FUNGAL ASSOCIATES OF KNOWN AND PUTATIVE VECTORS OF LAUREL WILT AND NUTRITIONAL ASPECTS OF THE LAUREL WILT PATHOGEN RAFFAELEA LAURICOLA By JOSÉ RAMÓN SAUCEDO CARABEZ 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 2018 © 2018 José Ramón Saucedo Carabez To the Creator, my parents Javier Saucedo Zuñiga † and Teresa Carabez Barragan and those who are persistent and follow their dreams ACKNOWLEDGMENTS I am deeply grateful for the unconditional love, patience and dedication of my parents, Javier Saucedo Zuñiga † and Teresa Carabez Barragan, without whom I would not have been able to achieve my dreams. I also thank Manuela Ángel Restrepo for her unconditional love and support. I thank the Consejo Nacional de Ciencia y Tecnologia (CONACYT) for a scholarship and NIFA grant # 2015-51181-24257 for supporting my research program. I appreciate the opportunity provided by Dr. Randy Ploetz to complete my Ph.D. degree as well as to Dr. Salvador Ochoa for his friendship, advice and motivation. I would also like to thank my committee members Dr. Jason Smith and Dr. Jeffrey Rollins for their contributions to my research project. I am grateful to José Pérez Martínez, Joshua Konkol and Randy Fernandez and all the people who made my experience at the University of Florida pleasant. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES ...........................................................................................................................7 LIST OF FIGURES .........................................................................................................................8 ABSTRACT .....................................................................................................................................9 CHAPTER 1 INTRODUCTION ..................................................................................................................11 2 REVIEW OF LITERATURE .................................................................................................14 Avocado ..................................................................................................................................14 Laurel Wilt ..............................................................................................................................15 Distribution ......................................................................................................................15 Symptoms ........................................................................................................................15 Disease Cycle ..................................................................................................................16 Host Range of Laurel Wilt ..............................................................................................17 Disease Management .......................................................................................................19 Raffaelea lauricola .................................................................................................................21 Nutritional Symbiosis of Ambrosia Fungi ..............................................................................23 Xyleborus glabratus and Vectors of Raffaelea lauricola .......................................................24 Tree Diseases Associated with Bark and Ambrosia Beetles ..................................................26 Ambrosia Beetles Associated with Avocado ..........................................................................29 Host Responses to Raffaelea lauricola ...................................................................................30 Plant Defenses Responses to Vascular Wilt Diseases ............................................................30 3 PARTNERSHIPS BETWEEN AMBROSIA BEETLES AND FUNGI: LINEAGE- SPECIFIC PROMISCUITY AMONG VECTORS OF THE LAUREL WILT PATHOGEN, RAFFAELEA LAURICOLA ............................................................................33 Background .............................................................................................................................34 Materials and Methods ...........................................................................................................37 Beetle sampling ...............................................................................................................37 Symbiont Isolation...........................................................................................................37 DNA Extraction, PCR Amplification and Sequencing ...................................................38 Phylogenetic Analyses.....................................................................................................39 Statistical Analysis ..........................................................................................................40 Results .....................................................................................................................................40 Isolation of R. lauricola from Ambrosia Beetles ............................................................40 Communities of Microbial Symbionts ............................................................................41 Discussion ...............................................................................................................................42 5 4 NUTRITIONAL SYMBIONTS OF A PUTATIVE VECTOR, XYLEBORUS BISPINATUS, OF THE LAUREL WILT PATHOGEN OF AVOCADO, RAFFAELEA LAURICOLA ...........................................................................................................................61 Background .............................................................................................................................62 Materials and Methods ...........................................................................................................66 Symbiont Recovery and Identification ............................................................................66 Artificial Media and Inoculation with Fungal Symbionts ...............................................66 Feeding of Unfertilized Females and Colony Initiation ..................................................67 Data Collection ................................................................................................................67 Statistical Analyses ..........................................................................................................68 Results .....................................................................................................................................69 Symbiont Identification ...................................................................................................69 Colony Initiation ..............................................................................................................70 Dissection of Artificial Diets ...........................................................................................70 Mycangia and Gallery Content and Location of Brood Chamber ...................................71 Discussion ...............................................................................................................................72 5 MINERAL CONTENT OF AMBROSIA FUNGI, XYLEM ENDOPHYTES FROM AVOCADO AND HOST AND NON-HOST XYLEM TISSUE ..........................................84 Background .............................................................................................................................84 Materials and Methods ...........................................................................................................87 Source of Samples ...........................................................................................................87 Identification of Fungi .....................................................................................................87 Fungal Biomass Production and Colonization Assays ....................................................88 Mineral Content ...............................................................................................................89 Statistical Analysis ..........................................................................................................89 Results .....................................................................................................................................89 Colonization of Avocado Wood ......................................................................................89 Mineral Content ...............................................................................................................90 Discussion ...............................................................................................................................93 6 CONCLUSIONS ..................................................................................................................102 LIST OF REFERENCES .............................................................................................................105 BIOGRAPHICAL SKETCH .......................................................................................................127 6 LIST OF TABLES Table page 3-1 Geographic location of different sampling sites in this study............................................49