Progress 10/01/12 to 09/30/13
Outputs
Progress Report 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. 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. This research was conducted in support of NP303 objective 3B "characterize natural host range, reservoirs and vectors of these viruses and develop strategies to minimize their impact on production" of the parent project. The National Clean Plant Network (NCPN)-Berry program at USDA-ARS in Corvallis, Oregon, working with Animal and Plant Health Inspection Service has developed a pathway that industry is using to bring strawberry and Rubus material from foreign sources into the U.S. with complete testing prior to distribution, propagation and planting. This program also responded to three international trades issues in 2012/ 2013. The first two involved the shipment of more than 30 million plants to Mexico and the Europian Union (EU) that needed to be tested and the other involved the shipment of plants from Canada to the U.S. In all cases, the testing capacity at NCPN-B in Corvallis allowed for the rapid testing of plants, allowing plants to be shipped to Mexico and the EU, and identifying viruses in plants shipped from Canada. The program also collaborates with multiple breeding programs in the U.S. for strawberry, raspberry, blackberry and blueberry has ensured that new materials being released to industry are free of targeted pathogens. The program works with public and private breeding programs to help protect small fruit production in the U.S. The program in Corvallis is the lead for the NCPN for Berries and collaborates with the Testing and Therapy Program at North Carolina State University, Raleigh, North Carolina, which also produces, tests, maintains and distributes Foundation plants of strawberry cultivars free of designated pathogens to nurseries in North Carolina and California. The program in Corvallis also collaborates with the NCPN-Berry program at the University of Arkansas, Fayetteville, Arkansas, in the development of improved diagnostic assays to enhance certification and quarantine programs. This project emphasizes the study of virus diversity to ensure that diagnostic assays are robust in terms of detecting a broad range of isolates before they are deployed.
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Progress 10/01/11 to 09/30/12
Outputs
Progress Report 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. 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. This project aims to develop improved diagnostic assays for virus diseases of small fruit crops and to eliminate targeted pathogens from commercial cultivars of berry crops prior to their release to industry. This program coordinates with breeders to test and clean up advanced selections prior to release. As a result, there are often 3-10 selections that go through the process for every cultivar that is released. During the past year the program had 40 Rubus (raspberry and blackberry) and 11 strawberry cultivars and advanced selections that have gone through heat therapy, meristem tip culture and complete virus indexing. We have also added four cultivars of elderberry and several grape clones to the clean up program. Additionally, experiments are underway to determine how to best eliminate viruses from blueberry, since there is very little information available on 'virus clean-up' in this crop. In 2011, ten named cultivars and 11 advanced selections were sent to nurseries. Nurseries have sold over 30 million plants propagated from plants obtained from this program, and fruit sales from these plants has exceeded 100 million dollars over the past five years (strawberries and Rubus, not including blueberries). Efforts to improve diagnostic assays have concentrated on examining virus diversity in an effort to develop tests that will detect all known strains of a virus. We now know that early tests developed for Blackberry yellow vein associated virus are only able to detect about 12% of the isolates now known. By sequencing several genes of 50 different isolates, a test that detects all known isolates of the virus has been developed. Similar efforts have been done to develop a reliable test for Blueberry necrotic ring blotch virus (55 isolates partially sequenced and a broad spectrum test developed), Blackberry chlorotic ringspot (30 isolates partially sequenced). Additionally, quantitative PCR tests have been developed for several of the berry viruses including (Raspberry bushy dwarf virus, Raspberry leaf mottle virus, Raspberry latent virus, Grapevine leafroll associated virus-3, Blueberry shock virus), which is a more rapid and sensitive test than standard PCR tests. We have used Deep or High Throughput sequencing to make the characterization of viruses of these crops more efficient. We have obtained sequences for new viruses in blueberry, raspberry, blackberry, elderberry, ribes, sugarberry, rose and redbud using this technology. We are now expanding this to use it as a means to identify possible threats to the berry crops by looking at viruses in native relatives of the important berry crops, and also to look at new diseases that are observed in these crops. With several virus-like diseases we have failed to detect a virus using this technology; thus, we will be looking at sequencing small RNAs from these plants. This will allow us to detect DNA viruses and viroids that would be missed by the method of using dsRNA templates, which is what we are doing currently. As new viruses are detected, diagnostic tests are developed and incorporated into the testing for the National Clean Plant programs to ensure material free of the viruses is being developed for commercial growers. This research was conducted in support of objective 3B of the parent project.
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Progress 10/01/10 to 09/30/11
Outputs
Progress Report 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. 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.
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