Brief Summary of Minutes of Annual Meeting

Minutes: H. Larsen, acting secretary. The 2008 annual meeting of WERA-20 was held Aug. 11-13, 2008 at the Holiday Inn in Grand Junction, CO. Participants: Guerra, Lauri (lguerra@prosser.wsu.edu) - WA State Dept. of Ag
Halbrendt, John (jmh23@psu.edu) - Penn St. Univ.
Hu, John (Johnhu@hawaii.edu) - Univ. of Hawaii
Larsen, Harold (Harold.Larsen@colostate.edu) - Colo. St. Univ.
Licha, Margarita (Margarita.F.Licha@aphis.usda.gov) - USDA-APHIS-, Beltsville
Mock, Raymond (Raymond.Mock@ARS.USDA.GOV) - USDA-ARS, Beltsville
Pokharel, Ramesh (Ramesh.Pokharel@Colostate.edu) - Colo. St. Univ.
Vrient, Albert (avrient@agdia.com) - AgDia

The meeting opened at 9:00 am, Monday, Aug. 11 with a welcome by H. Larsen (local host). In the absence of the official chairman for the meeting, the group decided to have H. Larsen serve as the chairperson for the meeting. The projected secretary also was unable to attend, so H. Larsen agreed to record the meeting for the minutes and to prepare the minutes after the meeting conclusion. State / Agency reports: Pokharel (CO) - See written report: He reported on his work with dagger nematodes and Cherry Rasp Leaf Virus (CRLV), one of the primary virus disease problems facing cherry and apple growers in Colorado. Dagger nematodes are very common in soils within the area, but species identification in Xiphinema is difficult. Collaborative work to look at molecular identification of dagger species from Colorado Soils is underway with A. Szalanski (Univ. of Ark.). Pot-in-pot studies were established in 2006 to examine comparative susceptibility of Bing cherry on Citation / Z-interstem rootstock and on Mazzard rootstock to CRLV infection. No symptoms have been observed in any of the scions or rootstock suckers to date. Observations continue on Van and Bing cherry on Gisela 5 and Gisela 7 rootstocks for susceptibility / resistance to CRLV. Nicotiana benthamiana mechanically inoculated with CRLV using common buffer and carborundum powder in 2007 failed to show any symptoms. Apple and peach inoculated with diseased cherry as the tissue source have not produced any symptoms to date.
An incidence of Fanleaf virus on Cabernet franc grapes was found in fall 2006. No spread was detected via ELISA tests in adjacent vines nor in an adjacent block of Cabernet sauvignon, despite the presence of a substantial dagger nematode population in these soils. The block was removed in winter 2006-07, fumigated in late summer 2007, and replanted in 2008. It and the neighboring block are being monitored for any sign of spread.
A lively discussion of nematode associated viruses and vector nematode identification followed. Morphological variability of dagger nematodes found in Colorado soils is a great difficulty for identification. Halbrendt (PA) suggested establishing single female colonies for ID purposes (minimize morphological and genetic variability). Hu (HI) - See written report. Pineapple mealybug wilt (PMW) research update: PMW has now been shown to be associated with four viruses (PMWaV-1, PMWaV-2, PMWaV-3, and PMWaV-4; all Ampeloviruses). Based on sequence available for PMWaV-4, it is distinctly different from the other three, PMWaV-1 toPMWaV-3. Badnavirus studies update: Cloning and sequencing of badnavirus-like particles in pineapple revealed the presence of four distinct clades. The phylogenetic relatedness of these clades to other badnaviruses, other Caulimoviridae members, and retro-like elements was examined. Grey pineapple mealybug had 100% transmission efficiency after a 3-day acquisition period and the PMWaV-2 virus persisted in these viruliferous mealybug vectors up to 3 days. Thus PMWaV-2 is transmitted by the grey pineapple mealybug in a semi-persistent manner. Longtailed mealybug also was shown to be a vector. Reverse transcription-polymerase chain reaction (RT-PCR) and PCR assays for PMWaV's and badnaviruses were used to determine the distribution and incidence of these viruses in two new commercially important pineapple hybrids and the germplasm accessions maintained at the ARS-USDA National Clonal Germplasm Repository in Hilo, HI. Differences were found within pineapple cultivars and hybrids with regard to incidence of PMWaV-2 and -3 and badnavirus clades A, C, & D. The high incidences of some PMWaVs and badnaviruses in newly-imported pineapple hybrids verify the importance of using virus screening assays to select virus-free plant material prior to mass propagation. Halbrendt (PA) - See written report. Reported on the Plum Pox virus (PPV) situation. The PPV eradication project in PA seems to be working at this time. The regulated areas in PA have decreased from a high of 349 sq. miles in 2003 to 51 sq. miles in 2008. The 2007 survey produced negative results for the first time since the detection of PPV in 1999. Two small areas in Adams County and one in Cumberland County remain under regulation for PPV, but no positive or suspect samples were found in 2007. In New York, samples positive for PPV have been reported in Niagara County and suspects in Wayne County. A total of 26.7 acres of orchard were destroyed in 2007 because of the positive detections in 2006 and 2007. NY currently is using a 50-meter minimum removal buffer around any positive detection due to the close proximity of the infections to the Canadian PPV infected area and the uncertain risk of reinfection via the natural movement of aphids between the two areas. The PPV infection found in Michigan was on a Pumiselect rootstock from OR. One aspect of concern was that trees on Pumiselect rootstock were also sold to WalMart which will make tracking more difficult. Guerra (WA) - See written report (from K. Eastwell, WA). Washington Dept. of Agric. work: Recently encountered a problem with CLS virus in some WA nursery material; he is looking at possibility of field transmission. There is a need for better serological detection methods for cherry mottle leaf (peaches can serve as symptomless hosts), little cherry (becoming an increasingly frequent problem), and western-X disease (seems to be on the increase now). He also is looking at peach latent mosaic viroid and prune dwarf virus (PDV, specifically interactions with other Prunus viruses).
