressing neuron npr-10 expressing neuron frpr-16 expressing neuron Sensory Neuron Interneuron Synapse Gap junction ASG BAG Male Tail Command Interneurons SPDL SPDR AVA RnB EF1 AVB AVD AVE AVK IL2 RMEL RMEV FLP-3 ADL AVF AIA AIB Forward Locomotion Backward Locomotion Turning Machinery Muscles IL1 FLP-3 DVC AIY GLUT CEM CEM CEM FLP-3-2 FLP-3-1 FLP-3-9 FLP-3-7T TPLGTMRF NPENDTPFGTMRF Sex-specific Avoidance SPLGTMRF NPLGTMRF SAEPFGTMRF SATPFGTMRF FLP-3-4 FLP-3-7 No sex-specific activity Sex-specific Attraction a b Fig. 7 Two FLP-3 NP/NPR modules mediate the male behavioral response to ascr#8. a Sequence speciﬁcity of FLP-3 peptide function. FLP-3-1, FLP-3-4, and FLP-3-7 exhibit no sex-speciﬁc effects on ascr#8 behavioral response. The threonine in the 9th position of FLP-3-2 and FLP-3-9, and in the mutated FLP-3-7T (red) is required for suppressing the sex-speciﬁc avoidance of ascr#8. The NPEND sequence of FLP-3-9 drives male attraction to ascr#8. b Schematic of the neural circuit regulating male responses to ascr#8 pheromone. ascr#8 is sensed by the male-speciﬁc CEM neurons. The neuropeptide gene ﬂp-3 is expressed in the IL1 neurons in the head and the SPD spicule neurons in the tail (teal). Following release of processed FLP-3 peptides, FLP-3-2 and FLP-3-9 are sensed by NPR-10 and FRPR-16 expressing neurons (blue and red, respectively) to mediate dwelling in the mating pheromone by modulating forward and reverse locomotion. Green neurons are connections inferred from the male synaptic connectome73. Gold denotes the command interneuron AVB and the forward locomotory circuitry. ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 FLP-3-2 FLP-3-1 FLP-3-9 FLP-3-7T TPLGTMRF NPENDTPFGTMRF Sex-specific Avoidance SPLGTMRF NPLGTMRF SAEPFGTMRF SATPFGTMRF FLP-3-4 FLP-3-7 No sex-specific activity Sex-specific Attraction a a CEM flp-3 expressing neuron npr-10 expressing neuron frpr-16 expressing neuron Sensory Neuron Interneuron Synapse Gap junction ASG BAG Male Tail Command Interneurons SPDL SPDR AVA RnB EF1 AVB AVD AVE AVK IL2 RMEL RMEV FLP-3 ADL AVF AIA AIB Forward Locomotion Backward Locomotion Turning Machinery Muscles IL1 FLP-3 DVC AIY GLUT CEM CEM CEM Sex-specific Avoidance No sex-specific activity Sex-specific Attraction b b Synapse Gap junction Fig. 7 Two FLP-3 NP/NPR modules mediate the male behavioral response to ascr#8. a Sequence speciﬁcity of FLP-3 peptide function. FLP-3-1, FLP-3-4, and FLP-3-7 exhibit no sex-speciﬁc effects on ascr#8 behavioral response. The threonine in the 9th position of FLP-3-2 and FLP-3-9, and in the mutated FLP-3-7T (red) is required for suppressing the sex-speciﬁc avoidance of ascr#8. Vector generation Our ﬁndings highlight the complexity of neuromodulators reg- ulating behavioral states in both invertebrates and vertebrates. Multiple hormonal receptors are involved in regulating the induc- tion of A. aegyptii ecdysteroid hormone production through two different neuropeptide signaling systems: ILP3 initiates digestion of the blood meal69, while OEH stimulates oocyte yolk uptake70. Melanin-concentrating hormone is a neuropeptidergic hormone that promotes appetite and feeding behaviors in mice in a sex- dependent manner71. Meanwhile, age-dependent changes in levels of Neuropeptide F result in the promotion of survival-beneﬁting appetitive memory in Drosophila, concurrent with the impairment of memories associated with insufﬁcient survival beneﬁts72. See Supplementary Table 2 for a complete plasmid list, and Supplementary Table 3 for primer and Ultramer sequences. Transgenic animals. CB1489 animals were injected with JSR#DKR18 (pﬂp-3::ﬂp- 3::GFP at 20 ng/µL), using punc-122::RFP (at 20 ng/µL) (kindly provided by k h h l h lk ) k Transgenic animals. CB1489 animals were injected with JSR#DKR18 (pﬂp-3::ﬂp- 3::GFP at 20 ng/µL), using punc-122::RFP (at 20 ng/µL) (kindly provided by Sreekanth Chalasani at the Salk Institute, CA) as a co-injection marker to generate JSR81 (him-8(e1489);worEx17[pﬂp-3::ﬂp-3::GFP; punc-122::RFP]). JSR81 