Updates on Grapevine virus diseases. Maher Al Rwahnih Ph.D. Foundation Plant Services University of California, Davis

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1 Updates on Grapevine virus diseases Maher Al Rwahnih Ph.D. Foundation Plant Services University of California, Davis

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3 Foundation Plant Services Produces, tests, maintains and distributes elite disease-tested plant propagation material Provides plant importation and quarantine services, virus testing and elimination Coordinates release of UC patented horticultural varieties Links researchers, nurseries, and producers College of Agricultural & Environmental Sciences

4 CROP PROGRAMS AT FPS Grape Strawberry Tree Rose Sweet potato Pistachio Olives

5 Why do you need clean plant material? Effects of Grapevine virus diseases: Causes substantial crop losses Reduces fruit quality Shortens the longevity of grapevines in the vineyard

6 Background Grapevine hosts more viruses than any other crop species in the world More than 63 known viruses have been reported in Grapevine (mostly from Europe) Not all the viruses we discover are causing problems Grapevine Leafroll Disease Grapevine Fanleaf Disease

7 1. Nepoviruses 2. Rugose Wood Viruses 3. Leafroll Viruses 4. New, Emerging Diseases Grapevine red Blotch disease

8 1. 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

9 GFLV - Leaf deformation Fanleaf Healthy Infected GFLV is transmitted specifically from grapevine to grapevine by the ectoparasitic nematode Xiphinema index

10 GFLV Yellow mosaic symptoms

11 GFLV Veinbanding Symptoms

12 GFLV Fruit symptoms Healthy infected

13 2. 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.

14 Foveavirus: Grapevine rupestris stem pitting associated virus GRSPaV Vitivirus: Grapevine Virus A Grapevine Virus B Grapevine Virus D Grapevine Virus E Grapevine Virus F GVA GVB GVD GVE GVF (recently reported)

15 Rupestris Stem Pitting Disease Rupestris Stem Pitting Associated Virus closely related to Apple Stem Pitting Virus RSP + RSP+ HEALTHY Healthy

16 Kober Stem Grooving (KSG) Grapevine Virus A (GVA)

17 3. Grapevine leafroll disease (GLRD) 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.

18 Grapevine leafroll disease symptoms in red-fruited varieties Red leaves in fall Leaf margins rolled under Reduced vigor Cabernet franc infected with GLRaV-3

19 Leafroll symptoms on White-fruited Varieties Chlorosis Leaf margins rolled under Reduced vigor Chardonnay, Healthy vs. leafroll infected

20 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 Healthy Emperor LR infected White Emperor

21 Leafroll virus infection in Cabernet Sauvignon Healthy (L) vs. Leafroll (R)

22 Grapevine Leafroll Associated Viruses 1. GLRaV-1 2. GLRaV-2 (includes GLRaV-2RG) 3. GLRaV-3 4. GLRaV-4 (includes GLRaV-4 strains 5, 6, 9, 11, Car, De, Pr) 5. GLRaV-7

23 Mealybugs spread Leafroll Viruses Obscure mealybug Pseudococcus viburni (= P. affinis) Grape mealybug, Pseudococcus maritimus Citrus mealybug, Planococcus citri (Risso) Vine mealybug, Planococcus ficus Longtailed mealybug, Pseudococcus longispinus Pink hibiscus mealybug, Maconellicoccus hirsutus

24 Leafroll incidence 20%

25 5 years later -leafroll incidence >60%

26 How can we minimize the potential for spread?

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29 Pathogen Elimination Meristem shoot tip culture 10.0 mm Cut to < 0.5 mm 7 months

30 Meristem dome Virus in protophloem Nicotiana benthamiana shoot apex infected with beet severe curly top virus. Photo courtesy Li-Fang Chen, UCDavis.

31 Evolution of Diagnostics at Foundation Plant Services

32 Standard Detection methods for identification of grapevine viruses Traditional screening methods for a range of suspected known viruses using a panel of specific tests: ELISA, RT-PCR and qrt-pcr

33 Herbaceous testing Virus infected Healthy 1. Chenopodium amaranticolor 2. C. quinoa 3. Cucumis sativus 4. Nicotiana benthamiana 5. N. clevelandii 6. N. occidentalis

34 Field indexing Healthy Virus infected Woody indicator St. George Cabernet franc LN-33 Kober 5BB Disease Fanleaf, Rupestris Stem Pitting Virus, Fleck Virus Leafroll viruses Corky Bark, Stem Grooving Rugose Wood Viruses

35 Next Generation Sequencing (NGS) This new technology involves high throughput DNA sequencing

36 NGS is like a FULL BODY SCANNER Comprehensive picture of the entire microbial profile.

37 We need to be ready to: Study new diseases of unknown etiology Study diseases caused by new strains of known viruses Further characterize existing undiagnosed diseases: Syrah decline diseases Graft-incompatibility symptoms Leafroll like diseases Grapevine Vein-Clearing Red blotch disease

38 4. New, Emerging Diseases Courtesy of Dr. M.R. Sudarshana Grapevine red Blotch disease

