An Investigation Into the Role of Genetics in the Tolerance
Total Page:16
File Type:pdf, Size:1020Kb
AN INVESTIGATION INTO THE ROLE OF GENETICS IN THE TOLERANCE OF TEXAS LIVE OAKS TO Ceratocystis fagacearum A Dissertation by MYRON CROWLEY GRAY Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2007 Major Subject: Plant Pathology AN INVESTIGATION INTO THE ROLE OF GENETICS IN THE TOLERANCE OF TEXAS LIVE OAKS TO Ceratocystis fagacearum A Dissertation by MYRON CROWLEY GRAY Submitted to the Office of Graduate Studies of Texas A&M University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Approved by: Chair of Committee, David A. Appel Committee Members, Bruce A. McDonald Heather H. Wilkinson Michael A. Arnold Head of Department, Dennis C. Gross May 2007 Major Subject: Plant Pathology iii ABSTRACT An Investigation into the Role of Genetics in the Tolerance of Texas Live Oaks to Ceratocystis fagacearum. (May 2007) Myron Crowley Gray, B.S., Colorado State University; M.S., Colorado State University Chair of Advisory Committee: Dr. David A. Appel The fungus Ceratocystis fagacearum (Bretz) Hunt causes the vascular disease of oak wilt and has been decimating live oaks (Quercus virginiana Mill. and Quercus fusiformis Small.) and red oaks (Quercus texana Small and Quercus marilandica Muenchh.) in Texas. The purpose of this research was to test the hypotheses that live oaks have heritable tolerance to oak wilt, and that allozyme markers are associated with this tolerance. One-year-old half-sib and two-year-old clonal progeny of live oaks (Q. fusiformis) were grown from acorns and ramets from a disease center and then challenged with C. fagacearum. Allozyme analyses were used to compare the pre- and post-epidemic populations in two natural disease centers to search for alleles associated with survivability and decreased crown loss. Half-sib and clonal challenge tests supported the hypothesis that heritable tolerance to the pathogen occurs in live oaks. The progeny tolerances seen in half-sib and clonal groups did not correlate with parental tree performance. This finding suggests that the tolerance of one-year-old seedlings in the greenhouse setting is not a good predictor of how mature trees will do in a natural setting. Seedlings may not be a good model for testing tolerance to a pathogen. The ability to survive this vascular pathogen is containment, and seedlings may be too small to test this type of tolerance. The clonal groups from post-epidemic trees performed better than the seedlings. They may have an increased resistance because they are mature or they may have a post- disease immunity. No significant allele frequencies between pre- and post-epidemic trees were consistent among sites or with previous research. The different disease sites had remarkably similar allele frequencies which indicate high levels of gene flow among iv sites. Both sites were found to contain significant numbers of clones, but the Izoro site had significantly larger clonal groups. Sites were in Hardy-Weinberg equilibrium which indicates substantial sexual reproduction and not just clonal reproduction is taking place. Several cases of linkage disequilibrium occurred at the Izoro site, but population structure was responsible in all but one case. v DEDICATION I owe a great debt to Margaret Mossman and Mary Burger for being mentors throughout my life. I am very lucky to have known such inspiring souls. vi ACKNOWLEDGEMENTS I would like to thank my committee chair, Dr. Appel, and my committee members, Dr. McDonald, Dr. Wilkinson, and Dr. Arnold, for their guidance and support throughout the course of this research. I want to thank to Dr. Starr for taking the time to serve as a substitute during my preliminary exams and Dr. Hart for help with data interpretation. I also want to thank Francis Pate for constructing the mist chamber that was central to this research and Tom Kurdyla for preparing fungal inoculum that was also central to this project. I would also like to thank the department faculty for making my time at Texas A&M University a positive experience. Your patience was much appreciated. I also thank my family for their encouragement and support vii TABLE OF CONTENTS Page ABSTRACT......................................................................................................... iii DEDICATION..................................................................................................... v ACKNOWLEDGEMENTS................................................................................. vi TABLE OF CONTENTS..................................................................................... vii LIST OF TABLES............................................................................................... x LIST OF FIGURES ............................................................................................. xi CHAPTER I INTRODUCTION.......................................................................... 1 II LITERATURE REVIEW............................................................... 4 Prior Research......................................................................... 4 Live oak taxonomy ............................................................ 4 Disease history, impact, and control.................................. 5 Epidemiology..................................................................... 8 Present Research ..................................................................... 13 Allozymes .......................................................................... 13 III SEEDLING AND CLONE EXPERIMENTS ............................... 16 Materials and Methods............................................................. 17 Acorn collection................................................................. 17 Growth of seedlings ........................................................... 20 Inoculation procedure ........................................................ 20 Half-sib analysis................................................................. 20 Evaluation of post-inoculation seedlings........................... 21 Half-sib group comparisons............................................... 21 Clonal group comparisons ................................................. 21 Comparative analyses performed....................................... 22 Statistical methods ............................................................. 23 viii CHAPTER Page Results...................................................................................... 25 Comparison among live oak half-sib groups for tolerance to oak wilt ...................................... 25 Comparison of live oak parents and half-sib progeny for tolerance to oak wilt.................................... 30 Comparison between pre- and post-epidemic live oak seedling tolerances to oak wilt .......................... 39 Comparison of stem inoculation versus root inoculation of live oak seedlings ............................. 39 Comparison of stem height among live oak half-sib seedling groups ........................................... 39 Comparison of clonal resistance to parental resistance of live oaks ....................................... 42 Comparison of latent period between inoculated live oak seedlings from pre- and post-epidemic parents ....................................... 48 Comparison of ramet production between live oak groups.................................................. 48 IV POPULATION EXPERIMENTS.................................................. 49 Materials and Methods............................................................. 50 Locations of live oak leaf collections ............................... 50 Tree Sampling................................................................... 51 Allozyme procedure.......................................................... 51 Gel evaluation ................................................................... 53 Statistical analyses ............................................................ 53 Results...................................................................................... 56 Allele frequencies of populations ..................................... 56 Alleles compared for average crown loss ......................... 60 Genotype diversity and additional genotype characteristics................................................. 61 Hardy-Weinberg (H-W) equilibrium ................................ 65 Linkage-disequilibrium..................................................... 66 F-statistics ......................................................................... 70 Genetic distance and similarity......................................... 70 Heterozygosity .................................................................. 71 ix CHAPTER Page V SUMMARY AND CONCLUSIONS .............................................. 73 Recommendations for Future Research.................................. 94 LITERATURE CITED ........................................................................................ 96 APPENDIX A...................................................................................................... 106 APPENDIX B ...................................................................................................... 107 APPENDIX C ...................................................................................................... 109 APPENDIX D...................................................................................................... 110 APPENDIX