WERA_OLD20: Virus and Virus-Like Diseases of Fruit Trees, Small Fruits, and Grapevines.

(Multistate Research Coordinating Committee and Information Exchange Group)

Status: Inactive/Terminating

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Date of Annual Report: 11/15/2012

Report Information

Annual Meeting Dates: 09/18/2012 - 09/20/2012
Period the Report Covers: 10/01/2011 - 09/01/2012

Participants

Zvezdana Pesic-VanEsbroeck, (zvezdana_pesic@ncsu.edu), North Carolina State University, Raleigh;
Erich Rudyj (and wife),(Erich.S.Rudyj@aphis.usda.gov), NCPN Riverdale, Maryland;
Mizuho Nita, (nita24@vt.edu), Virginia Tech, Winchester, VA;
Tammy Jones, (c-tajones@pa.gov), Pennsylvania;
Sarah Gettys, (sgettys@pa.gov), Pennsylvania;
Bindu Poudel, (bpoudel@g.clemson.edu), Clemson University;
Lauri Guerra, (lguerra@prosser.wsu.edu), Washington State Dept. of Agriculture;
Carol Masters, (Carol.Masters@inspection.gc.ca), Canadian Food Insp. Agency, Sidney, BC;
Ken Eastwell, (keastwell@wsu.edu), Clean Plant Center of the Northwest, Washington State University;
Gary Kinard, (Gary.Kinard@ARS.USDA.GOV), USDA-ARS National Germplasm Resources Lab, Beltsville, Maryland;
Simon Scott,(sscott@clemson.edu), Clemson University;
John Hu, (johnhu@hawaii.edu) University of Hawaii, Honolulu;
Annemiek Schilder, (schilder@msu.edu), Michigan State University;
Margarita Bateman, (Margarita.L.Bateman@aphis.usda.gov) USDA-APHIS, Beltsville, Maryland;
Keith Streigler, (keith@flintridgewinegrowingservices.com);
Yannis Tzanetakis, (itzaneta@uark.edu) University of Arkansas, Fayetteville;
Michael Kahn, (kahn@wsu.edu) AA for WERA-20, Washington State University;
Richard Kaitany, (kaitanyr@michigan.gov), Michigan Dept. of Agriculture, East Lansing, Michigan;
Shulu Zhang, (shulu@agdia.com) Agdia Inc., Elkhart, Indiana;
Michael Cunningham, (macunningham@ucdavis.edu), Foundation Plant Services, Davis, California;
Joseph Postman, (Joseph.Postman@ars.usda.gov) USDA-ARS, Corvallis, Oregon;
Phil Brannen, (pbrannen@uga.edu), University of Georgia
Ruth Welliver, (rwelliver@pa.gov), Pennsylvania

Brief Summary of Minutes

WERA-20: VIRUS AND VIRUS-LIKE DISEASES OF FRUIT TREES, SMALL FRUITS, AND GRAPEVINES.

Annual Meeting Minutes

September 18 - 20, 2012 Clemson University Conference Center and Inn

Clemson University, South Carolina.

Participants:Zvezdana Pesic-VanEsbroeck, Erich Rudyj (and wife), Mizuho Nita, Tammy Jones, Sarah Gettys, Bindu Poudel, Lauri Guerra, Carol Masters, Ken Eastwell, Gary Kinard, Simon Scott, John Hu, Annemiek Schilder, Margarita Bateman, Keith Streigler, Yannis Tzanetakis, Michael Kahn, Richard Kaitany, Shulu Zhang, Michael Cunningham, Joseph Postman, Phil Brannen, Ruth Welliver

Tuesday 18th September, 8:30 a.m.

Opening of meeting by host Simon Scott

Welcome by Dr. Joe Culin, Associate Dean of Clemson University College of Agriculture Forestry and Life Sciences.

Simon Scott relayed invitation from Naidu Rayapati to host 2013 meeting

Election of Secretary: Joseph Postman agreed to take minutes if Naidu or someone else would host in 2012.

Comments by Administrative Advisor Michael Kahn: Accepted administrative responsibilities from Ralph Cavalieri. Discussed status of Farm Bill

Reports Presented

Carol Masters (Carol.Masters@inspection.gc.ca) Canadian Food Insp. Agency, Sidney, BC.

Reported on summary of Canadian fruit tree & grape import and export testing for 2010 2012. Plum Pox Virus update: transition in 2012 to industry led management. CFIA monitored quarantine are in Ontario on both sides of the quarantine border. There is a ban on propagation in the quarantine zone. Talked about the possibility of relocation of lab activities from the Sydney quarantine lab.

Yannis Tzanetakis (itzaneta@uark.edu) University of Arkansas, Fayetteville
Reported on what is new and exciting in the world of berry viruses. Blackberry yellow vein disease is rampant in the SE. Attempting to determine the cause and vector. Blackberry leaf mottle associated virus is new Emara virus. This virus group is typically mite transmitted. Positive samples found in 6 southern states. Conventional and quantitative RT-PCR tests have been developed. A mite vector has not been identified, but an eriophyid mite is suspected. A new Ampelovirus in blackberry is closely related to GLRaV-A, a mealybug transmitted virus.

Blueberry Mosaic Disease has been known since 1957, and causes beautiful symptoms but the causal agent has never been identified. A new Ophiovirus has been detected in all mosaic infected plants. This virus group is soil-borne, and a fungus vector is a possibility. A summary was presented on strawberry viruses in North America. There is good industry support for a blueberry certification program, but hesitancy for Rubus certification.

Michael Cunningham (macunningham@ucdavis.edu), Foundation Plant Services, Davis, California.

FPS update. Building expansion in progress. History of Russel Ranch property and status of new grape plantings. Development of tree fruit, strawberry standard operating procedures (distributed copies). Made a presentation for Maher Al Rwahnih on isolation & characterizion of new vitivirus, tentatively named Grapevine virus F (GVF).

Phil Brannen (pbrannen@uga.edu), University of Georgia.

Georgia Virus Disease Report. Grape leafroll became prevalent in N. Georgia about 5-6 years ago. Newly planted vines seem to be more contaminated with virus than older vines, suggesting untrustworthy status of nursery stock used. Discussion about motivating growers & nurseries to participate in certification program. Recently, a new disease was observed on southern highbush blueberries. Initially observed in 2006, plants with symptoms reminiscent of a pathogen-induced disease were found in multiple locations in southeastern Georgia. The disease also has been observed in Florida, Mississippi, South Carolina, and North Carolina.

John Hu (johnhu@hawaii.edu) University of Hawaii, Honolulu.

Update on assorted viruses in Hawaii. Reviewed history of transformation with coat protein to engineer resistance to Papaya ringspot. Deregulated in Japan in 2011, opening the door for expanded exports. Hawaii papaya industry is attempting to get into China too. Banana bunchy-top is most important virus disease of this crop world-wide. Caused by the DNA virus BBTV. Attempts are underway to induce gene silencing with several constructs. Also discussed the status of Huanglongbing (Citrus Greening Disease).

Shulu Zhang (shulu@agdia.com) Agdia Inc., Elkhart, Indiana.

Reported on Development and evaluation of AmplifyRP and ImmunoStrip tests for plum pox virus. Summary provided by Shulu: Agdia Inc. has recently developed AmplifyRP and ImmunoStrip tests for plum pox virus (PPV). Both AmplifyRP exo and AmplifyRP nfo tests are nucleic acid-based isothermal detections of PPV using a recombinase polymerase amplification technology. In contrast, the ImmunoStrip is a protein-based test. Each of these three tests (AmplifyRP exo, AmplifyRP nfo or ImmunoStrip) can detect all currently known six strains (PPV-C, PPV-D, PPV-EA, PPV-M, PPV-Rec or PPV-W) of PPV either in purified plant total RNA preparations or in crude Prunus plant extracts. The AmplifyRP exo, AmplifyRP nfo and ImmunoStrip tests were evaluated and compared to ELISA and RT-PCR. The results demonstrated that the three new tests (AmplifyRP exo, AmplifyRP nfo or ImmunoStrip) are highly sensitive and reliable. In addition, the AmplifyRP exo, AmplifyRP nfo and ImmunoStrip tests are rapid and simple. The whole process from sample preparation to finish for these new tests can be done in as little as 20 minutes. All the new tests do not need thermal cycling and expensive equipment and can be performed in laboratories and in fields.

Annemiek Schilder (schilder@msu.edu), Michigan State University.

Reported on the results of surveys in Michigan for grapevine and blueberry viruses, using Agdia and Bioreba ELISA kits. Shared images of assorted symptoms in grape, seeking input on possible causes. After 2 years of surveys, it appears that Blueberry shock and scorch have been eradicated. Initial survey found BB scorch in a recent Legacy planting that traced back to the nursery source. Later survey found infections in older plantings. Infected plants (plantings?) were destroyed. Bronze leaf curl- a closterovirus?

Zvezdana Pesic-VanEsbroeck (zvezdana_pesic@ncsu.edu), North Carolina State University, Raleigh.

Reported on the Clean Plant Center for Berry Crops at NC State University. The program has a 50 year history working with strawberries. Discussed the protocol for production of G1 Foundation Plants. A new virus (Blueberry latent virus) was recently found in J. Ballingtons collection of plant materials (see: R. R. Martin, J. Zhouc, and I.E. Tzanetakis. 2011. Virus Research 155:175-180).

Simon Scott (sscott@clemson.edu), Clemson University.

Discussed a virus related to Cherry necrotic ring mottle that produced symptoms in rootstock sprouts beneath a tree with symptomless Shirofugen as the scion variety.

Bindu Poudel (bpoudel@g.clemson.edu), Clemson University.

Discussed her PhD project working with S. Scott on Blackberry yellow vein disease, which is associated with a complex of viruses in the SE United States. A new Ilarvirus was found in sentinel plants.

Joseph Postman (Joseph.Postman@ars.usda.gov) USDA-ARS, Corvallis, Oregon.
Reported on the genetic diversity, geographic origins, and virus status of several tree collections at the National Clonal Germplasm Repository in Corvallis. More than 500 tree accessions have been cleaned up at NCGR using heat therapy and meristem culture. Presently, 76% of the 800 Corylus accessions, 83% of 156 Cydonia accessions 79% of 42 Mespilus accessions and 81% of 2171 Pyrus accessions are virus negative.

Meeting Adjourned at 5:00 p.m. group dinner at Pixie and Bills

Wednesday 18th September, 8:30 a.m. - meeting resumes

Sarah Gettys (sgettys@pa.gov)

Reported on the Pennsylvania Fruit Tree Improvement Program. Summary of various aspects of the PA virus certified tree fruit program. Two certification levels. Developed Pest Management Manual covering procedures. Conduct annual visits (audits). (discussion about nursery practices and fumigation)

Ruth Welliver (rwelliver@pa.gov)

Reported on blueberry shock survey. Samples taken from 12 sites around PA. Found several sites with TomRSV, two farms positive/questionable for Shock and Scorch. Continuing to monitor for Plum Pox. No positive samples since 2006. 40,000 samples tested in 2012. Reviewed eradication efforts. Quarantines have been lifted against growing stone fruits. If all 2012 samples are negative remaining quarantines on nursery production will be lifted.

Margarita L. Bateman (Margarita.L.Bateman@aphis.usda.gov) USDA-APHIS, Beltsville, Maryland

Reported on administrative re-organization of APHIS and position of Plant Protection and Quarantine program. Focus of program is on incoming plant materials from foreign origins. Reviewed testing and therapy protocols and efforts to streamline.

Gary Kinard (Gary.Kinard@ARS.USDA.GOV), USDA-ARS National Germplasm Resources Lab, Beltsville, Maryland.

Development of multiplex TaqMan real-time RT-PCR assay for simultaneous detection of 3 Prunus viroids. Improved elimination of PNRSV from Prunus. Improved elimination of Rubus viruses. Factorial experiments looking at combinations of temperature and antiviral chemicals. Budget, International Treaty, GRIN-Global, Pathologist vacancy comments.

Ken Eastwell (keastwell@wsu.edu), Clean Plant Center of the Northwest, Washington State University.

Foundation programs for fruit trees, grapevines, hops and ELISA lab organized into single administrative "center". Discussion about Little Cherry, Cherry leaf roll, Cherry raspleaf, Betaflexiviridae in sweet cherry. Unusual expansion of little cherry, even in normally tolerant varieties like Bing. Cherry leafroll is spreading. Alternate reservoirs are in Elderberry and Rhubarb.

Mizuho Nita (nita24@vt.edu), Virginia Tech, Winchester, VA.

Virus diseases not as obvious when fungal diseases require attention. Reported on grape virus survey in Virginia. Leafroll 3 was most prevalent. Used cluster analysis to suggest vector, cultivar and virus relationships for several grape viruses.

Erich Rudyj (Erich.S.Rudyj@aphis.usda.gov) NCPN Riverdale, Maryland.
History and status of National Clean Plant Network. Farm Bill update relative to NCPN funding.

Keith Streigler (keith@flintridgewinegrowingservices.com).

NCPN Education and Outreach summary. Facilitates national working group to develop communication plans. Specialty crop brochures need regular updates. PowerPoint Presentation that can be used by the NCPN community.

Meeting concluded at noon.

Box lunches while traveling to Boiling Springs, SC to view blackberry viruses at Mr. James Cooleys Strawberry Hill Farm (cvs Ouachita and Navaho).

Travel to a recent planting of Primark 45 at Mr. Jose Landas Farm.

Thursday, 20 September

The group visited 3 wineries in Northeast Georgia: Blackstock Winery, Wolf Mountain Winery, and Frogtown Winery. Symptoms of Pierce's disease and plants displaying symptoms typical of infection by grapevine leaf roll virus were seen.

To view full 2012 individual state reports, go to WERA-20's Homepage under the Additional Documents Section at the following address: http://lgu.umd.edu/lgu_v2/homepages/attachs.cfm?trackID=13496

Accomplishments

Grapevines: Research by members of the project in Idaho, Washington, and Michigan on grapevines, and the viruses that infect them, provided evidence; 1) that Grape vine leaf roll associated virus 1 (GLRaV-1) occurs as genetically distinct variants in the three leading grapevine-producing states of the U.S.: California, New York and Washington, New York; 2) that Grapevine leaf roll disease (GLRD) - associated with a complex of Grape vine leaf roll associated viruses - has negative impacts on vine performance and the quality of grapes produced by own-rooted Merlot grapevines; and 3) Grapevine fleck virus (GFkV) previously identified in Washington is also present in Idaho and Michigan. Cherry: Cherry orchards in the northwest are affected by a number of different viruses of economic importance: Little cherry virus 2, Cherry leaf roll virus, Cherry Rasp Leaf virus, and the rusty mottle group of viruses. Research has identified Grape mealybug (Pseudococcus maritimusis) as a previously unsuspected vector of Little cherry virus 2 that may be contributing to the recent increase of the spread of this virus within the industry. Ongoing research demonstrates that the 'Colt' rootstock represents the best protection available to growers for Cherry leaf roll virus. Blue elderberry (Sambucus nigra) has been identified as a previously unrecognized reservoir host for Cherry rasp leaf virus and this may account for the persistence of the virus in the central Washington growing area where elderberry is abundant in valley vegetation. Molecular characterization of Cherry necrotic rusty mottle virus indicates that it may be part of a unique genus of viruses. Furthermore, the virus has been detected in a flowering cherry growing in South Carolina for the first time on. There is no cherry industry in this state but flowering cherries are frequently planted in the landscape which raises the question as to whether they act as a source of the virus that is readily moved around the nation. The majority of testing for viruses in the crops covered by WERA-20 is accomplished using PCR. Although most labs are equipped with thermocyclers, and PCR is routine in many locations, new technologies are being exploited to expand the scope of sensitive testing techniques. Recombinase polymerase amplification technology is a nucleic acid-based test completed at a single temperature, obviating the need for thermocyclers, and thus can be performed both in laboratories and in the field in as little as 20 minutes. Agdia has demonstrated the utility of this system with Plum Pox virus.