K. Eastwell's work: He has successfully produced monoclonal antibodies against Little cherry virus 1 (enhancing detection of this virus for quarantine / certification programs). Cherry leafroll virus (CLRV) is pollen-borne (successfully transmitted CLRV from pollen to the developing fruit), but controlled pollination of several thousand blossoms has not demonstrated transmission of the virus to the fruit-bearing tree; the virus gets as far as the sub-epidermal cell layer of the pedicel, but no further. Work on Foveaviruses in fruit trees and grapevines has focused on characterization, examination of molecular similarities, and development of polyclonal antibodies to the bacterial-expressed coat protein of a representative virus on one of the four distinct clades; this antibody reacts with all viruses within the same cluster. Another polyclonal antibody was developed for Rupestris stem pitting associated virus (RSPaV). Hop stunt viroid (HSVd) is of growing concern to hop producers; a hop root certification program has been revitalized to provide HSVd and virus-free roots to nurseries. The Washington Hop Commission has invested heavily in equipment and facilities for molecular detection of HSVd.
Main activities of NRSP-005 included: 1) rescuing the Mich. St. Univ. Peach breeding program after discovery of PPV at an MSU research station; 2) assisting USDA-APHIS-PPQ by providing therapy to virus-infected clones; & 3) distributing over 17,700 virus-tested buds to researchers & industry; & 4) virus testing and therapy of 185 new acquisitions of stone and pome fruit cultivars from nine foreign countries and eight U.S. states.
Creation of a National Clean Plant Network was mandated in the 2008 Farm Bill under "Te Food, Conservation, and Energy Act of 2008" and the legislation includes the establishment of clean plant centers for pathogen diagnosis and elimination. These will produce clean material for propagation and will maintain foundation blocks of pathogen-tested plant material throughout the USA. Organizational meetings have been held in Prosser, WA and Davis, CA to create a structural model for the network. The goal is to have the organization in place by Oct., 2008. Licha (USDA-APHIS-PGQP) - On Oct. 1, 2005, APHIS assumed responsibility for the plant quarantine program in Beltsville, MD. Program Supervisor - Dr. Joe Foster; Team Leader - Dr. Margarita Licha (Fruit Plant Pathologist - Plant Germplasm Quarantine Program). She reviewed the policy for importing budwood and described the testing protocols used to detect pathogens for both pome and stone fruits and then provided a summary of program activities, goals, and needs for the future. Mock (USDA-NGRL-PDRU) - See written report. The National Germplasm Resources Lab - Plant Disease Research Unit (NGRL-PDRU) performs research on viral and sub-viral pathogens of clonally propagated prohibited crop genera, emphasizing deciduous tree and small fruits, sugarcane, grasses, and sweet potoatoes. The mission of the unit is to characterize and investigate the etiology of poorly described diseases and pathogens of quarantine significance and to develop more reliable detection and elimination methods. Five current projects related to deciduous fruits include the following: 1) Development / adaptation of existing protocols for in vitro culture and therapy of infected stone and small fruits; 2) characterization of flowering cherry isolates of Cherry necrotic rusty mottle virus (CNRMV); 3) etiological investigations of several poorly characterized quarantine diseases of pome fruits (including flat limb/rubbery wood); 4) development of a detection method for six viroids that infect pome & stone fruits; and 5) etiological investigations of what is believed to be Stayman Blotch Virus in apple in the Shenandoah Valley, VA (w/ Dr. Keith Yoder, Va. Tech. Ag. Res. & Ext. Ctr., Winchester, VA). Results from PDRU research projects will benefit the USDA quarantine program by producing more effective pathogen detection methods, improving knowledge on etiology of poorly described pathogens, and developing therapeutic methods to eliminate pathogens from potentially valuable germplasm. An adjunct benefit is helping create a more effective quarantine program that encourages compliance with federal regulations regarding movement of germplasm and diminishes the temptations to smuggle plant material into the United States. Cavalieri (WERA-20 Administrative Advisor) - Phone conference call, covered the following administrative items:
1) WERA-20 is approaching the time for the required Mid-Point Assessment of the committee. He will send electronic files of two forms to H. Larsen to forward to all members to fill out in April, 2009. Please give these your attention when you get them. He needs to submit a summary of each of these by June 1, 2009. Specifically, he needs each member to provide a one sentence "Impact Nugget" and a one sentence statement of "Who Cares & Why".

