Environmental and cultural factors affecting the persistence and efficacy of fungicides on golf course turfgrass. By Paul Lawrence Koch A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Plant Pathology) at the UNIVERSITY OF WISCONSIN – MADISON 2012 Date of final oral examination: 7/26/2012 The dissertation is approved by the following members of the Final Oral Committee: James P. Kerns, Assistant Professor, Plant Pathology Patricia S. McManus, Professor, Plant Pathology Douglas I. Rouse, Professor, Plant Pathology John C. Stier, Professor, Horticulture Nancy P. Keller, Professor, Medical Microbiology i ABSTRACT Successful management of turfgrass diseases such as Microdochium patch and dollar spot on intensively-maintained golf course turf requires fungicide applications throughout the year. Repeat fungicide applications can have negative financial, environmental, and toxicological consequences and their use should be limited when possible. Research was conducted to determine the factors that influence degradation of the common turfgrass fungicides chlorothalonil and iprodione in a winter environment and under varying temperatures from 2009- 2012. Soil temperature, snow melt, and winter rains had the largest influence on fungicide degradation in a winter environment. Photodegradation, as influenced by the presence or absence of snow cover, did not impact fungicide degradation during winter. Temperature was directly related to degradation rates of both fungicides. The most likely mechanisms influencing degradation at higher temperatures were plant and bacterial metabolism. Strategies for reducing fungicide inputs were also explored. Alternative fungicide application timings in the spring, made well before the onset of dollar spot symptoms, delayed the onset of disease symptoms and reduced the annual number of fungicide applications by up to two applications without sacrificing turf quality. Disease-resistant creeping bentgrass cultivars such as ‘Declaration’ and ‘Memorial’ reduced dollar spot and Typhula blight severity compared to older cultivars such as ‘Penncross,’ though not to the degree where fungicide applications could be eliminated or drastically reduced. The cumulative effect of these studies has introduced new methods of studying and understanding the impact of the environment on turfgrass fungicides, and how simple strategies available today can reduce fungicide usage and enable more sustainable turfgrass management in the future. ii ACKNOWLEDGEMENTS There are numerous individuals I would like to thank for their contributions to the completion of this dissertation, and I will name just a few here. First, I would like to thank my major advisor Dr. Jim Kerns for his guidance and patience through the many peaks and valleys of my time as a Ph.D. candidate. In addition, I would like to thank the members of my Ph.D. committee for their guidance and excellent contributions to the direction of each research project; Dr. Nancy Keller, Dr. Patricia McManus, Dr. Doug Rouse, and Dr. John Stier. I would also like to thank the labs of Dr. Amy Charkowski and Dr. Jeri Barak for their guidance on ELISA methodology and bacterial quantification, respectively. Nick Keuler from the College of Agriculture and Life Sciences Statistical Consulting Service provided invaluable support in the analysis of many complicated sets of data. Of utmost importance have been my friends and family, both inside the department and out, who have provided support and needed relief from the everyday grind of scientific research. I would like to reserve my most sincere thank you to the members of the Kerns lab, especially our undergraduate student assistants. Tom Huncosky, Sam Soper, Ben van Ryzin, Jake Soper, P. J. Liesch, and Renee Rioux have provided assistance and support whenever and wherever needed, and completion of this project would have been impossible without their unwavering assistance. iii FOREWARD Each chapter in this dissertation has been developed with submission to a particular journal in mind, which has influenced the structure of each chapter. Chapter 1 was developed for submission into the journal Crop Protection. Chapter 2 and Chapter 3 were developed for submission into the journal Plant Disease. Chapter 4 was developed for submission into the International Journal of Turfgrass Research. Chapter 5 was developed for submission into Applied Turfgrass Science, and is the only chapter to deviate significantly from the traditional ‘Introduction’, ‘Materials and Methods’, ‘Results’, and ‘Discussion.’ For consistency, the ‘Literature Cited’ section in each chapter was formatted according to specifications outlined in Plant Disease. iv TABLE OF CONTENTS Abstract ............................................................................................................................i Acknowledgements ..........................................................................................................ii Foreward ..........................................................................................................................iii List of tables .....................................................................................................................v List of figures ...................................................................................................................viii INTRODUCTION ...........................................................................................................1 CHAPTER 1 ....................................................................................................................13 Modification of commercially-available ELISA kits to determine chlorothalonil and iprodione concentrations on golf course turfgrass. CHAPTER 2 ....................................................................................................................39 Effect of snow cover on the duration of Microdochium patch control provided by chlorothalonil and iprodione on golf course turfgrass. CHAPTER 3 ....................................................................................................................84 Influence of temperature on chlorothalonil and iprodione degradation and in vitro fungal sensitivity. CHAPTER 4 ....................................................................................................................123 Impact of novel fungicide timings on the development of snow mold and dollar spot on golf course turfgrass. CHAPTER 5 ....................................................................................................................144 Resistance of creeping bentgrass cultivars to dollar spot and snow mold. CONCLUSION ................................................................................................................158 v LIST OF TABLES CHAPTER 1: Table 1. Iprodione concentration as calculated using a modified Horiba SmartAssay® ELISA method and a gas chromatography/electron capture detection (GC/ECD) method. ELISA absorbance values at 450 nm were converted to ELISA fungicide concentrations using the equation of the linear regression of the absorbance of standard iprodione concentrations provided by Horiba. All turfgrass sampling was completed 1 h following iprodione application on creeping bentgrass (Agrostis stolonifera ‘Penncross’) maintained at a 1.2 cm height ................................................................................................. 34 Table 2. Absorbance values for high and low iprodione standard concentrations using the Iprodione Horiba SmartAssay® analysis kit. The regression equation produced from the high and low standards was used to convert absorbance values from each sample mg of iprodione per L ................................................................................ 35 Table 3. Chlorothalonil concentration as calculated using a modified Horiba SmartAssay® ELISA method and a gas chromatography/electron capture detection (GC/ECD) method. ELISA absorbance values at 450 nm were converted to ELISA fungicide concentrations using the equation of the linear regression of the absorbance of standard chlorothalonil concentrations provided by Horiba. All turfgrass sampling was completed 1 h following chlorothalonil application on creeping bentgrass (Agrostis stolonifera ‘Penncross’) maintained at a 1.2 cm height .................................................................................................................... 36 Table 4. Absorbance values of high and low chlorothalonil standard concentrations using the chlorothalonil Horiba SmartAssay® analysis. The regression equation produced from the high and low standards was used to convert absorbance values for each sample to mg of chlorothalonil per L ................ 37 Table 5. Time and cost comparison of ELISA SmartAssay® analysis versus gas chromatographic methods by the University of Wisconsin and Horiba, Ltd ................... 38 CHAPTER 2: Table 1. Analysis of variance (ANOVA) of Microdochium patch for turfgrass cores sprayed with iprodione and chlorothalonil and sampled from snow and non-snow covered plots at weekly or biweekly intervals during the winter of 2009-2010 in Verona, WI ....................................................................................................... 67 vi Table 2. Analysis of variance (ANOVA) of Microdochium patch for turfgrass cores sprayed with iprodione and chlorothalonil and sampled from snow and non-snow covered plots at weekly or biweekly