The Soybean Seedling Disease Complex: Pythium spp. and Fusarium graminearum and their Management through Host Resistance DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Margaret Lee Ellis, M.S. Graduate Program in Plant Pathology The Ohio State University 2011 Dissertation Committee: Dr. Anne E. Dorrance, Advisor Dr. Pierce A. Paul, Advisor Dr. M.A. Rouf Mian Dr. Thomas K. Mitchell Copyrighted by Margaret Lee Ellis 2011 Abstract Seedling diseases in soybean fields in Ohio have increased over the past decade. This study was conducted to better characterize some of these seedling pathogens, specifically Fusarium graminearum and two new pathogenic species of Pythium, as well as evaluate management strategies, particularly host resistance, for F. graminearum and Pythium irregulare. A rolled towel assay was developed to understand the potential impact of F. graminearum as a soybean pathogen by evaluating the effect of inoculum density, temperature parameters, and fungicide seed treatments on disease development. Inoculum concentrations of 2.5 × 104 macroconidia/ml or higher were necessary for disease development at temperatures 18 to 25oC, indicating that high levels of inoculum may be necessary for disease to occur. Seed treated with captan at 61.9 g a.i. or fludioxonil at 2.5 or 5.0 g a.i. per 100 kg developed smaller lesions than other seed treatments and the non- treated control. The rolled towel assay was used to screen 24 soybean genotypes for resistance to F. graminearum. Five genotypes had high levels of resistance to F. graminearum, including the cultivar Conrad, a major source of partial resistance to Phytophthora sojae. A population of 262 F6:8 recombinant inbred lines (RIL) derived from a cross of Conrad x Sloan (Susceptible) was evaluated for resistance and segregated as a quantitative trait. Four putative quantitative trait loci (QTLs) were identified from Conrad on chromosomes 8, 13, 15, and 16, and one putative QTL from Sloan on ii chromosome 19. The putative QTLs identified in this population did not map to the same regions that confer resistance to Ph. sojae, suggesting different mechanisms are required for these two seedling pathogens. In this study, two new species of Pythium, P. schmitthenneri and P. selbyi, were described using morphology and sequence analysis of the ITS1-5.8S-ITS2 region. These new species were recovered from 30% of fields surveyed which was focused on the identification of seedling pathogens; they are both pathogens to corn and soybean. Pythium irregulare is one of the most widespread Pythium species in Ohio soybean fields and has very high levels of pathogenicity. In a greenhouse assay, 105 soybean genotypes were evaluated for resistance to two isolates of P. irregulare. Isolate x genotype interaction for root weight and rot root score was not significant. The plant introduction (PI) 424354 had high levels of resistance to P. irregulare. Two BC1F2:3 populations were used to map the resistance including: 192 lines of OHS 303 (moderately susceptible) x (Williams (susceptible) x PI 424354) and 127 lines of Dennison (moderately susceptible) x (Williams x PI 424354). Both populations fit the model for quantitative resistance based on root weight and root rot score. Putative QTL were identified on chromosomes 1, 5, 6, 8, 10, 11, 13, 14, and 20. These results suggest PI 424354 can be an important source of partial resistance in developing germplasm for breeding new cultivars with more durable resistance to P. irregulare. iii Dedication I would like to dedicate this dissertation to my loving family, especially my father Mark Lee Ellis, mother Deborah Lynn Ellis, and my sister Elizabeth Stewart Ellis. They have been a great source of love, inspiration, and guidance throughout my life. I would also like to dedicate this dissertation to all my grandmothers who were/are remarkable women: Carol Epstein, Berenice Ellis, Dotty Appelbaum, and Ruby Bond. iv Acknowledgments I would like to thank my advisors Dr. Anne Dorrance and Dr. Pierce Paul for their constant support and guidance for the research presented in this dissertation. They have both helped me to develop both personally and professionally during my time at The Ohio State University. My knowledge on the Pythium can greatly be attributed to Dr. Dorrance and all the tools she provided me to study this amazing genus of plant pathogenic fungi. Dr. Paul’s statistical expertise has contributed greatly to this work and also my own personal knowledge. I would also like to thank my committee member Dr. Thomas Mitchell and Dr. Rouf Mian for their contributions and support. I had the privilege of being a teaching assistant for Dr. Mitchell’s mycology class, which was a great experience and helped me to confirm my own passion for teaching, and Dr. Mian, provided expertise in plant breeding. Dr. Leah McHale and Dr. Steven St. Martin contributed greatly to the last two chapters in my dissertation. Dr. St. Martin developed all of the populations used in this research. Thanks to all the members of the Soybean Pathology and Cereal Pathology Labs. Maria Ortega, Grant Austin, Sean Dawes, SelyAnn Headley, Michelle LaLonde, Colton Zody, Nikki Berry, Damitha Wickramasinghe, David Salgado, Wirat Pipatpongpinyo, Soledad Benitez, Lily Zelaya, Andika Gunadi, Freddy Cruz, Christian v Cruz, Matt Wallhead, Kate Gearhart, Chandra Phelan, Crystal Van Pelt, Zhifen Zhang, Sungwoo Lee, Alissa Kriss, and Kylea Odenbach for their help and friendships. A special acknowledgement to Kirk Borders, Hehe Wang, and Sue Ann Berry for their many contributions towards this research. This research was funded in part by the Ohio Soybean Council, OARDC SEEDS Grant graduate research competition, and Syngenta Crop Protection. vi Vita June 1980 .......................................................Born – Rockford, Illinois, USA 2003................................................................B.S. Biology, University of Illinois Urbana- Champaign 2007................................................................M.S. Plant Pathology, Michigan State University 2007 to present ..............................................Graduate Research Associate, Department of Plant Pathology, The Ohio State University Publications Ellis ML, Broders KD, Paul PA, Dorrance AE. 2011. Infection of soybean seed by Fusarium graminearum and effect of seed treatments on disease under controlled conditions. Plant Disease. 95:401-407. Zelaya-Molina, LX, Ellis, ML, Berry, SA, Dorrance, AE. 2010. First report of Phytophthora sansomeana causing wilting and stunting on corn in Ohio. Plant Disease. 94: 125. vii Broders, KD, Lipps PE, Ellis ML, Dorrance AE. 2009. Pythium delawarii-a new species isolated from soybean in Ohio. Mycologia. 101: 232-238. Ellis, ML. 2007. Distribution, identification, and population diversity of Armillaria spp. in Michigan cherry orchards. Thesis. Michigan State University. East Lansing, MI, USA. Fields of Study Major Field: Plant Pathology viii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgements ............................................................................................................. v Vita .................................................................................................................................... vii List of Tables ................................................................................................................... xiii List of Figures .................................................................................................................. xix Chapter Page 1. Introduction. ............................................................................................................. 1 2. Infection of soybean seed by Fusarium graminearum and effect of seed treatments on disease under controlled conditions ............................................................................. 32 Introduction ............................................................................................... 33 Objectives ................................................................................................. 36 Material and Methods ............................................................................... 37 Results ....................................................................................................... 42 Discussion ................................................................................................. 44 Acknowledgements .................................................................................. 48 ix List of References ..................................................................................... 59 3. Two new species of Pythium, P. schmitthenneri and P. selbyi pathogen of corn and soybean in Ohio ................................................................................................................ 63 Introduction ............................................................................................... 64 Material and Methods ............................................................................... 67 Results ......................................................................................................