BIOFERTILIZERS FOR SUSTAINABLE AGRICULTURE: ISOLATION AND GENOMIC CHARACTERIZATION OF NITROGEN-FIXING BACTERIA FROM SUGARCANE A Dissertation Presented to The Academic Faculty by Luz Karime Medina Cordoba In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in Biology in the School of Biological Sciences Georgia Institute of Technology May 2020 COPYRIGHT © 2020 BY LUZ KARIME MEDINA CORDOBA BIOFERTILIZERS FOR SUSTAINABLE AGRICULTURE: ISOLATION AND GENOMIC CHARACTERIZATION OF NITROGEN-FIXING BACTERIA FROM SUGARCANE Approved by: Dr. Joel E. Kostka, Advisor Dr. Jung H. Choi School of Biological Sciences School of Biological Sciences Georgia Institute of Technology Georgia Institute of Technology Dr. I. King Jordan, Co-advisor Dr. Leonard W. Mayer School of Biological Sciences School of Medicine Georgia Institute of Technology Emory University Dr. Frank Stewart School of Biological Sciences Georgia Institute of Technology Date Approved: December 10, 2019 To my family and friends ACKNOWLEDGEMENTS First, I would like to express my gratitude to my advisor, Joel Kostka, for the opportunity to complete my PhD under his guidance. I am extremely grateful for your contributions to my research, and the experiences in your laboratory, where I learned principles that have made me a better microbiologist. To my co-advisor, King Jordan, thank you for believing in me from the start of the PhD program. Your guidance through each part of this five-year journey has contributed to my growth as a scientist. I will always be grateful for your patience and dedicated effort in educating me on bioinformatics. Your love and dedication to science and your students have made a lasting impression on me. I want to thank my committee members, Dr. Frank Stewart, Dr. Leonard Mayer, and Dr. Jung H. Choi; your support, comments, and suggestions have been fundamental for my research and have helped me to become a better scientist. It would have been impossible to complete my research without my colleges and friends from the lab. Aroon Chande and Dr. Lavanya Rishishwar were extraordinary instructors during the last years of my studies. To Shashwat Deepali Nagar and Héctor Espitia, thank you for your friendship and all your help during my time in the laboratory. I would like to thank my country, Colombia, for providing resources to help further my education. I hope to one day make a difference in the lives of every Colombian through microbiology. Finally, thank you to my family, who have been the catalyst for my motivation to grow as a person and as a professional. To my parents, Dr. Miguel Medina iv and Professor Mabel Cordoba, thank you for your unconditional love and for teaching me early on to love science, and more importantly, to respect others. To my brother Miguel David Medina for being my best friend and always giving me good advice. And to Kai Parham for always being there when I needed encouragement and the strength to continue. v TABLE OF CONTENTS ACKNOWLEDGEMENTS iv LIST OF TABLES viii LIST OF FIGURES ix LIST OF SYMBOLS AND ABBREVIATIONS xi SUMMARY xii CHAPTER 1. INTRODUCTION: BIOFERTILIZERS FOR SUSTAINABLE AGRICULTURE 1 1.1 Sustainable agriculture 1 1.2 Biological fertilizers (biofertilizers) 2 1.3 Plant growth-promoting bacteria (biofertilizers) and their role in sustainable agriculture 2 1.3.1 Mechanisms of bacterial plant growth promotion 3 1.4 Sugarcane as a global crop for the study of plant growth-promoting bacteria 6 1.4.1 Plant growth-promoting (PGP) bacteria isolated from sugarcane 7 1.4.2 Cultivation based studies 8 1.4.3 Microscopy based studies 9 1.4.4 Genomic based studies 9 1.5 The most common strategies for the application of biofertilizers in sugarcane, and the impact of these strategies on plant growth and crop yield 10 1.6 Challenges to the use of biofertilizers 11 1.6.1 Biofertilizers for sustainable sugarcane agriculture in Colombia 12 CHAPTER 2. ISOLATION AND INITIAL CHARACTERIZATION OF NITROGEN-FIXING BACTERIA FROM COLOMBIAN SUGARCANE 13 2.1 Abstract 13 2.2 Introduction 14 2.2.1 Colombian sugarcane company INCAUCA 14 2.3 Materials and Methods 16 2.3.1 Sample site selection, design, and execution 16 2.3.2 Sample acquisition 22 2.3.3 Cultivation of putative nitrogen-fixing bacteria from sugarcane 23 2.3.4 Molecular screening and identification of nitrogen fixers 25 2.3.5 Initial genome characterization of putative nitrogen-fixing bacteria 26 2.4 Results 28 2.4.1 Cultivation of putative nitrogen-fixing bacteria from sugarcane 28 2.4.2 Initial genome characterization of putative nitrogen-fixing bacteria 28 vi CHAPTER 3. GENOMIC CHARACTERIZATION AND COMPUTATIONAL PHENOTYPING OF NITROGEN-FIXING BACTERIA ISOLATED FROM COLOMBIAN SUGARCANE FIELDS 31 3.1 Abstract 31 3.2 Introduction 32 3.3 Materials and methods 34 