Publications

Publications Janse, J.D., Valentini, F., Purcell, A.H. and Almeida, R.P.P. 2012. Detection and identification methods and news tests as used and developed in the framework of COST873 for bacteria pathogenic to stone fruits and nuts: Xylel la fastidiosa. Journal of Plant Pathology 94: S1.147-154. Tsai, C.W., Daugherty, M.P. and Almeida, R.P.P. 2012. Seasonal dynamics and virus translocation of Grapevine leafroll-associated virus 3 in grapevine cultivars. Plant Pathology 61: 977-985. Daane, K.M., Almeida, R.P.P., Bell, V.A., Botton, M., Fallahzadeh, M., Mani, M., Miano, J.L., Sforza, R., Walton, V.M. and Zaveizo, T. 2012. Biology and management of mealybugs in vineyards. p. 271-307. In: N.J. Bostanian et al. (eds.) Arthropod Management in Vineyards: Pests, Approaches, and Future Directions. Springer. 505p. Almeida, R.P.P., Killiny, N., Newman, K.L., Chatterjee, S., Ionescu, M. and Lindow, S.E. 2012. Contribution of rpfB to cell-cell signal synthesis, virulence, and vector transmission of Xylella fastidiosa. Molecular Plant-Microbe Interactions 25: 453-462. Killiny, N., Rashed, A. and Almeida, R.P.P. 2012. Disrupting the transmission of a vector-borne plant pathogen. Applied and Environmental Microbiology 78: 638-643. Daugherty, M.P., Gruber, B.R., Almeida, R.P.P., Anderson, M.M., Cooper, M.L., Rasmussen, Y. and Weber, E. 2012. Testing the efficacy of barrier plantings for limiting sharpshooter spread. American Journal of Enology and Viticulture 63: 139-143. Daane, K.M., Middleton, M.C., Sforza, R., Cooper, M.L., Walton, V.M., Walsh, D.B., Zaviezo, T. and Almeida, R.P.P. 2011. Development of a multiplex PCR for identification of vineyard mealybugs. Environmental Entomology 40: 1595-1603. Tsai, C.W., Bosco, D., Daane, K.M. and Almeida, R.P.P. 2011. Effect of host plant tissue on the vector transmission of Grapevine leafroll-associated virus 3. Journal of Economic Entomology 104: 1480-1485. Rashed, A., Daugherty, M.P. and Almeida, R.P.P. 2011. Grapevine genotype susceptibility to Xylella fastidiosa does not predict vector transmission success. Environmental Entomology 40: 1192-1199. Sharma, A.M., Wang, J., Duffy, S., Zhang, S., Wong, M.K., Rashed, A., Cooper, M.L., Daane, K.M. and Almeida, R.P.P. 2011. Occurrence of grapevine leafroll-associated virus complex in Napa Valley. PLoS ONE 6: e26227. Killiny, N. and Almeida, R.P.P. 2011. Gene regulation mediates host specificity of a bacterial pathogen. Environmental Microbiology Reports 3: 791-797. Daugherty, M.P., Rashed, A., Almeida, R.P.P. and Perring, T.M. 2011. Vector preference for hosts differing in infection status: sharpshooter movement and Xylella fastidiosa transmission. Ecological Entomology 36: 654-662. Plucinski, M.M., Starfield, R. and Almeida, R.P.P. 2011. Inferring social network structure from bacterial sequence data. PLoS ONE 6: e22685. Gutierrez, A.P., Ponti, L., Hoddle, M., Almeida, R.P.P. and Irvin, N.A. 2011. Geographic distribution and relative abundance of the invasive glassy-winged sharpshooter: effects of temperature and egg parasitoids. Environmental Entomology 40: 755-769. Kung, S.H. and Almeida, R.P.P. 2011. Natural competence and recombination in the plant pathogen Xylella fastidiosa. Applied and Environmental Microbiology 77: 5278-5284. Coletta-Filho, H.D., Bittleston, L.S. and Almeida, R.P.P. 2011. Spatial genetic structure of a vector-borne generalist pathogen. Applied and Environmental Microbiology 77: 2596-2601. Degnan, P.H., Bittleston, L.S., Hansen, A.K., Sabree, Z.L., Moran, N.A. and Almeida, R.P.P. 2011. Origin and examination of a leafhopper facultative endosymbiont. Current Microbiology 62: 1565-1572. Rashed, A., Killiny, N., Kwan, J. and Almeida, R.P.P. 2011. Background matching behaviour and pathogen acquisition: Plant site preference does not predict bacterial acquisition efficiency by vectors. Arthropod-Plant Interactions 5: 97-106. Wang, J., Sharma, A.M., Duffy, S. and Almeida, R.P.P. 2011. Genetic diversity in the 3' terminal 4.7-kb region of Grapevine leafroll-associated virus 3. Phytopathology 101: 445-450. Liming Lin, Ruhui Li, Ray Mock, and Gary Kinard, 2012. One-Step multiplex RT-PCR for simultaneous detection of four pome tree viroids. European Journal of Plant Pathology 133: 765-772. Wei-Li Rao, Fan Li, Ruijuan Zuo and Ruhui Li, 2011. First Report of Little cherry virus 2 in Flowering and Sweet Cherry Trees in China. Plant Disease 95: 1484. Schilder, A., and Brown-Rytlewski, D. Virus and Virus-like diseases in Grapes. MSU Extension, Michigan State University (in preparation). Schilder, A. 2011. Blueberry Scorch Virus. Regional Pest Alert, North Central IPM Center, 2 pp. Schilder, A. 2011. Blueberry Shock Virus. Regional Pest Alert, North Central IPM Center, 2 pp. Isaacs, R., Schilder, A., Zabadal, T., and Weigle. T. 2011. A pocket guide for grape IPM scouting in the North Central and Eastern U.S. Michigan State University Extension Bulletin E-2889. 4th Edition. Wise, J. C., Gut, L. J., Isaacs, R., Schilder, A. M. C., Sundin, G. W., Zandstra, B., Hanson, E., and Shane, B. 2011. Michigan Fruit Management Guide 2012. Extension Bulletin E-154. Michigan State University, East Lansing, MI. Eastwell KC, Druffel KL. 2012. Complete genome organization of American hop latent virus and its relationship to carlaviruses. Arch Virol 157:14031406 Eastwell KC, Mekuria TA, Druffel KL. 2012. Complete nucleotide sequences and genome organization of a cherry isolate of cherry leaf roll virus. Arch Virol 157:761764 Alabi, O. J., Al Rwahnih, M., Karthikeyan, G., Poojari, S., Fuchs, M., Rowhani, A., and Naidu, R. A. 2011. Grapevine leafroll-associated virus 1 occurs as genetically diverse populations. Phytopathology 101: 1446-1456. Rayapati, N.A., ONeil, S. and Walsh, D. 2011.Grapevine leafroll disease (la enfermedad del enrollamientode la hoja de parra) in Spanish (EB2027ES). Naidu, R.A. 2011. Virus Diseases. In: 2011 Pest Management Guide for Grapes in Washington. WSU Extension Bulletin EB0762, pp.25-28. Naidu, R.A. 2011. Virus update: The Status of Washington Vineyards. In: Viticulture and Enology Extension News Fall 2011. You Tube Videos: Grapevine Leafroll Disease Symptoms (http://wine.wsu.edu/research-extension/grape-growing/plant-health/virology/; http://www.youtube.com/watch?v=Es41Tdgd9L0&NR=1&feature=endscreen) Grapevine Disease: Be Clean, Stay Clean (http://www.youtube.com/watch?v=Vt_OyWpXohE&feature=related) How Grapevine Virus Diseases Spread: 4 propagation methods https://www.youtube.com/watch?v=605YHdIeSC8

Impact Statements

Information on the genetic diversity of GLRaV-1 and identification of GFkV in Idaho and Michigan continues to show the specific importance of virus-testing in order to meet the need for clean planting material to be available to the grape industry nationwide and also accurately reflects the situation that exists in the other crops covered by WERA-20.
The identification of GFkV in two previously undocumented locations indicates the importance of monitoring the viral status of plant material being moved from state-to-state.
The negative impact on yield and quality by the widely disseminated Grapevine leaf roll associated virus complex supports attempts to eliminate this virus from vineyards and replace infected vines with clean plants.
Outreach efforts are increasing awareness of virus diseases and their negative impacts on tree and vine health and fruit quality among industry stakeholders.
The science-based knowledge is being used for improving diagnostic assays in virus-indexing programs to prevent the spread of viruses and their genetic variants via planting materials.
Laboratory methods remain the only tool to detect the virus and provide information for stake-holders.
This need for laboratory methods to provide rapid, reliable, sensitive detection of viruses of economic importance is equally true for planting material of stone fruits, pome fruits and berries.
Information allows stake-holders to respond proactively either by demanding virus-tested plant material to establish new acreage or by modifying production processes to deal with previously unrecognized facets of disease epidemiology.
Efforts of the group to establish sources of virus-tested plant material for the benefit of stake-holders is clearly documented by the reports for Quarantine facility at Beltsville and the Germplasm Repository at Corvallis.

Date of Annual Report: 10/07/2013

Report Information

Annual Meeting Dates: 09/10/2013 - 09/12/2013
Period the Report Covers: 10/01/2012 - 09/01/2013

Participants

Jati Adiputra, WSU-Prosser, jati.adiputra@email.wsu.edu;
Segun Akinbade, WSDA, sakinbade@agr.wa.gov;
Gary Ballard, WSU-CPCNW, gballard@wsu.edu;
Sadanand Dhekney, University of Wyoming, sdhekney@uwyo.edu;
Bhanu Donda, WSU-Prosser, bhanu.donda@email.wsu.edu;
Ken Eastwell, WSU-Prosser, keastwell@wsu.edu;
Brooke Edmunds, Oregon Department of Agriculture, bedmunds@oda.state.or.us;
Lauri Guerra, WSDA, lguerra@agr.wa.gov;
Bill Howell, Northwest Nursery Improvement Institute, nwnurseryii@gmail.com;
John Hu, University of Hawaii, johnhu@hawaii.eduEunice Kanuya, WSU-Prosser;
Madhu Kappagantu, WSU-Prosser, madhu.kappagantu@email.wsu.edu;
Alexander Karasev, University of Idaho, akarasev@uidaho.edu;
Syam Kumar, WSU-CPCNW, syam_skumar@wsu.edu;
Ruhui Li, USDA-ARS, Ruhui.Li@ARS.USDA.GOV;
Bob Martin, USDA-ARS, Corvallis, OR, Bob.Martin@ARS.USDA.GOV;
Carol Masters, Canadian Food Inspection Agency, Carol.Masters@inspection.gc.ca;
Nathan McOwen, Agdia, Inc., nathanmcowen@agdia.com;
Jim Moyer, WSU-ARC, j.moyer@wsu.edu;
Tefera Mekuri, Agri-Analysis, tasamenew03@yahoo.com;
Mark Nakhla, USDA APHIS PPQ, Mark.K.Nakhla@aphis.usda.gov;
Olga Nikolaeva, University of Idaho, onikolaeva@uidaho.edu;
Sudarsana Poojari, WSU-Prosser, sudarsan@wsu.edu;
Josh Puckett, Foundation Plant Services, jmpuckett@ucdavis.edu;
Naidu Rayapati, WSU-Prosser, naidu.rayapati@wsu.edu;
Adib Rowhani, Foundation Plant Services, akrowhani@ucdavis.edu;
Erich Rudyj, USDA-APHIS NCPN, Erich.S.Rudyj@aphis.usda.gov;
Sead Sabanadzovic, Mississippi State University, ssabanadzovic@entomology.msstate.edu;
Vicky Scharlau, WA Wine Industry Foundation, vicky@501consultants.com;
Annemiek Schilder, Michigan State University, schilder@msu.edu;
Anna-Mary Schmidt, Canadian Food Inspection Agency, anna-mary.schmidt@inspection.gc.ca;
Simon Scott, Clemson University, SSCOTT@clemson.edu;
Susan Sim, Foundation Plant Services, stsim@ucdavis.edu;
Keith Striegler, NCPN Outreach. keith@flintridgewinegrowingservices.com;
Ioannis Tzanetakis, University of Arkansas, itzaneta@uark.edu;
Debbie Woodbury, WSU-CPCNW, deb.woodbury@wsu.edu

Brief Summary of Minutes

WERA-20: VIRUS AND VIRUS-LIKE DISEASES OF FRUIT TREES, SMALL FRUITS, AND GRAPEVINES

Annual Meeting Minutes, September 10-12, 2013

Tuesday, September 10, 2013

(Hampton Inn, 486 Bradley Blvd, Richland, WA 99352)

Part I. General session

Opening of meeting by the Chair of the WERA-020 group Dr. Naidu Rayapati, Department of Plant Pathology, Irrigated Agriculture Research and Extension Center (IAREC), Washington State University (WSU), Prosser, WA 99350.

Welcome by Dr. Jim Moyer, Administrative Advisor for WERA-020 and Associate Dean for Research & Director of the Ag Research Center, College of Agricultural, Human, and Natural Resource Sciences at Washington State University, Pullman

Welcome by Vicky Scharlau, the Executive Director of the Washington Association of Wine Grape Growers. She also provided an overview of the Washington grape industry

Two suggestions were proposed for the location of next year's meeting, NC and HI. The group finally decided to go to Hawaii next year. John Hu as the host of the next year's meeting will serve as the secretary for this year's meeting.

Jim Moyer reminded us that we will need to submit the final report within 30 days after the meeting. Naidu reminded the members that our reports should include an impact section and publications.

Part II - Summary of State Reports

Arkansas (Ioannis Tzanetakis):

Reported on the development of bioinformatics software to assist in evaluating data from next generation sequencing for the purposes of virus diagnosis that will require little training or supervision. It is being developed that will maximize ability of programs to analyze massive amount of data produced from high throughput sequencing processes; the potential of central server accessible to users is envisioned. Parameters for next generation sequencing such as the degree of multiplexing and sample preparation are being evaluated for reliability and cost effectiveness.

California (Adib Rowhani):

Reported on the conclusion of a multiyear trial to examine the effects of grapevine leafroll associated viruses (GLRaVs) on symptom development. The trial consisted of 19 viruses and mixes of viruses on 9 rootstocks. Grapevine leafroll associated viruses 1, 2, and 3 caused the most severe symptoms, GLRaV-4 caused mild symptoms and GLRaV-7 infections were symptomless. Rootstock AXR-1 was the most tolerant rootstock and mixed infections of GLRaV-1 with Grapevine virus A (GVA) were lethal on several rootstocks.

The Foundation Plant Services vineyards were being tested for Grapevine red blotch- associated virus (GRBaV). None was detected in the new Russell Ranch vineyard and only 9 vines were infected in the classic vineyard. Infected vines represented only one of several sibling vines suggesting there was some natural spread in the vineyard.

Canada (Anna-mary Schmidt):

Reported on the incidence of viruses that were detected in the import and export programs of the Canadian Food Inspection Agency. Several viruses are being detected on a regular basis on fruit trees, small fruits and grapevine material in both certified and non-certified material in the import/export program (see full report). GVA and Grapevine virus B (GVB) are regulated pests but detected in importations from France. A survey identified 2 sites in B.C. infected with GVA and 3 sites in Ontario infected with GVB. The regulatory status of GVA and GVB is being re-evaluated.

GRBaV was detected in 3 sites in Ontario but none detected in B.C. No GRBaV was detected in audit samples from Europe or U.S.A.

Canada (Carol Masters):

Reported on the phytoplasma detection in Nova Scotia. Symptoms are somewhat reminiscent of rubbery wood. Still at early stages of investigation but at this time, phytoplasma was detected in two trees imported from WA State in 2008 and 2009. The available sequence information is inadequate to determine if it is apple proliferation phytoplasma or another phytoplasma.

Hawaii (John Hu):

Reported on the characterization of two viruses found in hibiscus that are citrus leprosis-like viruses. One cilevirus found in hibiscus is very similar to citrus leprosis virus cytoplasmic type 2. A second cilevirus was found in both citrus and hibiscus and represents a potential new virus genus with the proposed name Higrevirus. Both viruses are associated with transmission by Brevipalpus mites.

Idaho (Alex Karasev):

A survey of Idaho vineyards consisting of 434 leaf samples was conducted from 2009 to 2011 and tested for a panel of 16 viruses and viroids. GLRaV-1, -3, and -4, and its strain -5 were detected, and GLRaV-3 was the most frequently detected virus occurring in 26% of the samples. Viruses of the rugose wood complex were also detected. GVA and GVB were the major viruses of the rugose wood complex and Grapevine rupestris stem pitting-associated virus was also detected in a few samples. Several viruses known to occur in Washington State were not detected suggesting that continued vigilance is warranted to keep these out of Idaho production areas.

Indiana (Nathan McOwen, Agdia, Inc.):

Reported on the development of an isothermal detection method based on recombinase polymerase assay for the detection of Candidatus asiaticus (Las) and Plum pox virus (PPV) The assay system is not as technically demanding as PCR and can be performed on site in 40 minutes. The sensitivity for detecting Las was comparable to qPCR, and detection of PPV was at least ten-fold more sensitive than ELISA and detected all representative strains. Both systems utilized crude extracts without further RNA or DNA enrichment.

Maryland (Mark Nakhla):

Reported on the activities of the Beltsville laboratories of the combined Plant Protection and Quarantine (PPQ) and the Center for Plant Health Science and Technology (CPHST). The organization develops operational diagnostics in CPHST laboratories with development and validation occurring in the Beltsville location. They also provide training to the Plant Diagnostic network and proficiency testing panels. Assays have been deployed for the detection of select agents at the ports. Also, several group specific PCR tests have been developed to support post entry quarantine activities.

Maryland (Ruhui Li):

Reported on the characterization of six isolates of Asian Prunus virus (APV). This virus consists of genetically diverse members that exhibit recombination. Sequence analysis also indicates that the previously described APV1 and APV3 are actually variants of the same virus species. Gooseberry vein banding associated virus is common in growing areas. In collaboration with J. Postman, developed and optimized molecular assays that provide rapid and sensitive detection; the virus is now detected in 6 host species in 12 countries.

The laboratory was charged to find a 'universal' media for the shoot tip culture of Prunus spp. Research revealed that the carbohydrate source has a significant influence of most species whereas no media was found to adequately support growth of almond. In related studies, no effective procedure was developed for the elimination of several important viruses of small fruits.

Michigan (Annemiek Schilder):

Reported on the results of the 2012 survey for grapevine diseases consisting of 65 samples from 33 vineyards. GLRaV-3 was found in approximately one-half the samples. GLRaV-1, -2, and -4 and its strain -9 were also detected. Tobacco ringspot virus (TRSV) and Tomato ringspot virus (ToRSV) were detected, frequently in combination with GLRaV-3. The effect of GLRaV3 was quantified. Shoot length, overall yield and clusters per vine were the most significantly affected parameters.

Blueberries are most notably infected with TRSV and ToRSV. However, a bronze leaf curl disease is becoming increasingly prevalent. The causal agent is not known but is under investigation in a collaborative research project with other berry scientists.

Mississippi (Sejo Sabanadzovic):

Presented data on characterization of two new blackberry viruses with elongated flexuous virions performed at Mississippi State University (MSU) in collaboration with scientists from University of Arkansas and USDA-ARS, Corvallis, OR. Rubus canadensis virus 1 was identified in a sample of smooth blackberry collected in the Great Smoky Mountains National Park. The virus is phylogenetically related to extant foveaviruses and apparently not widespread in cultivated blackberries. Blackberry vein banding-associated virus, originally isolated from a symptomatic cultivated blackberry in Mississippi, has a close relationship with members of the genus Ampelovirus (Family Closteroviridae), in particular with GLRaV-3. The virus was detected in ca 25% blackberry samples affected by yellow vein disease and its mealybug transmission was ascertained. In addition, he shared the results of his recent work on viruses affecting muscadines and the development of method for their detection. To date, MSU group has identified several viruses in this crop, some of which are reported economically important in Vitis vinifera.

Oregon (Bob Martin):

Reported on testing of archived RNA extracts from grapevines collected from 2009 to 2012 for GRBaV. Other studies are investigating the impact of GRBaV on fruit quality and quantity of Pinot gris and Pinot noir.

Raspberry latent virus is a phytoreovirus. In mixed infections with Raspberry leaf mottle virus and Raspberry bushy dwarf virus, a low percentage of aphids were found to acquire the Raspberry latent virus.

Blueberry necrotic ring blotch virus remains localized at site of necrotic rings, and may be transmitted by eriophyid mites. Several viruses including DNA viruses were reported in association with blueberry diseases. Sequencing is in progress. A carlavirus was reported from elderberry that appears to represent a new virus.

South Carolina (Simon Scott):

Reported on use of a peach interstock to delay bloom and protect peach crop from early spring frosts. TaTao5 delays flowering even when the interstock shoot was lost suggesting a graft transmissible agent. TaTao5 was shown to be infected with Apple chlorotic leaf spot virus, Asian Prunus virus-1, and Peach latent mosaic viroid. Isolates of these three agents did not delay bloom suggesting that a specific variant of one of them may be involved.

Raritan rose is a peach cultivar that has fallen out of favor with the industry. Screening of 4 different sources of Raritan Rose revealed ACLSV in one of the sources as well as the sample from Clean Plant Center North West (CPCNW). Distribution of the virus in the tree is erratic and was detected in only 40% of the samples from a single budstick.

