Program Chairs George Kennedy (North Carolina State University)

Diane Ullman (University of California, Davis)

Organizing Committee Scott Adkins (United States Department of Agriculture-Agricultural Research Service)

Robert Kemerait (University of Georgia)

Martha Mutschler (Cornell University)

Naidu Rayapati (Washington State University)

Dorith Rotenberg (Kansas State University)

John Sherwood (University of Georgia)

Anna Whitfield (Kansas State University)

Scientific Advisory Committee Adriano Cavalleri (Universidade Federal do Rio Grande do Sul Porto Alegre, Brasil)

Kleber del Claro (Universidade Federal de Uberlândia, Brazil)

Thomas German (University of Wisconsin)

Robert Gilbertson (University of California, Davis)

Elliot Watanabe Kitajima (Universidade de São Paulo, Brasil)

Richard Kormelink (Wageningen University)

Gerald Moritz (Martin-Luther-University Halle-Wittenberg)

Tatsuya Nagata (Universidade de Brasília, Brazil)

Hanu Pappu (Washington State University)

Denis Persley (Queensland Primary Industries and Fisheries, Australia)

Dick Peters (Wageningen University)

Stuart Reitz (Oregon State University)

Renato de Oliveira Resende (Universidade de Brasília)

Desley Tree (Queensland Primary Industries Insect Collection, Australia)

Massimo Turina (Institute of Plant Virology, Italy)

Shyi-dong Yeh (National Chung Hsing University, Taiwan) Encouraging New Scholars of Thysanoptera and Tospoviruses

Laurence Mound Fellows:

The Laurence Mound Endowment for International Research on Thysanoptera and Tospoviruses was founded in September, 2005 and presented to Laurence Mound on the occasion of his Plenary Address at the VIIIth International Symposium on Thysanoptera and Tospoviruses. The goal of the endowment is to foster research and interaction between scientists studying Thysanoptera and Tospoviruses with special attention on encouraging new scholars. Through a competitive application process, we selected eleven Laurence Mound Fellows to receive a scholarship to attend the Xth International Symposium on Thysanoptera and Tospoviruses, May 16th through May 20th, 2015 in Asilomar.

Luciana Ferrand Universidad Nacional de La Plata, Argentina Shirani Gamage The University of Queensland, Australia Romana Iftikhar Pakistan Institute of Engineering and Applied Sciences Stephanie Krueger Martin-Luther-University Halle- Wittenberg, Germany Elison Lima Universidade de São Paulo, Brazil Mariana Lindner Universidade Federal do Rio Grande do Sul, Brazil Alexander Muvea Leibniz Universität Hannover, Germany Duong Nguyen University of Western Sydney, Australia Pamella Ogada Gottfried Wilhelm Leibniz Universität Hannover, Germany Jonathan Oliver Kansas State University, United States Athos Silva de Oliveira Wageningen University, The Netherlands

Thrips Tospovirus Educational Network (TTEN) Fellows:

The Thrips Tospovirus Educational Network (TTEN) provides robust mentor training and experience to graduate students who, in partnership with their faculty mentors, provide undergraduate mentees with engaging and challenging research experiences cementing their interest in the sciences and preparing them professionally. For two years students from 7 institutions have participated in the program as mentors and mentees, conducting independent research projects and attending interactive sessions in a cyber-classroom through Adobe Connect. Through a competitive application process, twelve TTEN mentors and mentees were selected as TTEN Fellows, each receiving a scholarship to attend the Xth International Symposium on Thysanoptera and Tospoviruses, May 16th through May 20th, 2015 in Asilomar. The TTEN is funded by AFRI NIFA Coordinated Agricultural Project 2012-01785.

Raphael Adegbola Washington State University Ismael Badillo-Vargas Kansas State University and ARS-USDA Candice Stafford-Banks University of California, Davis Ozgur Batuman University of California, Davis Li-Feng Chen University of California, Davis Kory Herrington Abraham Baldwin Agricultural College Michelle Kwok University of California, Davis Spencer Marshall Washington State University Norma Ordaz University of California, Davis Derek Schneweis Kansas State University Anita Shrestha University of Georgia John Smeda Cornell University Blake Williams University of Georgia

Special Thanks to the Following Individuals and Groups

for Their Generous Contributions

• Agdia, Inc. • American Floral Endowment • California Cut Flower Commission • Citrus Research Board • DuPont Crop Protection • Georgia Tobacco Commission • Kee Kitayama Research Foundation • The Los Angeles Flower Market • Mellano & Company • Mike Mellano Sr • The Peanut Foundation, Inc. • Syngenta • United States Department of Agriculture, National Institute of Food and Agriculture Conference Award 2015-67013-23171 • AFRI NIFA Coordinated Agricultural Project 2012-01785 • University of California Davis, College of Agricultural and Environmental Sciences • The University of Georgia, Plant Pathology Department

TABLE OF CONTENTS

I Week at a Glance 1 II Program Saturday Registration and Hospitality Welcome 4 Saturday Opening Session and Reception 4 Sunday Plenary Session I: Looking to the Future 5 State of the Art Session I: Thrips Biology and Systematics 5 State of the Art Session II: Developing Host Plant Resistance 6 Against Tospoviruses and Thrips As Vectors State of the Art Session III: Integrated Management of 7 Tospoviruses and Thrips As Vectors State of the Art Session IV: Tospovirus Biology, Genetics, 7 and Diversity Poster Presentations, Informal Discussions and Reception 8 Monday Plenary Session II: Virus-Vector-Host Interactions 10 Field Trip 10 Facilitated Discussions and Reception 11 Tuesday Plenary Session III: Virus/Vector Biology and Emergence of 12 Diversity State of the Art Session V: Thrips/Tospovirus Ecology and 12 Management State of the Art Session VI: Virus Epidemiology and 13 Integrated Management Strategies State of the Art Session VII: Thrips Ecology and Behavior 14 State of the Art Session VIII: Thrips and Tospovirus 15 Management Banquet at the Monterey Bay Aquarium 15 Wednesday Final Plenary Session 16 III Abstracts Opening Conference Speaker 20 Plenary Session I: Looking to the Future 21 State of the Art Session 1: Thrips Biology and Systematics 25 State of the Art Session II: Developing Host Plant Resistance 36 and Integrated Management of Tospoviruses and Thrips As Vectors State of the Art Session III: Integrated Management of 45 Tospoviruses and Thrips As Vectors State of the Art Session IV: Tospovirus Biology, Genetics, 52 and Diversity Plenary Session II: Virus-Vector-Host Interactions 60 Plenary Session III: Virus/Vector Biology and Emergence of 65 Diversity State of the Art Session V: Thrips/Tospovirus Ecology and 71 Management State of the Art Session VI: Virus Epidemiology and 81 Integrated Management Strategies State of the Art Session VII: Thrips Ecology and Behavior 87 State of the Art Session VIII: Thrips and Tospovirus 95 Management Final Plenary Session 102 Poster Presentations, Informal Discussions, and Reception 103

WEEK AT A GLANCE Saturday Sunday Monday Tuesday Wednesday 16-May-15 17-May-15 18-May-15 19-May-15 20-May-15 Registration and Breakfast Breakfast Breakfast Breakfast Hospitality 7:30-8:30 7:30-8:30 7:30-8:30 7:30-8:30 Welcome 15:30-18:00

Dinner Plenary Plenary Plenary Final Plenary 18:00-19:00 Session I Session II Session III Session 8:30-12:00 8:30-11:45 8:30-12:00 9:00-12:00

Opening Session Lunch Field Trip Lunch Lunch and Reception 12:00-13:00 11:45-17:00 12:00-13:00 12:00-13:00 19:30-22:00

State of the Art Dinner State of the Art Sessions I/II 18:00-19:00 Session V/VI 13:00-15:45 13:00-16:00

State of the Art Facilitated State of the Art Sessions III/IV Discussions and Session VII/VIII 15:45-18:00 Reception 16:00-18:00 19:30-21:00

Dinner Monterey Bay 18:00-19:00 Aquarium Banquet 18:45-22:30

Poster Presentations, Informal Discussions and Reception 19:30-22:00

1 The Growing Standard

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2 Since 1961, AFE has funded more than $15 million in research, scholarships, internships and grants.

AFE Research Supports: AFE Scholarships Provide: n Insect and Disease Management of Floral n Hands-on training for the next generation Crops of floriculture industry leaders

n Biocontrol Practices n Opportunities for students to n Production Management learn tools of the trade from industry professionals n Postharvest, transport and retail handling practices, including control of n Knowledgeable students ready to join insects and diseases the industry workforce n Marketing Practices and Consumer Purchase Practices

Support the floriculture industry by making a donation at www.endowment.org/donate. By making a tax-deductible donation to the Endowment, you invest in the future of the industry—making it stronger and more sustainable for years to come.

Proud Supporter of the Xth International Symposium on Thysanoptera and Tospoviruses

www.endowment.org (703) 838-5211 | [email protected]

3 Program for the Xth International Symposium on Thysanoptera and Tospoviruses 16th May – 20th May, 2015; Asilomar Conference Grounds, Pacific Grove, California

SATURDAY 16 May-2015

Registration and Hospitality Welcome Room Title Time

Chapel Registration and Hospitality Welcome 15:30-18:00 Crocker Dinner 18:00-19:00 Dining Hall

Opening Session and Reception Room Title Presenting Time Author

Chapel Introduction Diane Ullman and 19:30-19:45 George Kennedy

Chapel Welcome to California Michael Parrella 19:45-20:00

Chapel Anticipating the Likely Effects of Climate Change on Virus Roger Jones 20:00-21:00 Vectors and Virus Disease Epidemics

Chapel Meet and Greet 21:00-22:00

4 SUNDAY 17 May-2015

Room Title Presenting Time Author

Crocker Breakfast 7:30-8:30 Dining Hall

Plenary Session I Looking to the Future Moderated by Jonathan Oliver Room Title Presenting Time Author

Chapel Introductory Remarks and Recognitions Diane Ullman and 8:30-8:45 George Kennedy

Chapel Current and Emerging Strategies in the War Against Thrips Anna Whitfield 8:45-9:30 and Tospoviruses

Chapel Genetic Modification of Insect Vectors: Innovations in Luke Alphey 9:30-10:15 Management

Chapel Coffee Break 10:15-10:30

Chapel Communicating with the Future – A Response to Gerald Moritz 10:30-11:15 Diminishing Taxonomic Expertise

Chapel Diversity of Tospoviruses and Thrips Vectors in Asia, and Shyi-Dong Yeh 11:15-12:00 Possible Control by Broad Spectrum Transgenic Resistance

Crocker Lunch 12:00-13:00 Dining Hall

CONCURRENT SESSIONS

State of the Art Session I Thrips Biology and Systematics Moderated by Mark Hoddle & Michael Parrella Room Title Presenting Time Author

Chapel What Has Prevented Bean Thrips, Caliothrips fasciatus, from Mark Hoddle 13:00-13:15 Becoming an Invasive Agricultural Pest?

The Utility of Soil Amendments in Suppressing Thrips Michael Parrella Populations

Chapel Thrips Biology and Systematics – a Tale of Two Cities? Laurence Mound 13:15-13:30

Chapel The Greenhouse Thrips, Heliothrips haemorrhoidalis: Complex Paul Rugman-Jones 13:30-13:45 Species or Species Complex?

Chapel What Happens when Ectoparasitic Thrips Get on Adriano Cavalleri 13:45-14:00 Auchenorrhynch Hemipterans? A Behavioral and Multitrophic Approach

Chapel Interactions Between Frankliniella bispinosa (Morgan) and Thomas Skarlinsky 14:00-14:15 Frankliniella cephalica (D.L.Crawford) in Florida

5 SUNDAY (CONTINUED) Room Title Presenting Time Author

Chapel Biotic Resistance Limits the Invasiveness of the Western Joseph Funderburk 14:15-14:30 Flower Thrips, Frankliniella occidentalis (Thysanoptera: Thripidae) in Florida

Chapel Dark and Pale Color Morphs of Common Blossom Thrips Matilda Gikonyo 14:30-14:45 Frankliniella schultzei (Trybom) - Are They Just Colour Forms?

Chapel Cardimium and Wolbachia Induce Cytoplasmic Incompatibility Duong Nguyen 14:45-15:00 and Sex Ratio Shifts in an Australian Species of Thrips

Chapel Detection of Wolbachia in Some Natural Populations of Thrips Alessandra 15:00-15:15 Species (Insecta: Thysanoptera) in Mediterranean Habitats De Grazia

Chapel DNA Barcode Analysis of Thrips (Thysanoptera) Diversity In Romana Iftikhar 15:15-15:30 Pakistan

Chapel Coffee Break 15:30-15:45

State of the Art Session II Developing Host Plant Resistance Against Tospoviruses and Thrips As Vectors Moderated by Martha Mutschler Room Title Presenting Time Author

Scripps Potential for Natural Plant Resistance for Control of Insects Martha Mutschler 13:00-13:30 and Insect-Vectored Viruses

Scripps Utilizing Acylsugar Chemistry for Optimal Thrips and John Smeda 13:30-13:45 Tospovirus Control in Tomato

Scripps Flower Power: The Potential Role of Acylsugars on Western Cassandre 13:45-14:00 Flower Thrips Oviposition in Tomato Flowers Kaplinsky

Scripps The Sw-5 Gene Cluster: Unravelling the Keys to Resistance Athos Silva de 14:00-14:15 to Tomato spotted wilt virus Oliveira

Scripps In Search of the Early Events in Sw-5 Mediated Resistance: Norma Ordaz 14:15-14:30 Optimizing Tomato spotted wilt virus Infection of Tomato

Scripps Harnessing Artificial MicroRNAs for Resistance to Neena Mitter 14:30-14:45 Tospoviruses: The Way Forward

Scripps An Eco-Metabolomic Approach to Study Host Plant Resistance Kirsten Leiss 14:45-15:00 to Western Flower Thrips

Scripps Field Resistant Peanut Cultivars for Management of Tomato Albert Culbreath 15:00-15:15 spotted wilt: History, Improvements, and Stability

Scripps Development of Methods for Maintenance of Lettuce-Infecting William 15:15-15:30 Tospoviruses and Germplasm Evaluation Wintermantel

Scripps Coffee Break 15:30-15:45

6 SUNDAY (CONTINUED) State of the Art Session III Integrated Management of Tospoviruses and Thrips As Vectors Moderated by Diane Alston Room Title Presenting Time Author

Chapel Field and Farmscape IPM for Onion Thrips and Iris yellow spot Diane Alston 15:45-16:15 virus

Chapel Integration of Peanut Genotypes Displaying Field Resistance Rajagopalbabu 16:15-16:30 to Tomato spotted wilt virus with other Management Tactics Srinivasan

Chapel Effect of Soil Potassium Levels on Iris yellow spot virus Claudia Nischwitz 16:30-16:45 Incidence and Symptom Expression in Onion

Chapel The Spread of Frankliniella occidentalis (Pergande) on Citrus Rita Marullo 16:45-17:00 Orchards into Specialized Growing Areas of Southern Italy

Chapel Thrips as Pests and Vectors of Maize Chlorotic Mottle Virus in Johnson Nyasani 17:00-17:15 Maize

Chapel An Efficient Serological Platform for Identification and Shyi-Dong Yeh 17:15-17:30 Detection of Tospoviral Species Prevalent in Different Geographic Regions

Chapel Interactions Between Temperature and Spinosyns Insecticides David Ben-Yakir 17:30-17:45 in the Control of Thrips tabaci

Chapel Discussion Moderator 17:45-18:00

Crocker Dinner 18:00-19:00 Dining Hall

State of the Art Session IV Tospovirus Biology, Genetics, and Diversity Moderated by Scott Adkins and Denis Persley Room Title Presenting Time Author

Scripps Tospovirus Biology, Genetics, and Diversity Session Denis Persley and 15:45-16:00 Overview Scott Adkins

Scripps Virus-Derived Small RNAs Associated with Tomato spotted Neena Mitter 16:00-16:15 wilt virus Infection in Peanut (Arachis hypogaea L.) and in Tobacco Thrips, Frankliniella fusca [Hinds]

Scripps The Functional Study of the Movement Protein (NSm) of Renato Resende 16:15-16:30 Distinct Tospovirus Species

Scripps Characterization of Australian Capsicum chlorosis virus Shirani Widana 16:30-16:45 Gamage

Scripps Homology Modeling of the Tospovirus Groundnut ringspot Rayane Nunes 16:45-17:00 virus Nucleoprotein: Intra-Molecular Interaction and RNA Lima Encapsidation

Scripps An Update on Sequence Diversity of Impatiens necrotic spot Raphael Adegbola 17:00-17:15 virus 7 SUNDAY (CONTINUED) Room Title Presenting Time Author

Scripps Dynamics of Genetic Diversity of Tomato spotted wilt virus in Spencer Marshall 17:15-17:30 the United States

Scripps Deciphering the Three Dimensional Structures of Tospoviral Hanu Pappu 17:30-17:45 Proteins

Scripps Discussion Moderators 17:45-18:00

Crocker Dinner 18:00-19:00 Dining Hall

Evening Session Poster Presentations, Informal Discussions and Reception Moderated by Hanu Pappu Room Title Presenting Time Author

Chapel Introductory Address Hanu Pappu 19:30-19:45

Chapel Poster Presentations, Informal Discussions and Reception 19:45-22:00

Chapel The Phylogeography of Tomato spotted wilt virus, Tomato Mariana Almeida chlorotic spot virus and Iris yellow spot virus

Chapel Tomato necrotic streak virus, a Novel Subgroup 2 Ilarvirus Ismael Badillo- Vargas

Chapel De novo Transcriptome Analysis of Thrips tabaci David Ben-Yakir Suggests the Involvement of Cytochrome P450 in Resistance to Spinosad

Chapel Functional Marker Assisted-Selection and Genetic Variability Leonardo Boiteux of Sw-5b Gene In Multi-Tospovirus Resistant Solanum (Section Lycopersicon) Germplasm Accessions Using Locus-Specific Primers

Chapel Tomato spotted wilt virus Resistance-Breaking Isolates Luciana Ferrand Infecting Pepper in Argentina

Chapel Use of Overwintering Thrips Species (Frankliniella sp.) and Kory Herrington Tomato spotted wilt virus - Susceptible Weed Species to Detect the Efficacy and Timing of Initial Inoculum Spread to Susceptible Crop Species.

Chapel An Update on Tospoviruses in Pakistan Romana Iftikhar

Chapel Complete Nucleotide Sequence of Iris yellow spot virus Satoshi Kagiwada Japanese Isolate on Lisianthus, Eustoma grandiflorum.

Chapel Tomato spotted wilt virus Nucleocapsid Protein Forms an Keisuke Komoda Asymmetric Trimeric Ring Structure, and Provides Insight into a Novel Genome Encapsidation Mechanism

Chapel Sternal Glands in Important Thrips Species Stephanie Krueger

Chapel Need a Drink? Western Flower Thrips Feeding from the Michelle Kwok Xylem 8 SUNDAY (CONTINUED) Room Title Presenting Author

Chapel Towards an Updated Checklist of Pest Thrips in Brazil Élison Lima

Chapel Diversity of Thrips (Thysanoptera, Thripidae) Associated Maria Lima with the Cultivation of Melon (Cucumis melo) in Northeastern Brazil

Chapel Species of Thrips (Insecta: Thysanoptera: Thripidae) Maria Lima Associated with Vegetable Cultivation in Baturité Mountain Range – Ceará/Brazil.

Chapel A Five-Year Survey of Tospoviruses Infecting Vegetable Mirtes Lima Crops in Main Producing Regions of Brazil

Chapel Current status of Tospoviruses (F: Bunyaviridae) infecting Basavaraju vegetables in India Mahesha

Chapel Molecular and Serological Diagnostics of Capsicum chlorosis Basavaraju virus (CaCV) (F: Bunyaviridae, G: Tospovirus) Infections on Mahesha Chilli (Capsicum annum L.)

Chapel Management of Tospovirus (Peanut bud necrosis virus) by Basavaraju Over-Expressing PBNV-NP Gene in Tomato (Solanum Mahesha lycopersicon L.): A Successful Antiviral Strategy

Chapel Polyploidy Versus Endosymbionts in Obligately Thelytokous Duong Nguyen Heliothrips haemorrhoidalis

Chapel Annotation of V-ATPase Gene Family Members in Jonathan Oliver Frankliniella occidentalis (Pergande)

Chapel Characterization of the Transcriptional Activity of Tomato Hanu Pappu spotted wilt virus RNAs

Chapel Evaluation of Acibenzolar-S-Methyl-Induced Resistance Hanu Pappu Against Iris yellow spot virus Chapel First Report of Tomato spotted wilt virus in India Hanu Pappu

Chapel Identification of Interacting Regions of the Movement Hanu Pappu Protein and Nucleocapsid Protein of Two Distinct Tospoviruses

Chapel Transcriptome-Wide Identification of Host Genes Targeted Hanu Pappu by a Tospovirus-Derived Small Interfering RNAs Reveals Host-Dependent Differential Down-Regulation

Chapel Using the i5K Frankliniella occidentalis Genome to Assemble Derek Schneweis a Reference Transcriptome for Analysis of Differential Gene Expression in First-Instar Larvae During Tospovirus Infection

Chapel Tomato Spotted Wilt Management in Peanut: Reduced Blake Williams Effects of Production Practices on Severity of Spotted Wilt in Current Variety Selections.

9 MONDAY 18 May-2015

Crocker Breakfast 7:30-8:30 Dining Hall

Plenary Session II Virus-Vector-Host Interactions Moderated by Derek Schneweis Room Title Presenting Time Author

Chapel Frontiers in Thrips Genomics: The Current Status of the Dorith Rotenberg 8:30-9:15 Frankliniella occidentalis Genome

Chapel Diversity of Tospoviruses and Their Thrips Vectors and Why Calum Wilson 9:15-9:45 it Can Be Hard to Generalize About Their Association

Chapel Coffee Break 9:45-10:00

Chapel Tomato spotted wilt virus Infection, Thrips Behavior and the Candice Stafford- 10:00-10:30 Salivary Gland Transcriptome of Frankliniella occidentalis Banks

Chapel Use of a Proteomics Approach to Identify Frankliniella Ismael Badillo- 10:30-11:00 occidentalis Proteins That Respond to Tomato spotted wilt Vargas virus Infection

Chapel A Biological Perspective on the Tospoviral Proteins and their Richard Kormelink 11:00-11:30 Roles During Infection and Dissemination in Plants and Thrips.

Prepare for Field Trip 11:30-11:45

Outside Pick Up Your Box Lunch for the Tour and Board the Buses 11:45-12:15 Chapel

On the Field Trip - We appreciate your cooperation in adhering to Led by Steve Koike 12:15-17:00 Road our field trip guidelines, participation in this field trip implies that you agree to the following:

Exercise caution when entering and leaving the vehicles. If you have special needs, inform workshop organizers prior to the field trip.

Exercise caution when walking on roads, crossing roads, and walking in fields. Be especially careful of vehicles that pass by on adjacent roads.

Wear appropriate clothing and sun protection (sunscreen, sunglasses, hats/caps, windbreakers or jackets, field shoes). Be aware that some sites may be dusty or muddy.

Do not collect or cut agricultural commodities (even diseased specimens) unless given permission by the field trip coordinator. During this field trip, eating any agricultural commodities from the field is strictly prohibited.

10 MONDAY (CONTINUED)

This is a group tour, so stay in the general vicinity of the group and do not leave the group.

Food safety requirements: Do not eat while in the field. Do not leave any papers, wrappers, food containers, etc. in or around fields. All personal hygiene must be completed in appropriate port-a-potty, restroom, bus restroom, or other appropriate facility. Thoroughly wash hands after completing personal hygiene. Do not spit in or near fields. If required by the host company, hair nets must be worn prior to entering fields.

The University of California Cooperative Extension, field trip coordinator, and workshop organizers are not responsible for any lost, stolen, or damaged items brought on the field trip.

Remember that we are guests at these locations and sites. We will adhere to all guidelines, restrictions, and rules set forth by our hosts. Violation of these guidelines and requirements may result in cancellation of the field trip.

Free Time 17:00-18:00

Crocker Dinner 18:00-19:00 Dining Hall

EVENING SESSION

Facilitated Discussions and Reception Room Title Presenting Time Author

Chapel Host Plant-Insect-Virus Interactions and Model Systems Facilitated by 19:30-21:00 James Moyer and Saskia Hogenhaut

Chapel Thysanoptera Systematics Facilitated by 19:30-21:00 Adriano Cavalleri and Gerald Moritz

Scripps Progress and Challenges Toward a Reverse Genetic System Facilitated by 19:30-21:00 Thomas German

Scripps Tospovirus Epidemiology and Management Facilitated by 19:30-21:00 Albert Culbreath

11 TUESDAY 19 May-2015

Crocker Breakfast 7:30-8:30 Dining Hall

Plenary Session III Virus/Vector Biology and Emergence of Diversity Moderated by Naidu Rayapati Room Title Presenting Time Author

Chapel Genetic Bottlenecks During Virus Infection and Host Santiago Elena 8:30-9:15 Colonization and Their Impact on Genetic Diversity and Evolution

Chapel Thrips/Tospovirus Relationships: Where Have They Come Laurence Mound 9:15-9:45 From, and Where Are They Going?

Chapel The Importance of Ecology, Species and Population Alana Jacobson 9:45-10:15 Interactions in Thrips-Tospovirus Coevolution Highlighted by Studies on Thrips tabaci

Chapel Coffee Break 10:15-10:30

Chapel Tospovirus Emergence, Interspecific Reassortment and Scott Adkins 10:30-11:00 Mixed Infections in North America

Chapel Tospovirus Emergence, Interspecific Reassortment and Renato Resende 11:00-11:30 Mixed Infections in Brazil: Advances in Breeding for Broad Spectrum Tospovirus Resistance

Chapel Resistance Breaking by TSWV and Its Relevance to Thrips George Kennedy 11:30-12:00 Transmission and Tospovirus Management

Crocker Lunch 12:00-13:00 Dining Hall

CONCURRENT SESSIONS

State of the Art Session V Thrips/Tospovirus Ecology and Management Moderated by Joe Funderburk and Rajagopalbabu Srinivasan Room Title Presenting Time Author

Chapel Challenges and Opportunities for Managing Thrips and Joseph Funderburk 13:00-13:15 Tospoviruses

Chapel Effects of Resistance to Tomato spotted wilt virus in Peanut Rajagopalbabu 13:15-13:30 Genotypes on Virus Population Genetics and Thrips Fitness Srinivasan

Chapel Seasonal Occurrence of Thrips Species Prevalent in Russell Groves 13:30-13:45 Soybean and the Economic Impact of Soybean vein necrosis virus in Wisconsin

Chapel Is Soybean vein necrosis virus (SVNV) Seed Transmitted? Thomas German 13:45-14:00

Chapel Bio-Ecology and Management of Thrips and Tospoviruses Sevgan Subramanian 14:00-14:15 in East Africa

12 TUESDAY (CONTINUED)

Room Title Presenting Time Author

Chapel Transmission of Tomato spotted wilt virus by Two Western Li-Fang Chen 14:15-14:30 Flower Thrips Populations in California and Evidence That Adult Thrips Emerging From Viruliferous Pupae in Soil Could be a Primary Inoculum Source

Chapel Transcriptional Changes Associated with Tomato spotted Anita Shrestha 14:30-14:45 wilt virus Infection in Various Life Stages of Its Thrips Vector, Frankliniella fusca [Hinds]

Chapel The Genetics of Vector Competence of Western Flower Pamella Ogada 14:45-15:00 Thrips (Frankliniella occidentalis) in Tomato spotted wilt virus Transmission

Chapel Enhancing Compatibility Between Entomopathogens and David Kupesa 15:00-15:15 Thrips Semiochemicals

Chapel Preference of Western Flower Thrips on the Arabidopsis Yasuhiro Tomitaka 15:15-15:30 Plants Infected With Thrips-Non-Transmissible Tomato spotted wilt virus

Chapel Discussion Led by Joseph 15:30-15:45 Funderburk and Rajagopalbabu Srinivasan

Chapel Coffee Break 15:45-16:00

State of the Art Session VI Virus Epidemiology and Integrated Management Strategies Moderated by Neil McRoberts and Robert Kemerait Room Title Presenting Time Author

Scripps Population Dynamics of Frankliniella occidentalis in the Neil McRoberts 13:00-13:30 Central Valley of California: Data, Demography and Differential Equations

Scripps Development of an Integrated Pest Management (IPM) Ozgur Batuman 13:30-14:00 Strategy to Control Thrips and Tomato Spotted Wilt Disease in Processing Tomato Fields in the Central Valley of California

Scripps A Thrips and Tomato spotted wilt virus (TSWV) Prediction George Kennedy 14:00-14:30 Tool to Aid TSWV Management Decisions in Tobacco

Scripps The Use of Viral Epidemiology to Better Manage Robert Kemerait 14:30-14:45 Tospoviruses in the Field

Scripps Management of Diseases Caused By Thrips-Transmitted Naidu Rayapati 14:45-15:00 Tospoviruses In Subsistence Agriculture: The Case Of Peanut bud necrosis virus In India

Scripps Biology, Epidemiology and Management of Iris yellow spot Hanu Pappu 15:00-15:30 virus: A Decade of Progress

13 TUESDAY (CONTINUED) Room Title Presenting Time Author

Scripps Discussion Moderated by Neil 15:30-15:45 McRoberts and Robert Kemerait

Scripps Coffee Break 15:45-16:00

State of the Art Session VII Thrips Ecology and Behavior Moderated by William Kirk Room Title Presenting Time Author

Chapel The Pheromones of the Western Flower Thrips and Their William Kirk 16:00-16:15 Use in Pest Management

Chapel Mating Behavior of the Western Flower Thrips Adeyemi Akinyemi 16:15-16:30

Chapel Aggregation Ecology in Bean Flower Thrips Megalurothrips Saliou Niassy 16:30-16:45 sjostedti Trybom (Thysanoptera: Thripidae)

Chapel Olfactory Responses of Western Flower Thrips (Frankliniella Mette-Cecilie Nielsen 16:45-17:00 occidentalis) Populations to a Non-Pheromone Lure

Chapel The Interplay of Cycad Cone Thermogenesis, Volatile Irene Terry 17:00-17:15 Emissions, Humidity, CO2 and Light in Mediating the Specialist Cycadothrips Pollinator Behavior

Chapel Offspring Sex Ratio and Duration of Ontogenesis are Stephanie Krueger 17:15-17:30 Determined by Copulation Activity in Echinothrips americanus (Thysanoptera: Thripidae)

Chapel Speciation in the Thrips tabaci Cryptic Species Complex and József Fail 17:30-17:45 Some of its Possible Consequences

Chapel Two Thrips Species Sharing Home: Interaction and Behavior Mariana Lindner 17:45-18:00 of the Galling Thrips and an Invader on Myrtaceae Galls

Free Time 18:00-18:45

“The Board Buses for the Monterey Bay Aquarium Banquet 18:45 Circle” Just West of Social Hall

Banquet at the Monterey Bay Aquarium 19:00-22:30

14 TUESDAY (CONTINUED) State of the Art Session VIII Thrips and Tospovirus Management Moderated by Candice Stafford-Banks Room Title Presenting Time Author

Chapel An Introduction to Integrated Pest Management Programs Candice Stafford- 16:00-16:15 for Thrips and Tospoviruses Banks

Chapel Can We Get off the Pesticide Treadmill for Managing Onion Stuart Reitz 16:15-16:30 Thrips and Iris yellow spot virus?

