ABSTRACT REEVES, ELLA ROBYN. Pythium spp. Associated with Root Rot and Stunting of Winter Field and Cover Crops in North Carolina. (Under the direction of Dr. Barbara Shew and Dr. Jim Kerns). Soft red winter wheat (Triticum aestivum) was valued at over $66 million in North Carolina in 2019, but mild to severe stunting and root rot limit yields in the Coastal Plain region during years with above-average rainfall. Pythium irregulare, P. vanterpoolii, and P. spinosum were previously identified as causal agents of stunting and root rot of winter wheat in this region. Annual double-crop rotation systems that incorporate winter wheat, or other winter crops such as clary sage, rapeseed, or a cover crop are common in the Coastal Plain of North Carolina. Stunting and root rot reduce yields of clary sage, and limit stand establishment and biomass accumulation of other winter crops in wet soils, but the role that Pythium spp. play in root rot of these crops is not understood, To investigate species prevalence, isolates of Pythium were collected from stunted winter wheat, clary sage, rye, rapeseed, and winter pea plants collected in eastern North Carolina during the growing season of 2018-2019, and from all crops except winter wheat again in 2019-2020. A total of 534 isolates were identified from all hosts. P. irregulare (32%), P. vanterpoolii (17%), and P. spinosum (16%) were the species most frequently recovered from wheat. P. irregulare (37% of all isolates) and members of the species complex Pythium sp. cluster B2A (28% of all isolates) comprised the majority of isolates collected from clary sage, rye, rapeseed, and winter pea. In experiments on winter wheat, highly aggressive isolates of P. irregulare, P. vanterpoolii, and P. spinosum at 14°C were identified. In vitro growth of these isolates was measured at 14°C and 20°C, and all isolates grew faster at the higher temperature. Experiments also investigated the influence of environment (3 nitrogen levels x 2 temperatures) and pathogen species (P. irregulare, P. spinosum, or P. irregulare + P. spinosum) on disease severity, plant height, root length, and biomass. All inoculation treatments caused severe root rot under all conditions tested, and disease was more severe at 12/14°C than at 18/20°C, but there was no effect of nitrogen application. In experiments on clary sage, P. irregulare and P. spinosum were aggressive pathogens at 18°C and caused moderate root necrosis at 28°C. Isolates representing Pythium sp. cluster B2A caused slight to moderate root necrosis on rapeseed and clary sage at 18°C, but no symptoms on rye or winter pea. Cultivation of winter wheat, clary sage, or rye may maintain or increase populations of P. irregulare and P. spinosum in crop rotation systems, whereas cultivation of clary sage, rye, rapeseed, or winter pea may maintain or increase populations of P. irregulare and Pythium sp. cluster B2A. P. irregulare and P. spinosum were aggressive pathogens of wheat and clary sage at temperatures ranging from 14°C to 18°C, which may explain their frequent recovery from multiple hosts during the winter. © Copyright 2020 by Ella Reeves All Rights Reserved Pythium spp. Associated with Root Rot and Stunting of Winter Field and Cover Crops in North Carolina by Ella Robyn Reeves A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the degree of Master of Science Plant Pathology Raleigh, North Carolina 2020 APPROVED BY: ____________________________ ____________________________ Dr. Barbara Shew Dr. Jim Kerns Committee Chair Committee co-chair ____________________________ ____________________________ Dr. Christina Cowger Dr. Lindsey Thiessen DEDICATION I am dedicating this work to my father, Jeff Reeves. He taught me to pay attention to the details of the world around me, and without his love and guidance I would not be where I am today. ii BIOGRAPHY Ella Robyn Reeves grew up on her family’s farm in Pulaski County, Virginia. Spending time outdoors with animals and plants sparked her interested in the natural sciences, which led her to study plant science at Virginia Tech. There, she obtained two degrees; one in biological sciences and one in environmental horticulture. While at Virginia Tech, Ella got a job in the Virginia Tech Plant Disease Clinic with Mary Ann Hansen and Elizabeth Bush, who inspired her to pursue a career in plant pathology. She accepted an assistantship at NC State University to pursue a M.S. in Plant Pathology under the guidance of Dr. Barbara Shew, which she began in the summer of 2018. iii ACKNOWLEDGEMENTS I have many people to thank but would like to begin by thanking Dr. Barbara Shew for being a wonderful graduate advisor, role model, and overall person. I have learned much under her guidance and am very grateful for my time working with her. I also thank my committee co- chair, Dr. Jim Kerns, for his guidance and support over the last two years. The knowledge, patience, and advice of my committee members, Dr. Christina Cowger and Dr. Lindsey Thiessen, have been integral to the completion of this work, and I have learned much during my time working with them. Multiple extension specialists, agents, and growers helped tremendously with this work and have been so kind while doing so. I would especially like to thank Dr. Rachel Vann and Richard Rhodes, and members of the Plant Disease and Insect Clinic for their help with sample collection. I have thoroughly enjoyed and gained much from my coursework at NC State, and would like to thank the multiple professors who have dedicated time to ensuring that students get a great education. I would especially like to thank Dr. Marc Cubeta for making PP 575 a fun and interesting course, and for being a great mentor. My fellow graduate students, including Alejandro Llanos Melo, Madison Stahr, and Johnny Balidion, have been great friends and mentors. Ian Mellon, Michael Elliot, and Christine Miller have been great lab mates and co-workers, and helped a lot with the completion of this project. I would especially like to thank Christine Miller for the many laughs shared while planting wheat in ~1,100 cone-tainers. I would also like to thank Mary Ann Hansen and Elizabeth Bush, who introduced me to the field of plant pathology and encouraged me to pursue graduate studies. Lastly, I would like to thank my family; Eli Reeves, Emma and Derek Brown, and Ann and Jeff Reeves. Their love and encouragement throughout my graduate career has meant so much. iv TABLE OF CONTENTS LIST OF TABLES ........................................................................................................................ vi LIST OF FIGURES ..................................................................................................................... vii CHAPTER 1: LITERATURE REVIEW ..................................................................................... 1 REFERENCES .................................................................................................................. 9 CHAPTER 2: Pythium spp. associated with root rot and stunting of winter wheat in North Carolina ........................................................................................................................................ 15 ABSTRACT ..................................................................................................................... 15 INTRODUCTION ........................................................................................................... 16 MATERIALS AND METHODS ..................................................................................... 18 RESULTS ........................................................................................................................ 24 DISCUSSION .................................................................................................................. 28 ACKNOWLEDGEMENTS ............................................................................................. 32 REFERENCES ................................................................................................................ 33 CHAPTER 3: Pythium spp. associated with root rot and stunting of winter crops in North Carolina ........................................................................................................................................ 51 ABSTRACT ..................................................................................................................... 51 INTRODUCTION ........................................................................................................... 52 MATERIALS AND METHODS ..................................................................................... 54 RESULTS ........................................................................................................................ 58 DISCUSSION .................................................................................................................. 61 ACKNOWLEDGEMENTS ............................................................................................. 65 REFERENCES ................................................................................................................ 66 v LIST OF TABLES Table 2.1 Species of Pythium isolated from wheat plants exhibiting symptoms of Pythium root rot in eight counties in North Carolina during the winter of 2018-2019 .......... 38 Table 2.2 Sequences of the ITS or COI barcode submitted to GenBank (National Center for Biotechnology Information)