ENHANCEMENT OF BIOLOGICAL CONTROL OF ANTHURIUM BLIGHT CAUSED BY Xanthomonas axonopodis pv. dieffenbachiae A THESIS SUBMITTED TO THE GRADUATE DIVISION OF THE UNIVERSITY OF HAW AI'I IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN TROPICAL PLANT AND SOIL SCIENCES MAY 2008 By PETER 1. TOVES Thesis Committee: Anne Alvarez, Chairperson Richard Criley Adelheid Kuehnle Hector Valenzuela We certify that we have read this thesis and that, in our opinion, it is satisfactory in scope and quality as a thesis for the degree of Master of Science in Tropical Plant and Soil Sciences. THESIS COMMITIEE Chairperson 12ubJJ. ~ 4J..eLIM A t.1I. QL b. I",,J.. ii To my grandmothers, Josefina G. Toves and Rosario Villamor iii ACKNOWLEDGEMENTS I would like to thank the Almighty for blessing me with the opportunity to achieve this degree. Thank you to my committee members who have provided resources, guidance, and suggestions to complete my research. My sincerest gratitude to Dr. Anne Alvarez for giving me the opportunity to not only learn about plant pathology, but also about life itself. I would like to thank my fellow lab mates for all the support, the late hours, and most especially the fun times. Special thanks to Wendy Sueno for helping me through the more difficult times during my graduate work. I thank Dr. Tessie Amore for all her help, patience, and expert advice on anthurium micropropagation. I would like to thank Drs. Mark Wright and Ian Pagano for helping me with the statistics. Thanks to Grayson Inouye for providing space to run our field experiments in Hilo. Thank you to the secretaries from TPSS and PEPS, but most especially to Ms. Susan Takahashi for looking out for me and keeping me on track with all my graduate requirements. I thank Emily Lloyd for reviewing my thesis at the last minute. I would like to thank the Toves and Calimlim families for all the moral support and encouragement through the duration of my research. iv ABSTRACT Anthuriums are Hawaii's signature cut flower, and optimal growth and protection of anthurium plants are crucial to the Hawaiian floriculture industry. Anthurium blight, caused by Xanthomonas axonopodis pv. dieffenbachiae (Xad), is the most destructive disease of anthurium worldwide. Beneficial bacteria have been identified for use as biological control agents (BCAs) against Xad and these strains have also been shown to stimulate growth of micro-propagated plants. Optimization of transplanted microplant growth was examined. Anthurium microplants grew better with the combination of inorganic fertilizer combined with BCAs than when grown in either fertilizer or BCAs alone. Biostimulation was observed on all anthurium cultivars treated with the beneficial strains. Populations of beneficial bacteria decline after foliar application on anthurium plants. Studies were focused on improving the efficacy of the BCAs with carbon sources that sustain beneficial bacterial populations on plant surfaces without stimulating pathogen growth. Valine and isoleucine were identified as amino acids that enabled growth of beneficial bacteria while inhibiting growth of the pathogen in vitro. In greenhouse and field studies, treatments with valine combined with the BCAs reduced disease incidence by 12 to 21 % compared to treatments with BCAs alone. Key words: Biological control agents, beneficial bacteria, feedback inhibition, amino acid v TABLE OF CONTENTS ACKNOWLEDGEl\1ENTS ........................................................................iv ABSTRACT ...........................................................................................v LIST OF TABLES .................................................................................. .ix LIST OF FIGURES ..................................................................................xi LIST OF ABBREVIATIONS ..................................................................... xiii CHAPTER 1: Literature Review .................................................................. 1 Anthurium ..................................................................................... 1 Economic importance ....................................................................... 2 Production practices ......................................................................... 3 Special practices for greenhouse grown anthuriums ........................... 5 Major diseases ................................................................................6 Fungal diseases .......................................................................6 Bacterial diseases ................................................................... 6 Previous studies on anthurium blight .......................................................8 Disease control of blight .......................................................................................... 8 Cultural practices for control ofblight... ..................................................... 8 Breeding anthuriums for resistance to anthurium blight ........................... l 0 Genetic Engineering for Resistance to Anthurium Blight ................... 10 Tissue cultured anthurium ....................................................... 11 Biological control of anthurium blight ......................................... 11 Feedback inhibition of amino acid pathways ........................................... 12 Literature cited .............................................................................. 13 vi CHAPTER 2: Use of Selected Amino Acids to Enhance Effectiveness of Beneficial Bacteria in Biocontrol of Anthurium Blight ............................................ 20 Abstract ......................................................................................20 Introduction ..................................................................................21 Materials and Methods .....................................................................23 Bacterial strains and inoculum preparation .....................................23 Bacterial growth on various carbon sources ...................................24 Greenhouse studies ................................................................25 Field Studies ....................................................................... 27 Results ........................................................................................28 Bacterial growth on various carbon sources ................................... 28 Greenhouse studies ................................................................ 31 Field studies ........................................................................31 Discussion ................................................................................... 32 Literature Cited ............................................................................. 44 CHAPTER 3: Optimizing Growth of Anthurium Microplants with Mineral Nutrients and Beneficial Bacteria .......................................................... .48 Abstract ..................................................................................... .48 Introduction ................................................................................ .48 Materials and Methods .....................................................................49 Plant materials and growth conditions ..........................................49 Bacterial strains and inoculum preparation .................................... 49 vii Effects of fertilizer treatments and beneficial bacteria on anthurium microplants .........................................................................50 Bacterial growth on various mineral solutions ................................ 51 Effects of mineral solutions on anthurium microplants ......................52 Results ....................................................................................... 53 Effects of fertilizer treatments and beneficial bacteria on anthurium microplants .........................................................................53 Bacterial growth on various mineral solutions .......................................... 54 Effects of mineral solutions on anthurium microplants ...................... 54 Discussion ....................................................................................54 Literature Cited .............................................................................61 APPENDICES A. Susceptibility ofAnthurium antioquense 'Cotton Candy' to anthurium blight and bioprotection by beneficial bacteria ......................................... 62 B. Bioprotection of five cultivars of .anthurium microplants ................................ 65 C. Comparison of anthurium microplants treated with beneficial bacteria and two inoculum levels ofXanthomonas axonopodis pv. dieffenbachiae .................... 69 D. Feedback inhibition of valine, leucine, and isoleucine biosynthesis ...................... 74 E. Overall conclusions ............................................................................. 75 viii LIST OF TABLES Tables 2-1 Relationship between viable cell counts and turbidity (measured in Klett units) for bacterial strains used in growth curve studies ....................................... 35 2-2 Oxidation and growth of beneficial bacteria on carbon sources not utilized by Xanthomonas axonopodis pv. dieifenbachiae ... ....................................... 36 2-3 Growth of beneficial bacteria and Xanthomonas axonopodis pv. dieifenbachiae on amino acids supplied as sole carbon sources in a standard mineral