Chapel Western Flower Thrips Management in Greenhouse Raymond Cloyd 16:30-16:45 Production Systems

Chapel Insecticide Program Influence on Tomato spotted wilt Tom Turini 16:45-17:00 virus Symptom Expression in Processing Tomatoes in Central California

Chapel Impact of DuPont Cyazypyr™ (cyantraniliprole, DPX-HGW86) Billy Annan 17:00-17:15 on the Biology, Behavior and Control of Thrips (Thysanoptera) in Agronomic Crops

Chapel Endophytic Colonization of Onions and Their Antagonistic Alexander Muvea 17:15-17:30 Impacts on Biology of Onion Thrips

Chapel The Impact of Cover Crops and Companion Plants on Thrips Stan Diffie 17:30-1745 Vectors and Tomato spotted wilt virus Spread in Vegetable Farmscapes

Chapel Discussion Moderated by 17:45-18:00 Candice Stafford- Banks

Free Time 18:00-18:45

“The Circle” Board Buses for the Monterey Bay Aquarium Banquet 18:45 Just West of Social Hall

Banquet at the Monterey Bay Aquarium 19:00-22:30

15 WEDNESDAY 20 May-2015

Crocker Breakfast 7:30-9:00 Dining Hall ***Important Notes: Those staying at Asilomar should check out of their rooms before 11:00. All plenary session speakers please see Michelle Early at the Registration Desk, which will open at 8:30.***

Final Plenary Session Moderated by George Kennedy Room Title Presenting Time Author

Chapel Understanding of Vector Pathogen Interactions: Where is it Saskia Hogenhaut 9:00-9:45 Taking Us

Chapel Highlights from the Xth International Symposium on Thomas German and 9:45-10:30 Thysanoptera and Tospoviruses: Perspectives on the Diane Ullman, Future of Tospovirus and Thrips Research Ismael Badillo- Vargas, Candice Stafford-Banks, Norma Ordaz

Chapel Coffee Break 10:30-11:00

Chapel Presentation of Venues and Selection of Lead Group and George Kennedy 11:00-12:00 Venue for the XIth ISTT (2018), Summary Discussion and Closing Address

Crocker Lunch 12:00-13:00 Dining Hall

16 Realize partnerships. Many hands make light work. And the same goes for many minds. With DuPont Crop Protection on your side, you have all the scientists of DuPont working for you. Creating the solutions that will help improve your yields, DuPont your profits and your future. Crop Welcome to The Global Collaboratory.™ Protection

Copyright © 2014 E. I. du Pont de Nemours and Company. All rights reserved. The DuPont Oval Logo, DuPont™, The miracles of science™ and all products denoted with ® or ™ are trademarks or registered trademarks of DuPont or its affiliates.

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Abstracts

19 Opening Conference Speaker

Anticipating the likely effects of climate change on virus vectors and virus disease epidemics

Roger A.C. Jones

School of Plant Biology and Institute of Agriculture, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia; and Department of Agriculture and Food, Locked Bag No.4, Bentley Delivery Centre, WA 6983, Australia In the current era of accelerating climate change and human population growth, insect-vectored virus diseases and insect vector feeding damage both threaten world food security. The important direct climate change parameters to consider are alterations in temperature, rainfall, relative humidity, wind and greenhouse gasses. However, indirect climate change parameters are important too as hosts, viruses and vectors are influenced by climate induced temporal and spatial shifts in crop, reservoir host and weed distributions, and alterations in cultivation systems. Comprehensive frameworks were developed for each (i) important direct and indirect climate change parameter, and (ii) significant biological (host, vector and virus) parameter. Then, (i) and (ii) were cross checked against each other. This approach revealed that alterations in climate are likely to modify diverse components of virus disease epidemics and vector behaviour in many different ways, including altering host morphology, physiology and resistance to vectors or viruses, and vector and virus life cycles, abundance, diversity, reservoirs and inoculum. In most instances climate change seems likely to enhance damaging virus epidemics and vector feeding damage, but could have the opposite effect sometimes, e.g. in drying mid-latitude regions without irrigation. Moreover, as temporal and spatial shifts in their distributions cause newly introduced crops and weeds to meet indigenous vegetation, new encounter scenarios between cultivated and wild plants would accelerate appearance of epidemics caused by emerging viruses. Furthermore, climate instability is likely to diminish effectiveness of control measures and make virus epidemics less predictable causing increasing difficulties in suppressing them successfully using current management technologies.

20 Plenary Session I Looking to the Future

Current and emerging strategies in the war against thrips and tospoviruses Anna E. Whitfield Department of Plant Pathology, 4024 Throckmorton Plant Sciences Center Kansas State University, Manhattan KS 66506, USA

The virus transmission cycle involves host-finding, acquisition of virus, transport and delivery of virus to a new host plant. Each step in the transmission process provides an opportunity for interdiction. Strategies for disrupting transmission include biotech-based approaches to reduce vectorial capacity and population reduction approaches that utilize the specificity of the virus– vector interaction to target insects. Due to the unique biology of the TSWV–thrips interaction, there is a narrow window of opportunity for virus acquisition during larval development that is a good target for blocking virus entry. Previous work has shown that the GN protein of Tomato spotted wilt virus (TSWV) serves as a viral attachment protein. Transgenic tomato plants expressing a soluble form of GN were made and thrips that fed on these transgenics had significantly lower virus titers and transmission efficiencies than thrips fed on TSWV-infected non-transgenic tomato plants. These results demonstrate that an initial reduction in virus infection of the larval insect midgut resulted in a significant decrease in virus titer and transmission over the life-span of the vector. Exploiting the specificity of viral attachment proteins represents a new strategy to disrupt transmission of plant viruses. On the insect side of the interaction, RNA interference (RNAi) is considered to be the basis for the next generation of transgenic plants designed for insect pest control. Effective RNAi targets for several insect vectors of plant viruses have been V-ATPase genes and they have been shown to reduce insect life-span and egg production. Genome and transcriptome resources will aid in the identification of additional RNAi gene targets unique to thrips, and the discovery of essential interactions for vector transmission may yield vector-specific targets for pest control and reduction of virus transmission by RNAi. The development of new biotechnology-based strategies to reduce transmission by vectors and to decrease vector populations is attractive because they target pathways in the transmission cycle. However, the long-term effectiveness of these control methods will depend on their judicious use and incorporation into existing tospovirus and thrips control regimes.

21 Plenary Session I Looking to the Future

Genetic Modification of Insect Vectors: Innovations in Management Luke Alphey The Pirbright Institute, UK Genetic control methods are being developed for several vector-borne diseases, based on genetic modification of the vector. These may aim to reduce the numerical size of the pest population (“population suppression”) or to reduce the vector competence of insects carrying the novel genetic trait (“refractory insects”). Much progress has been made for mosquitoes for each approach, with successful field trials completed in several countries with engineered “sterile” strains for population suppression. Such genetic control methods may also be feasible for thrips. Furthermore, the necessary molecular methods underpinning the development of genetic control strains – transgenesis and genome editing – would have many other uses, for example in basic science. I will describe the state of the art of pest insect genetic manipulation and genetic control and discuss the potential application to thrips.

22 Plenary Session I Looking to the Future

Communicating with the Future – a response to diminishing taxonomic expertise

G. Moritz

Martin Luther University Halle-Wittenberg, Faculty of Natural Sciences I, Institute of Biology, 06108 Halle (Saale), Germany

More than 5,500 thrips species of the order Thysanoptera (thrips) are currently recognized worldwide. Only a few thrips are key pests of staples and high value horticultural crops worldwide and inflict substantial qualitative and quantitative losses due to their feeding, transmission of pathogens and their relevance as quarantine pests. The cryptic feeding behaviour, small size and diverse character states make their detection and identification during phytosanitary inspections more difficult. Expanding trans-border trade of crop products has resulted in thrips emerging as a rapidly growing group of invasive organisms. Precise and timely identification is very critical for better understanding of thrips biology, diversity, and effective management strategies, often under limited pest management instructions and time pressure. Recently taxonomists have taken advantage of developments in information and identification technologies, microscope software solutions and recognition of different nucleotide sequences in mainly conservative DNA segments. The talk will give an introduction and an overview of a number of modern thrips identification tools (Lucid Phoenix, Lucid 3.51), Lucid Key Server, Lucid Mobile2) and molecular methods and service tools based on ITS-RFLP for investigations and identifications from embryo to adult) and their personal use, which is clearly dependent on the requirements and the experience of the identifier. Every identification leads to a fact sheet of the examined species with lots of biological data including original computerized pictures of the species for verification purposes. But the talk is also a desperate attempt against the gradual and critical loss of taxonomists and of taxonomic teaching load worldwide.

I am grateful to Laurence Mound, CSIRO Canberra, for many supportive comments, criticisms and important discussions over the last thirty years.

23 Plenary Session I Looking to the Future

Diversity of tospoviruses and thrips vectors in Asia, and possible control by broad- spectrum transgenic resistance

Shyi-Dong Yeh (1, 2, 3), Tsung-Chi Chen (4, 5), Jui-Chu Peng (6), & Yazhisai Uthaman (1, 3)

(1) Department of Plant Pathology, National Chung Hsing University (NCHU), Taichung, Taiwan; (2) NCHU-UCD Plant and Food Biotechnology Center, NCHU; (3) Agricultural Biotechnology Center, NCHU; (4) Department of Biotechnology, Asia University, Wufeng, Taichung, Taiwan; (5) Department of Medical Research, China Medical University Hospital, China Medical University, Taichung, Taiwan; (6) Tainan District Agricultural Research and Extension Station, Tainan, Taiwan

Based on the sequence homology of nucleocapsid proteins (NPs), the current 29 putative virus species in the genus Tospovirus are classified into six phylogenetic clades, in which individual members share cross reactions based on their NP serology. In Asia, the reported tospoviruses are classified in Watermelon silver mottle virus (WSMoV) clade (9 species), Iris yellow spot virus (IYSV) clade (4 species) and Groundnut yellow spot virus (GYSV) clade (2 species). These species are spread by the most prevalent thrips vector Thrips palmi (for WSMoV clade), and other vectors including T. tabaci (for IYSV clade) and Scirtothrips dorsalis (for GYSV clade). The Asia-type tospoviruses and other globally important tospoviruses have created serious threats to many important crops in Asia. During the last decade, we have been collecting tospoviruses from different parts of the world and have accumulated 20 species in our library. Moreover, we have developed detection tools based on serological and nucleic acid technology for species-specific, serogroup-specific or genus-specific detection of tospoviruses. Efficient control measures by traditional breeding are difficult due to scarcity of natural resistant resources and high divergence of tospoviruses. Transgenic resistance mediated by post-transcriptional gene-silencing (PTGS) has been reported for the control of tospoviruses, by siRNA targeting N gene, NSs gene, or NSm gene; and by artificial microRNA targeting the conserved regions of L gene. By targeting the highly conserved region of L replicase, we have generated transgenic resistance against most prevalent tospoviruses at the genus level. Moreover, the conserved fragment of NSs gene, a PTGS suppressor, enhances the level of the broad-spectrum resistance provided by the L fragment. Our transgenic approach provides a more practical and durable measure for controlling the devastating diseases caused by the prevailing and newly emerging tospoviral strains or species in different regions of the world.

24 State of the Art Session 1: Thrips Biology and Systematics What Has Prevented Bean Thrips, Caliothrips fasciatus, from Becoming an Invasive Agricultural Pest?

Mark S. Hoddle, Paul Rugman-Jones, and Richard Stouthamer Department of Entomology, University of California, Riverside, CA 92521 Bean thrips, Caliothrips fasciatus (Pergande) (Thysanoptera: Thripidae), is native to California USA and was once considered an important agricultural pest in this state. It is a regular contaminant of navel oranges that are exported globally from California. Overwintering adults aggregate deep inside the “navels” of navel oranges making detection difficult and destructive sampling is often necessary to confirm their presence. Despite regular interceptions in navel oranges shipped to Australia and New Zealand, for example, over long periods of time, this insect has failed to establish populations in areas that should be permissive to establishment. Experimental evidence suggests several factors may contribute to this lack of invasiveness. First, cold storage temperatures of fresh oranges during containerized oceanic shipment may be sufficiently low and of long enough duration to significantly reduce the reproductive potential of surviving females. Second, bean thrips have a very peculiar mating biology requiring a quorum ( i.e., a group of five females) of virgin females from the same population to initiate oviposition of unfertilized eggs that developed into males. Third, mixed mating groups made up of genetically distinct populations resulted in very low levels of reproduction, but individuals from the same populations produced offspring. Wolbachia was not present in tested populations and therefore did not influence observed reproductive outcomes. Studying “boring” pest species sometimes reveals extremely interesting and unexpected biology such as that discussed here.

25 State of the Art Session 1: Thrips Biology and Systematics The Utility of Soil Amendments in Suppressing Thrips Populations Daniel S. Klittich & Michael P. Parrella Department of Entomology and Nematology, University of California, Davis, One Shields Ave, Davis, CA 95616 (USA)

Western Flower Thrips (Frankliniella occidentalis (Pergande) (Thysanoptera; Thripidae) and the tospoviruses they transmit continue to be a major problem for growers of cut chrysanthemums (Chrysanthemum indicum L.) in California. Cut chrysanthemum production is concentrated in Santa Barbara county (Carpinteria) and over the past 20 years there has been a gradual shift from chrysanthemums to other less sensitive cut flower crops (e.g., gerbera daisy [Gerbera hybrida]) primarily due to the difficulty in managing thrips and tospovirus. The common scenario of constant reinfestation from crops/weeds adjacent to the greenhouses present a difficult challenge for the grower, especially for those who are trying to manage the overall pest complex using biological control. Managing thrips using natural enemies is intensive and expensive – typically this requires regular foliar application of predatory mites and entomopathogens (in addition to a food supplement for the predatory mites) and then releases of a different predatory mite into the soil as well as soil applied entompathogenic nematodes. Even with this barrage of natural enemies, the level of thrips control is often below what the grower expects. Additional management strategies are needed that impact thrips’ populations and are compatible with this suite of natural enemies. Recent studies using silicon as a soil supplement (@ 300-500 ppm) applied at irrigation have shown promise for suppressing another important pest of chrysanthemum – the leafminer Liriomyza trifolii Burgess (Diptera; Agromyzidae). Studies in greenhouses at UC Davis and with cooperating chrysanthemum growers in Carpinteria suggest that silicon may have a role to play in managing thrips populations.

26 State of the Art Session 1: Thrips Biology and Systematics Thrips Biology and Systematics – a Tale of Two Cities? Laurence Mound Australian National Insect Collection, CSIRO, Canberra, Australia, 2601 [email protected] “It was the best of times, it was the worst of times, it was the age of wisdom, it was the age of foolishness, it was the epoch of belief, it was the epoch of incredulity, it was the season of Light, it was the season of Darkness, it was the spring of hope, it was the winter of despair.” This talk will explore the commonly accepted paradigm that taxonomists describe species and biologists study the lives of species, and consider the possibilities of a more rational approach to the study of the diversity on which we are all dependent.

27 State of the Art Session 1: Thrips Biology and Systematics The greenhouse thrips, Heliothrips haemorrhoidalis: complex species or species complex? Paul Rugman-Jones (1), Mark S. Hoddle (1), Richard Stouthamer (1) (1) Department of Entomology, University of California, Riverside, CA 92521 The greenhouse thrips, Heliothrips haemorrhoidalis (Bouché) (Thysanoptera: Thripidae), is a significant economic pest of numerous crops, that has spread worldwide. Originally native to South America, invasive populations of this species demonstrate obligate thelytoky, under which males are completely absent. Thelytoky in H. haemorrhoidalis was long thought to be induced by the maternally inherited bacterium Wolbachia, but a recent study demonstrated very convincingly that bacterial reproductive manipulators were in fact not involved. Instead, polyploidy was implicated as a likely factor in the thelytokous reproduction of H. haemorrhoidalis. The incidence of males in H. haemorrhoidalis populations from Peru suggests that sexual reproduction still occurs in the native range, and points to the possible existence of an ancestral sexual population from which the global-conquering thelytokous form originated. Indeed, this idea is supported by recently published evidence from a geographically broad sample of thelytokous H. haemorrhoidalis populations that revealed very low levels of genetic diversity. Here we use a combination of mitochondrial and nuclear markers, to characterize invasive, thelytokous populations of H. haemorrhoidalis worldwide, and compare them with native sexual populations from Peru. In contrast to the earlier study, we found surprisingly high (and corroborating) levels of genetic divergence in both the mitochondrial and nuclear genes of individuals identified morphologically as H. haemorrhoidalis. Levels of divergence suggest as many as six species may currently be hiding under the guise of H. haemorrhoidalis, which are variously infected withWolbachia. The potential roles of polyploidy and bacterial reproductive manipulators in the evolution, maintenance, and success, of these “species” will be discussed.

28 State of the Art Session 1: Thrips Biology and Systematics What happens when ectoparasitic thrips get on auchenorrhynch hemipterans? A behavioural and multitrophic approach

Adriano Cavalleri & Milton de Souza Mendonça Jr. Department of Ecology, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, RS, Brazil

Aulacothrips dictyotus (Heterothripidae) was previously recorded ectoparasitising nymphs and adults of Aetalion reticulatum (Hemiptera: Aetalionidae) in Brazil, and this association was supposed to be singular amongst thrips. However, recent studies revealed a surprising diversity of ectoparasitic Aulacothrips species in South America, and five species are now known in this genus. Despite exhibiting different degrees of host range and specialization, all Aulacothrips species were found infesting gregarious hemipterans (Aetalionidae and Membracidae) which have mutualistic interactions with ants. In fact, this system involves a complex multitrophic association comprised by thrips, hemipterans, plants and ants. In most cases these ants provide protection against enemies, however, they do not attack Aulacothrips. Preliminary observations suggest that the presence of A. dictyotus in Aetalion aggregations affect host behaviour, which become agitated. We analyzed the effect of A. dictyotus presence on the behaviour of A. reticulatum through comparisons of behavioural repertories of thrips-infested versus non-infested individuals in the field. We excluded the ants from all aggregations and for each experimental group we made 43 hours of quantitative observations of nonsequential parameters. Our results indicated that A. dictyotus alter host behaviour, and infested bugs displayed a large number of behavioural acts related to self-cleaning, and they execute these activities at higher frequencies when compared to thrips-free hemipterans. The number of records related to feeding was also lower in infested A. reticulatum. Moreover, thrips-infested aetalionids showed more locomotion and dispersal records. Thrips ectoparasitism may also have an indirect effect on the hemipteran interaction with tending-ants by affecting host behaviour and physiology. Changes in the presence and intensity of this mutualism might affect the other participants of this multitrophic system as well. Investigating the impact of Aulacothrips in the quality and quantity of the honey-dew produced by host hemipterans seems a promising topic for answering this question.

29 State of the Art Session 1: Thrips Biology and Systematics

Interactions between Frankliniella bispinosa (Morgan) and Frankliniella cephalica (D.L. Crawford) in Florida

T. Skarlinsky(1) J. Funderburk (2) & A. Wallner (3) (1)(3) USDA, APHIS, PPQ, P.O. Box 660520, Miami, Florida 33266 USA (2) University of Florida, 155 Research Road, Quincy, Florida 32351 USA Frankliniella bispinosa (Morgan) and Frankliniella cephalica (D.L.Crawford) are morphologically similar species found on the flowers of Romerillo, Bidens alba (L.) DC. in Florida. In other Neotropical regions F. cephalica is reported to be widespread, but in Florida it is usually reported from this ubiquitous wild flower. The actual B. alba/flower thrips distribution in Florida is poorly understood as a result of misidentification of the two similar thrips species and near absence of previous study. Furthermore, the two flower thrips reproductive utilization of B. alba has not been studied. To evaluate the distribution of the two thrips species, and test the hypothesize that B. alba is a host of either thrips species, flowers of B. alba were sampled at 10 mile increments from the southern extreme of the Florida Keys to the northern border of Georgia. Adults and larvae were identified to species based on morphological differences. F. cephalica (~99%) was the predominant species identified from B. alba flowers between the 24° - 28° latitudes, mixed populations F. bispinosa (77%), F. cephalica (33%) between 28° - 30° and F. bispinosa (~99%) north of the 30° latitude. Moreover, the majority of larvae identified were F. cephalica (91%),suggesting that F. cephalica populations are utilizing B. alba as a host in Florida. These initial data suggest the widespread F. cephalica has become a specialist in Florida to avoid competition from F. bispinosa and other flower thrips species.

30 State of the Art Session 1: Thrips Biology and Systematics Biotic Resistance Limits the Invasiveness of the Western Flower Thrips, Frankliniella occidentalis (Thysanoptera: Thripidae), in Florida

Joe Funderburk (1) (1)Department of Entomology and Nematology, North Florida Research and Education Center, 155 Research Road, Quincy, FL 32351, USA The spread of the western flower thrips, Frankliniella occidentalis (Pergande), has resulted in the world-wide destabilization of established integrated pest management programs for many crops. It is hypothesized that frequent exposure to insecticides in intensive agriculture selected for resistant populations, which allowed invasive populations in the eastern USA to overcome biotic resistance from the native community of species. Research conducted in Florida to understand the role of biotic factors in limiting the abundance of the western flower thrips is reviewed. Orius spp. (Hemiptera: Anthocoridae) are effective predators that suppress populations of the common flower thrips species on many crop and non-crop hosts in southern and northern Florida. They are more effective predators of the western flower thrips than the native flower thrips, F. tritici (Fitch) and F. bispinosa (Morgan). These native species are competitors of the western flower thrips. Excessive fertilization and the use of broad-spectrum insecticides in crop fields further enhances populations of the western flower thrips. Biotic resistance clearly limits the abundance of western flower thrips in Florida, but populations are abundant in fertilized crop fields where application of insecticides excludes predation and interspecific competition.

31 State of the Art Session 1: Thrips Biology and Systematics Dark and pale color morphs of common blossom thrips Frankliniella schultzei (Trybom) – Are they just colour forms? M. W. Gikonyo (1, 2), S. Subramanian (1), S. Niassy (1), F. Khamis (1), E. Magiri (2), Villinger Jandouwe (1), I. Schütz (3) & G. B. Moritz (3) (1) Plant Health Division, International Centre of Insect Physiology and Ecology, P.O. Box 30772-00100, Nairobi, Kenya; (2) Department of Biochemistry, Jomo Kenyatta University of Agriculture and Technology (JKUAT), P.O. Box 62000-0200 Nairobi, Kenya; (3): Faculty of Natural Sciences, Institute of Biology, Martin Luther University Halle-Wittenberg, Domplatz 4 06108 Halle (Saale), Germany The common blossom thrips, Frankliniella schultzei (Trybom) is an important pest of ornamental and vegetable crops worldwide and a vector of Tospoviruses. They occur as two color morphs (dark and pale) which are morphologically very similar but with differences in vector competency and geographic spread. In Kenya, mixed populations of the color forms are observed with overlapping host range. To ascertain the taxonomic status of the two color forms, we characterized them based on morphological, molecular and reproduction characteristics. Results indicated that the third ocellar setae were longer in dark forms than the pale forms. The third ocellar setae of the dark form were also placed closer to the anterior tangent across the hind ocelli as compared to the pale forms. The two color morphs also differed widely in their ITS2- RFLP analysis banding patterns and this was also confirmed by ITS2 nucleotide sequences analysis. The offspring of dark form parents were morphologically identical to the parents, while laboratory colonies of pale forms conspicuously lacked males. Virgin pale females showed thelytokousparthenogenesis giving birth to females of the pale form while virgin females of dark forms exhibited arrhenotokous parthenogenesis giving birth to males of the dark form. Interbreeding of dark males with pale females consistently resulted only in pale females indicating absence of interbreeding between the two colour forms. Further field samples of F.schultzei males in different host plants indicated only presence of dark males. Hence dark forms reproduced sexually and asexually (arrhenotokous) whereas the pale forms reproduced only asexually (thelytokous). Lack of interbreeding, their distinct morphological features and molecular characteristics indicate that the two color forms of F.schultzei could be different species. The results also highlight the need to assess morphological, molecular and biological criteria for species nomenclature.

32 State of the Art Session 1: Thrips Biology and Systematics Duong is a Laurence Mound Fellow Cardinium and Wolbachia induce cytoplasmic incompatibility and sex ratio shifts in an Australian species of thrips Duong T Nguyen (1), Robert N Spooner-Hart (1,2), & Markus Riegler (1) (1) Hawkesbury Institute for the Environment, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia; (2)School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia Kelly’s citrus thrips, Pezothrips kellyanus (Thripidae), is an economically important pest of citrus in Australasia and the Mediterranean region. First recorded in Queensland in 1914, the species was later placed in the genus Pezothrips mostly restricted to the Palearctic. We investigated the genetic diversity of P. kellyanus collected from its current range in order to detect its most likely origin. In this way, we also detected Cardinium and Wolbachia for the first time and studied their roles in the reproduction of this species. Analyses of mitochondrial and nuclear genes revealed that P. kellyanus originated from Australia where these genes were more diverse. Haplotype analysis also suggested independent colonization of New Zealand and the Mediterranean region. This species is naturally co-infected by Cardinium and Wolbachia in its native Australian populations, while invasive populations appear to have lost Wolbachia, and harbour Cardinium only. We hypothesized that Wolbachia may impact host fitness and population dynamics more significantly than Cardinium through a sex ratio distortion that may be inhibiting invasion success. Through crossing experiments, we found that Wolbachia induced cytoplasmic incompatibility (CI) with female embryonic mortality, combined with larval mortality in incompatible crossing combinations. In contrast, Cardinium induced an intermediate expression of female mortality and male development with weaker larval mortality. Furthermore, both Cardinium and Wolbachia infections resulted in a female bias in compatible crosses. Transmission efficiency was complete for Wolbachia with some leakage for Cardinium. We concluded that both bacteria are reproductive parasites in this host species, however the effect of Wolbachia is more severe and may more strongly impact establishment of invasive populations. Our findings are the first report of CI for Thysanoptera, an insect order that has independently evolved haplodiploidy, and, thus, an ideal arena for the evolution of host sex ratio manipulations by reproductive parasites.

33 State of the Art Session 1: Thrips Biology and Systematics Detection of Wolbachia in some natural populations of thrips species (Insecta, Thysanoptera) in Mediterranean habitats

A. De Grazia *1, R. Marullo*1, G. Moritz*2 *1Department of Agriculture, Section of Entomology Mediterranean University of Reggio Calabria, Località Feo di Vito, 89060, Reggio Calabria, Italy; *2 Martin-Luther-Universität Halle-Wittenberg Naturwissenschaftliche Fakultät I, Biowissenschaften Institut für Biologie/Entwicklungsbiologie, Heinrich-Damerow-Str. 4A (TGZ III) D-06120 Halle (Saale), Germany Wolbachia, which belong to the α -protobacteria, are the most widely distributed bacterial endosymbionts in arthropods and nematodes. It is believed that they infect up to 70% of the world’s insect species (Stouthamer et al.,1999; Rowley et al.,2004; Baldo et al.,2007; Jacob et al.,2014). These bacteria cause a number of reproductive alterations in their hosts, including cytoplasmic incompatibility (CI) among strains (Clancy & Hoffmann, 1996; O’Neill & Karr,1990) and related species (Breeuwer et al.,1992; Breeuwer & Werren, 1990), parthenogenesis induction (PI) (Stouthamer et al.,1993), feminization of genetic males (Rousset et al., 1992)or they can be involved in the oocyte production (Dedeine et al.,2001). These modifications of host reproduction impart a selective advantage for the bacteria (Turelli,1994; Werren et al.,1995). This study was conducted using 16S rDNA and ftsZ gene primers in order to evaluate the presence or absence of the bacterium. Seven thrips species, i.e. Bagnalliella yuccae (Hinds), Echinothrips americanus Morgan, Frankliniella occidentalis (Pergande), Gynaikothrips ficorum (Marchal) Heliothrips haemorrhoidalis (Bouchè), Holarthrothrips tenuicornis Bagnall and Thrips tabaci Lindeman, were screened for the Wolbachia infection. The results obtained in this study support the earlier literature reports that assert E. americanus and G. ficorum as infected. Instead, Wolbachia was not found in T. tabaci and F. occidentalis , according with Kumm and Moritz (2008), who examined populations of both species. Contrary to the data obtained by Pointureau et al. (1999), the analysis carried on field strains of H. haemorrhoidalis collected in southern Italian habitats, revealed the absence of this bacterium. Moreover, the presence of Wolbachia was screened for the first time in populations of B. yuccae, living on wild and cultivated Yucca plants and H. tenuicornis from palm trees, which grow on the Mediterranean coast areas. The preliminary results suggest that further studies are necessary in order to clarify the reproductive ways of the natural thrips populations.

34 State of the Art Session 1: Thrips Biology and Systematics Romana is a Laurence Mound Fellow

DNA barcode analysis of thrips (Thysanoptera) diversity in Pakistan Romana Iftikhar (1), Muhammad Ashfaq (2), Akhtar Rasool (1) (1) National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore, Islamabad 45650, Pakistan; (2) Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada Although thrips are important crop pests and virus vectors, their diversity in most parts of the world is under explored. Correct identification of thrips is generally constrained due to their small size and inconspicuous morphological characters. Use of mitochondrial COI-5' (DNA barcode) for species identification and genetic diversity analysis in animals has proved very effective. We analyzed DNA barcode sequence (>500 bp) variation among 471 thrips collected from various plant hosts in north-central Pakistan. Sequence analysis revealed that the intraspecific and interspecific distances ranged from 0.0% to 7.5% and 2.3% to 22.3%, respectively. The Barcode Index Number (BIN) system assigned the 471 sequences to 55 BINs which were congruent with the recursive partitions revealed by Automatic Barcode Gap Discovery (ABGD). The Neighbor-Joining clustering and Bayesian inference supported the presence of 55 monophyletic lineages. Four important thrips species in the region including three pest/ disease vectors (Black flower thrips, Haplothrips reuteri (Karny), Melon thrips, Thrips palmi(Karny) and Onion thrips, Thrips tabaci (Lindeman) and one predator (Banded thrips, Aeolothrips intermedius (Bagnall) showed high intraspecific distances indicating that the species were cryptic complexes. Sequences analysis of COI-3' end (439 bp) from T. palmi and T. tabaci supported the occurrence of divergent lineages for these species. In conclusion, the DNA barcodes successfully discriminated thrips to their species. The study compiles the first DNA barcode reference library for thrips from Pakistan and connects regional lineages of four important pest and virus-vectors with those from other countries by haplotype networks.

