CHARACTERIZATION OF SYMBIOTIC MYCOFLORA OF NON-NATIVE AMBROSIA BEETLES By CRAIG CHRISTOPHER BATEMAN A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2014 © 2014 Craig Christopher Bateman To my parents ACKNOWLEDGMENTS I am greatly thankful to have received the guidance, support, time and patience of my committee chair Jiri Hulcr, and committee members Jason Smith and Matthew Smith. My committee has helped me grow as a scientist and person, while also providing welcome and enjoyment as a member of the lab and as a friend. For discussions, technical aids, and help navigating various analyses, I would like to thank the members of the Hulcr lab; Martin Kostovcik, Caroline Storer, Andrew Johnson, and Polly Harding, and members of the Forest Pathology lab; Adam Black, Tyler Dreaden, and Marc Hughes. I would like to thank Martin Sigut for his help extracting fungi from the black twig borer and for help with statistical analyses. I am also thankful for the advice and help on Fusarium from Kerry O’Donnell and Stacy Sink. For use of quarantined research facilities, I thank Dr. Timothy Schubert. For help with beetle collections, I am thankful for Dr. Paul Kendra and Chris Gibbard. I would also like to thank collaborators Wang Bo and Wisut Sittichaya for which work overseas would have not been possible. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................. 4 LIST OF TABLES ............................................................................................................ 7 LIST OF FIGURES .......................................................................................................... 8 ABSTRACT ..................................................................................................................... 9 CHAPTER 1 THE MYCANGIAL SYMBIONT OF THE BLACK TWIG BORER,Xylosandrus compactus (COLEOPTERA: CURCULIONIDAE, SCOLYTINAE) IS SPATIALLY SEGREGATED. ...................................................................................................... 11 Introduction ............................................................................................................. 11 Methods .................................................................................................................. 14 Sampling and Isolation ..................................................................................... 14 DNA Sequence Acquisition and Phylogenetic Analysis .................................... 15 Statistical Analysis ............................................................................................ 17 Results .................................................................................................................... 17 Fungi Associated with Xylosandrus compactus ................................................ 17 The Two Dominant Fungal Associates are Spatially Segregated during Transport ....................................................................................................... 18 Discussion .............................................................................................................. 19 2 FUNGI ASSOCIATED WITH EXOTIC XYLEBORINI AMBROSIA BEETLES IN FLORIDA ................................................................................................................ 27 Introduction ............................................................................................................. 27 Methods .................................................................................................................. 30 Beetle Collections and Fungal Isolations .......................................................... 30 DNA Sequence Acquisition and Analysis ......................................................... 31 Results .................................................................................................................... 32 Fungi Associated with Exotic Xyleborini ........................................................... 32 DNA Sequencing and Phylogenetic Analysis ................................................... 33 Discussion .............................................................................................................. 34 3 PREDICTING FUTURE INVADERS; ARE UNKNOWN PATHOGENS HIDING IN ASIA? ................................................................................................................. 44 Introduction ............................................................................................................. 44 The “Wait-and-see” Approach .......................................................................... 46 Preventing the Next Tree Epidemic .................................................................. 47 Methods .................................................................................................................. 49 5 Study Sites and Beetle Collection .................................................................... 49 Fungal Isolation ................................................................................................ 49 Molecular Identification and Phylogenetic Analysis .......................................... 50 Pathogenicity Test ............................................................................................ 52 Results .................................................................................................................... 53 Fungal Isolations and Identification .................................................................. 53 Pathogenicity Test ............................................................................................ 54 Discussion .............................................................................................................. 54 LIST OF REFERENCES ............................................................................................... 61 BIOGRAPHICAL SKETCH ............................................................................................ 68 6 LIST OF TABLES Table page 1-1 Primers used for PCR and DNA sequencing ...................................................... 23 1-2 Average number of fungal colonies from different parts of Xylosandrus compactus. ......................................................................................................... 23 1-3 Prevalence a of fungi isolated from different parts of Xylosandrus compactus. ... 24 1-4 Presence (+)/absence (-) of fungi isolated from beetle galleries from three localities in Gainesville, FL ................................................................................. 25 2-1 Prevalence and abundance of fungi isolated from exotic Xyleborini ................... 38 2-2 Combined reports of Xyleborini fungal symbionts in the literature and present study ................................................................................................................... 38 3-1 Prevalence and abundance of fungi associated with Asian bark beetles sampled. ............................................................................................................. 58 3-2 Mean number of weeks seedlings displayed resin production in loblolly pine genotype 1 inoculated with Asian fungi. ............................................................. 58 3-3 Mean number of weeks seedlings displayed resin production in loblolly pine genotype 2 inoculated with Asian fungi. ............................................................. 59 3-4 Mean number of weeks seedlings displayed resin production in slash pine inoculated with Asian fungi. ................................................................................ 59 3-5 Mean vertical xylem lesion length in loblolly pine genotype 1 inoculated with Asian fungi. ......................................................................................................... 59 3-6 Mean vertical xylem lesion length in loblolly pine genotype 2 inoculated with Asian fungi. ......................................................................................................... 59 3-7 Mean vertical xylem lesion length in slash pine inoculated with Asian fungi. ...... 60 3-8 Mean vertical phloem lesion length in loblolly pine genotype 1 inoculated with Asian fungi. ......................................................................................................... 60 3-9 Mean vertical phloem lesion length in loblolly pine genotype 2 inoculated with Asian fungi. ......................................................................................................... 60 3-10 Mean vertical phloem lesion length in slash pine inoculated with Asian fungi. ... 60 7 LIST OF FIGURES Figure page 1-1 Maximum likelihood-based identification of Xylosandrus compactus mycangial isolate of Ambrosiella xylebori with Raffaelea lauricola used as an outgroup. ............................................................................................................ 25 1-2 Principal Correspondence Analysis (PCA) ordination biplot with histogram of explained cummulative variation. PCA biplot shows beetle parts colonized by different fungi. ..................................................................................................... 26 2-1 Maximum likelihood-based identification of Xylosandrus amputatus mycangial isolate Ambrosiella beaveri with Microascus cirrosus used as an outgroup ............................................................................................................