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Identification of the Causal Agent of Bacterial Soft Rot of Potato and its Management 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 Ferdous-E-Elahi, M.S. Graduate Program in Plant Pathology The Ohio State University 2018 Dissertation Committee Sally A. Miller, Adviser Pierce A. Paul M. Soledad Benitez Ponce Melanie L. Lewis Ivey Copyright by Ferdous-E-Elahi 2018 Abstract Although commercial potato production started in 1920 in Bangladesh, potatoes are severely affected by diseases, resulting in poor yields. The demand for potato is growing day by day and many approaches are being implemented to increase yields. From December 2014 to January 2015, a total of 15 bacterial isolates were recovered from potato tubers with symptoms of soft rot collected in ten major potato- growing areas of Bangladesh. Based on biochemical and physiological assays, Pectobacterium carotovorum ssp. were identified as the causal agents of soft rot in potato tubers. The pathogens were further characterized with molecular methods including subspecies-specific PCR, 16s rRNA gene sequencing and multilocus sequence analysis (MLSA). Of the 15 isolates causing tuber soft rot, five of the isolates also caused blackleg symptoms in potato seedlings. PCR utilizing primer pair EXPCCF and EXPCCR resulted in the amplification of 550-bp DNA sequences of P. carotovorum ssp. carotovorum from the 15 soft rot isolates. 16s rRNA gene sequencing confirmed the identity of Pectobacterium carotovorum ssp. carotovorum from potato tubers. Phylogenetic analysis of concatenated gene sequences from six housekeeping genes (acnA, gapA, icdA, mdh, pgi and proA) from in MLSA revealed two distinct clades among the Bangladeshi strains. Although the sample size is relatively low, it is likely that P. carotovorum ssp. carotovorum is a principle cause of soft rot of potato tubers in Bangladesh. Blackleg has not yet been reported in Bangladesh. However, the finding that one third of the P. carotovorum ssp. carotovorum isolates recovered from potato tubers in Bangladesh could cause blackleg suggests that the disease could be a problem under appropriate environmental conditions. To develop an integrated blackleg management plan for potato producers in Bangladesh, several tactics were i evaluated. Chitosan, Trichoderma harzianum and gypsum fertilizer were applied alone or in combination to assess their ability to suppress the disease. The highest level of disease reduction was recorded for tubers treated with chitosan 3% + T. harzianum BTH-N1. Ten BARI-released potato varieties exhibited different levels of soft rot severity when they were inoculated with the highly virulent soft rot causing strain Pectobacterium carotovorum ssp. carotovorum Pki2. BARI Alu 25 was the most resistant to soft rot, while the recently released potato varieties BARI Alu 72 and BARI Alu 41 were the most susceptible. Calcium (p<0.05) but not dry matter percentage of tubers (p>0.05) was negatively correlated with soft rot ranking in these varieties. Potential antagonism of 41 previously characterized Pseudomonas spp. strains against Pectobacterium carotovorum ssp. carotovorum was investigated. Five Pseudomonas spp. were selected based on their performance in in vitro assays and previous history of disease reduction in other systems. However, there were no significant differences in reduction by any of the Pseudomonas strains in blackleg severity among the treatments (p<0.05). This may have been the result of physical separation of the Pseudomonas strains on tubers and the inoculation point for P. carotovorum ssp. carotovorum on stems. Further research is needed to determine the capacity of these Pseudomonas strains to suppress potato blackleg and tuber soft rot. A survey was undertaken to assess the knowledge of the growers regarding potato production practices, production challenges and blackleg and soft rot disease management. A total of 348 respondents were interviewed in six main potato-growing districts in Bangladesh in 2016. Data were collected using a structured questionnaire with direct interviews. Results showed that growers were very aware of potato diseases and considered disease the second most important constraint to potato ii production after market price. The majority of the growers identified late blight as the main field disease across the districts followed by yellow mosaic and soft rot as their main storage problem followed by dry rot. Potato growers across the districts cited fungicide application as the primary means of managing field diseases and sorting as the means of managing diseases in storage. None of the growers indicated that they used biological control methods to manage diseases in the field or in storage. Growers preferred high-yielding, disease-tolerant potato varieties. The results of this survey showed that an integrated blackleg and soft rot management program likely to be adopted by farmers in Bangladesh should integrate high yielding potato varieties, disease-free potato seeds, inoculum-free irrigation water, regular scouting, and roguing. iii Dedication To My supportive husband: Md. Mynul Islam My son: Muntahin Faiqul Islam And my entire family for their affection and love iv Acknowledgements I would like to express my sincere appreciation to my adviser Dr. Sally A. Miller for her advice, support and encouragement. Thanks for her continuous guidance and suggestions, without which it would have been very difficult to finish this work. I am also grateful to my committee members Dr. Pierce Paul, Dr. Soledad