EFFECT OF AZOXYSTROBIN ON TURFGRASS PHYLLOSPHERE MICROBIAL POPULATIONS AND DISEASE ENHANCEMENT A Thesis Presented to The Faculty of Graduate Studies of The University of Guelph by DANIEL BENEDETTO In partial fulfilment of requirements for the degree of Master of Science January, 2008 © Daniel Benedetto, 2008 Library and Bibliotheque et 1*1 Archives Canada Archives Canada Published Heritage Direction du Branch Patrimoine de I'edition 395 Wellington Street 395, rue Wellington Ottawa ON K1A0N4 Ottawa ON K1A0N4 Canada Canada Your file Votre reference ISBN: 978-0-494-41797-3 Our file Notre reference ISBN: 978-0-494-41797-3 NOTICE: AVIS: The author has granted a non­ L'auteur a accorde une licence non exclusive exclusive license allowing Library permettant a la Bibliotheque et Archives and Archives Canada to reproduce, Canada de reproduire, publier, archiver, publish, archive, preserve, conserve, sauvegarder, conserver, transmettre au public communicate to the public by par telecommunication ou par Plntemet, prefer, telecommunication or on the Internet, distribuer et vendre des theses partout dans loan, distribute and sell theses le monde, a des fins commerciales ou autres, worldwide, for commercial or non­ sur support microforme, papier, electronique commercial purposes, in microform, et/ou autres formats. paper, electronic and/or any other formats. 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Canada ABSTRACT EFFECT OF AZOXYSTROBIN ON TURFGRASS PHYLLOSPHERE MICROBIAL POPULATIONS AND DISEASE ENHANCEMENT Daniel Benedetto Advisor: University of Guelph, 2008 Professor T. Hsiang This study documents dollar spot enhancement months after application of azoxystrobin to Agrostis stolonifera and Poa pratensis. One possible cause of disease enhancement is the perturbation of foliar microbial populations. Dilution plating showed that foliar microbial populations on the grasses treated with azoxystrobin were reduced during spring and summer. This correlated with a five-fold increase of dollar spot incidence. Cultural morphology after 9 days distinguished 11 morphotypes, but longer culturing revealed 26 submorphotypes among yeasts, bacteria and filamentous fungi. DNA-RFLP revealed 111 ribotypes and rDNA-sequencing revealed 94 species from 450 culturable isolates. While identification by morphological characteristics was useful for filamentous fungi, molecular analysis was required for yeasts and bacteria. Thirty-eight microbial species were present only on untreated plots, while 24 species were only associated with azoxystrobin-treated plots. Reduction in microbial populations and absence or gain of some species due to azoxystrobin may help explain increases in dollar spot incidence. ACKNOWLEDGEMENTS I am very grateful with my advisor Dr. Tom Hsiang for giving me the opportunity to obtain a Master's degree. His guidance, patience and expertise were key components that encourage me to keep learning and working through my studies. I particularly appreciated his friendly suggestions, advice and understanding. I also extend my thanks to the members of my advisory committee, Dr. P. Godwin and Dr. M. Habash for their suggestions during this study and to the members of the examination committee Dr. G. Barron and Dr. G Stephenson. I would like to thank John McLaughlin, Lynn Tian, Champa Weijekoon and Xie Weilong for their assistance, useful advice and technical support offered unconditionally throughout this project. Special thanks to Alejandra Cortes for her support and charisma that cheer me up on through difficult times. Thanks to the people of Guelph Turfgrass Institute for allowing me to perform my experiments and prompt support. I would like to give my love and thanks to my mother for her endless love and encouragement and my father for giving me invaluable help and support. To my future wife Xiaoting (Cathreen) Wei, I thank you for your continuous love, support, encouragement and the sacrifices you have made. I thank you for everything. I TABLE OF CONTENTS ACKNOWLEDGEMENTS I TABLE OF CONTENTS II LIST OF TABLES VI LIST OF FIGURES VIII LIST OF APPENDICES XIII LIST OF ABBREVIATIONS AND ACRONYMS XIV CHAPTER 1. LITERATURE REVIEW 