DETECTION OF BOSCALID RESISTANCE AND THE H272R MUTATION IN THE SDHB GENE OF BLUMERIELLA JAAPII By Cory Alan Outwater A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Plant Pathology – Master of Science 2014 ABSTRACT DETECTION OF BOSCALID RESISTANCE AND THE H272R MUTATION IN THE SDHB GENE OF BLUMERIELLA JAAPII By Cory Alan Outwater Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii , is a major disease of tart cherry (Prunus cerasus ) trees, which uncontrolled leads to early defoliation. Pristine, a commonly-utilized fungicide for CLS management in Michigan, is a premix of boscalid, a succinate dehydrogenase inhibitor, and pyraclostrobin, a quinone outside inhibitor. Reduced efficacy of Pristine for CLS control observed in field trials and commercial orchards highlighted the importance of resistance monitoring. A total of 1,288 isolates from commercial orchards and 111 isolates from non-treated trees were collected in 2010 and 2011 and assayed on boscalid-amended media at concentrations of -1 0, 0.1, 0.5, 1, 2.5, 5, 10, and 25 µg ml . The minimum inhibitory concentration (MIC) of boscalid was determined after incubation at 23 ºC for 14 days . Isolates from non- -1 treated trees had MIC values ranging from 0.1 to 0.5 µg ml while isolates from -1 commercial orchards ranged from 0.1 to > 25 µg ml . Isolates with MIC values of > 25 -1 µg ml were considered resistant and comprised 22% and 35% of isolates in 2010 and 2011 respectively. Sequencing of the SDHB gene of resistant isolates led to the detection of the amino acid mutation H272R known to confer boscalid resistance. The occurrence of the H272R mutation in Michigan populations of B. jaapii is correlated with the reduction in sensitivity to boscalid observed in commercial orchards. To my grandpa Norman Schaub iii ACKNOWLEDGEMENTS I would like to thank Dr. George Sundin for giving me the opportunity to work in his laboratory and for his guidance through the process of obaining my master’s degree. Thank you Janette Jacobs for working with me during my undergraduate years and teaching me a wealth of techniques and skills that have proven extremely valuable through the years. Thank you to Dr. Tyre Proffer for guiding me through the world of fungi and for teaching me all the ins and outs of what has became one of my favorite fungal species, Blumeriella jaapii . Thank you to Kim Lesniak for inspiring me to go after a degree in plant pathology, for being there to mentor me through my graduate years, and finally for being a great friend and and source of support through this process. I would also like to thank Gayle McGhee, Gail Ehret and the entire Sundin lab for being an amazing group of people and for making this experience one that I will cherish for many years to come. I would like to thank the Michigan cherry growers and my funding sources including the Michigan Cherry Committee for allowing me to work on a very important disease affecting the Michigan cherry industry. iv TABLE OF CONTENTS LIST OF TABLES………………………………………….....…vii LIST OF FIGURES……………………………………………..viii LITERATURE REVIEW…………………………………………1 Introduction…………………………………………………………...1 Plant Pathogens Affecting the Michigan Tart Cherry Industry………5 Cherry Leaf Spot ( Blumeriella jaapii )………………………………..7 Development and Use of Plant Protection Materials in Fruit Production…………………………………………………………...16 The Development and Registration of Pristine……………………...20 Fungicides in the Age of Resistance………………………………...24 LITERATURE CITED……………………………………………...33 CHAPTER 1: DETECTION OF BOSCALID RESISTANCE AND THE H272R MUTATION IN THE SDHB GENE OF BLUMERIELLA JAAPII ………………………………………...45 Introduction………………………………………………………….45 Materials and Methods………………………………………………49 Fungicide efficacy trials……………………………………...49 Statistical analysis of field data………………………………50 B. jaapii isolate collection……………………………………50 In vitro sensitivity of B. jaapii to boscalid…………………...52 Amplification and identification of the succinate dehydrogenase subunit b gene ( SDHB ) of B. jaapii …………………………..53 Results……………………………………………………………….55 Efficacy of Pristine for control of cherry leaf spot in fungicide field trials at the NWMHRS 1999–2013……………………..55 Grouping of B. jaapii isolates into boscalid sensitivity groups………………………………………………………...57 Sensitivity of B. jaapii isolates to boscalid (2010)…………...59 Sensitivity of B. jaapii isolates to boscalid (2011)…………...62 v Amplification of the SDHB gene and molecular detection of the H272R mutation………………………………………………65 Discussion…………………………………………………………...69 LITERATURE CITED……………………………………………...76 APPENDIX………………………………………………………85 LITERATURE CITED…………………………………………….108 vi LIST OF TABLES Table 1. Codon and amino acid residue in the third cysteine-rich cluster of the SDHB gene of 33 B. jaapii