was then crossed with JSR99 to generate JSR109 (ﬂp-3(pk361);him-8(e1489);worEx17[pﬂp- 3::ﬂp-3::GFP; punc-122::RFP]). g µ ), g p ( g µ ) ( y p y Sreekanth Chalasani at the Salk Institute, CA) as a co-injection marker to generate JSR81 (him-8(e1489);worEx17[pﬂp-3::ﬂp-3::GFP; punc-122::RFP]). JSR81 was then crossed with JSR99 to generate JSR109 (ﬂp-3(pk361);him-8(e1489);worEx17[pﬂp- 3::ﬂp-3::GFP; punc-122::RFP]). ﬂp PS2218 animals were injected with JSR#DKR34 (pﬂp-3::ﬂp-3::SL2::mCherry at 25 ng/µL), using punc-122::GFP (at 50 ng/µL) as a co-injection marker to generate JSR119 (dpy-20(e1362);him-5(e1490);syls33[HS.C3(50 ng/µL) + pMH86(11 ng/ µL)];worEx21[pﬂp-3::ﬂp-3::SL2::mCherry; punc-122::GFP]). Sex-speciﬁc behaviors arise from processing specialized olfac- tory cues emitted by their conspeciﬁcs2. Odor processing within each sex is mediated by ﬂexible neural circuits and neuromodu- lation enables neural networks to adapt behaviors under ﬂuctu- ating external and internal environmental states. So how is this adaptability achieved? Our studies demonstrate that speciﬁc peptides encoded by a single-neuropeptide gene, activate evolu- tionarily divergent receptors resulting in ﬁne-tuned sex-speciﬁc behavioral responses to small-molecule pheromones. These NP/ NPR modules are expressed within speciﬁc neurons of the ner- vous system of the two sexes, mediating overlapping behavioral outputs, by simultaneously suppressing an avoidance response and driving an attractive response (Fig. 7b). Vector generation Peptide constructs. DNA oligos containing the sequence for the peptides of interest were generated using Integrated DNA Technologies’ Ultramer synthesis service. The DNA sequence encoding the peptide sequence was ﬂanked with sequences encoding EGL-3 cut sites (MRFGKR upstream, and KRK-STOP) downstream59. These sites were then ﬂanked with Gateway Cloning sites attB1 and attB2. Annealed oligos were then used to perform a BP reaction with pDONR p1-p2 to generate the pENTRY clones. These vectors where then recombined with pDEST- 527 (a gift from Dominic Esposito (Addgene plasmid # 11518) in LR reactions to generate the expression clones59. The SCRAMBLE control was generated in an identical manner, with the sequence between the cut sites being ampliﬁed from pL4440 (provided by Victor Ambros, University of Massachusetts Medical School, MA). While we only investigated the activity of single peptides in this study, FLP-3 is a complex gene encoding 10 discrete peptides. Not all peptides are sufﬁcient to rescue the ascr#8 behavior on their own, they may instead serve a synergistic role to “active” peptides. Our rescue-by-feeding approach makes it easy to perform com- binatorial studies of peptides59, allowing for the elucidation of such synergistic peptide function. However, other reasons for the lack of rescue are possible, including rapid rates of degradation of the rescue peptide RNA. Future studies should make note to carefully dissect negative results, using either peptide soaking29,65, or comparison to in vivo expression with partial null mutants. We pursued the second course of action, discerning that FLP-3-1 and FLP-3-4 are insufﬁcient to suppress ascr#8 avoidance in either the ﬂp-3(ok3625) allele (Supplementary Fig. 4), or in our rescue-by- feeding paradigm (Fig. 6). Fusion constructs. DNA for the ﬂp-3, npr-10, and frpr-16 promoter and coding regions were isolated from C. elegans genomic DNA via PCR. In generating the ﬂp-3 rescue product, PstI and BamHI restriction sites added onto the isolated fragments were introduced through primer design. PCR amplicons and the Fire GFP Vector, pPD95.75 (kindly provided by Josh Hawk, Yale University, CT), were digested with PstI and BamHI enzymes. Products were ligated together to generate JSR#DKR18 (pﬂp-3::ﬂp-3::GFP). The ﬂp-3 expression analysis construct, JSR#DKR34 (pﬂp-3::ﬂp-3::SL2::mCherry) was generated by Genewiz. The promoter-gene fragment of npr-10 was generated