Benitez Ponce and Dr. Melanie L. Lewis Ivey for their helpful comments and suggestions. I am especially thankful to Dr. Pierce Paul for helping me to design and execute the biocontrol experiment in the growth chambers and Dr. Christopher G. Taylor for providing me the biocontrol Pseudomonas strains. I thank the memebers of Dr. Miller’s lab. It has been really nice to know all of you. Thank you Angela Nanes, Anna L. Testen, Cláudio Vrisman, Mafruha Afroz, Md. Mynul Islam, Nitika Khatri, Xing Ma, Jhony Mera, Fulya Baysal-Gurel, Margaret Moodispaw, Nick Rehm, Hugo Pantigoso, Francesca Rotondo and Nagendra Subedi. I am grateful to my parents for the aspiration for my higher studies and continuous patience and support. I would like to thank A. K. M. Yousuf Harun, Deputy Director, Bangladeh Agriultural Development Corporation (BADC) for helping me to complete the survey section, collect potato seeds from BADC and for all of the relevant help during the last year of my PhD program. I thank Dr. Tapan Kumar Dey for his kind suggestions and help in conducting experiments in Bangladesh. I thank Dr. Shamsunnahar, Principle Scientific Officer, Horticultural Research Center (HRC), Bangladesh Agricultural Research Institute (BARI) for providing me the strains Trichoderma and Abul Kalam Azad, lab attendent of Plant Pathology lab, BARI. I would also like to acknowledge my funding agency Borlaug Higher Education Agricultural Research and Development (BHEARD) for financial support. v Vita 2007 B.S. Agriculture, Sher-E-Bangla Agricultural University, Dhaka, Bangladesh 2009 M.S. Plant Pathology, Sher-E-Bangla Agricultural University, Dhaka, Bangladesh 2010-present Scientific officer, Division of Plant Pathology, Bangladesh Agricultural Research Institute 2013 USDA Borlaug Fellow in Borlaug Agricultural Science and Technology Fellowship program 2013 Scholarship recipient for PhD in USAID Borlaug Higher Education Agricultural Research and Development program Publications Vrisman, C. M., Testen, A. L., Elahi, F., and Miller, S. A. 2017. First report of tomato brown root rot complex caused by Colletotrichum coccodesand Pyrenochaeta lycopersici in Ohio. Plant Disease. 101:247. Elahi, F., Mridha, M. A. U., and Aminuzzaman, F. M. 2013. Role of AMF on plant growth, nutrient uptake, arsenic toxicity and chlorophyll content of chili grown in arsenic amended soil. Bangladesh J. Agril. Res. 37:635-644. Elahi, F., Islam, M., Humauan, M. R., Akter, B., Khalequzzaman, K. M., and Dey, T. K. 2011. First report on club root disease (Plasmodiophora brassicae) on mustard in Bangladesh. Bangladesh J. Plant Pathol. 27:71-72. Elahi, F., Aminuzzaman, F. M., Mridha M. A. U., Begum, B., and Harun, A. K. M. Y. 2010. AMF inoculation reduced arsenic toxicity and increased growth, nutrient uptake and chlorophyll content of tomato grown in arsenic amended soil. Adv. Environ. Biol. 4:194-200. Booklet:(In Bengali) a. Cucumber diseases and its control strategies b. Soil borne diseases of crops and its integrated management Fields of study Major Field: Plant Pathology vi Table of Contents Abstract ........................................................................................................................... i Dedication ..................................................................................................................... iv Acknowledgements ........................................................................................................ v Vita ................................................................................................................................ vi List of Tables ................................................................................................................ ix List of Figures ............................................................................................................... xi Chapter 1: Introduction .................................................................................................
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    Final Report Review Bacterial Blackleg Disease and R&D Gaps with a Focus on the Potato Industry Project leader: Dr Len Tesoriero Delivery partner: Crop Doc Consulting Pty Ltd Project code: PT18000 Hort Innovation – Final Report Project: Review Bacterial Blackleg Disease and R&D Gaps with a Focus on the Potato Industry – PT18000 Disclaimer: Horticulture Innovation Australia Limited (Hort Innovation) makes no representations and expressly disclaims all warranties (to the extent permitted by law) about the accuracy, completeness, or currency of information in this Final Report. Users of this Final Report should take independent action to confirm any information in this Final Report before relying on that information in any way. Reliance on any information provided by Hort Innovation is entirely at your own risk. Hort Innovation is not responsible for, and will not be liable for, any loss, damage, claim, expense, cost (including legal costs) or other liability arising in any way (including from Hort Innovation or any other person’s negligence or otherwise) from your use or non‐use of the Final Report or from reliance on information contained in the Final Report or that Hort Innovation provides to you by any other means. Funding statement: This project has been funded by Hort Innovation, using the fresh potato and processed potato research and development levy and contributions from the Australian Government. Hort Innovation is the grower‐owned, not‐ for‐profit research and development corporation for Australian horticulture. Publishing details: