SEED-ASSOCIATED BACTERIAL ENDOPHYTES FROM TURF GRASSES PROMOTE SEEDLING GROWTH AND DEFEND PLANTS FROM DISEASE By QIANG CHEN A dissertation submitted to the School of Graduate Studies 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 James F. White, Jr. And approved by ___________________________________ ___________________________________ ___________________________________ ___________________________________ New Brunswick, New Jersey May 2020 ABSTRACT OF THE DISSERTATION Seed-Associated Bacterial Endophytes from Turf Grasses Promote Seedling Growth and Defend Plants from Disease by QIANG CHEN Dissertation Director: James F. White, Jr. Animals and plants harbor microbiomes providing benefits to hosts. In plants, both prokaryotic and eukaryotic microbiomes have been universally found and shown to promote plant growth, enhance disease resistance and abiotic stress tolerance. Fungal endophytes from grasses have been well characterized, but bacterial endophytes were rarely studied. This dissertation will present research that was conducted to test the hypothesis that cool-season turfgrass seeds bear bacterial endophytes providing beneficial effects on host, such as seed germination, growth promotion, and antifungal effects. Bacteria were isolated from seeds of different turfgrass species. Among them, Bacillus amyloliquefaciens, Bacillus pumilus, and Pantoea agglomerans were demonstrated to promote seed germination and seedling growth, and alter root architecture on host. B. amyloliquefaciens strains were shown to produce antifungal lipopeptides that suppress the growth of several fungal pathogens. Metagenomic analysis on the bacterial community associated with turfgrass seeds from low and high moisture climate revealed that moisture level influenced the community structure of the bacteria on/in turf seeds. The abundance of several bacterial groups at different taxonomy ranks on/in the seeds ii was either positively or negatively correlated with the seed germination rate. Overall, data supported that bacterial endophytes inhabiting turfgrass seeds benefited host by promoting seed germination and seedling development, as well as providing antifungal compounds. Moreover, moisture level was found to affect the structure of bacterial community on/in turfgrass seed, which further influenced the seed germination. iii DEDICATION To my maternal grandmother, Xiuyun, Hu, and in memory of my maternal grandfather, Shenggui Jiang, my paternal grandparents, Jitai Chen and Yafen Wang. iv ACKNOWLEDGEMENTS I would like to thank everyone in White Lab at Rutgers University for their help in the past few years. I would particularly like to thank Dr. James F. White for providing large amounts of help in guiding my research, and being exceptionally flexible in my PhD career. I thank the Plant Biology program for accepting me into the program, and providing assistantship. I also would like to thank my parents for your years of love and sacrifices for me to finish my prolonged education. At last, I thank my girlfriend, Chengyi Dan, for your love and support. It is acknowledged here that much of the research in Chapter 4 of this dissertation was published with some additions as: Chen, Q., Meyer, W. A., Zhang, Q., & White, J. F. (2020). 16S rRNA metagenomic analysis of the bacterial community associated with turf grass seeds from low moisture and high moisture climates. PeerJ, 8, e8417. DOI: 10.7717/peerj.8417 v TABLE OF CONTENTS ABSTRACT………………………………………………………………………….…...ii DEDICATION……………………………………………………………………………iv ACKNOWLEDGEMENT………………………………………………………………...v TABLE OF CONTENT…………………………………………………………………..vi LIST OF TABLES………………………………………………………………………...x LIST OF FIGURES………………………………………………………………………xi INTRODUCTION………………………………………………………………………...1 Chapter 1 Diversity and Functions Of Endophytes ............................................................ 2 1.1 Endophytes ................................................................................................................ 2 1.1.1 Definition of Endophytes ................................................................................... 2 1.1.2 Diversity of Endophytes .................................................................................... 3 1.1.3 Functions of Endophytes .................................................................................. 11 1.2 Association of Turfgrass and Endophytes .............................................................. 15 1.2.1 Colonization of Fungal Endophytes on Turfgrass ........................................... 15 1.2.2 Function of Fungal Endophytes ....................................................................... 16 1.3 References ............................................................................................................... 17 Chapter 2 Seed-Associated Bacterial Endophytes Promoted Seed Germination and Seedling Development of Cool Season Turf Grasses ....................................................... 29 2.1 Introduction ............................................................................................................. 29 2.2 Materials and Methods ............................................................................................ 30 2.2.1 Bacteria Isolation ............................................................................................. 30 2.2.2 Bacteria Identification ...................................................................................... 31 vi 2.2.3 Inoculation of Bacteria Isolates on Grasses ..................................................... 31 2.2.4 WinRhizo Analysis of Root Architecture ........................................................ 32 2.2.5 Root Dry Weights of Seedlings Under Pathogenic Stress ............................... 33 2.2.6 Seed Germination Test Under Salt Stress and Non-Stressed Conditions ........ 33 2.2.7 Microscopy Observation .................................................................................. 34 2.3 Results ..................................................................................................................... 34 2.3.1 Observation, Isolation, and Identification of Endophytic Bacteria in Cool- Season Turfgrasses .................................................................................................... 34 2.3.2 Colonization of Isolated Bacteria on Turfgrass Roots ..................................... 35 2.3.3 Growth Promotion of Root Hairs by Isolated Bacterial Endophytes ............... 39 2.3.4 Root Architecture Analysis .............................................................................. 39 2.3.5 Seed Germination Without Stress Treatment .................................................. 39 2.3.6 Seed Germination Under Salt Stress ................................................................ 43 2.3.7 Root Growth of Perennial Ryegrass and Tall Fescue Seedlings Under Pathogenic Stress ...................................................................................................... 45 2.4 Discussion ............................................................................................................... 46 2.4.1 Bacterial Endophytes Colonized Cool-Season Turfgrass ................................ 46 2.4.2 Bacterial Endophytes Increased Turf Seed Germination Rate under Both Salt Stress and Non-Stress Conditions ............................................................................. 47 2.4.3 Promotion of Seedling Root Growth by Bacterial Endophytes ....................... 47 2.5 Conclusion .............................................................................................................. 48 2.6 References ............................................................................................................... 48 vii Chapter 3 The Turfgrass-Associated Bacillus amyloliquefaciens Strains Producing Fengycin, Iturin and Surfactin Inhibit the Growth of Turf Fungal Pathogens .................. 51 3.1 Introduction ............................................................................................................. 51 3.2 Materials and Methods ............................................................................................ 53 3.2.1 Microorganisms and Growth Conditions ......................................................... 53 3.2.2 Effect of B. amyloliquefaciens Strains on Fungal Pathogens .......................... 54 3.2.3 Extraction of Lipopeptides from B. amyloliquefaciens ................................... 54 3.2.4 Mass Spectrometry Analysis ............................................................................ 55 3.2.5 Detection of Non-Ribosomal Peptide Synthetase Genes ................................. 55 3.3 Results ..................................................................................................................... 56 3.3.1 Inhibitory Effects of B. amyloliquefaciens Strains on Pathogenic Fungi ........ 56 3.3.2 Analysis of Lipopeptides Produced by B. amyloliquefaciens Strains .............. 61 3.3.3 PCR Detection of Non-Ribosomal Lipopeptide Synthetases .......................... 66 3.4 Discussion ............................................................................................................... 68 3.4.1 Antagonistic Effects of B. amyloliquefaciens Strains on Fungal Pathogens ... 68 3.4.2 Lipopeptides Production of B. amyloliquefaciens Strains ..............................