Brief Summary of Minutes of Annual Meeting

WERA-020: Virus and Virus-Like Diseases of Fruit Trees, Small Fruits, and Grapevines Committee - Annual Meeting May 29 to June 2, 2007 in Leamington, ON Chair and Local Organizer: Roberto Michelutti, Canadian Clonal Genebank, Agriculture & Agri-Food Canada, Harrow ON; Secretary: Ken Eastwell, Washington State University, Prosser, WA Meeting convened at 08:30 on May 30, 2007: Chair Michelutti welcomed the participants to the meeting; Cavalieri (Administrative Advisor) reviewed the renewed structure of WERA's to bring research and extension personnel together. Attendees represented the States and Provinces of British Columbia, California, Colorado, Hawaii, Manitoba, Michigan, Ontario, Oregon, Pennsylvania, and Washington STATE/PROVINCE REPORTS: Eastwell (WA - report attached on WERA-20 Homepage - Additional Documents section) reported on the development of a more robust assay Little cherry virus-1. There is growing concern that the milder symptoms caused by LChV-1 on current biological indicators may allow the virus to escape detection. On-going research on the cherry isolate of Cherry leafroll virus suggests that this virus can be transmitted to the fruit bearing tree by virus-infected pollen. Serological reagents are being developed to detect members of the genus Foveavirus that affect sweet cherry. Genetic analysis reveals a complex relationship between the members of this genus. The National Research Support Project-005 located at WSU-Prosser, continues to be the primary source of new virus tested fruit tree propagation material that serves both import and export of fruit tree propagation material.

Guerra (WA) indicated that Cherry leafroll virus had been detected in a certified seed block and increased monitoring is in place to minimize threats from this virus in the certification program. The tree fruit certification program is auditing the certification program to determine the status of Apple chlorotic leafspot virus and is finding some unexpected positives.

Halbrendt (PA - report attached on WERA-20 Homepage - Additional Documents section) reported on the status of the Plum pox virus eradication program initiated after the virus was confirmed in PA in 1999. The mandatory quarantines in large areas of the State have been rescinded since no plum pox virus has been detected in these areas for 3 years. Prunus species can now be replanted in these areas, although nursery activity is still prohibited. In 2006, six positive plants were identified and only one was in a commercial orchard. Trap plants and yellow traps continue to be analyzed for viruliferous aphids and possible unknown reservoirs of the virus.

Hu (HI - report attached on WERA-20 Hompage - Additional Documents section) indicated that a survey of citrus trees on the five major islands revealed an overall incidence of Citrus tristeza virus of 74%. Some infections could be detected with either serological or molecular assays, but not by both. Further analyses indicate that most trees are simultaneously infected with multiple strains of the virus. From this study, a strain that can be used as a cross protection agent was identified; field trials are in progress to evaluate this potential. Viruses associated with a citrus blight-like wilt disease in HI are under investigation to determine the nature and etiology of this disease. The long term goal is to develop a management strategy to allow the citrus industry to expand.

James (B.C.) has characterized Peach virus Agua (formerly identified as 4N6). This virus was identified through its reaction in ELISA with antiserum prepared to Plum pox virus. The virus also reacts in EM decoration assays with antiserum to Apple stem pitting virus. PcVA shares sequence and genome organization similar to ASPV, and share many peptide sequences in the C-terminus of the coat protein region. However, PcVA is a distinct member of genus Foveavirus in the Flexiviridae family.

Kalinina (ON - report attached on WERA-30 Homepage - Additional Documents section) examined the susceptibility of ornamental Prunus species to Plum pox virus. Generally, ELISA results of self-rooted plants were less definitive than those from tree grafted onto GF305. Inoculated P. americana, P. cistena, P. glandulosa, P. tomentosa developed distinct symptoms and the infection was confirmed by immunocapture RT-PCR, ELISA and by back inoculations. P. triloba yielded weak reactions whereas P. virginiana 'Schubert', P. serrulata 'Kwanzan' yielded inconsistent results that need further clarification. These are important potential host plants for PPV in North America.