2) Update on the "Clean Plant Network" - a) National structures have been established for tree fruits and for grapes; & b) funding for NRSP-005 has been set for this year (to bridge to future funding arrangements).

3) The "Specialty Crops Research Initiative" - connections (multi-state) will be very important in successful submissions for these.
4) Avoid August for future meetings (participation this time is one reflection of problems for this timing). Face-to-Face meetings (annual) are NOT required for the committee - could be done via conference call if travel expenses should become prohibitive in the future. This might be a possibility for future years.
5) Hatch funds for next year - Few of the appropriation bills are likely to be passed until the next Congress (do have continuing resolution funding until such, at the same levels as prior budget). [Ray Mock (USDA-NGRL-PDRU) - funding under continuing resolutions until next spring on a month-by-month basis, 1/12 of budget each month.] Vrient (AgDia) - Changes at AgDia are on the horizon. Mike Tiffany is contemplating retiring soon; fruit is being given increased importance in the restructuring as plant groups are being shuffled around. Albert Vrient is likely to assume stone fruit and ornamentals areas.
Experienced elevated PPV suspect detections (possibly elevated background levels) recently. Question to group as to possible causes: Halbrendt  weeds have lots of natural materials within them that react to the system. [AgDia: used antisera from Canada: rabbit source]. Guerra  possibly heatshock protein confounding results.
Possibilities for future? - Guerra - field detection strips for fruit (Immunostrip w/ dried reagent, immuno test using gold-labeled antibody; 30 min / test). Cost varies, GMO cheaper (viral/bacterial tests $2-5 each). Need for a good, simple tool for growers. TmRSV tests? - not currently available. Business Meeting: Chaired by H. Larsen.
* Next years location (for 2009 meeting): MSP (Mock/Hu) - to accept invitation from Michigan St. Univ. to host the meeting.
* 2010 meeting location: MSP (Mock/Hu) - to go to Pennsylvania (WSU-Prosser as a backup).
* Date for 2009: MSP - Recommendation to look at May / June (virus symptoms better for field trips).
* Chairperson for 2009: MSP - to have local host or his/her designee serve as chairperson.
* Secretary for 2009: MSP - Leave to decide at the meeting.

