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
0%
Research Effort Categories
Basic
(N/A)
Applied
100%
Developmental
(N/A)
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.