Washington (Naidu Rayapati):

Reported on the development of a distribution database for virus infections of grapevines in Washington State. GLRaV-3 is the most frequently detected virus. Of the vines infected with viruses, nearly 70% have single virus infections with most of them testing positive for GLRaV-3 and 28% are mixed infections. GLRaV-3 and GVA are the most commonly encountered combination in mixed virus infections. The sequence of Grapevine virus E detected in WA is similar to that obtained from other regions. In combination with asymptomatic strains of GLRaV-2 on Sangiovese, anthocyanin levels were reduced but there was no effect on yield.

GRBaV is a newly discovered virus that is transmitted by the Virginia creeper leafhopper. Infection by GRBaV decreased shoot length, pruning weight and yield of grapevines. There was a significant reduction in sugars of fruit on infected vines.

Washington (Ken Eastwell):

Reported that little cherry disease caused by Little cherry virus 2 re-emerged as a serious disease in Chelan and Douglas Counties. The most likely explanation is the increasing abundance of grape mealybug recently demonstrated to be a vector of the virus. In collaboration with Agdia, a simple assay based on recombinase-polymerase technology was developed. Little cherry disease caused by Western X phytoplasma is increasing in the southern portions of the state.

Cherry mottle leaf virus was confirmed as the agent that caused peach wart disease. The peach variant contains significant sequence variation in the open reading frame that encodes the coat protein. It is unknown is this is required for the development of peach wart disease. Symptomatic elderberry plants growing adjacent to a cherry orchard infected with Cherry rasp leaf virus was found to be infected with this virus also, a newly described host for Cherry rasp leaf virus. A carlavirus was also identified in the elderberry and is under further investigation.

Wyoming (Sadanand Dhekney):
Reported on the new program to develop genetically modified grapevines to increase tolerance to abiotic stresses such as drought, cold and salinity frequently encountered in the Wyoming grow area. An overview was provided of the transformation process and successful introduction of genetic markers into grapevines. The program will also screen existing grape cultivars and rootstock suitable for Wyoming conditions.

NCPN (Erich S. Rudyj):

Reported on the creation of the National Clean Plant Network. The Network now supports 23 programs at 19 institutions to provide propagation material free of economically important pathogens. The Network now supports diagnostics and therapeutics on specialty crops that are generally restricted for entry into the U.S. including temperate climate fruit and nut trees, grapevines, hops, citrus and berries. In the short four year life of the program it has been very successful in tapping into existing programs and facilitated the release of 350,000 clean propagation units to the industry for further increase. Emerging issues were identified for the future success of the network.

NCPN Outreach (Keith Striegler):

The goal of the outreach and education component of the National Clean Plant Network is to educate target groups about the mission and successes of the Network. Target audiences include industry, public, regulatory and political components. The NCPN provides websites to capture history and working documents, provides educational publications in print and electronic form, and provides PowerPoint presentations, newsletter articles and table top displays. The material is formulated for national consumption, but can be tailored for local audiences. A branding committee was established to ensure a uniform look and message to NCPN documents.

WA Clean Plant Campaign (Vicky Scharlau):

Reported on the Clean Plant Campaign funded by a WSDA Specialty Crop Block Grant. The initiative will survey Registered Mother Blocks for grapevine viruses in partnership with WSU (Naidu Rayapati). The results will be used to educate stakeholders and regulatory agencies. The information will provide basic information for the control, prevention and management of virus diseases of grapevines.

Formal meeting adjourned at 5:30 followed by a wine tasting and group dinner.

Wednesday 11th September, 8:45 am

A tour of the Clean Plant Center Northwest at WSU-IAREC, Prosser was provided. The programs for fruit trees, grapevines and hops were demonstrated including virus elimination procedures. The foundation plantings were shown as well as a tour of the orchards at WSU-IAREC to observe virus diseases. Research blocks on the use of different rootstocks was shown.

A meeting was convened in the WSU-IAREC conference room for a vigorous discussion of the impact of next-generation sequencing (NGS) on virus diagnostics and implications for NCPN centers.

Three issues emerged:

1. In considering adopting NGS, the impact of trade between countries and states must be recognized. For quarantine programs this is less of a consideration because agents identified by NGS will be eliminated. However, publication of novel agents found in commercial production must be handled carefully and thoughtfully.

2. Basic standard methodologies should be considered. Each specialty crop will have its individual nuanced needs, but general guidelines could be developed.

3. The output from NGS needs to be dealt with in a thoughtful manner. Outreach and education is necessary to explain the implications from some NGS results. WERA-020 will provide a venue for the continued development of a white paper regarding the use of NGS in this manner.

After a box lunch, the group left to tour Willow Drive Nursery in Ephrata, WA, to see production practices for rootstocks and finished trees. Hosted by Neil Manly.

Thursday 12th September, 8:45 am

Travel to Inland Desert Nursery in Benton City to see production methods of a large producer of certified grapevines. Hosted by Kevin Judkins. Then travel to Golden Gate Hop Ranch to see hop harvest, picking and processing. Hosted by Brad Studer. Wine tasting and winery tour at Mercer Wines in Prosser hosted by Rick Hamman.

After lunch, travel to diseased cherry block in Buena to see foliar symptoms and tree decline that appears to be associated with a specific rootstock. No fungal or bacterial agents could be detected in two diagnostic laboratories and the only virus detected is Cherry virus A. In an older portion of the orchard, symptoms of Cherry leaf roll virus were shown. Hosted by Sally Moffat.

Travel to Granger to see an orchard affected by Pear decline phytoplasma and a cherry orchard where symptoms of rusty mottle disease, Western X and Cherry rasp leaf virus were seen. Visits organized by Lauri Guerra. The group traveled to Prosser organized by Naidu Rayapati to observe a vineyard block infected with GLRaV3. The block is being monitored annually by Naidu to observe natural spread to new plantings and to determine impact of virus on berry quality.

Minutes prepared and submitted respectfully by John Hu and Kenneth Eastwell.

Full State reports can be viewed on the WERA-20 Project Homepage under the tab "Additional Documents" at: http://lgu.umd.edu/lgu_v2/homepages/attachs.cfm?trackID=13496

Accomplishments

Members of the WERA-020 from different states have made rapid advancements in the discovery of new viruses using state-of-the-art technologies such as next-generation sequencing methods. These approaches have resulted in documenting the occurrence of several new and emerging viruses and virus-like agents from tree fruits, small fruits, citrus, and grapevines across the US (including Hawaii) and Canada. In addition, the extensive sequence data generated by deep-sequencing technologies has been used to develop highly sensitive and reliable diagnostic assays for applications in National Clean Plant programs and improving the sanitary status of vineyards and orchards. Field surveys of tree fruits, grapevines and small fruits and testing suspected samples using sensitive and high throughput diagnostic methodologies have provided science-based information on the distribution of viruses across the continent. Members of the group have also examined impacts of virus diseases on crop yield and fruit quality attributes to better appreciate the economic significance of virus diseases. Members also pursued outreach activities to enhance awareness of virus diseases among various stakeholders and translate science-based knowledge for deploying disease management strategies.

Publications

1. Abou Ghanem-Sabanadzovic N., S. Sabanadzovic, P. Gugerli and A. Rowhani, 2012. Genome organization, serology and phylogeny of Grapevine leafroll-associated viruses 4 and 6: taxonomic implications. Virus Research 163: 120-128. 2. Alabi, O.J., Zheng, Y., Jagadeeswaran, G., Sunkar, R., and Naidu, R.A. 2012. High-throughput sequence analysis of small RNAs in grapevine (Vitis vinifera L.) affected by grapevine leafroll disease. Molecular Plant Pathology 13: 1060-1076. 3. Al Rwahnih, M., Sudarshana, M.R., Uyemoto, J.K., and Rowhani, A.. 2012. Complete genome sequence of a novel vitivirus isolated from grapevine J. Virol. 86:9545. 4. James, D., Varga, A. 2012. Sequence analysis of RNA 1 of lilac leaf chlorosis virus supports a close relationship to subgroup 3 ilarviruses. Archives of Virology. 157: 203-206. 5. Kanuya, E., Clayton, L.A., Naidu, R.A., and Karasev, A.V. 2012. First report of Grapevine fleck virus in Idaho grapevines. Plant Disease 96: 1705-1705. 6. Laney, A. G., Hassan, M. and Tzanetakis, I. E. 2012. An integrated badnavirus is prevalent in fig germplasm. Phytopathology 102: 1182-1189. 7. Li, R., Kinard, G., Mock, R., Forsline, P., Pooler, M., and Stover, E. 2012. A survey for viruses and virus-like pathogens in U.S. cherry genetic resources. Petria 22: 208220. 8. Lin, L., Li, R., Mock, R., and Kinard, G. 2012. One-Step multiplex RT-PCR for simultaneous detection of four pome tree viroids. European Journal of Plant Pathology 133: 765-772. 9. Martelli G.P., N. Abou Ghanem-Sabanadzovic, A.A. Agranovsky, M. Al Rwahnih, V.V. Dolja, C.I. Dovas, M. Fuchs, P. Gugerli, J.S. Hu, W. Jelkmann, N.I. Katis, V.I. Maliogka, M.J. Melzer, W. Menzel, M.E. Rott, A. Rowhani, S. Sabanadzovic & P. Saldarelli, 2012. Taxonomic revision of the family Closteroviridae with special reference to the grapevine leafroll-associated members of the genus Ampelovirus and the putative species unassigned to the family. Journal of Plant Pathology 94:7-19. 10. Martin, R.R., Polashock, J.J., and Tzanetakis, I.E. 2012. New and emerging viruses of blueberry and cranberry. Viruses 4: 2831-2852. 11. Melzer, M. J., Sether, D.M., Borth, W.B., and Hu, J.S. 2012. Characterization of a virus infecting Citrus volkameriana (Ten. & Pasq.) with citrus leprosis-like symptoms. Phytopathology 102: 122-127. 12. Melzer, M. J., Ayin, C., Sugano, J.S., Uchida, J.Y., Kawate, M.K., Borth, W.B., Sether, D,M., and Hu, J,S. 2013. Differentiation and distribution of Cordyline viruses 14 in Hawaiian plants (Cordyline fruticosa L.). Viruses 5:1655-1663. 13. Poudel, B., Sabanadzovic, S., Bujarski, J. and Tzanetakis I.E. 2012. Population structure of Blackberry yellow vein associated virus, an emerging crinivirus. Virus Research 169: 272275. 14. Schilder, A. 2012. Overview of grape diseases during the 2012 growing season. Michigan Grape and Wine Newsletter 2 (14): 4-5. 15. Schilder, A. 2012. Last chance to participate in grape virus diagnostic survey. Michigan Grape and Wine Newsletter 2 (13): 5. 16. Schilder, A. 2012. Recent observations of disease and other symptoms in grape vineyards. Michigan Grape and Wine Newsletter 2 (12): 3-4. 17. Schilder, A. 2012. Grape virus diagnostic support during the 2012 growing season. Michigan Grape and Wine Newsletter 2 (5): 5-6. 18. Sether, D.M., Melzer, M. J., Borth, W.B., and Hu, J.S. 2012. Characterization, Diversity, Distribution, and Transmission of a Pineapple Badnavirus in Commercial Pineapple Hybrids and Pineapple Germplasm Accessions in Hawaii. Plant Disease 96:1798-1804. 19. Sheveleva, A., Ivanov, P., Prihodko, Y., James, D.,and Chirkov, S. 2012. Occurrence and genetic diversity of Winona-like Plum pox virus isolates in Russia. Plant Disease 96:1135-1142. 20. Sheveleva, A., Ivanov, P., Prihodko, Y., James, D., and Chirkov, S. 2012. Plum pox virus W appears to be the most variable strain of the seven recognized strains of the virus. Petria 22: 226-232. 21. Wise, J. C., Gut, L. J., Isaacs, R., Schilder, A. M. C., Sundin, G. W., Zandstra, B., Hanson, E., and Shane, B. 2012. Michigan Fruit Management Guide 2013. Extension Bulletin E-154. Michigan State University, East Lansing, MI. 22. Abou Ghanem-Sabanadzovic, N., Tzanetakis, I.E., and Sabanadzovic, S. 2013. Rubus canadensis virus 1, a novel betaflexivirus identified in blackberry. Archives of Virology 158:445449. 23. Alabi, O.J., Poojari, S., Sarver, K., Martin, R.R., and Naidu, R.A. 2013. Complete Genome Sequence Analysis of an American isolate of Grapevine virus E. Virus Genes 46:563566. 24. Al Rwahnih, M., Daubert, S., Sudarshana, M.R. and Rowhani, A. 2013. Gene from a novel plant virus satellite from grapevine identifies a viral satellite lineage. Virus Genes 47: 114-118. 25. Al Rwahnih, M., Dave, A., Anderson, M. M., Rowhani, A., Uyemoto, J. K., and Sudarshana, M. R. 2013. Association of a DNA virus with grapevines affected by red blotch disease in California. Phytopathology 103:1069-1076. 26. Bahder, B. W., Alabi, O., Poojari, S., Walsh, D. B., and Naidu, R. A. 2013. A survey for grapevine viruses in Washington State Concord (Vitis × labruscana L.) vineyards. Online. Plant Health Progress doi:10.1094/PHP-2013-0805-01-RS. 27. Dhekney S.A., Vardiman J., Brock B., Fisher L., Kandel, R., Bergey D. 2013. Optimizing tissue culture protocols for cold-hardy grape cultivars and rootstocks. University of Wyoming, Agricultural Experiment Station, Field Days Bulletin, 113-115. 28. Dhekney S.A., Vardiman J., Brock B., Fisher L., Kandel, R., Bergey D. 2013. Production of disease-free grapevines using tissue culture technology. University of Wyoming, Agricultural Experiment Station, Field Days Bulletin, 117-118. 29. Dhekney S.A., Vardiman J., Kandel, R., Smith D. 2013. Screening grapevine cultivars for adaptability to soil and climatic factors in Wyoming. University of Wyoming, Agricultural Experiment Station, Field Days Bulletin, 119-120. 30. Finn, C.E., Moore, P.P., Yorgey, B.M., Strik, B.C., Kempler, C., Dossett, M. and Martin, R.R. 2013. Charm strawberry. HortScience (Accepted July 3, 2103). 31. James, D., Varga, A., and Sanderson, D. 2013. Genetic diversity of Plum pox virus: strains, disease and related challenges for control. Canadian Journal of Plant Pathology DOI:10.1080/07060661.2013.828100. 32. James, D., Varga, A., Jesperson, G.D., Navratil, M., Safarova, D., Constable, F., Horner, M., Eastwell, K., and Jelkmann, W. 2013. Identification and complete genome analysis of a virus variant or putative new foveavirus associated with apple green crinkle disease. Archives of Virology 158:1877-1887. 33. James, D., Sanderson, D., Varga, A., Sheveleva A., and Chirkov, S. 2013. Comparison of Plum pox virus strain W isolates to determine their relationships and further understand their genetic diversity. 2nd International Symposium on Plum Pox Virus, Olomouc Czech Republic, September 3 6, 2013. Book of Abstracts. Pg 14. (Abstr.). 34. James, D., Sanderson, D., Varga, A., Greig, N., and Stobbs, L. 2013. Relationships and genetic diversity of selected Canadian isolates of Plum pox virus. 2nd International Symposium on Plum Pox Virus, Olomouc Czech Republic, September 3 6, 2013. Book of Abstracts. Pg 15 (Abstr.). 35. Maree, H.J., Almeida, R.P., Bester, R., Chooi, K., Cohen, D., Dolja, V.V., Fuchs, M.F., Golino, D.A., Jooste, A.E., Martelli, G.P., Naidu, R.A., Rowhani, A., Saldarelli, P., and Burger, J. 2013. Grapevine leafroll-associated virus 3. Frontiers in Microbiology 4:82. 36. Martin, R.R. and Tzanetakis, I.E. 2013. High risk strawberry viruses by region in the United States and Canada: Implications for certification, nurseries and fruit production. Plant Disease 97: 1358-1362. 37. Martin, R.C., Glover-Cutter, K., Martin, R.R. and Dombrowski, J.E. 2013. Virus induced gene silencing in Lolium temulentum. Plant Cell Tissue Organ Cult. 113:163-171. DOI 10.1007/s11240-012-0257-z. 38. Martin, R.R., MacFarlane, S., Sabanadzovic, S., Quito-Avila, D.F., Poudel, B., and Tzanetakis, I.E. 2013. Viruses and virus diseases of Rubus. Plant Disease 97:168-182. 39. Mekuria, T., Druffel, K.L., Susaimuthu, J., and Eastwell, K,C. 2013. Complete nucleotide sequence of a strain of Cherry mottle leaf virus associated with peach wart disease in peach. Archives of Virology. DOI 10.1007/s00705-013-1698-3. 40. Mekuria, T., Smith, T.J., Beers, E.H., and Eastwell, K.C. 2013. Little cherry virus 2 is transmitted to sweet cherry by Pseudococcus maritimus (Ehrhorn), a new vector of this virus. Plant Disease 97:851. 41. Melzer, M.J., Sugano, J.S., Uchida, J.Y., Kawate, M.K., Borth, W.B., Sether, D.M., Hu, J.S. 2013. Molecular characterization of closteroviruses infecting Cordyline fruticosa (L.) in Hawaii. Frontiers in Microbiology doi: 10.3389/fmicb.2013.00039. 42. Melzer, M.J., Nelson Simbajon, N., Carillo, J., Borth, W.B., Freitas-Astúa, J., Kitajima, EW, Neupane, K.R., and Hu, J.S. 2013. A cilevirus infects ornamental hibiscus in Hawaii. Arch. Virol. Doi:10.1007/s00705-013-1745-0 43. Pallas, V.,Aparicio, F., Herranz , M.C., Sanchez-Navarro, J.A., and Scott, S.W. 2013. The Molecular Biology of ilarviruses. Advances in Virus Research 87:139 -182. 44. Peres, N.A., Whidden, A., Smith, H. and Martin, R.R. 2013. Aphid-borne viruses detected in strawberry plants shipped to Florida. Berry and Vegetable Times, Jan 2013, Pp5-6, Univ of Florida IFAS Extension. 45. Poojari, S., Alabi, O.J., Fofanov, V.Y., and Naidu, R.A. 2013. A leafhopper-transmissible DNA virus with novel evolutionary lineage in the family Geminiviridae implicated in grapevine redleaf disease by next-generation sequencing. PLoS ONE 8(6): e64194. 46. Poudel, B., Wintermantel, W.M., Cortez, A.A., Ho, T., Khadgi, A. and Tzanetakis, I.E. 2013. Epidemiology of Blackberry yellow vein associated virus. Plant Disease 97: 1352-1357. 47. Poudel, B., and Tzanetakis I.E. 2013. Population structure of blackberry chlorotic ringspot virus in the United States. Archives of Virology 158: 667-672. 48. Quito-Avila, D.F., Brannen, P.M., Cline, W.O., Harmon, P.F. and Martin, R.R. 2013. Genetic characterization of Blueberry necrotic ring blotch virus, a novel RNA virus with unique genetic features. J. Gen. Virol. 94:1426-14343. 49. Quito-Avila, D.F., Ibarra, M.A., Alvarez, R.A., Espinoza, L., Ratti, M.F., Peralta, E.L. and Martin, R.R. 2013. First report of Raspberry bushy dwarf virus in the Andean blackberry (Rubus glaucus) in central Ecuador. Plant Dis. 97:1003. 50. Rojas, P., Almada, R.D., Sandoval, C., Keller, K.E., Martin, R.R. and Caligari, P.D.S. 2013. Occurrence of aphidborne viruses in southernmost South American populations of Fragaria chilensis ssp. chiloensis. Plant Pathology 62:428-435. 51. Skinkis, P., Pscheidt, J., Peachy, E., Dreves, A., Walton, V., Sanchez, D., Zasada, I. and Martin, R.R. 2013. Pest Management Guide for Wine Grapes in Oregon. Oregon State Univ. Ext. Bull. 52. Villamor, D.E., Druffel, K.L., and Eastwell, K.C. 2013. Complete nucleotide sequence of a virus associated with rusty mottle disease of sweet cherry (Prunus avium). Archives of Virology 158:18051810. 53. Tzanetakis, I.E. and Martin, R.R. 2013. Expanding field of strawberry viruses which are important in North America. Intern. J. Fruit Sci. 13:184195. 54. Tzanetakis I.E., Martin, R.R., and Wintermantel W.M. 2013. Epidemiology of criniviruses, an emerging problem in world agriculture. Frontiers in Microbiology 4:119.

