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©2016 Lisa A. Beirn ALL RIGHTS RESERVED ©2016 Lisa A. Beirn ALL RIGHTS RESERVED MOLECULAR INSIGHTS INTO THE MICROBIAL COMMUNITY OF ANNUAL BLUEGRASS (POA ANNUA L.) PUTTING GREEN TURF By LISA A. BEIRN A dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Plant Biology Written under the direction of Dr. Bruce B. Clarke And approved by _____________________________________ _____________________________________ _____________________________________ _____________________________________ _____________________________________ New Brunswick, New Jersey January 2016 ABSTRACT OF THE DISSERTATION Molecular Insights into the Microbial Community of Annual Bluegrass (Poa annua L.) Putting Green Turf By LISA A. BEIRN DISSERTATION DIRECTOR: Dr. Bruce B. Clarke Annual bluegrass (Poa annua; ABG) putting green turf is a unique man-made environment that requires regular fertility inputs to maintain acceptable turfgrass quality and playability. While these inputs can affect foliar diseases such as anthracnose, caused by Colletotrichum cereale, little is known about their impact on microbial communities in this ecosystem. The objectives of this dissertation were to: 1) determine the frequency and distribution of C. cereale in the United States, 2) examine the resident microbial communities in the soil of ABG putting green turf over time using advanced molecular technologies, and 3) identify how nitrogen (N) and/or potassium (K) fertilization affects the distribution, diversity, and abundance of benign and pathogenic microorganisms in this system. More than 50 phyla, representing hundreds of species of archaea, bacteria, and fungi were identified. Above ground, this diversity was highlighted in the form of two distinct lineages of C. cereale, both able to cause anthracnose disease but exhibiting distinct host and geographic preferences. Below ground, the ABG rhizosphere supported a vast microbial community, despite high sand content and regular fertilization and pesticide applications. Few turfgrass pathogens were identified from the soil. However, ii tremendous variation was characterized within the nonpathogenic microbial community, with the rhizosphere of ABG hosting organisms capable of antibiotic production, fixing nitrogen, or serving as potential biocontrol agents or mycorrhizal partners. Over all, individual microbial groups were present in low abundance across all samples. Fertilization did not affect microbial diversity, but did alter the abundance of specific microbial groups. Changes associated with fertility treatments were limited to approximately 7% of the total archaea/bacteria and 23% of the total fungal community identified. In general, K and low rates of N increased abundance of archaea, bacteria, and fungi in the study sites. Seasonality also strongly influenced microbial communities, with samples collected in summer months clustering separately from those obtained in the spring. The research described here provides the first insight into the diverse microbial community residing in the soil of ABG putting green turf utilizing next- generation sequence-based analyses, and protocols developed to conduct this work should help facilitate future research examining the turfgrass microbiome. iii ACKNOWLEDGMENTS I would like to express my thanks to Drs. Bruce Clarke and Jo Anne Crouch for their guidance, expertise, and encouragement over the past several years. I am grateful to have had the opportunity to work with you both. I would also like to thank my committee members, Drs. Stacy Bonos, Michele DaCosta, and Peter Oudemans, for their time, helpful discussions, and contributing to my professional and intellectual development. I am also thankful to the numerous other faculty at Rutgers who have graciously contributed their intellect and advice. In particular, I wish to acknowledge Drs. James Murphy, Donald Kobayashi, William Meyer, Bradley Hillman, and Ann Gould. I would also like to extend my thanks to Mark Peacos, Joe Clark, and TJ Lawson for their assistance maintaining my studies, and for their ingenuitiy designing and constructing research equipment. I also wish to thank Marshall Bergen and Pradip Majumdar for their helpful suggestions and assistance in the lab and field. A very special thanks to Rich Buckley and Sabrina Tirpak for igniting that initial ‘spark’ and introducing me to the world of plant pathology as an undergraduate. I am also appreciative for the financial support received over the years. I wish to acknowledge the Rutgers Center for Turfgrass Science for their generous support of my research and graduate assistantships. I have had the pleasure of working with many exceptional graduate students throughout the years who have contributed their time and shared ideas. I wish to thank Chas Schmid, Greg Behringer, John Capik, James Hemfling, Wrennie Wang, Samantha Lee, Melissa AbdelBaky, Moustafa AbdelBaky, Dr. Richard Hung, Dr. Joseph Roberts, iv Dr. Karen Ambrose, Dr. Ari Novy, Dr. Elyse Rodgers-Vieira, and Dr. Robyn Barbato. I am proud to call you all colleagues and friends. I also wish to acknowledge the talented students who have worked for me over the years- Shamal Budhdev and Jospehine Roosli. I am extremely grateful for the unwavering support of my family and friends- you brought a smile to my face when I didn’t think it was possible. I especially wish to thank my parents for their love and encouragement and for teaching me what true dedication is. I am also grateful for my brother for our many adventures, his sense of humor and our friendship. Finally, I am indebted to my husband, Bryan. None of this would have been possible without his endless love and support. I am truly blessed to have him in my life. Words cannot express how much I love you all. Chapter 1 is published in the peer-reviewed journal PloS One. The full citation is: Beirn LA, Clarke BB, Crouch JA (2014) Influence of Host and Geographic Locale on the Distribution of Colletotrichum cereale Lineages. PLoS ONE 9(5): e97706. doi:10.1371/journal.pone.0097706. v TABLE OF CONTENTS ABSTRACT OF THE DISSERTATION ........................................................................... ii ACKNOWLEDGMENTS ................................................................................................. iv TABLE OF CONTENTS ................................................................................................... vi LIST OF TABLES ............................................................................................................. ix LIST OF FIGURES ........................................................................................................... xi LITERATURE REVIEW ....................................................................................................1 The Value and Benefits of Turfgrass: A Brief Introduction ....................................1 Putting Greens in the Northeastern U.S ...................................................................2 Colletotrichum cereale: A Common Pathogen of Annual Bluegrass Turf ..............3 The Golf Course Phytobiome ................................................................................11 Microbial Communities in Turfgrass .....................................................................12 Summary and Future Directions for Research Studying Microbial Communities in the Turfgrass Enivronment ....................................................................................22 Thesis Overview ....................................................................................................24 References ..............................................................................................................25 CHAPTER 1. Influence of Host and Geographic Locale on the Distribution of Colletotrichum cereale Lineages .......................................................................................34 Abstract ..................................................................................................................34 Introduction ............................................................................................................35 vi Materials and Methods ...........................................................................................38 Results ....................................................................................................................42 Discussion ..............................................................................................................48 References ..............................................................................................................54 Tables .....................................................................................................................58 Figures....................................................................................................................95 CHAPTER 2. Analysis of Microbial Communities in the Soil of Annual Bluegrass Putting Green Turf Highlights the Importance of Seasonality ..........................................98 Abstract ..................................................................................................................98 Introduction ..........................................................................................................100 Materials and Methods .........................................................................................105 Results ..................................................................................................................114
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