by Gibson Assembly to GFP (from pPD95.75) and as a linear fusion. The rescue-fusion construct of frpr-16 was achieved by fusing the promoter and gene sequence to mCherry isolated from JSR#DKR34 via Gibson Assembly. COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 This aversion likely arises from the absence of amphid NPR-10 leading to aberrant regulation of turning machinery (through ASG-AIA connections) and muscle innervation (via RME neu- rons). In the tail, lack of NPR-10 seems to prevent EF1 activation, and thereby allowing forward locomotion to continue. Mean- while, loss of only FRPR-16 will lead to the inability of FLP-3 to regulate backward locomotion machinery (Fig. 7b). However, the loss of both the FLP-3/NPR-10 and FLP-3/FRPR-16 modules abolishes the ability of the animal to response to ascr#8 at all, observed as neither attraction nor aversion (Fig. 5). We post that the absence of both receptors results in the inability of FLP-3 to suppress forward locomotion (through NPR-10) nor drive backwards locomotion (via FRPR-16) in response to acsr#8. Future studies incorporating cell-speciﬁc rescue of both NPR-10 and FRPR-16 will further elucidate this circuitry. Methods Strains. Strains were obtained from the Caenorhabditis Genetics Center (Uni- versity of Minnesota, MN), the National BioResource Project (Tokyo Women’s Medical University, Tokyo, Jagan), Chris Li at City University of New York, Paul Sternberg at the California Institute of Technology, Ding Xue at University of Colorado Boulder, and Maureen Barr at Rutgers University. The novel allele of frpr-16 was generated via CRISPR editing using previously discussed methods58. Strains were crossed with either him-5 or him-8 worms to generate stable males prior to testing. See Supplementary Table 1 for a comprehensive list of strains used in this study. ARTICLE ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 Conversely, while FRPR-16 is conﬁned to a small, yet biologically speciﬁc subset of neurons in the head of the animals, NPR-10 exhibits more promiscuous expression that innervates both forward and reverse locomotion circuitries (Fig. 3e, f). As such, while the FLP-3/FRPR-16 module speciﬁcally modulates reversals, the FLP-3/NPR-10 module may instead serve to balance both forward and backwards locomotion in response to ascr#8, allowing the animal to interrogate its sur- roundings more thoroughly. Interestingly, these two high-potency FLP-3 receptors are extremely divergent in their evolutionary history. NPR-10 is most related to other “NPR” C. elegans receptors that evolved from the same family as the Drosophila melanogaster Neuropeptide F Receptor family55,67, and exhibits predicted NPY activity. FRPR- 16, however, is more closely related to the ﬂy FMRFamide Receptor67. Interestingly, while some C. elegans FRPR receptors function as FLP receptors19,64,65, at least one receptor within the same evolutionary clade (DAF-37) acts as a chemosensor for pheromones68. The evolutionary distance between NPR-10 and FRPR-16 suggests that these two receptors have undergone convergent evolution. g g y We argue that the loss of both of these modules that underlies the behavioral response in npr-10;frpr-16 double mutant animals to ascr#8. The loss of only one module results in a skewed behavioral response, observed as aversion to ascr#8 (Figs. 3, 5). COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio 12 COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio ARTICLE To transform the data, the log of this fold-change was taken, and the average log(fold-change) was used for statistical analyses and graphical display. Generation of a Null frpr-16 mutant by CRISPR mutagenesis. The frpr-16 CRISPR/Cas9 knockout was provided by the Vancouver node of the International C. elegans Consortium. The mutation was generated following previously described techniques58. In short, a 1685 bp region containing the coding sequence, as well 52 bp upstream and 60 bp downstream, was