39 Association of a Circular DNA Virus in Grapevines Affected by Red Blotch Disease in California M. Al Rwahnih, A. Dave 1,2, M. Anderson 3, J. K. Uyemoto 2 and M.R. Sudarshana 1,2 1 Dept. of Plant Pathology, 2 USDA-ARS, 3 Dept. of Viticulture and Enology, University of California, Davis, CA 95616, USA

40 Grapevine red blotch on Cabernet Franc Courtesy of Dr. M.R. Sudarshana

41 Courtesy of Dr. M.R. Sudarshana

42 Grapevine red Blotch Grapevine leafroll Courtesy of Dr. M.R. Sudarshana

43 Fall, 2010 Dormant canes collected from: - Vineyard 1 Cab Franc 214/ Vineyard 2 Cab Sauvignon 337/ Vineyard 3 Zinfandel 1A/AXR

44 qrt-pcr assays using Taq Man probes for grapevine viruses Virus Cab Franc 214 / Vineyard 1 Vineyard 2 Vineyard 3 Cab Sauv337 / Zinfandel1A /AXR1-1 GRSPaV GLRaV

45 Spring 2011 Next generation Sequencing Grapevine red Blotch Isolated dsrna cdna Synthesis Bioinformatics analysis

46 NGS results Virus Vineyard 1 Vineyard 2 Vineyard 3 Cab Franc214 / Cab Sauv337 / Zinfandel1A /AXR1-1 GRSPaV GLRaV DNA virus The discovery of a new circular DNA virus: Grapevine red blotch-associated virus (GRBaV)

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48 Chip bud inoculations Source vines PCR +ve Tested Petioles Roots a) Grapevines Cabernet Franc Cab Sauvignon Zinfandel b) Controls Healthy Ungrafted Detection of Grapevine red blotch-associated virus in graftinoculated plants of Cabernet Franc test plants inoculated with chip buds from source vines.

49 Red Blotch: What do we know? 1. Red Blotch is recently identified disease 2. Fruit ripening of diseased vine can be delay with Lower sugar accumulation 2-5 Brix 3. A DNA virus with a genome of 3206 nt is associated with diseased grapevines. 4. Grapevine red blotch-associated virus (GRBaV) is graft-transmissible. 5. GRBaV is detectable by PCR in symptomatic old and young grapevines.

50 What we don t know? Fulfill Koch s Postulates: Can we reproduce the red blotch symptoms? Will the grafted plants show disease symptoms? Generate infectious clones and inoculate grapevines. Localize the virus in infected grapevines and visualization of virus particles (tissue sampling)

51 What we don t know? Is the disease/virus spread by a vector or is it just clonally propagated? Does mixed infection by other grapevine viruses and GRBaV influence disease symptoms? Effect of Red Blotch Disease on Different Grapevine Rootstocks and scions Genetic variability of GRBaV? How do we manage this disease?

52 Virginia creeper leafhopper (Erythroneura ziczac Walsh) GRBaV was transmitted by the Virginia creeper leafhopper from grapevine-to-grapevine under greenhouse conditions.

53 Projects on GRBaV ( Funding Cycle) American Vineyard Foundation: $69,600 for Biology and Spread of GRBaV to lead researcher Dr. Mark Fuchs of Cornell University. $38,300 for Evaluating the Effects of GRBaV on Symptom Development and Fruit Maturity, to lead researcher Rhonda Smith, UC Cooperative Extension viticulture advisor for Sonoma County. $28,600 for Virginia Creeper Leafhopper (VCLH) Control. to lead researcher Dr. Kent Daane, entomologist at UC Berkeley. USDA-Agricultural Research Service: $178,000 for Study the vector of Red Blotch Disease to lead researcher Dr. Mysore Sudarshana, USDA-ARS, Department of Plant Pathology, University of California, Fruit Tree, Nut Tree and Grapevine Improvement Advisory Board: $51,000 for Study of the Effects of Red Blotch Disease on Different Grapevine Rootstocks and Different Vitis vinifera Plants to lead researcher Dr. Adib Rowhani of UC Davis Foundation Plant Services.

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57 What about Red Blotch in Russell Ranch or the Classic Foundation Vineyard?

58 Classic Foundation Vineyard

59 1620 VINES TESTED: 3 POSITIVES Variety Location qpcr Result Ruby Cabernet 02 NYL D3V3 Positive Thomcord 02 BKN E8 V3 Positive Chardonnay 68 BKN D3V8 Positive

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62 The National Clean Plant Network

63 New Russell Ranch Foundation Vineyard established 2010, Davis California October, 2012

64 Qualification of Russell Ranch Foundation Vines Grapevine Disease Testing Protocol ) Microshoot tip tissue culture therapy 2) Negative test results - long list of pathogens - index, herbaceous, ELISA and PCR tests 3) Agrobacterium vitis testing at Cornell univesity Q: What is clean grapevine material? A: Budwood that has gone through microshoot tip tissue culture therapy and tested negative for targeted pathogens.

65 What about Red Blotch in Russell Ranch Foundation Vineyard? 1102 VINES TESTED: All were NEGATIVE

66 Thank you