DOWNY MILDEW OF SUNFLOWERS: ESTABLISHMENT OF BASELINE SENSITIVITY TO AZOXYSTROBIN AND MONITORING FOR THE DEVELOPMENT OF FUNGICIDE RESISTANCE AND PLASMOPARA HALSTEDII VIRULENCE PHENOTYPE CHANGES A Thesis Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Michelle Arbogast Gilley In Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE Major Department: Plant Pathology May 2017 Fargo, North Dakota North Dakota State University Graduate School Title Downy mildew of sunflowers: Establishment of baseline sensitivity to azoxystrobin and monitoring for the development of fungicide resistance and Plasmopara halstedii virulence phenotype changes By Michelle Arbogast Gilley The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of MASTER OF SCIENCE SUPERVISORY COMMITTEE: Dr. Samuel Markell Chair Dr. Julie Pasche Dr. Herman Kandel Dr. William Underwood Approved: 5/1/2017 Dr. Jack Rasmussen Date Department Chair ABSTRACT Downy mildew, caused by Plasmopara halstedii (Farl.) Berl. and de Toni, is an economically important disease in cultivated sunflowers, Helianthus annuus L. The objectives of this study were to determine disease pressure in North Dakota and South Dakota, determine the virulence phenotypes in the pathogen population, determine the baseline sensitivity to azoxystrobin and evaluate select isolates for fungicide insensitivity. While downy mildew was present in many fields, incidence was typically low. To determine virulence phenotypes, selected isolates were evaluated on an expanded set of differential lines. New virulence was found to the Pl8 resistance gene, but no virulence was observed on the PlArg, Pl15, Pl17 and Pl18 genes. Using a discriminatory dose of 10 ug ai azoxystrobin/seed, no isolate approached infection levels found in inoculated, nontreated controls; therefore, the pathogen is considered sensitive to azoxystrobin in the greenhouse and azoxystrobin should still suppress downy mildew in the field. iii ACKNOWLEDGEMENTS I would like to thank and acknowledge all of the special people who have helped me complete my graduate studies. I would like to express my gratitude to my major advisor, Dr. Sam Markell, for his patience, his guidance, his encouragement, his time and his insight. I have learned so much from him. Thank you for making sure I had time and resources to work on my research and thesis. I would like to thank Dr. Tom Gulya for getting me started with sunflowers, for providing me with pathogen isolates, research materials and advice throughout my thesis. Your enthusiasm for this project was contagious. I would like to express my sincere thanks to my committee members: Dr. Julie Pasche for helping me with project design, statistics and greenhouse space; Dr. Hans Kandel for sharing data with me and supporting my project and Dr. William Underwood for giving me flexibility in my work schedule, encouragement, advice and greenhouse and laboratory space. I would especially like to thank Chris Misar at USDA-ARS for his advice and help and for always being willing to look at downy mildew infected sunflower plants with me. I would like to thank Dr. Gerald Seiler for his support and interest in my project. A special thanks to Ryan Humann for sharing his tips on various classes and for sharing his knowledge of working with Plasmopara halstedii and fungicides and to Jessica Halvorson for answering so many questions and encouraging me during the writing of my thesis. I would like to thank Scott Meyer, Brandt Berghuis, Morgan Bruhn, Ian Gilley, Bryan Hansen, Isaac Jensen, Jacob Lardy, Tristan Marette, Presley Mosher and Trent Thomasson for all their cheerful assistance in the greenhouse and the laboratory. I want to thank Jan Knodel for sharing her iv survey knowledge. I would like to thank Shashi Kiran Reddy, Dr. Luis del Rio, Sarah Budde, Claudia Munoz and Mitch Bauske for encouragement and help with statistics. I want to thank the funding sources that made this project possible: National Sunflower Association, NDSU Agriculture Experiment Station and NDSU Extension Service. Finally, my deepest gratitude is extended to my husband, my sons and my extended family for their support and encouragement of my graduate studies. Thank you to my husband for many evenings spent helping in the greenhouse and laboratory. Knowing my family was proud of me made everything possible. v TABLE OF CONTENTS ABSTRACT ................................................................................................................................... iii ACKNOWLEDGEMENTS ........................................................................................................... iv LIST OF TABLES ......................................................................................................................... ix LIST OF FIGURES ........................................................................................................................ x LIST OF APPENDIX TABLES ................................................................................................... xii LITERATURE REVIEW ............................................................................................................... 1 Introduction: Importance ............................................................................................................ 1 Sunflower .................................................................................................................................... 2 Origin and History ................................................................................................................... 2 Sunflower Production in the United States ............................................................................. 3 Plasmopara halstedii – Downy Mildew ..................................................................................... 4 Taxonomy ................................................................................................................................ 4 Host Range .............................................................................................................................. 4 Life Cycle ................................................................................................................................ 5 Symptoms and Signs ............................................................................................................... 6 Epidemiology .......................................................................................................................... 7 Management of Downy Mildew ................................................................................................. 8 Genetic Resistance................................................................................................................... 8 Virulence Phenotypes ............................................................................................................ 11 Fungicidal Seed Treatments .................................................................................................. 14 Literature Cited ......................................................................................................................... 17 CHAPTER 1. ASSESSMENT OF DISTRIBUTION, INCIDENCE AND PREVALENCE OF DOWNY MILDEW IN THE NORTH CENTRAL GREAT PLAINS ....... 28 Introduction ............................................................................................................................... 28 vi Materials and Methods .............................................................................................................. 30 Results ....................................................................................................................................... 31 Discussion ................................................................................................................................. 42 Literature Cited ......................................................................................................................... 47 CHAPTER 2. DETERMINATION OF VIRULENCE PHENOTYPES OF PLASMOPARA HALSTEDII IN THE NORTH CENTRAL UNITED STATES ......................... 49 Introduction ............................................................................................................................... 49 Materials and Methods .............................................................................................................. 55 Pathogen Material and Increase from Field Collection ......................................................... 55 Pathogen Increase from Storage ............................................................................................ 57 Determination of Virulence Phenotypes ............................................................................... 58 Results ....................................................................................................................................... 61 Isolates ................................................................................................................................... 61 Standard Set of Differential Lines ......................................................................................... 62 Proposed INRA Lines............................................................................................................ 64 Supplemental Differential Lines...........................................................................................