Brief Summary of Minutes of Annual Meeting

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

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.

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