Submitted to: Phytopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 12/29/2008
Publication Date: 4/1/2009
Citation: Martin, F.N., Coffey, M., Berger, P., Hamelin, R.C., Tooley, P.W., Garbelotto, M., Hughes, K., Kubisiak, T. 2009. Evaluation of molecular markers for Phytophthora ramorum detection and identification; testing for specificity using a standardized library of isolates. Phytopathology 98:390-403. Interpretive Summary: This experimentation evaluates different molecular detection methods for identification of the pathogen Phytophthora ramorum, which is responsible for sudden oak death.
Technical Abstract: A number of molecular techniques have been developed for detection of Phytophthora ramorum from infected tissue. These have been based on spacer regions (the rDNA ITS region, the spacer region between the cox I and II gene) or specific genes (beta tubulin, elicitin) and have been configured for use by both conventional and real-time PCR. Techniques based on PCR-RFLP of the cox 1 and 2 gene cluster or single strand conformational polymorphism of the ITS region also have been proposed for isolate identification. In an effort to make a uniform comparison of specificity among the techniques a standard library of DNA was extracted from 317 isolates representing 60 described species and 22 isolates with ambiguous taxonomic classification. Aliquots were adjusted to 10ng/µl, coded and sent blind to all participants (a total of 457 samples were sent). For all extracts the ITS region and cox2 gene were also amplified and sequenced to confirm the identity of the samples. A total of 12 marker systems were evaluated (conventional PCR: cox 1 and 2 spacer, ITS; real-time PCR: cox 1 and 2 spacer, ITS, beta tubulin, elicitin), many by the labs in which they were developed. The results of these evaluations and their implications for effective diagnostics of P. ramorum were discussed.