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Download/Cacao.Pdf What is Inside of the Cacao Seed We eat? Approach to Mining the Cacao Microbiome (Theobroma cacao) Aidee Ximena Flórez Buitrago Student ID: 260865418 Department of Plant Sciences Faculty of Agricultural and Environmental Sciences McGill University Montreal Quebec, Canada December, 2020 A thesis submitted to McGill University in partial fulfillment of the requirements for the degree of Masters of Science Ó Ximena Florez-Buitrago2020 II DEDICATION For all people in science, who have inspired me and transmitted me the admiration for the processes that articulate the complexity of the interactions of living organisms. With special admiration for my undergraduate professors Dr. Pedro Adolfo Jimenez and Dr. Liliana Franco. I dedicate this work to my parents Elsa and Ricardo and my older small sister Catalina, my indefatigable motivators. To my friends Carmen Schlöder, Maria Fernanda Castillo, Diana Barrera, Cindy Celis, Julian Martinez and Francis van Oordt La Hoz, for the shared experiences and for being an example of courage, determination, and perseverance. I would also like to especially highlight the motivational support of Mamta Rani, Meha Sharma, Annis Fatmawati, Bikram Poudel and Nathan Liang during my M.Sc, for creating with me remarkable memories. III TABLE OF CONTENTS LIST OF TABLES .......................................................................................................... V LIST OF FIGURES ........................................................................................................ VI ABSTRACT .................................................................................................................. VII RÉSUMÉ ..................................................................................................................... VIII ACKNOWLEDGEMENTS ............................................................................................. IX CONTRIBUTION OF AUTHORS .................................................................................. XI Chapter 1 ........................................................................................................................ 1 1. Introduction ............................................................................................................ 1 1.1. Objectives ........................................................................................................ 3 1.2. Hypothesis ........................................................................................................ 3 Chapter 2 ........................................................................................................................ 4 2. Literature Review ................................................................................................... 4 2.1. Morphological Description of Theobroma cacao ......................................... 4 2.2. Cacao Fermentation Process ......................................................................... 5 2.3. Life Cycle MoniLiophthora roreri .................................................................... 6 2.4. Cultural and Biological Control of MoniLiophthora roreri ............................ 6 2.5. General Mechanisms Employed by Plant Growth Promotion Bacteria in Plant Protection ......................................................................................................... 8 2.5.1. Direct Mechanisms ........................................................................................ 9 2.5.2. Indirect Mechanisms .................................................................................... 12 2.6. Figures ........................................................................................................... 17 Chapter 3 ...................................................................................................................... 21 3. Identification and Biochemical Characterization of Culturable Bacterial Endophytes Associated with Cacao Seeds............................................................... 21 3.1. Biological Material......................................................................................... 21 3.2. Surface Sterilization and Recovery of microbes Using Culture Dependent Methods .................................................................................................................... 21 3.3. DNA Extraction and Molecular Identification .............................................. 22 3.4. Phylogenetic Tree ......................................................................................... 23 3.5. Biochemical Tests ......................................................................................... 23 3.5.1. Plant Colonization Traits .............................................................................. 23 3.5.2. Plant Growth Promotion ............................................................................... 24 3.5.3. Test for Antimicrobial Properties .................................................................. 26 IV 3.6. Results ........................................................................................................... 27 3.6.1. Diversity and abundance of isolated bacteria .............................................. 27 3.6.2. Biochemical characterization of the isolates ................................................ 28 3.7. Tables ............................................................................................................. 30 3.8. Figures ........................................................................................................... 42 Chapter 4 ...................................................................................................................... 46 4. Identification of Microbes Associated with Susceptible and Tolerant Varieties of Cacao Against Frosty Pod Rot. .............................................................................. 46 4.1. Plant Material ................................................................................................. 47 4.2. Collection sites .............................................................................................. 47 4.3. Microbial isolation and Culture .................................................................... 48 4.4. DNA Extraction and Molecular Identification .............................................. 49 4.5. Results ........................................................................................................... 49 4.5.1. Diversity and abundance of isolated bacteria .............................................. 49 4.6. Tables ............................................................................................................. 51 4.7. Figures ........................................................................................................... 57 Chapter 5 ...................................................................................................................... 63 5. Discussion ............................................................................................................ 63 Chapter 6 ...................................................................................................................... 69 6. ConcLusions and Future Perspectives ............................................................... 69 Chapter 7 ...................................................................................................................... 71 7. Bibliography ......................................................................................................... 71 Chapter 8 ...................................................................................................................... 93 Appendices .................................................................................................................. 93 Appendix I. List of culture media used to isolate endophytic bacteria associated with cacao seeds .................................................................................. 93 Appendix II. Biochemical tests performed on isolated endophytic bacteria. .... 93 Appendix III. Composition of culture media used to determine the ability of endophytic bacteria to produce lytic enzymes. .................................................... 94 Appendix IV. Stock solutions for performing the ACC deaminase test. ............ 94 Appendix V. Stocks and working solutions of tested antibiotics. ..................... 95 V LIST OF TABLES Chapter 3 Table 3.7.1. Taxonomic groups isolated from cacao seeds……………………………….30 Table 3.7.2. Screening of endophytes associated with cacao seeds for colonization traits……………………………………………………………………………………………..31 Table 3.7.3. Antibiotics resistance tests from all microbes isolated from the cacao seeds……………………………………………………………………………………………34 Table 3.7.4. Antimicrobials traits of bacterial endophytes isolated from cacao seeds…..37 Table 3.7.5. IAA and ACC deaminase production of endophytes associated with seeds……………………………………………………………………………………………40 Table 3.7.6. Selected strains with exceptional biochemical traits, mostly based on the production of IAA and siderophore…………………………………………………………...41 Chapter 4 Table 4.6.1. List of bacterial species isolated from the cacao variety CATIE-R6………...51 Table 4.6.2. List of the species of microbes isolated from the cacao variety CATONGO……………………………………………………………………………………..53 Table 4.6.3. Role of the endophytic bacteria isolated from cacao layers reported in other plant species……………………………………………………………………………………55 VI LIST OF FIGURES Chapter
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