New Business:

- L Guerra: There is a need for a manual of pome / stone fruit virus / virus-like diseases in order to capture info from people who are retiring. It needs to be peer reviewed and might best be in electronic media format. It is something to think about for next year. He will prepare a format suggestion to consider at the 2009 meeting.

Meeting was adjourned at 4:30 pm.

Field trips:

Tues: Field trip to Delta County orchards; return via the Grand Mesa scenic highway.
Wed: Field trip to Mesa County orchards (morning); tour of the Colorado National Monument (afternoon).

Reports submitted by WERA-20 members unable to attend 2008 meeting:
See Meeting Minutes Attachment section for full texts of individual reports.

Almeida (CA; UC-Berkeley)> - Transmission of grapevine leafroll-associated virus 3 by the vine mealybug: Grapevine leafroll disease is caused by grapevine leafroll-associated viruses (GLRaVs). Within this virus complex, GLRaV-3 is the predominant species in the world. Several GLRaVs have been shown to be transmitted from vine to vine by mealybugs although a detailed characterization of transmission biology is lacking. The introduction of the vine mealybug (Planococcus ficus) in California and other regions of the world may result in increasing disease incidence of established GLRaVs. We studied the characteristics of GLRaV-3 transmission by the vine mealybug. Our results indicate that the vine mealybug transmits GLRaV-3 in a semipersistent manner. First instars were more efficient vectors than adult mealybugs. GLRaV-3 transmission lacked a latent period in the vector. Virus transmission occurred with a 1-h acquisition access period (AAP) and peaked with a 24-h AAP. Mealybugs inoculated GLRaV-3 with a 1-h inoculation access period (IAP), and transmission efficiency increased with longer plant access period up to 24 h, after which transmission rate remained constant. After an AAP of 24 h, mealybugs lost GLRaV-3 and infectivity 4 days after virus acquisition. In addition, GLRaV-3 was not transovarially transmitted from infected females to their progeny as detected by reverse transcription polymerase chain reaction. In summary, we systematically analyzed transmission parameters of GLRaV-3 by the vine mealybug and showed that transmission of this virus occurs in a semipersistent manner. This research fills in important gaps in knowledge of leafroll virus transmission, which is critical for development of leafroll disease management practices. Rowhani (CA; UC-Davis) - Real time TaqMan® RT-PCR (TaqMan® RT-PCR) assays were developed to detect the viruses associated with Rugose wood complex of grapevines. The viruses detected were Rupestris stem pitting-associated virus (RSPaV) in the genus Foveavirus, Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine virus D (GVD) in the genus Vitivirus. The coat protein was found to be the most conserved gene within the viral species, therefore, the primers and probes for TaqMan® RT-PCR assays were designed from the multiple alignment of the coat protein sequence of various isolates of each virus. Comparisons were also made between the conventional one step RT-PCR and TaqMan® RT-PCR for the detection of these viruses using four fold serial dilutions of both purified RNA and crude extract prepared from grapevine tissue. Results showed that TaqMan® RT-PCR was more sensitive and could detect viruses at 32 and 256 fold higher dilutions for purified RNA and crude extract, respectively, compared to RT-PCR.