removed from the genome, and replaced with a trackable cassette containing pmyo-2::GFP and a neomycin resis- tance gene (Fig. 5a, b). The ﬂanking sequences of the mutated sequence are TCA TAATTGTTTGTTTGACAAAAACCGGGA and GGTGGAAACGGAAATGAAA GAAAAAACCGA. PCR conﬁrmation of gene replacement with cassette was performed via four sets of PCR reactions checking the upstream insertion site and the downstream insertion site in the mutant strain, and a test for wild-type sequence in both mutant and wild-type strains. A band is present on the gel in wild-type samples, with no band present in the mutant, as a primer sequence is removed with the cassette insertion. Visit count. The number of visits per-worm was calculated, and the average visit count determined per quadrant, and per plate. The average visit count across ﬁve plates was calculated and used for statistical analyses and graphical display. Percent attractive visits. An “attractive visit” was ﬁrst determined for each plate as any visit greater than two standard deviations above the mean dwell time within the vehicle control for that plate. Any individual visit meeting this threshold was scored as a “1”, and any below was scored a “0”. The percent visits per-worm that were attractive was determined, and the average of each quadrant taken. The four- quadrant values were then averaged to generate plate averages. The average percent of attractive visits across ﬁve plates was calculated and used for statistical analyses and graphical display. See Supplementary Table 3 for primer sequences. Avoidance assay. Assays were performed as described previously4,11,41. Fifty to sixty L4 worms were segregated by sex and stored at 20 °C for 5 h to overnight to be assayed as young adults. One to four hours prior to the assay, the lids of unseeded plates were tilted to allow any excess moisture to evaporate off the plates. At the time of the assay, 10 or more animals were transferred onto each of the dried, unseeded plates. ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 containing a camera and recorded for 20 m. Each strain and sex were assayed over ﬁve plates per day on at least three different days. transformed by taking the log (base 10) of the fold-change. Comparisons were then made by one-way ANOVA followed by multiple comparisons using Dunnett’s correction. p values are deﬁned in respective ﬁgure captions, with thresholds set as: p < 0.05, p < 0.01, p < 0.001, *p < 0.0001. Single worm assay. The outer forty wells of a 48-well suspension culture plate (Olympus Plastics, Cat #: 25-103) were seeded with 200 µL of standard NGM agar. To prepare the plates for the assay, they were acclimated to room temperature, at which point each well was seeded with 65 µL of OP50 E. coli. The assay plates were then transferred to a 37 °C incubator with the lid tilted for 4 h to allow the bacterial culture to dry on the agar. Once the bacterial culture dried, the lid was replaced the plate was stored at 20 °C until used in the assay. Fifty to sixty L4 worms were segregated by sex and stored at 20 °C for 5 h to overnight to be assayed as young adults. 0.8 µL of either vehicle control or ascaroside #8 was placed in the center of the well corresponding to that condition within the quadrant being tested, fol- lowing a random block design (Fig. 1a). A single worm was placed in each of the 10 wells to be assayed, and the plate was transferred to a light source and camera and recorded for 15 m. This process was repeated for all four quadrants. Each strain and sex were assayed over ﬁve plates assayed on at least three different days. Avoidance assay. Statistical comparisons within each strain were made by paired t- test against a signiﬁcance level set at 0.05. For comparisons between strains/con- ditions, comparisons were made by One-Way ANOVA, followed by multiple comparisons using Bonferroni correction. p values are deﬁned in respective ﬁgure captions, with thresholds set as: p < 0.05, p < 0.01, p < 