In April 2002, when APHIS-USDA-PPQ sent out the guidelines for the Sudden Oak Death national nursery survey, it seemed obvious that it would be in Oklahoma’s best interest to participate in the survey. Funding was obtained through the Western Region beginning in July 2002. Although all ornamental nursery stock meeting the survey criteria will be surveyed in May-June of 2003, a pilot survey of the two largest ornamental nurseries was conducted during late July 2002 to get the sampling and assay systems in place. PCR technology was chosen as the primary means of detecting P. ramorum in plant samples. Dr. Matteo Garbelotto, University of California-Berkeley, supplied us with the specific protocols, primer sequences, and enough extracted P. ramorum DNA to be able to run the PCR assays. Cultural methods were also used on some samples to establish what other Phytophthora spp. were present. In June 2002, a training session was held for all personnel that would be involved in the surveys, including the Oklahoma Department of Agriculture nursery inspectors, who are responsible by law for the actual sampling of all nursery stock in Oklahoma. However, we assisted the inspectors with sampling of the two pilot nurseries so that the sampling methods and data handling procedures could be established for the 2003 survey. During August 2002 assaying of the survey samples was initiated.
A number of challenges were encountered in this pilot study. The first set of these concerned the sampling of the nursery material. It is always difficult to survey for something that is not expected to be found, but it is especially difficult when information on symptoms, plant parts affected, and the disease cycle is preliminary. Should only symptomatic above-ground parts be sampled and tested? Several Phytophthora spp. cause shoot disease on rhododendrons in Oklahoma, so inspectors should be familiar with these and should be able to take appropriate samples. But what do P. ramorum symptoms really look like on viburnum? Only one poor photo from Europe was available as a guide. Viburnum is a very common nursery plant in Oklahoma nurseries so this is no small question. What tissues should be sampled? To detect P. ramorum, it is critical that the small amounts of tissue selected for the PCR assays be those most likely to have the fungus in them. The second set of challenges concerned the PCR assays. Unlike the simpler PCR assays that were part of the lab’s repertoire, the P. ramorum protocol was a considerably more complicated, nested assay requiring two DNA amplifications. Work programs had to be adapted to the much larger volume of assays as compared to that encountered for diagnostic samples. It took some time to get the procedures working and to maximize efficiency. One frustration of being outside the quarantine area is not having reference cultures of P. ramorum to work with and not being able to include P. ramorum infected plant material in assays to increase confidence.
No P. ramorum was been detected in any of the samples and no cross reactions with other Phytophthora spp. have been encountered, even in samples shown by culturing to have other Phytophthora spp. present.
