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1 Possible Drivers in Endophyte Diversity and Transmission in The Possible Drivers in Endophyte Diversity and Transmission in the Tomato Plant Bacterial Microbiome Thesis Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Ana María Vázquez, B.S. Graduate Program in Plant Pathology The Ohio State University 2020 Thesis Committee Dr. María Soledad Benítez-Ponce, Advisor Dr. Christine Sprunger Dr. Jonathan M. Jacobs 1 Copyrighted by Ana María Vázquez 2020 2 Abstract It has been documented that beneficial plant-associated bacteria have contributed to disease suppression, growth promotion, and tolerance to abiotic stresses. Advances in high-throughput sequencing have allowed an increase in research regarding bacterial endophytes, which are microbes that colonize the interior of plants without causing disease. Practices associated with minimizing the use of off-farm resources, such as reduced tillage regimes and crop rotations, can cause shifts in plant-associated bacteria and its surrounding agroecosystem. Integrated crop–livestock systems are an option that can provide environmental benefits by implementing diverse cropping systems, incorporating perennial and legume forages and adding animal manure through grazing livestock. It has been found that crop-livestock systems can increase soil quality and fertility, reduce cost of herbicide use and improve sustainability, especially for farmers in poorer areas of the world. This work explores how crop-livestock systems that integrate chicken rotations can impact tomato plant growth, as well as soil and endophytic bacterial communities. Tomato plants were subjected to greenhouse and field studies where biomass was assessed, and bacterial communities were characterized through culture- dependent and -independent approaches. ii In greenhouse experiments, the greater percent of chicken grazed soil incorporated in the planting substrate, the greater the stunting of tomato seedlings. In the field study, bacterial communities differed significantly by sample origin and plant development stage, regardless of chicken grazing history. Our findings suggest stronger contribution of agricultural management practices during early plant stages on endophytic microbiome, as opposed to later on in the host lifecycle. Taxonomic composition of dominant groups of recovered endophytic bacterial isolates were consistent with those found by amplicon sequencing. Plots with history of chicken grazing had a significant increase of soil fertility and differentially abundant ASVs. Dominant phyla (Actinobacteria, Proteobacteria, Bacteroidetes and Firmicutes) of bacterial taxa identified were uniform between culture-dependent and -independent approaches from both greenhouse and field experiments. Most of the recovered isolates were found to be phylogenetically similar to formerly cultured plant endophytes and bacteria found in soil. However, some isolates were similar to bacteria recovered in human clinical settings. Further directions should focus on identification and elucidation of transmission of possible plant growth promoting bacteria, as well as human pathogens, found in crops grown in this type of agricultural management strategy. Moreover, the influence of stage in plant lifecycle on bacterial community composition under specific management strategies remains to be explored. This work provides insight into the influence of crop- livestock rotations on soil- and tomato plant-associated bacterial communities. iii Dedication This thesis is dedicated to the memory of my biggest fan, my grandmother Carmen Leticia Cabrera Amador. Regardless of adversities, Abuela lived by seeing beauty in science, art and human connections. I will always honor her by emulating her tenacity and persistence. iv Acknowledgments First, I would like to acknowledge my advisor, María Soledad Benítez-Ponce for her support and commitment in mentoring me during these past two years. Thank you for always stimulating an intellectually challenging and collaborative environment where I could thrive as a scientist. In addition, thank you for encouraging me to integrate other aspects to my professional skillset. Moreover, I would like to thank the members of my student advisory committee, Christine Sprunger and Jonathan Jacobs, for their valuable and constructive suggestions. I would also like to thank Suranga Basnagala for his advice in setting up my first field experiment at Mellinger Research Farm, as well as his assistance in looking out for my tomato plants and answering all of my questions. I would like to offer my special thanks to Leslie Taylor for all her help with my experiments and much needed cheer during this journey. Through countless emails exchanged, assistance with computational analysis provided by former lab member Matthew Willman was greatly appreciated. In addition, I would like to thank lab members and fellow graduate students Daowen Huo and Ranjana Rawal for their inspiring motivation and persistence. A special thanks to the whole Benitez lab, current v and former members, for making my time in Wooster memorable and cherished. Thank you to undergraduates Nedas Matulionis, Emmily Moses and Yesenia Velez for helping in different aspects of my research project, in and outside the lab. My grateful thanks are also extended to three very special people that have helped me make Wooster a home away from home, Marlia Bosques, Madeline Horvat and Edwin Navarro. In addition, a big thanks to all my friends, back home and in different places, that have encouraged me from afar. Finally, the biggest thanks are for the first supporters and mentors I ever had, my family. Mamá, Michelle, Berto, Carlos, José, Tommy and Tito. Thanks to my abuela for being my biggest and, arguably, my most outspoken fan. vi Vita 1995………………………………………… Born, Arecibo, Puerto Rico 2013-2018……………………………………B.S. Biology and B.S. Industrial Microbiology, University of Puerto Rico at Mayagüez 2018 to present………………………………Graduate Research Associate, Department of Plant Pathology, The Ohio State University Fields of Study Major Field: Plant Pathology vii Table of Contents Abstract ............................................................................................................................... ii Dedication .......................................................................................................................... iv Acknowledgments ............................................................................................................... v Vita .................................................................................................................................... vii List of Tables .................................................................................................................... xii List of Figures .................................................................................................................. xvi Chapter 1. Literature Review .............................................................................................. 1 Introduction ..................................................................................................................... 1 The Plant Microbiome .................................................................................................... 3 Bacterial Endophytes in Aboveground Plant Tissues ..................................................... 5 Bacteria in Soil and Rhizosphere of Plants ..................................................................... 6 Transmission and Colonization of Plant-Associated Bacterial Communities ................ 8 Role of Bacterial Endophytes ....................................................................................... 12 Summary and Conclusions ........................................................................................... 15 viii References ..................................................................................................................... 18 Chapter 2. Characterization of the Bacterial Endophytic Microbiome of Tomato Seedlings Grown in a Soil Diversity Gradient Using Chicken-Grazed Soils ................... 35 Introduction ................................................................................................................... 35 Materials and Methods .................................................................................................. 40 Field Site Description ............................................................................................... 40 Soil Sampling ............................................................................................................ 41 Planting Substrate, Plant Materials and Growth Conditions .................................... 42 Seedling Sampling and Evaluation ........................................................................... 44 Plant Tissue Sampling and Isolation of Endophytic Bacteria from Tomato Seedling Stem .......................................................................................................................... 45 DNA Extraction, PCR Amplification and Sequencing of the 16S rRNA and rpoB Gene Regions from Individual Bacterial Isolates ..................................................... 45 Isolate Sequence Data Analysis ................................................................................ 48 Plant Growth Data Analysis and Statistics ............................................................... 49 Results ..........................................................................................................................
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