35 State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

Potential for Natural Plant Resistance for Control of Insects and Insect-Vectored Viruses Martha A. Mutschler (1) Darlene De Jong (1), Brian Leckie (1), John Smeda (1) (1) Department of Plant Breeding and Genetics, Cornell University, 301 Bradfield Hall Ithaca, NY 14853

Sustainable insect and disease control is needed to ensure reliable production of marketable vegetables. Tomato, Solanum lycopersicum L. is a particularly good system for the study of natural plant resistance and resistance mechanisms, due its widespread growth globally, and the wealth of information available on its insect pests, as well as prior work on insect resistance in wild tomato species. Insect resistance has been associated with several types of secondary metabolites including compounds such as monoterpenes, sesquiterpenes, flavonoids and glycoalkaloids, as well as induced systems such as jasmonate-regulated proteinase inhibitors. While several of these mechanisms might be of eventual use, perhaps the most promising system in wild tomato involves the production of acylsugars, which have an unusual breadth of defensive activity across insect species. Acylsugars produced by several wild Solanum species, including Solanum pennellii Correll, are strongly associated with insect resistance. Previous work with purified S. pennellii acylsugars demonstrated that acylsugars act through deterrence, thereby reducing feeding and oviposition by many insect species without selecting for resistance. Using S. pennellii LA716, the Cornell tomato breeding program created acylsugar-producing tomatoes, first CU97FL and later CU071026. These lines which produce acylsucroses with defined fatty acid constituents provide >95% control of Silverleaf Whitefly (Bemisia tabaci Gennadius) in field cages and under natural infestation in field. Additional acylsugar- producing lines that vary in acylsugar level, or that also possess virus resistance genes such as Sw-5 (for TSWV) orTy-3 (for TYLCV control) have also been created and are being used in interdisciplinary research to test for control of insects and the viruses they vector. Strategies for testing these lines, and use of such lines for research and eventually for sustainable insect control in tomato production will be discussed.

36 State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

John is a TTEN Fellow

Utilizing Acylsugar Chemistry for Optimal Thrips and Tospovirus Control in Tomato John Smeda (1), Brian Leckie (1), Martha A. Mutschler (1) Diane Ullman (2), George Kennedy (3)

(1) Department of Plant Breeding and Genetics, Cornell University, 301 Bradfield Hall Ithaca, NY 14853; (2) Department of Entomology, University of California, Davis, One Shields Ave. Davis, Ca 95616, USA; (3) Department of Entomology, North Carolina State University, 2301 Gardner Hall, Raleigh, NC 27695, USA Tomatoes are attacked by many pests, causing losses directly through feeding damage and indirectly through virus transmission. The control of insects relies heavily on pesticides, a practice that is increasingly crippled by development of pesticide-resistant insects and increased health and environmental concerns; therefore, alternative means of insect control are necessary. Acylsugars produced by some wild Solanum species, including Solanum pennellii Correll, are associated with strong insect resistance. Work with purified S. pennellii acylsugars demonstrated that acylsugars reduce feeding and oviposition by many insect species. S. pennellii accessions collected from different regions of the species native range have unique acylsugar chemotypes. This diversity among S. pennellii accessions raises questions regarding their functional differences and whether this diversity can be leveraged to optimize acylsugars as a defense strategy. Using S. pennellii LA716, Mutschler’s breeding program created an acylsugar- producing tomato, CU071026, which produces acylsucroses with defined fatty acid constituents. Using a BC1F1 population derived from CU071026 and S. pennellii LA716, a series of QTL were identified that modify acylsugar chemotype. I will present data that when these QTL were introgressed into CU071026 the resulting sister lines differed for acylsugar chemotype as predicted, confirming the function of the QTL. These lines are being used to evaluate the efficacy of the chemotype shift in planta on control of critical tomato pests. I will present recent work where lines with altered acylsugar chemotype were evaluated in field trials with natural thrips and virus pressure. Data was taken on thrips feeding and virus incidence, as well as on acylsugar level and acylsugar chemistry through gas chromatography characterization. These data will help elucidate not only the effect of the environment on acylsugar level and chemistry, but also the effect of acylsugar chemotype on insect feeding and oviposition, and also inform the breeding effort on optimal acylsugar producing lines for thrips control.

37 State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

Flower Power: The Potential Role of Acylsugars on Western Flower Thrips Oviposition in Tomato Flowers C. Kaplinsky (1), C. Stafford-Banks (2), M. Mutschler (3), J. Smeda (4), G. Kennedy (5), T. Chappell (6), D. Ullman (7) (1, 2, 7) Department of Entomology, University of California, Davis, One Shields Ave. Davis, Ca 95616, USA, (3,4) Department of Plant Breeding and Genetics, Cornell University, 240 Emerson Hall, Ithaca, NY 14853, USA, (5, 6) Department of Entomology, North Carolina State University, Research Annex West, Raleigh, NC 27695, USA Western flower thrips (Frankliniella occidentalis) and its ability to transmit Tomato spotted wilt virus (TSWV) poses an important threat to agricultural and horticultural industries. Leaves of tomato breeding lines developed by M. Mutschler at Cornell University have type IV glandular trichomes that produce the secondary compounds known as acylsugars. Acylsugars are complex molecules with diverse compositions that act as deterrents to many insects, including thrips. Thrips feeding and oviposition on leaves are deterred by acylsugar production, but the impact of acylsugars in altering thrips behavior in the flowers of acylsugar-producing tomato lines has not been characterized. In our experiments, we used lines known from previous research to vary in the level and/or composition of acylsugars produced by type IV trichomes on the leaves. We hypothesized that acylsugar-producing type IV glandular trichomes would occur and produce acylsugars on some floral structures of these lines, and that the level and composition of these acylsugars would affect Western flower thrips oviposition. For each entry tested, we examined floral tissue for the presence of acylsugar-producing glandular trichomes and counted eggs on sepal, petal, stamen, and pistil tissues. This work was performed using tomato flowers at four different developmental stages. Acylsugar-producing glandular trichomes were present on all four stages of tomato flower, primarily occurring on sepal tissue and to a lesser extent on petal tissue. Thrips oviposition rate was highest on fully developed and open flowers and in petal tissue and significantly decreased on lines with higher acylsugar levels. These results show that acylsugar-mediated resistance is expressed in flowers, as well as leaves. Our findings have implications for tomato breeding aimed at use of this natural resistance for control of thrips and tospoviruses, as well as for future research on the effectiveness of acylsugars as a potential sustainable management mechanism for thrips-transmitted tospoviruses.

38 State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

Athos is a Laurence Mound Fellow

The Sw-5 gene cluster: Unravelling the keys to resistance to Tomato spotted wilt virus Athos Silva de Oliveira (1,2); Renato Oliveira Resende (2); Richard Kormelink (1) (1) Laboratory of Virology, Department of Plant Sciences, Wageningen University, Wageningen, Netherlands; (2) Laboratory of Virology, Department of Cell Biology, Institute of Biological Sciences, University of Brasília, Brasília, Brazil Tomato spotted wilt virus (TSWV) causes substantial losses on crop production around the world. So far only two natural resistance sources are available for commercial plant breeding against TSWV and other tospovirus species. One of them is the Sw-5b gene which encodes a CC-NB-ARC-LRR protein able to halt tospovirus infections in Solanum peruvianum L. and bred S. lycopersicon L., wild and commercial tomato species, respectively. Here we show that the cell-to-cell moment protein (NSM) of TSWV has been identified as the avirulence determinant (Avr) of the Sw-5b-mediated resistance. The transient expression of the NSMprotein triggers a clear hypersensitive response (HR) in tomato and Nicotiana benthamiana L. harboring the Sw-5bgene. Moreover, it is shown that a high Sw-5b accumulation in N. benthamiana leaves, achieved by co-expression of theSw-5b protein with RNA silencing suppressors (RSS), leads to auto-HR in the absence of NSM. In a similar approach Sw-5a, the highest conserved paralog of Sw-5b from S. peruvianum, also triggered auto-HR while the highest conserved ortholog from susceptible S. lycopersicon, namedSw-5aS, did not. None of those last two homologs, however, were able to trigger a NSM-dependent HR. Truncated and mutated versions of Sw-5 homologs revealed that the NB-ARC domain is sufficient for HR-triggering and seems to be suppressed by the CC domain. Furthermore, a single mutation was sufficient to restore auto-HR activity within the NB-ARC domain of the Sw-5aS homolog. When the latter was fused to the Sw-5b LRR domain, NSM-dependent HR triggering was regained, but not in the presence of its ownSw- 5aS LRR domain. A model for the activation of Sw-5band the functionality of other homologs will be discussed.

39 State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

Norma is a TTEN Fellow In Search of the Early Events in Sw-5 Mediated Resistance: Optimizing Tomato spotted wilt virus Infection of Tomato Norma Ordaz1, Candice Ann Stafford-Banks2, S. P. Dinesh-Kumar3, Diane E. Ullman2 1Department of Plant Pathology, 2Department of Entomology and , 3Department of Plant Biology and The Genome Center University of California, Davis, One Shields Ave. Davis, CA 95616, Tomato spotted wilt virus (TSWV) infects a large number of important crops worldwide and is the type member of the genus Tospovirus, the only plant infecting member of the family Bunyaviridae. Economically, TSWV results in extensive damage in tomatoes worldwide. TSWV is transmitted by several thrips species, the most important of which is Frankliniella occidentalis, the Western flower thrips. The most commonly deployed gene for TSWV resistance in tomato is Sw-5. The long term goal of our study is aimed at revealing early and discernable transcriptional and proteomic changes triggered by Sw-5-mediated recognition of TSWV. Because there is no infectious clone for TSWV, a prerequisite for conducting these experiments is a robust system for optimizing TSWV infection in tomato through mechanical and thrips inoculation. We hypothesized that isolate and plant developmental stage would be significant components in optimizing infections across a large number of plants. With mechanical inoculations we demonstrated that tomato cv. Celebrity plants at developmental stage 3 (3 expanding leaves) were an optimum size, reached 100% infection and showed symptoms earlier than plants at developmental stages 2, 4, 5, 6 and 7. Enzyme-linked immunosorbent assay (ELISA) showed that virus could be detected in thrips larvae 24 hours post-acquisition. Lower ELISA readings resulted during the two pupal stages, increasing again in the adult stage. The relationship between ELISA results and transmission of TSWV will be discussed.

40

State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

Harnessing artificial microRNAs for resistance to Tospoviruses: The Way Forward N. Mitter (1), Ying Zhai (2), A.X. Bai (1), K. Chua (1), S. Eid (2), R. Mitchell (1), & H.R. Pappu (2) (1) Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, QLD, Australia; (2) Department of Plant Pathology, Washington State University, Pullman, WA, USA; (3) Department of Agriculture and Fisheries, QLD, Australia. Host-mediated gene silencing against virus infections involves the siRNA pathway by deliberate production of sequence-specific dsRNA. Another approach is the artificial miRNA (amiRNA)- mediated antiviral resistance that imitates the natural miRNA pathway to degrade the viral target. It uses endogenous miRNA precursors as a backbone in which the 21-nt region of a mature miRNA is replaced with a specific sequence complementary to the target. Compared to siRNA- mediated resistance, the amiRNA approach offers many advantages: it utilizes only 21-nts from the viral genome compared to siRNAs from longer hairpin sequences (>300 nt) thereby reducing the extent of off-target effects, facilitates targeting conserved regions common to multiple vial strains/viruses to deliver broad-spectrum resistance and is stable at wider temperatures. We developed an artificial micro RNA (amiRNA) strategy against Tomato spotted wilt virus (TSWV), targeting the nucleoprotein (N) and silencing suppressor (NSs) genes. The amiRNA constructs replaced the natural miRNA in a shortened Arabidopsis 173 nucleotide miR159a precursor backbone (athmiR159a) without the stem base extending beyond the miR/miR* duplex. Agro-infiltration-mediated transient expression in Nicotiana benthamiana followed by challenge inoculation with TSWV, it was found that the plants expressing N-specific amiRNAs remained asymptomatic and were negative for TSWV by ELISA. In contrast, plants expressing the NSs-amiRNAs were symptomatic and accumulated high levels of TSWV. Similar results were obtained in stably transformed N. tabacum plants. Our results show that a shortened 173-nt athmiR159a backbone is sufficient to express amiRNAs and that the presence of mismatch at position 12-13 does not influence amiRNA expression or conferring of resistance. We also show that selection of target gene and positional effect are critical in amiRNA-based approach for introducing resistance. We are progressing on employing the amiRNA approach for broad- spectrum resistance to tospoviruses by targeting both virus and vector sequences.

41 State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

An eco-metabolomic approach to study host plant resistance to Western flower thrips

K. Leiss (1), Y. Choi (2), P. Klinkhamer (1) (1)Plant Ecology and Phytochemistry, Institute of Biology, Leiden University, Sylvisusweg 72, 2333 BE Leiden, The Netherlands; (2)Natural Products Laboratory, Institute of Biology, Leiden University, Sylvisusweg 72, 2333 BE Leiden, The Netherlands Western flower thrips, Frankliniella occidentalis (Pergande) is a key agricultural and horticultural pest worldwide. Its control proved to be difficult and demands the use of multiple tactics in the framework of an integrated pest management (IPM) programme. Chemical host plant resistance, based on secondary plant metabolites, which form a tremendous potential of natural crop protection, is an essential part of IPM. Therefore, we developed an eco- metabolomic approach to identify candidate compounds for host plant resistance to Western flower thrips (Leiss et al., 2011). We classify resistant and susceptible plants using in- vivo bioassays. Subsequently, we establish NMR (Nuclear Magnetic Resonance Spectroscopy) profiles which are compared by multivariate statistics to identify metabolites involved in host plant resistance. The negative effect of the candidate compounds is validated with in- vitro bioassays. We applied the approach in different host systems including Senecio as a wild plant, chrysanthemum and gladiolus as ornamentals and tomato and carrot (Leiss et al., 2013) as vegetables. In all systems the metabolomic profiles of resistant and susceptible plants were significantly different leading to a range of different metabolites involved in thrips resistance. The majority of these compounds were phenylpropanoids and flavanoids. Interestingly, these did not only show a negative effect on thrips, but as anti-oxidants, were also linked to positive human health effects such as prevention of cancer and cardio-vascular diseases. As such these compounds may not only inhibit thrips but also improve human health. Therefore, our eco- metabolomic approach constitutes a significant advance in the study of plant-insect relationships providing key information for the development of thrips pest management such as the implementation of thrips plant resistance breeding programmes. Leiss, K.A., Choi, Y.H., Verpoorte, R. & Klinkhamer, P.G.L. (2011) An overview of NMR- based metabolomics to identify secondary plant compounds involved in host plant resistance. Phytochemistry Reviews 10: 205-216. Leiss, K.A., Cristofori, G. van Steenis, R., Verpoorte, R. & Klinkhamer P.G.L. (2013) An eco- metabolomic study of host plant resistance to Western flower thrips in cultivated, biofortified and wild carrots. Phytochemistry 93: 63-70.

42 State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

Field Resistant Peanut Cultivars for Management of Tomato spotted wilt: History, Improvements, and Stability A.K. Culbreath (1), W.D. Branch (2), R. Srinivasan (3), C.C. Holbrook (4), & B.L. Tillman (5) (1) Department of Plant Pathology, The University of Georgia, 2360 Rainwater Rd. Tifton, GA 31793, USA; (2) Department of Crop and Soil Science, The University of Georgia, 2360 Rainwater Rd. Tifton, GA 31793, USA; (3) Department of Entomology, The University of Georgia, 2360 Rainwater Rd. Tifton, GA 31793, USA; (4) USDA-ARS, Crop Genetics and Breeding Research Unit, Tifton, GA 31793, USA; (5) Agronomy Department, University of Florida, Marianna, FL 32446, USA. In the early 1990s, tomato spotted wilt caused by Tomato spotted wilt virus (TSWV) emerged as a major problem in peanut (Arachis hypogaea) production areas of the southeastern U.S. Predominant cultivars grown at that time were very susceptible to TSWV, but a moderate level of partial field resistance was found in the cultivar Southern Runner. Although Southern Runner was never planted extensively, it was a parent of Georgia Green, released in 1995. Georgia Green has a level of field resistance similar to that of Southern Runner, and was the predominant cultivar grown in the southeastern U.S. for over 10 years. The level of field resistance in Georgia Green was not sufficient to provide adequate control of spotted wilt in severe situations, but the cultivar responded well to cultural and chemical practices that also suppressed the disease. Field resistance to TSWV has been an essential area of emphasis of university and USDA peanut breeding programs in Georgia and Florida, and several cultivars and breeding lines have been developed with better field resistance than that of Georgia Green. In the mid-2000s, cultivars Georgia-06G, Georgia-07W, Florida-07, Tifrunner, and Tifguard were released with much better field resistance to TSWV than Georgia Green. These cultivars have allowed more flexibility regarding other practices that suppress spotted wilt, and have excellent yield potential. Even higher levels of field resistance were found in the cultivar Georganic, and breeding lines NC 94022 and NC 94002. Newer cultivars Georgia-10T, Georgia-12Y, TifNV-High O/L, and several breeding lines from multiple programs have even greater levels of field resistance. Although field resistance is not complete, cultivars and breeding lines available now have sufficient resistance to allow much less dependence on other suppressive practices. Field resistance in these cultivars and breeding lines has been stable.

43 State of the Art Session II: Developing Host Plant Resistance and Integrated Management of Tospoviruses and Thrips As Vectors

Development of methods for maintenance of lettuce-infecting tospoviruses and germplasm evaluation W.M. Wintermantel and I. Simko USDA-ARS, Salinas, CA Tomato spotted wilt virus (TSWV) and Impatiens necrotic spot virus (INSV) (family Bunyaviridae, GenusTospovirus) have become problematic for California lettuce production due to a dramatic increase in thrips populations that has resulted in severe thrips damage and more importantly, virus transmission to a wide range of crops, including not only lettuce (Lactuca sativa) but also many crops grown in proximity to lettuce. Management of tospoviruses in lettuce is difficult, and there is little information on resistance to either TSWV or INSV. Furthermore, field evaluations for identification of resistance in lettuce are challenging due to the inconsistent and intermittent prevalence of these viruses and their thrips vectors. Transmission of TSWV and INSV can be accomplished by either using the thrips insects to deliver these viruses to test plants, or by mechanical transmission in which virus-infected sap is rubbed on the leaf of a test plant. The latter method is preferable because it does not require the production and management of large thrips populations that can cause feeding damage and spread tospovirus to other experiments in nearby greenhouses. However, studies have shown that sequential mechanical inoculation of plants with tospoviruses without passage through the thrips vector in which the virus replicates results in loss of infectivity. Therefore, efforts were initiated to develop a viable thrips-free method for evaluation of tospovirus resistance in lettuce. Studies compared effectiveness of mechanical transmission of each virus from different infected source plants and with different virus and/or thrips propagation strategies. Although numerous methods were explored, effective mechanical transmission was only possible when virus and thrips are propagated together, followed by mechanical passage of each virus from newly established infected plant tissue to lettuce. Optimization is continuing but methods to date indicate and effective strategy for resistance evaluation that can be used for large scale evaluations.

44

State of the Art Session III: Integrated Management of Tospoviruses and Thrips As Vectors

Field and Farmscape IPM for Onion Thrips and Iris Yellow Spot Virus Diane Alston (1), Claudia Nischwitz (1), Bonnie Bunn (1), Daniel Drost (2), Jennifer Reeve (2), Corey Ransom (2), and Kristine Buckland (2) (1) Department of Biology, Utah State University, 5305 Old Main Hill, Logan, UT 84322; (2) Department of Plants, Soils, and Climate, Utah State University, 4820 Old Main Hill, Logan, UT 84322 Onion thrips, Thrips tabaci Lindeman, is an economically important insect pest of onion production worldwide, and the primary vector of the Tospovirus Iris yellow spot virus (IYSV). We have investigated key drivers of interactions among crop and pest management factors on the field- and farmscape-scale in onion bulb production systems in Utah. Surveys of 88 onion fields in four years found high leaf tissue nitrogen levels and high onion thrips densities in June and July were significant positive predictors of IYSV incidence. Likewise, onion thrips densities responded positively to high onion leaf nitrogen levels. In contrast, high levels of soil inorganic nitrogen and dehydrogenase, a measure of microbial activity, were associated with fewer thrips and reduced virus occurrence. IYSV incidence was reduced by a greater number of insecticide applications. Four years of replicated plot studies consistently found higher thrips densities on onions fertilized with a standard as compared to reduced nitrogen rate (350 and 120 lb N per acre). We hypothesize that onion plants with higher nitrogen levels may have greater apparency, reduced plant defense compounds, and increased digestibility to thrips. Evaluation of landscape- scale factors found that onion thrips densities were consistently higher near field edges than in the interior, suggesting an influence of neighboring crops and/or weedy ditchbanks. Neighboring alfalfa fields and perennial, biennial, and winter annual weeds supported development of onion thrips populations and may serve as ‘green-bridge’ hosts between growing seasons. Weed species that favored onion thrips reproduction included common mallow, field bindweed, flixweed, prickly lettuce, and shepherdspurse. Our results support that onion IPM should include avoidance of planting next to alfalfa and good weed control to reduce dispersal of onion thrips into onion, as well as good management of soil and plant nitrogen levels to reduce attractiveness to thrips, and thus the risk for IYSV transmission.

45 State of the Art Session III: Integrated Management of Tospoviruses and Thrips As Vectors Integration of peanut genotypes displaying field resistance to Tomato spotted wilt virus with other management tactics Rajagopalbabu Srinivasan (1), Kathleen Marasigan (1), Pin-Chu Lai (1), Robert Kemerait (2), & Albert K. Culbreath (2) (1)Department of Entomology; (2)Department of Plant Pathology, University of Georgia, Tifton, GA 31793, USA Thrips transmitted Tomato spotted wilt virus (TSWV) is a major constraint to peanut production in the Southeast. The most important management option is planting peanut genotypes displaying field resistance to TSWV. However, these genotypes only display moderate levels of field resistance, succumb to TSWV infection under pressure, and are generally susceptible to thrips, Frankliniella fusca (Hinds). Therefore, TSWV-resistant genotypes are often used in conjunction with chemical and cultural tactics. Insecticides such as aldicarb (Temik®) and phorate (Thimet®) are routinely used to manage thrips and consequently spotted wilt. Recently, aldicarb production was halted and will be phased out by 2018. Phorate and aldicarb are organophosphate and carbamate insecticides and possess broad-spectrum toxicity. Field evaluations over three years identified alternatives such as neonicotinoids that could be used with TSWV-resistant genotypes without compromising yields. Furthermore, neonicotinoids were also amenable as seed, at-crack, and in-furrow treatments than just in-furrow applications of aldicarb and phorate. Over four years, newly released TSWV-resistant genotypes were evaluated with variations of cultural practices such as strip vs conventional tillage, single vs twin row patterns, and varying seeding rates and planting dates. In general, fewer thrips and reduced thrips feeding injuries were associated with strip tillage and twin-row planting than conventional tillage and single-row planting. TSWV incidence was also reduced under strip tillage than conventional tillage, but no substantial differences were observed between twin rows than single rows. Increased seeding rates also helped suppress TSWV incidences. However, yields were not significantly influenced by variations in cultural practices, particularly in recently released genotypes. In general, the additive effects of cultural practices with genotypes displaying field resistance were less in recently released genotypes than in previously released (before 2000) genotypes. The increased field resistance and/or better yield potential in recently released genotypes could be offsetting additive benefits typically obtained with varying cultural practices.

46 State of the Art Session III: Integrated Management of Tospoviruses and Thrips As Vectors Effect of soil potassium levels on IYSV incidence and symptom expression in onion Claudia Nischwitz (1), Howard Schwartz (2), Diane Alston (1), Dan Drost (3) (1)Utah State University, Dept. of Biology, Logan, UT 84322 ; (2)Colorado State University, Dept. of Bioagricultural Sciences and Pest Management, Fort Collins, CO 80523; (3) Utah State University, Dept. of Plant, Soil and Climate, Logan, UT 84322 Iris yellow spot virus (IYSV) is the most important disease problem for Utah and Colorado onion growers. The virus, transmitted by onion thrips (Thrips tabaci) (Lindeman), is very difficult to manage. Research on other crops and pathogens has shown that mineral nutrients in soil and/or tissue either by themselves or in ratios with other nutrients affect diseases. To determine if nutrition could be used to manage IYSV incidence and symptom expression, soil samples and IYSV incidence data were collected from onion fields in Utah and Colorado in 2013. Regression analysis showed that soil potassium (K) by itself and in ratios with phosphorus (P) and zinc (Zn) affected IYSV incidence in fields. The higher the soil K levels the more likely IYSV occurred. A field trial in Utah in summer 2014 with four levels of soil potassium showed a clear trend between increased levels of K and higher numbers of symptomatic plants and a higher incidence of infected plants. Plants in a treatment with 170 ppm K in the soil had 9.5% symptomatic plants compared to the treatments with 350 ppm, 580 ppm, 1,016 ppm which had 17, 17 and 19 percent IYSV respectively. With increasing available soil K levels, available soil P, Mn, Cu, Na and Fe levels also increased. Comparing leaf tissue levels showed that increasing soil K resulted in increased tissue levels of K, Mn, Zn and Fe. Manganese levels between the lowest and highest K treatments doubled. IYSV symptomatic plants and IYSV incidence increased with increasing tissue levels of the four nutrients. Interestingly, the average number of adult onion thrips was higher on plants in the low K treatment compared to the other treatments. The mechanism behind the increased IYSV symptomatic plants and IYSV incidence with increasing soil K levels is currently unknown.

47 State of the Art Session III: Integrated Management of Tospoviruses and Thrips As Vectors The spread of Frankliniella occidentalis (Pergande) on citrus orchards into specialized growing areas of southern Italy Rita Marullo* & Alessandra De Grazia* *Department of Agriculture, Mediterranean University of Reggio Calabria, Località Feo di Vito, I-89060 Reggio Calabria, Italy Since its introduction into Europe, at the end of 1980s through the horticultural trade in living plants, Frankliniella occidentalis (Perg.) (Thysanoptera, Thripidae) has become one of the most harmful pest to economic importance crops, in greenhouses and in open fields. The species is highly polyphagous and this characteristic is responsible for its impact, as a new introduced pest, on the native thrips fauna in the Mediterranean Basin crops, and its influence on the agricultural systems of southern European regions. As concerns citrus crops, F.occidentalis has not yet been reported in the past literature, as the main pest thrips, compared to the other indigenous thrips species, such as Pezothrips kellyanus (Bagnall), Thrips flavus Schrank, T.major Uzel and T.tabaci Lindeman. Only recently, the results of surveys carried on in some specialized citrus areas in Spain, Turkey and Cyprus have shown the spreading and colonization of citrus crops by F.occidentalis and the reduction of infestations by the native thrips species. The aim of the present study is to demonstrate the spreading on citrus crops by F.occidentalis in some intensive growing areas of the Calabrian region (South Italy), the role of wild plants and the impact on infesting activity by indigenous pest thrips. Surveys have been carried on monthly - from March to November - during 2012 and 2013 – and the statistical analysis has been performed considering the adults of F.occidentalis, P.kellyanus, T.flavus and T.major . The results obtained show the abundance of F.occidentalis on lemon and bergamot crops while kellyanus is significantly reduced on both crops. However, only P.kellyanus can be considered as a pest on citrus for the severe damages due to the scars produced on rind of the little fruits. T.major was predominant only in the orange thrips fauna. The spread of WFT is largely favoured by some wild plants such as Amaranthus retroflexus , Chaenopodium album, Chrysanthemum segetum and Portulaca oleracea.

48 State of the Art Session III: Integrated Management of Tospoviruses and Thrips As Vectors Thrips as pests and vectors of Maize chlorotic mottle virus in maize J. Nyasani (1), E. Kusia (1), & S. Subramanian (1) (1) Plant Health Division, International Centre of Insect Physiology and Ecology, P O Box 30772-00100 Nairobi, Kenya A serious maize disease diagnosed as maize lethal necrosis (MLN) was first reported in Kenya in September 2011. The disease has spread rapidly to other eastern Africa countries. Yield losses of up to 100% have been reported in severely affected fields posing a threat to food security. The disease is caused by a co-infection of maize with Maize chlorotic mottle virus (MCMV), and Sugarcane mosaic virus (SCMV). Of the two viruses, SCMV was first reported in Kenya in 1970’s while MCMV is presumably a recent introduction and is thought to be key in the epidemiology of MLN. Despite the rapid spread, the role of potential vectors in MLN epidemiology is poorly understood in eastern Africa. Hence we aimed to identify potential vectors of MCMV and their host range. Field surveys to determine the seasonality and diversity of potential vectors of MCMV were carried out in MLN hotspots in Kenya. The competence for transmission of MCMV by the key thrips species such as corn thrips, Frankliniella williamsi (Hood); onion thrips, Thrips tabaci (Lindeman); common blossom thrips (pale forms), Frankliniella schultzei (Trybom); and western flower thrips, Frankliniella occidentalis (Pergande) encountered on cereals and grasses were determined in laboratory and greenhouse bioassays. An insect was designated as a transmitter if specific MCMD symptoms developed on the test plants it fed on and MCMV was detected by either DAS-ELISA or RT-PCR in the test plant. The results indicated that corn thrips, common blossom thrips and onion thrips transmitted MCMV but with varying efficiencies. The role of these thrips species and their host plants in the spread of MCMV will be discussed.

49 State of the Art Session III: Integrated Management of Tospoviruses and Thrips As Vectors An efficient serological platform for identification and detection of tospoviral species prevalent in different geographic regions Tsung-Chi Chen (1, 2), Wan-Chu Chien (1), Wei-Ting Tsai (3), Ya-Chi Kang (3), Yu-Han Chen (1), Yun-Chi Wang (1), Yi-Jhen Ou (1), & Shyi-Dong Yeh (3, 4, 5) (1) Department of Biotechnology, Asia University, Wufeng, Taichung 41354, Taiwan; (2) Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 40402, Taiwan; (3) Department of Plant Pathology, National Chung Hsing University (NCHU), Taichung 40227, Taiwan; (4) NCHU-UCD Plant and Food Biotechnology Program, Biotechnology Center, NCHU; (5) Agricultural Biotechnology Center, NCHU The S RNA-encoded nucleocapsid protein (NP) is the key target for identification and detection of a tospovirus. Based on the sequence homology of NPs, the known 29 virus species in the genus Tospovirus can be classified into six phylogenetic clades. Our previous studies proved that the species classified in the same clade are also serologically related based on NP serology using antisera containing polyclonal antibodies. Most prevalent tospoviruses in different geographic regions are classified in serogroups with Tomato spotted wilt virus (TSWV), Watermelon silver mottle virus (WSMoV) and Iris yellow spot virus (IYSV) as individual type members. The NP monoclonal antibodies (MAbs) recognizing specific epitope are useful to distinguish individual tospovirus species. Another S RNA-encoded NSs protein, acting as the gene silencing suppressor, is also abundant in infected cells and can be used as a target for detection of tospoviruses. Noteworthily, the MAbs recognizing a common epitope of the NSs protein of WSMoV (WNSscon) broadly react with all tested members of WSMoV and IYSV serogroups that have been collected by our laboratory. Recently, MAbs against the bacterial-expressed NSs proteins of TSWV (TNSs) or Impatiens necrotic spot virus (INSV) (INSs) were also produced. The obtained MAb to TNSs is TSWV-specific, while the MAb to INSs reacts with INSV, Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV). The two-in- one combination of MAbs to TNSs or INSs reacts with members of TSWV serogroup and INSV serotype. The three-in-one combination of MAbs to WNSscon, TNSs or INSs can be used as a prompt tool for detection of 12 tested tospoviruses prevalent in Europe, America and Asia. Thus, our serological method using NP and NSs MAbs for detecting the common or specific epitopes is a highly efficient platform for identification and detection of important tospoviruses prevalent in different geographic regions.

