A High Throughput System for the Detection of Phytophthora ramorum in Susceptible Plant Species: A Preliminary Report

Phytophthora ramorum is a pathogen of regulatory concern in North America and Europe. In 2004, potentially infected plants were shipped from large, wholesale nurseries on the West Coast (California, Oregon, and Washington) throughout the U.S. This prompted a nationwide survey effort and the adoption of a federal order requiring mandatory inspection and testing of all West Coast nurseries shipping P. ramorum host and associated host plants (HAP) interstate. In Oregon, this required the testing of 51,645 samples from 1,034 growing areas and 79,930 samples from 1,394 growing areas in 2005 and in 2006, respectively. Because all testing must be completed before nurseries can ship HAP interstate, the Oregon Department of Agriculture developed a high throughput system using a 96-well format to enable testing of large numbers of samples in accordance with federally validated protocols (ELISA, nested PCR, and qPCR). To verify the efficacy of the system, healthy leaves from four HAP species were wounded and then artificially inoculated with P. ramorum; healthy control leaves were wounded and then inoculated with a sterile agar plug. Samples were collected from the inoculated and control leaves and placed into 10 X 96 collection microtubes. Subsamples from each HAP were bulked five per microtube in varying ratios of inoculated to noninoculated tissue (5:0, 1:4, and 0:5). Sample tissues were macerated and tested with ELISA according to the manufacturer’s protocol. The OD readings of all inoculated samples were consistently 5X the negative control. All non-inoculated controls were below the 2X threshold with one exception. In the second replicate, the OD reading of the Pieris japonica noninoculated control was >2X the negative control. DNA was then extracted from the remaining sample tissue in the GEB2 buffer. All inoculated samples were positive using nested PCR while non-inoculated controls were negative. Sample DNA was then tested with qPCR. All inoculated samples were positive while all non-inoculated controls were negative with one exception. In the first replicate, the Camellia non-inoculated control had a Ct value of 40.08 for the P. ramorum-specific probe. According to USDA protocol, this sample would be tested with nested PCR to confirm this negative test result. The high throughput, 96-well format system was also used successfully with environmental samples. Samples from four HAP species and six HAP genera were identified as positive with ELISA and subsequently as P. ramorum-positive by nested PCR and/or qPCR. USDA officially confirmed these test results. These preliminary results indicate that the high throughput system successfully detected P. ramorum in infected plant tissue using the USDA-validated ELISA, nested PCR, and qPCR protocols.