1a.Objectives (from AD-416):
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, retest existing G1 material in the block.
4. Develop laboratory-based diagnostic tests for uncharacterized viruses of the berry crops.
5. Develop new technologies for detection, emphasis will be based on deep sequencing as a method to do broad spectrum testing on clean plants.
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):
Extracted dsRNAs will be shipped (under permit approval) to the University of Arkansas where they will be converted to cDNA. Sequence analysis will be done at the USDA-ARS-HCRL and University of Arkansas. For deep sequencing both Illumina and 454 technologies will be evaluated for detection of all viruses in a plant. We will also be using Illumina to sequence micro RNAs from plants, since that has been shown recently to be useful in detecting RNA and DNA viruses. Detection primers for RT-PCR will be evaluated at USDA-ARS-HCRL for viruses under permit and at both locations for viruses present in the areas. Virus clean-up will be done at- and virus-free plants will be maintained at USDA-ARS-HCRL.
This research was conducted in support of NP303 objective 2B of the parent project. The National Clean Plant Network (NCPN)-Berry program at USDA-ARS in Corvallis, Oregon, carries out several aspects of the NCPN for Berries. The primary activity is the collaboration 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. We also have an NCPN import permit from APHIS and provide 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. We are currently working with nurseries to determine Critical Control Points in keeping plant material from NCPN clean during the propagation cycles in screenhouse and field propagation. 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 has two major goals: first 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, and; second the evaluation of Next-Generation sequencing as a tool to determine virus status of G1 plants, the top tier plants in certification programs.