50 State of the Art Session III: Integrated Management of Tospoviruses and Thrips As Vectors Interactions between temperature and spinosyns insecticides in the control of Thrips tabaci D. Ben-Yakir, G. Lebedev, M. Chen, M. Ghanim Department of Entomology, ARO, the Volcani Center, P.O. Box 6, Bet-Dagan, Israel 50250 The onion thrips (OT), Thrips tabaci Lindeman (Thysanoptera: Thripidae) is a major polyphagous pest infesting many Allium crops and other crops worldwide. Chemical insecticides are the main means used for reducing OT infestation levels and its damages. In recent years, spinosyns (spinosad and spinetoram) are the major insecticides used for OT control. In greenhouses of chives in Israel, OT populations resistant to spinosyns were found most often during the cooler part of the year (November to March; mean daily temperature 12°-17°C). In these greenhouses, bi-weekly application of spinosyns insecticides during the cool months had no effect on OT infestation levels but it induced resistance after 2-3 months. Resistance was also induced in the laboratory by exposing an OT population to 5 bi-monthly applications of 0.1 ppm spinosad at 16°C. Under controlled conditions, mortality of OT 48 hours after exposure to 1 ppm of spinosad, increased from 4% to 62% as temperature was raised from 15°C to 25°C, respectively. Mortality of spinosyns' resistant OT, 24 hours after exposure to the recommended field dose of spinosad (375 ppm), increased from 10% to 68% as temperature was raised from 25°C to 35°C, respectively. In a greenhouse, an effective control of spinosyns' resistance OT was achieved by applying spinosyns insecticides in combination with high temperatures (35°- 40°C; produced by solarization). It appears that the efficacy of spinosyns insecticides in controlling OT is clearly affected by temperature. Application of these insecticides below 20°C was ineffective for OT control and it induced resistance within 2-3 generations (sub-lethal effect). The efficacy of OT control by these insecticides increased significantly as temperature was raised from 25°C to 40°C. This was probably due to the combined effect of the two stressors, the pesticide and the high temperature.

51 State of the Art Session IV: Tospovirus Biology, Genetics, and Diversity Tospovirus Biology, Genetics, and Diversity Session Overview Denis Persley (1) and Scott Adkins (2) (1) DAF-ASQ, Brisbane, QLD, Australia; (2) USDA-ARS, Fort Pierce, FL 34945, USA Impatiens necrotic spot virus (INSV) was reported in 1990, thus removing Tomato spotted wilt virus (TSWV) as the sole species within the Tospovirus genus of the Bunyaviridae family. In a comparatively short time, some 20 tospovirus species have now been described. This is a reflection of the increased capacity of virologists to detect and differentiate viral species and, more importantly, the diversity and genetic variation present in this important and complex group of plant viruses. TSWV likely remains the single most economically important tospovirus worldwide and remains the workhorse for basic molecular studies and thrips transmission experiments. However, species such as Iris yellow spot virus (IYSV) and members of the tropically-adapted Watermelon silver mottle virus group are of considerable economic importance and of increasing interest to virologists. Lack of durable resistance sources among many seriously impacted crops and the capacity of tospoviruses to generate variants virulent towards available resistance genes are major reasons for the economic impact of tospoviruses. A more detailed understanding of the molecular basis for virulence/avirulence with, for example, studies on the NSs protein role in the virus- host interaction. Novel means of durable, broad spectrum resistance are being developed through technologies such as microRNA. The basis for the marked diversity within tospoviruses is becoming more apparent with identification of recombination and reassortment of genome segments. Persistent propagative transmission of tospoviruses by thrips is an integral part of the host-virus-vector interaction. Virus genetic determinants for thrips transmission have been investigated with the GN protein shown to play a major role. A greater understanding of genetic diversity, both within a country and internationally, will ideally lead to more efficient management systems.

52 State of the Art Session IV: Tospovirus Biology, Genetics, and Diversity Virus-derived small RNAs associated with Tomato spotted wilt virus infection in peanut (Arachis hypogaea L.) and in tobacco thrips, Frankliniella fusca [Hinds] S. Fletcher (1), A. Shrestha (2), R. Srinivasan (2), H.R. Pappu (3) & N. Mitter (1) (1) Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St. Lucia, Australia; (2) Department of Entomology, University of Georgia, Tifton, GA, USA; (3) Department of Plant Pathology, Washington State University, Pullman, WA, USA Virus-derived small RNAs (vsiRNA) are produced in plants in response to virus infection and play an important role in the antiviral defense system. In this study, we investigated Tomato spotted wilt virus (TSWV; (Tospovirus:Bunyaviridae)-specific vsiRNAs in TSWV-peanut (Arachis hypogaea L.) pathosystem. Thrips-transmitted TSWV is an economically important virus of several field and horticultural crops. TSWV consists of large (L), medium (M), and small (S) RNA segments. Studies on TSWV-specific vsiRNAs in TSWV host plants and thrips vectors could provide valuable insights into the interactions between TSWV and its hosts and vectors. We compared vsiRNA profiles between TSWV-infected peanut plants and tobacco thrips, Frankliniella fusca [Hinds]. Initially, small RNA fractions from TSWV-infected peanut plants and adult thrips were deep-sequenced. As expected, the size distribution of sRNAs differed markedly between the two organisms, with abundant sRNAs ranging from 18-25 nt in plants, and 17-33 nt in thrips. We identified unique vsiRNA profiles in plants and thrips by aligning reads to the TSWV genome. In thrips, the largest portion of vsiRNA reads aligned to the S RNA (40%), followed by the M (37%) and L (24%) RNAs. In contrast, in TSWV-infected peanut plants, 40% of vsiRNAs aligned to the L RNA, followed by M (35%) and S (25%) RNAs, more closely matching the expected alignment ratios based on the RNA size. Among vsiRNA reads complementary to each TSWV segment, 21 and 22 nt were the dominant classes in both samples, with a greater relative abundance of 22 nt sRNAs in thrips, and 21 nt sRNAs in the plant. In contrast, the number of antisense derived vsiRNAs was higher than sense-derived vsiRNAs for NSs and NSm in both samples, and lower in the remaining genes. A comparative analysis of small RNA profiles of TSWV in plants vs thrips vector will be presented.

53 State of the Art Session IV: Tospovirus Biology, Genetics, and Diversity The functional study of the movement protein (NSm) of distinct Tospovirus species. Mikhail Leastro (1); Vicente Pallás (2), Jesús Sánchez-Navarro (2), Renato O. Resende (1) (1) Universidade de Brasília, Instituto de Biologia 70910-900 Brasília, Brazil; (2) IBMCP (CSIC-UPV) Av. De los Naranjos S/N, 46022 Valencia, Spain. Tospovirus is the only genus of the family Bunyaviridae that includes plant-infecting viral species. The M RNA segment of tospoviruses encodes a non-structural protein (NSm) involved in the virus movement. Comparison among the 28 tospovirus species reported so far, reveals significant differences in amino acid sequences of viral proteins and in their biological features, such as host range, possibly due to restrictions in viral infection including the cell-to-cell and/or systemic movement. The aim of this work was to study the involvement of four distinct movement protein (NSm) on virus infection using the Alfafa mosaic virus expression system. As results we observed that: i) all tospovirus NSm analyzed herein are associated with the cytosolic surface of the biological membranes, in which the central hydrophobic and coiled-coil regions were submerged in the periphery of the lipid membrane and the N-and C-termini oriented to the cytoplasm; ii) all NSm and N were able to form dimers; the NSm interacted with the cognate N and heterologous interaction between NSm, N and NSm-N were observed; iii) differences in the efficiency in cell-to-cell and systemic movement were observed among the tospovirus species; iv)in vivo analyses confirmed that the machinery of tospovirus movement is via tubule-guided transportation; v) C-terminal deletion revealed that this end of BeNMV NSm (highly diverse tospovirus showing a narrow host range) was shown not to be essential for virus movement, in contrast toCSNV, TCSV and TSWV-NSm which required the entire NSm proteins for cell-to- cell movement; vi) The NSm protein of BeNMV is not functional as an avirulence determinant, in contrast with the “American clade” species for which the NSm triggers HR reaction. These results may indicate that mixed infections among tospovirus species and heterologous interaction between movement and nucleocapsid proteins may allow virus synergism, facilitating virus infection and/or evolution.

54 State of the Art Session IV: Tospovirus Biology, Genetics, and Diversity Shirani is a Laurence Mound Fellow

Characterization of Australian Capsicum chlorosis virus Shirani Widana Gamage (1), Denis M. Persley (2), Colleen M. Higgins (3) & Ralf G. Dietzgen (1)

(1) Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia, QLD 4072, Australia; (2) Queensland Department of Agriculture, Fisheries and Forestry, AgriScience Queensland, Ecosciences Precint,GPO Box 267, Brisbane QLD 4001; (3) Institute of Applied Ecology, School of Applied Sciences, Auckland University of Technology, Private Bag 92006, Auckland 1142, New Zealand Capsicum chlorosis virus (CaCV) that infected capsicum and tomato crops was first discovered in Southern Queensland, Australia in 1999. Subsequently, CaCV was reported from South-East Asia, and the genomes of two isolates from each Thailand and Taiwan have been completely sequenced. In Australia, only nucleocapsid (N) gene data of several isolates were available. Hence, we determined the first complete genome sequence of an Australian CaCV isolate using a combination of Illumina HiSeq RNA and Sanger sequencing technologies. Australian CaCV contained the typical tripartite genome structure of tospoviruses with large, medium and small RNA segments of 8913, 4846 and 3944 nucleotides (nt) in length, respectively. The intergenic region (IGR) of S RNA contained 1663 nt, being the largest IGR of all CaCV isolates sequenced so far. When comparing available CaCV N gene amino acid sequences, this isolate was 98% identical to the previously reported Australian isolate Burdekin-1043. Comparison of Australian CaCV genome with complete CaCV genome sequences from other geographic regions showed highest sequence identity with Ph-Taiwan isolate. Genome sequence comparisons and phylogeny of all available CaCV isolates provided evidence for two highly diverged groups of CaCV isolates that may warrant re-classification of AIT-Thailand and CP-China isolates as unique tospoviruses separate from CaCV. We further characterized CaCV proteins for intracellular localization by transient expression as green fluorescent protein fusions in planta. CaCV suppressor of silencing (NSs) and Gn glycoprotein had unique nuclear localization compared to analogous cytoplasmic proteins of other tospoviruses. Bimolecular fluorescence complementation showed unique interactions of NSs with itself and with N protein. CaCV NSm movement protein interacted with itself and with N protein similar to interactions observed for TSWV proteins. The functions of CaCV NSs as both local and systemic RNA silencing suppressor, similar to TSWV NSs were experimentally validated in N. benthamiana using a GFP reporter assay.

55 State of the Art Session IV: Tospovirus Biology, Genetics, and Diversity Homology Modeling of the tospovirus Groundnut ringspot virus nucleoprotein: intra- molecular interaction and RNA encapsidation Rayane Nunes Lima (1); Muhammad Faheem (2,3); João Alexandre Ribeiro Gonçalves Barbosa(2,3); Fernando Lucas Melo (1); Renato Oliveira Resende (1). (1) Laboratório de Virologia Vegetal. Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil; (2) Laboratório de Biofísica. Departamento de Biologia Celular, Instituto de Ciências Biológicas, Universidade de Brasília, Brasília, DF, Brazil; (3) Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil. The rapid progress in the understanding of protein folding mechanisms and the advances in the bioinformatics field have provided reliable tools to modeling and predict three dimensional structures of plant virus proteins. Recently, the nucleoprotein (NP) crystal structures of related RNA virus families (Arena/Orthomyxo/Bunyaviridae) were elucidated and despite having different sizes and distinct NP-folding structures, these proteins share common features and architectural principles when forming NP-NP multimers and NP–RNA complexes. Therefore, due to their genetic relationship, the La Crosse virus (LACV-Orthobunyavirus) crystal structure in complex with ssRNA (PDB ID 4BHH) was selected as template for a homology modeling approach to predict a three dimensional model for the NP of the tospovirus Groundnut ringspot virus (GRSV). The GRSV NP monomer was predicted to possess thirteen helical segments and two small beta-sheets organized in a globular core domain (33-223 aa) containing a deep positively charged groove with the two terminal chains forming a N-terminus arm (1-32 aa) and a C-terminus arm (224-258 aa). Both N- and C-arms extend outwards from the globular core domain and they interact with the globular core domain of neighboring monomers to mediate the multimerization, supporting the “head-to-tail” model. The RNA is primarily bound at the central RNA-binding groove and the key residues for this interaction are mainly located in this groove. RNA is strongly bent at each NP–NP interface and is largely solvent-inaccessible in the tetramer structure. The dimensions of the groove allow accommodation of ssRNA and further analysis showed that the majority of residue-nucleotide interactions occur with the ribose and the phosphate moiety, suggesting a non-sequence-specific ssRNA interaction. Importantly, most of the key residues are highly conserved among all tospoviruses. Multiple copies of the NP form oligomers that interact with the viral RNAs to build ribonucleoprotein complexes (RNPs) that are proposed to be transported via plasmodesmata and are functional templates for RNA replication and transcription. Thus, the proposed model may shed light on the mechanisms of RNP shaping and could allow the identification of essential amino acid residues as potential targets for tospovirus control strategies.

56 State of the Art Session IV: Tospovirus Biology, Genetics, and Diversity Raphael is a TTEN Fellow

An Update on Sequence Diversity of Impatiens necrotic spot virus Adegbola, R.O. (1), Marshall, S.H. (1), Batuman, O.(2), Ullman, D.(3), Gilbertson, R.(2), Adkins, S. (4) & Naidu, R.A. (1) (1) Department of Plant Pathology, Washington State University Irrigated Agriculture Research and Extension Center, Prosser, WA. 99350; (2) Department of Plant Pathology, UC-Davis, CA 95616, (3) Department of Entomology, UC-Davis, CA 95616; (4) USDA-ARS, USHRL, 2001 S. Rock Rd., Fort Pierce, FL 34945 Impatiens necrotic spot virus (INSV; genus Tospovirus, family Bunyaviridae) is an economically important viral pathogen for a wide range of plants, including greenhouse ornamental and vegetable crops. In many cases, symptoms induced by INSV are similar, though not identical, to those induced by Tomato spotted wilt virus (TSWV). Although much attention has been paid to TSWV, many reports in recent years have underscored the economic significance of INSV to agricultural and horticultural sectors in the US and other countries. INSV can be disseminated via vegetative propagation of nursery plants and transmitted by thrips species that also spread TSWV and other tospoviruses. Improved knowledge about INSV biology and genetics will expand our grasp on its epidemiology and the factors contributing to disease outbreaks. As a first step, we began studies to explore the genetic diversity of INSV collected from nursery and vegetable crops. We have cloned the complete S-RNA segment of INSV isolates from five plant species from California and Washington and compared the nucleotide sequences with corresponding sequences available in GenBank. The S-RNA sequences obtained in this study showed 97 to 99% similarity with those previously characterized. Phylogenetic analyses of S- RNA sequences of INSV obtained in this study with corresponding sequences from GenBank showed segregation into two distinct clades, with those from the US clustering into one whereas those from Asia were into another clade, suggesting geographic structuring of global isolates of INSV. However, isolates from the Netherlands and Italy grouped with US and Asian clades, respectively. Similar studies are underway with cloning and sequencing of the complete M-RNA segment of INSV. An improved knowledge of sequence diversity will guide us in understanding the origins of bioinvading tospoviruses, evaluating virus evolution and aide growers in developing detection systems to prevent the spread of infected stock plants and other materials.

57 State of the Art Session IV: Tospovirus Biology, Genetics, and Diversity Spencer is a TTEN Fellow

Dynamics of Genetic Diversity of Tomato spotted wilt virus in the United States Marshall, S.(1), Adegbola, R.(1), Batuman, O.(2), Ullman, D.(3), McGrath, M.(4), Gilbertson, R.(2); Kennedy, G.(5), Hanson, S.F.(6), Adkins, S.(7) and Naidu, R.A.(1) (1) Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and Extension Center, Prosser, WA. 99350; (2) Department of Plant Pathology, UC-Davis, CA 95616; (3) Department of Entomology, UC-Davis, CA 95616; (4) Cornell University, Long Island Horticultural Research & Extension Center, Riverhead, NY 11901; (5) Department of Entomology, North Carolina State University, Raleigh, NC 2769; (6) New Mexico State University, Las Cruces, NM 88003, (7) USDA-ARS, USHRL, 2001 S. Rock Rd., Fort Pierce, FL 34945 Among known tospoviruses, Tomato spotted wilt virus (TSWV; genus Tospovirus, family Bunyaviridae) continues to be the major viral disease affecting a wide range of agricultural and horticultural crops worldwide. Like many other RNA viruses, TSWV is known to maintain heterogeneous and divergent populations with individual isolates that have distinct biological and virulence characteristics. Adaptation of TSWV isolates to new hosts and development of resistance-breaking isolates capable of overcoming Tsw and Sw5 resistance genes are challenging the sustainability of management systems against the virus. As we strive to develop better management strategies with greater sustainability, it is critical to gain a deeper understanding of genetic diversification of the virus. Towards this objective, we have begun to analyze genetic diversity among S- and M-RNA segments of field isolates of TSWV collected from different plant species in California, North and South Carolina, New Mexico, New York, Virginia and Washington. We have developed improved methods to amplify, clone and sequence the complete S- and M-RNA genome segments from several TSWV isolates. A global phylogenetic analysis of these sequences and corresponding sequences of TSWV isolates available in public databases revealed segregation of virus isolates into distinct clades. Interestingly, the results indicated segregation of CA isolates into several variant groups compared to isolates collected from other states. Further studies are in progress to elucidate the genetic landscape of TSWV isolates for an improved knowledge about biological and molecular genetic properties of the virus. Results will also provide a better understanding of TSWV epidemiology and the factors contributing to disease outbreaks.

58 State of the Art Session IV: Tospovirus Biology, Genetics, and Diversity Tospovirus Genetics and Diversity Deciphering the Three Dimensional Structures of Tospoviral Proteins C. Olaya (1), B. Adhikari (2), J. Cheng (2) & H.R. Pappu (1) (1) Department of Plant Pathology, Washington State University, Pullman, WA, USA;(2) Department of Computer Science, University of Missouri, Columbia, MO 65211, USA. Recently, our lab identified two motifs that are critical for the suppressor activity of the TSWV- NSs protein. While much is known about the genome of TSWV, little is known about the structure of its coded proteins. Understanding the structures of the viral proteins and the critical reactive residues, it might be possible to devise novel virus suppression strategies. The objective of this study was to decipher the 3D structures, based on the primary amino acid sequence, of the NSs and N proteins of TSWV, and to predict the location of the conserved motifs that play a role in the silencing suppressor activity and viral encapsidation, respectively. We utilized the structure prediction servers MULTICOM-CLUSTER, MULTICOM-NOVEL, and MULTICOM- CONSTRUCT to predict in silico 3D models of TSWV-NSs and N proteins. For the NSs protein, seven conserved domains were identified and two of them were mutated. For N protein, 13 conserved residues were identified and their positions were determined in the predictive models for future mutations. The two conserved motifs GKV/T and YL were found to be critical for the suppressor activity of the NSs.The model showed beta sheets and alpha helices, and both mutated sites were exposed. However the structure was not severely affected by any of the mutations. Each server predicted five models for N protein, and the conserved residue LGI was referenced. Interestingly, most of the 13 conserved residues were exposed in the tertiary structure. These results are the first step to gain a better understanding of the structure-function relationships of TSWV proteins and could provide important clues to developing novel strategies for engineering virus resistance.

59

Plenary Session II: Virus-Vector-Host Interactions

Frontiers in Thrips Genomics: The current status of the Frankliniella occidentalis genome Dorith Rotenberg Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center, Manhattan, KS 66506, USA In 2012, Frankliniella occidentalis (Pergande) was selected by the prioritization committee of the i5k Genome Sequencing Initiative for Insects and Other Arthropods (http://arthropodgenomes.org/wiki/i5k) to be the first thysanopteran genome to be sequenced as part of the i5k pilot project at the Baylor College of Medicine-Human Genome Sequencing Center (BCM-HGSC; http://www.hgsc.bcm.tmc.edu/content/i5k-western-flower-thrips). The source of genomic DNA for the i5k sequencing project was a 10th generation sibling-sibling breeding line of F. occidentalis derived from a lab colony reared at Kansas State University that originated from an isolate collected from Hawaii. The genome size of F. occidentalis was determined to be 415 Mb and the resulting whole genome assembly (Focc 1.0) consists of 6,263 scaffolds (N50 = 948 Kb) containing 17,553 predicted genes. To improve the automated genome annotation (MAKER) performed by BCM-HGSC, an international effort was launched to recruit community members to participate in manually annotating gene sets of interest. To simplify the process, the United Stated Department of Agriculture National Agriculture Library created a genome portal called i5k Workspace@NAL (http://i5k.nal.usda.gov/home) to house and support the F. occidentalis genome and the other pilot arthropod genome projects by providing community annotators with a central data repository for genome and transcriptome resources, genome-viewing capabilities (Web Apollo), and analysis tools (e. g., BLAST). Several international arthropod community members interested in particular gene sets (e. g., insect development and structure) have committed time and expertise to manually curating these genes for F. occidentalis using the i5k genome portal and available transcriptome resources provided by my team. The current status of these community annotation efforts and the utility of these genomic and transcriptomic resources for F. occidentalis will be discussed.

60 Plenary Session II: Virus-Vector-Host Interactions Diversity of tospoviruses and their thrips vectors and why it can be hard to generalize about their association C.R. Wilson Tasmanian Institute of Agriculture, University of Tasmania, New Town Research Laboratories, 13 St Johns Avenue, New Town, Tas 7008, Australia Both thrips and tospoviruses occur globally causing significant damage and loss to crops. Tomato spotted wilt virus (TSWV) was first described in 1915. TSWV became the type species of the tospoviruses which was, until Impatiens necrotic spot virus was described in 1990, considered monotypic. Since this time an increasing number of new tospoviruses have been reported. This expansion of species has coincided with a resurgence of TSWV epidemics associated with the global spread of Western flower thrips. Changes in thrips vector populations may have enabled local virus populations to expand their natural host range. Molecular tools have provided evidence for both reassortment and mutation in tospovirus evolution. Selection pressures posed by deployment of resistance genes against tospoviruses has led to emergence of resistance breaking isolate populations. Resistance to vector thrips and/or tolerance to tospovirus infections may decrease the selection pressures on the virus and lead to improved resistance durability. The thrips vectors of tospoviruses also show considerable diversity. Several thrips species are known to vector one or more tospoviruses. The increasing use of molecular tools has identified variation within species. Biological studies have shown these cryptic species may possess distinct biological traits including variation in their capacity to vector Tospoviruses. As an example, some studies have suggested Onion thrips may be a poor (or even non) vector of TSWV. However various data has shown that distinct biotypes within this species have profound differences in their ability to vector TSWV. This means when we consider thrips vector species we should not assume biological traits carefully defined in one region are necessarily transferable to another.

61 Plenary Session II: Virus-Vector-Host Interactions Candice is a TTEN Fellow TSWV infection, thrips behavior and the salivary gland transcriptome of Frankliniella occidentalis

Candice A. Stafford-Banks and Diane E. Ullman Department of Entomology, University of California, Davis, One Shields Ave. Davis, CA, 95616, USA. The Western flower thrips (Frankliniella occidentalis) is an important omnivorous insect vector of Tomato spotted wilt virus (TSWV), which infects over 1,000 different species of plants and its insect vector. Infection of F. occidentalis with TSWV effects many aspects of thrips plant feeding and predatory behaviors. Saliva is known to play a crucial role in both insect feeding behavior and virus transmission. To better understand thrips saliva and salivary gland functions we sequenced the transcriptome of the primary salivary glands of F. occidentalis using short read sequencing (Illumina) technology. Our de novo-assembled transcriptome identified 31,392 high quality contigs with an average size of 605 bp. A high percentage (61.24%) of contigs had no apparent protein or nucleotide hits, however a total of 12,166 contigs had significant BLASTx or tBLASTx hits (E ≤ 1.0E-6) to known proteins. To annotate the F. occidentalis salivary gland transcriptome (sialotranscriptome) we compared our sequences against a published F. occidentalis full body transcriptome assembled from Roche-454 reads. This comparison revealed several contigs with putative annotations associated with salivary gland functions. A KEGG pathway analysis of the sialotranscriptome also revealed that the majority (18 out of the top 20 predicted KEGG pathways) of the salivary gland contig sequences match proteins involved in metabolism. Several genes likely to be involved in detoxification and inhibition of plant defense responses were identified, including aldehyde dehydrogenase, metalloprotease, glucose oxidase, glucose dehydrogenase, and regucalcin. Several genes were also identified that may play a role in the extra-oral digestion of plant structural tissues including β-glucosidase and pectin lyase; as well as the extra-oral digestion of sugars, including α-amylase, maltase, sucrase, and α- glucosidase. This analysis of the F. occidentalis sialotranscriptome provides a foundational tool to further our understanding of how thrips interact with their plant hosts and the viruses they transmit.

62 Plenary Session II: Virus-Vector-Host Interactions Ismael is a TTEN Fellow Use of a proteomics approach to identify Frankliniella occidentalis proteins that respond to Tomato spotted wilt virus infection I.E. Badillo-Vargas, D. Rotenberg, and A.E. Whitfield Department of Plant Pathology, Kansas State University, 4024 Throckmorton Hall Manhattan, KS 66506, USA. Tomato spotted wilt virus (TSWV) is the type species of the Tospovirus genus and the best characterized tospovirus with regard to the interaction with its insect vector. The western flower thrips, Frankliniella occidentalis (Pergande), is the most efficient vector of TSWV. Once TSWV is acquired by F. occidentalis, it journeys through the insect body and replicates in the midgut and salivary gland tissues for transmission to occur. Despite the well-known biological features of this intricate interaction, very little is known of the molecular basis that lead to acquisition, replication, dissemination, and transmission of this or any tospovirus by an efficient vector. The goal of this work was to use a proteomics approach to identify larval thrips proteins that respond to the virus infection. Using two-dimensional gel electrophoresis, we identified 26 protein spots that displayed differential abundances in response to TSWV infection, most of which (62%) were down-regulated during the early stages of the infection process. Mass spectrometry coupled with Mascot searches resulted in the identification of 37 thrips proteins within the 26 spots. Thirty two of these proteins had gene ontologies representing biological roles of proteins associated with infection cycles of other plant- and animal-infecting viruses and antiviral defense responses. For example, the vacuolar ATPase-E that is part of a holoenzyme involved in clathrin-coated vesicle trafficking, a known mechanism of entry of animal-infecting bunyaviruses, was up-regulated during TSWV infection. Up- and down-regulation of thrips proteins associated with innate immune defenses further suggest a complex response. A thioredoxin-dependent peroxidase that among several functions acts as an inhibitor of apoptosis was for instance down-regulated. Ultimately, identification of thrips molecules putatively involved in entry, midgut barrier escape and antiviral defense responses during tospovirus infection of the insect vector provides new insights into the molecular basis of this interaction and novel targets for control.

63 Plenary Session II: Virus-Vector-Host Interactions A biological perspective on the Tospoviral proteins and their roles during infection and dissemination in plants and thrips. Richard Kormelink Laboratory of Virology, Department of Plant Sciences, Wageningen University, Droevendaalsesteeg 1, 6708PB Wageningen, The Netherlands Tomato spotted wilt virus (TSWV) presents the type species of the genus Tospovirus within the arthropod-borne Bunyaviridae, a large family of primarily animal infecting viruses that are classified into the four genera Orthobunyavirus, Phlebovirus, Nairovirus and Hantavirus and a fifth one, the Tospovirus genus, containing the plant-infecting members. From all these viruses some are listed as potential agents for bioterrorism, or belong to the most devastating plant viruses worldwide (Plyusnin & Elliott, 2011; Kormelink et al., 2011). Like for all bunyaviruses, tospovirus particles are spherical and membrane bound (~80-120 nm) and contain a tripartite RNA genome tightly encapsidated by the nucleo(capsid)protein (N) and small amounts of the viral RNA-dependent RNA polymerase (RdRP, or L protein). The structural proteins N and two glycoproteins (Gn and Gc) play a major role in particle assembly during which they interplay with elements from the cytoskeleton and endomembrane system. For the acquisition and transmission of the virus from plants by its thrips vector the glycoproteins are indispensable. The tospovirus genome additionally encodes two non-structural proteins, NSM and NSS, that are involved in viral spread (NSM) respectively counter defense against antiviral RNAi, but also trigger two intracellular innate immunity sensors in plants. During an overviewing presentation the roles of the tospoviral proteins in the “life cycle” of these viruses in plants and thrips will be described and their modes of action discussed compared to/from the perspective of their animal- infecting counterparts. R.M. Elliott and A. Plyusin (eds., 2011). “The Bunyaviridae”, New York: Plenum Press. Kormelink, R., Garcia, M.L., Goodin, M., Sasaya, T. and Haenni, A.-L. (2011). Negative-Strand RNA Viruses: The plant-infecting counterparts. Virus Research 162, 184– 202

64 Plenary Session III:Virus/Vector Biology and Emergence of Diversity Genetic bottlenecks during virus infection and host colonization and their impact on genetic diversity and evolution Santiago F. Elena (1,2), Nicolas Tromas (1), Guillaume Lafforgue (1), & Mark P. Zwart (1) (1) Instituto de Biología Molecular y Celular de Plantas, CSIC-UPV, Campus UPV CPI 8E, 46022 València, Spain; (2) The Santa Fe Institute, Santa Fe, NM 87501, USA For virus infections of multicellular hosts, the occurrence of narrow genetic bottlenecks during horizontal transmission appears to be widespread. Furthermore, genetic bottlenecks can also occur within the host and during vertical transmission (parent-to-offspring). These bottlenecks will affect the maintenance of genetic variation in a virus population and the prevalence of mixed-strain infections, hereby modulating the impact of different levels of selection and ultimately determining the strength with which different random forces act during evolution. Here we first introduce key concepts from evolutionary genetics central to understanding the effects of genetic bottlenecks. Next we consider different approaches to estimating bottleneck sizes – many of which are reliant upon models of virus infection kinetics – and weigh the merits of these different approaches based on theoretical and empirical considerations. We then contrast estimates of bottleneck sizes for different plant viruses at different stages of infection. For both horizontal transmission and within-host expansion, genetic bottlenecks appear to be dependent on virus concentration, and – with the notable exception of multipartite viruses – approach a single virus particle when very low doses are used. On the other hand, only a single estimate of the genetic bottleneck during vertical transmission has been recently made, precluding general conclusions. Finally, we consider the evolutionary implications of genetic bottlenecks during virus infection. Although on average strong bottlenecks will lead to declines in fitness, we consider a number of scenarios in which bottlenecks could also be advantageous for viruses.