Impact Statements

The research-outreach efforts pursued by members of the WERA-020 have impacts to bear on the sustainability of fruit crops in the US.
Sharing knowledge among colleagues from across the US (including Hawaii) and Canada promoted collaborative efforts to tackle virus and virus-like disease problems as a unified approach for mitigating negative impacts of economically important diseases affecting perennial fruit crops and to ensure reliable supply and propagation of clean plant materials benefiting growers.
The project members have shared science-based information via several dissemination pathways that led to increased knowledge of virus diseases among industry stakeholders and regulatory agencies.
Planting healthy fruit crops will lead to: (i), production of quality fruits with desirable health benefits and increased opportunities for global competitiveness of fruits and fruit products produced by American growers, and (ii), reduction in usage of pesticides to control insect vectors of plant viruses and virus-like agents thereby contributing to reduced environmental pollution and human health risks.

Date of Annual Report: 06/25/2014

Report Information

Annual Meeting Dates: 05/29/2014 - 06/03/2014
Period the Report Covers: 09/01/2013 - 10/01/2014

Participants

Jim Moyer (AA), WSU, Director-Agricultural Research Center, j.moyer@wsu.edu

Margarita Bateman, USDA-APHIS-PPQ
Margarita.L.Bateman@aphis.usda.gov

Wayne Borth, University of Hawaii, borth@hawaii.edu

Jay Bost, University of Hawaii, jbost@hawaii.edu

Kishore Dey, University of Hawaii, kishore@hawaii.edu

Sadanand Dhekney, University of Wyoming, sdhekney@uwyo.edu

Steven Ferreira, University of Hawaii, stephenf@hawaii.edu

Deborah Golino, Foundation Plant Services, University of California, dagolino@ucdavis.edu

Lauri Guerra, WA State Department of Agriculture, lguerra@agr.wa.gov

Richard Hoenisch, Department of Plant Pathology, UC Davis, rwhoenisch@ucdavis.edu

John Hu, University of Hawaii, johnhu@hawaii.edu

Alexander Karasev, University of Idaho, akarasev@uidaho.edu

Richard Manshardt , University of Hawaii, manshard@hawaii.edu

Mark Nakhla, USDA APHIS PPQ, Mark.K.Nakhla@aphis.usda.gov

Adib Rowhani, Foundation Plant Services, Univ. of California, akrowhani@ucdavis.edu

Erich Rudyj, USDA-APHIS NCPN, Erich.S.Rudyj@aphis.usda.gov

Maher Rwahnih, Foundation Plant Services. Univ. of California, malrwahnih@ucdavis.edu

Sead Sabanadzovic, Mississippi State University, ssabanadzovic@entomology.msstate.edu

Anna-Mary Schmidt, Canadian Food Inspection Agency, anna-mary.schmidt@inspection.gc.ca

Ioannis Tzanetakis, University of Arkansas, itzaneta@uark.edu

Dan Villamor, Washington State University, dvillamor@wsu.edu

Alan Wei, Agri-Analysis LLC, West Sacramento, CA, ceos07@gmail.com

Shulu Zhang, Agdia Inc. Indiana, shulu@agdia.com

Brief Summary of Minutes

Thursday, May 29, 2014

(Ala Moana Hotel, 410 Atkinson Dr, Honolulu, HI 96814)

Part 1. General session

Opening of meeting by the Chair of the WERA 20 group, Dr. John Hu.

Welcome by Dr. Kenneth J. Grace, Interim Associate Dean of College of Tropical Agriculture and Human Resources at University of Hawaii

Part 2. State Reports

Wyoming (Sadanand Dhekney):

Reported on the identification of promising grape cultivars with increased tolerance to different abiotic stresses, especially cold tolerance; as part of this screening process, sampled cultivars are also tested for the presence of different viruses. An overview on Precision Breeding was presented, that is defined as using sequences from the grape genome rather than from other species (cis-genic rather than trans-genic). In this regard, functionally equivalent segments of the grape genome to replace the left and right borders of the Agrobacterium transformation vector was reported along with using anthocyanin grape sequences as reporter gene.

A group photo was taken during the coffee break time.

Washington (Kenneth Eastwell and Dan Edward Villamor):

Reported on the update of the little cherry disease (LCD) outbreak in Douglas and Chelan counties of the state. Approximately 400 acres of sweet cherry block have been eradicated due to the disease and an estimated 300 acres will be eradicated this year. A recombinase-polymerase amplification assay (RPA) was developed for Little cherry virus 2, the main virus associated with LCD. This assay, which can be used in the field as a rapid and sensitive assay for the virus, is now commercially available as a kit. A total of 242 grapevines from the screenhouse and foundation block of the Clean Plant Center Northwest (CPCNW) were indexed for Grapevine red blotch associated virus (GRBaV) and Grapevine vein clearing associated virus (GVCV). None except one recent accession was tested positive for GVCV. This accession has been removed from the foundation block. Data on deep sequencing of 30 grapevine and 31 stone and pome fruit tree accessions showed the potential of deep sequencing as tool in grape and fruit tree virus certification programs.

Washington (Naidu Rayapati):

The genetic diversity of Grapevine virus A in California (with Maher Rwahnih) and Washington vineyards was analyzed using two functional classes of virus genes, a transcription-related (RNA-dependent RNA polymerase [RdRp]) and a structural (CP) gene. The data showed presence of divergent variants of GVA in both Washington and California vineyards. In many cases, distinct variants of GVA were found as mixtures in individual grapevines. Survey of juice grape (Vitis × labruscana L. ‘Concord’) vineyards indicated the presence of three grapevine leafroll-associated viruses (GLRaVs) and Grapevine fanleaf virus. Among the GLRaVs, GLRaV-2, -3, -4 and its strain -9 were documented so far in ‘Concord’ vineyards. GLRaV-3 was found to be widely distributed in ‘Concord’ vineyards. In collaboration with entomology (D. Walsh), we demonstrated that grape mealybug (Pseudococcus maritimus Ehrhorn) and European fruit lecanium scale (Parthenolecanium corni Bouché) are capable of transmitting GLRaV-3 between wine grapes (V. vinifera) and juice grapes. The results were disseminated through extension and outreach presentations for the benefit of grape growers, certified nurseries, regulatory agencies and other industry stakeholders.

Mississippi (Sead Sabandzovic):

Reported on the identification of six viruses associated with Blackberry yellow vein disease (BYVD), two of which are newly described. Viruses that infect muscadines were also reported, which are the following: Grapevine Syrah virus 1, Grapevine virus B, Grapevine leafroll associated virus 2, a new marafivirus and two other new viruses that are still being characterized. In addition, he shared the results of the screening of Muscadines and other grape cultivars for Rupestris stem pitting-associated virus (RSPaV); RSPaV amplicons that were sent for sequencing corresponded to host sequences, highlighting the need to further investigate PCR results and proceed to downstream sequencing when the PCR data seems questionable. A petition for NCPN support through the creation of a grape center in Mississippi was mentioned.

Maryland (Erich Rudyj):

Reported on the history of the National Clean Plant Network (NCPN); its creation, roles and responsibilities and source of funding. Originally under 2008 Farm Bill (2008-2014), the funding for NCPN was renewed for five years from 2014-2018. The US House of Representatives recognized the value of NCPN and made it permanent by including the network in the Plant Protection Act. A consequence of such change is the surrendering of the no-year funding option for the network but a guaranteed funding of no less than $5M annually. Several critical and emerging issues on NCPN activities were discussed, among which include specialty crop expansion such as roses and sweet potato and the adoption of next generation sequencing for pathogen detection.

Maryland (Margarita Bateman):

Reported on the activities of the USDA-APHIS Plant Germplasm Quarantine Program, particularly on the quarantine program for stone fruits. She mentioned that the Post-entry Quarantine is now known as Controlled Import Permits and reported on the development of a tissue culture system for Prunus as the major accomplishment of the program last year. The review of the French Fruit Certification Program was also reported, citing the similarities and differences by which plant material is accepted in the US and Canada. The results of the visit revealed that the French Fruit Tree Certification Program meets the requirements of both US and Canada.

Maryland (Mark Nakhla):

Reported on the activities and functions of the Center for Plant Health Science and Technology (CPHST). The organization certifies laboratories, develops and validates molecular diagnostic methods, provide training on diagnostics and evaluate new diagnostic technologies. He also reported on Biosensors for rapid detection of regulated plant pathogens called CANARY (Cellular Analysis and Notification of Antigen Risks and Yield). These cells are available for Ralstonia, Potyvirus, Phytophthora, Citrus leprosis virus C (CiLV-C) and citrus variegated disease pathogen, Xyllela fastidiosa.

Indiana (Shulu Zhang):

Reported on the development and availability of AmplifyRP kits for the detection of several pathogens such as Plum pox virus PPV), Candidatus liberibacter asiaticus and recently, Little cherry virus 2 (LChV-2). This technology is an isothermal detection based on recombinase-polymerase assay. The kits provide rapids tests for these pathogens, which take only 15 minutes to achieve the result, and are amenable to crude sap preparations, unlike PCR that require purified nucleic acid material. The kits are available as lyophilized reagents containing buffers, enzyme mix and primers for end-point detection and real time detection. A basic kit called Discovery kit does not contain the primers for the pathogen is also available, allowing flexibility for users to design and use their own primers.

Idaho (Alex Karasev):

The survey of Idaho vineyards revealed the presence of Grapevine leafroll associated viruses 1, 3, 4 and 5, Rupestris stem pitting aassociated virus (RSPaV), Grapevine virus A and B and Grapevine fleck virus (GFkV). The most prevalent among these viruses is GLRaV-3 followed by GLRaV-1. The commercially available antibody against GLRaV-3 has been consistently reliable in detecting the virus but not for GLRaV-1. In his laboratory, antibody was produced against GLRaV-1 using bacterially expressed coat protein of the virus as antigen for immunization. The resulting antibodies were optimized for triple antibody sandwich enzyme-linked immonosorbent assay (TAS-ELISA). This assay is now used in the laboratory for detection of GLRaV-1.

Hawaii (Steve Ferreira):

Presented an overview of the papaya industry in Hawaii, with particular emphasis of the devastating effect of the Papaya ringspot virus (PRSV) and how the genetically modified papaya came to rescue the industry. The transgenic papaya cultivar ‘Rainbow’ was developed by inserting a fragment of the coat protein gene of PRSV. The mechanism of resistance operates through RNA silencing.

Hawaii (Richard Manshardt):

Reported on the transgene flow of genetically modified (GM) papaya to non GM papaya through pollen drift study. This study showed that about 22% of GM papaya plants were observed in feral papaya populations, female papaya plants are the only ones susceptible to transgene flow and transgene flows primarily by seed. Overall, transgene flow into feral papaya populations will be slow and gradual.

Formal meeting adjourned at 5:45pm followed by a group dinner.

Friday 30th May, 8:30am (Oahu, HI)

Field trips were provided. The first stop was at the Waimanalo Research Station, College of Tropical Agriculture and Human Resources (CTAHR), University of Hawaii Manoa (UHM). A field project on the screening of banana germplasm for resistance to Banana bunchy top virus (BBTV), the transgenic papaya block, and the sweet potato germplasm collection were shown. The second stop was the tour of the subtropical fruit tree orchard of the Pearl City Urban Garden Center where collections of citrus, mango, guava, breadfruit, avocado and many others were shown. Group lunch was provided followed by the third stop at Dole Plantation. The group started at the visitor center followed by a visit to the cacao and coffee plantations.

Monday 2nd June, 8:30am (Hilo, HI)

Field trips on the Big Island were provided. The group started at Mauna Loa Visitor Center to check the macadamia nut orchards and the nut and chocolate processing plant. A tour of the facilities as well as the subtropical fruit tree germplasm collection of the National Clonal Germplasm Repository of the United Stated Department of Agriculture-Agricultural Research Service at the Pacific Basin Agricultural Research Center (USDA-ARS-PBARC) and Waiakea Research Station, CTAHR, UHM were shown. After group lunch, the group visited Plant it Hawaii to see production methods of a wholesale nursery of subtropical fruit trees. The final stop was at the Volcano winery. A tour of the winery’s vineyard and tea plantation was provided. Foliar symptoms typical of Grapevine leafroll disease was observed in some wine grape cultivars in the vineyard. This was followed by wine and tea tasting at the winery.

Tuesday 2nd June, 8:30am

(University of Hawaii at Hilo, 200 W. Kawili St, Hilo, HI 96720)

Comments by Administrative Advisor, Dr. Jim Moyer, Director of Agricultural Research Center
and Associate Dean for Research for the College of Agricultural, Human and Natural Resource Sciences at Washington State University. He reminded that the level of accountability for each one is increasing and stressed the importance of regional projects. He also reminded to be mindful of the submission of final report within thirty days and underscored the impact statement in the report as well as evidence of collaboration among peers.

Two suggestions were proposed for the location of next year’s meeting, Arkansas and Maryland.
The group decided to go to Maryland next year. Margarita Bateman will be the host of the next
year’s meeting and the Chair of WERA-20 next year. John Hu will be the Vice-chair.

Continuation of Part 2: State Reports

Hawaii (Mike Melzer and John Hu):

Reported that the economic potential of growing fig in Hawaii is huge. Viruses infecting fig has
not yet been reported in Hawaii but virus-like symptoms on fig were observed. Next generation
sequencing using 454 platform and dsRNAs from symptomatic fig revealed the presence of
virus-like contigs that showed high identities with several closteroviruses, an umbra-, myco-,
badna- and emaravirus, and Tobacco necrosis statellite virus. Characterization of these
virus-like contigs is currently being pursued.

Canada (Anna-Mary Schmidt):

Reported on the activities of Canadian Food Inspection Agency (CFIA). The agency is the
national post quarantine facility for grapevines, tree fruit and small fruits in Canada which
provide testing activities (bioassays, ELISA, PCR and next generation sequencing), virus
elimination, tissue culture, maintenance of generation 1 virus tested grapevine and tree fruit
repository. She also reported on the grapevine virus status in Canadian vineyards. GLRaV -3 is
most prevalent in British Columbia (BC) followed by GLRaV-2, mixed infection of GLRaV-2
and -3 and GLRaV-1. GLRaV-1, -2 and -3 is also prevalent in Ontario as well as Grapevine red
blotch associated virus (GRBaV).

California (Deborah Golino):

Reported on the progress of Grape Tier II of NCPN. The goals and objectives of NCPN were
briefly mentioned including the specialty crop groups that are included. Progress on the grape
clean plant centers throughout the country was summarized. For each grape center, this included
the number of cultivars that were indexed for viruses, number of cuttings released as well as the
number of vines planted in the screenhouses and foundation blocks. The renewal of the NCPN
funding for 2014 was also mentioned.

California (Maher Al Rwahnih and Adib Rowhani):

Reported on the development and application of next generation sequencing (NGS) as a new
technology for testing new grapevine accessions. The advantages and disadvantages of NGS
were mentioned. The cost of NGS has significantly decreased over the last seven years from
$4K to $500 per sample, making this technology amenable for large scale use. Several grapevine
viruses were characterized through NGS such as Grapevine Syrah virus, GRBaV, Grapevine
virus F (GVF), Grapevine vein clearing virus (GVCV). These viruses reacted poorly or none at
all with known field woody indicators and would not have otherwise been discovered or would
take longer time to be discovered using the current tools if not for NGS. Careful attention,
however, must be made as virus/viruses discovered by NGS still require the fulfillment Koch’s
postulate.

California (Alan Wei):

Reported on the work funded by a USDA SBIR project. They made a new antibody reagent for
GLRaV-3 which showed broader detection spectrum than the mostly commonly used antibody
reagent from BioReba. Their antibody reacted positively to several samples which the BioReba
antibody did not. After testing these samples by RT-PCR, sequencing revealed that the
amplicons were possibly divergent strain of GLRaV-3. This discovery should have potential
impact on certification programs because most certification programs Bioreba reagent which
may have let some strains of GLRaV-3 escape detection. The preliminary data for GLRaV-3
nanobodies was also presented. Nanobodies consist of a 15K single monomeric variable
antibody domain. Like regular antibodies, nanobodies show high affinity to their target antigen
but are more thermally stable than regular antibodies.

California (Richard Hoenisch):

Reported on the activities of the National Plant Diagnostic Network (NPDN). The network is a
consortium of plant diagnostic laboratories all over the country and is subdivided into five
regions. The networks primary purpose is to provide a system that quickly detects and identifies
pests and pathogens that are of concern to a particular area. This is mainly achieved by
providing laboratory infrastructure and training and network development of first detectors. Pest
risk assessment was also discussed in order to mitigate or avoid potential outbreak in a particular
area. The importance of this activity was highlighted by providing historical case studies of pest
and diseases that have devastated agricultural crops in the country. Present day examples of high
risk pests were also enumerated.

