University of Tennessee, Knoxville TRACE: Tennessee Research and Creative Exchange Masters Theses Graduate School 5-2018 Identification, characterization, and genetic comparison of Fusarium species isolated from switchgrass (Panicum virgatum L.) Tamara Annette Collins University of Tennessee, [email protected] Follow this and additional works at: https://trace.tennessee.edu/utk_gradthes Recommended Citation Collins, Tamara Annette, "Identification, characterization, and genetic comparison of Fusarium species isolated from switchgrass (Panicum virgatum L.). " Master's Thesis, University of Tennessee, 2018. https://trace.tennessee.edu/utk_gradthes/5097 This Thesis is brought to you for free and open access by the Graduate School at TRACE: Tennessee Research and Creative Exchange. It has been accepted for inclusion in Masters Theses by an authorized administrator of TRACE: Tennessee Research and Creative Exchange. For more information, please contact [email protected]. To the Graduate Council: I am submitting herewith a thesis written by Tamara Annette Collins entitled "Identification, characterization, and genetic comparison of Fusarium species isolated from switchgrass (Panicum virgatum L.)." I have examined the final electronic copy of this thesis for form and content and recommend that it be accepted in partial fulfillment of the equirr ements for the degree of Master of Science, with a major in Entomology and Plant Pathology. Bonnie H. Ownley, Major Professor We have read this thesis and recommend its acceptance: Denita Hadziabdic-Guerry, Darrell D. Hensley, Robert N. Trigiano Accepted for the Council: Dixie L. Thompson Vice Provost and Dean of the Graduate School (Original signatures are on file with official studentecor r ds.) Identification, characterization, and genetic comparison of Fusarium species isolated from switchgrass (Panicum virgatum L.) A Thesis Presented for the Master of Science Degree The University of Tennessee, Knoxville Tamara Annette Collins May 2018 Copyright © 2017 by Tamara Collins All rights reserved. ii Dedication I dedicate this research to my family. To my remarkable parents, Timothy and Susan Collins for being there for me throughout my life to encourage me to strive for the best, never settle, and never give up. To my precious children, Gideon and Killian Collins- Figgs for giving me a reason to work hard to make the world better for generations to come. To my incredible boyfriend, Tyler and his parents Keith and Leslie Marcum for being extra strength and support through my graduate school process. iii Acknowledgments Thank you to my advisor Dr. B. Ownley for giving me the opportunity to be part of the Entomology and Plant Pathology graduate program. Her guidance and avocation for diversity, specifically women in science related fields, gave me the extra confidence and drive I needed to succeed. Thank you to Dr. D. Hensley for hiring me as a graduate assistant on the cornelian cherry dogwood grant. I would like to thank my other committee members, Dr. D. Hadziabdic, Dr. R. Trigiano, for their assistance and insight during this project, and Dr. K. Gwinn for extra encouragement and guidance above and beyond. Thank you to my lab mates Sara Collins, Alex Bruce, Shalini Yerukala, Mary Dee, and adopted lab mates from the Dr. R. Trigiano’s Molecular Genetics Lab: Sarah Boggess, Chris Wyman, Tyler Edwards, Marcin Nowicki, and Dr. Romina Gazis for all your valuable insight and humorous moments. For her aid in bioinformatics and computer software, I would like to thank Dr. Margaret Staton. I would like to thank the Tennessee Department of Agriculture, the U.S. Department of Agriculture, and The University of Tennessee for funding this research. Finally, to my family, thank you for being here to support me and push me to never give up, and for putting up with the stressful and heartbreaking moments that we encountered the last two years. iv Abstract Fusarium spp. are ubiquitous, soilborne and seedborne pathogens that affect over 100 economically important plant hosts annually, resulting in billions of dollars in economic losses. They cause a variety of plant disease symptoms including, crown, root, and stem rots, wilts, and foliar necrosis. Furthermore, they produce harmful mycotoxins that are detrimental to the health of animals and humans. Members of this genus have been isolated from diseased tissues of switchgrass. The objective is to correctly identify and classify Fusarium isolates to species using both classic phenotypic characterization and current genetic technologies of DNA extraction and polymerase chain reaction and create microsatellite loci that will give more insight into the genetic template of Fusarium species. This will enable us to better understand the role these fungi play in agriculture systems and how to control the pathogenic ones more effectively. Whole genome sequences were developed for four Fusarium species (F. equiseti, F. graminearum, F. oxysporum, and F. sporotrichioides) isolated from commercial switchgrass seed from 11 geographic locations across the United States. From each genome, 15 dinucleotide and 15 trinucleotide (n = 30) microsatellite loci were evaluated for amplification and polymorphism. An additional 30 microsatellite loci were developed for F. graminearum. The percentage amplification of the microsatellites from each species was 93% for F. equiseti, 42% for F. graminearum, 50% for F. oxysporum and 36% for F. sporotrichioides. The sample size of F. equiseti isolates was small (n = 6), therefore this species was not included in further study. Microsatellite loci from the Fusarium species were considered polymorphic if they yielded one clear peak and the base pair (bp) length difference among individual alleles was greater than 3 bp. Polymorphic v microsatellite loci were evaluated for cross amplification of F. graminearum, F. oxysporum, and F. sporotrichioides. Microsatellite loci Fg057 (from F. graminearum) and Fr006 (F. sporotrichioides) did not amplify products from F. oxysporum isolates, and Fo006 and Fo012 (from F. oxysporum) did not amplify against F. sporotrichioides. Fusarium oxysporum isolates were nonpathogenic endophytes of switchgrass, while isolates of F. graminearum and F. sporotrichioides were pathogenic on this host. vi Table of Contents Chapter 1 Literature Review .......................................................................................... 1 Switchgrass .............................................................................................................. 1 General information ............................................................................................... 1 Economic uses ...................................................................................................... 2 Pathogens of switchgrass ...................................................................................... 4 Fusarium .................................................................................................................. 4 Classification ......................................................................................................... 4 Plant pathogens ..................................................................................................... 5 Fusarium equiseti .................................................................................................. 8 Fusarium graminearum ....................................................................................... 10 Fusarium oxysporum ........................................................................................... 12 Fusarium sporotrichioides ................................................................................... 15 Plant Pathogen Characterization Techniques ........................................................ 16 Traditional taxonomy ........................................................................................... 16 Genetic characterization ...................................................................................... 17 Molecular techniques for Fusarium identification ................................................ 18 Microsatellite Loci ................................................................................................... 19 Current uses ........................................................................................................ 20 Research Objectives and Justification ................................................................... 20 Research Definitions .............................................................................................. 22 Chapter 2 Identification, characterization, and genetic comparison of Fusarium spp. isolated from switchgrass (Panicum virgatum L.) ............................................ 24 Abstract .................................................................................................................. 24 Introduction ............................................................................................................. 25 Materials and Methods ........................................................................................... 31 Culture collection ................................................................................................. 31 Phenotypic characterization ................................................................................ 31 Molecular characterization ................................................................................... 31 Polymerase chain reaction amplification ............................................................