Diversity and Management of Sclerotinia sclerotiorum in Brassica spp. in Bangladesh Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University by Md. Mynul Islam, MS Graduate Program in Plant Pathology The Ohio State University 2018 Dissertation Committee: Sally A. Miller, Advisor Anne Dorrance Pierce A. Paul Christopher Taylor Copyright by Md. Mynul Islam 2018 Abstract Sclerotinia sclerotiorum is an important pathogen of many crops including rapeseed- mustard (Brassica spp.). The pathogen has emerged relatively recently in Bangladesh and there is no information available regarding its population structure in mustard-growing regions of the country. A series of experiments were conducted to determine the variability of S. sclerotiorum isolates from different regions of Bangladesh and from Ohio, USA. In 2014, a total of 132 S. sclerotiorum isolates were collected from 11 locations in Bangladesh and Ohio. Morphological characteristics including mycelial radial growth and number and fresh weight of sclerotia were measured. Genetic variability was also assessed by Internal Transcribed Spacer (ITS) rDNA sequencing, microsatellite markers and mycelial compatibility grouping. Overall, isolates from Bangladesh showed low variability based on morphological and molecular characteristics. Mycelial radial growth of isolates from one location in Bangladesh was significantly higher than the radial growth of isolates from Ohio. No significant difference was observed in the number of sclerotia produced by isolates from the 11 locations. The weight of sclerotia produced by isolates from Tangail, Mirzapur was smaller than that of isolates from Ohio and from Tangail, Ghatail and Shirajganj, Chalakpara, Bangladesh. No significant variation was observed among isolates from any location based on their ITS rDNA sequences. Based on eight informative microsatellite loci, 91% of the variation was within the isolates and 9% was due to location, indicating low divergence among the populations from the 11 locations. Principle Component Analysis (PCA) separated Ohio isolates from isolates from nine locations in Bangladesh; however, isolates from Jamalpur were in the same PCA quartile as isolates from Ohio. Twenty-seven microsatellite haplotypes were identified from 118 isolates from 11 locations, and one haplotype (haplotype 1) ii was predominant in ten locations in Bangladesh. The Ohio population contained two isolates with two separate haplotypes (haplotype 25 and haplotype 26). Thirty-four mycelial compatibility groups (MCGs) were identified among 80 S. sclerotiorum isolates; those from Ohio formed four groups while the remaining 30 groups were from Bangladesh. Fifty-one isolates from ten Bangladesh populations were in haplotype 1, which shared 14 MCGs. On the other hand, four MCGs contained more than one haplotype. Based on morphological and genetic characters, the S. sclerotiorum populations from Bangladesh and from Ohio were different, however, populations from Bangladesh had low variability. Fungicide application is the primary tactic widely used to manage white mold in mustard and other crops. However, no information is available on sensitivity of S. sclerotiorum to fungicides in Bangladesh. Sensitivity of S. sclerotiorum to iprodione, propiconazole, fluazinam and penthiopyrad was determined using isolates collected from 11 locations in Bangladesh and Ohio, USA in 2014. Sensitivity was assessed using discriminatory doses and concentrations and 50% mycelial inhibition (EC50) values were determined. Compared with the EC50 of the fungicides to S. sclerotiorum from the published literature, none of the tested S. sclerotiorum isolates were resistant to iprodione, propiconazole, fluazinam or penthiopyrad. However, some isolates of S. sclerotiorum exhibited reduced sensitivity to propiconazole. The EC50 values obtained in the first experiment ranged from 0.18 - 0.50 ppm, 0.12 - 0.78 ppm, 0.0019 - 0.0044 ppm and 0.012 - 0.429 ppm for iprodione, propiconazole, fluazinam and penthiopyrad, respectively. In the second experiment, EC50 values ranged from 0.16 - 0.36 ppm, 0.02 - 0.93 ppm, 0.0024 - 0.0050 ppm and 0.08 - 0.83 ppm for iprodione, propiconazole, fluazinam and penthiopyrad respectively. Relative toxicity index (RTI) values, using iprodione as the standard, were 103.2 and 67.6 (experiments 1 and 2, respectively) for fluazinam, and 6.0 and 1.6 iii (experiments 1 and 2, respectively) for penthiopyrad. Propiconazole was similar to iprodione in toxicity to S. sclerotiorum. Fluazinam and penthiopyrad are not registered in Bangladesh. Iprodione and propiconazole are registered for other diseases, but not resigtered for white mold management in mustard, therefore the EC50 values of fluazinam and