BIOLOGICAL CONTROL OF Imperata cylindrica IN WEST AFRICA USING FUNGAL PATHOGENS By ALANA DEN BREEYEN 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 2007 1 © 2007 Alana Den Breeyen 2 To my parents, Henk and Pathia Den Breeÿen 3 ACKNOWLEDGMENTS I thank my major professor, Dr. Raghavan Charudattan, for his belief in me even at times when it didn’t seem justified and for giving me the opportunities to grow professionally. I thank Dr. Fen Beed, for helping me realize that if I’m not part of the solution then I am part of the problem and for his generous hospitality and friendship during my time at IITA, Cotonou, Benin. I also thank my committee members, Dr Jeffrey Rollins, Dr. Greg MacDonald, and Dr. Fredy Altpeter for their time, interest and support. My thanks go to the many people who have helped me with my research: Jim DeValerio, for always being a willing ’driver’ and one of my first friends in Gainesville, Mark Elliott, whose love and friendship has made life here well worth the journey, to Gabriella Maia, for her love and friendship and willingness to sit in the midday sun harvesting cogongrass rhizomes, and to Adolphe Avocanh who willing shared his office, expertise and friendship during my time at IITA, Benin. My thanks go to Eldon Philman and Herman Brown for the help they provided during the course of my experiments. Thanks go to my former labmates, Jennifer Cook and Jonathan Horrell. My special thanks go to my lab mate, Abby Guerra, who often put up with more than he should have and whose special friendship has brightened my days. To the students and staff at IITA, Benin who were always willing to help with my research and for reminding me to slow down, merci beaucoup! Thanks to Ms. Gail Harris, Ms. Donna Perry, Ms. Lauretta Rahmes, Ms. Jan Sapp, Ms. Sherri Mizell and Ms. Dana Lecuyer who helped me in their own special way. My thanks go to Mark and Maria Minno for allowing me access to their cogongrass collection and Meghan Brenan for help with my statistical data analysis. My special thanks to Sarah Clark Selke, Linley Smith, Tara Taranowski, Heidi HansPetersen, and Amanda Watson, the amazingly talented women who have become my friends. 4 My special thanks go to my Mom and Dad, who made the realization of the dream that much easier, and to my brother and sister-in-law, Ashley and Izelle for their love and support. To all my friends, back home and around the world, thank you for your special blend of friendship. 5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................8 LIST OF FIGURES .......................................................................................................................10 ABSTRACT...................................................................................................................................12 CHAPTER 1 INTRODUCTION ..................................................................................................................14 Biology and Morphology........................................................................................................15 Cogongrass in the United States.............................................................................................16 Cogongrass in West Africa.....................................................................................................18 Biological Control Strategies for Forest Weeds .....................................................................20 Biological Control Strategies for Cogongrass ........................................................................26 Potential Insect and Nematode Biocontrol Agents..........................................................27 Potential Plant Pathogenic Biocontrol Agents ................................................................28 Mycoherbicides ...............................................................................................................30 Genetic Variation in Invasive Weeds: The Implication for Biological Control.....................31 Biological Control and Genetic Markers................................................................................33 2 AN EXAMINATION OF GENETIC DIVERSITY OF Imperata cylindrica BASED ON INTER-SIMPLE SEQUENCE REPEATS AND SEQUENCE ANALYSIS OF INTERNAL TRANSCRIBED AND CHLOROPLAST TRNL-F SPACER DNA ................36 Introduction.............................................................................................................................36 Materials and Methods ...........................................................................................................40 Population Sampling .......................................................................................................40 Extraction of Genomic DNA and PCR Amplification of ISSR Population Markers......40 Results.....................................................................................................................................44 Analysis of ISSR Markers...............................................................................................44 Analysis of ITS................................................................................................................45 Analysis of trnL-F ...........................................................................................................47 Discussion...............................................................................................................................47 3 EVALUATION OF FUNGAL PATHOGENS AS BIOLOGICAL CONTROL AGENTS OF Imperata cylindrica (L.) BEAUV. FROM THE USA AND BENIN..............60 Introduction.............................................................................................................................60 Materials and Methods ...........................................................................................................62 Propagation of Test Plants...............................................................................................62 Inoculum Production .......................................................................................................63 6 Imperata cylindrica Inoculations ....................................................................................64 Efficacy of Bipolaris sacchari and Drechslera gigantea Applied Singly or as a Mixture.........................................................................................................................64 Statistical Analysis ..........................................................................................................65 Results.....................................................................................................................................65 Efficacy of Bipolaris sacchari and Drechslera gigantea Applied Singly or as a Mixture.........................................................................................................................65 Florida Imperata cylindrica Accessions and Fungal Isolates..........................................65 Benin Imperata cylindrica Accessions and Fungal Isolates............................................68 Discussion...............................................................................................................................69 4 EVALUATION OF OBLIGATE BIOTROPHIC FUNGI ASSOCIATED WITH Imperata cylindrica IN SOUTH AFRICA AS POTENTIAL BIOCONTROL AGENTS FOR COGONGRASS IN WEST AFRICA............................................................................92 Introduction.............................................................................................................................92 Materials and Methods ...........................................................................................................95 Propagation of Test Plants...............................................................................................95 Source of Pathogens and Inoculum .................................................................................95 Treatments and Experimental Design .............................................................................96 Results.....................................................................................................................................96 Discussion...............................................................................................................................97 5 SUMMARY AND CONCLUSIONS...................................................................................103 APPENDIX A FIELD EVALUATION OF THE INDIGENOUS FUNGUS, Colletotrichum caudatum, ASSOCIATED WITH COGONGRASS IN BENIN ...........................................................108 Introduction...........................................................................................................................108 Materials and Methods .........................................................................................................111 Inoculum Production .....................................................................................................111 Inoculation.....................................................................................................................111