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Rapid methods for detection of Phytophthora ramorum in nursery water


<div><em>Phytophthora ramorum</em>, causal agent of sudden oak death (SOD), continues to threaten U.S. forest ecosystems and the U.S. nursery industry. Presently, USDA APHIS relies upon water baiting for <em>P. ramorum</em> diagnosis and confirmation in nurseries inside the boundaries of regulated zones. USDA’s national water baiting program utilizes the Bottle of Bait (BOB) recovery method for <em>P. ramorum</em>, which requires collecting water from a source stream, baiting with healthy rhododendron leaves for a 3-day incubation/infection period, followed by plating on semi-selective media. More rapid methods are needed for recovery and detection of <em>P. ramorum</em> propagules from nursery water sources. We have developed and refined rapid water filtration methods for recovery of <em>P. ramorum</em> propagules using tandem filters with defined pore sizes. Novel antibodies, raised against <em>P. ramorum</em> -specific peptide epitopes in cell wall, flagellar and secreted proteins, were applied for detection of propagules by immunofluorescence directly on filters and in immunoassays with filter extracts. Results indicate that <em>P. ramorum</em> propagules recovered by filtration from spiked nursery water samples can be detected in immunoassays in 24 h or less.</div>