Ferguson (ON) reported on the status of efforts in Canada to contain Plum pox virus. No PPV has been detected in two sites in Nova Scotia or three areas in Ontario for three years, so these areas are now removed from the quarantine area. However, the major quarantined area in Ontario remains. Results of the 2006 survey revealed 18 positives in the St. Catherines area. Eradication will only be possible with aggressive testing and enforcement. A group discussion followed regarding strategies that might be feasible to reduce the aphid transmission of PPV.

Larsen (CO - report attached on WERA-20 Homepage - Additional Documents section) established in-pot trials to determine the potential resistance of new rootstocks to transmission of Cherry raspleaf virus. Soil containing Bing trees on mazzard and 'Citation/Z-stem' rootstocks were infested with viruliferous nematodes in spring 2006. No symptoms are evident yet on the inoculated trees. Grape fanleaf virus was detected in a block of 'Cabernet franc' in 2006. The infection appeared to be localized although significant populations of dagger nematodes were detected. Studies will continue to determine if the CO populations of nematodes are vectors of GFLV.

Tiffany (Agdia) reported that a new ELISA test is available that recognizes all known strains of Plum pox virus.

Mock (USDA-NGRL-PDRU - report attached on WERA-20 Homepage - Additional Documents section) indicated that reorganization of the unit is complete and they are now working in close associated with USDA-APHIS to develop methods for the safe introduction of germplasm. A molecular assay has been reported to supplement the biological indicators currently used for the detection of Gooseberry vein banding virus. This will shorten time requirements for virus testing. A molecular assay for Cherry green ring mottle virus and Cherry necrotic rusty mottle was also developed. Some isolates of Cherry necrotic rusty mottle do not produce symptoms on the standard biological indicator 'Sam'. A RT-PCR assay for black currant reversion has been developed but validation is still required.

Lemmon (ON) indicated that audits of programs in France and Germany cause concern about phytoplasma diseases and led to the detection of phytoplasma (Bois noir) in a single shipment of vines imported into the Okanagan Valley. It is believed that grapevines are a dead end host for the phytoplasma, and that it moves into grapevines from weedy reservoirs. The known vector of the disease does not occur in B.C. Therefore, it is believed that removal of the two infected vines effectively eradicated the Bois noir. Future importations require hot water treatment to eliminate potential Bios noir infestations.

Results from Rowhani (CA - report attached on WERA-20 Homepage - Additional Documents section) indicate that Plum bark necrosis and stem pitting associated virus shares 97% with Apricot stem pitting virus in Italy, and has been found in cherry and plum. Sequence analysis indicates that this virus has a close relationship to Pineapple mealybug wilt associated virus and some of the grapevine leafroll associated viruses.

Msikita (PA - report attached on WERA-20 Homepage - Additional Documents section) is investigating the occurrence of Tomato ringspot virus in apple trees where it causes apple decline and eventual death. ELISA detection of ToRSV has been inconsistent and the more sensitive RT-PCR has revealed a much higher incidence of ToRSV. This underscores the need to re-examine current virus detection methods and the need for robust detection methods.

Anderson (AC Diagnostics) indicated that ELISA reagents are available for PVY-n and that AC Diagnostics will also perform contract research.

Schilder (MI) detailed the discovery of Plum pox virus in July 2006 at the South West Michigan Research and Extension Center. The infected 5-year old tree was an European plum on Pumiselect® rootstock and did not exhibit any symptoms. All Pumiselect trees in the NC-140 rootstock trial have been removed. Testing of all hosts within a 5 mile radius did not reveal any other infected trees. Based on these results, fruit production is allowed in the regulated area, but no nursery production within the 7 mile area. This included advanced selection of the breeding program and 58 elite selections were sent to NRSP-005 for virus testing and retention.

Nurseries in the MI blueberry certification programs are testing for shoestring, mottle, tobacco ringspot virus and tomato ringspot virus, and will include blueberry scorch.