Arkansas (Ioannis Tzanetakis):

Reported on the bioinformatics pipelines to follow in analyzing NGS data. The software
developed in his laboratory, called Virfind, is already available which can be used by outside
users with little training and supervision. The software also allows changing analysis parameters
depending on user’s requirement. The value of NGS in small fruit virus detection was
underscored by the fact that of the 30 viruses already discovered in blueberries, most do now
induce symptoms on known indicators as typified by Blueberry mosaic virus. The
overview of the certification programs for blueberry and Rubus was also described and a
proposal for the process of certification was presented.

State reports finished at 12:00 pm followed by a group lunch.

Group discussion about the issues pertaining to NGS:

(2:00 pm, USDA-ARS-PBARC 64 Nowelo St. Hilo, Hawaii 96720)

Welcome by Dr. Marissa Wall, Acting Director of USDA-ARS-PBARC at Hilo, HI. The group
was given a brief overview of the research center. Its main objective is to make sure invasive
pest species do not enter the state and, if by any chance they get in, prevent at any circumstance
dissemination of the pest out of the state. Another purpose is to help farmers transition from
plantation to diversified crops. A tour of the laboratory facilities of the center was provided
before the group discussion started.

During the group discussion on the impact of NGS on virus diagnostics, it was suggested that
separate working group on NGS that focuses on the (1) technical/scientific aspect of the
technology and (2) legal/policies involved with it be created.

(1) The technical working group will provide workshop regarding the standard methodologies that come with different NGS platforms. This group will be responsible for organizing dry laboratory training on the proper analysis and interpretation NGS sequence data. Case studies regarding the development and use of the technology for different crop specialty groups, especially for fruit trees, grapes and berries, will be helpful so that protocols for each crop can be compiled and validated.
(2) The legal working group will be responsible for convincing regulators to approve the use of NGS technology in certification programs. This will require substantial amount of data, complied from the technical working group, showing the robustness of the NGS over current technology (PCR, ELISA and biological indexing on indicators) in detecting viruses from different crops. An important component of this endeavor is to show the process and speed by which NGS could assist in releasing a crop accession on a shorter period of time.

The group recognized issues that arose with the current technology. The output of NGS has already revealed new viruses which do not either have strong association with a disease or no association with a disease at all. In scenarios like this, whether these types of virus or virus-like agents will be ignored in certification programs are still not clear. Another issue is the international acceptance of the technology by other countries. Data generated by NGS could be used by regulators from other countries to restrict/prohibit the entry of planting material or produce into their respective countries. This is less of an issue with quarantine programs since new viruses detected by NGS will eventually be eliminated. But careful attention must be made in publishing new viruses in commercial orchards. Outreach and education is necessary to address the implications of NGS results.

The meeting ended with a proposed agenda for next year’s meeting in Maryland, which will be composed of three parts:

(1) NGS workshop (1 day)
(2) Field trip and state reports (3 days)
(3) Policy discussion with some invited regulators on NGS (1 day)

Formal annual meeting of WERA-20 adjourned at 4:00 pm.

Minutes prepared and submitted respectfully by Dan Villamor and John Hu.

Accomplishments

Collaborative efforts by the members of the WERA-20 have brought together unique experiences and cutting-edge technologies to advance the detection and characterization of new and emerging viruses and virus-like agents from fruit trees, small fruits, citrus, and grapevines in the US and Canada. Powerful next-generation sequencing (NGS) technology was used extensively in the past year to identify new viruses and virus-like agents from grapevines, blueberries, pome and stone fruit, and edible fig. Comprehensive studies have been conducted in various crop systems to compare the current bioassay systems with NGS for their relative sensitivity and reliability. Progress has been made in the application of NGS technology in quarantine systems that regulate trade between states and countries. Sensitive and reliable diagnostic assays that have been developed by WERA-20 members have been shared with labs of various federal and state agencies as well as centers of the National Clean Plant Network. Novel virus detection systems including recombinase polymerase amplification and nanobody technology have been developed and applied for the rapid detection and reliable diagnosis of viral diseases. The molecular diversity of viruses has been revealed using sensitive and high throughput diagnostic methodologies. Science-based information on the distribution of viruses and transmission of new viruses by insect vectors were obtained and shared with industries to develop practical and environmentally sound strategies for managing the virus and virus-like diseases of fruit trees, small fruits, citrus, and grapevines. Latest information on viruses and vectors was also shared with federal and state regulatory agencies for revising policies to contain spread of viruses.

Publications

Abou Ghanem-Sabanadzovic N., Tzanetakis, I.E. and Sabanadzovic, S. 2013. Rubus canadensis virus 1, a novel betaflexivirus identified in blackberry. Archves of Virology 158: 445-449. Al Rwahnih, M., Daubert, S., Sudarshana, M., and Rowhani, A. 2013. Gene from a novel plant virus satellite from grapevine identifies a viral satellite lineage. Virus Genes 47: 114-118. Al Rwahnih, M., Dave, A., Anderson, M., Rowhani, A., Uyemoto, J. K., and Sudarshana, M. 2013. Association of a DNA virus with Grapevines affected by Red 1 Blotch disease in California. Phytopathology 103:1069-76. Al Rwahnih, M., S. Daubert, C. Islas, D.A. Golino, and A. Rowhani. 2014. Description of a fifth Vitivirus in grapevine. Journal of Plant Pathology. 96: 219-222. Alabi, O. J., Al Rwahnih, M., Mekuria, T. A., Rowhani, A., and Naidu, R. A. 2014. Genetically diverse isolates of Grapevine virus A are present in Washington and California vineyards. Phytopathology. 104: 548-560 Alabi, O.J., Al Rwahnih, M., Mekuria, T., and Naidu, R.A. 2014. Genetic diversity of Grapevine virus A in Washington and California vineyards. Phytopathology 104:548-560. Bahder, B.W., Naidu, R.A., Daane, K.M., Millar, J.G., and Walsh, D.B. 2013. Pheromone-based monitoring of Pseudococcus maritimus (Hemiptera: Pseudococcidae) populations in Concord grape vineyards. Journal of Economic Entomology 106:482-490. Bahder, B. W., Poojari, S., Alabi, O.J., Naidu, R.A., and Walsh, D. B. 2013. Pseudococcus maritimus (Hemiptera: Pseudococcidae) and Parthenolecanium corni (Hemiptera: Coccidae) are capable of transmitting Grapevine leafroll-associated virus 3 between Vitis labruscana and Vitis vinifera. Environmental Entomology 42: 1292-1298. Dhekney S.A., Kandel, R., Garcia y Garcia A. 2014. University of Wyoming-Grape Growers Partnership may Improve Sustainable Grape Production in Wyoming. University of Wyoming, Agricultural Experiment Station, Field Days Bulletin, In Press. Doan, H.K., Zhang, S. and Davis, M.R. 2014. Development and Evaluation of AmplifyRP Acceler8 Diagnostic Assay for the Detection of Fusarium oxysporum f. sp. vasinfectum Race 4 in Cotton. Plant Health Progress 15(1):48-52. Ghimire., P., Abou Ghanem-Sabanadzovic, N., Tzanetakis, I.E. and Sabanadzovic, S. 2013. Partial characterization of a novel flexivirus from blackberry. 90th Meeting of the Southern Division of the American Phytopathological Society, February 8-10, 2013, Baton Rouge, LA Golino, D.A., Rowhani, A. and Uyemoto, J.K. 2013. “Grapevine Virus Diseases”. In : Grape Pest Management, third edition. Pp: 157-173. Ed: Larry J. Bettiga, University of California, Agriculture and Natural Resources publication #3343. Gray, D.J., Li, Z.T. and Dhekney, 2014. S.A. Precision breeding of grapevine (Vitis vinifera L.) for improved traits. Plant Science, <a href="http://dx.doi.org/10.1016/j.plantsci.">http://dx.doi.org/10.1016/j.plantsci. </a> Hintz, W.E., Carneiro, J.S., Kassatenko, I., Varga, A., and James, D. 2013. Two novel mitoviruses from a Canadian isolate of the Dutch elm pathogen Ophiostoma novo-ulmi (93-1224). Virology Journal 10:252. James, D. 2013. Challenges and benefits of standardized diagnostic protocols – International Plant Protection Convention’s perspective. Symposium on “Science and Technology Tools Supporting Phytosanitary Work”, 37th NAPPO Annual Meeting, Guelph, Ontario, Canada, October 31, 2013. Book of Abstracts. (Abstr.). James, D., Sanderson, D., Varga, A., Greig, N., and Stobbs, L. 2013. Relationships and genetic diversity of selected Canadian isolates of Plum pox virus. 2nd International Symposium on Plum Pox Virus, Olomouc Czech Republic, September 3 – 6, 2013. Book of Abstracts. Pg 15 (Abstr.). James, D., Sanderson, D., Varga, A., Sheveleva A., and Chirkov, S. 2013. Comparison of Plum pox virus strain W isolates to determine their relationships and further understand their genetic diversity. 2nd International Symposium on Plum Pox Virus, Olomouc Czech Republic, September 3 – 6, 2013. Book of Abstracts. Pg 14. (Abstr.) James, D., Varga, A., and Sanderson, D. 2013. Genetic diversity of Plum pox virus: strains, disease and related challenges for control. Canadian Journal of Plant Pathology 35(4):431-441. Leal I., Allen, E., Anema, J., Reisle, C., Foord, B., Uzunovic, A., Varga, A., and James, D. 2013. Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect living pinewood nematode, Bursaphalenchus xylophilus, in wood. In:Schroder, T (ed), Pine Wilt Disease Conference 2013, Brunschweig, Germany, ISSN: 1866-590X, pp 39-40. Leal I., Allen, E., Reisle, C., Anema, J., Uzunovic, A., Varga, A., and James, D. 2013. Development of a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method to detect living pinewood nematode, Bursaphalenchus xylophilus, in wood. Canadian Journal of Plant Pathology. (Abstr.) 35(1):92. Lu, Q., Abou Ghanem-Sabanadzovic, N., Ghimire, G., Tzanetakis, I.E., Lawrence, A. and Sabanadzovic, S. 2013. New marafivirus identified ib yellow vein disease-affected blackberries. Abstracts Annual Meeting of the American Phytopathological Society, Austin, TX, USA. Phytopathology 103: S2.87 Mekuria, T.A,, Zhang S. and Eastwell, K.C. (2014) Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification. Journal of Virological Methods 205:24-30. Melzer M.J., Ayin, C., Sugano, J.S., Uchida, J.Y., Kawate, M.K., Borth, W.B., Sether, D.M. and Hu, J.S. 2013. Differentiation and Distribution of Cordyline Viruses 1–4 in Hawaiian ti Plants (Cordyline fruticosa L.). Viruses 5:1655-1663. Melzer, M.J., Nelson Simbajon, N., Carillo, J., Borth, W.B., Freitas-Astúa, J., Kitajima, E.W., Neupane, K.R. and Hu, J.S. 2013. A cilevirus infects ornamental hibiscus in Hawaii. Archives of Virology doi:10.1007/s00705-013-1745-0 Osman, F., Al Rwahnih, M. and Rowhani, A. 2013. Improved Detection of Ilarviruses and Nepoviruses Affecting Fruit Trees Using Quantitative PCR. Journal of Plant Pathology (in press). Osman, F., Hodzic, E., Omanska-Klusek, O., Olineka, T. and Rowhani, A. 2013. Development and validation of a multiplex quantitative PCR assay for the rapid detection of Grapevine virus A, B and D. Journal of Virological Methods 194:138-145. Poojari, S., Alabi, O.J., and Naidu, R.A. 2013. Molecular characterization and impacts of a strain of Grapevine leafroll-associated virus 2 causing asymptomatic infection in a wine grape cultivar. Virology Journal 10:324. Poudel, B., Ho, T., Laney, A., Khadgi, A. and Tzanetakis, I.E. 2014. Epidemiology of Blackberry chlorotic ringspot virus. Plant Disease 98:547-550. Sabanadzovic S. and Abou Ghanem-Sabanadzovic, N. 2013. Cryptic viruses in the flora of the Great Smoky Mountains National Park. Abstracts Annual Meeting of the American Phytopathological Society, Austin, TX, USA. Phytopathology 103: S2.125 Thekke-Veetil, T., Aboughanem Sabanadzovic, N., Keller, K.E., Martin, R.R., Sabanadzovic S. and Tzanetakis, I.E. 2013. Molecular characterization and population structure of Blackberry vein banding associated virus, new ampelovirus associated with yellow vein disease. Virus Research 178: 234-240. Thekke-Veetil, T., Ho, T., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2014. A new ophiovirus associated with blueberry mosaic disease. Virus Research 189: 92-96. Villamor, D.E.V, Ward, K.F., Collman, S.J. and Eastwell, K.C. 2014. First report of infection of cherry rusty mottle associated virus in Portuguese Laurel (Prunus lusitanica) in Washington State. Plant Disease 98: 699. Villamor, D.E. and Eastwell. K.C. 2013. Viruses associated with cherry twisted leaf and the rusty mottle group of diseases of sweet cherry represent distinct species within the family Betaflexiviridae. Phytopathology 103:1287-1295. Zhang S, Russell P, McOwen N, Bohannon S and Vrient A. 2014. AmplifyRP Acceler8 – a rapid isothermal test for plum pox virus using reverse transcription-recombinase polymerase amplification (Abstract). American Phytopathological Society Annual Meeting, August 2014, St. Paul, Minnesota. Video Presentations Deborah Golino, UCDavis, “Red Blotch Status of the FPS Classic Foundation and the Russell Ranch Foundation” <a href="http://lecture.ucanr.org/Mediasite/Play/486e1db1ad5348eaada05ed570cc8c971d">http://lecture.ucanr.org/Mediasite/Play/486e1db1ad5348eaada05ed570cc8c971d </a> Marc Fuchs, Cornell UNIVERSITY “Red Blotch: Challenges and Opportunities” <a href="http://lecture.ucanr.org/Mediasite/Play/7e6250539e5e4676ad4cd8880c51164c1d">http://lecture.ucanr.org/Mediasite/Play/7e6250539e5e4676ad4cd8880c51164c1d</a>

Impact Statements

Virus and virus-like diseases are the major limiting factors for production of fruit trees, small fruits, citrus, and grapevines in the US and Canada. The establishment of sensitive, reliable, and robust detection systems for viruses and virus-like agents by members of WERA-20 has advanced efforts to identify several viruses and advanced efforts to eliminate them in clean planting stock available for growers.
The WERA-20 working group, comprising members representing several states in the US, provinces in Canada, public and private universities, the USDA, and public and private sectors, brought together unique experiences and cutting-edge technologies for the detection, characterization, and management of important virus diseases of perennial fruit crops.
Research-outreach efforts and advances in providing virus-tested planting materials to growers pursued by members of the WERA-20 have significant impacts on the sustainability of fruit crops in the US and Canada.
The WERA 20 has taken lead in applying cutting-edge science and technologies including NGS for rapid identification of new and emerging viruses and deploying this information for improving quarantine and certification rules and regulations to prevent the spread of exotic/emerging viruses across state and national borders.
Collaborative efforts by members of WERA-20 have facilitated the reliable supply and propagation of clean, virus-free plant materials for enhancing global competitiveness of agricultural industries in the US and Canada.
Science-based information generated by WERA-20 members and shared with industry stakeholders and regulatory agencies is strengthening national efforts in safeguarding perennial fruit crops from a wide range of new and emerging virus and virus-like diseases, thereby advancing sustainable growth of an important agricultural sector in the US.

Date of Annual Report: 11/25/2015

Report Information

Annual Meeting Dates: 07/06/2015 - 07/10/2015
Period the Report Covers: 10/01/2014 - 09/01/2015

Participants

Al Rwahnih, Maher (malrwahnih@ucdavis.edu) - Univ of California-Davis

Almeyda, Christie - NC State University

Bateman, Margarita (margarita.f.licha@aphis.usda.gov) - USDA-APHIS-PPQ

Cao, Mengji - USDA-ARS

Cheong, Eun Ju - USDA-ARS

Eastwell, Ken (keastwell@wsu.edu) - Washington State Univ

Foster, Joseph - USDA-APHIS-PPQ

Fuchs, Mark (marc.fuchs@cornell.edu) - Cornell Univ.

Golino, Deborah (dagolino@ucdavis.edu) - Univ. of California-Davis, Foundation Plant Services

Grinstead, Sam - USDA-ARS

Guerra, Lauri (lguerra@agr.wa.gov) - WA State Dept Agriculture

Howell, Bill - Northwest Nursery Institute

Hu, John (johnhu@hawaii.edu) - University of Hawaii

Jones, Robert - USDA-APHIS-PPQ

Jordan, Ramon - USDA-ARS

Karasev, Alexander (akarasev@uidaho.edu) - University of Idaho

Kinard, Gary - USDA-ARS

Li, Ruhui (Ruhui.Li@ars.usda.gov) - USDA-ARS

Liu, Zhaowei - USDA-APHIS-CPHST

Martin, Bob (bob.martin@ars.usda.gov) - USDA-ARS

Mavrodieva, Vessela - USDA-APHIS-PPQ

Melzer, Michael (melzer@hawaii.edu) - University of Hawaii

Mollov, Dimitre - USDA-ARS

Nakhla, Mark - USDA-APHIS-CPHST

Pappu, Hanu - (hrp@wsu.edu) - Washington State Univ

Pesic-VanEsbroeck, Zvezdana (zvezdana@ncsu.edu) - NC State University

Postman, Joseph (Joseph.Postman@ars.usda.gov) - USDA-ARS

Poudyal, Dipak - Oregon Dept. Agriculture

Rabindran, Shailaja - USDA-APHIS-PPQ

Rascoe, John - USDA-APHIS-PPQ

Rayapati, Naidu (naidu.rayapati@wsu.edu) - Washington State Univ

Rott, Michael - Canadian Food Insp. Agency

Rowhani, Adib (akrowhani@ucdavis.edu) - Univ. of California-Davis

Rudyj, Erich - USDA-APHIS-NCPN

Schilder, Annemiek (schilder@msu.edu) - Michigan State Univ

Schmidt, Anna-mary (anna-mary.schmidt@inspection.gc.ca) - Canadian Food Insp. Agency

Scott, Simon (sscott@clemson.edu) - Clemson Univ.

Sharma-Poudyal, Dipak (dpoudyal@oda.state.or.us) - Oregon Dept. of Ag

Sivasankarapillai, Syamkumar - Washington State Univ

Sohrab, Bodaghi - Univ. California-Riverside

Sudarshana, Mysore (mysore.sudarshana@ars.usda.gov) - USDA-ARS

Susaimuthu, James - Driscolls, Inc

Tahir, Nouman - USDA-ARS

Tzanetakis, Ioannis (itzaneta@uark.edu) - University of Arkansas

Villamor, Dan - Washington State Univ

Welliver, Ruth (rwelliver@pa.gov) - PA Dept of Agriculture

Zhang, Shulu (shulu@agdia.com) - Agdia, Inc.

