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The Bacterial Microbiomes of Saskatchewan Crops and Potential for Plant Growth Promotion

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The Bacterial Microbiomes of Saskatchewan Crops and Potential for Plant Growth Promotion THE BACTERIAL MICROBIOMES OF SASKATCHEWAN CROPS AND POTENTIAL FOR PLANT GROWTH PROMOTION 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 Soil Science University of Saskatchewan Saskatoon By Jorge Cordero Elvia © Copyright Jorge Cordero Elvia, January 2019. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfillment 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 or 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 uses of materials in this thesis in whole or part should be addressed to: Head of the Department of Soil Science 51 Campus Drive, Room 5D34 University of Saskatchewan Saskatoon, Saskatchewan, S7N 5A8 Canada OR Dean College of Graduate and Postdoctoral Studies University of Saskatchewan 116 Thorvaldson Building, 110 Science Place Saskatoon, Saskatchewan, S7N 5C9 Canada i DISCLAIMER Reference in this thesis was prepared by the author to meet the requirements for the degree of Doctor of Philosophy at the University of Saskatchewan. Reference in this thesis to any specific commercial products, process, or service by trade name, trademark, manufacturer, or otherwise, does not constitute or imply its endorsement, recommendation, or favoring by the University of Saskatchewan. The views and opinions of the author expressed herein do not state or reflect those of the University of Saskatchewan, and shall not be used for advertising or product endorsement purposes. ii ABSTRACT Bacteria associated with the rhizosphere and plant tissues may contribute to host crops by providing nutrients, stimulating growth and/or controlling phytopathogens. These beneficial plant × bacteria associations may have a key role in the establishment and production of agricultural ecosystems. This research thesis investigated the bacterial communities associated with canola (Brassica napus L.), wheat (Triticum aestivum L.), lentil (Lens culinaris L.) and field pea (Pisum sativum L.) grown in agricultural fields in Saskatchewan and assessed the potential of some of these bacteria for plant growth promotion. Analysis of bacterial communities associated with these crops suggested a selection of the root endophytic microbiome from the rhizosphere. In contrast, endophytic bacteria associated with aboveground plant organs varied greatly among crops, soils and plant organs. Furthermore, Denaturing Gradient Gel Electrophoresis (DGGE) and high throughput-sequencing analyses of baterial profiles in wheat and canola suggested that each crop may select specific bacterial taxa at different plant growth stages and within different plant compartments. Pseudomonas and Stenotrophomonas were predominant genera in the rhizosphere and root interior of all crops, suggesting a generalist distribution of these bacteria. Relative abundance of specific bacterial groups in the rhizosphere, as well as bacterial Phospholipid Fatty Acids (PLFA) in the bulk soil, were significantly correlated with soil pH, silt and organic matter content. There was, however, no correlation between soil properties and the most abundant endophytic bacterial genera, thus suggesting that soil characteristics may not influence bacterial communities within plant roots. Culturable bacteria (n=157) isolated from the root interior of the studied crops were tested for their effect on seed germination, root elongation and plant growth promotion. A total of nine bacterial strains, isolated from the root interior of field grown crops, stimulated seed germination and root elongation when inoculated to canola and wheat plants, and five of these bacterial strains also promoted shoot growth in canola. Overall, these results suggest the bacterial microbiomes of the rhizosphere and plant tissues were modulated by soil properties and host crop, respectively. In addition, several endophytic bacteria demonstrated potential for use as bioinoculants in agriculture. iii ACKNOWLEDGMENTS Foremost, I would like to thank to my mother Margarita Elvia for her love, sacrifice and inspiration. I am extremely grateful to my supervisors Drs. Jim Germida and Renato de Freitas for their mentorship, support, guidance and encouragement during my Ph.D. studies. I would like to acknowledge the valuable inputs from my advisory committee members, Drs. Bobbi Helgason, Fran