Thompson (BC - report attached on WERA-20 Homepage - Additional Documents section) is evaluating the use of the 'TriFoCap' primers for the detection of tricho-, fovea-, and capilloviruses. These primers successfully detected 20 virus isolates from these genera and, with only one exception, detected them throughout the year. Cherry necrotic crook disease could only be detected in dormant wood. It was reported that of 247 Prunus samples, 3 false positive reactions were obtained from cherry, and from 181 pome fruit samples, 3 false positive were obtained. Therefore, caution in interpretation of results is necessary, the case for all PCR assays.

Tian (ON) is investigating the use of transgenic plants for resistance to Plum pox virus. The HC-Pro segment is an effective activator of post transcriptional gene silencing in Nicotiana benthamiana, and transformation of plum indicates that a high percentage of transformants are resistant to infection by the D strain of PPV. The advanced selections are sent to France for evaluation for resistance to other strains.

Tzanetakis (OR - report attached on WERA-20 Homepage - Additional Documents section) summarized the determination of the viruses associated with the rapid decline of strawberry in CA (predominantly Strawberry pallidosis virus). This disease situation was resolved through aggressive management techniques. Similar disease symptoms occur in B.C. but the aphid-borne Strawberry mild yellow edge virus is the underlying cause of decline. Laboratory tests for more than 20 viruses affecting strawberry have been developed to aid in the management of strawberry diseases. Raspberry bushy dwarf is re-emerging as an important disease in raspberry and more recently, in 'Marion' blackberry which develops severe fruit loss in response to infection. Transgenic plants of 'Meeker' red raspberry have been developed and preliminary testing showed that several lines offer robust field resistance. Blueberry scorch has become an important virus, particularly with the discovery that cranberry and huckleberry are latent hosts of the virus. Virus elimination programs are underway in OR. Status of several blackberry viruses was also described.

Michelutti (ON) expanded the indexing program of the Canadian Clonal Genebank collection of more than 3300 unique accessions. Indexing on 'Kwanzen' gave 14 positive reactions last year and 25 this year. In addition, 224 Prunus and 134 Fragaria were tested for nepoviruses. In parallel, 6 Fragaria were found to be infected with Strawberry mild yellow edge virus. Rubus accessions in the screenhouse (91) were tested for nepoviruses, Raspberry bushy dwarf virus and Tobacco streak virus. Three accessions were positive for RBDV and one was positive for TSV. Eighty-five Rubus accessions in the field were tested and 4 were infected with RBDV and 1 for TSV. Nineteen representatives of the 'Jesuit pears' have been transferred to the germplasm collection for genetic analysis at a later date. Last year, symptoms developed on Ribes similar to aphid damage or Gooseberry vein banding virus. Virus infection was confirmed by PCR, and a second virus was also detected. Tissue print blots in cooperation with Palles and Flores are being used to evaluate the viroid status of the collection.

Reports (attached on WERA-20 Homepage - Additonal Documents section) were submitted by WERA-020 participants unable to attend: Deborah Golino, Ray Johnson, Simon Scott, and Aiming Wang.

Photographs attached to WERA-20 Homepage area. Business meeting:

Cavalieri indicated that the project was revision was submitted in 2005 and authorized in 2006. The project will submit its next revision in 2010.

Eastwell gave a brief summary of the planning meeting of the National Clean Plant Network that was held in Greenbelt, MD on May 9-10. The successful creation and funding of the network would be an important step forward in controlling virus diseases of perennial fruit crops in the U.S. However, it will require several fundamental changes in the way industries and government agencies interact. WERA-020 participants may have a larger role in developing some of the guidelines for network operations.

Larsen offered to host the nest meeting of WERA-020 in Grand Junction, CO. Michelutti moved and Rowhani seconded to hold the meeting in Grand Junction during the middle two weeks of June 2008 - motion passed unanimously.

Larsen moved and Hu seconded that the WERA-020 meeting be held in MI in 2009 - motion passed unanimously.

