Source: WASHINGTON STATE UNIVERSITY submitted to NRP
AGRICULTURAL BIOSECURITY: A VERSATILE, HIGH-THROUGHPUT, MOLECULAR DIAGNOSTIC PROTOCOL TO DETECT PLANT PATHOGENS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032206
Grant No.
2024-68013-42671
Cumulative Award Amt.
$999,656.00
Proposal No.
2023-08042
Multistate No.
(N/A)
Project Start Date
Jul 1, 2024
Project End Date
Jun 30, 2027
Grant Year
2024
Program Code
[A1181]- Tactical Sciences for Agricultural Biosecurity
Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
(N/A)
Non Technical Summary
Accurate diagnosis and detection methods are foundational for managing plant diseases. The long-term goal of this integrated agricultural biosecurity project is to develop a complete "field-to-report" system founded on a simplified sample collection protocol that integrates high-throughput molecular diagnostics and sample archiving for rapid and effective plant disease diagnostics. With potato growers supporting the initial sample collection process, we propose capacity building activities to equip a National Plant Diagnostic Network (NPDN) laboratory for virus testing of large volumes of potato tuber samples (Objective 1). A series of greenhouse, laboratory, and field sampling activities will be performed to expand the use of the diagnostic protocol to detect pathogens of economic importance to U.S. agriculture (Objective 2). The project will also create educational and workforce training opportunities on high-throughput molecular detection methods that leverage the efficiencies of the developed diagnostic protocol (Objective 3). The proposed project aims to safeguard the U.S. potato industry by detecting multiple potato pathogens more quickly along with transitioning the diagnostic protocol to a NPDN laboratory, and then further disseminating this protocol to other diagnostic labs.
Animal Health Component
100%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
80713101160100%
Knowledge Area
807 - Disaster Preparedness, Mitigation, Response, and Recovery;

Subject Of Investigation
1310 - Potato;

Field Of Science
1160 - Pathology;
Goals / Objectives
The long-term goal of this integrated agricultural biosecurity project is to develop capacity for a high-throughput molecular-based diagnostic protocol for bacterial, fungal, oomycete, and viral/viroid pathogens of agricultural and horticultural crops. To meet this overarching goal, the three objectives of this project are to: i) Establish a model for integrating simplified plant sample storage and high-throughput PCR assays in a National Plant Diagnostic Network laboratory; ii) Expand the high-throughput PCR assay workflow developed for potato viruses to detect multiple pathogens of potato and other commodity crops; and iii) Create educational and workforce training opportunities on high-throughput molecular detection methods that leverage the efficiencies of the diagnostic protocol.
Project Methods
Objective 1: Establish a model for integrating simplified sample storage and high-throughput PCR assays with a National Plant Diagnostic Network (NPDN) laboratory.Objective 1a: Validation of standards for potato virus Y (PVY) detection at a NPDN lab.For this sub-objective, we will perform validation assays in two labs to generate additional performance statistics and to confirm the process performs as expected in these labs. We will include studies on sensitivity, limit of detection, specificity, accuracy, precision, and a contamination assessment for detection of PVY from potato samples pressed onto Whatman Flinders Technology Associates Plantsaver (FTA) cards using a reverse transcription quantitative real-time PCR (RT-qPCR) assay. All samples will be extracted and amplified using an established protocol.Objective 1b: Implementation of a high-throughput molecular diagnostic protocol.We will collect tuber samples from 10-15 farms in project years 2 and 3, working with potato growers who will support the sampling process with their farm crews. Tuber cores will be pressed onto FTA cards, nucleic acids will be extracted, and RT-qPCR assays will be completed. The data will be analyzed, and a report will be provided to the grower cooperators. To identify logistical barriers for adoption at the field level, we will develop a questionnaire for each cooperator to identify their pros and cons for on-farm sampling, interpretation of results, and use of results from the testing lab for decision-making.Objective 2: Expand the FTA card-based diagnostic protocol developed for viruses of potato to detect multiple pathogens of potato and other commodity crops.Objective 2a: Detection and validation of multiple potato pathogens from inoculated tubers.Greenhouse studies involving artificial inoculations of potato plants with pathogens causing Verticillium wilt, soft rot and late blight will be conducted. Diagnostic protocols developed in Objective 1 will be used to collect samples on FTA cards and qPCR assays will be conducted to detect respective pathogens.Objective 2b: Detection and validation of multiple potato pathogens from field samples.For this sub-objective, we will expand the diagnostic protocol to test field samples. We will obtain 50 tubers/plants from each of three fields exhibiting symptoms of Verticillium wilt, late blight, and/or bacterial soft rot. Leaf, tuber, and shoot samples will then be tested for the respective pathogens by pressing the samples onto FTA cards and pathogen isolations will be conducted from each host tissue sample. In addition to the real-time qPCR assays for the three pathogens, we will screen samples for PVY and PVS, viruses prevalent in all potato growing regions in the U.S. We will develop one unified diagnostic protocol for detecting multiple pathogens from potato field samples.Objective 2c: Expansion of FTA card-based diagnostic protocol to other commodity crops.We will test the protocol with viruses infecting other commodity crops such as wheat and blueberry. Accuracy, specificity, and sensitivity will be evaluated for FTA card and PCR assay with each pathogen-host combination.Objective 3: Create educational and workforce training opportunities on high-throughput molecular detection methods that leverage the efficiencies of the diagnostic protocol.Objective 3a: Technology transfer and protocol dissemination.For this sub-objective, the Colorado State University Plant Diagnostic Clinic will share the diagnostic protocol established in Objective 1 and provide necessary training to NPDN and other interested diagnostic labs. We will provide an overview of the protocol to NPDN members through the Great Plains Diagnostic Network webinar series, which is attended by the broader NPDN community. We will create short videos and guides on FTA card use and applications, molecular detection protocols adapted to multiple pathogens, data analysis, and interpretation, and statistical considerations for this high-throughput model.Objective 3b: Educational resources and workforce training opportunities.Complementary to the lab protocol training, we will provide training on FTA card preparation and shipping for field personnel, growers, crop consultants, and others involved in sample collection. We will develop videos and written documents with sampling and FTA card handling guidelines and provide in-person training for sample preparation and processing. We will train students/interns on the scientific/technical aspects of the project.

