1a.Objectives (from AD-416)
The major objectives of this proposal are to:
1. Develop improved virus elimination procedures for the berry crops.
2. Perform virus elimination therapy and produce virus-tested material.
3. Establish a foundation block in a screenhouse with virus-tested planting.
4. Develop laboratory-based diagnostic tests for uncharacterized viruses of the berry crops.
5. Develop a diagnostic membrane-based macroarray procedure for the simultaneous detection of berry crop viruses; either a single array or a separate array for each crop genus.
6. Develop serological (ELISA) assays for key viruses that are most easily vectored in different regions of the US.
7. Inform nurseries and growers about clean plant activities and promote clean plants and the NCPN activities through a website to reach out to a broader audience, including stakeholders throughout the US.
1b.Approach (from AD-416)
Extract dsRNAs will be converted to cDNA and will be deep sequenced. Sequence analysis will be performed and primers will be designed and macroarray developed. Detection primers for RT-PCR will be evaluated for viruses under permit and in Oregon and Arkansas for viruses present in the area. Virus clean-up will be performed and virus-free plants will be maintained in Oregon.Documents Reimbursable with APHIS. Log 42070. Formerly 5358-22000-033-16R (5/2011).
This is part of a national effort to develop Clean Plant Programs for major vegetatively propagated fruit crops, including tree fruit, citrus, grapes, berries and hops. The program for developing clean plants for berry crops in Oregon has been active since the mid-1960s. Thus, this program has been designated as the center for the National Clean Plant Network for Berries with auxillary programs currently at North Carolina State University, University of Arkansas (U of A) and University of California at Davis. During the initial year of this project through heat treatment, meristem (0.5 mm) propagation and virus testing we have produced six strawberry and 18 rubus clones free of known viruses. In addition, we initiated studies using a chemotherapy combined with thermal therapy to determine if this will increase efficiency of clean-up for some of the viruses that are more difficult to eliminate. The collection of virus-tested material currently includes: 73 Rubus cultivars and 50 clones of Rubus from the breeding programs in Oregon, Washington and Arkansas and 35 cultivars and clones of Strawberry. We also work with industry partners to identify clones of interest to the U.S. producers from outside of the country that will be brought into the program to minimize the risk of introducing potentially new viruses or other pathogens into the United States. We retested the entire blueberry collection for all known viruses of blueberry as well as for blueberry stunt phytoplasma and Xylella, this was the first time the collection was screened for Blueberry necrotic ring blotch virus and Xylella. The testing of materials for the national clean plant program requires from 10-50 virus tests per sample depending on the crop; blueberry, strawberry, raspberry or blackberry. In collaboration with University of Arkansas, we are working to make this process more efficient and are examining the possibility of using Deep or High Throughput sequencing as a tool to determine the virus status of plants entering the National Clean Plant Network for Berries. In addition, virus assays are being evaluated over a large number of isolates to develop confidence that a test will detect all strains of the virus in question. As an example, over 50 isolates of Blueberry necrotic ring blotch virus have been obtained, and we are in the process of determining variability and identification of conserved regions for developing broad spectrum detection assay. We have done this with Raspberry bushy dwarf virus and Rubus yellow net viruses as well. We are also developing Real-Time PCR tests that are being developed with a Plant Pathologist at the University of Arkansas to be sure they are robust and work well with our isolates in another laboratory setting. The plan is to then expand this validation process to other labs in the United States and beyond so that these assays will be recognized as the standard assay worldwide.