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Foundation Plant Services Clean Stock Program

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Foundation Plant Services Clean Stock Program FOUNDATION PLANT SERVICES CLEAN STOCK PROGRAM MAHER AL RWAHNIH DIAGNOSTIC AND RESEARCH LABORATORY DIRECTOR, FOUNDATION PLANT SERVICES Foundation Plant Services (FPS) Mission • Produce, test, maintain and distribute elite disease-tested plant propagation material. • Provide plant importation and quarantine services, virus testing and virus elimination. • Coordinate release of UC- patented varieties. • Link researchers, nurseries, and producers. http://fps.ucdavis.edu Background •Grapevine hosts more viruses than any other crop species in the world •More than 75 known viruses have been reported in Grapevine (mostly from Europe) •Not all the viruses we discover are causing problems Types of Viruses • Nepoviruses • Rugose Wood Viruses • Leafroll Viruses • New Emerging Viruses • Grapevine red blotch virus • Grapevine Pinot gris virus Nepoviruses: “Nematode Transmitted Polyhedral Virus” • Artichoke Italian latent AILV • Arabis mosaic ArMV • Blueberry leaf mottle BBLMV • Bulgarian latent GBLV • Grapevine chrome mosaic GCMV • Grapevine fanleaf GFLV • Grapevine Tunisian ringspot GTRV • Peach rosette mosaic PRMV • Raspberry ringspot RRV • Tobacco ringspot TRSV • Tomato ringspot ToRSV • Tomato black ring TBRV • Strawberry latent ringspot SLRSV GFLV – Yellow mosaic symptoms GFLV – Fruit symptoms Healthy Infected Rugose wood complex (RW) • Rupestris stem pitting • Kober stem grooving • LN33 stem grooving • Corky bark RW affects grapevines on the woody cylinder causing pitting and grooving of the zone underneath the bark. Foveavirus: • Grapevine rupestris stem pitting-associated virus GRSPaV Vitivirus: • Grapevine Virus A GVA • Grapevine Virus B GVB • Grapevine Virus D GVD • Grapevine Virus E GVE • Grapevine Virus F GVF • Grapevine Virus H GVH (recently reported) • Grapevine Virus G GVG (recently reported) • Grapevine Virus J GVJ (in progress) Rupestris Stem Pitting Disease Rupestris stem pitting-associated virus (GRSPaV) • closely related to Apple stem pitting virus Kober Stem Grooving (KSG) Grapevine Virus A (GVA) Corky bark symptoms on LN33 Grapevine Virus B (GVB) Healthy CB-infected Grapevine Leafroll Disease (GLD) • GLRD is one of the most economically damaging and widespread diseases of Vitis vinifera throughout the world. • This disease can cause up to 40% yield losses • All of the GLRaVs can be graft-transmitted, some are also transmitted by mealybugs and soft scale insects. Grapevine leafroll disease symptoms in red-fruited varieties • Red leaves in fall • Leaf margins rolled under • Reduced vigor Cabernet franc infected with GLRaV-3 Leafroll symptoms on white-fruited varieties • Chlorosis • Leaf margins rolled under • Reduced vigor Chardonnay: healthy vs. leafroll-infected Effects of grapevine leafroll virus Reduced berry yield • Reduces sugar • Increases acidity • Delays ripening • Reduces fruit pigments • Reduces yield • Dependent on variety, clone, rootstock, site, year and leafroll type and strain Leafroll-infected ‘white’ Emperor Healthy cv. Emperor Proposed New Taxonomy for the Leafroll Viruses Genus Virus Acronym Closterovirus Grapevine leafroll associated virus 2 GLRaV-2 Ampelovirus Grapevine leafroll associated virus 1 GLRaV-1 (subgroup I) Grapevine leafroll associated virus 3 GLRaV-3 Grapevine leafroll associated virus 13 GLRaV-13 Grapevine leafroll associated virus 4 GLRaV-4 Grapevine leafroll associated virus 4 strain 5 GLRaV-4 strain 5 Ampelovirus (subgroup II) Grapevine leafroll associated virus 4 strain 6 GLRaV-4 strain 6 Grapevine leafroll associated virus 4 strain 9 GLRaV-4 strain 9 Grapevine leafroll associated virus 4 strain Car GLRaV-4 strain Car Velarivirus Grapevine leafroll associated virus 7 GLRaV-7 New Emerging Diseases: Grapevine red blotch disease New Emerging Diseases: Europe Grapevine Pinot gris virus 2016 2012 Several reports of GPGV GPGV identification HTS 2003 Symptom appearance in Trentino vineyards (North Italy) chlorotic mottling, leaf deformation and stunting Grapevine Virus Management • Plant disease-free, certified propagative material • Control virus vectors • Plant grapevines resistant to viruses and their vectors Registration and Certification of Grapevines 3 CCR § 3024-3024.8 Last updated 2010 The Grapevine Registration & Certification Program is a voluntary program that provides for the testing of source vines for significant grape pathogens. Registered sources and certified nursery stock are then inspected by CDFA staff and maintained by the participant in a manner to protect them from exposure to regulated diseases. California Registration and Certification Programs Grapes Fruit and Nut Trees Strawberries Economic Benefits of Grapevine Certification Programs • Napa and Sonoma Counties and Northern