65 Plenary Session III:Virus/Vector Biology and Emergence of Diversity Thrips/Tospovirus relationships: Where have they come from, and where are they going? Laurence Mound Australian National Insect Collection, CSIRO, Canberra, Australia, 2601 [email protected]

For any evolutionary biologist studying a dependency between unrelated organisms the primary consideration is the origin of the association. No tospovirus can exist without one or more of scarcely 0.2% of the known thrips species. In contrast, thrips do not need Tospovirus. This stark asymmetry in dependency remains curiously ignored. Students of thrips biology rarely show any interest in tospoviruses. Most tospovirus research is carried out by leaf manipulation in the absence of a vector, thus virus workers have limited interests in thrips biology. As a result, the thrips/tospovirus association remains poorly studied. Most thrips species cannot vector any tospovirus, and not all individuals within a vector species seem equally effective as vectors. Tospoviruses are presumed to have evolved from one or more Bunyaviridae species, but what was the pathway by which a bunyavirid was converted into a tospovirus. With no evidence of the origin and mechanism, any possibility of genetically engineering the system seems remote.

66 Plenary Session III:Virus/Vector Biology and Emergence of Diversity Alana is a TTEN Fellow The importance of ecology, species and population interactions in thrips-tospovirus coevolution highlighted by studies on Thrips tabaci A.L. Jacobson1 and G.G. Kennedy2 1Department of Entomology and Plant Pathology, Auburn University, Auburn, AL 36849, 2Department of Entomology, North Carolina State University, Raleigh, NC 27695-7630 Thrips-tospovirus interactions driving the epidemiology, evolution and emergence of these viruses in the landscape involve complex biological and ecological factors that include abundance and distribution of vector species and host plants for both vector and virus, as well as variation in competency of thrips species to transmit tospoviruses. Complex interactions among these factors likely contribute to coevolution of the virus and vector resulting in spatial and temporal variation in vector competency. Our limited understanding of the influence that thrips species- and population-level structuring has on thrips-tospovirus interactions also complicates our ability to understand the biology and ecology driving epidemiology of tospoviruses. Thrips tabaci (Lindeman) is a Tomato spotted wilt virus vector of variable importance throughout the world and a putative cryptic species comprised of three genetically distinct groups. Results from recent studies examining transmission efficiency in relation to geographic location, population genetic structuring, and host plant utilization will be presented. Implications of these findings for other thrips species and tospoviruses will be discussed.

67 Plenary Session III:Virus/Vector Biology and Emergence of Diversity Tospovirus emergence, interspecific reassortment and mixed infections in North America Scott Adkins (1) (1) USDA-ARS, Fort Pierce, FL 34945, USA Groundnut ringspot virus (GRSV) and Tomato chlorotic spot virus (TCSV) emerged in Florida in 2009 and 2012, respectively, joining Tomato spotted wilt virus (TSWV), as economic threats to horticultural and agronomic crops in the southeastern U.S. Currently, GRSV, TCSV and TSWV infect solanaceous vegetable crops and occur sympatrically in south Florida. All U.S. GRSV isolates characterized to date have a reassorted genotype (LGMTSG) with GRSV S and L RNAs and a TCSV M RNA; this is the first known instance of an interspecies tospovirus reassortant. Methods were developed to detect reassortants in natural mixed infections and a study was initiated to examine reassortment in experimental mixed infections. A better understanding of these processes will enhance rational disease management and forecasting strategies.

68 Plenary Session III:Virus/Vector Biology and Emergence of Diversity Tospovirus Emergence, Interspecific Reassortment and Mixed Infections in Brazil: Advances in Breeding for Broad Spectrum Tospovirus Resistance Renato O. Resende (1); Leonardo S. Boiteux (2) (1) Departament of Plant Pathology and Cell Biology, University of Brasilia (UnB), 70910-900, Brasília-DF, Brazil; (2) National Center for Vegetable Crops Research (CNPH)/Embrapa Hortaliças, CP 0218, 70359-970 Brasília-DF, Brazil. The Neotropical South America is a Tospovirus diversity center with many endemic species. The co-existence of different tospovirus species, alternative hosts, and vector species may allow the emergence of new species, virus reassortants, and facilitates mixed infections. Our studies demonstrated that mixed infections among tospovirus species and heterologous interaction between movement and nucleocapsid proteins may allow virus synergism, facilitating virus infection and/or evolution. However, extensive surveys in Brazil revealed that reassortants may represent rare events and/or reassortants have a low fitness in the field. The combination of high diversity and synergic interactions represent a threat for obtaining broad tospovirus resistance. In tomatoes the Sw-5 locus is yet the best genetic source of resistance. This locus contains at least five paralogues of which Sw-5bis the effective copy. Novel sources of tospovirus resistance were found in Solanum(section Lycopersicon: Solanaceae). Seven S. peruvianumL. accessions displayed a broad-spectrum resistance. A polymorphic amplicon was found encompassing the Sw-5b gene, resulting in a specific, co-dominant polymorphism. The subset of S. peruvianum accessions having wide spectrum resistance displayed minor Sw-5b gene sequence variation, thus representing distinct alleles at the Sw-5 locus. Sources of multi-resistance to four Tospovirus species were also found in accessions of S. pimpinellifoliumL.; S. chilenseDunal; S. arcanum Peralta; S. habrochaitesKnapp; S. corneliomuelleriMacbr; and S. lycopersicum L. The introgression/incorporation of these genetic factors from these distinct Solanum species into elite germplasm might be useful to expand the genetic basis and to allow the selection of multi-resistant tomato lines. The relatively large number of accessions with broad-spectrum Tospovirus resistance in Solanum (Lycopersicon) is in sharp contrast with the current situation reported in the genus Capsicum where only sources of TSWV-specific resistance (Tsw gene) have been found so far.

69 Plenary Session III:Virus/Vector Biology and Emergence of Diversity Resistance Breaking by TSWV and Its Relevance to Thrips Transmission and Tospovirus Management

George G. Kennedy (1), Thomas Chappell (2,) & Jessica L. Houle (3) (1) Department of Entomology, North Carolina State University, Raleigh, NC 27695-7630, USA; (2) Department of Entomology, North Carolina State University, Raleigh, NC 27695- 7630, USA; (3) Department of Entomology, North Carolina State University, Raleigh, NC 27695-7630, USA Resistant cultivars are a mainstay in the management of Tomato spotted wilt virus (TSWV) in pepper (Capsicum annuum) and tomato (Solanum lycopersicum) for which resistance alleles have been identified. However, the emergence of TSWV variants able to break resistance conferred by the Sw-5 allele in tomato and the tsw allele in pepper has compromised the effectiveness of these resistances where they occur. Effective stewardship of TSWV-resistance alleles to enhance their durability can be advanced by understanding the patterns of genetic variation associated with resistance-breaking that exist in the virus populations, and the ways in which crop production and protection measures select on that variation to enhance or diminish the frequency and spread of resistance-breaking variants. Following an overview of resistance- breaking by TSWV for the Sw-5 allele in tomato and the tsw allele in pepper, we describe the results from a series of studies examining factors that may influence selection for emergence and persistence of resistance-breaking variants in the landscape. One is an experimental evolution study examining changes in tsw resistance-breaking and genetic variation through time. In this study a native TSWV isolate collected at a location in which TSWV-resistant crops had never been grown was challenged with repeated selection in TSWV resistant (tsw+) and TSWV susceptible (tsw-) pepper. In another study, the patterns of genetic variation of TSWV from tomato fruit produced by plants expressing the Sw-5allele were compared. Symptomatic fruit are frequently observed on Sw-5+ plants in the field but only a portion of these fruit infections are accompanied by systemic infection of the plant. The implications of these results for stewardship of these TSWV-resistance alleles and for TSWV management will be discussed.

70 State of the Art Session V: Thrips/Tospovirus Ecology and Management Challenges and Opportunities for Managing Thrips and Tospoviruses Joe Funderburk (1) (1) Department of Entomology and Nematology, North Florida Research and Education Center, 155 Research Road, Quincy, FL 32351, USA Flower thrips attributes of polyphagy, rapid development, high fecundity, an ability for rapid dispersal, and rapid development of insecticide resistance makes management a serious challenge. This is further complicated in situations where tospviruses are pests. Successful strategies involve define pest status (economic thresholds); increase biotic resistance (natural enemies and competition); integrate preventive and therapeutic tactics (scouting, ultraviolet- reflective technologies, biological control, compatible insecticides, companion plants, and fertility); and vertically integrate with other pests. Such systems are effective, economical, and sustainable. Such programs are developed from large investments in research and require trained technologists to implement.

71 State of the Art Session V: Thrips/Tospovirus Ecology and Management Effects of resistance to Tomato spotted wilt virus in peanut genotypes on virus population genetics and thrips fitness Rajagopalbabu Srinivasan (1), Sivamani Sundaraj (1), David G. Riley (1), Albert K. Culbreath (2), &Hanu Pappu(3). (1)Department of Entomology; (2) Department of Plant Pathology, University of Georgia, Tifton, GA 31793, USA; (3) Department of Plant Pathology, Washington State University, Pullman, WA 66506, USA. Spotted wilt caused by Tomato spotted wilt virus (TSWV) remains a peanut production constraint in southeastern United States. Breeding efforts over three decades have yielded several peanut genotypes with field resistance to TSWV. The magnitude of field resistance in these genotypes has increased steadily with time. However, the mechanism of resistance, and their interactions with the vector and TSWV remain largely uncharacterized. As a first step, TSWV- resistant and susceptible genotypes were subjected to thrips-mediated and mechanical inoculation of TSWV. The incidence of TSWV infection was up to 90% and did not differ significantly between susceptible and resistant genotypes. Resistant and susceptible genotypes exhibited typical TSWV symptoms upon infection. However, estimation of TSWV N-gene copies suggested that some resistant genotypes accumulated fewer copies than the susceptible genotype. To assess if resistant genotypes imparted any selection pressure on TSWV, ~80 virus isolates from resistant and susceptible peanut genotypes were partially sequenced. N-gene from all isolates was completely sequenced and compared with old Georgia isolates. Phylogenetic analyses conducted using translated amino acid sequences indicated consistent differences between old (collected in 1998) and new isolates (2010) but not between isolates from resistant and susceptible genotypes. There was evidence for overabundance of non-synonymous substitutions but not for positive selection. Population genetics analyses indicated that purifying selection, population expansion, and differentiation seem to have influenced TSWV populations temporally rather than positive selection induced by host resistance. These results suggest there is little likelihood of development of highly virulent isolates/strains of TSWV. A series of experiments were also conducted to evaluate the effects of TSWV-resistant genotypes on thrips. Choice and no-choice tests indicated that resistant and susceptible genotypes differentially affected thrips, Frankliniella fusca (Hinds) fitness. Thrips feeding and survival were suppressed on some resistant genotypes than on a susceptible genotype.

72 State of the Art Session V: Thrips/Tospovirus Ecology and Management Seasonal Occurrence of Thrips Species Prevalent in Soybean and the Economic Impact of Soybean Vein Necrosis Virus in Wisconsin Russell L. Groves1, Chris Bloomingdale1 and Damon Smith2 1Department of Entomology and2Department of Plant Pathology, University of Wisconsin, Madison, Wisconsin 53706 Soybean vein necrosis disease (SVND) is caused by Soybean vein necrosis virus (SVNV), which is a relatively new tospovirus first discovered in the southern U.S. in 2008. Very little is known about the impact and importance of SVNV on soybeans. To better understand SVND and the vector (soybean thrips, Neohydatothrips variabilis), field experiments were established in 2013 and 2014 to investigate the seasonality of thrips populations in Wisconsin soybean fields and the impact of SVND on soybean yield and quality. Yellow sticky card traps were used to sample thrips present in soybean fields on a weekly basis in both years. Sampling was performed at five locations across the major soybean-growing regions of Wisconsin. Peak captures in 2013 suggest two major movements of thrips into sampled fields, the first of which occurred in mid-July and the second in late August. Similar trends were observed in 2014. The diversity of thrips species captured in 2013 was low. Eastern flower thrips represented 91% of total captures, while soybean thrips represented 2%. The magnitude of captures did differ by week and location (P<0.01). Preliminary data from 2014 suggest similarities in the species composition to those in 2013. The yield studies conducted in both years revealed a weak negative correlation between disease severity and yield and pod quality. Symptoms of SVND were not observed until late August in either year, suggesting SVND may be more problematic in years or climates conducive to early vector arrival.

73 State of the Art Session V: Thrips/Tospovirus Ecology and Management Is Soybean Vein Necrosis Virus (SVNV) Seed Transmitted? C. Groves (1) T. German (2), R. Dasgupta (2), D. Mueller (3) and D. Smith (1) (1) Department of Plant Pathology, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, USA; (2) Department of Entomology, University of Wisconsin, 1630 Linden Drive, Madison, WI 53706, USA ; (3) Department of Plant Pathology and Microbiology, Iowa State University, 351 Bessey St., Ames, IA, 50011, USA Soybean vein necrosis virus (SVNV; genus Tospovirus; Family Bunyaviridae) is a virus that has been detected across the United States and in Ontario, Canada. Symptoms of SVNV on soybean (Glycine max) start as yellowing along leaf veins that transition to brown, irregular lesions, eventually becoming necrotic. In 2013, a seed lot of a commercial soybean variety with a high percentage of discolored, deformed and undersized seed was obtained. In April of 2014 a random sample of this seed was planted in a growth room under standard conditions. Germination was greater than 90% and the resulting seedlings looked normal. Four bulk samples of six plants each were tested by reverse transcription polymerase chain reaction (rtPCR) using published primers complimentary to the N gene of SVNV. Two bulk samples yielded amplicons with a size and sequence predictive of SVNV. Twelve individual plants were further tested; of these, two individuals were positive for SVNV. Because there was no source of inoculum near these plants we concluded that the infected plants must be due to infected or infested seed. Experiments were repeated by growing seed from the same seed lot in an isolated room inside a thrips-proof cage to further insure no external source of infection or PCR contamination. Additional positive plants were found using these measures. We also utilized primers for the M and L RNAs of SVNV to strengthen the diagnosis of viral infection. In experiments, by three scientists, in two different labs all three genomic RNAs of SVNV were amplified in these plant materials. Preliminary data from Western Blot experiments suggests that the N protein is also present in these samples. These data and the methods used will be presented to explore the hypothesis that infested or infected seed can transmit SVNV and generate further discussion.

74 State of the Art Session V: Thrips/Tospovirus Ecology and Management Bio-ecology and management of thrips and tospoviruses in East Africa Sevgan Subramanian*, Sunday Ekesi, Nguya Kalemba Maniania Plant Health Division, International Centre of Insect Physiology and Ecology, Nairobi, Kenya Email: [email protected] Agriculture sector in East Africa is characterized by both widespread cultivation by smallholders of staple food crops and a rapidly growing horticultural sub-sector which caters to both the domestic and export market needs. Key pest thrips species such as the Western Flower thrips, Frankliniella occidentalis (Pergande); Onion thrips, Thrips tabaci (Lindeman); Yellow flower thrips, F. schultzei Trybom;Bean flower thrips, Megalurothrips sjostedti (Trybom) and thrips-transmitted tospoviruses, Tomato spotted wilt virus and Iris yellow spot virus seriously constrain the productivity of both staple food crops and horticultural crops with yield losses ranging from 20 – 80%. In spite of its economical importance, information on the bio-ecology and the integrated pest management (IPM) of thrips were largely unknown in the region. In this regard, research efforts spearheaded by icipe with national, international and private sector partners focused on developing and implementing IPM strategies for thrips and tospoviruses in East Africa. IPM tools/strategies such as effective thrips monitoring tools, attractants, habitat management strategies, entomopathogens, endophytes and resistant cultivars are developed and disseminated. Innovative biopesticides application strategy such as “Lure and infect” are under development. Recently our focus has also been on understanding the role of thrips in the epidemiology of the maize lethal necrosis (MLN) disease in East Africa. The significance of the above activities and outcomes for the management of thrips and tospoviruses in East Africa and beyond will be discussed.

75 State of the Art Session V: Thrips/Tospovirus Ecology and Management Li-Fang is a TTEN Fellow Transmission of Tomato spotted wilt virus by two western flower thrips populations in California and evidence that adult thrips emerging from viruliferous pupae in soil could be a primary inoculum source L-F. Chen (1), D.C. Mackie (1), B.A. Bazor (1), O. Batuman (1), D.E. Ullman(2) and R.L. Gilbertson (1) (1) Department of Plant Pathology, University of California, Davis, One Shields Ave. Davis, CA 95616, USA; (2) Department of Entomology, University of California, Davis, One Shields Ave. Davis, CA 95616, USA Tomato spotted wilt virus (TSWV) causes serious diseases in many economic important crops and ornamentals in California, including tomato. Transmission efficiency of TSWV by the predominant vector, western flower thrips (Frankliniella occidentalis), collected from Fresno and Yolo counties was compared with two virus isolates from these counties (TSWV-F and TSWV-Y). The TSWV isolates were maintained on Datura stramonium, and this host plant was used for acquisition and transmission experiments. In the transmission assays, individual viruliferous adult male or female thrips were placed in a vial with a healthy datura leaf and maintained for six days at room temperature in dark. ELISA was then used to determine the presence of virus in inoculated leaves. The overall mean transmission rate of the Fresno thrips population was greater (41%) than Yolo thrips (28%) and this was regardless of the isolate. For both populations, male thrips transmitted TSWV more efficiently (39%) than female thrips (31%). To test if thrips pupae can overwinter in soil and emerging adult thrips can serve as a primary source of inoculum for TSWV in California, rates of emergence of adult thrips were tested by maintaining nonviruliferous and viruliferous thrips pupae at 4 °C over a period of 0 to 8 weeks. The rates of emergence for nonviruliferous and viruliferous adult thrips were similar, and declined over time from 50% (1 week) to 4% (8 weeks). For all time points, adult thrips emerging from viruliferous pupae transmitted TSWV. Viruliferous thrips pupae were then incubated at 10 °C (a typical winter soil temperature in California) over an eight week period. Adult thrips emerged at an average rate of 45%, with peak emergence at the third and fourth weeks. Most importantly, emerging adult thrips transmitted TSWV over the course of the entire experiment.

76 State of the Art Session V: Thrips/Tospovirus Ecology and Management Anita is a TTEN Fellow Transcriptional changes associated with Tomato spotted wilt virus infection in various life stages of its thrips vector, Frankliniella fusca [Hinds] Anita Shrestha (1), Donald E. Champagne (1), Gaelen R. Burke (1), Albert K. Culbreath (2), Dorith Rotenberg (3), Anna E. Whitfield (3), & Rajagopalbabu Srinivasan (1) (1) Department of Entomology; (2) Department of Plant Pathology, University of Georgia, Tifton, GA 31793, USA; (3) Department of Plant Pathology, Kansas State University, Manhattan, KS 66506, USA. Thrips transmitted Tomato spotted wilt virus (TSWV) is a type member of the genus Tospovirus in the family Bunyaviridae. TSWV is an economically important pathogen of agricultural and horticultural crops and is transmitted by thrips in a persistent propagative manner. Also, TSWV acquisition during early larval stages is requisite for TSWV transmission, indicating intricate thrips-TSWV interactions. Fitness and behavioral studies conducted on tobacco thrips, Frankliniella fusca [Hinds] and TSWV at macro levels revealed that TSWV infection could affect thrips fitness and feeding behavior. To gain insights into TSWV-induced changes on F. fusca at molecular levels, a transcriptomics approach was undertaken. Transcriptomes of viruliferous and nonviruliferous F. fusca were developed by deep sequencing and compared at larval, pupal, and adult stages. Using ‘Trinity’ software, reads were assembled into 72, 366 contigs. A total of 1,161 transcripts were identified as differentially expressed transcripts at three life stages. Functional annotations of differentially expressed transcripts associated with virus-vector interactions alone are discussed here. Transcripts associated with virus replication and movement, vector response to virus infection, and effect of virus infection on vector fitness were examined in depth. As expected, analyses indicated that transcripts associated with virus replication and movement such as lipid metabolic process and clathrin-mediated endocytosis were upregulated in viruliferous thrips. Also, transcripts pertaining to five immune pathways including apoptosis, toll pathway, and RNAi were upregulated in viruliferous thrips. Upregulation of these transcripts was specific to the adult life stage. Further, in viruliferous thrips, transcripts associated with reproduction and embryo development were upregulated. However, transcripts associated with multicellular organismal development were also downregulated in viruliferous thrips. This study provides transcriptional evidence for TSWV-induced positive and negative effects on thrips observed at macro levels. Gene knockout experiments have to be conducted to further validate or confirm the putative functions of several transcripts of interest.

77 State of the Art Session V: Thrips/Tospovirus Ecology and Management Pamella is a Laurence Mound Fellow The Genetics of Vector Competence of Western Flower Thrips (Frankliniella occidentalis) in Tomato Spotted Wilt Virus Transmission P. A. Ogada and H. -M. Poehling Institute of Horticultural Production Systems, Department of Phytomedicine, Gottfried Wilhelm Leibniz Universität Hannover, Germany The interaction between virus, vector and host plant has been found to be specific, but complex and in some kind mutualistic often favouring virus transmission and spread. The main shortcoming of all the interaction studies e.g. between Frankliniella occidentalis and Tomato spotted wilt virus (TSWV), is the missing information on the intrinsic potential of individual thrips to serve as efficient vector, both at phenotypic as well as genotypic level, as most of these studies were performed at population level. Furthermore, detailed analysis of vector competence heredity such as monitoring the splitting of phenotypes in filial generations obtained from individual crossings of parent thrips with different vector competence status have not been reported. In this study, using the model systems F. occidentalis and TSWV, we examined the influence of individual vector genetic composition in the virus transmission efficiency. We hypothesized that vector competence of F. occidentalis in the transmission of TSWV, is a trait controlled by a recessive heredity mechanism. Crossings of individually characterised (vector competent or non-competent) partners were carried out, and the competence of all resulting individual offspring per couple determined. Moreover offspring from virgin females, also individually characterised for vector competence, were evaluated. For the basic crossing experiment, the offspring were evaluated up to F2. Virgin females produced 100% male offspring parthenogenetically, allowing evaluation up to F1 only. Our results showed that over 81% of the TSWV-transmitting male offspring inherited their competence status maternally, while from the non-competent mothers; over 90% of the offspring were also non-competent. These findings suggest that vector competence of F. occidentalis is a heritable trait controlled by a recessive allele. From an application point of view, an improved knowledge of thrips vector competence at individual level may help to model more precisely the risks of virus spread through plant populations.

Keywords: Vector competence, Genetic, Inheritance, Recessive gene, Frankliniella occidentalis, thrips, Tomato Spotted Wilt Virus

78 State of the Art Session V: Thrips/Tospovirus Ecology and Management Enhancing compatibility between entomopathogens and thrips semiochemicals K. Mfutia,b, S. Subramaniana; S. Niassya, H. du Plessisb, S. Ekesia and N.K. Manianiaa* a International Centre of Insect Physiology and Ecology, PO Box 30772-00100, Nairobi, Kenya; b Unit for Environmental Sciences and Management, North-West University, Private Bag X6001, Potchefstroom, 2520, South Africa Grain legumes are among the key economical crops in Africa. Entomopathogenic fungi (EPF) are among the most promising alternatives to synthetic chemical pesticides being explored. However, in an attempt to address shortcomings surrounding fungal innundative application (high amount of innoculum and short persistence), we first investigated the effect of spatial separation of the semiochemical Lurem-TR, which has been found to inhibit conidia of EPF when put together. Parameters such as persistence of conidia of Metarhizium brunneum and M. anisopliae in the greenhouse and field were evaluated in an autodissemination strategy for the management of Megalurothrips sjostedti a grain legume pest. Influence of spatial separation of the semiochemical on thrips attraction and conidial acquisition by thrips from the autoinoculation device was also investigated in the field. Persistence of conidia of both fungal species increased with distance of separation of Lurem-TR. Direct exposure of fungus without separation from Lurem-TR recorded the lowest conidial germination. Attraction of thrips to the device also varied significantly according to distance between device and semiochemical, with a higher number of thrips attracted when Lurem-TR was placed in a container below the device and at 10cm distance. There was no significant difference in conidial acquisition between spatial separation treatments of conidia and Lurem- TR. In a second experiment, we screened several thrips attractant for their compatibility with M. anisopliae, and attraction to BFT in the field. Results showed that Methyl anthranilate was compatible with M. anisopliae and its attractiveness was similar to Lurem-TR. We concluded that spatial separation of fungal conidia from Lurem-TR in an autoinoculation device could provide a low-cost strategy for effective management of BFT on grain legume cropping systems. Methyl anthranilate can be recommended as an alternative thrips attractant. Keywords: Bean Flower thrips, grain legumes, lure and infect, Metarhizium, attractant.

79 State of the Art Session V: Thrips/Tospovirus Ecology and Management Preference of western flower thrips on the Arabidopsis plants infected with thrips-non- transmissible Tomato spotted wilt virus. Yasuhiro TOMITAKA(1), Hiroshi ABE(2), Tamito SAKURAI(1), Shinya TSUDA(1) (1)National Agricultural Research Center, Tsukuba 305-8666, Japan; (2) Experimental Plant Division, RIKEN BioResource Center, Tsukuba 305-0074, Japan. The western flower thrips (Frankliniella occidentalis [Pergande]) is one of the important herbivores that causes serious damages on many agricultural plants and also transmits tospoviruses, such as Tomato spotted wilt virus(TSWV). Therefore, both feeding damage and viral disease are serious problems in many countries including Japan. Our previous study showed that thrips was attracted to the TSWV-infected Arabidopsis plants, and its attractiveness was increased in jasmonic acid (JA) insensitive coi1-1mutants as compared to wild type (WT) plants. In addition, salicylic acid (SA) application to WT plants enhanced attractiveness of the thrips, like the case of TSWV infection. The results suggest the mechanism of virus strategy to attract vector thrips to virus-infected plants taking advantage of antagonistic SA-JA plant defense system. In this meeting, we report the relationship between the vector transmissibility of TSWV and attractiveness of thrips on the virus-infected Arabidopsis plants. First, a thrips-non- transmissible TSWV isolate, named TSWV-Mo, was obtained by serial mechanical transfer in plants, which was made sure that was never transmitted by three thrips species, F. occidentalis, Thrips tabaci, T. palmi. Expression levels of genes relating to plant defense in the infected plant were similar between wild-type TSWV (TSWV-wt) and TSWV-Mo. The preference assay revealed the higher number of F. occidentalis on either TSWV-wt- or TSWV- Mo-infected plant than that on the uninfected plant, significantly. In conclusion, our results showed that thrips-non-transmissible TSWV-infected plants attracted the vector thrips.

80 State of the Art Session VI: Virus Epidemiology and Integrated Management Strategies Population dynamics of Frankliniella occidentalis in the Central Valley of California: Data, demography and differential equations Neil McRoberts1, Ozgur Batuman1, A.J. Campbell1, Bob Gilbertson1, Frank van den Bosch2, Michael J. Jeger3 1Plant Pathology Department, University of California, Davis, One Shields Avenue, Davis, CA 95616, USA; 2Computational and Systems Biology Department, Rothamsted Research, Harpenden, Hertfordshire, AL5 2JQ, UK; 3Imperial College, London, Silwood Park Campus, Buckhurst Road, Ascot, Buckinghamshire, SL5 7PY, UK As the dominant vector of Tomato Spotted Wilt Virus (TSWV) in the Central Valley of California the Western Flower thrips, Frankliniella occidentalis, is one of the primary determinants of the severity of the spotted wilt diseases in the many host crops that are produced in the region annually. The population dynamics of the vector drive the population dynamics of the virus diseases and so understanding thrips populations and their interaction with the virus inoculum is a key issue in managing TSWV-related diseases. In the paper we present three different views of the dynamics seen through the lenses of field data collection, a degree-day demography model and an evolutionary population dynamics model expressed in differential equations. We examine the extent to which the three different views agree, and highlight aspects of the dynamics that each view provides. In examining the differential equation model we highlight an apparent evolutionary trade-off which it suggests between juvenile acquisition of the virus and adult visiting rate to host plants. The existence of the trade-off embedded in the dynamics of the model raises hypotheses about the relationship between vector and the virus which could be investigated by experimental work.

81 State of the Art Session VI: Virus Epidemiology and Integrated Management Strategies Ozgur is a TTEN Fellow Development of an integrated pest management (IPM) strategy to control thrips and tomato spotted wilt disease in processing tomato fields in the Central Valley of California Ozgur Batuman1, Thomas A. Turini2, Michelle LeStrange3, Scott Stoddard4, Gene Miyao5, Brenna Aegerter6, Li-Fang Chen1, Neil McRoberts1, Diane E. Ullman7 and Robert L. Gilbertson1 1Department of Plant Pathology, University of California, Davis; 2University of California Cooperative Extension (UCCE), Fresno County; 3UCCE, Kings and Tulare Counties;4UCCE, Merced County; 5UCCE, Yolo, Solano and Sacramento Counties; 6UCCE, San Joaquin County;7Department of Entomology, University of California, Davis Tomato spotted wilt virus (TSWV; genus Tospovirus; family Bunyaviridae) is a thrips- transmitted virus causing substantial losses in many crops, including tomato. Since 2005, TSWV has emerged as an important virus of processing tomatoes in the Central Valley of California, in part due to increased populations of the primary vector, the Western flower thrips (WFT; Frankliniella occidentalis). To better understand the epidemiology of TSWV in this region, WFT population densities and TSWV incidence were monitored in processing tomato transplant-producing greenhouses and associated fields (2007-2013). Relatively low thrips populations were detected on transplants in greenhouses, and no TSWV infection was found in transplants, indicating that transplants were not a major inoculum source. In processing tomato fields, thrips populations began to increase in March-April depending on temperature, and high thrips populations persisted throughout the growing season. Thrips populations declined substantially in late September-October and remained low November through February. Thrips identified in monitored processing tomato fields were WFT, and numbers of females were three- fold higher than males. Consistent presence of larva in tomato flowers indicated that WFT reproduced on tomato. Symptoms of TSWV infection first appeared in April-May and eventually were observed in all monitored processing tomato fields. Incidences ranged from 1-20%, and were always highest in late-planted fields. Inoculum sources (e.g., weeds and bridge crops) for thrips/TSWV varied depending on location. The results were used to develop a model for TSWV infection of processing tomatoes in the Central Valley of California. Specifically, initial low levels of TSWV inoculum are amplified in early-planted susceptible crops, with highest levels of infection in later-planted fields, especially those with high thrips populations. An integrated pest management (IPM) strategy for TSWV in processing tomatoes in California has been developed and provided to growers.

