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ARS Home » Pacific West Area » Corvallis, Oregon » Horticultural Crops Research » Research » Research Project #434816

Research Project: Testing, Therapy, Clean-up, and Diagnostics for Berry Crops

Location: Horticultural Crops Research

Project Number: 2072-21220-003-24-I
Project Type: Interagency Reimbursable Agreement

Start Date: Oct 1, 2017
End Date: Sep 30, 2018

Objective:
1. Introduce, index and release pathogen-tested domestic and foreign berry (Fragaria, Rubus, Vaccinium) plant material to meet the needs of the industries in the U.S. Perform virus elimination therapy on material that has tested free of targeted pathogens and add to G1 blocks. Expand G1 block for Vaccinium. 2. Expand ring-test to non-National Clean Plant Network (NCPN) laboratories to increase testing capacity. 3. Validation of Next Generation Sequencing (NGS) technology for routine virus testing of G1 plants. 4. Evaluate the use of RNAi therapy for viruses that are difficult to eliminate using thermal or chemotherapy. 5. Retest the G1 blocks of Rubus and Vaccinium. 6. Improve diagnostics for the following viruses: blueberry virus A, blueberry shoestring virus, blueberry shock virus, blackberry virus Y, black raspberry ringspot virus, strawberry crinkle virus, strawberry pallidosis associated virus. 7. Disseminate information about NCPN-Berries efforts: Participation in regional and national meetings.

Approach:
High throughput sequencing (HTS), also known as Next generation Sequencing (NGS), provides a rapid and robust alternative approach for the identification of viral pathogens. All genomic DNA and RNA sequences in the sample are produced by HTS and bioinformatic analysis. Pathogens are identified by comparisons of the information in the data set against the database of all known pathogen genomic sequences. HTS detection of viruses is not limited by viral strain differences and does not change with environmental conditions. Most importantly, HTS is not dependent on prior knowledge of the pathogen sequence. Current applications of HTS include studying diseases of unknown etiology, resequencing known viruses, and determining host pathogen interactions. HTS is also used as a diagnostic tool in post entry quarantine and in the identification of known viruses. Recent studies have found HTS to be superior to the standard conventional methods for the detection of viruses of economic significance in grapevine and fruit trees. While HTS remains powerful new technology with significant benefits, there are technical challenges associated with HTS technology and establishing economic significance for viruses identified via HTS analysis remains a consideration. Faced with these challenges, an inter-laboratory collaboration to establish guidelines for HTS use is foundational to future progress in the field. The collaboration will be used to coordinate the development and adoption of standardized HTS technologies for the detection of viral diseases. A standardized protocol will be validated for use as an official test method. This standardized protocol will increase the efficiency of diagnostic units and improve the quality of results. Added benefits of a standardized protocol include optimal use of resources, reduction in the duplication of efforts, and providing a method to share material, protocols, and information. Specifically, the protocol would include details on sample collection, nucleic acid preparation, and library construction. Common bioinformatics pipelines will be adapted or developed, protocols will be validated by inter-laboratory comparisons, and reference sequence data will be added to GenBank. Viral diseases are major agricultural economic problem and the establishment of standardized protocols is vital to protect agricultural production in the United States.