Walley, Steven Siciliano and Vladimir Vujanovic. I want to express my gratitude to many friends from the Soil Microbiology Lab 5E25, who made this journey with me. Special thanks to Eduardo Mitter, Panchali Katalunda, Avanthi Wijesinghe, Zayda Morales, Bethany Templeton, Natalie Blain, Mortuza Reza and Anisha Biswaray. I would also like to thank Marc St. Arnaud, Kim Heidinger and Dr. Ken Van Rees from the Department of Soil Science for their help and support. Thanks to all friends, professors and personnel at the College of Agriculture and Bioresources for contributing to my academic development. I would like to thank the Crop Development Centre and the research station of Agriculture Agri-Food Canada (AAFC) in Melfort for their help with plant sampling for this thesis. I am very thankful to Dr. Jeff Schoenau for providing me with his valuable advice, as well as the soil and plant samples from his farm. I am grateful for the financial support received from the University of Saskatchewan. I would like to thank my family for always being there for me and inspiring me to follow my dreams. Thanks to Adriana, Leslie, Dale, Dayana, Veriozka, David, Sadiel and Sebastian for being such amazing friends. iv TABLE OF CONTENTS PERMISSION TO USE….…………………………………………………………………..……i DISCLAIMER……..…………………………………………………………………………..….ii ABSTRACT…...……………………………………...………………………………………..…iii ACKNOWLEDGMENTS……………………………………………………………………….iv TABLE OF CONTENTS…….…………………………………………………………..……….v LIST OF TABLES…………………..……………………………………………………..…..…ix LIST OF FIGURES………..…………………………………………...………………..……….xi LIST OF ABBREVIATIONS…….…....……………………………...…………………..…..xviii 1. GENERAL INTRODUCTION..……….……...……………………………………………….1 1.1. Organization of the Thesis………………..…...……………...……………………………..4 2. LITERATURE REVIEW………………….……...………..………………………………….5 2.1. Plant × Bacteria Interactions: Implications for Crop Production and Agroecosystem Functioning…….....…………………....………...…………………………………………6 2.2. Bacterial Microbiome Associated with Crops…………..…..………………………………8 2.2.1. Rhizosphere……………………………….……………………..……………………8 2.2.2. Phyllosphere…………………………………….……..……………..……………...10 2.2.3. Spermosphere…………………………….…………………………………...…..…11 2.2.4. Endosphere…………………………………….…………………………….....……12 2.3. Factors Affecting the Diversity of Bacterial Communities Associated with Crops…....…...15 2.3.1. Soil Physical and Chemical Characteristics…………………..……….….………...15 2.3.2. Crop Species and Plant Development Stages…….………………………..….…….17 2.4. Plant Growth Promotion by Bacterial Endophytes Associated with Crops….....……...….19 2.4.1. Mechanisms of Plant Growth Promotion…...…………...……………………….....19 2.4.2. Perspectives for Crop Production in Saskatchewan………………......………..…...22 3. THE BACTERIAL MICROBIOMES ASSOCIATED WITH THE RHIZOSPHERE AND ROOT INTERIOR OF CROPS IN SASKATCHEWAN, CANADA……………..….28 3.1. Preface……..………………………………...………………………………………..…...28 3.2. Abstract……...………………………………………………………..…………………...29 3.3. Introduction…………..……………………………………………..…..…………………30 3.4. Materials and Methods………………………………………………………..…………...32 v 3.4.1. Sampling and Processing………………………...…………………..……………..32 3.4.2. Analysis of Soil Physical and Chemical Properties….…………………...…...……32 3.4.3. Phospholipid Fatty Acids (PLFA) Analysis of Soil Microbial Communities…….....35 3.4.4. Bacterial Isolation and Identification using Culture Dependent Methods…..……....35 3.4.5. Survey of Rhizosphere and Root Endophytic Bacterial Communities.....……...…...37 3.4.6. Analysis of Bacterial Communities using Denaturing Gradient Gel Electrophoresis (DGGE)…………………………………………………..……….37 3.4.7. Analysis of Bacterial Communities using 16S rRNA High-Throughput Sequencing...........………………...………………………………………………..38 3.4.8. Bioinformatics and Statistical Analyses……..………...………...………………….39 3.5. Results………….………………………………………………………...…..……………40 3.5.1. Culturable Bacteria Associated with the Rhizosphere and Root Interior of the Crops...……………………...…………………………………………………...…40 3.5.2. PLFA Profiles of Soil Bacterial Communities……………………………..….…....43 3.5.3. Community Structure of Endophytic, Rhizosphere and Bulk Soil Bacteria Associated with Crops using DGGE………..……..……………………………......45 3.5.4. Community Structure and Diversity of Bacterial Communities Associated with Crops assessed by 16S rRNA High-Throughput Sequencing……………………....51 3.5.5. Taxonomic Analysis of Bacterial Communities Associated with Crops assessed by 16S rRNA High-Throughput Sequencing………..…..…………..…………...…65
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