2008 Annual Report 1a.Objectives (from AD-416) The long term objectives of this project are to identify causal agents, develop diagnostic assays, identify virus vectors and develop management strategies for controlling virus diseases of small fruit crops. Control strategies will range from improving certification programs with better diagnostics, managing vectors and virus sources, and working with breeders to identify resistant germplasm and cultivars as well as developing resistance using pathogen derived approaches. The priority of diseases to be addressed is determined by their economic impact for growers or processors of these fruits. 1b.Approach (from AD-416) DsRNA analysis will be used to: 1. Look for viruses in new diseases of small fruit crops as a way to initially determine if a virus(es) is present in symptomatic plants; 2. Re-examine known diseases to look for the presence of additional viruses that may have been overlooked using bioassays and electron microscopy; 3. Determine if mixed infections are responsible for symptom variation in a single cultivar in different growing areas. Native plants and agricultural crops can serve as important inoculum for viruses studied in this project. Native vegetation and weeds in and adjacent to fields with virus infections will be tested for the presence of the viruses being studied using the tests developed in subobjective 1a and those already available. We will use standard molecular biology techniques to develop full length clones of the three RNAs of RBDV and test them for infectivity after generating RNA in various transcription systems. Replacing 5358-22000-023-00D (1/03). Replacing 5358-22000-028-00D (1/07). 3.Progress Report Developed a protocol for the extraction of double-stranded RNA from Ericaceous plants. This plant family is recalcitrant to dsRNA extraction using standard protocols, and thus, it has been difficult to characterize viruses in blueberry and cranberry that may be responsible for new diseases in these crops. With the new method, dsRNA was purified from cranberry exhibiting ‘Funky Flower’ disease in New Jersey and Massachusetts. The dsRNA was cloned and sequenced in collaboration with USDA-ARS in Beltsville and the Univ. of Massachusetts. The sequence information showed that Cucumber mosaic virus (CMV) was present in the diseased cranberries. In immuno-capture reverse-transcription polymerase chain reaction (IC-RT-PCR) assays it was shown that CMV was present in all symptomatic plants examined but not in healthy plants. Additionally, dsRNA was purified, cloned and sequenced from blueberries in the Pacific Northwest that exhibited early fruit drop symptoms. A unique virus was present in the symptomatic bushes. In collaboration with colleagues at Arizona State Univ., Louisiana State Univ., Mississippi State Univ. and the Univ. of Arkansas it was shown that this virus is very closely related to a new disease causing virus in tomato that was also sequenced in our laboratory. Two new viruses associated with blackberry yellow vein disease were cloned and sequenced from dsRNA. Either virus by itself is symptomless in blackberry but in mixed infections they cause a severe yellow vein disease and death of blackberry fruiting canes. Efforts at identifying the vectors of these two viruses are continuing. Crumbly fruit in red raspberry is being reexamined to study the affect of mixed virus infections since we identified two new viruses in plants with severe crumbly fruit. This work suggests that crumbly fruit symptoms may be more severe in mixed infections and that there may be a means to reduce the impact of crumbly fruit in raspberry and blackberry by controlling the vectors these other viruses. RBDV is pollen-borne, and controlling the vector results in poor fruit set since pollination is essential for fruit set. The two new viruses should be aphid-transmitted based on their sequence and relationship to known viruses, thus, vector control may be an option. To further study this interaction, infectious clones of Raspberry bushy dwarf are being developed to facilitate creating mixed infections and to be sure that we are working with single infections in these studies. Established a vineyard to study the impact of grapevine leafroll virus 1, 2, and 3 singly and in mixed infections with Rupestris stem pitting on plant establishment, fruit yield and fruit and wine quality. Three new scientists were hired in the unit during the past year, two are new to ARS. Mentors have been identified for these two scientists and initial meetings have taken place. Each of the 16 scientists in the unit has obtained extramural funding from various sources, industry, CSREES – NRI and USDA-ARS Northwest Centers. NP303, Component 1, Problem b. 4.Accomplishments 1. Identified Cucumber mosaic virus in cranberry showing ‘Funky Flower’ symptoms. Funky flower is a new disease of cranberry that is spreading in Massachusetts and New Jersey, where the flowers are misshapen and the plants do not produce fruit. ARS scientists in the Horticultural Crops Research Unit in Corvallis, OR extracted dsRNA from the diseased plants that were cloned by ARS in Beltsville, MD. The sequence showed that the plants were infected with Cucumber mosaic virus and we developed PCR primers that, together with immunocapture, allowed for the detection of CMV in cranberry. This test will be used to monitor the spread of CMV in cranberry and in studies to complete Koch’s postulates to confirm that CMV is the causal agent. At that point, specific control measures can be implemented to reduce the population of the vectors of CMV. This project supports NP 303, Component 1, Problem Statement B and Componenet 2, Problem Statement A. 2. Characterized a virus associated with Fruit Drop disease in blueberry. Blueberry fruit drop disease has been observed in the Pacific Northwest since the late 1990’s and its pattern of spread within fields and within plants suggested a virus as the likely causal agent. DsRNA was purified by ARS scientists in the Horticultural Crops Research Unit in Corvallis, OR from diseased bushes and cloned and sequenced. The virus is most closely related to a new virus recently characterized from tomato showing a chlorosis, necrosis and decline in Mexico and the southern US. Diagnostic RT-PCR tests have been developed, and vector studies are underway with the Blueberry fruit drop associated virus. This project supports NP 303, Component 1, Problem Statement B and Component 2, Problem Statement A. 5.Significant Activities that Support Special Target Populations Many of the nurseries and farmers that benefit from the research done in the small fruit virology lab at HCRL are small businesses (less than $250,000) and the benefits to growers in terms of $/acre are independent of farm size. In addition, many of the stakeholders that make use of the research results from the unit are of Hispanic or Russian decent. Eleven presentations have been given at grower oriented meetings this year, which is an efficient means of technology transfer to “Special Target Populations”. 6.Technology Transfer Review Publications Mekuria, T., Martin, R.R., Naidu, R.A. 2008. First report of the occurrence of Grapevine fanleaf virus in the Pacific Northwest region vineyards. Plant Disease. 92:1250. Malowicki, S.M., Qian, M.C., Martin, R.R. 2008. Comparison of aroma-active compounds in Raspberry bushy dwarf virus-resistant transgenic 'Meeker' red raspberries using stir bar sorptive extraction-gas chromatography-mass spectrometry. Journal of Agricultural and Food Chemistry. 56:6648-6655. Kraus, J., Tzanetakis, I.E., Putnam, M.L., Martin, R.R. 2008. Complete nucleotide sequence of an isolate of Coleus vein necrosis virus from Verbena. Archives of Virology. 152:381-384. Tzanetakis, I.E., Martin, R.R. 2008. Nucleotide Sequence of the tripartite Fragaria chiloensis cryptic virus and presence of the virus in the Americas. Virus Genes. 36:267-272. Tzanetakis, I.E., Martin, R.R. 2008. A new method for extraction of double-stranded RNA from plants. Journal of Virological Methods. 149:167-170. Walton, V.M., Dreves, A., Gent, D.H., James, D.G., Martin, R.R., Chambers, U., Skinkis, P.A. 2007. Relationship between rust mites, Calepitrimerus vitis (Acari: Eriophyidae), bud mites Colomeris vitis (Acari: Eriophyidae) and short shoot syndrome in Oregon vineyards. International Journal of Acarology. 33:307-318. Susaimuthu, J., Tzanetakis, I.E., Gergerich, R., Martin, R.R. 2007. A member of a new genus in the Potyviridae infects rubus. Virus Research. 131:145-151.