In a different experiment, Low Density Arrays (LDA) were designed based on the real-time RT-PCR (TaqMan®) assays for the specific detection of 13 viruses that infect Grapevines. The viruses included in the study were Grapevine leafroll associated viruses (GLRaV) types 1, 2, 3, 4, 5, and 9, Grapevine leafroll associated virus 2-Redglobe strain (GLRaV-2RG), RSPaV, GVA, GVB, Grapevine Fanleaf Virus (GFLV), Tomato Ringspot virus (ToRSV), and Grapevine Fleck virus (GFkV). The LDAs were evaluated against a wide range of geographically distributed isolates. Geographical locations included Africa, Europe, Australia, Asia, Latin America and the United States. High throughput detection of these viruses using LDAs was compared to RT-PCR and real time TaqMan® RT-PCR. The efficiency of different RNA extraction methodologies and buffers were compared for use in low density array detection. In addition improving the RNA extraction technique and testing the quality of the RNA using the 18S ribosomal RNA TaqMan® assay as an RNA specific internal control proved to generate better diagnostic assays. This is the first report on use of LDA for the detection of plant viruses.

Scott (SC) - Research on the bloom delay associated with the peach germplasm Ta Tao 5 and characterization of the virus and virus-like agents associated with this phenomenon has continued. Ta Tao 5 is infected with Peach latent mosaic viroid (PLMVd), Apple chlorotic leaf spot virus (ACLSV), and Asian prunus virus (APV). The complete nucleotide sequences of the isolate of PLMVd and ACLSV (GenBank Accession EU223295) have been determined. APV has been partially purified and approx 50% of the sequence of the virus has been determined.

Ta Tao 5 that had been heat-treated at NRSP-5, Prosser, WA has been shown to contain only APV according to repeated PCR tests. In 2008 trees of heat-treated T a Tao 5 exhibited no delay in bloom compared to other peach cultivars commonly grown in South Carolina. Trees of Ta Tao 5 bloomed at least 10 days after the heat-treated material.

A trial involving trees of the peach cultivar Springprince inoculated with different sources of ACLSV, PLMVd, and APV (heat-treated Ta Tao 5) and compared with trees that had been inoculated by transferring the mixture contained in Ta Tao 5 has been established. In 2008, only those treatments which contained PLMVd displayed bloom delay.

The APV infecting Ta Tao 5 appears to be a mixture of at least APV-1 and APV-3 (Marais et al., 2006. Virus Research 120: 176-183) and further research is needed to confirm this. A second trial involving the cultivar Juneprince and refined treatment mixtures based on the preliminary data from the trial with Springprince has been established and will be monitored for the next 5 -7 years.

See Meeting Minutes Attachment section for full texts of individual reports.

1. High incidences of some PMWaVs and badnaviruses were found in newly-imported pineapple hybrids; the discovery verifies the importance of using virus screening assays to select virus-free plant material prior to mass propagation. 2. Systematic analysis of transmission parameters for GLRaV-3 by the vine mealybug showed transmission of this virus occurs in a semipersistent manner. This research fills important knowledge gaps for leafroll virus transmission that are critical for development of leafroll disease management practices. 3. TaqMan® RT-PCR assays were found to be more sensitive and could detect viruses at 32 and 256 fold higher dilutions for purified RNA and crude extract, respectively, compared to RT-PCR, for viruses associated with Rugose wood complex of grapevines. The viruses detected were Rupestris stem pitting-associated virus (RSPaV) in the genus Foveavirus, Grapevine virus A (GVA), Grapevine virus B (GVB) and Grapevine virus D (GVD) in the genus Vitivirus. The enhanced efficiency of detection will help reduce risk of release of grape materials infected with these viruses to plant nurseries and thus lower incidence of such in plant materials purchased by grape growers.