0.001, **p < 0.0001. In vitro GPCR activation assay. The GPCR activation assay was performed as previously described66,74. Brieﬂy, npr-10 and frpr-16 cDNAs were cloned into the pcDNA3.1 TOPO expression vector (Thermo Fisher Scientiﬁc). Statistical analyses Imaging. Animals were mounted on a 2% agar pad and paralyzed using 1 M sodium azide on a microscope slide, as described previously11. Spot retention assay. Statistical comparisons within each strain were made by Paired t-tests. For comparisons between strains/conditions, the data was trans- formed as described previously38. In short, the data were transformed to have only non-zero data for the calculation of fold-changes. This was done using a Base 2 Exponentiation (2n, where n is equal to the dwell time). The log (base 2) of the fold-changes of these transformed values was used to allow for direct comparisons between strains of the same background (i.e., him-5 and osm-3;him-5) using a Student’s t-test. p values are deﬁned in respective ﬁgure captions, with thresholds set as: p < 0.05, p < 0.01, p < 0.001, ***p < 0.0001. Images for amphid ﬂp-3 expression were acquired using a Zeiss LSM700 confocal microscope. Final images were obtained using a ×63 oil objective with a ×1.4 digital zoom, for a ﬁnal magniﬁcation of ~×90. Tail images were acquired using a Zeiss Apotome using a ×40 oil objective. Images of npr-10 and frpr-16 expression were acquired using a Zeiss LSM510 Meta inverted confocal microscope. Final images of npr-10 were obtained using a ×63 oil objective with a ×0.8 digital zoom, for a ﬁnal magniﬁcation of ~×50. Final images of frpr-16 were obtained at either ×20 (air objective) or ×63 (oil) with a 2× digital zoom, for a ﬁnal magniﬁcation of ~×125. Single worm assay. Statistical comparisons within each strain/sex (spatial, vehicle, ascaroside) were made by Repeated Measured ANOVA with the signiﬁcance level set at 0.05, followed by multiple comparisons using Bonferroni correction. For comparisons between strains/sexes, the spatial control dwell times were compared using a one-way ANOVA followed by a Dunnett’s correction to conﬁrm that mutations of interest had no effect on the amount of time animals naturally spent in the center of the well. To directly compare strains, a fold-change was calculated by dividing the ascaroside by vehicle dwell times for each assay. This was then Reporting summary. Further information on research design is available in the Nature Research Reporting Summary linked to this article. ARTICLE A drop of either water or 1 µM ascr#8 was placed on the tail of forward moving animals, and their response was scored as either an avoidance response, or no response. The total number of avoidances was divided by the total number of drops to generate an avoidance index for that plate. This was repeated for at least 10 plates over at least three different days. Peptide rescue. SCRAMBLE control or FLP-3 peptide constructs were grown overnight in LB media containing 50 µg/µL ampicillin at 37 °C and diluted to an OD600 of 1.0 prior to seeding on NGM plates containing 50 µg/µL ampicillin and 1 mM IPTG. The 75 µL lawn was left to dry and grow overnight at room tem- perature before three L4 animals were placed on the plates. Males were selected for testing in the same manner as described above but were isolated onto plates also seeded with the same peptide on which they had been reared59. Animals were then assayed using either the avoidance assay or single worm assay. Vector generation The CEM neurons, which act as a primary site of ascr#8 sensation, synapse onto both forward (AVB) and backward (AVD) locomotory neurons, the activity of which are modulated by these NP/NPR modules both indirectly and directly, respectively (Fig. 7b)73. These ﬁndings highlight the complexity of peptidergic modulation of the nervous system, wherein individual peptides either from a single gene or multiple genes modulate opposing behaviors, through multiple NP/NPR modules. Using