PARASITIC FITNESS OF SDHI-SENSITIVE AND -RESISTANT ISOLATES OF ALTERNARIA SOLANI A Dissertation Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Mitchell James Bauske In Partial Fulfillment of the Requirements for the Degree of DOCTOR OF PHILOSOPHY Major Department: Plant Pathology April 2017 Fargo, North Dakota North Dakota State University Graduate School Title Parasitic Fitness of SDHI-Sensitive and -Resistant Isolates of Alternaria solani By Mitchell James Bauske The Supervisory Committee certifies that this disquisition complies with North Dakota State University’s regulations and meets the accepted standards for the degree of DOCTOR OF PHILOSOPHY SUPERVISORY COMMITTEE: Dr. Neil C. Gudmestad Chair Dr. Gary A. Secor Dr. Julie S. Pasche Dr. Andrew P. Robinson Dr. Kirk A. Howatt Approved: April 28, 2017 Dr. Jack Rasmussen Date Department Chair ABSTRACT Early blight of potato (Solanum tuberosum L.) is caused by Alternaria solani Sorauer. The single-site mode of action chemistries of the succinate dehydrogenase inhibitors (SDHIs) and quinone outside inhibitors (QoIs) have been widely used for early blight control but resistance has developed rapidly to a number of fungicide chemistries. QoI resistance in A. solani has been attributed to the F129L mutation, or the substitution of phenylalanine with leucine at position 129. Resistance to SDHI fungicides in A. solani is conferred by five known point mutations on three AsSdh genes. Over 1,300 A. solani isolates collected from 11 states in 2013 through 2015 were characterized for the presence of mutations associated with QoI and SDHI resistance through real- time, SDH multiplex, and mismatch amplification analysis (MAMA) polymerase chain reaction (PCR). Approximately 95% of isolates collected from 2013 to 2015 possessed the F129L mutation. Additionally, 95% of the A. solani population was determined to be SDHI-resistant, with the most prevalent mutation being on the AsSdhC gene. All A. solani isolates collected that were characterized as possessing the D123E mutation, or the substitution of aspartic acid for glutamic acid at position 123 in the AsSdhD gene, were evaluated for boscalid and fluopyram sensitivity in vitro. Furthermore, 15 isolates characterized as being SDHI-sensitive or -resistant, including five D123E-mutant isolates, were evaluated in vivo for percentage disease control to boscalid and fluopyram. Sensitivity of D123E-mutant isolates to fluopyram ranged from 0.8 to 3 µg/ml, and were found to be sensitive or only slightly higher than those of baseline isolates, ranging from 0.1 to 0.6 µg/ml. Disease control of all five D123E isolates evaluated in vivo was reduced significantly with the application of fluopyram compared to SDHI-sensitive isolates. Fitness was compared among 120 SDHI-sensitive and -resistant A. solani isolates using the parameters of spore germination and mycelial growth in vitro and aggressiveness in vivo. Spore iii germination and mycelial growth between SDHI-sensitive and -resistant isolates was not significantly different. However, D123E-mutant isolates were significantly more aggressive in in vivo assays compared to other SDHI-resistant and SDHI-sensitive isolates. These results illustrate the importance of implementing fungicide resistance management strategies. iv ACKNOWLEDGEMENTS First and foremost, I would like to extend a sincere thank you to my major professor, Dr. Neil C. Gudmestad, for his guidance, support, advice, and patience throughout this graduate program and the completion of this dissertation. Your leadership over the last four years has allowed me to achieve beyond what I thought possible. I will be forever grateful for the years spent learning as your student. I would also like to thank Dr. Gary A. Secor, Dr. Julie S. Pasche, Dr. Andrew P. Robinson, and Dr. Kirk A. Howatt for their valuable assistance and insight as members of my graduate committee. Acknowledgement is also extended to Bayer CropScience and Syngenta for financial assistance and cooperation during the course of this research. Special thanks are given to multiple past and present members of the Neil C. Gudmestad Laboratory: Dr. Shashi Yellareddygari, Dr. Raymond J. Taylor, and Dr. Francisco Bittara Molina for your mentorship, friendship, and countless hours of assistance. Ipsita Mallik, Dean Peterson, and Russel Benz for your expertise, technical assistance, and endless support. Dimitri Lakshan Fonseka, Dr. Owusu Domfeh and Sarah Budde for your friendship and lending a helping hand in the completion of this research. Appreciation also goes to Dr. Sam Markell and Dr. Andrew Friskop for your mentorship, advice, and constructive criticism over the years of this graduate program. Finally, above all, I wish to express a very special thanks to my wife, Elizabeth, for the inspiration to complete this dissertation and degree. v TABLE OF CONTENTS ABSTRACT ..................................................................................................................................... iii ACKNOWLEDGEMENTS ............................................................................................................. .v LIST OF TABLES ............................................................................................................................ x LIST OF FIGURES ......................................................................................................................... xi LIST OF APPENDIX TABLES .................................................................................................... xiii INTRODUCTION AND LITERATURE REVIEW ........................................................................ 1 Introduction ........................................................................................................................... 1 Pathogen Introduction ........................................................................................................... 2 Disease Cycle and Symptomatology .................................................................................... 3 Disease Management ............................................................................................................ 4 Host resistance .......................................................................................................... 4 Cultural practices ...................................................................................................... 5 Chemical control ....................................................................................................... 6 SDHI Fungicides. .................................................................................................................. 7 Parasitic Fitness .................................................................................................................... 9 Literature Cited ................................................................................................................... 19 CHAPTER 1. SPATIAL AND TEMPORAL FREQUENCY DISTRIBUTION OF MUTATIONS CONFERRING QOI AND SDHI RESISTANCE IN ALTERNARIA SOLANI ACROSS THE UNITED STATES. ................................................................................. 23 Abstract ............................................................................................................................... 23 Introduction. ........................................................................................................................ 24 Materials and Methods ........................................................................................................ 27 A. solani isolate collection. ..................................................................................... 27 DNA extraction ....................................................................................................... 28 Detection of the F129L mutation ............................................................................ 29 vi Molecular detection of SDHI-resistant isolates. ..................................................... 30 Genotype characterization. ..................................................................................... 32 Statistical analysis. .................................................................................................. 32 Results. ................................................................................................................................ 33 Determination of overall prevalence of mutations conferring SDHI and QoI resistance. ................................................................................................................ 33 Determination of temporal frequency distribution of mutations conferring QoI and SDHI resistance in A. solani ..................................................................... 35 Determination of spatial frequency distribution of mutations conferring QoI and SDHI resistance in A. solani ............................................................................ 42 Prevalence of genotypes I and II in a diverse population of A. solani across the United States ..................................................................................................... 44 Discussion ........................................................................................................................... 45 Literature Cited. .................................................................................................................. 51 CHAPTER 2. POTENTIAL