Larsen moved and Eastwell seconded that WERA-020 participants formally extend appreciation for the efforts made by the hosts at Agriculture & Agri-Food Canada and the local organizer Roberto Michelutti for making the meeting both informative and enjoyable - motion passed unanimously.

Meeting adjourned.

Plum bark necrosis and stem pitting associated virus causes severe necrosis of bark tissues, and necrotic pitting on the woody cylinders of Prunus salicina. PBNSPaV was alsdo identified in other Prunus species and cultivars: almond cv. 'Filippo Ceo', peach cvs. 'Springcrest' and 'May Crest', plum cv. 'Black Beaut', apricot cv. 'Castlebrite', and sweet cherry cv. 'Ferrovia'. The virus was detected in samples from Italy, Morocco, and Turkey.

A robust molecular assay for Little cherry virus-1 from disperse geographic locations was developed and verified.

Several ornamental Prunus species were identified as hosts for Plum pox virus. Plants on their own roots supported a lower virus titer and therefore may elude detection.

Group specific primers for the detection of tricho-, fovea-, and capilloviruses were evaluated and found to be a reliable and valuable tool for rapid detection of any one of many viruses that affect temperate fruit trees.

It was demonstrated that the rapid decline of strawberries in CA and BC was caused by two different viruses emphasizing the need to develop site-specific disease management tools.

Chisholm, J., G. Zhang, A. Wang, and H. Sanfacon. 2007. Peripheral association of a polyprotein precursor form of the RNA-dependent RNA polymerase of Tomato ringspot virus with the membrane-bound viral replication complex. Virology. (In Press)

Eastwell, K.C., P.L. Sholberg, and R.J. Sayler. 2006. Characterizing potential bacterial biocontrol agents for suppression of Rhizobium vitis, causal agent of crown gall disease in grapevines. Crop Protection. 25:1191-1200.

Gibson, P.G., G.L. Reighard, S.W. Scott, and D. Marini. 2006. Phenotypical Variation in Peach Trees Inoculated with Defined Mixtures of Viruses and Peach Latent Mosaic Viroid. Acta Horticulturae.

Halgren, A.B., I.E. Tzanetakis, and R.R. Martin. 2007. Identification, characterization, and detection of Black raspberry necrosis virus. Phytopathology. 97:44-50.

Jones, A.T., W.J. Mcgavin, V. Gepp, S.W. Scott, and M.T. Zimmerman. 2006. Purification and properties of Blackberry chlorotic ringspot, a new virus species in Subgroup 1 of the genus Ilarvirus found naturally infecting blackberry in the UK. Annals of Applied Biology. 149:125-135.

Joyce, P.A., F.E. Constable, J. Crosslin, K. Eastwell, W.E. Howell, and B.C. Rodini. 2006. Characterisation of Pear blister canker viroid isolates from Australian pome fruit orchards. Australasian Plant Pathology. 35:465-471.

Lima, M.F., R. Alkowni, J.K. Uyemoto, D. Golino, F. Osman, and A. Rowhani. 2006. Molecular analysis of a Californian strain of Grapevine rupestris stem pitting associated virus isolated from declining Syrah grapevines. Archive of Virology. 151:1889-1894.

Liu, Z., R. Scorza, J-M Hily, S.W. Scott, and D. James. 2007. Engineering Resistance to Multiple Prunus Fruit Viruses Through Expression of Chimeric Hairpins. Journal of the American Society for Horticultural Science. 132:407-414.

Martin, R.R. and I.E. Tzanetakis. 2006. Characterization, detection and management of strawberry viruses. Plant Disease. 90:384-396.

Martin, R.R., I.E. Tzanetakis, M. Sweeney, and L. Wegener. 2006. A virus associated with blueberry fruit drop disease. Acta Horticulturae. 715:497-501.

Melzer, M.J., W.B. Borth, F. Zee, M.E. Hilf, S.M. Garnsey, and J.S. Hu. 2006. Incidence and diversity of Citrus tristeza virus in the Hawaii. Phytopathology. 96:77.

