Oomycete Community Diversity and Pathogenicity Associated with Soybean in Ohio DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Krystel A. Navarro-Acevedo Graduate Program in Plant Pathology The Ohio State University 2019 Dissertation Committee: Dr. Anne E. Dorrance, Advisor Dr. Jason Slot, Member Dr. Maria Soledad Benitez-Ponce, Member Dr. Feng Qu, Member Dr. Andrew Michel, Member Copyrighted by Krystel A. Navarro-Acevedo 2019 Abstract The oomycetes, Phytophthora (Ph.), Phytopythium (Pp.), and Pythium (Py.) cause detrimental effects to soybean yields when susceptible cultivars are planted, and conducive conditions occur. Soils with high clay content found in many soybean production areas of Ohio paired with heavy rainfall, allows for water retention for longer periods of time. This enables the germination of oospores and mobility of zoospores towards soybean roots. Subsequently, infections occur and early symptoms include pre-and post- emergence damping-off of soybean seed and seedlings. In addition, mid-season infections lead to root rots but one species, Phytophthora sojae causes stem rot which results in plant death. Within the genus Phytophthora, only Ph. sojae and Ph. sansomeana have been recovered from soybean seedlings and reported as pathogens. In contrast, a vast diversity of Pythium species have been recovered from soybean seedlings across major soybean producing states in the U.S. and Canada. Pythium sylvaticum, Py. irregulare, Py. ultimum var. ultimum, Py. ultimum var. sporangiiferum and Py. heterothallicum are among the most frequently recovered species. More importantly, more than one species has been recovered from symptomatic seedlings or roots, suggesting that these occur as species complexes. To better manage this root pathogen complex, an integrated disease management approach is recommended. This includes a combination of fungicide seed treatments paired with host resistance. Fungicide efficacy is variable among species and host resistance has shown to be the best means of management in regions with high levels of inoculum and conducive environments. The distribution and species diversity of Phytophthora, ii Phytopythium and Pythium among soybean and corn producing states have been documented in several previous surveys that used different isolation techniques. These surveys suggested that soil edaphic factors may play a role in species distribution and diversity. However, how community composition and species diversity change when soil edaphic factors remain constant was unexplored. Thus, the first objective of this study was to test the effects of temperature and agronomic practices on the community composition and species diversity of Phytophthora, Phytopythium and Pythium. A soil baiting technique was used to identify the pathogen complex in soils from five fields with different rotation schemes and tillage practices but with similar soil edaphic factors. Soils were incubated at 15 and 25oC and seed of the susceptible cultivar Sloan was used as bait. Symptomatic seedlings were collected for direct isolation of pathogens onto oomycete selective media. Additionally, rhizosphere soil was collected for metabarcoding approach in which DNA was extracted, followed by amplification with oomycete primers of the ITS1 region and sequencing. Regardless of temperature or agronomic practice, Py. sylvaticum, and Py. ultimum were isolated through the baiting procedure and these along with Py. acrogynum, Py. attrantheridium, and Py. heterothallicum were detected with amplicon sequencing at all temperatures and all fields and were considered as the core species associated with soybean. More importantly, there were distinct communities between fields with different agronomic practices. Pythium arrhenomanes and Py. inflatum, were found in greater abundance in soil from the field under continuous corn while Ph. sojae was higher in soils from fields planted to a soybean corn rotation. There were also differences in community composition due to temperature, six Pythium species were abundant at 15oC while another iii six were favored by 25oC. These findings demonstrated that the diversity of Phytophthora, Phytopythium, and Pythium, associated with soybean roots can be influenced by temperature and potentially production practices which may explain in part the differences in species composition among the many surveys that have taken place as well as the importance previous production practices may be having on the community composition within a field. The second objective was to evaluate if soybean genotype could impact the abundance of Phytophthora, Phytopythium, and Pythium. Here, three cultivars (Kottman, Lorain, and Sloan) with different levels and types of resistance towards these three genera were planted across eleven environments with high disease pressure in Ohio over a two- year period. At soybean growth stage V1-V3, seedlings from each environment were collected for direct isolation, and rhizosphere soil used for a metabarcoding approach. In addition, data for early plant population and yield was collected from each environment to determine the performance of the cultivars under natural field conditions. Based on both a metabarcoding approach and direct isolation, the environment played a significant role on the Phytophthora, Phytopythium, and Pythium communities and these were also influenced by different soybean growth stages. In addition, cultivar significantly affected the abundance of Phytophthora species in the rhizosphere of soybean seedlings. Here the greater number of Ph. sojae reads were recovered from rhizosphere soil and symptomatic seedlings of the moderately susceptible cultivar Sloan compared to Kottman with race- specific resistance towards Ph. sojae. Two species, Py. periilum and Pythium sp. CAL, not reported previously in Ohio, were detected in the soybean rhizosphere using a iv metabarcoding approach. Pythium periilum was only detected in the rhizosphere while the undescribed Pythium sp. CAL. was only recovered from seedlings retrieved from one environment although it was detected in 9 environments. When tested against different soybean cultivars this species was highly pathogenic and optimal growth was approximately 25oC. In culture, oospores and sporangia were observed however, zoospores were not produced. This species was highly abundant across soybean fields in Ohio and further studies should be conducted to classify this species among the Pythium clades. Furthermore, Py. periilum was not isolated from seedlings suggesting that temperature during isolation or type of media should be modified to recover this species from the field. These results provide evidence that disease, when observed in the field, is often caused by more than one pathogen. In addition, the effect of cultivar was observed for Ph. sojae providing further evidence that host resistance is still an effective management strategy in environments conducive for disease development. These results will also allow for the development of targeted disease management approaches for fields in which conducive conditions for disease development are often encountered. Finally, species of Phytophthora, Phytopythium, and Pythium should be continuously monitored since populations can change and novel species can emerge that can be detrimental to soybean production. v Dedication Dedicate to my family; my father Victor Navarro and my mother Carmen Acevedo for their support throughout my life and academic career. My grandmother Ana M. Villanueva for her never-ending love and advice. To my brother Yarom Navarro and sister Surhail Navarro for their mentorship and love. I would also dedicate this thesis to my husband Alexander Orellana for his support and understanding in this journey from Puerto Rico to Ohio. To my mother in law Mercedes Sanchez for her unconditional support and willingness to help when most needed. And to my lovely daughter Alejandra K. Orellana that came to this world in 2016, this work is for you. Thank you all for believing in me. vi Acknowledgments I would like to first thank my advisors Dr. Anne E. Dorrance for her support and opportunity to develop my research skills. Also, for her advises, guidance and encouragement to become a better professional and person. To Dr. Jason Slot and Dr. Maria Soledad Benitez-Ponce for their guidance and for providing critical comments and suggestions to my research work. To Dr. Andrew Michel, Dr. Feng Qu for the open-door policy always encouraged and for guidance and advise. I will also like to thank the present and past members of the Dorrance lab: Jonell Winger, Amilcar Vargas, Kelsey Scott, Linda Hebb, Felipe Sartori and Carlos Bolanos. To all thanks for your constant support, technical assistance and knowledge taught. Special thanks to Deloris Veney for the many technical guidance, advises and for making this journey enjoyable. To my friends Saranga Wijeratne, Jaqueline Huzar Novakowiski, Cassidy Gedling, Francesca Rotondo and Amine Batnini for their constant support, friendship, brainstorming talks, advice, and for always pushing me forward in the process. Finally, to my husband for always believing in me and for your unconditional love. Salaries and research support were provided in part by State and Federal Funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. This project was funded in part by the Ohio’s Soybean Producers’