EPIPHYTIC SEED MICROBIOMES OF WHEAT, CANOLA, AND LENTIL A Thesis Submitted to the College of Graduate and Postdoctoral Studies In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Food and Bioproduct Sciences University of Saskatchewan Saskatoon By Zayda Piedad Morales Moreira © Copyright Zayda Piedad Morales Moreira, June, 2021. All rights reserved. Unless otherwise noted, copyright of the material in this thesis belongs to the author PERMISSION TO USE In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying, publication, or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Food and Bioproduct Sciences University of Saskatchewan 51 Campus Drive University of Saskatchewan Saskatoon, Saskatchewan, S7N 5A8 Canada OR Dean of the College of Graduate and Postdoctoral Studies University of Saskatchewan 107 Administration Place Saskatoon, Saskatchewan S7N 5A2 Canada i ABSTRACT Microorganisms are found colonizing all plant organs including seeds. Seeds are reproductive structures able to carry and transfer microorganisms from one plant generation to the next acting as an initial source of microbial inocula for the next generation of plants. Seed- associated microbial communities offer the potential of improving crop production and yield through protection against abiotic and biotic stresses. Despite their agricultural relevance, seed- borne bacterial and fungal communities as well as factors influencing their assemblage remain largely unknown. Therefore, the purpose of this study was to: i) characterize the seed-associated microbiomes of three agricultural crops important for food security; wheat, canola, and lentil, ii) explore genetic and environmental factors influencing the assembly of the microbiota carried by seeds, and iii) examine the preservation and transmission of seed microbiomes. To achieve these objectives seed samples of different lines harvested from different field locations, years, and generations were subjected to high-throughput amplicon sequencing of the bacterial 16S rRNA and the fungal internal transcribed spacer (ITS) regions. My results suggest recruitment, transmission, and preservation of seed-associated microbiota are determined mainly by the environment in which the plants are grown and to some extent by the host. In addition, a shared set of microorganisms (i.e., core microbiome) was found when seed microbiomes of different crops, lines, and from different sources (i.e., produced in different fields and years) were analyzed together. The existence of this core microbiome implies that plants recruit and carry bacterial and fungal species that could interact with further generations, affecting their adaptation and establishment in novel environments. Some members of this core including Sphingomonas sp., Pantoea agglomerans, and Vishniacozyma victoriae are reported to be beneficial to their hosts. In my study I also found evidence suggesting seed-associated microbial communities are vertically transmitted from the mother plant to the offspring. Cutibacterium, Methylobacterium, Sphingomonas, Streptococcus, and Tepidimonas were found across multiple generations of lentil seeds irrespective of the soil in which they were grown. These findings represent an important step toward the advancement of sustainable breeding and agricultural strategies to utilize microbial communities carried by seeds for their potential contribution to plant health and productivity. ii ACKNOWLEDGMENTS First and foremost, I would like to thank the person who brings happiness and joy to my days, my little nephew José, love you JG. Special thanks to Tobías, Amparito, Jane, Nena, and Gabo for your continued support and encouragement. I am so grateful God chose you to be my family. “Distance means so little when someone means so much”. I would like to express my sincere gratitude to my supervisors Dr. Jim Germida and Dr. Bobbi Helgason for giving me the opportunity to be part of their fantastic research team. Thank you for your mentorship, motivation, kindness, and for constantly sharing your knowledge and work ethic with me. I would not be here without your support and guidance. Muchas gracias! I would also like to acknowledge the members of my advisory committee, Dr. Melissa Arcand, Dr. Timothy Sharbel, Dr. Leon Kochian, Dr. Vladimir Vujanovic, and Dr. Takuji Tanaka. Thank you for your feedback and suggestions during my whole PhD journey. I really appreciate it. I could not have done this research without the support of many scientists who shared knowledge and advice with me: Dr. Eric Lamb, Dr. Steven Siciliano, Dr. Sally Vail, Dr. Isobel Parkin, Dr. Curtis Pozniak, Dr. Kirstin Bett, Dr. Diane Knight, Dr. Jeff Schoenau, Dr. Renato de Freitas, Dr. Jennifer Town, Dr. Steven Mamet, Dr. Navid Bazghaleh, and Dr. Charlotte Norris. Many thanks to Kira Blomquist, Jaelyn Dietz, and Devini De Silva for all your help with the growth chamber experiment. Thanks to all Soil Micro lab (5E25) members, specially to Jorge, Eduardo, Claire, Panchali, Jesse, and Kim. Big thanks to Shanay, Alix, and Steve Ryu for all the lab and field camaraderie. This project was possible due to the support of the Plant Phenotyping and Imaging Research Centre (P2IRC), funded by the Canada First Research Excellence Fund (CFREF), managed by the Global Institute for Food Security (GIFS) and the Discovery Grant Program of the Natural Sciences and Engineering Research Council of Canada (NSERC). Special thanks to Agriculture and Agri- Food Canada (AAFC), SaskCanola, Saskatchewan Pulse Growers, and the College of Agriculture and Bioresources at University of Saskatchewan. Thank you to my dear friends Natalia, Claudia, Serena, Jennifer, Rafa, María, and the Batuque members: Emiliana, Carolina, Sue, Bruno, Priscila, Laís, and Sara. Thanks for being my family away from home. Love you guys. iii TABLE OF CONTENTS PERMISSION TO USE ................................................................................................................... i ABSTRACT .................................................................................................................................... ii ACKNOWLEDGMENTS .............................................................................................................. iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF TABLES ....................................................................................................................... viii LIST OF FIGURES ........................................................................................................................ xi LIST OF ABBREVIATIONS ...................................................................................................... xiii 1. INTRODUCTION ....................................................................................................................... 1 2. LITERATURE REVIEW ............................................................................................................ 3 2.1. Food Security ........................................................................................................................ 3 2.2. Agricultural crops .................................................................................................................. 3 2.2.1. Wheat ............................................................................................................................. 4 2.2.2. Canola ............................................................................................................................. 4 2.2.3. Lentil .............................................................................................................................. 5 2.3. Sustainable crop production .................................................................................................. 5 2.4. The plant microbiome ........................................................................................................... 6 2.5. Seeds ...................................................................................................................................... 8 2.6. The seed microbiome ............................................................................................................ 8 2.6.1. Origin of seed-associated microbial communities ......................................................... 9 2.6.2. Microbial niches within the seeds ................................................................................ 12 2.6.3. Characteristics of seed-borne microorganisms ...........................................................