3.3.1 Genome sequencing, assembly, and annotation 34 3.3.2 Comparative genomic analysis 36 3.3.3 Computational phenotyping 38 3.3.4 Experimental validation 48 3.4 Results 50 3.4.1 Genome characterization of putative nitrogen-fixing bacteria 50 3.4.2 Comparative genomic analysis 52 3.4.3 Computational phenotyping 59 3.4.4 Virulence comparison 64 3.4.5 Experimental validation of prioritized isolates 66 3.5 Discussion 68 3.5.1 Computational phenotyping for the prioritization of potential biofertilizers 69 3.5.2 Virulence profiling for the prioritization of potential biofertilizers 70 3.5.3 Potential for the use of computational phenotyping in other microbiology applications 71 CHAPTER 4. DEVELOPMENT OF A nifH DATABASE TO FACILITATE THE TAXONOMic ASSIGNMENT AND DIVERSITY ANALYSIS OF NITROGEN- FIXING BACTERIA 73 4.1 Nitrogen fixation 73 4.2 Need for a nifH gene database 75 4.3 Problems with previous nifH gene databases 78 4.4 Creation and evaluation of the nifH gene reference sequence database 81 4.4.1 Automated database pipeline: sequence retrieval and validation 82 4.5 A natural genetic classification system for nifH 87 CHAPTER 5. CONCLUSIONS AND FUTURE PROSPECTS 96 APENDIX A. SUPPLEMENTARY TABLES FOR CHAPTER 2 100 APENDIX B. SUPPLEMENTARY TABLES FOR CHAPTER 3 101 APENDIX C. SUPPLEMENTARY TABLES FOR CHAPTER 4 112 PUBLICATIONS 202 REFERENCES 203 vii LIST OF TABLES Table 1. Physicochemical characterization of INCAUCA sugarcane soil fields.............. 19 Table 2. Genome assembly statistics for the isolates characterized here. ........................ 29 Table 3. List of genome sequence analysis software used in this study. .......................... 35 Table 4. Custom gene panel for nitrogen fixation genes. ................................................. 40 Table 5. Custom gene panels for plant growth promotion genes. .................................... 42 Table6. Genome sequences for clinically-associated isolates from closely related Klebsiella species. ............................................................................................................. 47 Table 7. Genome sequencing assembly and functional annotations. ................................ 51 Table 8. Identity of the most closely related species (genus) for the isolates characterized here. ................................................................................................................................... 55 Table 9. Agro ecological zones sampled for this project. ............................................... 100 Table 10. Sugar cane varieties sampled for this project. ................................................ 100 Table 11. Presence/absence calls for nitrogen fixation genes panel. .............................. 102 Table 12. Presence/absence calls for plant growth promotion genes panel. ................... 104 Table 13. Presence/absence calls for virulence factor genes panel. ............................... 106 Table 14. Presence/absence calls for predicted antibiotic resistance profiles genes panel. ......................................................................................................................................... 110 Table 15. Lis of all full length sequences used for the genetic classification for nifH ... 113 viii LIST OF FIGURES Figure 1. INCAUCA sugarcane fields. ............................................................................. 15 Figure 2. INCAUCA sugarcane growing zones and farms............................................... 16 Figure 3. Triangular field sampling design. ...................................................................... 17 Figure 4. Sugarcane sampling. .......................................................................................... 18 Figure 5. Sample preparation and shipping system. ......................................................... 22 Figure 6. Platform for high-throughput enrichment of nitrogen-fixing bacteria. ............. 24 Figure 7. Cultivation and storage of nitrogen-fixing bacterial isolates. ........................... 25 Figure 8. Molecular screening of putative nitrogen-fixing bacterial isolates. .................. 26 Figure 9. Intial whole genome sequencing and analysis pipeline. .................................... 27 Figure 10. Screenshot of the BioProject PRJNA418312 available in the NCBI. ............. 30 Figure 11. Phylogeny of the bacterial isolates characterized here (SCK numbers) together with their most closely related bacterial type strains. ....................................................... 53 Figure 12. Phylogeny of the 16S rRNA genes for the bacterial isolates characterized here (SCK numbers) together with their most closely related bacterial type strains ................ 54 Figure 13. Comparison