CHARACTERIZATION OF SENSITIVITY OF SCLEROTINIA SCLEROTIORUM ISOLATES FROM NORTH CENTRAL US TO THIOPHANATE-METHYL AND METCONAZOLE A Thesis Submitted to the Graduate Faculty of the North Dakota State University of Agriculture and Applied Science By Gazala Ameen In Partial Fulfillment for the Degree of MASTER OF SCIENCE Major Department: Plant Pathology May 2013 Fargo, North Dakota North Dakota State University Graduate School Title CHARACTERIZATION OF SENSITIVITY OF SCLEROTINIA SCLEROTIORUM ISOLATES FROM NORTH CENTRAL US TO THIOPHANATE-METHYL AND METCONAZOLE By Gazala Ameen 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. Luis E. del Rio-Mendoza Chair Dr. Berlin D. Nelson Dr. Mohamed F. R. Khan Dr. Juan M. Osorno Approved: 05-28-2013 Dr. Jack B. Rasmussen Date Department Chair ABSTRACT Sclerotinia sclerotiorum (Lib.) de Bary causes Sclerotinia stem rot on canola and many other crops of economic importance in the U.S. SSR is primarily controlled with fungicides applied at flowering time. Most fungicides currently used to control SSR can promote resistance buildup in their target populations making monitoring of sensitivity important. In this study the reaction of S. sclerotiorum to thiophanate-methyl (TM) and metconazole (MTZ) was characterized. Samples collected in several states of north central U.S. were used. Three and ten isolates were considered to be moderately insensitive to TM and MTZ, respectively. Greenhouse trials indicated, however, that diseases caused by these isolates could be effectively controlled using currently recommended doses of each compound. In vitro sensitivity to TM was temperature dependent. A previously unreported mutation at codon E111D in the β-tubulin gene of a TM-moderately insensitive isolate was identified. iii ACKNOWLEDGMENTS I would like to express my deep and sincere gratitude to my major advisor Dr. Luis E. del Rio Mendoza, under whose excellent supervision, logical thinking, constructive criticism and support have been of great value for me. His guidance, understanding and encouragement have given a good basis for not only research but also for present thesis. I was blessed with a great opportunity of working with him and learning different aspects of plant pathology and disease control. I owe my sincere thanks to my co-advisor Dr. Berlin D. Nelson, who not only provided research material used in this thesis but also for guidance me throughout the research. I extend my gratitude to Dr. Mohamed Khan and Dr. Juan M. Osorno for time and effort throughout the preparation of this thesis. I want to thank Dr. Kishore Chittem for his help with analysis and effort throughout the research and thesis. I would like to extend thanks to the funding agency, National Sclerotinia Initiative. iv DEDICATION This thesis is dedicated to my parents Mr. V.T. Haneef and Mrs. S. Bano who believed in me and supported my every decision and encouraged me to be a better person every day. This thesis is also dedicated to would be husband, Shyam Solanki, who has inspired, encouraged and motivated me to succeed. Last but not the least, this thesis is dedicated to all who have love of learning. v TABLE OF CONTENTS ABSTRACT… ............................................................................................................................... iii ACKNOWLEDGMENTS ............................................................................................................. iv DEDICATION ................................................................................................................................ v LIST OF TABLES .......................................................................................................................... x LIST OF FIGURES ...................................................................................................................... xii CHAPTER 1. LITERATURE REVIEW ........................................................................................ 1 Introduction ................................................................................................................................ 1 Canola .................................................................................................................................... 1 Sclerotinia stem rot ................................................................................................................ 1 Taxonomy of the pathogen .................................................................................................... 2 Pathogen biology and disease cycle ........................................................................................... 4 Disease cycle ......................................................................................................................... 4 Signs and symptoms of sclerotinia stem rot .......................................................................... 5 Disease management .................................................................................................................. 5 Chemical control ................................................................................................................... 6 Fungicide resistance ................................................................................................................... 7 Fungicide description and mechanism of resistance ............................................................. 8 Resistance to thiophanate-methyl ..................................................................................... 8 Resistance to metconazole .............................................................................................. 10 Literature cited ......................................................................................................................... 11 CHAPTER 2. CHARACTERIZATION OF SENSITIVITY OF SCLEROTINIA SCLEROTIORUM ISOLATES FROM NORTH CENTRAL U.S. TO THIOPHANATE- METHYL AND METCONAZOLE ............................................................................................. 18 Abstract .................................................................................................................................... 18 vi Introduction .............................................................................................................................. 18 Thiophanate-methyl and problem of resistance .................................................................. 20 Metconazole and problem of resistance .............................................................................. 21 Materials and methods ............................................................................................................. 22 Isolates ................................................................................................................................. 22 Fungicide concentrations ..................................................................................................... 23 Thiophanate-methyl ........................................................................................................ 23 Metconazole ................................................................................................................... 24 Data analyses ....................................................................................................................... 24 Results ...................................................................................................................................... 25 Thiophanate-methyl ............................................................................................................ 25 Metconazole ........................................................................................................................ 28 Discussion ................................................................................................................................ 31 Literature cited ......................................................................................................................... 34 CHAPTER 3. EFFECT OF ENHANCED INSENSITIVITY OF SCLEROTINIA SCLEROTIORUM ISOLATES TO THIOPHANATE-METHYL AND METCONAZOLE ON EFFICACY OF DISEASE CONTROL ................................................................................. 37 Abstract .................................................................................................................................... 37 Introduction .............................................................................................................................. 37 Material and methods ............................................................................................................... 41 Plant materials ..................................................................................................................... 42 Inoculum production ........................................................................................................... 42 Thiophanate-methyl ........................................................................................................ 42 Metconazole ................................................................................................................... 43 Inoculation and spraying procedures ................................................................................... 44 vii Data collection and analyses