Vidalakis, Georgios - Univ. California-Riverside

Brief Summary of Minutes

WERA-20 Meeting
July 6-9, 2015
Please see the attached meeting minutes.

Individual member reports will be uploaded to the "Additional Documents" section on the WERA-20 Homepage once it is again available.

Minutes Attachment:

Mtg. Rpt - WERA-20 2015 State Report.pdf

Minutes Attachment:

Attachment

Accomplishments

The latest information on viruses and vectors, and new detection technologies was shared with federal and state regulatory agencies for revising policies to contain spread of viruses. Collaborative efforts by the members of the WERA-20 have brought together a diverse group of scientists from policy, academia, State, federal and private industry to discuss the possibilities of the use of new technologies for diagnostic purposes to start a conversation on how to improve and shorten the current processes of quarantine using these new technologies. Progress has been made in the conversation related to the application of NGS technology in quarantine systems that regulate trade between states and countries. Sensitive and reliable diagnostic assays that have been developed by WERA-20 members have been shared with labs of various federal and state agencies as well as centers of the National Clean Plant Network. Scientific collaborative efforts by members of the WERA-20 have brought together cutting-edge technologies to advance the detection and characterization of new and emerging viruses and virus-like agents from fruit trees, small fruits, citrus, and grapevines in the US and Canada. Comparative studies have been conducted in various crop systems to compare and contrast the current bioassay systems with NGS for their relative sensitivity and reliability. This has resulted in finding molecular diversity of viruses using sensitive and high throughput diagnostic methodologies. Next-generation sequencing (NGS) technology has been used extensively in the past two years to identify new viruses and virus-like agents from grapevines, blueberries, pome and stone fruits.

Publications

Akinbade SA, Mojtahedi H, Guerra L, Eastwell K, Villamor DEV, Handoo ZA, Skantar AM. 2014. First Report of Xiphinema rivesi (Nematoda, Longidoridae) in Washington State. Plant Disease 98: 1018. Al Rwahnih, M., Rowhani, A., Golino, D., Islas, C., Preece, J., and Sudarshana, M.R. 2014. Detection and genetic diversity of Grapevine red blotch-associated virus isolates in table grape accessions in the National Clonal Germplasm Repository in California. Can. J. Plant Pathology 37:130–135. Al Rwahnih, M., Daubert, S., Golino, D., Islas, C., and Rowhani, A. 2015. Comparison of Next Generation Sequencing vs. Biological Indexing for the Optimal Detection of Viral Pathogens in Grapevine. Phytopathology, in press. Al Rwahnih, M., Rowhani, A., and Golino, D. First Report of Grapevine red blotch-associated virus in archival grapevine material from Sonoma County, California. Plant Disease, in press. Al Rwahnih, M., Daubert, S., Golino, D., Durvasula, A., and Rowhani, A. 2015. Description and Detection of a novel Reovirus species in Cabernet grapevine in California. APS meeting, Pasadena CA, August 1-15, 2015. Al Rwahnih, M., Daubert, S., Golino, D., and Rowhani, A. 2015. Next Generation Sequencing for the optimal detection of viral pathogens in Grapevine. APS meeting, Pasadena CA, August 1-15, 2015. Al Rwahnih, M., Daubert, S., Golino, D., and Rowhani, A. Characterization of a Novel Reovirus Species in Cabernet Grapevine in California. Proceedings of the 18th Congress of ICVG, Ankara, Turkey. September 7-11, 2015 Al Rwahnih, M., Daubert, S., Golino, D., and Rowhani, A. Next-Generation Sequencing poised to replace Biological Indexing as the Gold Standard for Virus Detection in Grapevine. Proceedings of the 18th Congress of ICVG, Ankara, Turkey. September 7-11, 2015. Al Rwahnih, M., Daubert, S., Islas, C., Golino, D. and Rowhani, A. 2014. Characterization of a fifth vitivirus in grapevine. J. Plant Pathol. 96: 219-222. Alvarez, R.A., Martin, R.R. and Quito-Avila, D.F. 2015. First report of Pineapple mealybug wilt associated virus-1 in Ecuador. Plant Pathology 31:15. Bag, S., Al Rwahnih, Li, A., Gonzalez, A., Rowhani, A., Uyemoto, J.K., and Sudarshana, M.R. 2015. Detection of a new luteovirus in imported nectarine trees: A case study to propose adoption of metagenomics in post-entry quarantine. Phytopathology 105:840-846. Cao, Mengji, Lingling Pu, Margarita Bateman, Gary Kinard, Changyong Zhou and Ruhui Li. Simultaneous identification and molecular characterization of viruses associated with an apple tree. 2015. Abstract presented at the ICVF meeting in Japan, June 8-12, 2015 Cheong, Eun Ju, Chan-Soo Kim, Gary Kinard and Ruhui Li, 2015, Evaluation of the virus and viroid infection status of flowering cherry (Prunus yedoensis) collections in Korea and the U.S. J. Plant Path. 97: 155-160. Cheong, Eun Ju, Ae Rin Jeon, Ray Mock, Gary Kinard and Ruhui Li. 2014. Elimination of Gooseberry vein banding associated virus by in vitro therapy. USDA-ARS-NGRL- PDRU-TT/2014-1. Cheong, Eun Ju, Ae Rin Jeon, Jun Won Kang, Ray Mock, Gary Kinard and Ruhui Li. 2014. In vitro Elimination of Black raspberry necrosis virus from black raspberry (Rubus occidentalis). Hort. Sci. 41: 95–99. Cheong, Eun Ju, Gary Kinard and Ruhui Li. 2014. Effect of carbohydrate sources on in vitro shoot growth of various Prunus species. USDA-ARS-NGRL-PDRU-TT/2014-3. Dey, KK, Borth, WB, Melzer, MJ, Hu, JS. 2015. Application of circular polymerase extension cloning to generate infectious clones of a plant virus. Journal of Applied Biotechnology 3:34-44 Dey, K, Borth, W, Melzer, M, Wang, ML, Hu JS. 2015. Analysis of Pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors. Viruses 7:969-995 Diaz-Lara, A., Mosier, N.J., Keller, K.E. and Martin, R.R. 2015. A variant of Rubus yellow net virus with altered genomic organization. Virus Genes 50:104-110. Finn, C.E., Strik, B.C., Yorgey, B. and Martin, R.R. 2013. ‘Vintage’ red raspberry. HortScience 48:1181-1183. Finn, C.E., Moore, P.P., Yorgey, B.M., Strik, B.C., Kempler, C., Dossett, M. and Martin, R.R. 2013. ‘Charm’ strawberry. HortScience 48:1184-1188. Finn, C.E., Strik, B.C., Yorgey, B.M., Moore, P.P., Dossett, M., Kempler, C., Martin, R.R., Jamieson, A.R. and Galletta, G.J. 2014. ‘Sweet Sunrise’ strawberry. HortScience accepted 6.11.14 Fuchs, M., Marsella-Herrick, P., Hessler, S., Martinson, T. and Loeb, G. 2015. Seasonal acquisition of viruses by the grape mealybug, Pseudococcus maritimus (Erhorn), in a leafroll-diseased vineyard. Journal of Plant Pathology, in press. Gergerich, R.C., Welliver, R., Gettys, S., Osterbauer, N.K., Kamenidou, S., Martin, R.R., Golino, D., Eastwell, K., Fuchs, M., Vidalakis, G. and Tzanetakis, I.E. 2015. Safeguarding fruit crops in the age of agricultural globalization. Plant Disease 99: 176- 187. Golino, D., Rowhani, A., Klaassen, V., Sim, S., and Al Rwahnih, M. Grapevine Leafroll Associated Virus 1 Effects on Different Grapevine Rootstocks. Proceedings of the 18th Congress of ICVG, Ankara, Turkey. September 7-11, 2015. Gottula, J., Lewis, R. Saito, S. and Fuchs, M. 2014. Allopolyploidy and the evolution of plant virus resistance. BMC Evolutionary Biology, 14:149. Ho, T. and Tzanetakis, I.E. 2014. Developing a virus detection and discovery pipeline using next generation sequencing. Virology 471-473:54-60 James, D. 2014. Plum pox (Sharka); the disease and variability of the virus. University of California Plum Pox International Meeting, September 29 - October 1, 2014. UC-Davis, Davis, California, USA. <a href="http://ucanr.edu/sites/plumpox2014/Abstracts/James_Plum_pox_virus_variability/">http://ucanr.edu/sites/plumpox2014/Abstracts/James_Plum_pox_virus_variability/ </a> James, D., Varga, A., Lye, D. 2014. Analysis of the complete genome of a virus associated with twisted leaf disease of cherry reveals evidence of a close relationship to unassigned viruses in the family Betaflexiviridae. Archives of Virology. 159: 2463-2468. James, D., Phelan, J., Varga, A., Rott, M., Berube, J.A. 2015. First Report of Rose Cryptic Virus 1 in Rosa Plants in Canada. Plant Disease. 99: 558. James, D. 2014. Plum pox virus (PPV) detection: the standardized IPPC diagnostic protocol. University of California Plum Pox International Meeting, September 29 - October 1, 2014. UC-Davis, Davis, California, USA. <a href="http://ucanr.edu/sites/plumpox2014/Abstracts/James_Plum_pox_virus_detection/">http://ucanr.edu/sites/plumpox2014/Abstracts/James_Plum_pox_virus_detection/ </a> James, D., Green, C., and Wierenga, E. 2014. Plum Pox Monitoring and Management Program (PPMMP) in Canada. University of California Plum Pox International Meeting, September 29 - October 1, 2014. UC-Davis, Davis, California, USA. <a href="http://ucanr.edu/sites/plumpox2014/Abstracts/James_et_al_Program_in_Canada/">http://ucanr.edu/sites/plumpox2014/Abstracts/James_et_al_Program_in_Canada/ </a> James, D., Phelan J. 2015. Detection and analysis of a filamentous virus isolated from black currant (Ribes nigrum cv. Baldwin) showing symptoms of leaf chlorosis and deformity. 23rd International Conference on Virus and Other Graft Transmissible Diseases of Fruit Crops, Morioka City, Japan, June 8 – 12, 2015. Book of Abstracts. Pg 67 (Abstr.). James, D., Sanderson, D., Varga, A., Greig, N. and Stobbs, L.W. 2015. Analysis of the genetic diversity and relationships of selected Canadian isolates of Plum pox virus. Acta Hort. (ISHS) 1063:33-40. James, D., Sanderson, D., Varga, A., Sheveleva, A., Chirkov, S. 2015. Recombination events may play an important role in the evolution of Plum pox virus (PPV). 23rd International Conference on Virus and Other Graft Transmissible Diseases of Fruit Crops, Morioka City, Japan, June 8 – 12, 2015. Book of Abstracts. Pg 52 (Abstr.). Jinbo Song; Eric P. Benson; Patricia A. Zungoli; Patrick Gerard; Simon W. Scott. 2015. Using the DAS-ELISA test to establish an effective distance between bait stations for control of Linepithema humile (Hymenoptera: Formicidae) in natural areas. Journal of Economic Entomology doi: 10.1093/jee/tov152 Jones, T.J., Naidu, R.A., and Nita, M. 2015. Occurrence of Grapevine leafroll associated virus- 2, ?3 and Grapevine fleck virus in Virginia, U.S.A., and factors affecting virus infected vines. European Journal of Plant Pathology 142: 209-222. Kalinowska, E., Marsella-Herrick, P. and Fuchs, M. 2015. Genetic variability of Blueberry scorch virus isolates from highbush blueberry in New York. Archives of Virology, 160:1537-1542. Krenz, B., Thompson, J.R., McLane, H.L., Fuchs, M. and Perry, K.L. 2014. Grapevine red blotch-associated virus is widespread in the United States. Phytopathology, 104:1232- 1240. Leal, I., Allen, E., Foord, B., Anema, J., Reisle, C., Uzunovic, A., Varga, A., James, D. 2014. Detection of living Bursaphelenchus xylophilus in wood, using reverse transcriptase loop-mediated isothermal amplification (RT-LAMP). Forest Pathology. 45: 134-148. Lin, Liming, Ruhui Li, Ray Mock and Gary Kinard. 2014. Simultaneous Detection and Differentiation of Four Pome Viroids by RT-PCR. USDA-ARS-NGRL-PDRU-TT/2014- 2. Long, MH, Ayin, C, Li, R, Hu, JS, Melzer, MJ. 2014. First report of taro vein chlorosis virus infecting taro [Colocasia esculenta (L.) Schott] in the United States of America. Plant Disease 98:1160 Martin, R.R. and Tzanetakis I.E. 2014. Control of virus diseases of berry crops. Advances in Virus Research 91:44-81. Martin, R.R. and Tzanetakis, I.E. 2013. High risk strawberry viruses by region in the United States and Canada: Implications for certification, nurseries and fruit production. Plant Dis. 97:1358-1362. Martin, R.R. and Tzanetakis I.E. 2015. Control of virus diseases of berry crops. Advances in Virus Research 91:271-309. Mekuria, TA, Zhang S, Eastwell KC. 2014. Rapid and sensitive detection of Little cherry virus 2 using isothermal reverse transcription-recombinase polymerase amplification. Journal of Virological Methods 205:24-30. Melzer MJ, Shimabukuro, JK., Long, M., Nelson, S., Alvarez, A., Borth, WB, Hu, JS. First report of Capsicum chlorosis virus infecting waxflower (Hoya calycina Schlecter) in the United States of America. 2014 Plant Disease 98:1160. Moore, P.P., Barritt, B., Sjulin, T., Robbins, J.A., Finn, C.E., Martin, R.R. and Dossett, M. 2014. ‘Cascade Gold’ raspberry. HortScience 49:358-460. Moore, P.P., Hoashi-Erhardt, W., Finn, C.E., Martin, R.R. and Dossett, M. 2015. ‘Cascade Harvest’ red raspberry. HortScience 50:24-627. Naidu, R.A., 2014. Grapevine viruses and clean plants. In: Vine to Wine DVD. Edited by Gwen Hoheisel and Michelle Moyer (in press). Naidu, R.A. 2014. Virus Diseases. In: 2015 Pest Management Guide for Grapes in Washington. WSU Extension Bulletin EB0762, pp.28-31. Naidu, R.A., Maree, H.J., and Burger, J. 2015. Grapevine leaf roll disease and associated viruses – A unique pathosystem. Annual Review of Phytopathology 53 (in press). Naidu, R.A., Rowhani, A., Fuchs, M., Golino, D., and Martelli, G.P. 2014. Grapevine leafroll: A complex viral disease affecting a high-value fruit crop. Plant Disease 98:1172-1185. Naidu, R.A., Scharlau, V. 2014. Why ‘clean’ plants – Fact sheet (4 pages). Naidu, R.A. and Walsh, D. 2015. Is ‘grape virus tax’ hitting your pocketbook? Good Fruit Grower May 15, 2015. Vol. 66, No. 10, pages 10-11. Osman, F., Al Rwahnih, M. and Rowhani, A. 2014. Improved detection of ilarviruses and nepoviruses affecting fruit trees using quantitative RT-qPCR. 2014. J. Plant Pathol. 96:577-583. Postman, J.D. and H.R. Cameron. 1987. Apple mosaic virus in U.S. filbert germplasm. Plant Disease 71:944-945. Postman, J.D. and S.A. Mehlenbacher. 1994. Apple mosaic virus in hazelnut germplasm. Acta Horticulturae 351:601-609. Poudel, B., Ho, T., Laney, A., Khadgi, A. and Tzanetakis, I.E. 2014. Epidemiology of Blackberry chlorotic ringspot virus. Plant Disease 98:547-550. Quito-Avila, D., Alvarez, R.A. and Martin, R.R. 2015. An umbra-like virus of papaya discovered in Ecuador: detection, occurrence and phylogenetic relatedness. EJPP (Dec. 9, 2014) Quito-Avila, D.F., Brannen, P.M., Cline, W.O., Harmon, P.F. and Martin, R.R. 2013. Genetic characterization of Blueberry necrotic ring blotch virus, a novel RNA virus with unique genetic features. J. Gen. Virol. 94:1426-1434. Quito-Avila, D.F., Ibarra, M.A., Alvarez, R.A., Espinoza, L., Ratti, M.F., Peralta, E.L. and Martin, R.R. 2013. First report of Raspberry bushy dwarf virus in the Andean blackberry (Rubus glaucus) in central Ecuador. Plant Dis. 97:1003. Quito-Avila, D.F, Lightle, D. and Martin, R.R. 2014. Effect of Raspberry bushy dwarf virus, Raspberry leaf mottle virus, and Raspberry latent virus on plant growth and fruit crumbliness in ‘Meeker’ red raspberry. Plant Dis. 98:176-183. Quito-Avila, D.F., Peralta, E.L., Ibarra, M.A., Alvarez, R. and Martin, R.R. 2014. A Raspberry bushy dwarf virus isolate from Ecuadorean Rubus glaucus contains an additional RNA that is a rearrangement of RNA 2. Arch. Virol. 159:2519-2521. Rayapati, N., Rowhani, A., Fuchs, M., Golino, D. and Martelli, G.P. 2014. Grapevine leafroll: a complex viral disease affecting a high-value fruit crop. Plant Dis. 98: 1172-1185. Ricketts, K.D., Gomez, M.I., Atallah, S.S., Fuchs. M.F., Martinson, T., Smith, R.J., Verdegaal, P.S., Cooper, M.L., Bettiga, L.J. and Battany, M.C. 2015. Reducing the economic impact of grapevine leafroll disease in California: identifying optimal management practices. American Journal of Enology and Viticulture, 66:138-147. Rott, Michael, Mark Belton, Ian Boyes, Heidi Rast. Development of Next Generation Sequencing methods for plant virus diagnostics in grapevine and tree fruits. 11th International Conference of the European Foundation for Plant Pathology, Krakow, Sept 8-13, 2014. Rott, Michael, Yurit Xiang, Michael Bernardy, Mark Belton, Ian Boyes, Heidi Rast, Cindy Tu, Edward Clarke, Bari Befeh Aadum. Analysis of Grapevine and Tree Fruit virus collections using Next Generation Sequencing. 11th International Conference of the European Foundation for Plant Pathology, Krakow, Sept 8-13, 2014. Rowhani, A., Golino, D., Klaassen, V., Sim, S., Gouran, M., and Al Rwahnih, M. Grapevine Leafroll Associated Virus 3: Effects on Rootstocks, Vine Performance, Yield and Berries. Proceedings of the 18th Congress of ICVG, Ankara, Turkey. September 7-11, 2015. Roy, A., Hartung, JS, Schneider, WL, Shao, J, Leon, MG, Melzer, MJ, Beard, JJ, Otero-Colina, G, Bauchan, GR, Ochoa, R, and Brlansky, RH. 201X. Role bending: complex relationships between viruses, hosts, and vectors related to citrus leprosis, and emerging disease. Phytopathology (in press). Scott, S.W., MacFarlane, S.A. McGavin, W.J. and Fargette, D. 2014. Cassava Ivorian Bacilliform virus is a member of the genus Anulavirus. Archives of Virology 159: 159:2791–2793 Seguin, J., Rajeswaran, R., Malpica-López, N., Martin, R.R., Kasschau, K., Dolja, V.V., Otten, P., Farinelli, L. and Pooggin, M.M. 2014. De novo reconstruction of plant RNA and DNA virus quasispecies from siRNAs. PLoS One at <a href="http://dx.plos.org/10.1371/journal.pone.0088513">http://dx.plos.org/10.1371/journal.pone.0088513 </a> Skinkis, P., Pscheidt, J., Peachy, E., Dreves, A., Walton, V., Sanchez, D., Zasada, I. and Martin, R.R. 2014. Pest Management Guide for Wine Grapes in Oregon. Oregon State Univ. Ext. Bull. <a href="http://ir.library.oregonstate.edu/xmlui/bitstream/handle/1957/45975/em8413.pdf">http://ir.library.oregonstate.edu/xmlui/bitstream/handle/1957/45975/em8413.pdf </a> Sudarshana, M.R., Perry, K.L., and Fuchs, M.F. 2015. Grapevine red blotch-associated virus, an emerging threat to the grapevine industry. Phytopathology 105: (In print). Thekke-Veetil, T., Ho, T., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2014. A new ophioivirus is associated with blueberry mosaic disease. Virus Research 189:92-96. Thekke-Veetil, T., Ho, T., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2014. A new ophiovirus associated with blueberry mosaic disease. Virus Research 189: 92-96. Thompson, J.R., Fuchs, M., McLane, H., Toprak-Celebi, F., Fischer, K., Potter, J. and Perry, K.L. 2014. Profiling viral infections in grapevine using a randomly primed reverse transcription-polymerase chain reaction/macroarray multiplex platform. Phytopathology, 104:211-219. Tzanetakis, I.E., Martin, R.R. and Wintermantel, W.M. 2013. Epidemiology of criniviruses, an emerging problem in world agriculture. Front. Microbiol. 4:119:1-15. doi: 10.3389/fmicb.2013.00119 Accepted April 26, 2013 Thekke-Veetil, T., Polashock, J.J., Marn, M.V., Plesko, I.M., Schilder, A.C., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2015. Population structure of blueberry mosaic associated virus: Evidence of reassortment in geographically distinct isolates. Virus Research 201: 79-84. Thekke-Veetil, T., Polashock, J., Marn, M.V., Plesko, I.M., Schilder, A., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. 2015. Population structure of blueberry mosaic associated virus: Evidence of genetic exchange in geographically distinct isolates. Virus Res. 201:79-84. Thekke-Veetil, T., Sabanadzovic, N. A-G., Keller, K.E., Martin, R.R., Sabanadzovic, S. and Tzanetakis, I.E. 2013. Molecular characterization and population structure of Blackberry vein banding associated virus, a new ampelovirus associated with blackberry yellow vein disease. Virus Res. 178:234-240. Villamor DEV, Susaimuthu J, Eastwell KC. 2015. Genomic analyses of cherry rusty mottle group and cherry twisted leaf associated viruses reveal a possible new genus within the family Betaflexiviridae. Phytopathology 105:399-408. Walker, L., Bagewadi, B., Schultz, A., and Naidu, R.A. 2015. First report of Tobacco ringspot virus associated with fanleaf disease in a Washington State vineyard. Plant Disease (in press). Walker M, Chisholm J, Wei T, Ghoshal B, Saeed H, Rott M, Sanfaçon H. 2015. Complete genome sequence of three tomato ringspot virus isolates: evidence for reassortment and recombination. Arch Virol. 160(2):543-7. Wallingford, A.K., Fuchs, M.F., Hessler, S., Martinson, T.M. and Loeb, G.M. 2015. Slowing the spread of grapevine leafroll-associated viruses in commercial vineyards with insecticide control of the vector, Pseudococcus maritimus (Erhorn) (Hemiptera: Pseudococcidae). Journal of Insect Science, in press. Ward, N., Polashock, J., Thekke-Veetil, T., Martin, R.R. and Beale, J. 2015. First report of blueberry mosaic disease caused by Blueberry mosaic associated virus in Kentucky. Plant Dis. 99:421 Zhang S, Ravelonandrob M, Russell P, McOwen N, Briard P, Bohannon S, Vrient A (2014) Rapid diagnostic detection of plum pox virus in Prunus plants by isothermal AmplifyRP® using reverse transcription-recombinase polymerase amplification. Journal of Virological Methods 207:114–120. Zhang S, Ravelonandrob M, Chambers M, Briard P, Masson M , Amato M, Vrient A (2015) Rapid diagnostic detection of plum pox virus by isothermal AmplifyRP® and by ImmunoStrip®. Acta Hort 1063:167-172. Zhang S, Russell P, McOwen N, Bohannon S, Davenport B (2015) Development of a novel isothermal AmplifyRP method combining both real-time and endpoint assays in single tubes for rapid detection of plant pathogens. American Phytopathological Society Annual Meeting, August 1-5, 2015, California.