isolates grouped by boscalid sensitivity groups. Amino acid residues were changed from a histidine to arginine for all resistant isolates sequenced………….68 vii LIST OF FIGURES Figure 1. Efficacy trial results of Pristine treatments for control of CLS. Pristine was evaluated as a full season treatment from 2001 to 2003 and 2009 to 2011. Pristine was evaluated following two applications of chlorothalonil at petal fall and shuck split in 2005 and 2012. Trials were conducted in an experimental orchard of Prunus cerasus cv . Montmorency located at the Northwest Michigan Horticulture Research Center near Suttons Bay, Michigan. CLS incidence and defoliation was assessed in late August or early September of each year. Defoliation data for 2005 was not determined due to low disease pressure. Leaf spot infection and defoliation is scaled as the disease incidence or defoliation divided by the disease incidence or defoliation on unsprayed controls. Scaled infection is represented by black bars and scaled defoliation is represented by hash marked bars………………………………………………………………………………56 Figure 2. Isolates plated on boscalid amended media at concentrations of 0.1, 0.5, 1, -1 2.5, 5, 10, and 25 µg ml . Each colony on plates within a row represents a separate B. jaapii isolate. There are eight different isolates in the sensitive group, ten isolates in the reduced sensitivity group and ten isolates in the resistant group. Minimum inhibitory concentrations were determined as the lowest concentration that completely inhibits mycelial growth following a 14 day incubation period. -1 a. Minimum inhibitory concentration (0.1 µg ml ) of an isolate classified as sensitive. -1 b. Minimum inhibitory concentration (5 µg ml ) of an isolate classified as reduced sensitive. -1 c. Minimum inhibitory concentration (>25 µg ml ) of an isolate classified as resistant..............................................................................................................................58 Figure 3. Boscalid sensitivity distribution of 2010 non-treated and commercial B. jaapii isolates. Baseline isolates are represented by white bars and commercial isolates are represented by black bars………………………………………………………………60 Figure 4. Distribution of 2010 Orchard or site sensitivity groups. Orchards or sites classified as sensitive had average Minimum Inhibitory Concentrations between 0.1 and -1 0.5 µg ml and are identified by white bars. Orchards or sites classified as reduced -1 sensitivity had average Minimum Inhibitory Concentrations between 1 and 10 µg ml and are identified by hatched bars. Orchards or sites classified as resistant had average viii -1 Minimum Inhibitory Concentrations between 25 and > 25 µg ml and are identified by black bars………………………………………………………………………………...61 Figure 5. Boscalid sensitivity distribution of 2011 non-treated and commercial B. jaapii isolates. Non-treated isolates are represented by white bars and commercial isolates are represented by black bars………………………………………………………………63 Figure 6. Distribution of 2011 Orchard or site sensitivity groups. Orchards or sites classified as sensitive had average Minimum Inhibitory Concentrations between 0.1 and -1 0.5 µg ml and are identified by white bars. Orchards or sites classified as reduced -1 sensitivity had average Minimum Inhibitory Concentrations between 1 and 10 µg ml and are identified by hatched bars. Orchards or sites classified as resistant had average -1 Minimum Inhibitory Concentrations between 25 and > 25 µg ml and are identified by black bars………………………………………………………………………………...64 Figure 7. PCR amplification of the SDHB gene from 7 B. jaapii isolates utilizing the primer pairs SDHB-FL and SDHB-R, SDHB-FS and SDHB-R, and SDHBFEL and SDHB-R. B. jaapii baseline isolates 10PEBJ-1, 3, 4, 9, 11 and B. jaapii boscalid resistant isolates 10BWPB-21, 22 were used for each primer pair. Lanes 1 and 17, DNA ladder; lanes 2, 10 and 19, water control; lanes 3-9, amplification with primer pair SDHB-FL and SDHB-R; lanes 11-16 and 18, amplification with primer pair SDHB-FS and SDHB-R; lanes 20-26, amplification with primer pair SDHBFEL and SDHB-R………………………………………………………………………………….66 Figure 8. Sequences obtained from the second and third cysteine-rich clusters of the SDHB gene of B. jaapii . Both cysteine-rich clusters (shaded areas) were amplified from sensitive, reduced sensitive and resistant isolates. Amino acids highlighted in red have been previously associated with boscalid resistance. No amino acid mutations were detected from the second cysteine-rich cluster from the three sensitivity groups. An amino acid mutation from histidine to arginine was detected for resistant isolates in the third cysteine rich cluster………………………………………………………………...67 Figure A1. Fluopyram