Location: Foreign Disease-Weed Science Research
Project Number: 8044-22000-052-009-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2018
End Date: Jul 31, 2023
Objective:
The specific objectives of this research are to:
1. Develop new or refine current methods for filtration and concentration of P. ramorum propagules in spiked nursery water samples.
2. Test antibodies reactive against P. ramorum with water samples containing infectious propagules of P. ramorum, focusing on detection in irrigation and surface water samples from the National Ornamental Research Site -Dominican University of California (NORS-DUC) mock nursery site.
3. Refine immunoassay diagnostic protocols for nursery water samples.
The primary goal of this agreement is to develop improved detection technologies targeting P. ramorum in nursery irrigation water, reducing detection time, cost and effort needed to demonstrate that nurseries within CA, OR and WA quarantine zones are free of P. ramorum.
Approach:
At the National Ornamental Research Site -Dominican University of California (NORS-DUC) mock nursery site, we will test sampling methods for recovery and enrichment of P. ramorum propagules, including zoospores, sporangia and chlamydospores, aimed toward development of simplified protocols for rapid ELISA assays. Water samples will be taken from ongoing funded projects at NORS-DUC to include: 1) Direct runoff from P. ramorum-infected plants; 2) top, middle, and bottom strata from the NORS-DUC retention pond (not currently recirculating through the nursery), and a large retention tank system (separate, ongoing research project) that feeds and recirculates irrigation water through infected plants. We will use the standard rhododendron leaf baiting technique and compare with filtration through porous, protein-binding membranes, microfiltration technologies vs. centrifugation of water samples for direct recovery of P. ramorum propagules, assessed by plating on PARPH and PCR.
The experiments will focus upon development of methods for rapid filtration of nursery water samples containing suspended soil, potting mixes, plant debris, microorganisms, etc. We will then use filtered nursery water residues for refinement of a direct ELISA immunoassay, testing protocols on zoospores, sporangia and chlamydospores, using fluorescent tag-conjugated monoclonal antibodies (mAbs). For these experiments, we will use un-spiked water as well as samples spiked with P. ramorum zoospores, sporangia, chlamydospores. For spiked water samples, we will deliver a dilution series of cells to ELISA plate wells and identify the lower threshold (number of P. ramorum cells and protein) of the mAbs, ground-truthing the assays by plating on PARPH media and comparing against the APHIS-validated P. ramorum PCR assay.
In addition to development of a direct plating assay, we will test commercially available plant sample extraction buffers (Agdia Inc., Elkhart, IN), pulverizing leaf tissue sections in sample bags (Agdia) to process leaf baits from water samples after the standard 3-day APHIS incubation period. We will test the various commercially available buffer formulations in ELISA formats, using spiked vs. un-spiked samples, testing for sensitivity and specificity as described above.
Assays will employ antibodies to capture P. ramorum sporangia and zoospores from filtrates of nursery runoff and irrigation sources from the NORS-DUC mock nursery, and employ mAbs as the detection reagent. We will use dot blots to vary the titer of target antigen and antibody to test specificity and sensitivity of the mAbs. We will use the mAbs to test applicability and reactivity in mock diagnostic samples, extracted using field extraction buffer kits with 6.5 cm2 leaf samples (Agdia).