82 State of the Art Session VI: Virus Epidemiology and Integrated Management Strategies A Thrips and Tomato Spotted Wilt Virus Prediction Tool to Aid TSW Management Decisions in Tobacco George Kennedy and Thomas Chappell Department of Entomology, North Carolina State University, Raleigh, NC 27695-7630 USA Tomato spotted wilt virus is a major threat to tobacco production in North Carolina, South Carolina and Georgia, where it is spread spread by Frankliniella fusca from winter annual weeds to tobacco. Primary spread during the first five -six weeks following transplanting accounts for the vast majority of the final incidence of TSW in tobacco. To manage this problem, tobacco growers apply the maximum allowed rate of imidacloprid to tobacco prior to transplanting and/or apply one or more applications of the plant activator acilbenzolar-S methyl. Both chemicals carry significant risks of phytotoxicity, and applications of the latter must be timed properly, before virus spread occurs. Because TSW prevalence varies greatly among locations and years, these applications are often unnecessary. Building on research documenting that winter and spring temperatures and precipitation are critical determinants of TSWV inoculum abundance in the landscape and spread by F. fusca into the crop, a series of weather-based models were developed describing F. fusca development and dispersal and TSW incidence in tobacco. These models have been incorporated into a TSWV and Thrips Risk Forecasting Tool [accessible on- line at http://www.nc-climate.ncsu.edu/thrips/index.php] to provide a TSWV management decision aid tobacco growers. This tool and the relationships that underlie it will be described.

83 State of the Art Session VI: Virus Epidemiology and Integrated Management Strategies The Use of Viral Epidemiology to Better Manage Tospoviruses in the Field R.C. Kemerait (1), B. Williams (2), A. K. Culbreath (3), M. Abney (4) & R. Srinivasan (5) (1) Department of Plant Pathology, University of Georgia, 2360 Rainwater Road, Tifton, GA 31793, USA; (2) Department of Plant Pathology, University of Georgia, 2360 Rainwater Road, Tifton, GA 31793, USA; (3) Department of Plant Pathology, University of Georgia, 2360 Rainwater Road, Tifton, GA 31793, USA; (4) Department of Entomology, University of Georgia, 2360 Rainwater Road, Tifton, GA 31793, USA; (5) Department of Plant Pathology, University of Georgia, 2360 Rainwater Road, Tifton, GA 31793, USA Since the mid-1990’s, farmers growing peanut (Arachis hypogaea L.) in the southeastern United States have had to make significant efforts to manage tomato spotted wilt caused by the Tomato spotted wilt virus (TSWV). Significant yield losses continue to be associated with this disease and if not for release of more-resistant cultivars, beginning with ‘Georgia Green’, and the risk management tool “University of Tomato Spotted Wilt Index”, the peanut industry in the region would have struggled to survive. Tactics for management of tomato spotted wilt on peanut are now presented to growers in the “Peanut Rx Disease Risk Index” as a series of steps, to include variety selection, planting date, tillage practices, row spacing, at-plant use of insecticides and seeding rate which can be combined to lower risk to TSWV. However, in following a series of simple steps, most growers fail to grasp the impact of these factors on the epidemiology of the viral disease and the associated thrips vector. This paper will provide a specific account of how decisions based upon risk-factors found in Peanut Rx actually affect the disease epidemic or the dynamics of the thrips populations. For example, selection of more resistant cultivars slows the development of the epidemic as does use of the at-plant insecticide phorate. Manipulating planting date impacts the thrips population present when the peanut crop is most vulnerable. Current efforts are underway to provide increased education to the peanut growers as to the biology behind the effective risk management tool they have adopted.

84 State of the Art Session VI: Virus Epidemiology and Integrated Management Strategies Management of diseases caused by thrips-transmitted tospoviruses in subsistence agriculture: the case of Peanut bud necrosis virus in India Gandhi Karthikeyan (1), William Turechek (2), Scott Adkins (2), & Rayapati Naidu (3) (1) Department of Plant Pathology, Center for Plant Protection Studies, Tamil Nadu Agricultural University, Coimbatore 641 003, Tamil Nadu, India; (2) USDA ARS USHRL, 2001 South Rock Road, Fort Pierce, FL 34945, USA; (3) Department of Plant Pathology, Irrigated Agriculture Research and Extension Center, Washington State University, Prosser, WA 99350, USA. Among the tospoviruses (genus Tospovirus, family Bunyaviridae) reported in India, Peanut bud necrosis virus (PBNV) is by far the most economically significant for tomato production in subsistence agriculture. Presence of PBNV was also confirmed in Bangladesh and Indonesia, indicating the likely distribution of the virus across many countries in Asia. Previous studies in India have shown that PBNV can infect a broad range of legume and solanaceous crops and non- crop species. Since subsistence farmers in India grow different crops that are susceptible to the virus and perpetuate its principal vector, Thrips palmi, in contiguous seasons, the practice of overlapping cultivation provides continuous sources of inoculum year round to infect tomato crops. The lack of resistance to PBNV in cultivated tomato is offering little hope to control the virus. Indiscriminate application of pesticides by farmers is contributing to the buildup of pesticide resistance in thrips, making vector control least effective. To circumvent these practical challenges, we have sought alternative strategies to minimize impacts of PBNV. Studies on spatio-temporal spread of PBNV indicated that the virus was introduced initially into farmers’ fields via infected transplants obtained from nurseries and secondary spread occurred to neighboring plants within rows rather than between rows. Using this information, transplanting virus-free tomato seedlings and roguing of symptomatic seedlings during transplanting and until 45 days post-planting were found to be simple and affordable tactics for reducing virus incidence and minimizing crop losses in farmer's fields. By incorporating these measures into IPM packages, farmers were able to reduce PBNV incidence and its impacts on tomato yield without applying pesticides to control thrips vectors. Thus, similar environmentally benign IPM strategies can be implemented as an alternative to pesticide-based measures for mitigating negative impacts of tospoviruses in subsistence agriculture in developing countries.

85 State of the Art Session VI: Virus Epidemiology and Integrated Management Strategies Biology, Epidemiology and Management of Iris yellow spot virus: A Decade of Progress H.R. Pappu Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA Iris yellow spot virus (IYSV) presents an interesting case study in epidemiology in the USA and several parts of the World. In the US, from a relatively obscure virus of onion beginning in late 1990s, IYSV became an emerging threat beginning in early 2000s, and an endemic viral pathogen that caused total crop loss in some regions of the country and continues to be a constraint to both bulb and seed onion crops. IYSV is now reported from many onion growing states in the US and several countries from the Americas, Asia, Australia, and Europe. The ever increasing globalization and trade of plants, setts and bulbs, and the small size of its primary vector, Thrips tabaci, have likely contributed to the spread of IYSV to many onion growing regions. The continued spread and increasing economic impact of the virus on an economically important crop such as onion, and threat to other Allium crops, have provided impetus to gain a better understanding of its biology, epidemiology, and molecular biology, and to develop management options. Progress included improved virus diagnostics, complete genome characterization, better understanding of the role of thrips, onion volunteers, and weeds in virus epidemiology, effect of soil and irrigation management on disease incidence, multi-location and multi-year field trials of onion cultivars and germplasm for virus resistance, combined with breeding programs focused on identifying resistance/tolerance to thrips and IYSV. A risk-index that consists of factors that might positively or negatively affect the final disease incidence was developed. A co-ordinated, multi-state, multi-disciplinary and multi-institutional US-wide effort facilitated significant progress toward an integrated disease management program.

86 State of the Art Session VII: Thrips Ecology and Behavior The pheromones of the western flower thrips and their use in pest management W. D. J. Kirk School of Life Sciences, Keele University, Newcastle-under-Lyme, Staffordshire ST5 5BG, UK The western flower thrips, Frankliniella occidentalis (Pergande) has been the subject of much research because it is a major crop pest. This has led to the identification of three pheromones so far. An alarm pheromone (decyl acetate and dodecyl acetate) was discovered by Teerling in 1992; it is produced by larvae and induces a defensive response in other larvae and affects take- off, landing and oviposition in adults, as well as arresting predators. An aggregation pheromone (neryl (S)-2-methylbutanoate) was discovered in 2005; it is produced by adult males and increases activity in adults as well as increasing trap catches of female and male adults. A contact pheromone (7-methyltricosane) was discovered in 2013; it is produced by adult males and is detected by adult females and males. Other male-produced compounds are being studied and these may prove to be further pheromones. The number of pheromones found in this species probably just reflects the amount of research on it, so it is likely that other species of thrips will also have several pheromones. Aggregation pheromones have now been identified in other thrips species, such as the melon thrips, Thrips palmi Karny, and identifications are almost complete for some further species. The increase in activity stimulated by alarm pheromone and aggregation pheromone has the potential to increase the effectiveness of insecticides and biological control. Experiments in commercial strawberry crops in the UK showed that mass trapping with blue sticky roller traps baited with aggregation pheromone reduced the number of adult thrips in flowers by 73% and the amount of bronzing damage to fruit by 68%. A cost- benefit analysis showed that the treatment was cost-effective. Pheromones play a critical role in the biology of the western flower thrips and offer potential for novel biocontrol-compatible approaches to pest management, particularly in high-value crops.

87 State of the Art Session VII: Thrips Ecology and Behavior Mating behavior of the western flower thrips A. O. Akinyemi, W. D. J. Kirk School of Life Sciences, Keele University, Staffordshire ST5 5BG, UK The western flower thrips Frankliniella occidentalis (Thysanoptera: Thripidae) is an important pest of agriculture and horticulture for which novel control methods are sought. The mating behaviour of the western flower thrips has been studied previously, before the identification of any adult-produced pheromones in this species. A male-produced aggregation pheromone (neryl (S)-2-methylbutanoate), a contact pheromone (7-methyltricosane) and a compound ((R)- lavandulyl acetate) whose function is not yet clear have been identified since then in this species. The aim of this research is to investigate the possible roles of pheromones in the mating behaviour of the western flower thrips. Male and female thrips are placed in an arena and the behaviour of virgin and mated females can be compared in the presence of virgin males, with or without added synthetic pheromones. The behaviour was recorded with high definition video using a digital camera mounted on a dissecting microscope. Receptive females stayed still and lifted the tip of the abdomen slightly as the male climbed on her back. Unreceptive females flipped the abdomen up and down to dislodge the male attempting to climb her. Understanding of the role of pheromones in this mating behaviour may allow the development of novel control methods.

88 State of the Art Session VII: Thrips Ecology and Behavior Aggregation ecology in Bean Flower thrips Megalurothrips sjostedti Trybom (Thysanoptera: Thripidae) Saliou Niassy1, Sunday Ekesi1, Nguya K. Maniania1, Gerald B. Moritz2, Willem Jan de Kogel3 and Sevgan Subramanian1* 1: International Centre of Insect Physiology and Ecology, P.O. Box 30772-00100, Nairobi, Kenya; 2: Martin Luther University Halle-Wittenberg, Faculty of Natural Sciences,Institute of Biology, Domplatz 4 06108 Halle (Saale), Germany; 3: Plant Research International, PO Box 69, 6700AB Wageningen, The Netherlands We studied the bioecology of the bean flower thrips (BFT),Megalurothrips sjostedti Trybom (Thysanoptera: Thripidae), a key pest of grain legumes in sub-Saharan Africa. The study was conducted first on cowpea plants at different time of the day and crop stages to evidence BFT aggregation behaviour. We also compared BFT aggregation in various cowpea growing regions in Kenya (Nairobi, Matuu and Mbita). Results indicated that for all the stages, the density of BFTs varied across the blocks and time of the day, with significantly more insects being collected at 10.00hrs, 13.00hrs and 16.00hrs than at 07.00hrs (P<0.005). Dispersion indices suggested that both male and female BFTs were aggregated. Male aggregation varied across blocks, crop stages and time of the day and locations and occurred only on green plant parts. Female numbers in flowers increased significantly with crop stage (P<0.0001) and had a distribution different from that of a random population. The degree of aggregation varied with locations. To understand the mechanisms that govern the observed aggregation, we studied the behavioural response of M.sjostedti to male and female conspecific odour in a four-arm olfactometer bioassay. Results showed that both sexes were attracted to male odour. Moreover, female M. sjostedti spent more time in the male odour-treated arm than males. In contrast, odour collected from female BFT was not attractive to conspecifics. This study is the first report on BFT aggregation. BFT aggregation on bean plant parts differed between the sexes and was influenced by time of the day, crop stage and location. Although the presence of male sterna glands is being investigated, the study suggests that male odours are responsible for the observed aggregation in M. sjostedti. The identification of the active compounds could offer an excellent opportunity to develop new tools for grain legume pest management in Africa, where the pest causes widespread damage. Keywords: Aggregation behaviour, sternal glands, pheromone, grain legumes, legumes

89 State of the Art Session VII: Thrips Ecology and Behavior Olfactory responses of western flower thrips (Frankliniella occidentalis) populations to a non-pheromone lure M-C. Nielsen (1), S.P. Worner (2), M. Rostás (2), R.B. Chapman (2,3), R.C. Butler (1), W.J. de Kogel (4) and D.A.J Teulon (1) (1) The New Zealand Institute for Plant & Food Research Limited, Private Bag 4704, Christchurch, New Zealand; (2) Bio-Protection Research Centre, Lincoln University, PO Box 84, Canterbury, New Zealand; (3) Insect Science Solutions, 830 Selwyn Road, Christchurch 7678, New Zealand; (4) Wageningen UR, Biointeractions and Plant health, P.O. Box 16, 6700 AA Wageningen, The Netherlands The search for alternative pest management techniques to mitigate the impact of western flower thrips, Frankliniella occidentalis (Pergande) (Thysanoptera: Thripidae) has resulted in increasing interest in the use behaviour-modifying chemicals. In this study, we determined whether the origin of populations, feeding history and/or genotype influence the response of western flower thrips (WFT) populations to the thrips kairomone lure methyl isonicotinate (MI) in a Y-tube olfactometer study. Four New Zealand populations were tested: 1) thrips from a commercial glasshouse capsicum crop, 2) thrips from a long-established laboratory colony (>222 generations) kept on chrysanthemums, 3) thrips from a laboratory colony (6–9 generations) kept on French dwarf beans, and 4) thrips thought to be a separate cryptic non-pest species from outdoor yellow tree lupins (Lupinus arboreus). In laboratory tests significantly more WFT from all four populations chose the MI-laden arm of a Y-tube olfactometer when it contained 1 µl methyl isonicotinate (61.3–73.2%) compared with the blank no-odour arm. No differences in response to MI were found between the two laboratory and the one glasshouse WFT populations. Both laboratory populations and the greenhouse population belonged to the ‘glasshouse pest’ genotype of WFT. However, the cryptic non-pest WFT genotype responded more strongly to methyl isonicotinate than any of the other populations, although the response was only significantly stronger than that of the long established laboratory population. Significant differences were also found among populations in the average time taken for thrips to make a choice to enter either arm of the Y-tube olfactometer, with the cryptic non-pest lupin genotype taking the shortest time, followed by thrips from the capsicum glasshouse. The results are discussed with respect to variability of olfactory perception and olfactory behaviour within a species and the relevance to the use of such a kairomone lure in pest management programmes.

90 State of the Art Session VII: Thrips Ecology and Behavior

The interplay of cycad cone thermogenesis, volatile emissions, humidity, CO2 and light in mediating the specialist Cycadothrips pollinator behavior I. Terry (1), R. Roemer (2), D. Booth (3), D. Brooks (3) and G. Walter (3) (1) University of Utah, Department of Biology, 257 South 1400 E, Salt Lake City, UT 84112 USA; (2) University of Utah, Department of Mechanical Engineering, 50 S. Central Campus Dr.,2202 Merrill Engineering Bldg., Salt Lake City, UT 84112 USA; (3) The University of Queensland, School of Biological Sciences, Brisbane, 4072 Queensland, Australia Many of the Australian endemic Macrozamia cycad species in eastern Australia are pollinated solely by the thrips, Cycadothrips chadwicki Mound, and are disjunctly distributed from central Queensland to southern New South Wales. These thrips thrive only on male cones, where they mate, lay eggs, and adults and larvae feed on pollen. Cones of two species, M. lucida and M. macleayi, have a daily thermogenic event during the mid-day that is associated with dramatic increases in ß-myrcene, CO2, humidity, and thrips depart cones en masse at this time. Later in the day, thrips return to cones, including some ovulate cones, where thrips carrying pollen reach the micropyle. We investigated the effects of these thermogenic signals on their potential to induce thrips to leave cones in the dark and light with a suite of Y-tube behavioral experiments. The results suggest that ambient light, high cone temperatures, humidity, and β-myrcene levels, but not CO2, each induce Cycadothrips to leave cones. At typical overnight springtime temperatures (14°C) thrips were relatively inactive and negatively phototactic. At typical daytime ambient temperatures, 22-26°C (lower than typical thermogenic temperatures), thrips were active and positively phototactic. Thrips moved away from or avoided thermogenic temperatures and high concentrations of β-myrcene, and they preferred dry to humid air. Even though several signals alone induce thrips to leave a cone towards the daytime light, these signals are presented simultaneously during thermogenesis, creating an enhanced or redundant signaling that enforces thrips departure from cones. In contrast, low concentrations of β-myrcene attract thrips to cones later in the day. We also report on the differences found in the thrips’ molecular genetics (CO1 mitchondrial and 18s nuclear genes and microsatellite data) that suggest several cryptic Cycadothrips species are aligned along a north to south distribution of Macrozamia species that also vary in their cone traits.

91 State of the Art Session VII: Thrips Ecology and Behavior Stephanie is a Laurence Mound Fellow Offspring sex ratio and duration of ontogenesis are determined by copulation activity in Echinothrips americanus (Thysanoptera: Thripidae) S. Krueger & G. Moritz Institute of Biology, Martin-Luther-University Halle- Wittenberg, Heinrich-Damerow-Str. 4, 06120 Halle, Germany The costs of the reproductive modes, arrhenotoky and thelytoky, are more or less unknown in thrips. However, integrated pest management strategies require a full understanding of temporal and spatial life history variations and sex ratios. Hence, different circumstances with regard to mating /copulation and its effect on the life history of the adults and their progeny in E. americanus were tested. Five different test conditions were examined: (1) one female and one male with permanent access to each other, (2) one female and one male with limited access to each other (single access for 24 hours), (3) one virgin male only, (4) one virgin female only, and (5) two virgin females only. The objectives of investigation were the survivability, the oviposition rate and the number/proportion of larvae hatched. Furthermore, the time for ontogenetic development of the offspring and the sex ratio were analyzed. Mating or not mating has a significant effect on the longevity of both females and males, and also on female fecundity. As a result of the condition (2) tested, limited access has a positive effect on the survivability of the male. On the other hand, permanent access (1) or no access to a female (3) leads to shorter longevity of the male. For a female with permanent access to a male (1), longevity and oviposition rate are reduced. However, the presence of a second virgin female (5) has a positive impact on both traits. In addition, the test conditions of parents have a significant effect on developmental time of the offspring and the sex ratio. Compared to a female under condition (1), a female with limited access to a male (2) delivers offspring with a more male biased sex ratio, independent of the period of time after last male access. Concerning the time for development of the offspring, females under condition (2) and under condition (4) deliver an F1 generation with a shorter developmental time than those under condition (1). E. americanus seems to have a feedback system, i.e. offspring in low male biased populations develop faster and generate an adapted sex ratio. Hence, the assumption of a lower infestation risk in arrhenotokous than in thelytokous should be reconsidered.

92 State of the Art Session VII: Thrips Ecology and Behavior Speciation in the Thrips tabaci cryptic species complex and some of its possible consequences József Fail Department of Entomology, Corvinus University of Budapest, 44 Ménesi út, Budapest, 1118, Hungary Based on DNA sequences of the mitochondrial COI gene, Thrips tabaci Lindeman, 1889 (Thysanoptera: Thripidae) has been divided into three lineages: a tobacco-associated (T) and two leek-associated types (L1, L2). It is believed that the arrhenotokous leek-associated type (L1) is the ancient form of T. tabaci and the T type diverged from it and adapted to solanaceous host plants. The third lineage of T. tabaci, the so-called thelytokous leek-associated type (L2) and the L1 form share many host plants. The distribution of T. tabaci lineages cannot be established with certainty because there is no known morphological difference between the adults of the three lineages in this species complex. Since molecular genetic studies identifying the forms have been published only in the past 10-15 years and much of the literature regarding the geographical distribution of T. tabaci was generated before this time period, it is not always clear which form of T. tabaci was reported in the vast majority of studies. Some of the published host associations are potentially incorrect as well, due to potentially invalid conclusions from the presence of adult thrips only on host plants. The divergence of the three known forms of T. tabaci have most likely led to the development of different ecological traits. The most obvious one is the appearance of thelytoky in the L2 form. Although endosymbiotic microorganisms are well-known to modify the sex ratio in the progeny of arthropods, and thus induce thelytokous reproduction in otherwise arrhenotokous species, thelytoky in T. tabaci seems to be genetically inherited and not microbe- induced. Adaptation to different primary host plants might have also been driving speciation in this cryptic complex. Some of the reported ecological differences between the lineages are discussed in more detail.

93 State of the Art Session VII: Thrips Ecology and Behavior Mariana is a Laurence Mound Fellow Two thrips species sharing home: interaction and behavior of the galling thrips and an invader on Myrtaceae galls M. F. Lindner* (1), A. Cavalleri (1) & M. S. Mendonça Jr. (1) (1) Departamento de Ecologia, Instituto de Biociências, Universidade Federal do Rio Grande do Sul, Bento Gonçalves Ave. 9500, 91501-970 Porto Alegre, Brazil A new species of Holopothrips (Phlaeothripidae) was found inducing galls on leaves of Myrcia guianensis (Myrtaceae) in southern Brazil. However, an invader thrips, belonging to a new genus and species of Haplothripini, was observed breeding inside the galls and co-occurring with the galling thrips. The abundance of both thrips species inside the galls showed a great variability, with the number of Holopothrips ranging from zero to 75. The morphology of the invader thrips is remarkable, with females bearing swollen forelegs with well-developed tarsal tooth, suggesting a predatory behavior. To identify the factors influencing the abundance of both thrips species, measurements of leaf and gall size were taken, as presence of perforations, and compared with thrips’ abundances. To assess the interaction between both thrips species, observations ad libitum were made, and an attack test with a fine brush was performed, with the thrips’ responses being later classified into categories. Our results showed no correlation between leaf size and thrips abundance. However there was marginally significant negative influence of perforations on thrips abundance and a positive correlation between gall size and thrips abundance, in galls that both species were present. No negative direct interactions between both thrips species were observed, but when the abundance of one species was high, the abundance of the other was low. Moreover, the invader was observed feeding on leaf tissue during laboratory observations. The most frequent behavior in the galling thrips when disturbed was to raise the abdomen quickly, sometimes producing an anal droplet, while the most frequent response of the invader thrips was to escape. We suggest that the invader thrips is possibly an inquiline, and both species should be competing for resources and space, which might explain the negative influence of the high abundance of one species on the other thrips’ abundance.

94 State of the Art Session VIII: Thrips and Tospovirus Management Candice is a TTEN Fellow An introduction to integrated pest management programs for thrips and tospoviruses Candice Ann Stafford-Banks (1) (1) Department of Entomology, University of California, Davis, One Shields Ave. Davis, CA 95616, USA

Over 5,000 species comprise the order Thysanoptera; however, only a small fraction of these species are considered economically important pests. One of the most important Thysanopteran pests is the Western flower thrips (WFT, Frankliniella occidentalis), which has spread around the world wreaking havoc directly through feeding and oviposition damage, and indirectly through tospovirus transmission. WFT has circumvented both established chemical control and integrated pest management programs in horticultural and agricultural crops. Several behavioral and ecological factors contribute to the elite pest status of WFT including: extremely high fecundity and dispersal; an extensive host range of over 1,090 plant species; an omnivorous feeding strategy enabling WFT to utilize alternative food sources when on unsuitable host plants; thigmotactic behaviors which make it difficult for predators and chemical control measures to reach thrips; and the ability to develop resistance to multiple insecticide modes of action with little effect on overall fitness. It is essential to use our understanding of the biology and ecology of WFT to develop effective and sustainable integrated pest management programs. Management strategies that have proven successful against WFT incorporate several of the following techniques: monitoring and identification of pest and non-pest thrips species; use and establishment of economic thresholds specific to cropping systems; identification, maintenance, and establishment of important natural enemies; use of repellants including ultraviolet-reflective mulches, kaolin clay, and bio-pesticides; use and development of host plant resistance against thrips and tospoviruses; and selective use of insecticides with special attention paid to insecticide resistance management techniques. It is important to continuously evaluate the effectiveness of these strategies, while developing novel techniques to stave off the constant threat of WFT.

95 State of the Art Session VIII: Thrips and Tospovirus Management Can We Get Off the Pesticide Treadmill for Managing Onion Thrips and Iris Yellow Spot Virus? Stuart Reitz Department of Crop and Soil Science, Oregon State University, Malheur County Extension, 710 SW 5th Ave., Ontario, OR 97914, USA Onion thrips, Thrips tabaci Lindeman and thrips-transmitted Iris yellow spot virus are the most significant pest complex affecting onion production in the Treasure Valley of eastern Oregon and southwestern Idaho. Thrips feeding damage and virus infection significantly reduce onion bulb size and economic returns for this high value crop. The high concentration of onion fields in the Treasure Valley and the long, hot, dry growing season present a number of challenges for managing onion thrips and Iris yellow spot virus (IYSV). Insecticides are the primary tool that growers use to manage this pest complex, as there are no biological or cultural controls, or resistant cultivars that have been shown to substantially reduce thrips damage. With the long growing season, there is a great risk for insecticide resistance to develop if sound insecticide resistance management programs are not employed. A short term challenge is to design insecticide resistance management programs that identify insecticides that are effective and when during the season different products may be most effectively used. A longer term challenge is identify means to employ biological and cultural controls in this intensely farmed region. Research on the development of improved IPM programs in the Treasure Valley and limitations of IPM in this environment will be discussed.

96 State of the Art Session VIII: Thrips and Tospovirus Management Western Flower Thrips Management in Greenhouse Production Systems Raymond A. Cloyd Department of Entomology, Kansas State University, 123 Waters Hall, Manhattan, KS 66506 USA Western flower thrips, Frankliniella occidentalis (Pergande), is a major insect pest of greenhouse-grown horticultural crops that producers worldwide deal with using a variety of plant protection strategies, including insecticides and biological control. However, continual use of insecticides, especially those with narrow-spectrum modes of action, may lead to the development of resistance. In addition, the introduction of insecticides with new active ingredients is limited due to the costs to manufacturers of development and registration. Therefore, it is important for greenhouse producers to judiciously use what is currently available and also utilize insecticides with broad modes of activity. Although the use of biological control is increasing worldwide, there are issues associated with the effectiveness of specific natural enemies including predatory mites, predatory bugs, and entomopathogenic nematodes. In addition, because western flower thrips can vector viruses, the tolerance for their presence in production systems is extremely low. The future prospects of effectively managing western flower thrips populations on greenhouse-grown horticultural crops will involve an integrated or holistic approach that entails implementing typical strategies including scouting, cultural practices, sanitation, physical impediments, insecticides, and biological control. Furthermore, greenhouse producers will have to develop a new mind-set in that the goal of managing western flower thrips is not complete elimination. So, in order to obtain long-term suppression of western flower thrips populations in greenhouse production systems, not only do the typical strategies need to be utilized but alternative strategies need to be considered such as 1) entomopathogenic fungi, 2) selective insecticides, 3) selective insecticides with natural enemies, 3) combinations of natural enemies, and 4) push-pull systems (e.g., trap plants and plant volatiles). These alternative strategies, however, cannot be too expensive to implement as less than 2% of the total cost of production is associated with plant protection. Moreover, these alternative strategies must not only be inexpensive but also easy to implement and effective greenhouse producers will simply return to relying on insecticides.

97 State of the Art Session VIII: Thrips and Tospovirus Management Insecticide Program Influence on Tomato spotted wilt virus Symptom Expression in Processing Tomatoes in Central California T. Turini1, M. LeStrange2, O. Batuman3, and Robert Gilbertson3 1 University of California Cooperative Extension, Fresno County, 550 E. Shaw Ave., Suite 210, Fresno, CA 93710, USA, 559-375-3147, [email protected]; 2 University of California Cooperative Extension, Tulare and Kings counties, 4437-B S. Laspina St., Tulare, CA 93274, USA, (559) 684-3320, [email protected]; 3 University of California, Department of Plant Pathology, One Shields Avenue - Davis, CA 95616, USA, (530)752-3163, [email protected] Tomato spotted wilt virus (TSWV) incidence and severity increased from negligible levels in 2002 to levels inflicting substantial economic damage in the highly productive Fresno area in 2005. Growers in this production area had little understanding of the virus at the onset of the increased damage. As a component of a comprehensive research project on the disease, insecticide program evaluations were conducted. To address questions regarding the utility of insecticide programs in managing this disease, efficacy of insecticides against thrips were compared in trails conducted from 2007 to 2012 and insecticide programs were evaluated in trials conducted from 2009 to 2012. Main plot treatments were drip applied programs: Thiamethoxam 3-4 weeks after planting both with and without a subsequent application of dinotefuron 3-4 weeks later, plus an untreated control. Sub-plot treatments were rotations of foliar applied insecticides, which were spinetoram and dimethoate rotations applied 2 or 5 times at 10 day intervals. Reductions of TSWV incidence were documented in 2009, 2011 and 2012 due to foliar programs. No differences between the treatments with neonicotinoid injections and untreated were observed and the influence of the transplant treatment was inconsistent. Two to 5 foliar applications were similar. In 2010, TSWV incidence in commercial fields nearby was very high and no differences among treatments were observed possibly due to inundation by TSWV- infected thrips from external sources. Effective and sustainable management will depend upon multiple tactics, which include prudent insecticide treatments.