Progress 07/01/24 to 06/30/25

Outputs
Target Audience:Our efforts to reach target audience and make them aware of the project progress include giving presentations at local grower conferences in Washington, Oregon, Wisconsin; a national grower conference (Potato Expo), a regional meeting of the American Phytopathological Society, and a national potato industry conference (Annual Meeting of the Potato Association of America). We also initiated efforts to adapt the protocol for use in virus testing of a public orchid collection composed of over 3,000 plants, with the goal of improving efficiency in their routine screening. Changes/Problems:A major challenge encountered during this reporting period was the moving of one of the Co-PIs (Dr. Ana Cristina Fulladolsa) from Colorado State University to the University of Maryland in December 2024. This led to additional paperwork for transferring sub-contract funds to the new institution. Now that Dr. Fulladolsa has assumed full responsibilities as Director of the Plant Diagnostic Clinic at the University of Maryland, we will continue to show progress on the objectives led by her. Due to the USDA hiring freeze we were unable to hire technical support. What opportunities for training and professional development has the project provided?This project has supported a graduate student, Francely Flores, through coursework towards her PhD degree in plant pathology. How have the results been disseminated to communities of interest?Presentations: Mattupalli, C., and Loganathan. D. G. 2025. The Ag Biosecurity Project: pathogen detection and tuber sampling robot. WSU Potato Field Day (June 26, 2025; audience size = 60). Fulladolsa, Ana C. 2025. The plant diagnostic lab: a model of the land-grant mission - research and education. 81st Annual Potomac Division Meeting of the American Phytopathological Society. Dowell, MD (March 26-28; audience size = 30). Mattupalli, C. 2025. Lessons learned from a grower supported mail away PVY test project. 2025 Wisconsin Potato and Vegetable Grower Association and University of Wisconsin Division of Extension, Grower Education Conference, Stevens Point, WI (February 5, 2025; audience size = 100). Mattualli, C. 2025. Direct tuber testing for detection of potato pathogens. 2025 Washington and Oregon Potato Conference, Kennewick, WA (January 30, 2025; audience size = 200). Mattpalli, C., and Sklarczyk, D. 2025. A field-to-report workflow for improved detection of potato pathogens. 2025 Potato Expo, Orlando, FL (January 10, 2025; audience size = 30). Matupalli, C., Ingram, J. T., Charkowski, A. O., and Filiatrault, M. J. 2024. Direct tuber testing for detecting potato pathogens. 2024 Lynden Ag Show, Lynden, WA (December 4, 2024; audience size = 50). Ingram, J. T., Mattupalli, C., Charkowski, A. O. and Filiatrault, M. J. 2024. Lessons learned from a grower supported mail-away PVY test project. 108th Annual Meeting of the Potato Association of America, Portland, OR (July 22, 2024; audience size = 50). What do you plan to do during the next reporting period to accomplish the goals? We anticipated that a NPDN member lab would be positioned for high throughput processing of grower submitted samples by the end of year 1. This was not accomplished due to moving of one of the co-PIs (Dr. Fulladolsa) to another institution. As Dr. Fulladolsa has assumed new role at the University of Maryland, we will accomplish this objective in year 2. In year 2, we will give at least one presentation at a grower meeting. By the end of year 2, we will complete at least one greenhouse experiment for detecting Verticillium, Pectobacterium, and Phytophthora infestans from potato samples. In year 2, we will train one staff member and one graduate student in sample processing, FTA card use, and molecular detection of potato pathogens. Develop one webinar/workshop in year 2 to NPDN members, field personnel, and public. In year 2, we will design one brochure on the FTA card-based diagnostic protocol.

Impacts
What was accomplished under these goals? The overall goal of this project is to develop capacity for a high throughput, molecular-based pathogen detection workflow for bacterial, fungal, oomycete, and viral pathogens of agricultural and horticultural crops. The primary scientific challenges being addressed are: • How to integrate validated, FTA card-based sample storage, and high throughput molecular assays with a National Plant Diagnostic Network laboratory. • Can we expand the FTA card-based pathogen detection workflow developed for viruses of potato to detect multiple pathogens of potato and other agronomic and horticultural crops? • How to create educational and workforce training opportunities for faster adoption of FTA card-based pathogen detection workflow. During this reporting period, the Plant Diagnostic Laboratory at the University of Maryland is being equipped with machines (e.g., real-time PCR; automated nucleic acid extraction system) that will facilitate the implementation of high-throughput molecular tests. A greenhouse experiment was conducted to study the applicability of the FTA card-based workflow to detect Verticillium dahliae, an important soilborne pathogen causing Verticillium wilt of potato. Potato tubers showing symptoms of soft rot were obtained from grower fields and the feasibility of detecting soft rot pathogens from FTA cards was tested. A graduate student was provided educational opportunity to develop knowledge and skills on high-throughput detection of plant pathogens. Since this is the first year of the project, we are planning experiments and gathering data and therefore no measurable changes have resulted from this project.

Publications