Impact Statements

This year, the WERA-20 working group, comprising members representing several states in the US, Canada, public and private universities, the USDA, and public and private sectors, brought together two very unique perspectives by having two additional day sessions. The first session was a summary of the current new technologies by scientists of the group, including comparative studies in which Next Generation Sequencing was compared with the current set of standard testing protocols.
The second session was a conversation between scientists and regulators with the purpose of requesting guidelines from the regulators for the scientists to be able to implement the use of Next Generation Sequencing for quarantine purposes for the detection, characterization, and management of important virus diseases of perennial fruit crops.
Virus and virus-like diseases are the major limiting factors for production of fruit trees, small fruits, citrus, and grapevines in the US and Canada. In 2015 The WERA 20 members have taken the lead in applying cutting-edge science and technology, including NGS for rapid identification of new and emerging viruses and deploying this information for increasing the information available so that this information may be used in the future for the purpose of improving quarantine and certification rules and regulations that will prevent the spread of exotic/emerging viruses across state and national borders.
Members of the WERA-20 group have advanced the efforts towards the establishment of sensitive, reliable, and robust detection systems for viruses and virus-like agents by to identify viruses and worked on advancing efforts to eliminate pathogens in clean planting stock available for growers. Research efforts as well as advances in providing virus-tested planting materials to growers pursued by members of the WERA-20 have an important impact on the sustainability of fruit crops in the United States and Canada.
In summary, state- of-the- art science and technology and science-based information generated by WERA-20 members shared with industry stakeholders and regulatory agencies is strengthening national efforts in safeguarding American agriculture for fruit crops from a wide range of virus and virus-like diseases, advancing sustainable growth of an important agricultural sector.

Date of Annual Report: 09/12/2016

Report Information

Annual Meeting Dates: 07/11/2016 - 07/13/2016
Period the Report Covers: 10/01/2015 - 09/30/2016

Participants

Participants Attachment:

WERA-20 - Participants List.doc

Brief Summary of Minutes

WERA - 20
Virus and Virus-like Diseases of Fruit Trees, Small Fruit and Grapevines

Foundation Plant Services
Davis, CA
July 11-13, 2016

July 11

8:30 - Welcome

Hanu Pappu (Washington State University)
Hanu opened the meeting with welcome remarks.

Deborah Golino (Foundation Plant Services, UC Davis)
Deborah discussed the location for next year’s meeting. Joseph Postman motioned to consider 
North Carolina for the next meeting location and Simon Scott seconded the motion.

Maher Al Rwahnih (Foundation Plant Services, UC Davis)
Maher welcomed everyone to the meeting and introduced the first speaker.

9:00 - Scientific presentations

Allison Gratz and Anna-mary Schmidt (Tree Fruit Diagnostics Program, Canadian Food 
Inspection Agency - CFIA)
Update on the Grapevine and Tree Fruit Diagnostic Programs at CFIA's Centre for Plant 
Health
Allison provided as overview of the program and diagnostic methods, including laboratory and 
biological tests, tissue culture and virus elimination. Their G1 virus-tested repository has over 
550 varieties and is the only G1 repository in Canada. Grapevine audits have found no Canadian 
regulated virus detected during audits from 2011-2015. Regulated tree fruit viruses have been 
detected in tree fruit audits. Grapevine Pinot gris virus (GPGV) has been detected in Canada 
in British Columbia and Ontario.

Margarita Bateman (USDA, Fruit Tree Quarantine Program)
WERA 20 2016 USDA APHIS Annual Quarantine Report
Margarita provided updates on the program for 2016 including total releases and accessions 
received. The program routinely intercepts a variety of important viruses for tree fruits. 
Margarita provided an overview of program personnel and discussed the flow of plant material 
through the program.

Dimitre Mollov (USDA, National Germplasm Resources)
Detection of viruses infecting Prunus spp. using HiSeq analysis
Ruhui Li could not attend the meeting. Dimitre Mollov gave her talk in her absence. There are 
challenges in detecting tree fruit viruses in quarantine programs. Dimitre provided an overview 
of the application of RNA-Seq to detect viruses. NGS is the way to go, but one should proceed 
carefully with the data analysis.

Joseph Postman (USDA ARS, Corvallis, Oregon)
Impact of Xylella fastidiosa on Corvallis, Oregon USDA Genebank
Xylella fastidiosa was causing significant problems in olive trees in Europe. It was first identified 
in pear in Oregon. Pierce’s disease of grape is caused by a different isolate. Xylella is widely 
distributed in a variety of hosts and there are at least four different isolates that have been 
identified. Joseph provided an overview of the investigation of the pear trees that were found 
positive, including testing, potential sources (Sambucus is a possible reservoir host), and 
precautions to prevent movement of Xylella.

Syamkuma Pillai (Washington State University, Prosser)
Clean Plant Center Northwest: New Virus Findings and Program Update
Syam provided an update of the CPCNW program in fruit trees, grapes and hops. He included 
information on new selections, virus elimination, and the adoption of high throughput 
sequencing (HTS) for pathogen detection. Dan Villamor continued Syam’s talk and focused on 
the new viruses detected with HTS. Using HTS, the group discovered and investigated: Cherry 
necrotic rusty mottle virus (CNRMV), a fabavirus in cherry; Prunus Virus F (PrVF), a fabavirus 
in Prunus; and Nectarine virus M (NeVM), a marafivirus/Nectarine stem-pitting-associated virus 
(NSPaV), a luteovirus (interaction between these two viruses).

Naidu Rayapati (Washington State University, Prosser)
An Update on Grapevine Viruses in Washington State
Naidu provided and update on the sanitary status of grapevines in the five Washington State 
certified nurseries. Composite samples from vines of mother blocks were tested by RT-
PCR/PCR for Grapevine leafroll associated virus-3 (GLRaV-3) and Grapevine rupestris stem 
pitting- associated virus (GRSPaV). HTS was used to test random composite samples of mother 
blocks. RT-PCR and HTS results were very similar for detecting GLRaV-3. So far, samples have 
tested negative for red blotch.

12:00 - LUNCH

Simon Scott (Clemson University, South Carolina)
Report from South Carolina
Simon described the existing Southeastern Budwood Program of testing the 34 cultivars of 
commercial significance for Prunus necrotic ringspot virus (PNRSV), Prune dwarf virus (PDV) 
and Plum pox virus (PPV) by ELISA since 2000. They are working on detecting other viruses, 
including Little cherry virus-1 (LCV-1), Cherry virus A (CVA), NSPaV, and NeVM. Simon has 
detected a new virus of unknown origin present in Yoshino cherry on the university campus. He 
provided an overview of their work on blackberry viruses.

Ruth Welliver (Pennsylvania Department of Agriculture, Harrisburg)
Phytoplasmas in Pennsylvania tree fruit
Pennsylvania fruit tree improvement program is a state-level certification program that currently 
has three nurseries. They test for PNRSV, PDV, ToRSV, and PPV. They conducted a survey for 
PPV (Pennsylvania has been PPV-free for 10 years). They also surveyed for exotic 
Monilinia/Molilia and phytoplasmas. CVA was found in material sent to Prosser for the CVA 
survey. Pennsylvania apple orchard decline was investigated.

Bob Martin (USDA-ARS, Horticulture Crops Research Unit, Corvallis, Oregon)
Update from Oregon
Bob provided an update for Oregon. His update included his investigation on Rubus yellow net 
virus (RYNV) which is occasionally seen in the northwest. They are also investigating Blueberry 
fruit drop virus (BFDaV-a caulimovirus) which has been observed since the 1990s. His study has 
shown the symptoms strongly correlate with presence of the virus. Bluecrop is the only variety 
exhibiting BFDaV fruit drop symptoms. Other research includes studies on GRBaV in vineyards.

Wenping Qiu (Missouri State University, Mountain Grove)
Current status of Grapevine vein clearing virus
Wenping discussed the current status of Grapevine vein clearing virus (GVCV) which was first 
discovered on Chardonnay. He performed biological investigations and viral characterization 
studies. The disease has been found in other regions (AR, IL, MO, and IN) and on other 
varieties. The correlation of symptoms with virus is 99%. Four new GVCV isolates were 
discovered in wild Vitis and Ampelopsis plants.

Joseph Postman (USDA-ARS, Corvallis, Oregon)
GRIN-Global Demo: Public access to USDA Germplasm; potential use for NSPN Centers
Joseph introduced and demonstrated the GRIN-Global database to the group. The database 
contains records for more than 500,000 accessions.

Annemiek Schilder (Michigan State University, Lansing)
Small fruit virus update for Michigan
Annemiek provided an update on viral diseases affecting small fruits in Michigan.

Shulu Zhang (Agdia, Inc., Elkhart, Indiana)
Rapid detection of viruses and viroids in fruit crops using AmplifyRP
Shulu described the rapid detection of viruses and viroids in fruit crops using isothermal 
AmplifyRP. Agdia's different formats for AmplifyRp can be used for different endpoint 
applications and for different pathogens. The kits are fast, simple, sensitive and specific.

John Hu (Plant and Environmental Protection Sciences, University of Hawaii at Manoa)
Deregulation of papaya in China
John shared the history of Papaya ringspot virus (PRSV) investigations using pathogen-derived 
resistance to produce transgenic plants. He also explained the process and difficulties 
encountered in the deregulation of papaya in China.

Georgios Vidalakis (Citrus Clonal Protection Program, University of California, Riverside)
Developments on the detection of graft-transmissible pathogens of citrus
Georgios provide and update on the developments on Asian citrus psyllid and Huanglongbing in 
California, including a description of symptoms, vector control, vector distribution, 
monitoring/detection efforts, and plan to “flood the market” with clean budwood. He also shared 
the success story of the California Citrus Nursery Stock Pest Cleanliness Program requiring 
mandatory testing of mother trees.

Erich Rudyj (USDA APHIS PPQ, National Clean Plant Network)
Erich honored Yannis Tzanetakis for his contributions to NCPN with a medallion. Ken Eastwell 
was honored with a letter for his service supporting the mission of NCPN. Erich then gave an 
overview of the NCPN program.

5:30 - End of presentations

July 12

8:30 - Scientific Presentations

Kari Arnold (Foundation Plant Services, University of California, Davis)
Interpreting a north coast multi-virus survey to assess the influence of disease management
Kari described her survey of nine viruses in California’s north coast vineyards. Older (heritage) 
vineyards had a relatively even distribution of a variety of viruses. Viruses detected in newer 
vineyards are vectored viruses, previously unknown viruses or viruses not controlled by the 
certification program (RSPaV).

Mike Rott (CFIA)
Application of VirTool and NGS for the detection of tree fruit and grapevine viruses
Mike described the general workflow and procedures for processing samples for NGS. He 
provided a comparison of test results for conventional testing versus NGS. Mike gave an 
overview of the use of VirTool.

Kristian Stevens (Foundation Plant Services, University of California, Davis)
Intro to Bioinformatics at FPS
Kristian provided an overview of the application of bioinformatics for the identification of an 
unknown putative virus. He discussed the processes, analyses, challenges, and future 
opportunities of HTS/genomics.

Yannis Tzanetakis (University of Arkansas, Fayetteville)
Update for Arkansas/Understanding blackberry virus movement in the field scale
Yannis provided an overview of his investigations on berry viruses in Arkansas, including his 
approach to new discoveries from HTS. He provided an update on his work on Blueberry green 
mosaic virus (BGMaV) and his progress on certification guidelines for blueberry and Rubus.

Deborah Golino requested a volunteer for secretary for next year who will host the meeting for 2018.

Maher Al Rwahnih (Foundation Plant Services, University of California, Davis)
Establishment of Biological Significance of HTS Findings
Maher provided an example of a new virus, Grapevine fabavirus (GFabV), for which biological 
significance needs to be established as for any new virus identified by HTS. Graft transmission, 
Koch’s postulates and spread and distribution studies must be performed; and agronomic 
significance must be assessed. These steps were performed for GRBaV which is still considered 
an emerging disease and is now included in certification by CDFA. A discussion followed that 
focused on the impact of HTS findings on certification and regulation.

11:00 - Regulatory Discussion

The group discussed the need for coming to a consensus for careful wording and handling of our 
scientific results in a reasonable manner. While we want to share our new virus discoveries, we 
need to also come with solutions. Multiple people focused on the use of the wording virus-tested, 
not virus-free. Others shared their methods for cleaning material. The question was raised 
whether or not to share publicly the viruses that come on plant material prior to cleaning. Yannis 
Tzanetakis suggested (credit to Bob Martin) we build a library of samples for each commodity so 
we can access the source material in the library. Regulators can only regulate viruses of which 
they are knowledgeable. The regulators have a system of checks and balances that need to be 
performed for a disease to be regulated.

Deborah Golino will work on securing a location for the 2018 meeting.

