University of New Hampshire University of New Hampshire Scholars' Repository Master's Theses and Capstones Student Scholarship Fall 2015 THE DYNAMIC HOST-PATHOGEN INTERACTIONS OF VERTICILLIUM DAHLIAE: COMPARATIVE GENOMICS OF V. DAHLIAE AND ITS INTERACTIONS IN COVER CROP SPECIES AND WILD STRAWBERRY (FRAGARIA VESCA) Garrett Gleeson University of New Hampshire, Durham Follow this and additional works at: https://scholars.unh.edu/thesis Recommended Citation Gleeson, Garrett, "THE DYNAMIC HOST-PATHOGEN INTERACTIONS OF VERTICILLIUM DAHLIAE: COMPARATIVE GENOMICS OF V. DAHLIAE AND ITS INTERACTIONS IN COVER CROP SPECIES AND WILD STRAWBERRY (FRAGARIA VESCA)" (2015). Master's Theses and Capstones. 1037. https://scholars.unh.edu/thesis/1037 This Thesis is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Master's Theses and Capstones by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. THE DYNAMIC HOST-PATHOGEN INTERACTIONS OF VERTICILLIUM DAHLIAE: COMPARATIVE GENOMICS OF V. DAHLIAE AND ITS INTERACTIONS IN COVER CROP SPECIES AND WILD STRAWBERRY (FRAGARIA VESCA) BY Garrett Gleeson BS, Allegheny College, 2012 THESIS Submitted to the University of New Hampshire in Partial Fulfillment of the Requirements for the Degree of Master of Science September, 2015 This thesis has been examined and approved in partial fulfillment of the requirements for the degree of Maters in Microbiology by: Thesis Director, Dr. Kirk Broders, Assistant Professor (Biological Sciences) Dr. Thomas Davis, Professor of Plant Biology (Biological Sciences) Dr. Louis Tisa, Professor (Molecular, Cellular, & Biomedical Sciences) On June 25th, 2015 Original approval signatures are on file with the University of New Hampshire Graduate School ii ACKNOWLEDGMENTS I would like to express my deep gratitude to Kirk Broders for his roles as thesis director and mentor. Kirk dedicated countless hours to bring invaluable insight and guidance to this project. I would like to offer my special thanks to my committee members, Louis Tisa and Tom Davis, who provided necessary materials and critique. I would like to thank all of my current and former lab mates: Andre Boraks, Matthew Wallhead, Franz Lichtner, Kate Blasioli, Stephen Wyka, Taruna Aggarwal, Gloria Broders, and the numerous undergraduates that have come through the lab for their support and friendship. I would also like to thank those who assisted with this project outside the Broder’s lab: Mark Townley, Jonathan Ebba, David Goudreault, Lise Mahoney, Kelley Thomas, Jordan Ramsdell, and Sheldon Hurst. To the following organizations that provided funding towards my research: the United States Department of Agriculture, University of New Hampshire Agriculture Experiment Station, and the Molecular, Cellular and Biomedical Sciences Department. Finally I would like to thank my friends and family, especially my best friend Sabah, for all of their support. iii TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................ iii FIGURES ........................................................................................................................ vi TABLES ......................................................................................................................... viii ABSTRACT .................................................................................................................... ix INTRODUCTION ........................................................................................................... 1 1.1 Scope of Thesis .............................................................................................. 1 1.2 Disease Management in Strawberry ............................................................. 3 1.3 Verticillium dahliae ....................................................................................... 4 1.4 Objectives ...................................................................................................... 6 CHAPTER 2: COVER CROP INFECTION ................................................................. 7 2.1 Introduction ................................................................................................... 7 2.2 Methods ......................................................................................................... 8 2.2.1 Cover Crop Germination ............................................................... 8 2.2.2 Verticillium Preparation/Inoculation ............................................ 9 2.2.3 Confocal Imaging ........................................................................... 9 2.3 Results ............................................................................................................ 10 2.3.1 Greenhouse Infection Assay ........................................................... 10 2.3.2 Confocal Imaging ........................................................................... 11 2.4 Discussion ...................................................................................................... 11 2.4.1 Infection of Tolerant Hosts ............................................................. 11 2.4.2 Future Work .................................................................................... 13 CHAPTER 3: FRAGARIA VESCA-VERTICILLIUM DAHLIAE INTERACTIONS ......................................................................................... 16 3.1 Introduction ................................................................................................... 16 3.2 Methods ......................................................................................................... 17 3.2.1 Strawberry Growth Conditions ...................................................... 17 3.2.2 Verticillium Growth Conditions ..................................................... 17 3.2.3 Inoculation Assay ........................................................................... 18 3.2.4 Visual Disease Assessment ............................................................. 18 3.2.5 Confocal Microscopy ..................................................................... 19 3.3 Results ............................................................................................................ 20 3.3.1 Lateral Root Infection ..................................................................... 20 iv 3.3.2 Primary Root and Stem Infection ................................................... 20 3.3.3 Ordinal Rating ................................................................................ 21 3.4 Discussion ...................................................................................................... 21 3.4.1 Resistant Phenotypes ...................................................................... 21 3.4.2 Lateral Root Infection ..................................................................... 22 CHAPTER 4: COMPARATIVE GENOMICS OF VERTICILLIUM DAHLIAE .......................................................................................... 31 4.1 Introduction ................................................................................................... 31 4.2 Methods ......................................................................................................... 34 4.2.1 Isolates Information ....................................................................... 34 4.2.2 De novo Assembly ........................................................................... 34 4.2.3 Making Reads Compatible with BWA MEM .................................. 34 4.2.4 Variant Discovery ........................................................................... 35 4.2.5 Generating Phylogenetic Tree ........................................................ 35 4.2.6 Mauve Analysis ............................................................................... 36 4.3 Results ............................................................................................................ 36 4.3.1 De novo Assemblies ........................................................................ 36 4.3.2 Variant Discovery ........................................................................... 36 4.3.3 Phylogenetic Analysis ..................................................................... 37 4.3.4 SNP Distribution ............................................................................. 37 4.3.5 Mauve Analysis ............................................................................... 38 4.4 Discussion ...................................................................................................... 38 4.4.1 Synteny ............................................................................................ 38 4.4.2 Host Specificity ............................................................................... 39 REFERENCES ................................................................................................................ 52 v FIGURES Figure 2.1: Infection of Cover Crops by V. dahliae. Confocal images of GFP-expressing V. dahliae overlaid with transmitted light images of Buckwheat (A&B), Field Pea (C), Crimson Clover (D), and Winter Rye (E) roots ten days post-inoculation. Figure 3.1: Strawberry growth conditions. Strawberry plants used in inoculation assays were initially harvested as plantlets from runners (A), then rooted