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University of Vermont Scholarworks@ UVM Graduate College Dissertations and Theses Dissertations and Theses 2007 Epizootiology and Phylogenetics of Entomopathogenic Fungi

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University of Vermont Scholarworks@ UVM Graduate College Dissertations and Theses Dissertations and Theses 2007 Epizootiology and Phylogenetics of Entomopathogenic Fungi University of Vermont ScholarWorks @ UVM Graduate College Dissertations and Theses Dissertations and Theses 2007 Epizootiology and Phylogenetics of Entomopathogenic Fungi Associated with Fiorinia externa ferris(Hemiptera: Diaspididae) in the Northeastern USA Jose A. P. Marcelino University of Vermont Follow this and additional works at: https://scholarworks.uvm.edu/graddis Recommended Citation Marcelino, Jose A. P., "Epizootiology and Phylogenetics of Entomopathogenic Fungi Associated with Fiorinia externa ferris(Hemiptera: Diaspididae) in the Northeastern USA" (2007). Graduate College Dissertations and Theses. 148. https://scholarworks.uvm.edu/graddis/148 This Dissertation is brought to you for free and open access by the Dissertations and Theses at ScholarWorks @ UVM. It has been accepted for inclusion in Graduate College Dissertations and Theses by an authorized administrator of ScholarWorks @ UVM. For more information, please contact [email protected]. EPIZOOTIOLOGY AND PHYLOGENETICS OF ENTOMOPATHOGENIC FUNGI ASSOCIATED WITH FIORINIA EXTERNA FERRIS (HEMIPTERA: DIASPIDIDAE) IN THE NORTHEASTERN USA A Dissertation Presented by José A. P. Marcelino to The Faculty of the Graduate College of The University of Vermont In Partial fulfillment of the Requirements for the Degree of Doctor of Philosophy Specializing in Insect Pathology October, 2007 Accepted by the Faculty of the Graduate College, The University of Vermont, in partial fulfillment of the requirements for the degree of Doctor of Philosophy, specializing in Plant and Soil Sciences. Date: August 24th, 2007 1 Abstract The eastern hemlock [Tsuga canadensis (L.) Carrière] is one of the native dominant forest components of northeastern US. At present, these valuable stands face an alarming decline, in part due to the Fiorinia externa, elongate hemlock scale (EHS), (Hemiptera: Coccoidea: Diaspididae). The armored shield of F. externa provides an excellent defense against insecticides, natural enemies and adverse conditions. Chemical and classical biocontrol methods have been unable to stop the spread of this pest. Recently, the occurrence of an epizootic within the F. externa population in the Mianus River Gorge Preserve in Bedford, NY revealed a promising opportunity for control of this scale. Entomopathogenic fungi represent a valuable, although under-utilized, group of organisms with unique capabilities for self-sustaining pest management. Given the significant impact of this epizootic on F. externa, we have conducted extensive research on the biology, genetics and biological control potential of this epizootic. We molecularly identified a complex of entomopathogenic, phytopathogenic, and endophytic fungi associated with the epizootic in 36 localities within the states of New York, Pennsylvania, Connecticut and New Jersey. One fungus, Colletotrichum sp., was the most commonly isolated organism in populations of F. externa within areas of the epizootic. The host range of this Colletotrichum species comprised both insects and plants, although diverse life cycles occured in the different hosts. Endophytic growth was observed in 28 species of plants comprising 18 families (52% of the sampling), whereas in F. externa biotrophic and necotrophic growth was detected. Colletotrichum is a widely known phytopathogenic genus and reports of entomopathogenic activity are extremely rare. In order to understand the biological processes involved in the host-pathogen interactions we quantified the pathogenicity and virulence of this Colletotrichum sp. to four insect families and six plants families as well as the occurrence of sexual recombination in this Colletotrichum sp., both in vitro and in planta. We observed that this Colletotrichum sp. displays a propensy to induce rapid disease and mortality in F. externa hosts. Phylogenetic analysis comprising six of the most commonly studied nuclear genes in molecular phylogenetics (D1/D2 domain of the 28 rDNA gene, ITS region, β-Tubulin 2, GPDH gene, GS gene and HMG box at the MAT1-2 mating-type gene) and RAPDs showed this fungus is closely related to phytopathogenic strains of Colletotrichum acutatum and that it may represent a single population lineage of this species (i.e., Colletotrichum acutatum forma specialis fiorinia). Though a large body of information exists regarding the phytopathogenic genus Colletotrichum, ours is only the second reported entomopathogenic strain. It is not clear whether the colonization of an insect by this fungus is truly rare or a common but undetected event. Sexual recombination, observed in planta and in vitro, could be the means by which new genetic variants are generated leading to new biotypes with a selective advantage to colonize new hosts, which in this case is a novel host in a different kingdom. 