Melzer, M.J., W.B. Borth, F. Zee, M.E. Hilf, S.M. Garnsey, and J.S. Hu. 2006. Incidence, distribution, and diversity of Citrus tristeza virus in the Hawaiian islands. In: Proc. 16th Conf. IOCV. Pages 179-186.

Osman, F., C. Leutenegger, D. Golino, and A. Rowhani. Real-time RT-PCR (TaqMan®) assays for the detection of grapevine leafroll associated virus 1, 2, 2-Redglobe strain, and 3. J. Virol. Methods. (In Press)

Raghupathy, M.B., J.S. Griffiths, L.W. Stobbs, D.C.W. Brown, J.E. Brandle, and A. Wang. 2006. Transfection of Arabidopsis protoplast with a Plum pox virus (PPV) infectious clone for studying early molecular events associated with PPV infection. J. of Virological Methods. 136:147-153.

Scott, S.W. and M.T. Zimmerman. 2006. The complete nucleotide sequence of the genome of Humulus japonicus latent virus. Archives of Virology. 151:1683-1687. DOI 10.1007/s00705-006-0771-6.

Scott, S.W. and M.T. Zimmerman. 2006. Production of Full-length Copies of the Genomic RNAs of Prunus necrotic ring spot virus. Acta Horticulturae.

Soule, M.J., K.C. Eastwell, and R.A. Naidu. 2006. First report of Grapevine leafroll-associated virus-3 in American Vitis grapevines in Washington State. Plant Disease. 90:1461.

Susaimuthu, J., I.E. Tzanetakis, R.C. Gergerich, and R.R. Martin. 2006. Yellow vein-affected blackberries and the presence of a novel Crinivirus. Plant Pathology. 55:607-613.

Tzanetakis, I.E., and R.R. Martin. 2007. Strawberry chlorotic fleck: Identification and characterization of a novel Closterovirus associated with the disease. Virus Research. 124:88-94.

Tzanetakis, I.E., J.D. Postman, and R.R. Martin. 2007. First report of Blackberry chlorotic ringspot virus in Rubus sp. in the United States. Plant Disease. 91:463.

Tzanetakis, I.E., A.B. Halgren, N. Mosier, and R.R. Martin. 2007. Identification and characterization of Raspberry mottle virus, a novel member of the Closteroviridae. Virus Research. 127:26-33.

Tzanetakis, I.E., J.D. Postman, R.C. Gergerich, and R.R. Martin. 2006. A virus between families: nucleotide sequence and evolution of Strawberry latent ringspot virus. Virus Research. 121:199-204.

Tzanetakis, I.E., J. Suisumathu, R.C. Gergerich, and R.R. Martin. 2006. Nucleotide sequence of blackberry yellow vein associated virus, a novel member of the Closteroviridae. Virus Research. 116:196-200.

Tzanetakis, I.E., W.M. Wintermantel, A.A. Cortez, J.E. Barnes, S.M. Barrett, M.P. Bolda, and R.R. Martin. 2006. Epidemiology of Strawberry pallidosis associated virus and occurrence of pallidosis disease in North America. Plant Disease. 90:1343-1346.

Wang, A., H. Sanfacon, L.W. Stobbs, D. James, D. Thompson, A.M. Svircev, and D.C.W. Brown. 2006. Plum pox virus in Canada: Progress in research and future prospects for diseases control. Canadian J. of Plant Pathology. 28:1-10.

Wegener, L.A., R.R. Martin, M.G. Bernardy, L. Macdonald, and Z.K. Punja. 2007. Epidemiology and strain identification of Blueberry scorch virus on highbush blueberry in British Columbia. Canadian Journal of Plant Pathology. 28:250.

Wegener, L.A., Z.K. Punja, and R.R. Martin. 2007. First report of Blueberry scorch virus in black huckleberry in British Columbia. Plant Disease. 91:328.