98 State of the Art Session VIII: Thrips and Tospovirus Management

Impact of DuPont Cyazypyr™ (cyantraniliprole, DPX-HGW86) on the Biology, Behavior and Control of Thrips (Thysanoptera) in Agronomic Crops

I. Billy Annan (1), Hector E. Portillo (1), Juan M. Alvarez (1) and Sanjay Sharma (2)

(1) DuPont Crop Protection, Stine-Haskell research Center, Elkton Rd., Newark DE 19711. USA; (2) E.I. DuPont India Pvt. Ltd., 7th Floor, DLF Cyber Greens, Gurgaon 122002, Haryana, India

Cyazypyr™ (cyantraniliprole, DPX-HGW86) is the second anthranilic diamide discovered and commercialized by DuPont™. It is the first product in the IRAC Group 28 (diamides) insecticide class to control a cross-spectrum of species and multiple life stages of a complex of rasping pests such as thrips, piercing-sucking pests including whiteflies, aphids, psyllids, leafhoppers and planthoppers, and biting-chewing pests like caterpillars and leafminers. Therefore, Cyazypyr™ offers a new and unique mode of action for control of sucking, and rasping pests. Cyazypyr™ is an ingestion toxicant that selectively activates the ryanodine receptor in insect muscles causing rapid cessation of pest feeding, impairment of locomotion, mating disruption, and other muscle- controlled activities, and ultimately to mortality in the affected individuals. Thus, the rapid feeding cessation enables Cyazypyr™ to provide excellent pest control, outstanding plant protection from pest feeding damage, significant reductions in the incidence and severity of pest- transmitted virus diseases, and to improved crop growth and yield characteristics. While exhibiting the excellent pest control attributes, the product also has minimal impact on beneficial arthropods due to their limited exposure. So, Cyazypyr™ is useful in the conservation of predators and parasitoids of crop pests and also shows minimal to no impact on other non-target organisms such as earthworms, crustaceans, molluscs, fish, birds and mammals, and is not persistent in the environment. It is not cross-resistant with other known insecticide modes of action for the control of sucking pests. Overall, Cyazypyr™ is an ideal complement to insecticide resistance management (IRM), integrated pest management (IPM) and integrated crop management (ICM) programs, and other sustainable agricultural practices. A summary of the key product attributes and features, results of laboratory, greenhouse and field studies and the agronomic implications for thrips pest management will be discussed. Keywords: Cyazypyr™, cyantraniliprole, Thrips, Thysanoptera, rapid feeding cessation, disease reduction, anthranilic diamides, ryanodine receptor Corresponding Author: Dr.Isaac Billy Annan, DuPont Crop Protection, Stine-Haskell Research Center, 1094 Elkton Rd., Newark DE, USA. [email protected]

99 State of the Art Session VIII: Thrips and Tospovirus Management Alexander is a Laurence Mound Fellow

Endophytic colonization of onions and their antagonistic impacts on biology of onion thrips Alexander M. Muvea (1, 2), Rainer Meyhöfer (1), Sevgan Subramanian (2), Hans-Michael Poehling (1), Sunday Ekesi (2) & Nguya K. Maniania (2) (1) Institute of Horticultural Production Systems, Dept. Phytomedicine, Leibniz Universität Hannover, Hannover, Germany; (2) Plant Health Division, IPM cluster, International Centre of Insect Physiology and Ecology, Nairobi, Kenya. Fungal endophytes, which live within host plant tissues without causing any visible symptom of infection, are important mutualists that mediate plant–herbivore interactions. Onion thrips, Thrips tabaci (Lindeman) is one of the key pests of onion, Allium cepa L., an economically important agricultural crop cultivated worldwide. However, information on potential integrated pest management (IPM) tools such as endophytic fungi with ability to colonize onions, and impacting on the biology of onion thrips feeding on them, is lacking. We tested the colonization of onion plants by selected fungal endophyte isolates using two inoculation methods. The effects of inoculated endophytes on T. tabaci infesting onion were also examined. Seven fungal endophytes used in our study were able to colonize onion plants either by the seed or seedling inoculation methods. Seed inoculation resulted in 1.5 times higher mean percentage post inoculation recovery of all the endophytes tested as compared to seedling inoculation. Fewer thrips were observed on plants inoculated with Clonostachys rosea ICIPE 707, Trichoderma asperellum M2RT4, Trichoderma atroviride ICIPE 710, Trichoderma harzianum 709, Hypocrea lixii F3ST1 and Fusarium sp. ICIPE 712 isolates as compared to those inoculated with Fusarium sp. ICIPE 717 and the control treatments. Onion plants colonized by C. rosea ICIPE 707, T. asperellum M2RT4, T. atroviride ICIPE 710 and H. lixii F3ST1 had significantly lower feeding punctures as compared to the other treatments. Among the isolates tested, the lowest numbers of eggs were laid by T. tabaci on H. lixii F3ST1 inoculated plants. These results extend the knowledge on colonization of onions by fungal endophytes and their effects on T. tabaci.

100 State of the Art Session VIII: Thrips and Tospovirus Management The impact of cover crops and companion plants on thrips vectors and Tomato spotted wilt virus spread in vegetable farmscapes Stan Diffie (1), Stuart Reitz (2), Kristen Bowers (3), & Rajagopalbabu Srinivasan (1) (1) University of Georgia Department of Entomology, Tifton GA 31793 (2) Oregon State University Department of Crop and Soil Science, Ontario, OR 97914 (3) USDA ARS Insect Behavior and Biocontrol Research, Tallahassee, FL 32308 Thrips-transmitted Tomato spotted wilt virus (TSWV) is a major constraint to vegetable production in the Southeast. Tobacco thrips (Frankliniella fusca Hinds) and western flower thrips (F. occidentalis Pergande) are important TSWV vectors. Thrips and TSWV management is heavily dependent upon insecticides. However, heavy insecticide usage has led to unintended consequences such as insecticide resistance, suppression of natural enemies, and secondary pest outbreaks. Such an insecticide-dependent strategy is not cost-effective and is unsustainable. The proposed alternative is a holistic approach aimed at promoting biotic resistance in vegetable farmscapes. Cover crops and companion plants could act as reservoirs for beneficials, a trap crop for vector thrips, and refuges for non-vector thrips that compete with vectors. From 2012 to 2014, tomato, pepper, and cucumber were planted in spring in plots with cover crop, blue lupin (Lupinus augustifolius L.)/ sunn hemp (Crotolaria juncea L.), residues. In addition, in some plots, a companion plant, bidens (Bidens pilosa L.), was included. Vegetable crops in conventional raised beds with plastic served as controls. A split plot design was adopted with three replications for each sub-plot. Counts of vector thrips, non-vector thrips, natural enemies, and TSWV incidence were recorded from all plots for 8 weeks. Regardless of the cover crop, more non-vector thrips were found on crop bloom samples than vector thrips. In some instances, more vector thrips were found on vegetable blooms from cover crop plots than conventional plots. Though there were instances of TSWV incidence reduction in vegetables with cover crop and companion plants, the effect was not robust. More data will be presented at the meeting. Thus far, results suggest that cover and companion crops could promote sustainability in vegetable farmscapes by enhancing biotic resistance.

101 Final Plenary Session Understanding of Vector Pathogen Interactions: Where is it Taking Us Saskia A. Hogenhout Department of Cell and Developmental Biology, John Innes Centre, Norwich Research Park, Norwich, Norfolk, NR4 7UH, UK Plants are sessile, and this presents a challenge to plant pathogens by limiting their dispersal. Many pathogens have circumvented this by adopting herbivorous insects as vectors, allowing the pathogens to travel great distances. Insects are abundant and have evolved a diverse range of plant feeding and colonization strategies that enable pathogens to adapt in various ways to utilize these herbivores as vectors for their transmission. Indeed, insects transmit the majority of plant viruses; a single insect species can transmit over a 100 different plant viruses. In contrast, relatively few viruses use other organisms for transmission, such as fungal pathogens and nematodes. Even for plant pathogens that use other dispersal strategies, for instance water drops, insects may nonetheless aid their spread in nature as passive or facultative carriers. Recent studies have advanced our understanding of the mechanisms of physical association between plant pathogens and insect vectors. Furthermore, it was demonstrated that the transmission of plant pathogens goes beyond a physical association with the insect, and involves active modulation of plant processes by pathogens to promote insect herbivore attraction, colonization and pathogen transmission. However, mechanisms by which insects modulate plant processes have remained largely elusive. Insects secrete an arsenal of molecules during feeding and egg laying that trigger or modulate plant defenses and some of these could promote colonization within host plants of the plant pathogens. In addition, plant pathogens may carry virulence factors that have direct effects on insect vector (feeding) behavior and promote specific processes in the insect vector for enhanced transmission during feeding. The elucidation of mechanisms by which insects modulate plant processes and the means by which plant pathogens alter insect physiology and behavior should further enhance our understanding of how plant pathogens and insect herbivores have co-evolved.

102 Poster Presentations, Informal Discussions, and Reception The Phylogeography of Tomato spotted wilt virus, Tomato chlorotic spot virus and Iris yellow spot virus M.M.S. Almeida (1), F.A. Ferreira (2), A.S de Oliveira (1,3), F.L. Melo (1) & R.O. Resende (1) (1) Department of Cellular Biology, University of Brasilia, Brazil; (2) Division of Medical Sciences, Harvard Graduate School of Arts and Sciences; (3) Wageningen University The diversity of the genus Tospovirus comprises 11 recognized species with a variable host range and geographic distribution, besides the brief reports of new species. Decipher the global trends in tospoviral spread is an economic necessity, since it may be permit the formulation of better quarantine methods. Phylogeografic reconstructions of three selected species were performed: TSWV is the type member of the genus; IYSV occurs occasionally in most locations that is reported, but has few endemic occurrences all over the world; TCSV was reported with GRSV in the early 1990's. Nowadays, GRSV is present in both new and the old world, whereas TCSV could be find only in the Americas. Through BEAST v.1.6.2 we carried out extensive Bayesian phylogeografic analyses of the RNA S with coding (N protein) and no-coding regions of sequences of these three tospovirus, sampled in different countries and available in the GeneBank. For TSWV there were more than five hundred sequences. For IYSV around eighty sequences were utilized and for TCSV thirty-three sequences were downloaded from database. The resulting trees were summarized in the maximum clade credibility (MCC) tree using TreeAnnotator v.1.6.2 and the ancestral reconstruction was visualized with FigTree v.1.3.1. The supported routes were then compared to the level of export/import of an important susceptible crop of involved countries. Analyses of the phylogenetic trees allow noting that viral migration for all three species intensified beginning in the mid-1980s, correlating with trade liberation policies of the time. Despite this similarity, the dynamics of viral trade and the countries involved were distinct to each specie. IYSV seems to be much more mobile than the others. TCSV disappeared for some years and now reemerged in the Caribbean and USA. TSWV has two different lines, one is circulating in USA and the other between Europe and Asia.

103 Poster Presentations, Informal Discussions, and Reception Ismael is a TTEN Fellow

Tomato necrotic streak virus, a novel subgroup 2 ilarvirus I. E. Badillo-Vargas (1), C. A. Baker (2), J. E. Funderburk (1), and S. Adkins (3) (1) University of Florida, NFREC, Quincy, FL 32351, USA; (2) FDACS-DPI, Gainesville, FL 32608, USA; (3) USDA-ARS, Fort Pierce, FL 34945, USA A novel plant virus has been identified infecting fresh market tomato plants in south and southeast Florida. The virus causes necrosis of tomato leaves, petioles and stems, and necrotic rings or spots on tomato fruits. Symptomatic tomato plant tissue was used to mechanically inoculate tomato plants and reproduced symptoms observed in the original field samples. A portion of the replicase protein 1a gene from RNA1, and the complete movement protein (MP) and coat protein (CP) genes from RNA3 were amplified and sequenced. Sequence analysis showed that these three viral genes are most closely related to, but distinct from, Tulare apple mosaic virus and other subgroup 2 ilarviruses. Considerably less identity was observed with previously reported subgroup 1 tomato-infecting ilarviruses including Tobacco streak virus, Parietaria mottle virus, and Tomato necrotic spot virus. These results indicate that the tomato symptoms observed in Florida in fall 2013, and subsequently in spring and fall 2014, are caused by a novel subgroup 2 ilarvirus, for which the name Tomato necrotic streak virus (TomNSV) is proposed. Potential TomNSV transmission by pollen, seed and/or insect vectors, which may include thrips similar to other ilarviruses, is being investigated.

104 Poster Presentations, Informal Discussions, and Reception De Novo transcriptome analysis of Thrips tabaci suggests the involvement of cytochrome P450 in resistance to spinosad Ran Rosen(1), David Ben-Yakir(2), Murad Ghanim(2) (1) Department of Entomology, The Hebrew University of Jerusalem, Rehovot, Herzel st., 76100, Israel; (2) Agricultural Research Organization (ARO),Bet Dagan, 5025001, The Volcani Center, Israel The onion thrips (OT), Thrips tabaci(Thysanoptera: Thripidae) is a major polyphagous pest attacking wide range of economically important crops. Thrips' damage can result in severe yield loss in onion (Allium cepa) and other crops. In the past two decades, resistance of OT to insecticides with various modes of actions had been documented. Lately, OT resistance to spinosad has been reported from Israel. Little is known about the molecular mechanisms underlying spinosad resistance in OT. We attempted to characterize the mechanisms involved in OT resistance to spinosad, using quantitative transcriptomics with the illumina platform. We used susceptible (LC50=0.6ppm) and resistant (LC50=23,258ppm) OT populations collected in Israel. A partly resistant population (LC50=117ppm) was produced by selection in the laboratory from the susceptible population. De Novo transcriptome analysis of the resistant and the susceptible populations was done to detect differently expressed genes (DGEs) which may be involved in the resistance mechanism. 25,552 genes were sequenced, assembled and functionally annotated. About 1500 DGEs were identified. The expression levels of 3 selected genes, including a Cytochrome P450 (known to involved in metabolic resistance), were examined using qRT-PCR. Results indicate that Cytochrome P450 gene was up regulated significantly (2 to 7 folds) in the resistant populations. This suggests that Cytochrome P450 is involved in OT resistance to spinosad. This is the first reported De Novo transcriptome analysis of spinosad resistant or susceptible OT. The genetic information obtained in this study may be useful in future studies of OT and its close relatives.

105 Poster Presentations, Informal Discussions, and Reception Functional marker assisted-selection and genetic variability of Sw-5bgene in multi- tospovirus resistant Solanum (section Lycopersicon) germplasm accessions using locus- specific primers Maria Esther N. Fonseca (1), Érico C. Dianese (2), Renato O. Resende (2), Matias González- Arcos (3), Ana Arruabarrena (3) & Leonardo S. Boiteux (1) (1) National Center for Vegetable Crops Research (CNPH)/Embrapa Hortaliças, CP 0218, 70359-970 Brasília-DF, Brazil; (2) Departaments of Plant Pathology and Cell Biology, University of Brasilia (UnB), 70910-900, Brasília-DF, Brazil; (3) National Agricultural Research Institute (INIA), Estación Experimental INIA Salto Grande, CP50000, Salto, Uruguay. The most important breeding source for Tospovirus resistance reported in tomatoes thus far is the Sw-5 locus, which was introgressed into cultivated Solanum lycopersicum L. accessions from an unidentified S. peruvianum L. accession. This locus contains at least five paralogues (denoted Sw-5a through Sw-5e), of which Sw-5bis the effective copy involved with the resistance response to distinct Tospovirus species. A polymorphic amplicon was found encompassing a sequence region of the functional Sw-5b gene, resulting in a specific, co-dominant polymorphism between germplasm accessions with and without this copy. The objective of the present work was to evaluate the conservation of this Sw-5b-specific polymorphism in a range of wild tomato species and breeding lines aiming to identify potential allelic variation of the locus in accessions of Solanum (section Lycopersicon) germplasm. A subset of accessions previously identified as having wide spectrum resistance against four Tospovirus species (Tomato spotted wilt virus, Tomato chlorotic spot virus, Groundnut ringspot virus, and Chrysanthemum stem necrosis virus) was also included in the evaluation. Three distinct amplicon patterns were observed with two of them being associated with susceptible accessions. Distinct insertion/deletion events were associated with these amplicon size differences. These indels generated multiple allele variants in the wild species. However, minor sequence variation was observed among S. peruvianum accessions, thus representing distinct Sw-5b alleles. The only exception was S. peruvianum ‘PI 128660’,which individual plants displayed either one SNP or they were 100% identical to the original Sw-5b gene.

106 Poster Presentations, Informal Discussions, and Reception Luciana is a Laurence Mound Fellow

TSWV resistance-breaking isolates infecting pepper in Argentina L. Ferrand (1,3), M.M. Severo de Almeida (2), A. F. Orílio (2), M. L. García (3), P. A. Balatti (1), R.O. Resende (2) & E. Dal Bó (1) [email protected] (1) Centro de investigaciones de Fitopatología, Universidad Nacional de La Plata, CIC, Buenos Aires, Argentina; (2) Dpt. de Biología Celular, Universidade de Brasília, Brasil; (3) Instituto de Biotecnología y Biología molecular, Universidad Nacional de La Plata-CONICET, Buenos Aires, Argentina. The disease caused by Tomato spotted wilt virus (TSWV) is endemic on vegetable crops in Buenos Aires green belt, the most important vegetable production area in Argentina. Half of the greenhouse surface planted with sweet pepper (Capsicum annuum L.) is located in this area. In November 2014, peppers harboring the Tsw resistance gene showing typical tospovirus symptoms were observed. In early spring and by midsummer, symptoms appeared 20 days after transplanting in 100% of the plants in most of the greenhouses, causing important economic losses. Samples from symptomatic plants were analyzed by ELISA with antisera against TSWV, Groundnut ringspot virus (GRSV),Tomato chlorotic spot virus (TCSV), Potato virus Y (PVY),Cucumber mosaic virus (CMV) and Tobacco mosaic virus(TMV). All samples were positive for TSWV and negative for the rest. Presence of TSWV was confirmed by RT-PCR with primers designed from conserved region of the N gene and by molecular hybridization using DIG-labeled RNA single stranded probe complementary to the N gene. To test the ability to overcome the resistance, this greenhouse-isolate named TSWV-A2, was mechanically transmitted to commercial pepper cultivars carrying the Tsw gene. After two weeks, all plants were systemically infected showing the typical symptoms of TSWV infection. RT-PCR from total RNA extracted from symptomatic leaves, confirmed that TSWV-A2 was able to overcome Tsw gene resistance. Further to amplify the complete N gene of TSWV-A2 isolate, a new RT-PCR was carried out. A specific 800 bp product was cloned and sequenced. Comparison of this sequence with TSWV sequences in the GenBank showed the closest identity (99% in the nucleotide and in the amino acid sequence) to the isolates EF195230 and EF195224 from South Korea. In addition, TSWV-A2 showed to share common characteristics with the resistant- breaking isolates infecting resistant sweet pepper cultivars, previously reported in Italy and Spain.

107 Poster Presentations, Informal Discussions, and Reception Kory is a TTEN Fellow Use of overwintering thrips species (Frankliniella sp.) and TSWV-susceptible weed species to detect the efficacy and timing of initial inoculum spread to susceptible crop species. K. Herrington (1), B. Williams (2), and R.C. Kemerait (3) (1)TTEN Student, Abraham Baldwin Agricultural College, (2) Graduate Research Assistant, Department of Plant Pathology, The University of Georgia, Tifton, GA 31793, and (3) Professor, Department of Plant Pathology, The University of Georgia, Tifton, GA 31793. In this study, different species of winter weeds susceptible to tomato spotted wilt virus (TSWV) were sampled and assayed for presence of the virus using ELISA. Objectives were to determine the likelihood of each species to serve as a viral reservoir and a subsequent source of inoculum for spread to agricultural crops. Also, it was to be determined if infection frequency was affected by field cropping history. It was hypothesized that TSWV-susceptible, over-wintering weed species have different infection frequencies and that field history significantly affects infection frequency. Samples of four different weed species (Stellaria media, Geranium carolinianum, Coronopus didymus, Oenothera laciniata) were collected biweekly beginning in Jan 2015 at 13 locations and tested for infection using ELISA. Crops planted in 2014 included cotton, corn, peanut and tobacco. It was hypothesized that weed species found in a field where the previous year’s crop was susceptible to TSWV would have a higher percentage of TSWV incidence, thus an increased risk for local TSWV infection. Whole plant weed species were collected biweekly from each location. Enzyme-linked immunosorbent assays (ELISA) using tissue and root composites were performed to detect TSWV in individual plant samples. TSWV has only been found to date in very few samples and thus far, field history does not seem to affect incidence. Information from this study will be useful in the determining the intensity of weed management through the winter months to reduce the amount of available TSWV inoculum.

108 Poster Presentations, Informal Discussions, and Reception Romana is a Laurence Mound Fellow

An update on tospoviruses in Pakistan Romana Iftikhar (1), S.V. Ramesh (2), M. Ashfaq (3), and H.R. Pappu (2) (1) National Institute for Biotechnology and Genetic Engineering, Faisalabad, Pakistan Institute of Engineering and Applied Sciences (PIEAS), Nilore, Islamabad 45650, Pakistan; (2) Department of Plant Pathology, Washington State University, Pullman, WA, USA; (3) Biodiversity Institute of Ontario, University of Guelph, Guelph, ON N1G 2W1, Canada Tospoviruses exhibit a great degree of diversity in Asia. Within the Indian subcontinent, several tospoviruses were reported mainly from India. Little or no information is available from Pakistan with the exception of one report of occurrence of Groundnut bud necrosis virus by ELISA. A survey to determine the incidence of selected tospoviruses was carried out in onion-growing regions that included thirteen administrative districts in the Punjab province of Pakistan. Plants with symptoms suggestive of Iris yellow spot virus(IYSV) infection were collected and tested for the presence of the virus by ELISA and RT-PCR. Sequence analysis of RT-PCR amplified nucleocapsid (N) gene confirmed IYSV infection of onion. This was the first report of IYSV infecting onion in Pakistan. A global genetic analysis of known IYSV nucleocapsid gene (N gene) sequences was carried out to determine the comparative population structure, spatial and temporal dynamics with reference to its genetic diversity and evolution. A total of 98 complete N gene sequences available in GenBank and reported from 23 countries were characterized by in silico RFLP analysis. Based on RFLP, 94% of the isolates could be grouped into NL or BR types while the rest belonged to neither group. The relative proportion of NL and BR types was 46% and 48%, respectively. A temporal shift in the IYSV genotypes with a greater incremental incidence of IYSVBR was found over IYSVNL before 2005 compared to after 2005. The virus population had at least one evolutionarily significant recombination event, involving IYSVBR and IYSVNL. Genetic differentiation studies showed inherent differentiation and infrequent gene flow between IYSVBR and IYSVNL genotypes corroborating the geographical confinement of these genotypes.

109 Poster Presentations, Informal Discussions, and Reception Complete nucleotide sequence of Iris yellow spot virus Japanese isolate on lisianthus, Eustoma grandiflorum. S. Kagiwada (1), K. Endo (1), H. Kanzaki (1), T. Ono (2), & H. Hoshi (2) (1) Faculty of Bioscience and Applied Chemistry, Hosei University, 3-7-2 Kajino-cho, Koganei, Tokyo 184-8584, Japan; (2) Tokyo Metropolitan Agriculture and Forestry Research Center, 3-8- 1 Fujimi-cho, Tachikawa, Tokyo 190-0013, Japan In July 2011, lisianthus, Eustoma grandiflorum, exhibiting characteristic symptoms of necrotic spot or ring spot on the leaves in Tokyo, Japan was observed. The plant samples were tested by multiplex RT-PCR to detect five tospoviruses. As a result, Iris yellow spot virus (IYSV) was detected in the samples, and the partial sequences of nucleocapsid (N) gene shared 94-98% identities with that of American, Texas isolate on onion. There are two representative IYSV isolates; the Netherlands isolate and the Brazilian isolate (Pozzer et al. 1999: Plant Dis 83: 345- 350), and the nucleotide sequences of N gene in the Tokyo isolate indicated that they were closely related to Brazilian isolate. Although a lot of sequences of N gene of IYSV isolates in the world have been reported, a few complete sequences of S, M and L segments of the genome have been analyzed. The complete nucleotide sequence of a Japanese isolate on lisianthus was determined using primers based on the reported sequences. The full length nucleotide sequences of S and M segments of Japanese isolate shared 87% identities with those of Netherlands isolate, and M and L segments shared 97% and 95% identities with those of American isolates, belonging to Brazilian isolate group, respectively. This report is the first sequences of complete set of a IYSV isolate genome.

110 Poster Presentations, Informal Discussions, and Reception Tomato spotted wilt virus nucleocapsid protein forms an asymmetric trimeric ring structure, and provides insight into a novel genome encapsidation mechanism K. Komoda (1)(2), M Narita (1), K. Yamashita (1), I. Tanaka (1), & M. Yao (1) (1) Graduate School of Life Sciences, Hokkaido University, Sapporo, Hokkaido, 060-0810, Japan (2) Present Address: Department of Plant Pathology, Iowa state University, Ames, IA 50011, USA Like all other negative-strand RNA viruses, Tomato spotted wilt virus (TSWV) nucleocapsid (N) protein plays very important roles in their life cycle. N protein encapsidates to protect genomic RNAs and forms a viral ribonucleoprotein complex (vRNP) with an RNA-dependent RNA polymerase. We elucidated the crystal structure of N protein from TSWV, the first example of the structure of Tospovirus N protein. Protomer of TSWV N protein consists of three domains (N arm, C arm, and core domain). Unlike N proteins of other negative-strand RNA viruses, TSWV N protein forms an asymmetric trimeric ring. To form the trimeric ring, N and C arms of N protein interact with core domains of two adjacent N proteins. By solving crystal structures of TSWV N protein with nucleic acids (DNA- and RNA-binding form), we showed that an inner cleft of the asymmetric trimeric ring is an RNA-binding site. The structures of apo, DNA- and RNA-binding form showed that one N protein trimer interacts with another trimer using the terminus of C arm of one N protein of each trimer. Based on these observations, we propose a novel model that the building unit of TSWV vRNP is an asymmetric trimeric ring, and that RNA encapsidation occurs without dissociation of trimeric rings.

111 Poster Presentations, Informal Discussions, and Reception Stephanie is a Laurence Mound Fellow

Sternal glands in important thrips species S. Krueger1, S. Subramanian2, S. Niassy2, G. Moritz1 1 Institute of Biology, Martin-Luther-University Halle- Wittenberg, Heinrich-Damerow- Str. 4, 06120 Halle, Germany; 2 Division Plant Health, International Centre of Insect Physiology and Ecology (icipe), Duduville (PO) Po. Box 30772-00100, Kasarani, Nairobi, Kenya Extensive knowledge about species morphology and anatomy is a key prerequisite in understanding reproduction behavior and strategy. Many males of thrips species have sternal glands on sternite II/III to VI/VII. Area of releasing (areae porosae, pore plate) vary in shape, size and distribution even between species of one genus. Therefore pore structures are important features in identification of male thrips, especially within the Thripinae. Glands probably produce sex- or aggregation pheromones. However sexual aggregations are not reported in Echinothrips americanus, with known presence of large pore plates, while we observed aggregations in Megalurothrips sjostedti with reported absence of sternal pores. With light- and electron microscopic techniques sternal glands of different species were examined. Based on this new data distribution and function of sternal glands are further discussed.

112 Poster Presentations, Informal Discussions, and Reception Michelle is a TTEN Fellow

Need a Drink? Western Flower Thrips Feeding From the Xylem M. Kwok, C. Ann Stafford-Banks, M. Yang, and D. Ullman Department of Entomology, University of California, Davis, One Shields Ave. Davis, Ca 95616, USA Frankliniella occidentalis (Western flower thrips) is a major agricultural pest with an extensive host range of over 1,000 plant species. Electrical penetration graph (EPG) recordings of thrips feeding show that thrips engage in several different feeding behaviors including sustained ingestion from an unknown source during long ingestion. It has been assumed that western flower thrips only feed from epidermal and mesophyl tissues; however, the sustained ingestion we observed with EPG suggested vascular feeding. Furthermore, we observed that WFT prefers low sucrose concentrations in feeding solutions. Based on these data, we hypothesized that thrips may enhance their water intake by ingesting from the xylem. To test this hypothesis, we infiltrated Emelia sonchifolia leaf material with xylem-limited, fluorescently labeled (fluorescene) dextran. Thrips were allowed to feed on the fluorescene infiltrated plant material and then compared to thrips that fed on non-labelled plant material. A fluorescent dissecting scope was used to examine thrips gut contents post feeding for the presence of flourescene. We found evidence of flourescene in the intestinal tract of thrips feeding on treated plants indicating that these thrips fed in the xylem. No fluorescence was observed in the intestines of WFT fed on non-treated plants. Our future plans including using EPG of flourescene treated plants to determine if long ingestion waveforms indicate xylem feeding. These findings indicate that WFT may include vascular feeding in their repertoire, which may be more complex than previously thought.

113 Poster Presentations, Informal Discussions, and Reception Elison is a Laurence Mound Fellow

Towards an updated checklist of pest thrips in Brazil E.F.B. Lima (1) & R.A. Zucchi (1) (1) Departamento de Entomologia e Acarologia, Escola Superior de Agricultura “Luiz de Queiroz”, Universidade de São Paulo, Piracicaba, SP, 13418-900, Brazil Out of more than 550 thrips species recorded in Brazil, 38 are considered pests of cultivated plants. Twenty-two species are native(1), and most of the exotic pests(2) was introduced since the 1990’s, when vegetal transportation has increased in the Country. Five species are vectors of Tospovirus(3). Thrips attack 61 plant species, among agricultural, horticultural, ornamental and forests. In spite of the large number of pest species, not all causes significant economic losses and sometimes have restricted distribution, mainly in south and southeast, but probably because thrips fauna is poorly known in northward Brazilian regions. A checklist of the pest thrips in Brazil is extended from the 19 species listed by Monteiro et al. (1999) with data gathered from literature and two new records(4): Aurantothrips orchidearum (Bondar)1; Bradinothrips musae (Hood)1;Caliothrips phaseoli (Hood)1; Chaetanaphothrips orchidii(Moulton)2; Corynothrips stenopterus Williams1; Echinothripsaff. mexicanus; Danothrips trifasciatus Sakimura2;Dendrothripoides innoxius (Karny), Dolichothrips indicus(Hood)2,4; Elixothrips brevisetis (Bagnall)2; Enneothrips flavens Moulton1; Frankliniella brevicaulis Hood1; F. condeiJohn1; F. gemina Bagnall1; F. insularis (Franklin)1; F. occidentalis (Pergande)2,3; F. parvula Hood1,4, F. schultzei(Trybom)1,3; F. williamsi Hood1; F. zucchini Nakahara & Monteiro1,3; Gynaikothrips ficorum (Marchal)2; G. uzeli(Zimmermann)2; Heliothrips haemorrhoidalis (Bouché)1;Hercinothrips bicinctus (Bagnall)1; Holopothrips ananasiLima1; Hol. claritibialis Cavalleri & Kaminski1; Hoodothrips lineatus (Hood)1; Liothrips adisi Zur Strassen1;Neohydatothrips samayunkur (Kudô)2; Pseudophilothrips ichini (Hood)1; Psydrothrips kewi Palmer & Mound1;Retithrips syriacus Mayet2; Scirtothrips manihoti (Bondar)1;Selenothrips rubrocinctus (Giard)2; Stenchaetothrips minutus(Deventer)2; Thrips palmi Karny2,3; Thrips simplex (Morison)2and Thrips tabaci Lindeman2,3. Dolichothrips indicus was collected from Acacia sp. in São Luís, Maranhão and Ochroma pyramidale in Teresina, Piauí (northeast), whereas F. parvula was collected from Musa ornata in Manaus, Amazonas (north). For thirteen other species recorded in Brazil, and known as pests abroad, no damages were observed in Brazil.