pethiopyrad determined in this study can be considered baseline sensitivity levels for future efforts to monitor development of resistance to these fungicides in S. sclerotiorum in Bangladesh. Development of rapeseed-mustard varieties partially or fully resistant to S. sclerotiorum would enhance the disease management toolbox and reduce or eliminate the need for fungicides to control this disease. Fourteen varieties and one breeding line developed by the Bangladesh Agricultural Research Institute (BARI) were screened to determine their reactions to S. sclerotiorum. Twenty S. sclerotiorum isolates were pre-evaluated for virulence and a highly virulent isolate was selected. Isolate SCS1 caused large lesions 24 h after inoculation in a detached leaf assay. This isolate was used in cotyledon and petiole inoculation assays. In screening with cotyledon inoculation, the smallest lesions were observed in BARI Sharisa 14. There were no significant differences among the varieties/line in percentage of infected cotyledons. In petiole inoculation screening, variation in the reactions of the rapeseed-mustard varieties/line to S. sclerotiorum was insignificant, except for breeding line SS 75 in both experiments. This line showed significantly higher resistance to S. sclerotiorum than BARI Sharisa 10 in first experiment and Tori 7 in the second experiment. Although the results obtained using two inoculation methods were inconsistent, both BARI Sharisa 14 and SS 75 may prove to be useful as sources of resistance to S. sclerotiorum upon more extensive evaluation. Integrated management is the most durable management strategy. Two experiments were conducted at Rangpur and Jamalpur to evaluate different treatments separately and in iv combination to control white mold disease of mustard. The fungal biocontrol agent Trichoderma harzianum isolate BHT-N1 (ThBHT-N1) and five fungicides in different groups (carbendazim, thiophanate-methyl, propiconazole, iprodione and azoxystrobin + difenoconazole) were tested separately and in combination with ThBHT-N1 in natural field conditions. In Burirhut, Rangpur, the incidence of white mold disease was low. However, azoxystrobin + difenoconazole-treated plots had significantly lower disease incidence and higher yield than non-treated control plots. In Jamalpur, white mold was not observed, but Alternaria blight was recorded. All fungicide treatments and ThBHT-N1 significantly reduced disease severity compared to the non-treated control, but azoxystrobin + difenoconazole and iprodione treatments were more effective than the other treatments. Information generated from this study will enhance our understanding of population structure of S. sclerotiorum in Bangladesh, its diversity and sensitivity to fungicides and sources of resistance to S. sclerotiorum. This information will be helpful for increasing production and ultimately will contribute to food security of Bangladesh, a developing country. v Dedication In memory of my parents vi Acknowledgements I would like to express my sincere thanks and appreciation to my advisor Dr. Sally Miller for her insightful scholarly guidance, encouragement, partial research funds and many other opportunities provided me for my development as a plant pathologist since the last 4 years. I am very much grateful to the members of my advisory committee, Dr. Anne Dorrance, Dr. Pierce Paul and Dr. Christopher Taylor for their cooperation and suggestions to understand many topics related to my research and to improve the quality of my thesis research. Thanks to all members of the Miller lab, Fulya Baysal-Gurel, Francesca Rotondo, Angela Nanes, Jhony Mera, Cláudio Vrisman, Anna Testen, Xing Ma, Nagendra Subedi, Ferdous- E- Elahi, Mafrua Afroz, Loïc Deblais, Ram Khadka, Andres Sanabria, Nick Rehm, Margaret Moodispaw, and Luis Huezo for their cordial cooperation. I would like to thank Ken Nanes, Bob James, Lee Wilson, Monica Lewandowski, Pat Rigby, and Beau Ingle for their help in facilitating research work, processing my trips to USA and continuous coordination with funding agencies. I would like to thank all the students of the Department of Plant Pathology, OSU for their support and friendly cooperation. Also thanks to Dr. Tapan Kumar Dey for his valuable suggestions on the design and set up of field experiments in Bangladesh. I am grateful to my funding agencies, USAID mission Bangladesh, Borlaug Higher Education for Agricultural Research and Development (BHEARD) team in Michigan State University, and Bangladesh Agricultural Research Institute (BARI). Without their support, it would be impossible to complete the program. Finally I would like thank my family members for their cooperation and help during the time of my dissertation.