2009 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; and 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).
The plant family Ericaceae is recalcitrant to dsRNA extraction using standard protocols, so 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 colleagues at the 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.
Double-stranded RNA 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 virus in tomato that we also sequenced in our laboratory. It now is clear that the virus we have from blueberry is not the causal agent of Fruit Drop; it may be involved, but we have identified a number of plants with this virus that do not show any Fruit Drop symptoms.
In collaboration with colleagues in the southeastern United States, we have cloned and sequenced dsRNA from blueberries exhibiting a necrotic ring blotch symptom in that region. Initial studies suggest the virus is related to the disease (present in 8 of 8 plants tested in June of 2009 and not in symptomless plants). More testing will continue throughout the summer. DsRNA patterns suggest at least one additional virus present in symptomatic plants; efforts to clone this are ongoing.
Crumbly fruit in red raspberry is being reexamined to study the effect 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 of these other viruses. RBDV is pollen-borne and controlling the vector results in poor fruit set since pollination is essential for fruit set. One of the new viruses, a member of the Closterovirus genus, should be aphid transmitted based on its sequence and this was borne out with transmission studies carried out in the greenhouse. This virus has been shown to be the causal agent of Raspberry leaf mottle and Raspberry leaf spot diseases. The other virus, a unique member of the Reoviridae family, should be leafhopper transmitted and the transmission studies are underway.
A vineyard was established 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. The vines were grafted with the various virus combinations in the spring of 2009.
Identified a virus associated with Blueberry necrotic ring blotch disease in the southeastern United States. Blueberry necrotic ring blotch (BNRB) is an emerging disease that was first observed in Georgia in late 2007 and by the end of 2008 was detected through out the southeastern United States. ARS scientists in Corvallis, OR have cloned and sequenced dsRNA extracted from diseased plants and identified a new virus in blueberry. From the partial sequence we have obtained, Polymerase Chain Reaction (PCR) primers were developed that can be used to successfully detect the virus. The sequence obtained thus far suggests an eriophyid mite as the most likely vector of the virus but this needs to be confirmed by transmission studies. The ability to detect the virus provides the tool needed to ensure the virus is not being spread in planting stocks, identify native sources of the virus and assist in the vector transmission studies.
Identified a new Reo-like virus in raspberry associated with crumbly fruit in mixed infections with raspberry bushy dwarf virus. Crumbly fruit in red raspberry and blackberry significantly decreases the fruit quality in that the fruit is not useable for the fresh or whole berry markets, which are the most profitable for the grower. ARS scientists in Corvallis, CA extracted, cloned and sequenced dsRNA from the diseased plants. The sequence information showed the presence of three viruses, Raspberry bushy dwarf virus, Raspberry leaf mottle virus and an unusual Reo-like virus and based on the sequence, several diagnostic tests were developed by ARS scientists in Corvallis, OR for detection of the latter two viruses. The Polymerase Chain Reaction (PCR) test is now being used to identify the vector and native hosts of the Reo-like virus. In addition, plants singly infected with the each of the three viruses are being developed to study the role of each virus individually and in various combinations in the crumbly fruit symptom. These diagnostic tests have been made available to laboratories carrying out virus clean-up programs to ensure the plants produced are free of these viruses and to laboratories responsible for quarantine and certification to improve their programs.
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. I have given 17 presentations at grower oriented meetings this year, which is an efficient means of technology transfer to “Special Target Populations”.
Martin, R.R., Pinkerton, J.M., Kraus, J.E. 2009. The use of collagenase to improve the detection of plant viruses in vector nematodes by RT/PCR. Journal of Virological Methods. 155:91-95.
Susaimuthu, J., Tzanetakis, I.E., Gergerich, R., Kim, K.S., Martin, R.R. 2008. Viral interactions lead to decline of blackberry plants. Plant Disease. 92(9):1288-1292.
Sabanadzovic, S., Vlaverde, R.A., Brown, J.K., Martin, R.R., Tzanetakis, I.E. 2009. Southern tomato virus: the link between the families Totiviridae and Partitiviridae. Virus Research. 140(1-2):130-137.