San Joaquin Valley: Economic cost of Grapevine Leafroll Disease $29,902 - $226,405/ha (Ricketts et al. 2015) • California North Coast: starting with clean planting stock benefits of $50 million/year (Fuller et al. 2015) • New York Finger Lakes Region: net economic returns of $9,693 - 16,014 per acre over 25-year life of vineyard by reducing grapevine leafroll virus (Atallah et al. 2012) The Dangers of Samsonite Importation Pierce’s Disease apsnet.org, 2016 Plum pox virus on stone fruit National Grapevine Importation Program: Foreign Imports • Housed at FPS • Largest nationally- recognized program for importing grape selections into the US • Serves as both an importation and quarantine facility APHIS – Animal Plant Health Inspection Service Plant Health (PPQ) • APHIS’ Plant Protection and Quarantine (PPQ) program safeguards U.S. agriculture and natural resources against the entry, establishment, and spread of economically and environmentally significant pests, and facilitates the safe trade of agricultural products. National Clean Plant Network (NCPN) • A national network of clean plant centers, scientists, educators, state and federal regulators, and growers and nurseries. • Focused on providing healthy planting stock of vegetatively propagated specialty crops to nurseries and growers. Participating Crops 1. Fruit Trees 2. Grapes 3. Berries 4. Hops 5. Citrus 6. Sweet potatoes 7. Roses www.nationalcleanplantnetwork.org The NCPN Clean Plant Centers START CLEAN Production Vineyards Registered Increase Blocks Foundation FPS Nurseries Growers STAY CLEAN FPS testing protocol Release time 2-6 years Disease Testing at FPS • Biological Indexing • ELISA – Enzyme-linked immunosorbent assay • PCR – Polymerase Chain Reaction • HTS – High Throughput Sequencing Tests required by APHIS and CDFA for certification Standard detection methods for a range of suspected ‘known’ viruses using a panel of specific tests: ELISA, RT-PCR and RT-qPCR ELISA RT-PCR RT-qPCR Drawbacks Requires prior knowledge of the pathogen Is incapable of detecting variants, such as new strains or ‘unknown’ agents Move to broader techniques (If all negative…) Herbaceous Index Procedure • Dust herbaceous host indicators with carborundum powder • Inoculate indicators with ground leaf tissue with nicotine buffer • Rinse leaves with water 1. Chenopodium amaranticolor 2. C. quinoa 3. Cucumis sativus 4. Nicotiana benthamiana 5. N. clevelandii 6. N. occidentalis Herbaceous Index Procedure • Read symptoms between 7 days and 21 days post inoculation Healthy Virus infected Healthy Virus infected Cucumis Nicotiana benthamiana sativus Woody Index = Field Index • Woody index done at FPS since 1950’s. • Developed by Austin Goheen, USDA, UC Davis. • Index, heat treatment and distribution of clean stock were the primary reasons FPS was formed. • Goheen found specific grape varieties that were good indicators of virus by trial and error. Woody Index Procedure • Disbud, wax, root and pot up indicator plants • Chipbud 2 buds/plant, 6 plants/variety Woody Index Procedure Woody Indicator Disease(s) Detected Rupestris St. George Grapevine fanleaf virus . Grapevine fleck virus Grapevine rupestris stem pitting-associated virus LN33 Corky bark Cabernet franc Leafroll disease, Red blotch Kober 5BB Kober stem grooving Healthy Virus infected Biological Indexing Advantages: Broad spectrum • Able to detect the majority of agronomically significant pathogens • Able to detect previously unknown agents Disadvantages: • Does not detect all pathogenic viruses, e.g., can miss GLRaV-2RG, GLRaV-7, some vitiviruses • Time consuming: takes 2-3 years to complete • High cost in greenhouse and field space, and labor • Cannot specifically identify pathogenic viruses (further steps required) • Woody Indexing Reliability Reliability is variable… it can be conditional on: 1. The success of bud-take 2. The transmission of the virus from the bud to the indicator vine 3. Effects of climatic conditions on symptom expression 4. The virus species, and strain All Standard Detection tests results were negative Test Type/ # Cab Franc 01 Ruby Cabernet 03 pathogens ELISA/ 12 negative negative pathogens Conventional negative negative PCR/33 pathogens qPCR/21 negative negative pathogens Move to broader techniques This new technology involves high throughput (HTS) DNA sequencing Why use HTS technologies? Because it’s rapid, accurate and efficient HTS gives a comprehensive picture of the entire microbial profile in a sample HTS Sequencing Revolution • Millions of short sequences generated for a given DNA sample • Comprehensive picture of the entire microbial profile. • Can sequence 1.6-1.8 Tb of DNA per run - Enough to fully sequence complete genomes. High throughput sequencing (HTS) Infected plant material Isolated RNA, siRNA, dsRNA Library preparation Category Input 7,105,243 Reads Total
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