114 Poster Presentations, Informal Discussions, and Reception Diversity of thrips (Thysanoptera, Thripidae) associated with the cultivation of melon (Cucumis melo) in Northeastern Brazil A. S. de Carvalho (1), E. M. da Costa (2), E. L. de Araujo (2) & M. G. A. de Lima (3) (1) Laboratory of Phitossanity, Federal Institute of Education, Science and Technology of Rio Grande do Norte (IFRN)/Campus Ipanguaçu, RN 118, Base Física, Ipanguaçu, RN, Brazil. (2) Laboratory of Entomology Applied Federal Rural University of (UFERSA), BR 110, Km 47, Presidente Costa e Silva, CEP 59.625-900, Mossoró, RN, Brazil. (3) Laboratory of Ecology, Departament of Biology, University of Ceará (UECE)/Campus Itaperi, Av. Paranjana, 1700, CEP 60.740-903, Fortaleza, CE, Brazil. Thrips are cosmopolitan pests that cause damage the many diverse cultures of economic importance. However, there are few studies about the diversity of thrips in the melon crop, especially in the Semiarid in Northeast of Brazil. The objective of this study was to identify the species of thrips found in a commercial melon production area in the state of Rio Grande do Norte, a region that stands out as the main producer of melon of the Brazil. Collections were performed during the crop cycle, in the period between August and September 2012, in a commercial area of production of melon with 2.5 ha, in the municipality of Baraúna (4° 59' 06.40"S and 37° 33' 13.90" W), RN, using 20 Moericke traps installed in the center of the area and fulfilled with 400 ml of water + detergent solution (2%).The collected specimens were sent to the Ecology Laboratory of the State University of Ceará (UECE) held assemblies microscope slides, the identifications according to Mound & Marullo (1996) and Mound & Kibby (1998) and were deposited in the entomologic collection of Ecology Laboratory of UECE, Department of Biology. Were captured a total of 62 adult females, where 54 were tomato thrips or common thrips flower, Frankliniella schultzei (Thripinae), most of the dark form (47) (brown) and few of clear form (7) (yellow), the others being goldtipped tubular thrips, Haplothrips gowdeyi (Phlaeothripitidae) (4), South American flower thrips, F. insularis (Thripinae) (2), Mexican grass thrips, Arorathrips mexicanus (Thripinae) (1) e American bean thrips, Caliothrips phaseoli (Panchaetothripinae) (1).This work was the first record of occurrence of these species in agroecosystems of melons in Rio Grande do Norte Semiarid, Brazil. Despite of the Thysanoptera diversity found, was not captured the melon thrips, Thrips palmi (Thripinae), causers of severe damage to the melon crop.

115 Poster Presentations, Informal Discussions, and Reception Species of Thrips (Insecta: Thysanoptera: Thripidae) Associated with Vegetable Cultivation in Baturité Mountain Range – Ceará/Brazil. Maria Goretti Araújo de Lima (1), Isabelly Maria Barros de Lima (2), Marcos Adelino Almeida Filho (3) & Rebeca Veras Araújo (4). (1)(2)(3)(4) Health Sciences Center CCS, State University of Ceará – UECE, Dr. Silas Munguba Ave., 1700, Itaperi Campus, Fortaleza-CE. The mountain range of Baturité is one of the main vegetable producers in the State of Ceará, Brazil. This agricultural activity has an important role in generating income for local farmers. One of the obstacles to vegetable production is the occurrence of thrips that affect those crops directly, with damage to those plants, and the transmission of viruses. In order to choose and adopt control strategies of such pest it is essential to understand the species of thrips that affect those vegetables. The aim of this research was to identify the species of thrips associated with vegetable cultivation. The samples were taken from the flowers and leaves, which were lightly tapped over a white tray to remove the thrips from 8 farms in the municipalities of Aratuba (S 4º 25’’ 06’ W 39º 02’’ 42’, 830m), and Mulungu (S 4º 18’’ 20’ W 38º 59’’ 47’, 790m) from June to December of 2014, in the mountain range of Baturité – CE. A total of 489 adult subjects (males and females) were collected, which were identified in association with the following vegetables: Arorathrips mexicanus(Crawford) in lettuce (n=1); Frankliniella insularis (Franklin) in green pepper (n=2), pepper (n=1), and tomato (n=5); F. occidentalis (Pergande) in lettuce (n=351), tomato (n=36), green pepper (n=15), and okra (n=1); F. schultzei (Trybom) in lettuce (n=7), tomato (n=10), green pepper (n=4), pepper (n=6), and okra (n=5);Haplothrips gowdeyi (Franklin) in lettuce (n=2), and pepper (n=1); Microcephalothrips abdominalis(Crawford) in tomato (n=1); Thrips tabaci (Lindeman), in scallion (n=35), broccoli (n=5), and pepper (n=1). It was concluded that, among the species found, F. occidentalis was the most abundant species mainly in the cultivation of lettuce. This preliminary result shall contribute to the correct adoption of control measures and appropriate management of pests in those vegetables.

116 Poster Presentations, Informal Discussions, and Reception A five-year survey of tospoviruses infecting vegetable crops in main producing regions of Brazil Mirtes Freitas Lima (1), Camila Costa Barriolli (2), Maria Geane Fontes (3), Leonardo Silva Boiteux (1), Julyane Marques Almeida (4) (1) Embrapa Hortaliças, Cx. Postal 218, Brasília-DF 70351-970, Brazil; (2) Universidade Paulista, SGAS Q. 913 – Conj.B, 70390-130 Brasília-DF, Brazil; (3) Universidade de Brasília, Campus Universitário Darcy Ribeiro, Av. L3 Norte, 70.910-900, Brasília-DF, Brazil; (4) Universidade Católica, QS.07 – L.01 – EPCT, Águas Claras, Brasília-DF, 71966-700, Brazil. The "spotted wilt" disease caused by tospoviruses (genus Tospovirus; family Bunyaviridae) transmitted by thrips (Thysanoptera: Thripidae) is a major cause of economic losses in many vegetable crops in Brazil, including tomato, peppers, and lettuce. The present work describes a five-year survey (2010–2014) of tospoviruses infecting these vegetable crops under Brazilian conditions. A total of 318 samples from tomato (Solanum lycopersicon L. – 285), lettuce (Lactuca sativa L. – 14), pepper (Capsicum L. species – 11), gilo (Solanum aethiopicum var. gilo Raddi – 5) and pigweed (Amaranthus L. species – 5) were collected from plants displaying typical tospovirus symptoms. Samples were obtained across 13 counties in four major vegetable-producing regions (Goiás, Minas Gerais, and São Paulo States and, in the Federal District). Viral infection was detected by two-step RT-PCR using total RNA as template. The primer J13 (encompassing the eight terminal nucleotides conserved in all tospovirus RNA termini) was used in the reverse transcription step, and the primer set BR060 and BR065 (targeting the non-translated region from the 3' terminal portion of the S RNA and the protein N- coding gene, respectively) was used in the conventional PCR assays. Tospoviruses were detected in 79.6% (253) of the samples, encompassing all host plants. A set of 184 randomly selected samples (175 from tomato and nine from lettuce) were tested using species-specific primers for Tomato spotted wilt virus – TSWV (TSWV722/TSWV23) and Groundnut ringspot virus – GRSV (GRSVNv/GRSVNvc). GRSV was identified in 97, while TSWV was detected in 66 samples, indicating the prevalence of these two tospovirus species. These data reinforced the economic importance and the widespread incidence of tospovirus infection in vegetable crops in Brazil and the need of identifying and/or incorporating resistance factors into commercial cultivars and hybrids.

117 Poster Presentations, Informal Discussions, and Reception Current status of Tospoviruses (F: Bunyaviridae) infecting vegetables in India *B. Mahesha, A.B.Rai, B. Singh and P.S.Naik Division of Crop Protection, Plant Virology Laboratory, Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India, 221305. *e-mail: [email protected] Tospoviruses (F: Bunyaviridae) are identified as second most problematic plant viruses in the world next to Tomato leaf curl viruses (G: Begomovirus, F: Geminiviridae). Till date there are 28 type species of tospoviruses naturally transmitting by 14 different thrips species (F: Thripidae, O: Thysanoptera) are reported across the globe. So far, In India five tospoviruses are reported; namely, Capsicum chlorosis virus (CaCV), Groundnut bud necrosis virus (GBNV), Iris yellow spot virus (IYSV), Groundnut yellow spot virus(GYSV) and Watermelon bud necrosis virus (WBNV); among all the five viruses, GBNV is having wide host range that infects field crops, horticultural and ornamental crops compared to others. CaCV is most predominant in chilli (Capsicum annum L. and Capsicum frutescens L); IYSV is quite common in onion and garlic. But GBNV (wide host range), GYSV (On groundnut / peanut) and WBNV (on watermelon) are most commonly infecting tospoviruses. There is an economic yield losses ranging from 30-100% in fruits, ornamentals and vegetable crops in India. The survey data of Varanasi and nearby villages (Farm fields) reveals the same statistics. The severity of the disease is picks up in kharrif months and aggravates in spring season/ summer months (Jan-March). Early invasion of tospoviruses leads to 80-100% yield and quality losses. Survey data reveals that thrips became resistant to major and even broad spectrum insecticides at our region (Varanasi). Over and multiple insecticidal sprays might be the reason; but that is also not producing any impacts in the management of either tospoviruses or thrips species. We have diagnosed and characterized these viruses through serological, molecular and biological techniques. Unfortunately, farmers and most of the stake holders are not aware about either pathogen/vector or its management. Hence, there is an urgent need to capture and strengthen the tospovirus research in India. Key words: Tospoviruses, Epidemiology, Diagnosis and Management.

118 Poster Presentations, Informal Discussions, and Reception Molecular and serological diagnostics of Capsicum chlorosis virus (CaCV) (F: Bunyaviridae, G: Tospovirus) infections on chilli (Capsicum annum L.) *B. Mahesha (1), V. Venkataravanappa (2), A.B.Rai (3), B. Singh (4) and P.S.Naik (5) Division of Crop Protection, Plant Virology Laboratory, Indian Institute of Vegetable Research, Varanasi, Uttar Pradesh, India, 221305. *e-mail: [email protected] Capsicum chlorosis virus (CaCV) (F: Bunyaviridae, G:Tospovirus) is emerging as most problematic viruses next to leaf curl viruses (F: Geminiviridae, G: Begomovirus) by affecting the chilli production both in India and the world. These viruses are naturally transmitting by thrips species (O: Thysanoptera, F: Thripidae). In the current investigation, we have detected and confirmed the symptomatic CaCV infections on chilli/pepper through serological and molecular techniques. However, we have conducted the direct antigen coating Enzyme Linked Immuno Sorbent Assay (DAC-ELISA),Dot Immuno Binding Assay (DIBA) with virus specific antibody (CaCV) and confirmed the tospovirus (CaCV) infections; further, samples were subjected for molecular confirmation through reverse transcriptase polymerase chain reaction (RT-PCR) with CaCV gene specific primer. The RT-PCR results released the amplicon of 770bp. This confirms the first report of CaCV infections at Varanasi, India. Key words: Chilli, Tospovirus, Diagnosis, Serology, RT-PCR.

119 Poster Presentations, Informal Discussions, and Reception Management of Tospovirus (Peanut Bud Necrosis Virus) by over-expressing PBNV-NP gene in tomato (Solanum lycopersicon L.): A Successful antiviral strategy *B. Mahesha (1), K.T. Rangaswamy (2), +Rohini Sreevathsa (3) and M. Udaya Kumar (4) Authors (1) and 2): Department of Plant Pathology, Gandhi Krishi Vignana Kendra (G.K.V.K.), University of Agricultural Sciences (U.A.S.), Bangalore, India, 560065. (*Current address: Scientist (Plant Pathology), Division of Crop Protection, Plant Virology Laboratory, Indian Institute of Vegetable Research, Varanasi, India, 221305. e-mail:[email protected]; Authors (3) and (4): Department of Crop Physiology, G.K.V.K., U.A.S., Bangalore, 560065. (+Current address: Senior Scientist, National Research Center for Plant Bio-Technology, New Delhi, 110012). Tospoviruses (F: Bunyaviridae) are emerging as one of the major problems in horticultural crop production both in India and the world. Till date there are 28 type species of tospoviruses are existing in the world; but under Indian conditions Groundnut (=Peanut) bud necrosis virus(GBNV/PBNV) is the predominant one; naturally transmitted by Thrips (F: Thripidae and O: Thysanoptera) with a yield losses of 30-100%. Due to lack of antiviral drugs and development of insecticidal resistance by thrips; management of both virus and vector is became one of the major constraints in tomato production (Solanum lycopersicon L.). Therefore, we made an attempt to develop reliable and robust management strategy of the virus by over-expressing the PBNV nucleo-capsid protein gene (PBNV-NP) in tomato (NSPLT-1) by Agrobacterium- mediated in planta method. Further, putative plants were subjected for serological (DAC- ELISA), molecular (PCR, Nested PCR, RNA, cDNA and RT-PCR analysis) and biological (Sap/mechanical transmission) characterization. The results confirmed the over-expression of protein (PBNV-NP gene) in tomato plants; which confers the resistance against PBNV compared to wild types (Control). To our knowledge PBNV management in tomato by over-expressing PBNV-NP gene through in-planta method is first report from India. Key words: Tomato, Tospovirus, PBNV-NP gene, Management.

120 Poster Presentations, Informal Discussions, and Reception Duong is a Laurence Mound Fellow

Polyploidy versus endosymbionts in obligately thelytokous Heliothrips haemorrhoidalis – Duong T Nguyen (1), Robert N Spooner-Hart (1, 2), & Markus Riegler (1) (1) Hawkesbury Institute for the Environment, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia; (2) School of Science and Health, University of Western Sydney, Locked Bag 1797, Penrith, NSW 2751, Australia Thelytoky, the parthenogenetic development of females, has independently evolved in several insect orders yet the study of its mechanisms has so far mostly focused on haplodiploid Hymenoptera, while alternative mechanisms of thelytoky such as polyploidy are far less understood. In haplodiploid insects, thelytoky can be encoded in their genomes, or induced by maternally inherited bacteria such as Wolbachia or Cardinium. Microbially facilitated thelytoky usually results in complete homozygosity due to gamete duplication and can be reverted into arrhenotoky, the parthenogenetic development of males, through treatment with antibiotics. In contrast, genetically encoded thelytoky cannot be removed and may result in conservation of heterozygosity due to gamete fusion. We probed the obligate thelytoky of the greenhouse thrips, Heliothrips haemorrhoidalis (Bouché) (Thripidae), a significant cosmopolitan pest and a model species of thelytoky in the haplodiploid insect order Thysanoptera. Earlier studies suggested terminal fusion as a mechanism for thelytoky in this species, while another study reported presence of Wolbachia; subsequently it was speculated that Wolbachia may play a role in this thrips’ thelytokous reproduction. Using PCR and sequence analysis, we demonstrated that global population samples of H. haemorrhoidalis were not infected with Wolbachia, Cardinium or any other known bacterial reproductive manipulators. In addition, antibiotic treatment of this thrips did not result in male production. Some individuals carried two different alleles in two nuclear loci, histone 3 andelongation factor 1 alpha, suggesting heterozygosity. However, the majority of individuals had three different alleles, indicating that they were polyploid. This suggests that polyploidy may be involved in the thelytokous reproduction of this thrips species. Genetic diversity across both nuclear loci was low across all populations, and was absent from mitochondrial cytochrome oxidase I, indicating that this species had experienced genetic bottlenecks, perhaps due to its invasion biology or a switch to thelytoky.

121 Poster Presentations, Informal Discussions, and Reception Jonathan is a Laurence Mound Fellow

Annotation of V-ATPase gene family members in Frankliniella occidentalis (Pergande) J. E. Oliver (1), A. Whitfield (1), & D. Rotenberg (1). (1) Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center, Manhattan, KS 66506, USA. The genome of the western flower thrips, Frankliniella occidentalis (Pergande), was sequenced by the Baylor College of Medicine – Human Genome Sequencing Center (http://www.hgsc.bcm.tmc.edu/content/i5k-western-flower-thrips) as part of a pilot study for an international initiative to sequence 5,000 arthropod species (i5k). In the current assembly, there are 415 Mb of sequence with 17,553 predicted genes distributed across 6,265 scaffolds. Annotation of this genome is currently underway, and as the first genomic resource available for the insect order Thysanoptera it is expected to provide valuable tools for thrips research. To examine the usefulness of the genome assembly, a search was carried out for members of the highly conserved vacuolar ATP synthase (V-ATPase) multigene family within the F. occidentalis genome assembly. The V-ATPase family of genes code for the individual subunits of the multi- subunit vacuolar ATPase complex, which functions as an ATP-driven proton pump in a wide range of organisms to acidify intracellular compartments and the extracellular environment. Nine members of the V-ATPase multigene family were identified and manually-annotated for the i5K F. occidentalis genome housed at the National Agricultural Library i5K Workspace using the Web Apollo interface to view predicted gene models within the original assembly and Roche- 454 Newbler-assembled contigs previously generated from a cDNA library prepared from isolated F. occidentalis mRNA. Identified members of this family were found to be present in single copies within the assembly, with the exception of V-ATPase subunit c, for which three copies were identified, each located on a different scaffold. Previous work in our laboratory with F. occidentalis documented that silencing V-ATPase subunit B resulted in decreased fertility and increased mortality, and the annotation of other members of this gene family will enable additional functional studies of thrips genes. Overall, this work illustrates that these new genomic tools can be utilized for gene annotation and will undoubtedly open new avenues for F. occidentalis research.

122 Poster Presentations, Informal Discussions, and Reception Characterization of the Transcriptional Activity of Tomato spotted wilt virus RNAs G. Raikhy (1), Y. Zhai (1), N. Mitter (2) & H.R. Pappu (1) (1) Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA; (2) Queensland Alliance for Agriculture and Food Innovation, the University of Queensland, QLD, Australia. Tomato spotted wilt virus, the type member of the tospovirus group, is a cosmopolitan virus with a wide host range that includes many crops and weeds. The genome consists of three single- stranded RNAs. The large RNA is in negative sense, while the medium (M) and small (S) RNAs have ambisense genome organization. The RNAs have varying lengths of un-translated regions (UTR). The promoter elements that drive the expression of various viral genes of TSWV are poorly understood. Sequence analysis of these UTRs showed the presence of the TATA box and the CAAT box - signatures of promoters. Using GUS as a reporter gene, we used a transient assay to identify the presence of promoter elements in the 5’ and 3’ UTRs of the M and S RNAs. The 5’ and 3’ UTRs of each RNA were separately cloned in virus genome sense and reverse complementary sense orientations upstream of a promoter-less GUS vector and the constructs were agroinfiltrated into Nicotiana benthamiana plants. GUS expression was quantified. Initial results revealed that out of eight UTR constructs tested, three showed significantly higher GUS expression compared to a Cauliflower mosaic virus (CaMV) 35S-driven GUS construct. These included one UTR construct, in viral sense from the S RNA, and two from M RNA - one each in viral and viral complementary sense, respectively. Compared to the CaMV 35S promoter, the highest GUS activity was found for the UTR upstream of the nucleocapsid gene of S RNA followed by the UTR regions of M RNA. Ongoing work is focused on dissecting regions to identify the critical elements within these UTRs.

123 Poster Presentations, Informal Discussions, and Reception Evaluation of Acibenzolar-S-Methyl-Induced Resistance against Iris yellow spot virus D. Tripathi, & H.R. Pappu Department of Plant Pathology, Washington State University, Pullman, WA 99164, USA Acibenzolar-S-Methyl (ASM) is a functional analog of salicylic acid (SA) that activates local and systemic acquired resistance (SAR) responses in plants against a wide variety of pathogens. Iris yellow spot virus (IYSV) is an economically important tospovirus of onion that causes severe economic losses to both bulb and seed crops. IYSV resistant onion cultivars are not available to date and there are limited control options. To explore the possibility of utilizing SAR as a control option, we first used two different hosts of IYSV, Datura stramonium and Nicotiana benthamiana, to study the ability of ASM in triggering SAR against IYSV infection. Quantitative descriptors based on both symptom expression and relative levels of IYSV nucleoprotein and viral small RNA were developed and used to determine the SAR in ASM- and buffer-treated plants. A significant reduction in virus levels in ASM-treated plants was noticed by ELISA and PCR. The level of SAR response was also assessed by measuring the IYSV lesion size and number on the inoculated leaves of ASM-treated plants. ASM-treated plants showed reduced symptoms compared to buffer-treated plants. This study could be useful in potentially developing novel SAR-based options for virus management.

124 Poster Presentations, Informal Discussions, and Reception First Report of Tomato spotted wilt virus in India P. Renukadevi (1), K. Nagendran (1), S. Nakkeeran (1), G. Karthikeyan (1), M. Jawaharlal (1), D. Alice (1), V.G. Malathi (1) & H.R. Pappu (2) (1) Center for Plant Protection Studies, Department of Plant Pathology, Tamil Nadu Agricultural University,Coimbatore-641 003, Tamil Nadu, India; (2) Department of Plant Pathology, Washington State University, Pullman,WA99164-6430, USA A severe necrosis disease was noticed in chrysanthemum (Dendranthema grandiflora Tzvelev)plants grown in the Nilgiris district of Tamil Nadu state, India during August, 2013. The plants showed veinal necrosis and necrotic spots on leaves, browning of flower petals, malformed flowers and stem necrosis. In severe cases, extensive necrosis, complete drying and death of the plants were also observed. Disease incidence was estimated to be 30%. The virus was mechanically transmitted from chrysanthemum plants to cowpea (Vigna unguiculata, cv.C152) and pigweed (Trianthema portulacastrum). Inoculated plants produced local chlorotic concentric rings which later turned to necrotic spots six days post inoculation. Symptoms on cowpea resembled the lesions caused by tospoviruses. Total RNA was extracted from the symptomatic cowpea and chrysanthemum plants and RT-PCR was performed using the Tospovirus degenerate primers corresponding to the L protein gene of tospoviruses. An amplicon of ca. 750 bp was amplified from the symptomatic samples which was sequenced. The sequence had 98% identity with the large RNA of Tomato spotted wilt virus(TSWV). RT-PCR was performed with two more sets of TSWV-specific primer pairs. The 870 bp and 1000 bp PCR products obtained for the N and NSm genes, respectively were sequenced. Sequences obtained had the highest nucleotide identity of 98% with the corresponding regions of TSWV isolates from different hosts and countries. While there are several tospoviruses known to infect economically important crops in India, TSWV is not known to occur. Our finding of TSWV in a widely grown ornamental crop such as chrysanthemum is of great concern considering the wide host range of TSWV. Surveys to determine its incidence and thrips vectors involved needs to be undertaken. To our knowledge this is the first report of TSWV occurring in India.

125 Poster Presentations, Informal Discussions, and Reception Identification of interacting regions of the movement protein and nucleocapsid protein of two distinct tospoviruses D. Tripathi, G. Raikhy and H.R. Pappu Department of Plant Pathology, Washington State University, Pullman, WA99164, USA Some of the tospoviruses are known to exist as mixed infections in the same host plant. When present together in mixed infections, Tomato spotted wilt virus (TSWV) was shown to facilitate the movement of Iris yellow spot virus(IYSV). Recently, we reported that N and NSm proteins of IYSV and TSWV multimerize and interact with each other in infected Nicotiana benthamiana plants. To identify the specific interacting regions of N and NSm proteins, N and NSm genes were divided into N-terminal, middle, and C-terminal regions, amplified, and cloned into LexA-based Yeast-2-hybrid (Y2H) vectors. Y2H results were confirmed by beta- galactosidase assay of interacting partners. Identified interacting N- and C-terminal regions have been reported to play critical roles in virus movement and pathogenesis. Findings of this research could facilitate a better understanding of the virus-virus interactions within host plants and could help develop efficient and durable control strategies for these viruses.

126 Poster Presentations, Informal Discussions, and Reception Transcriptome-wide identification of host genes targeted by a Tospovirus-derived small interfering RNAs reveals host-dependent differential down-regulation S.V. Ramesh(1), S. Williams(2), N. Mitter(2) and H.R. Pappu (1). (1) Department of Plant Pathology, Washington State University, Pullman, WA, USA;(2) Queensland Alliance for Agriculture and Food Innovation, University of Queensland, St. Lucia, QLD, Australia. A subset of highly expressed siRNAs derived from the Tomato spotted wilt virus (TSWV; Tospovirus: Bunyaviridae) TSWV genome was analyzed in silico for their propensity to down regulate tomato transcriptome. VsiRNAs were found to target gamut of host genes involved in basal cellular activities including enzymes, transcription factors, membrane transporters, cytoskeletal proteins. KEGG pathway annotation of targets revealed that they were mapped to secondary metabolites biosynthesis, amino acids, starch and sucrose metabolism, carbon and purine metabolism etc. However protein processing, hormone signaling, plant- pathogen interaction transcripts were the most targeted from the genetic, environmental information processing and organismal systems respectively. qRT-PCR validation of the predicted target transcripts revealed the transcripts are generally down regulated in tomato cv. Marglobe whereas cv Red defender exhibited differential regulation of transcripts involved in defense and abiotic stress signaling. The interactome scenario is a first report on the interaction between Tospovirus genome derived siRNAs and tomato transcripts.

127 Poster Presentations, Informal Discussions, and Reception Derek is a TTEN Fellow

Using the i5K Frankliniella occidentalis genome to assemble a reference transcriptome for analysis of differential gene expression in first-instar larvae during tospovirus infection

D. J. Schneweis (1), A. E. Whitfield (1), & D. Rotenberg (1)

(1) Department of Plant Pathology, Kansas State University, 4024 Throckmorton Plant Sciences Center, Manhattan, KS 66506, USA

The interplay between a persistent-propagative virus and its insect vector is dependent on interactions between both virus and vector molecules. Much is known about Tomato spotted wilt virus proteins and the roles they play in virus replication, infection, and movement within the plant host. While the western flower thrips vector, Frankliniella occidentalis (Pergande), is also undoubtedly an important player with respect to molecular interactions, there is very little known about thrips molecules and their role in the virus life-cycle. Our research provides an investigation of thrips gene expression changes during virus infection. With the advent of the i5K genome for Frankliniella occidentalis, we are now able to explore molecular interactions at a new level. The i5K genome provides, for the first time, a direct reference comparison with transcriptomics data from Frankliniella occidentalis. The genome currently consists of 6,265 scaffolds (N50 = 948.9kb). We constructed a genome-reference-based transcriptome assembly from thrips coding RNA derived from sequence reads of 8 Illumina RNA-Seq libraries from four independent biological replications of TSWV-exposed and non-exposed young larval (L1) thrips. RNAseq reads were aligned to the genome using Bowtie2 as a short read aligner and subsequently Tophat2 to identify splice junctions between exons. Cufflinks2 was exploited to construct a reference-based transcriptome assembly that effectively represented the larval insect stage. Differential gene expression due to TSWV infection was quantified, and 35 differentially- expressed contigs were identified. This necessarily large-scale transcriptomics is one of few methods available to explore host molecules involved in virus-host interactions on a global scale, and it is important for establishing an understanding of the dynamic and complex molecular interaction between TSWV and F. occidentalis. We are excited to report the use of the i5K genome for differential gene expression analysis, and we hope that many more discoveries arise from its creation.

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Poster Presentations, Informal Discussions, and Reception Blake is a TTEN Fellow Tomato spotted wilt management in peanut: reduced effects of production practices on severity of spotted wilt in current variety selections. B.W. Williams (1), R.C. Kemerait, Jr. (2), A.K. Culbreath (2), R.S. Tubbs (3), R. Srinivasan (4) (1) Department of Plant Pathology, University of Georgia, Tifton, GA 31793; (2) Department of Plant Pathology, University of Georgia, Tifton, GA 31793; (3) Department of Crop and Soil Science, University of Georgia, Tifton, GA 31793; (4) Department of Entomology, University of Georgia, Tifton, GA 31793 Tomato spotted wilt virus (TSWV), the causal agent of spotted wilt disease of peanut, is an important virus affecting peanut production in the southeast United States. Current management is targeted at minimizing spotted wilt severity and includes an assortment of production practices including use of varieties with moderate to high TSWV resistance and implementation of chemical and cultural controls. Each factor contributes a component of a disease risk assessment index. At the introduction of the risk index in 1996, the effects of these production practices in reducing spotted wilt were significant; however, as newer levels of host resistance became available, diminishing effects have been observed. The objective of this study was to evaluate the effects of production practices with varieties containing varying levels of TSWV resistance. Using historical data, spotted wilt severity based on chemical and cultural practices was evaluated. Susceptible peanut variety ‘Georgia Green’, moderately resistant ‘Georgia-06G’, and highly resistant ‘Georgia-12Y’ were used in this analysis. In a compiled dataset covering multiple years, the use of phorate significantly reduced spotted wilt severity in cultivars ‘Georgia Green’ (p = <.0001) and ‘Georgia-06G’ (p = 0.0387), but not highly-resistant variety ‘Georgia- 12Y’ (p = 0.9445). Using a similar dataset, twin row pattern significantly reduced spotted wilt severity in ‘Georgia Green’ (p = <.0001) and ‘Georgia-06G’ (p = 0.0003), but no significance was seen in ‘Georgia-12Y’ (p = 0.6432). Results conclude that the effects of cultural and chemical production practices have been reduced with the introduction of greater TSWV- resistant varieties.

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With faculty members located on three campuses (Athens, Tifton, and Griffin), we provide high-quality graduate and undergraduate education; conduct innovative research into the causes, nature, and management of plant disease; and help growers in Georgia and across the nation manage diseases through relevant and dynamic extension and outreach efforts. We are especially proud of our vibrant graduate program. In addition to the research-based MS and PhD degrees in Plant Pathology, an interdisciplinary, non-thesis Master of Plant Protection and Pest Management (MPPPM) is offered. We provide exceptional opportunities for students who are interested in understanding how basic biological principles are applied to the science of plant pathology so diseases can be understood and managed for the benefit of agriculture, industry, science, and society. To learn more about graduate opportunities in Plant Pathology please see: http://plantpath.caes.uga.edu/. We are pleased to provide significant support for the success of the Xth International Symposium on Thysanoptera & Tospoviruses.

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College of Agricultural and Environmental Sciences University of California, Davis

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 For the past three years (2013–2015), UC Davis Distinctions is ranked the No. 1 university in the world for National teaching and research in agriculture and forestry Program ranking  The College of Agricultural and Environmental Agricultural sciences 1 Sciences has more than 5,000 acres, with 3,700 Agriculture / Agronomy 1 devoted to agricultural teaching and research Agriculture and forestry 1 (international ranking)  360 faculty members

Entomology 1  6,600 undergraduate students and 30 majors Food science and nutrition 1 (24% of UC Davis students; 2014 data)

Plant and animal science 1  1,000 graduate students in 45 graduate Soil science 1 programs (25% of UC Davis graduate students) Agricultural economics 2  15 academic departments covering agricultural, Agricultural engineering 2 environmental, and human/social sciences Plant pathology 2 Addressing critical issues Food security and safety UC Davis excellence Eliminating hunger  World-class research facilities Agricultural sustainability

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 Technology transfer Environmental stewardship Climate change  Leaders in biotechnology and genomics Clean energy, air, and water  International programs and partnerships Population growth  Education of top students from California, Healthy families and communities the U.S., and the world Rural revitalization Preventing global disease Contact us Biodiversity Economic growth telephone (530) 752-0108 International development www.caes.ucdavis.edu

May 2015

131 Special Thanks to the Following Individuals and Groups

for Their Generous Contributions

• Agdia, Inc. • American Floral Endowment • California Cut Flower Commission • Citrus Research Board • DuPont Crop Protection • Georgia Tobacco Commission • Kee Kitayama Research Foundation • The Los Angeles Flower Market • Mellano & Company • Mike Mellano Sr • The Peanut Foundation, Inc. • Syngenta • United States Department of Agriculture, National Institute of Food and Agriculture Conference Award 2015-67013-23171 • AFRI NIFA Coordinated Agricultural Project 2012-01785 • University of California Davis, College of Agricultural and Environmental Sciences • The University of Georgia, Plant Pathology Department