CHARACTERIZATION OF THE STRUCTURE AND FUNCTION OF THE MICROBIOME IN CITRUS RHIZOSPHERE By UTPAL HANDIQUE A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2017 © 2017 Utpal Handique To my family, for their love To my advisors, for guiding the direction of my research ACKNOWLEDGMENTS To those who helped me through the years I stayed at the University of Florida to finish my Ph. D. program. First, I would like to thank my advisors Dr. Nian Wang and Dr. Jude William Grosser, who supported me during my whole doctoral study. They gave the opportunity to me to visit multiple citrus groves and guided me to see the science behind plant disease and understand Huanglongbing. Understanding the microbiome work that was done previously and continuing to extend the research was an opportunity for me to improve. I would like to thank Dr. Larry Duncan, Dr. James Graham, and Dr. Tripti Vasisth for precious suggestions during the committee meeting. Their advice helped me to understand the importance of being an expert on a specific topic as well as having a wider global view of scientific problems. I would like to thank all my labmates, who helped make this project practical to accomplish. They were always friendly and would take time to help if I had problem in my experiments. I would also thank my wife, Jian Wu who have supported me emotionally to achieve my goal. I am happy to have all my friends with whom I have spent my time during my research program in Gainesville and Lake Alfred. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ................................................................................................................... 10 CHAPTER 1 LITERATURE REVIEW .......................................................................................... 12 1.1 Rhizosphere Microbiome .................................................................................. 12 1.2 Soil-Microbes Interaction ................................................................................... 16 1.3 Plant-Microbe Interactions ................................................................................ 18 1.4 Huanglongbing .................................................................................................. 20 1.4.1 HLB Disease Development ..................................................................... 21 1.4.2 HLB Management ................................................................................... 22 1.5 Citrus ................................................................................................................. 25 1.5.1 Citrus Rootstock Breeding and Disease in Florida .................................. 25 1.5.2 HLB Impact on Citrus Roots .................................................................... 27 1.6 Beneficial Bacteria ............................................................................................ 28 1.6.1 Beneficial Bacteria Use in Agriculture ..................................................... 28 1.6.2 Advantage and Disadvantage of Microbial Application. .......................... 29 1.7 Approaches to Investigate Microbiome ............................................................. 29 1.8 Project Goals and Objectives ............................................................................ 33 2 STRUCTURE AND FUNCTION OF THE MICROBIOME OF THE CITRUS RHIZOSPHERE AFFECTED BY HUANGLONGBING ........................................... 35 2.1 Introduction ....................................................................................................... 35 2.2 Materials and Methods ...................................................................................... 38 2.2.1 Tree Selection and Sampling .................................................................. 38 2.2.2 Quantification of Candidatus Liberibacter asiaticus ................................ 39 2.2.3 RNA Extraction from Rhizosphere Soil ................................................... 39 2.2.4 Bioinformatics Analysis ........................................................................... 40 2.3 Results .............................................................................................................. 41 2.3.1 Metatranscriptome Annotation. ............................................................... 41 2.3.2 Taxonomic Assignment of Expressed Gene Function in Healthy Sample .......................................................................................................... 44 2.3.3 Comparative Taxonomic Assignment ..................................................... 46 2.3.4 Genes Associated with Nitrogen Cycle, Carbon Cycle, Phosphorus,Sulfur and Aromatic Compounds. ............................................. 47 2.3.5 Comparative Functional Profiles ............................................................. 52 5 2.4 Discussion ......................................................................................................... 54 2.5 Conclusion ........................................................................................................ 63 3 APPLICATION OF BENEFICIAL BACTERIA FOR THE IMPROVEMENT OF ROOT GROWTH .................................................................................................... 83 3.1 Introduction ....................................................................................................... 83 3.2 Materials and Methods ...................................................................................... 85 3.2.1 Selection of Groves ................................................................................. 85 3.2.2 Isolation of Beneficial Bacteria ................................................................ 85 3.2.3 Test of Beneficial Traits .......................................................................... 86 3.2.4 Bacterial Identification ............................................................................ 88 3.2.5 Graft Inoculation with Las and Microbe Application ................................ 88 3.2.6 Candidatus Liberibacter asiaticus Detection ........................................... 89 3.2.7 Statistical Analysis .................................................................................. 90 3.3 Results .............................................................................................................. 90 3.3.1 Bacterial Isolation and Beneficial Trait Analysis ...................................... 90 3.3.2 Root Growth Responses for Carrizo Citrange and Grapefruit ................ 92 3.4 Discussion ......................................................................................................... 93 4 SUMMARY ........................................................................................................... 108 APPENDIX: PHYLOGENETIC TREE OF BACTERIAL STRAINS .............................. 110 LIST OF REFERENCES ............................................................................................. 113 BIOGRAPHICAL SKETCH .......................................................................................... 133 6 LIST OF TABLES Table page 2-1 Summary of sequencing and annotation of citrus rhizosphere metatranscriptome .............................................................................................. 64 2-2 Clean Sequence data for five samples from the rhizosphere of healthy Hamlin orange trees on Cleopatra rootstock. ..................................................... 67 2-3 The 15 most transcribed gene functions in Citrus rhizosphere metatranscriptome .............................................................................................. 76 3-1 The list of citrus groves and locations with respective scion/rootstock and soil type description. ................................................................................................. 99 3-2 Combination of treatment applied in greenhouse experiment.. ........................ 100 3-3 Bacterial isolates from the healthy citrus rhizosphere. ...................................... 101 3-4 Strains with IAA production, siderophore production and PO4 solubilization ... 102 3-5 Mean of new, fibrous, total root length of Carrizo citrange seedlings ............... 104 3-6 Mean of new, fibrous, total root length of grapefruit seedlings. ........................ 105 A-1 Mean of root and above ground biomass of Las-inoculated grapefruit seedlings in the greenhouse study. .................................................................. 111 A-2 Mean of root and above ground biomass of Las-inoculated Carrizo citrange seedlings in the greenhouse study ................................................................... 112 7 LIST OF FIGURES Figure page 2-1 Principal Component Analysis (PCA) of the 3 healthy Cleopatra rhizosphere samples and 3 HLB Cleopatra rhizosphere samples .......................................... 65 2-2 Rarefaction curve of annotated species richness. ............................................. 66 2-3 Taxonomic assignment of healthy Cleopatra rhizosphere metatranscriptome.