our rescue-by-feeding paradigm59, we can unravel the function of discrete peptides, enhancing our understanding of pathways of extra synaptic information ﬂow in the complex functional connectome. JSR102 animals were injected with a linear fusion product (pnpr-10::npr- 10::GFP at 25 ng/µL), alongside punc-122::RFP (at 50 ng/µL) as a co-injection marker to generate JSR126 (npr-10(tm8982);him-8(e1489);worEx37[pnpr-10::npr- 10::GFP, punc-122::RFP]). This was crossed with PT2727 (myIS20 [pklp- 6::tdTomato + pBX], a gift from Maureen Barr) to generate JSR138 (myIS20 [pklp- 6::tdTomato, pBX]; npr-10(tm8982);him-8(e1489);worEx37[pnpr-10::npr-10::GFP, punc-122::RFP]). JSR103 animals were injected with a linear fusion product (pfrpr-16::frpr- 16::SL2::mCherry at 25 ng/µL), alongside punc-122::GFP (at 50 ng/µL) as a co- injection marker to generate JSR133 (frpr-16(gk5305[loxP + pmyo-2::GFP::unc-54 3′ UTR + prps-27::neoR::unc-54 3′ UTR + loxP]);him-8(e1489);worEx41[pfrpr- 16::frpr-16::SL2::mCherry, punc-122::GFP]). Injections for JSR81 were generously performed by the Alkema Lab at UMass Medical School. Injections for JSR119, JSR126, and JSR133 were performed by In Vivo Biosystems (formerly NemaMetrix). Chemical compounds. The ascarosides ascr#3 and ascr#8 were synthesized as described previously28,33. Peptides used in in vitro GPCR activation assays were synthesized by GL Biochem Ltd. Spot retention assay. Assays were performed as described previously14,33. Fifty to sixty larval-stage 4 (L4) males were segregated by sex and stored at 20 °C for 5 h to overnight to be assayed as young adults. For hermaphrodite trials, young adult hermaphrodites were segregated 1.5 h prior to testing. 0.6 µL of vehicle control or ascaroside #8 was placed in each scoring region (Supplementary Fig. 1a). As the working stock of ascaroside #8 was made in MilliQ-puriﬁed ultrapure H2O, this was used as the vehicle control. Five animals were placed on each “X” the assay plate (Supplementary Fig. 1a), which was then transferred to a microscope 13 COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio ARTICLE A CHO-K1 cell line (PerkinElmer, ES-000-A24) stably expressing apo-aequorin targeted to the mitochondria (mtAEQ) and human Gα16 was transiently transfected with the receptor cDNA construct or the empty pcDNA3.1 vector using Lipofectamine LTX and Plus reagent (Thermo Fisher Scientiﬁc). Cells were shifted to 28 °C one day later and allowed to incubate for 24 h. On the day of the assay, cells were collected in BSA medium (DMEM/Ham’s F12 with 15 mM HEPES, without phenol red, 0.1% BSA) and loaded with 5 mM coelenterazine h (Thermo Fisher Scientiﬁc) for 4 h at room temperature. The incubated cells were then added to synthetic peptides dissolved in DMEM/BSA, and luminescence was measured for 30 s at 496 nm using a Mithras LB940 (Berthold Technologies) or MicroBeta LumiJet luminometer (PerkinElmer). After 30 s of readout, 0.1% triton X-100 was added to lyse the cells, resulting in a maximal calcium response that was measured for 10 s. After initial screening, concentration-response curves were constructed for HPLC-puriﬁed FLP-3 peptides by subjecting the transfected cells to each peptide in a con- centration range from 1 pM to 10 µM. Cells transfected with an empty vector were used as a negative control. Assays were performed in triplicate on at least two independent days. Concentration-response curves were ﬁtted using Prism v. 7 (nonlinear regression analysis with a sigmoidal concentration-response equation). Raw dwell time. Raw dwell time values were calculated by subtracting the time a worm exited the cue (center of the well in spatial controls), from the time it entered, as in the SRA14. This was determined per visit, and the average dwell time was calculated for each animal in the quadrant. Averages of the four-quadrant means were determined per plate, and a minimum of ﬁve plates were assayed per strain/condition. The mean raw dwell time across ﬁve plates was calculated and used for statistical analyses and graphical display. Log(fold-change). The average dwell time in the ascaroside was divided by the average dwell time within the vehicle control per plate to generate a fold- change. To transform the data, the log of this fold-change was taken, and the average log(fold-change) was used for statistical analyses and graphical display. Log(fold-change). The average dwell time in the ascaroside was divided by the average dwell time within the vehicle control per plate to generate a fold- change. 