DISCRIMINATION AND TRANCRIPTIONAL RESPONSE ANALYSIS OF HEMIPTERAN PHYTOPATHOGEN VECTORS By SHARON ANDREASON Bachelor of Science in Biology University of Texas at Tyler Tyler, Texas 2009 Submitted to the Faculty of the Graduate College of the Oklahoma State University in partial fulfillment of the requirements for the Degree of DOCTOR OF PHILOSOPHY July, 2016 DISCRIMINATION AND TRANSCRIPTIONAL RESPONSE ANALYSIS OF HEMIPTERAN PHYTOPATHOGEN VECTORS Dissertation Approved: Astri Wayadande, Ph.D. Dissertation Adviser Jacqueline Fletcher, Ph.D. Francisco Ochoa-Corona, Ph.D. Ulrich Melcher, Ph.D. ii ACKNOWLEDGEMENTS I would first like to extend my deepest thanks to my advisor, Dr. Astri Wayadande, for all of her years of support. Always full of positivity, encouragement, and advice, her enthusiasm for research and effort devoted to others are attributes that I aspire to cultivate in my future career. I am forever grateful for her guidance throughout earning my degree. I would also like to thank my committee members, Drs. Jacqueline Fletcher, Francisco Ochoa-Corona, and Ulrich Melcher, each of whom have not only offered me thoughtful advice through my research, but have also educated and inspired me in unique and invaluable ways. I am deeply appreciative of their guidance over the years. I want to thank Dr. Judith Brown, without whom I would not have had the opportunity to work on such an important vector system. I am sincerely grateful to Dr. Mohammad Arif, who provided exceptional expertise and advice through my whitefly projects. I would also like to extend my thanks to Dr. Trenna Blagden for all her help and guidance on my projects, particularly during my work with EDNA, as well as for the professional advice she has provided. I want to thank Dr. William Schneider, who welcomed me onto ‘Team EDNA’ and provided me with excellent opportunities through that work. I also want to thank Jon Daniels for all his help with learning EDNA. Thank you especially to Dr. Brian Couger, whose help with my transcriptome analysis has been absolutely indispensable. Finally, I want to thank my friends and family, especially my parents, both of whom have supported me, encouraged me, and inspired me, and my sister Faith, my bestfriendsister and comic relief. I could not have made it to this day without each of you. iii Acknowledgements reflect the views of the author and are not endorsed by committee members or Oklahoma State University. Name: SHARON ANDREASON Date of Degree: JULY, 2016 Title of Study: DISCRIMINATION AND TRANSCRIPTIONAL RESPONSE ANALYSIS OF HEMIPTERAN PHYTOPATHOGEN VECTORS Major Field: PLANT PATHOLOGY Abstract: Insect vectors play a prominent role in the epidemiology of the pathogens they transmit, often acting as vehicles of pathogen movement into new areas. With the globalization of world economies, pathogen and vector introductions increase; however, the impact can be minimized with use of rapid detection strategies at ports of entry. The development and application of novel, reliable tools that accurately detect pests before they enter the country can facilitate interventions that prevent introductions of exotic invasive species. The work described herein provides new discrimination strategies to this end. Bemisia tabaci is an agriculturally damaging whitefly pest species that transmits over 200 viruses to hundreds of susceptible host plants, many of agricultural importance. Compounding the problems caused by B. tabaci, is its genetic and biological diversity. As a cryptic species, one with genetically distinct, non-interbreeding variants but morphological homogeneity, highly problematic, non-native biotypes can be distinguished from the native biotype only by molecular tools. As such, three discriminatory techniques were designed for two high-consequence biotypes (B and Q), and the native biotype (A), as well as another whitefly virus vector species, Trialeurodes vaporariorum. A reliable multiplex PCR protocol, that incorporates the use of sensitivity- enhancing 5’ A/T-rich overhang sequences, was designed and validated by testing over 125 whiteflies from over 55 populations and an inclusivity panel of 17 non-target organisms. Primers were also designed for real-time PCR-based and isothermal amplification discrimination. Melting temperature analysis and helicase dependent amplification protocols were developed and tested for identification of B. tabaci B, Q, and A biotypes, and T. vaporariorum. Leafhoppers are important vectors of phytopathogenic mollicutes. As pathogens can be carried and introduced undectected via their insect vectors, use of e-probe diagnostic nucleic acid analysis (EDNA) was tested for phytopathogen detection in an insect. The vector Exitianus exitiosus harboring Spiroplasma kunkelii was used to validate pathogen detection in an insect-derived Next Generation Sequencing dataset background. Finally, the effects of S. kunkelii infection in E. exitiosus were explored by transciptome sequencing and differential espression analysis. As more is discovered about pathogen and vector interactions, more innovative strategies for pest prevention and management can be devised. iv TABLE OF CONTENTS Chapter Page I. INTRODUCTION ......................................................................................................1 II. REVIEW OF LITERATURE....................................................................................5 Agricultural Biosecurity...........................................................................................5 Bemisia tabaci ..........................................................................................................6 History and Distribution ....................................................................................6 Biology ...............................................................................................................7 Plant Damage .....................................................................................................9 Virus Transmission ..........................................................................................10 Mode of Transmission .....................................................................................11 Biotypes ...........................................................................................................15 Biotype Discrimination ....................................................................................18 Next Generation Sequencing .................................................................................19 Metagenomics ........................................................................................................20 E-probe Diagnostic Nucleic Acid Analysis ...........................................................21 Leafhopper Transmission of Plant Pathogens........................................................25 Viruses .............................................................................................................25 Phytopathogenic Spiroplasmas ........................................................................28 Spiroplasma-Vector Molecular Interactions ....................................................29 Vector System of Interest.......................................................................................30 Exitianus exitiosus ...........................................................................................30 Spiroplasma kunkelii ........................................................................................31 Research Justification ............................................................................................33 References ..............................................................................................................36 III. RELIABLE SINGLE-TARGET AND MULTIPLEX DISCRIMINATION OF HIGH-CONSEQUENCE BEMISIA TABACI BIOTYPES AND TRIALEURODES VAPORARIORUM WITH FINE TUNING OF MODIFIED OLIGONUCLEOTIDE PRIMER THERMODYNAMICS ...................................51 Abstract ..................................................................................................................51 Introduction ............................................................................................................52 Materials and Methods ...........................................................................................54 Results ....................................................................................................................62 Discussion ..............................................................................................................67 References ..............................................................................................................73 v Chapter Page IV. MELTING TEMPERATURE ANALYSIS AND HELICASE DEPENDENT AMPLIFICATION DISCRIMINATION OF BEMISIA TABACI BIOTYPES AND TRIALEURODES VAPORARIORUM ..........................................................76 Abstract ..................................................................................................................76 Introduction ............................................................................................................77 Materials and Methods ...........................................................................................79 Results ....................................................................................................................84 Discussion ..............................................................................................................93