1 1.1 FUNGICIDES AND TURFGRASS 1 1.2 STROBILURINS 3 1.2.1 History 4 1.2.2 Structure 5 1.2.3 Mode of action 6 1.2.4 Fungicide resistance 7 1.2.5 Environment persistence and animal safety 7 1.3 DOLLAR SPOT 9 1.3.1 Hosts and symptoms 10 1.3.2 Causal agent 11 1.3.2.1 Taxonomy 11 1.3.2.2 Morphology 12 1.3.2.3 Control 13 1.3.3 Dollar spot and fungicides 15 1.4 PHYLLOSPHERE 17 1.4.1 Microbial Biodiversity 18 1.4.1.1 Bacteria phyllosphere communities 23 1.4.1.2 Yeast phyllosphere communities 24 II 1.4.1.3 Fungal phyllosphere communities 25 1.5 ASSESSMENT OF MICROBIAL DIVERSITY 26 1.5.1 Traditional approaches 26 1.5.2 Molecular approach 27 1.6 HYPOTHESES AND OBJECTIVES 32 1.6.1 Hypotheses 32 1.6.2 Objectives 32 CHAPTER 2. EFFECT OF AZOXYSTROBIN ON DOLLAR SPOT DISEASE DEVELOPMENT 44 2.1 INTRODUCTION 44 2.2 MATERIALS AND METHODS 47 2.2.1 Study site 47 2.2.2 Field Experiments 50 2.2.2.1 Trials 51 2.2.2.2 Weather data 52 2.2.3 Statistic analyses 52 2.3 RESULTS 53 2.3.1 Weather data 53 2.3.2 Plant injury 54 2.3.2.1 Native sand green multi-season trial of 2005-2006 54 2.3.2.2 Poa pratensis multi-season trial of 2005-2006 55 2.3.2.3 Native sand fairway multi-season trial of 2006-2007 56 2.3.2.4 Pathology green multi-season trial of 2006-2007 57 2.3.3 Dollar spot 58 2.3.3.1 Native sand fairway multi-season trial of 2005-2006 58 2.3.3.2 Poa pratensis multi-season trial of 2005-2006 58 2.3.3.3 Native sand green multi-season trial of 2006-2007 58 2.3.3.4 Pathology green multi-season trial of 2006-2007 59 2.3.3.5 Summer plots of 2006 60 2.4 DISCUSSION 60 III CHAPTER 3. EFFECT OF AZOXYSTROBIN ON THE PHYLLOSPHERE MICROBIAL COMMUNITIES OF TURFGRASSES 93 3.1 INTRODUCTION 93 3.2 MATERIALS AND METHODS 96 3.2.1 Study site 96 3.2.2 Sample collection 97 3.2.3 Microbial screening 98 3.2.3.1 Leaf washings 98 3.2.3.2 Media preparation 98 3.2.3.3 Microbial pre-screening 99 3.2.3.4 Microbial screening 100 3.2.4 Leaf weight 101 3.2.5 Colony counting 102 3.2.6 Statistic analyses 104 3.3 RESULTS 105 3.3.1 Green leaves 107 3.3.2 Yellow leaves 110 3.4 DISCUSSION 112 CHAPTER 4. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS AND SEQUENCE ANALYSES OF MICROBIAL ISOLATES FROM TURFGRASS PHYLLOSPHERES 132 4.1 INTRODUCTION 132 4.2 MATERIALS AND METHODS 138 4.2.1 Microbial isolation 138 4.2.1.1 Leaf samples 138 4.2.1.2 Epiphyte isolation 139 4.2.1.3 Endophyte isolation 141 4.2.2 DNA extraction 142 4.2.2.1 Epiphytes 142 4.2.2.2 Endophytes 144 4.2.3 DNA concentration 145 IV 4.2.4 PCR amplifications 146 4.2.4.1 Internally transcribed spacer (ITS) amplification for fungi 146 4.2.4.2 Ribosomal 16S amplification for bacteria 146 4.2.4.3 Restriction Fragment Length Polymorphism (RFLP) 147 4.2.4.4 Restriction enzymes digestions 147 4.2.4.5 Band recording and analysis 149 4.2.5 DNA sequencing and alignment 150 4.3 RESULTS 151 4.3.1 Epiphyte collection 152 4.3.2 Restriction Fragment Length Polymorphism 153 4.3.2.1 Bacterial RFLP 154 4.3.2.2 Yeast RFLP 154 4.3.2.3 Fungal RFLP 155 4.3.3 Sequencing 156 4.3.3.1 Bacteria 156 4.3.3.2 Yeast 158 4.3.3.3 Filamentous fungi 159 4.4 DISCUSSION 161 CHAPTER 5. GENERAL DISCUSSION 191 REFERENCES 201 LIST OF APPENDICES 220 v LIST OF TABLES Table 1.1. Common phyllosphere bacteria of grasses. 34 Table 1.2. Common phyllosphere yeast of grasses. 35 Table 1.3. Common phyllosphere filamentous fungi of grasses. 36 Table 3.1. Colony forming units (CFU) per gram of leaves of microbial morphotypes obtained from the Agrostis stolonifera native sand fairway multi-season trial of 2005-2006 at the Guelph Turfgrass Institute, Guelph, Ontario. 118 Table 3.2. Colony forming units (CFU) per gram of leaves of microbial morphotypes obtained from the Poa pratensis multi-season trial of 2005-2006 at the Guelph Turfgrass Institute, Guelph, Ontario. 119 Table 3.3. Colony forming units (CFU) per gram of leaves of microbial morphotypes obtained from the Agrostis stolonifera pathology green multi-season trial of 2006-2007 at the Guelph Turfgrass Institute, Guelph, Ontario. 120 Table 3.4. Comparisons between microbial populations on green leaves of treated and untreated samples of Agrostis stolonifera of the native sand fairway multi-season trial of 2005-2006.