Location: Emerging Pests and Pathogens Research
Project Number: 8062-22000-023-004-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 15, 2019
End Date: Aug 14, 2023
RESEARCH OBJECTIVES: The overall objective of this project is to provide seed certification with protocols for determining virus disease incidence estimates using dormant tuber testing and to help with the implementation of the new procedures based on industry needs and risk tolerances. This will also aid in the development of other pathogen-specific assays. Specific objectives are: YEAR 1: 1. Compare virus detection efficiencies in tubers using the recently developed PCRbased assays versus visual or ELISA-based assessments of foliar disease. 2. Obtain information from growers regarding their willingness to accept changes to seed certification with an emphasis on cost, effort, information availability, and risk tolerances. YEAR 2: 1. Optimize PCR-based diagnostics for important viruses and bacteria using the DNA/RNA extracted from the FTA cards. 2. Compare the results obtained from the new assay with current protocols used by seed certification. YEAR 3: We will refine methods to automate dormant tuber testing of on-farm collected FTA cards prepared by collaborating seed growers. In addition to determining how these sample results compare with winter grow-out results, another component will include identifying problems related to scaling up sampling and the turnaround time for results. Transitioning development to a partnership with growers sampling will require a new “How to” user’s manual and grower input. Another component seeks to safeguard the continued utility of PCR testing for potato viruses. In summary, Dormant Direct Tuber Testing work will focus on: “Automate, Forecast, Expedite, and Educate”. Specific objectives for the third year are: 1. Automate - Continue to optimize and automate RT-PCR/PCR-based diagnostics for important viruses and bacteria using the DNA/RNA extracted from the FTA cards. 2. Forecast- Explore using Next Generation sequencing (NGS) from FTA samples to confirm the PCR methods used for screening for viruses remain effective. 3. Expedite - Continue to work with grower sampled FTA cards and seed certification programs to compare results obtained from the new assay with current protocols used by seed certification and timeliness. 4. Educate - Transition from Development to a “How To Owners Manual” for on farm success.
EXPERIMENTAL APPROACH: YEAR 1: Objective 1. PVY incidence is, for the most part, the only virus data that seed certification programs routinely use for certifying seed lots. No program currently tests for TRV or PMTV, and it's not possible to visually assess incidence of these viruses. Visual assessment is the primary means to determine PVY incidence although several certification programs follow-up with ELISA testing of foliage. Only one program, Maine, is testing tubers directly using ELISA testing of sprouts. Our experiments will focus on newly harvested dormant tubers as the starting testing material. Preliminary experiments have identified seed or commercial potato fields in CO and WI that are infested with viruliferous vectors of TRV and PMTV and generate a potato crop with high incidence of virus infected tubers. Objective 2. Implementation of new testing protocols will only be successful if the potato industry, notably seed growers and seed buyers, advocate for the change. This requires a campaign that can provide the industry with solid data that new protocols: a) are as good or better than current protocols, b) they will provide high quality seed, and c) that information is provided in a timely manner that facilitates seed purchases and farm planning. We plan to work with the Cornell University Institute for Social and Economic Research to survey growers and pertinent industry reps to understand needs and tolerance for changes in protocols including sample collection, sample processing, data distribution and costs. These data will facilitate the development of final diagnostic protocols and the design of the outreach campaign needed to implement formal changes in seed certification practices. The project has the potential to guide a transformative change in U.S. potato seed certification programs & enable the programs to provide more reliable virus and other disease incidence estimates to growers shortly after harvest without the need for winter grow out plots that are subject to weather. Growers would have the option to collect and provide tuber flesh samples on the farm and avoid the cost of shipping tubers. Once samples are saved on the FTA cards they can be tested for an array of pathogens that will vary from year to year. Samples can be stored for months and retested as needed. Current seed certification practices cannot reliably determine incidence of many emerging or chronic pathogens responsible for the degradation of seed quality. Competitive and sustainable seed potato certification programs need new options to manage and maintain a reliable supply of high-quality seed potatoes for domestic use and for international trade. YEAR 2: Objective 1: Further validating of testing protocols by expanding PCR-based testing to include other viruses, bacteria, and pathogens and optimizing PCR-based detection assay using real-time quantitative PCR (qPCR) or high throughput sequencing (HTS). Based on grower surveys, other pathogens, including bacteria, were reported as grower concerns. Objective 2: To compare direct tuber testing with visual/ELISA protocols now in use by using two seed-certification program lots as test subjects.