1:00 – Local field trip of the FPS Facility
2:30 – Field trip to Russell Ranch and Armstrong
6:00 – Dinner at Season’s Restaurant

July 13

Field Trip to Napa
 
8:00 - Bus leaves Davis
8:30 - Arrive at Wolfskill Experimental Orchards

```
Coffee service
```

History and Mission of the Davis National Clonal Germplasm Repository – Bernie Prins, Horticulturist NCGR

A Virus Repository – Maher Al Rwahnih, FPS Laboratory Director

9:30 - Bus heads to Napa Valley
10:30 - Arrive Oakville Experimental Vineyard

History of the Oakville vineyards – Mike Anderson, Oakville Vineyard Manager

The Story of Oakville and Red Blotch – Mysore Sudarshana, USDA-ARS Research Plant Pathologist

11:45 - Bus leaves Oakville and heads to St. Helena
Noon - Lunch at Cindy’s Backstreet Kitchen cindysbackstreetkitchen.com
3:00 - Bus leaves for winery
3:20 -  Trinchero Family Estates Winery
5:00 - Bus returns to FPS in Davis

Minutes Attachment:

WERA-20 2016 Combined State Reports.pdf

Accomplishments

The latest information on viruses and vectors, and new detection technologies was shared with federal and state regulatory agencies for revising policies to contain spread of viruses. Collaborative efforts by the members of the WERA-20 have brought together a diverse group of scientists from policy, academia, state, federal and private industry to discuss the possibilities of the use of new technologies for diagnostic purposes to start a conversation on how to improve and shorten the current processes of quarantine using these new technologies. Progress has been made in the conversation related to the application of high throughput sequencing (HTS) technology in quarantine systems that regulate trade between states and countries. Sensitive and reliable diagnostic assays that have been developed by WERA-20 members have been shared with labs of various federal and state agencies as well as centers of the National Clean Plant Network. Scientific collaborative efforts by members of the WERA-20 have brought together cutting-edge technologies to advance the detection and characterization of new and emerging viruses and virus-like agents from fruit trees, small fruits, citrus, and grapevines in the US and Canada. Comparative studies have been conducted in various crop systems to compare and contrast the current bioassay systems with HTS for their relative sensitivity, reliability, and rapidity. This technology has been used extensively in the past few years to identify new viruses and virus-like agents from grapevines, blueberries, pome and stone fruits. As the use of this technology becomes more widespread, the biological significance of newly identified viruses and virus-like agents must be thoroughly investigated.

Publications

Adiputra, J., Swamy, P., Donda, B., Bagewadi, B., Natra, N. and Naidu, R.A. 2016. The prevalence of grapevine leafroll and red blotch diseases in Washington vineyards. 2016 American Phytopathological Society Annual Meeting, June 30-August 3, 2016, Tampa, FL. Adiputra, J., Swamy, P., Donda, B.P., Bagewadi, B., Natra, N., and Naidu, R.A. 2016. The Relative Distribution of Leafroll and Red Blotch Diseases in Washington Vineyards. Washington Association of Wine Grape Growers 2016 Annual Meeting, Convention and Trade Show, February 9-11, 2016, Kennewick, WA.  Alabi, O.J., Casassa, L.F., Gutha, L.R., Larsen, R.C., Henick-Kling, T., Harbertson, J.F., and Naidu, R.A. 2016. Impacts of grapevine leafroll disease on fruit yield and grape and wine chemistry in a wine grape (Vitis vinifera L.) cultivar. PLoS ONE 11: e0149666.  Alabi, O. J., Al Rwahnih, M., Gregg, L., Jifon, J. L. Crosby, K. M., Mirkov, T. E. 2015. First report of Pepper vein yellows virus infecting pepper (Capsicum spp.) in the United States. Plant Dis. 99(11):1656.  Alabi, O. J., Al Rwahnih, M., Brown, J. K., Idris, A., Gregg, L., Kmieciak, E., Sétamou, M., Jifon, J. L. 2016. First report of papaya (Carica papaya L.) naturally-infected with the introduced Tomato yellow leaf curl virus-Israel. Plant Dis. 100(9):1959.  Alabi, O. J., Al Rwahnih, M., Isakeit, T., Gregg, L., Jifon, J. L. 2016. First Report of Rottboellia yellow mottle virus Infecting Sorghum Sudangrass Hybrid (Sorghum bicolor× Sorghum bicolor var. sudanense) in North America. Plant Dis. 100(6):1255.  Alderman, S.C., Martin, R.C., Gilmore, B.S., Martin, R.R., Hoffman, G.D., Sullivan, C.S. and Anderson, N.P. 2016. First Report of Cocksfoot mottle virus Infecting Dactylis glomerata in Oregon and the United States. Plant Dis. 100:1030.  Al Rwahnih, M., Alabi, O.J., Westrick, N.M., Golino, D., Rowhani, A. Description of a novel monopartite geminivirus and its defective subviral sequence in grapevine (Vitis vinifera L.). Phytopathology 00:000-000 (Accepted).  Al Rwahnih, M., Golino, D., Rowhani, A. 2016. First Report of Grapevine Pinot gris virus Infecting Grapevine in the United States. Plant Dis. 100(5):1030.  Al Rwahnih, M., Alabi, O.J., Westrick, N.M., Golino, D., Rowhani, A. 2016. Near-Complete Genome Sequence of Grapevine Fabavirus, a Novel Putative Member of the Genus Fabavirus. Genome Announc 4(4):e00703-16. doi:10.1128/genomeA.00703-16.  Bagewadi, B., Ocampo, C., Movva, A., Hottell, D., Garza, M., Natra, N. and Naidu, R.A. 2016. Improving the Sanitary Status of Certified Mother Blocks in Washington State. Washington Association of Wine Grape Growers 2016 Annual Meeting, Convention and Trade Show, February 9-11, 2016, Kennewick, WA.  Barba, M. and James, D. 2016. Certification and quarantine for viroids and viroid diseases. Chapter XX. In Viroids and Plant Viral Satellites. (Eds. Hadidi A., Flores, R., Randles, J., and Palukaitis, P.) Elsevier Publishing. pp. 000 000 (in press).  Dey, K. Borth, W.B., Melzer M.J., Wang, M.L., Hu, J.S. 2015 Analysis of Pineapple mealybug wilt associated virus -1 and -2 for potential RNA silencing suppressors and pathogenicity factors. Viruses 7:969-995.  Dey, K. Borth, W.B., Melzer M.J., Hu, J.S. 2015 Application of circular polymerase extension cloning to generate infectious clones of a plant virus. Journal of Applied Biotechnology 3:34-44.  Diaz-Lara, A. and Martin, R.R. 2016. Blueberry fruit drop associated virus: A new member of the family Caulimoviridae isolated from blueberry exhibiting fruit drop symptoms.  Plant Disease Accepted June 27.  Diaz-Lara, A., Gent, D.H. and Martin, R.R. 2015. Identification of extrachromosomal circular DNA in hop via rolling circle amplification. Cytogenetic and Genome Research 148:237-240.  Di Bello, P.L., Laney, A.G., Druciarek, T., Ho, T., Gergerich, R.C., Keller, K.E., Martin, R.R. and Tzanetakis, I.E. A novel Emaravirus is associated with redbud yellow ringspot disease. Virus Res. 222:41-47.  Eves-van den Akker et al. 2016. The genome of the yellow potato cyst nematode, Globodera rostochiensis, reveals insights into the basis of parasitism and virulence. Genome Biology. 17:124-147.  Finn, C.E., Strik, B.C., Mackey, T., Hummer, K.E. and Martin, R.R. 2015. ‘Perpetua’ Ornamental Reflowering Blueberry. HortScience 50:1828-1829.  Finn, C.E., Strik, B., Yorgey, B., Mackey, T., Hancock, J., Lee, J. and Martin, R.R. 2016 'Baby Blues' Highbush Blueberry. HortScience 51:761-765.  <a href="http://apsjournals.apsnet.org/action/doSearch?Contrib=Gergerich%2C+R+C">Gergerich</a>, R.C., et al. 2015. Safeguarding Fruit Crops in the Age of Agricultural Globalization. <a href="http://apsjournals.apsnet.org/loi/pdis">Plant Disease</a> 99 (2): 176-187.  Gergerich, R.C., Tzanetakis, I.E. and Martin, R.R. 2016. Towards a national certification scheme for Rubus in the United States. Acta Horticulturae 1133: 483-486. Golino, D., Waclawa, P., Khuu, N., Shoulders, J., Aldamrat, R., & Sim, S. T. (2016, June). The Use of Micro-Shoots Tip Culture to Eliminate Virus for the Clean Plant Programs at Foundation Plant Services, University of California at Davis. In: IN VITRO CELLULAR & DEVELOPMENTAL BIOLOGY-ANIMAL (Vol. 52, pp. S21-S21). 233 SPRING ST, NEW YORK, NY 10013 USA. SPRINGER.  Hammond, R.W., Zhang, S. 2016. Development of a rapid diagnostic assay for the detection of Tomato chlorotic dwarf viroid based on isothermal reverse-transcription-recombinase polymerase amplification. Journal of Virological Methods (Accepted).  Ho, T., Quito-Avila, D., Keller, K.E., Postman, J.D., Martin, R.R. and Tzanetakis, I.E. 2016. Evidence of sympatric speciation of elderberry carlaviruses. Virus Research 215: 72-75.  Hottell, D., Garza, M., Ocampo, C., Bagewadi, B., Swamy, P. and Naidu, R.A. 2016. Gambling with Grafting: Do’s and Don’ts. Washington Association of Wine Grape Growers 2016 Annual Meeting, Convention and Trade Show, February 9-11, 2016, Kennewick, WA.  James, D. and Phelan, J. 2016. Complete genome sequence of a strain of Actinidia virus X detected in Ribes nigrum cv. Baldwin showing unusual symptoms. Archives of Virology 161(2): 507-511.  James, D. and Phelan, J. 2016. Detection and analysis of a filamentous virus isolated from black currant (Ribes nigrum cv. Baldwin) showing symptoms of leaf chlorosis and deformity. Plant Virus Disease Workshop Report, PSJ, Japan 00: 000-000 (in press).  James, D., Sanderson, D., Varga, A., Sheveleva, A., and Chirkov, S. 2016. Genome sequence analysis of new isolates of the Winona strain of Plum pox virus (PPV W) and the first definitive evidence of intra-strain recombination events. Phytopathology 106:407-416. James, D., Cieslinska, M., Pallas, V., Flores, R., Candresse, T., and Jelkmann, W.   2016. Viruses, Viroids, Phytoplasmas and Genetic Disorders of Cherry. Chapter 16. In: Cherries: Genetics, Ecophysiology, Production, Protection and Uses. (Eds. Garcia, J.Q., Iezzoni, A., Lang, G. and Pulawska, J.) Centre for Agriculture and Biosciences International (CABI) Press, Oxfordshire, UK. pp. 000 – 000 (in press). Kesanakurti, P., Belton, M., Saeed, H., Rast, H., Boyes, I., and Rott, M. 2016. Screening for plant viruses by next generation sequencing using a modified double strand RNA extraction protocol with an internal amplification control. Journal of Virological Methods. 236: 35-40.  Lanning, K.K., Moore, P.P. and Martin, R.R. 2016. First report of a resistance-breaking strain of Raspberry bushy dwarf virus in red raspberry (Rubus idaeus) in North America. Plant Dis. 100:868.  Li, R., Fuchs, M.F., Perry, K.L., Mekuria, T., Zhang, S. 2016. Development of a rapid AmplifyRP Acceler8 test for Grapevine red blotch-associated virus in grapevines. American Phytopatholocal Society Annual Meeting. July 30-Agust 3, 2016, Tampa, Florida.  Lightle, D., Dossett, M., Ebert, T., Finn, C.E., Martin, R.R, Lee, J.C. 2015. Effects of three novel resistant black raspberries on Amphorophora agathonica feeding behavior and performance. J. Arthropod-Plant Interactions 9:487-496.  Martin, R.R., Constable, F. and Tzanetakis, I.E. 2016. Quarantine regulations and the impact of modern detection methods. Annu. Rev. Phytopathol.  54:189-205.  Naidu, R.A., Maree, H.J., and Burger, J. 2015. Grapevine leafroll disease and associated viruses – A unique pathosystem. Annual Review of Phytopathology 53:613-634.  Naidu, R.A. 2016. Managing viruses in Washington vineyards. WAVE 2016 Washington Advancements in Viticulture and Enology. WSU’s Ste. Michelle Wine Estates Wine Science Center, Richland, WA. July 14, 2016.  Naidu, R.A. 2016. Rational Mealybug Control Programs vs. Irrational Exuberant Programs. Washington Association of Wine Grape Growers 2016 Annual Meeting, Convention and Trade Show, February 9-11, 2016, Kennewick, WA.  Naidu, R.A., Natra, N., Akinbade, S., Bagewadi, B., Swamy, P., Adiputra, J., Hottell, D., Garza, M., Ocampo, C. and Schultz, A. 2016. Tackling emerging soil-borne virus diseases in Washington vineyards. Washington Association of Wine Grape Growers 2016 Annual Meeting, Convention and Trade Show, February 9-11, 2016, Kennewick, WA.  Ornamental and fruit tree virus certification program. 2015. Oregon Department of Agriculture, Salem, OR. 2pp.  Phelan, J., and James, D. 2016. Complete genome sequences of a putative new alphapartitivirus detected in Rosa spp. Archives of Virology XX: 000-000. (in press)  Poojari, S., Lowery, T., Rott, M., Schmidt, A., DeLury, N., Boulé, J., and Urbez-Torres, J. R. 2016. First report and prevalence of Grapevine fleck virus in grapevines (Vitis vinifera) in Canada. Plant Disease. 100: 1028.  Poojari, S., Lowery, T., Rott, M., Schmidt, A., and Urbez-Torres, J. R. 2016. First report of Grapevine Pinot gris virus in British Columbia, Canada.  Plant Disease. 100: 1513.  Poojari, S., Alabi, O.J., Okubar, P.A. and Naidu, R.A. 2016. SYBR® Green-based real-time quantitative reverse-transcription PCR for detection and discrimination of grapevine viruses. Journal of Virological Methods 235: 112–118.  Quito-Avila, D.F., Martin, R.R., Alvarez, R.A., Insuasti, M. and Ochoa, J. 2016. First report of Potato virus Y, Potato virus V and Peru tomato mosaic virus in tamarillo orchards of Ecuador. Plant Dis. 100:868.  Shahid, M.S., Tzanetakis, I.E. Aboughanem-Sabanadzovic, N. and Sabanadzovic, S. 2016. An integrating badnavirus infects blackberry. Acta Horticulturae 1133: 507-510. Sharma-Poudyal, D., Osterbauer, N. K., Putnam, M. L., and Scott, S. W. 2016. First report of Lilac ring mottle virus infecting lilac in the United States. Plant Health Prog. 17:158-159. Sharma-Poudyal, D., Lane, S., Grant, J., Osterbauer, N. 2015. Latent virus survey in pome and stone fruit nurseries in Oregon. Phytopathology 551-P.  Swamy, P. and Naidu, R.A. 2016. Impacts of grapevine leafroll and redblotch diseases in Washington vineyards. 67th American Society for Enology and Viticulture (ASEV) National Conference, June 27-30, 2016, Monterey, CA.  Swamy, P. and Naidu, R.A. 2016. Impacts of grapevine leafroll and redblotch diseases in commercial vineyards. Washington Association of Wine Grape Growers 2016 Annual Meeting, Convention and Trade Show, February 9-11, 2016, Kennewick, WA.  Thekke-Veetil, T., Polashock, J., Plesko, I.M., Keller, K.E., Martin, R.R., Ho, T. and Tzanetakis, I.E. 2016. Blueberry mosaic associated virus - a putative new member of the Ophioviridae. Acta Horticulturae 1117: 103-110. Thekke-Veetil, T., Ho, T., Moyer, C., Whitaker V.M. and Tzanetakis, I.E. 2016. Detection of Strawberry necrotic shock virus using conventional and TaqMan® quantitative RT-PCR. Journal of Virological Methods 235:176-181. Thekke-Veetil, T. and Tzanetakis, I.E. 2016. First report of strawberry polerovirus-1 in strawberry in the United States. Plant Disease 100: 867.  Vargas-Ascencio, J., Al Rwahnih, M., Rowhani, A., Celebi-Toprak, F., Thompson, J. R., Fuchs., M., Perry, K. L. 2016. Limited Genetic Variability Among American Isolates of Grapevine virus E from Vitis spp. Plant Dis. 100:159-163.  Walker, L., Bagewadi, B., Schultz, A., and Naidu, R.A. 2015. First report of Tobacco ringspot virus associated with fanleaf disease in a Washington State vineyard. Plant Disease 99:1286.  Welliver, R., et al. 2014. Expelling a Plant Pest Invader: The Pennsylvania Plum Pox Eradication Program, A Case Study in Regulatory Cooperation. Posted online September 2014. <a href="http://www.agriculture.pa.gov/Protect/PlantIndustry/PlumPox/Documents/PA%20PPV%20Eradication%209-2014.pdf">http://www.agriculture.pa.gov/Protect/PlantIndustry/PlumPox/Documents/PA%20PPV%20Eradication%209-2014.pdf</a>  Xiao H. Shabanian, M., McFadden-Smith, W., and Meng, B. 2016.  First report of Grapevine Pinot gris virus in commercial grapes in Canada.  Plant Disease.  100: 1030.  Xu, Y., Wang, S., Li, Y., Tao, H., Huang, Y., Wu, B., Dong, Y., Hu, J., Liu, Y. 2016. Complete genome sequence of a distinct Calla lily chlorotic spot virus isolated from Mainland China. Arch. Virol. 161:219-222.  Zhang, J., Borth, W.B., Sether, D., Wang, I., Lin, B, Melzer, M.J., Shen, H., Pu, X, Nelson, S., Hu, J.S. 2016. Deep Sequencing of Banana Bract Mosaic Virus from Flowering Ginger (Alpinia purpurata) and Development of an Immunocapture RT-LAMP Detection Assay. Arch. Virol 161 (7): 1783-1795.  Zhang S, Ravelonandro M, Chambers M, Briard P, Masson M, Amato M, Vrient A (2015). Rapid diagnostic detection of Plum pox virus by isothermal AmplifyRP® and by Immunostrip®. Acta Hort. (ISHS) 1063:167-172.  <a name="_Toc332991048"></a>Zhang S, Russell P, McOwen N, Davenport B, Li R (2016). Development of a novel isothermal AmplifyRP® assay for rapid detection of plum pox virus - a real-time and endpoint assay in a single PCR tube. The 3rd International Symposium on Plum Pox Virus, May 9-13, 2016, Antalya,Turkey.

Impact Statements

In summary, state- of-the- art science and technology and science-based information generated by WERA-20 members shared with industry stakeholders and regulatory agencies is strengthening national efforts in safeguarding American agriculture for fruit crops from a wide range of virus and virus-like diseases, advancing sustainable growth of an important agricultural sector.