Almeida, R.P.P. 2008. Ecology of emerging vector-borne plant diseases. pp. 70-77. In: Institute of Medicine Forum on Vector-borne diseases: Understanding the environmental, human health, and ecological connections. The National Academies Press, Washington, D.C. 350p. Almeida, R.P.P., Bennett, G.M., Anhalt, M.D., Tsai, C.W. and O'Grady, P. 2008. Spread of an introduced vector-borne banana virus in Hawaii. Molecular Ecology (accepted). Almeida, R.P.P., Nascimento, F.E., Chau, J., Prado, S.S., Tsai, C.W., Lopes, S.A. and Lopes, J.R.S. 2008. Genetic structure and biology of Xylella fastidiosa causing disease in citrus and coffee in Brazil. Applied and Environ. Microbiology 74:3690-3701. Anhalt, M.D. and Almeida, R.P.P. 2008. Effect of temperature, vector life stage and plant access period on transmission of Banana bunchy top virus to banana. Phytopathology 98:743-748. Chatterjee, S., Almeida, R.P.P. and Lindow, S.E. 2008. Living in two worlds: the plant and insect lifestyles of Xylella fastidiosa. Ann. Rev. of Phytopath. 46:243-271. Gibson , P.G. Reighard, G.L. Scott, S.W. , and Marini, D. 2008. Phenotypical Variation in Peach Trees Inoculated with Defined Mixtures of Viruses and Peach Latent Mosaic Viroid. Acta Horticulturae,781:541-546. Hooks, C.R.R., Wright, M.G., Kabasawa, D.S., Manandhar, R. and Almeida, R.P.P. 2008. Effect of Banana bunchy top virus infection on morphology and growth characteristics of banana. Ann. of Applied Biol. 153:1-9. Hu, J.S., D. M. Sether, M.J. Melzer, C. V. Subere, K. Cheah, Y. Chen, Qi Li, W. Borth, I. C. Wang, C. Nagai, and M.L. Wang. 2007. Characterization and Management of Pineapple Mealybug Wilt Associated Viruses. VI International Pineapple Symposium in Brazil. Hu, J.S., M.J. Melzer, D. M. Sether, C.V. Subere, W. Borth, and A.V. Karasev. 2008. The nucleotide sequence and genome organization of Pineapple mealybug wilt associated virus-1 and PMWaV-3 support a new genus in the family Closteroviridae. ASVAnnual Meeting at Cornell University. Jarugula, S., Soule, M.J., Rowhani, A. and Naidu, R.A. 2008. First report of Grapevine leafroll-associated virus-9 in Washington State vineyards. Plant Disease 92:485. Li, R. and Mock, R. 2008. Characterization of a flowering cherry strain of Cherry necrotic rusty mottle virus. Arch. Virology 153:973-978. Marini, D.B., Gibson, P.G. and Scott, S.W. 2008. The complete nucleotide sequence of an isolate of apple chlorotic leaf spot virus from peach (Prunus persica (L.) Batch). Arch. Virol. 153:1003-1005. Mekuria, T., Martin, R.R. and Naidu, R. A. 2008. First report of the occurrence of Grapevine fanleaf virus in Washington State vineyards. Plant Disease 92:1250. Melzer, M.J., Sether, D.M., Karasev, A.V. and Hu, J.S. 2008. Complete nucleotide sequence and genome organization of Pineapple mealybug wilt-associated virus-1. Arch. Virology. 153:707-714. Prado S.S. and Almeida, R.P.P. 2008. Phylogenetic placement of pentatomid stink bug gut symbionts. Current Microbiology. (in press) Prado, S.S., Lopes, J.R.S., Demetrio, C.G.B., Borgatto, A.F. and Almeida, R.P.P. 2008. Host colonization differences between citrus and coffee isolates of Xylella fastidiosa in reciprocal inoculation. Scientia Agricola 65:251-258. Sether, D.M., Melzer, M.J., Subere, C.V.Q., Borth, W.B., and Hu, J.S. 2008. Genome organization, phylogenetic relationships, and distribution of Pineapple mealybug wilt associated virus-3 in Hawaii. Virus Genes. (Manuscript submitted) Subere, C.V.Q., Sether, D.M., Zee, F. and Hu, J.S. 2007. Diversity and distribution of pineapple mealybug wilt-associated viruses (PMWaVs) and pineapple badnaviruses in Hawaii. American Phytopathological Society Meeting. Phytopathology 97:S112. Tsai, C.W., Chau, J., Fernandez, L., Bosco, D., Daane, K.M., and Almeida, R.P.P. 2008. Transmission of Grapevine leafroll-associated virus 3 by the vine mealybug (Planococcus ficus). Phytopathology 98:1093-1098.