Location: Emerging Pests and Pathogens Research
Project Number: 8062-21000-048-010-A
Project Type: Cooperative Agreement
Start Date: Jul 1, 2022
End Date: Sep 30, 2023
Objective:
The design of this project is based on the premise that high throughput laboratory-based detection of viruses from tubers directly would facilitate more accurate and faster delivery of results to seed potato growers than conventional approaches followed by state seed potato certification agencies. Two long-term goals of this project are:
1. Improved decision-making support for seed potato certification so the potato industry can respond quickly to emerging pathogens and reduce spread of tuber-borne viruses.
2. Generate awareness towards harmonization of seed potato certification rules across all potato producing states in the US.
To meet these goals, the project is divided into three objectives:
a. Develop a workflow for commercial high throughput PCR testing of seed potatoes.
b. Establish a model for the adoption of PCR-based seed potato testing by the potato industry.
c. Identify and mitigate barriers for wide scale adoption related to farm level tuber sampling and PCR-based seed potato testing approach.
Approach:
A molecular test for potato virus Y in seed potato tubers will be validated. ARS will provide the cooperator with cards with known positive and negative Potato virus Y controls to develop protocols and a workflow that will facilitate high throughput processing of samples in accordance with the ISO 17025 accreditation standards. The cooperator will perform a validation, including a detailed testing plan meeting accreditation requirements prior to initiation of any work, adequate training for testing personnel, an organized validation data binder and standard operating procedures for audit purposes that demonstrates compliance with all accreditation guidelines. The validation plan will require the following studies: sensitivity/viral limit of detection, comparison to current testing methods, specificity of the selected primers, accuracy and precision (reproducibility and repeatability) of the assay with known samples, contamination/false positive assessment.
Once the assay is validated, testing of seed potato tubers will begin using the validated work-flow. Grower cooperators will sample tuber cores on paper cards. The field crew will be trained on-site by participating seed certification inspectors and researchers or through stand alone instructions. Sample from many different potato producing states will be obtained to maximize geographic footprint across states and the farms ability to track and provide meta-data about agronomic conditions of the seed lots grown. The field crews provided by each farm or cooperator will sample four tuber cores (heel, rose, and two eyes) from each tuber using a 3mm diameter biopsy tool and placed onto one square in the cards. Tuber cores from 25 tubers will be placed on one paper card and pressed using a hydraulic shop press at a minimum of 12 tons for one-minute.
The potato solids will be removed, cards dried and then shipped for further processing. Cooperator will carry out testing for virus using the validated protocol. Data will be analyzed and organized into graphs and tables for the validation summary. Excel or other IT tools for tracking samples through the laboratory process and conversion of the final results to a client report template will be performed. Detailed standard operating procedures will be created based on the validation for both laboratory processing and interpretation criteria. Training of testing personnel will include lectures on both the scientific virus background and laboratory techniques, laboratory demonstrations and supervised testing sets and final independent competency testing. Results will be compared with seed potato certification decisions to determine if the method used for determining virus incidence has a significant impact on seed lot fate. Information will be obtained from our grower cooperators on seed potato certification data for comparison to the laboratory data generated in this project, including vine kill dates, and regional aphid data to assess additional uses of early and accurate information about Potato virus Y.
