2012 Annual Report
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.
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.