2 Citations Material from this dissertation has been submitted for publication to Forest Science, Mycologia and Journal of Insect Science on September 15th 2007 in the following form: José A. P. Marcelino, Svetlana Gouli, Rosanna Giordano, Vladimir V. Gouli, Bruce L. Parker, Margaret Skinner. Fungi associated with a natural epizootic in Fiorinia externa Ferris (Hemiptera: Diaspididae) populations. Forest Science. Marcelino J.A.P., Giordano R., Gouli S., Gouli, V. V., Parker B. L., Skinner, M., Cesnik R., TeBeest D. Colletotrichum acutatum f. sp. fiorinia infection of a scale insect, Fiorinia externa Ferris (Hemiptera: Diaspididae). Mycologia. José A. P. Marcelino, Svetlana Gouli, Bruce L. Parker, Margaret Skinner, Rosanna Giordano, Lora Schwarzberg. Host Plant Associations with an Entomopathogenic Strain of Colletotrichum acutatum from Elongate Hemlock Scale. J. Insect Science. José A. P. Marcelino, Svetlana Gouli, Vladimir V. Gouli, Bruce L. Parker, Margaret Skinner. Entomopathogenic Activity of a Colletotrichum acutatum Strain Recovered from Elongate Hemlock Scale. J. Insect Science. ii Acknowledgments I am very grateful to a number of people for their assistance and encouragement during my studies at the University of Vermont, to whom I feel indebt. I would like to thank my supervisor, Dr. Bruce L. Parker, for his guidance in this project and to Drs. Svetlana Gouli and Rosanna Giordano for sharing their vast knowledge in fungi and genetics with me, as well as, for their kind friendship and perseverance (and food!). I would also like to thank the valuable ideas and suggestions given by Drs. Margaret Skinner, Vladimir Gouli, David TeBeest, Felipe Neri Soto-Adames, Brent Teillon and Dale Bergdahl during the course of this project. I must thank as well all the staff of the Entomology Research Laboratory and the Plant and Soil Science Department, for their help and valuable technical assistance and Mr. Jon Turmel, for being an example of what it means to be an excelent teacher. Also, I have to thank Lora Schwarzberg, which was instrumental in achieving some of the goals in this work. My task would have not been possible without their help and professional expertise. I would also like to thank the encouragement and support of my friends. Luisa, Dina, Duarte, Felipe and Carla who, from a distance, lived through much of my experiences during this journey, and to my roommate Trevor which patiently listen to me every day. Also to Ana Melo, which watched me from the distance and then from the stars. Finally I would like to thank the unique opportunity that the University of Vermont has given me to grow as a professional and as a person in the US. iii Table of Contents Citations .............................................................................................................................. ii Acknowledgments..............................................................................................................iii List of Tables ..................................................................................................................... vi List of Figures................................................................................................................... vii CHAPTER 1. LITERATURE REVIEW ....................................................................................... 2 1. Introduction............................................................................................................... 2 1.1. Importance and decline of eastern hemlock........................................................... 4 1.2. The Fiorinia externa (Hemiptera: Coccoidea: Diaspididae) armored scale.......... 9 1.2.1. Life-cycle .................................................................................................... 12 1.2.2. Damage ....................................................................................................... 14 1.2.3. Management................................................................................................ 15 1.2.3.1. Chemical control............................................................................. 16 1.2.3.2. Natural enemies: parasites and predators........................................ 17 1.2.3.3. Entomopathogenic fungi................................................................. 18 1.3. Entomopathogenic fungi as biological control agents......................................... 21 1.4. The genus Colletotrichum.................................................................................... 26 1.4.1. Taxonomic considerations .......................................................................... 27 1.4.2. rDNA analyses to discern Colletotrichum spp............................................ 30 1.4.3. Hosts
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