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Nucleus accumbens melanin-concentrating hormone signaling promotes feeding in a sex-speciﬁc manner. Neuropharmacology 178, 108270 (2020). Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/ licenses/by/4.0/. 72. Tonoki, A., Ogasawara, M., Yu, Z. & Itoh, M. Appetitive memory with survival beneﬁt is robust across aging in Drosophila. J. Neurosci. 40, 2296–2304 (2020). 73. Cook, S. J. et al. Whole-animal connectomes of both Caenorhabditis elegans sexes. Nature 571, 63–71 (2019). 74. Peymen, K. et al. Myoinhibitory peptide signaling modulates aversive gustatory learning in Caenorhabditis elegans. PLoS Genet. 15, e1007945 (2019). Author contributions D.K.R. performed behavioral assays, contributed to the single worm assay design, per- formed all molecular biology, performed and designed peptide rescue experiments, performed the statistical analyses, and led manuscript writing and revision. E.J.M., H.T.N., and A.N.R. contributed to the behavioral assays and as well as manuscript revisions. A.N.R. and C.S.M. generated the materials for and performed experiments related to FLP-3-7. E.V. and I.B. performed the GPCR activation assays, as well as contributed to manuscript revisions. I.B. also provided funding for the GPCR activation studies. W.J. contributed to injections of the ﬂp-3 rescue, while M.J.A. provided com- ments on manuscript. R.J.G. assisted in the development of the single worm assay design. J.S. and D.K.R. wrote the manuscript with input from M.A. and R.J.G. 61. Barrios, A., Nurrish, S. & Emmons, S. W. Sensory regulation of C. elegans male mate-searching behavior. Curr. Biol. 18, 1865–1871 (2008). 62. Bentley, B. et al. The multilayer connectome of Caenorhabditis elegans. PLoS Comput. Biol. 12, e1005283 (2016). p 63. Rojo Romanos, T., Petersen, J. G., Riveiro, A. R. & Pocock, R. A novel role for the zinc-ﬁnger transcription factor EGL-46 in the differentiation of gas- sensing neurons in Caenorhabditis elegans. Genetics 199, 157–163 (2015). 64. Chew, Y. L., Grundy, L. J., Brown, A. E. X., Beets, I. & Schafer, W. R. Neuropeptides encoded by nlp-49 modulate locomotion, arousal and egg- laying behaviours in Caenorhabditis elegans via the receptor SEB-3. Philos. Trans. R. Soc. Lond. B Biol. Sci. https://doi.org/10.1098/rstb.2017.0368 (2018). ARTICLE COMMUNICATIONS BIOLOGY \| https://doi.org/10.1038/s42003-021-02547-7 55. Cardoso, J., Felix, R., Fonseca, V. & Power, D. Feeding and the rhodopsin family G-protein coupled receptors in nematodes and arthropods. Front. Endocrinol. https://doi.org/10.3389/fendo.2012.00157 (2012). Boulder), and Dr. Maureen Barr (Rutgers) for providing strains. The synthetic ascr#8 utilized in this study was generously provided by Frank Schroeder (Cornell University). We want to thank Dr. Don Moerman (UBC; C. elegans Knockout Facility) for providing the frpr-16 knockout. We thank InVivo Biosciences for generating transgenic animals by injection. We thank Kate Pearce and Suzanne Scarlatta for assistance in confocal ﬂuorescent imaging the npr-10 and frpr-16 reporter strains, as well as Dr. Jeremy Flor- man of Mark Alkema’s lab for assistance in ﬂp-3 reporter imaging. We also would like to thank Dr. Victor Ambros (UMass Medical School), Dr. Shreekanth Chalasani (Salk Institute), Dr. Josh Hawk (Yale), and Dr. Dominic Esposito (Addgene) for providing us with the necessary plasmid reagents. We acknowledge and thank Profs. Frank Schroeder and Shreekanth Chalasani, as well as members of the Srinivasan lab for their feedback on this manuscript. Funding for this work was provided under the Research foundation Flanders (FWO) project grant G0C0618N (I.B.), and the National Institutes of Health grants R01 NS107475 (M.J.A.), and R01 DC016058 (J.S.). p g 56. Gershkovich, M. M., Groß, V. E., Kaiser, A. & Prömel, S. Pharmacological and functional similarities of the human neuropeptide Y system in C. elegans challenges phylogenetic views on the FLP/NPR system. Cell Commun. Signal. 