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Using Molecular Analysis to Investigate Phylogenetic Relationships

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Using Molecular Analysis to Investigate Phylogenetic Relationships USING MOLECULAR ANALYSIS TO INVESTIGATE PHYLOGENETIC RELATIONSHIPS IN TWO TROPICAL PATHOSYSTEMS: WITCHES’ BROOM OF CACAO, CAUSED BY MONILIOPHTHORA PERNICIOSA, AND MANGO ANTHRACNOSE, CAUSED BY COLLETOTRICHUM SPP. By TARA LUANA BARRETT TARNOWSKI 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 2009 1 © 2009 Tara Luana Barrett Tarnowski 2 To David, for the endless love and support 3 ACKNOWLEDGMENTS There are so many people that contribute support to a student during dissertation research, and I want to thank everyone who pitched in. I would especially like to thank my committee members Dr. Aime, Dr. Palmateer, Dr. Ploetz, Dr. Rollins, and Dr. Soltis for their instruction, advice, and support. I also appreciate all the help I received from José Pérez, Patricia Lopez, and Gail Harris. For the financial support I received for research, I thank IFAS, the Florida Mango Forum, and the Redland’s Citizen Association. I also received assistance for travel to professional meetings from the Graduate School, the Department of Plant Pathology, and the Mycological Society of America. Many people also contributed material support for my research. Drs. Harry Evans and Robert Barreto donated Moniliophthora perniciosa isolates for phylogenetic analysis. Limeco, LLC and Brooks Tropicals, Inc. donated fruit for pathogenicity experiments. Lastly I want to thank all the friends and family for the love and support they gave me. I could not have completed all of this without them. I want to especially thank David, Trevor, Mom, and Dad, for always believing in me and giving me strength when I need it the most, and for always reminding me what is important in life. 4 TABLE OF CONTENTS page ACKNOWLEDGMENTS ...............................................................................................................4 LIST OF TABLES...........................................................................................................................8 LIST OF FIGURES .......................................................................................................................10 CHAPTER 1 INTRODUCTION ..................................................................................................................14 Current State of Fungal Systematics.......................................................................................14 Classification of Different Phyla of Fungi ......................................................................15 Species Characterization in Fungi...................................................................................16 Species defined by morphology ...............................................................................16 Biological species concept .......................................................................................19 Phylogenetic species concept ...................................................................................21 Evolutionary species concept ...................................................................................24 The Role of Plant Pathology in Fungal Systematics .......................................................25 Witches’ Broom of Cacao ......................................................................................................29 Colletotrichum-induced Anthracnoses: Plagues to Tropical Fruit .........................................36 Taxonomic History of Colletotrichum ............................................................................36 Colletotrichum gloeosporioides as the Cause of Mango Anthracnose ..........................40 2 INVESTIGATING THE RELATIONSHIPS AMONG BIOTYPES OF MONILIOPHTHORA PERNICIOSA USING PHYLOGENIES FROM MULTIPLE DNA REGIONS .....................................................................................................................50 Introduction.............................................................................................................................50 Materials and Methods ...........................................................................................................56 Isolate Selection...............................................................................................................56 DNA Extraction and PCR of ITS, IGS, and RPB1 Regions ...........................................56 Phylogenetic Analysis .....................................................................................................58 Congruence of Molecular Data Sets and Combined Analysis ........................................59 Morphological Description..............................................................................................61 Results.....................................................................................................................................62 Phylogenetic Analysis .....................................................................................................62 Congruence Tests and Combined Data Analysis ............................................................65 Morphological Characterization......................................................................................66 Discussion...............................................................................................................................67 3 USE OF PHYLOGENIES FROM MULTIPLE DNA REGIONS TO ASSESS THE DIVERGENCE OF LINEAGES OF COLLETOTRICHUM GLOEOSPORIOIDES SENSU LATO THAT ARE ASSOCIATED WITH MANGO ANTHRACNOSE IN SOUTH FLORIDA.................................................................................................................96 5 Introduction.............................................................................................................................96 Materials and Methods ...........................................................................................................99 Collection and Storage of Isolates...................................................................................99 DNA Extraction and PCR of the ITS, MAT1-2, and CGTT5 Regions.........................100 Phylogenetic Analyses...................................................................................................102 Congruence of Molecular Data Sets and Combined Analysis ......................................103 Morphology Studies ......................................................................................................104 Pathogenicity Studies ....................................................................................................104 Blossom blight........................................................................................................105 Leaf anthracnose ....................................................................................................105 Fruit pathogenicity .................................................................................................106 Results...................................................................................................................................107 Phylogenetic Analyses...................................................................................................107 Congruence Tests and Combined Analysis ...................................................................109 Morphology Studies ......................................................................................................110 Pathogenicity studies.....................................................................................................111 Discussion.............................................................................................................................112 4 INVESTIGATING THE ROLE PLAYED BY COLLETOTRICHUM ACUTATUM IN THE DEVELOPMENT OF BLOSSOM BLIGHT AND LEAF AND FRUIT ANTHRACNOSE OF MANGO IN SOUTHERN FLORIDA ............................................153 Introduction...........................................................................................................................153 Materials and Methods .........................................................................................................154 Leaf Anthracnose Survey ..............................................................................................154 Pathogenicity Tests........................................................................................................157 Blossom blight tests ...............................................................................................157 Leaf pathogenicity..................................................................................................158 Fruit pathogenicity .................................................................................................158 Results...................................................................................................................................159 Survey Results...............................................................................................................159 Pathogenicity of C. acutatum and C. gloeosporioides on Mango Organs ....................160 Blossom blight........................................................................................................160 Leaf anthracnose ....................................................................................................160 Fruit anthracnose....................................................................................................161
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