2010 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).
Characterization of viruses this year included our first attempts at high density sequencing to identify multiple viruses in a single source plant. We have become familiar with the multitude of software programs and we have identified as many as five viruses in a single plant using this technology. However, we have not been able to identify all genome segments of a new virus in raspberry. We did get sequence for nine of the 10 genomic segments with this technology, but the 10th segment was identified using standard cloning strategies. The problem with the last segment is that it did not have a close match in the database, thus the software for the high throughput analysis disregarded this sequence. Using traditional methods, we worked with the genomic segment, and when it did not come up with a match in the database we lowered the search stringency and were able to pick out a very weak relationship to an animal reovirus. Once we had that, we went ahead and finished sequencing the fragment and showed it had the same ends as the other nine genomic pieces of the virus, indicating in was part of the total virus genome. We then developed detection primers for this piece of the genome and showed that it was present in infected plants but not healthy plants, confirming the sequence we had was indeed part of the viral genome.
We have used our genomic data to identify vectors of several viruses. With two viruses where sequence data suggested an aphid vector, we found that indeed they were aphid transmitted in greenhouse experiments. However, one virus where the sequence information suggested a leafhopper vector proved to be aphid transmitted, and the virus replicates in the aphid. This information will change our strategies in developing control measures for several reasons; for example, aphids are much less mobile than leafhoppers in the field. Also, with virus replication in the vector, once a vector acquires the virus it will be able to transmit the virus for the rest of its' life. This means that we will need to know when this aphid disperses in the field before we can plan a suitable control strategy for this virus.
In an effort to identify a virus or viruses associated with a new disease of blueberry in British Columbia, Washington and Oregon, we have characterized a new virus in blueberry. As it turns out, this virus is widespread in blueberry in all areas tested, including the Northeast, Southeast, Midwest and Pacific Northwest, but it is not associated with blueberry fruit drop disease.
Partial sequence and characterization of a new virus from blueberry infected with Blueberry necrotic ring blotch suggest this is a novel virus that could well be transmitted by eriophyid mites. Since this virus does not occur in our area, the vector transmission studies are being done at the University of Georgia. In a collaborative project with colleagues in Alaska, we have tentatively identified Grapevine virus E in black currant in Alaska. Previously, this virus has been reported from grapevine in Japan and South Africa.
Diagnostic tests for viruses in small fruit crops. Virus detection in small fruit crops is still largely dependent on graft transmission assays to indicator hosts, which is time consuming and in some cases good indicators are unknown. ARS researchers in Corvallis, Oregon together with a colleague at the University of Arkansas have developed PCR based tests for a number of viruses in these crops. These tests are more sensitive and much faster than graft indexing and are currently being used in parallel with graft indexing to ensure they are 'as good as or better' than the currently used tests. The new methods improve the reliability of testing of plant material before it is released to industry for commercial production, increasing the quality of plants used in fruit production, thereby improving the profitability of the growers.
Sequence of new reovirus from raspberry, raspberry latent virus. Mixed virus infections were identified in red raspberry in northern Washington and British Columbia that exhibited severe crumbly fruit. A new virus was detected in red raspberry in northern Washington and shown to be widely distributed in plantings within one year of establishment. ARS Researchers in Corvallis, Oregon together with a graduate student at Oregon State University extracted, cloned and sequenced the dsRNA, developed a detection method for the virus and demonstrated the virus is transmitted by aphids. The virus test is being used in certification and virus clean up programs to ensure plants free of the virus are being made available to the industry. With it's rapid dispersal in the field, starting with clean plants and vector control will be required to minimize the impact of this virus on raspberry production in the future.
5.Significant Activities that Support Special Target Populations
The production of blackberry, raspberry and strawberry plants free of known viruses. Having high quality plants that produce high quality fruit enhances the profitability of small as well as large farmers, it is size neutral
Mekuria, T.A., Karasev, A.V., Martin, R.R., Naidu, R.A. 2009. First Report of Grapevine leafroll-associated virus-3 in wine grape cultivars in Idaho. Plant Disease. 93:1218.
Brown, J.K., Rogan, D., Idris, A.M., Martin, R.R., Rehman, M. 2010. First report of "Candidatus Liberibacter psyllaurous" (synonym "Ca. L. solanacearum") associated with 'tomato vein-greening' and 'tomato psyllid yellows' diseases in commercial greenhouses in Arizona. Plant Disease. 94(3):376.
Tzanetakis, I.E., Tsai, C., Martin, R.R., Dreher, T.W. 2009. A tymovirus with an atypical 3´-UTR illuminates the possibilities for 3´-UTR evolution. Virology. 392:238-245.
Alabi, O.J., Martin, R.R., Naidu, R.A. 2010. Sequence diversity, populationgenetics and potential recombination events in Rupestris stem pitting-associated virus in Pacific Northwest vineyards. Journal of General Virology. 91:265-276.
Tzanetakis, I.E., Martin, R.R., Scott, S.W. 2010. Genomic sequences of Blackberry chlorotic ringspot virus and Strawberry necrotic shock virus and the phylogeny of viruses insubgroup 1 of the genus Ilarvirus. Archives of Virology. 155:557-561.
Jarugula, S., Martin, R.R., Naidu, R.A. 2010. Molecular diversity of Grapevine leafroll-associated virus-2 isolates in Pacific Northwest vineyards. Phytopathology. 100:698-707.
Mekuria, T., Gutha, L.R., Martin, R.R., Naidu, R.A. 2009. Genome diversity and intra- and inter-species recombination events in Grapevine fanleaf virus . Phytopathology. 99(12)1394-1402.