17, 123 (2019). 57. Beets, I., Lindemans, M., Janssen, T. & Verleyen, P. Deorphanizing G protein- coupled receptors by a calcium mobilization assay. Methods Mol. Biol. 789, 377–391 (2011). 58. Au, V. et al. CRISPR/Cas9 methodology for the generation of knockout deletions in Caenorhabditis elegans. G3 (Bethesda) 9, 135–144 (2019). g 59. DiLoreto, E. M., Reilly, D. K. & Srinivasan, J. Non-transgenic functional rescue of neuropeptides. bioRxiv https://doi.org/10.1101/2021.05.10.443513 (2021). 60. Lipton, J., Kleemann, G., Ghosh, R., Lints, R. & Emmons, S. W. Mate searching in Caenorhabditis elegans: a genetic model for sex drive in a simple invertebrate. J. Neurosci. 24, 7427–7434 (2004). The authors declare no competing interests. 66. Van Sinay, E. et al. Evolutionarily conserved TRH neuropeptide pathway regulates growth in Caenorhabditis elegans. Proc. Natl Acad. Sci. USA 114, E4065–e4074 (2017). Additional information Supplementary information The online version contains supplementary material available at https://doi.org/10.1038/s42003-021-02547-7. 67. Hobert, O. The neuronal genome of Caenorhabditis elegans. WormBook 1–106 https://doi.org/10.1895/wormbook.1.161.1 (2013). Correspondence and requests for materials should be addressed to J.S. 68. Park, D. et al. Interaction of structure-speciﬁc and promiscuous G-protein- coupled receptors mediates small-molecule signaling in Caenorhabditis elegans. PNAS 109, 9917–9922 (2012). Peer review information Communications Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Primary Handling Editors: Karli Montague-Cardoso. Peer reviewer reports are available. g 69. Gulia-Nuss, M., Robertson, A. E., Brown, M. R. & Strand, M. R. Insulin-like peptides and the target of rapamycin pathway coordinately regulate blood digestion and egg maturation in the mosquito Aedes aegypti. PLoS ONE 6, e20401 (2011). Competing interests 65. Nelson, M. D. et al. FRPR-4 is a G-protein coupled neuropeptide receptor that regulates behavioral quiescence and posture in Caenorhabditis elegans. PLoS ONE 10, e0142938 (2015). References Defective processing of neuropeptide precursors in Caenorhabditis elegans lacking proprotein convertase 2 (KPC-2/EGL-3): mutant analysis by mass spectrometry. J. Neurochem. 98, 1999–2012 (2006). 26. Bhardwaj, A., Thapliyal, S., Dahiya, Y. & Babu, K. FLP-18 functions through the G-protein-coupled receptors NPR-1 and NPR-4 to modulate reversal length in Caenorhabditis elegans. J. Neurosci. 38, 4641 (2018). length in Caenorhabditis elegans. J. Neurosci. 38, 4641 (2018). 54. Thacker, C., Srayko, M. & Rose, A. M. Mutational analysis of bli-4/kpc-4 reveals critical residues required for proprotein convertase function in C. elegans. Gene 252, 15–25 (2000). 27. Yu, Y., Zhi, L., Guan, X., Wang, D. & Wang, D. FLP-4 neuropeptide and its receptor in a neuronal circuit regulate preference choice through functions of ASH-2 trithorax complex in Caenorhabditis elegans. Sci. Rep. 6, 21485 (2016). 15 Acknowledgements We thank the Caenorhabditis Genetics Center, which is funded by the NIH Ofﬁce of Research Infrastructure Programs (P40 OD01044), as well as Dr. Paul Sternberg (Cal- Tech), Dr. Chris Li (CUNY), the National BioResource Project, Dr. Ding Xue (UC © The Author(s) 2021 16 COMMUNICATIONS BIOLOGY \| (2021) 4:1018 \| https://doi.org/10.1038/s42003-021-02547-7 \| www.nature.com/commsbio

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