Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 10, 2004
Publication Date: June 20, 2004
Citation: Martin, F.N., Tooley, P.W., Blomquist, C. 2004. Molecular detection of phytophthora ramorum and a phytophthora ilicis-like species associated with sudden oak death in california. Phytopathology. v. 94. p. 621-631. Interpretive Summary: Sudden Oak Death has been an emerging disease problem in coastal California and has caused significant losses in forest ecosystems in some regions of the state. A molecular marker system based on mitochondrial DNA sequences was developed for identification of Phytophthora species from symptomatic host tissue as well as the pathogen responsible for sudden oak death, P. ramorum,. Molecular markers also were developed for identification of a P. ilicis-like species that is also commonly recovered from plant tissue exhibiting symptoms similar to P. ramorum.
Technical Abstract: Sudden oak death is a disease currently devastating forest ecosystems in several coastal areas of California. A molecular marker system was developed based on mitochondrial sequences for detection of Phytophthora species in general and P. ramorum and a P. ilicis-like isolate in particular. The first round multiplex amplification contained two primer pairs, one for amplification of plant sequences to serve as an internal control to ensure that extracted DNA was of sufficient quality to allow for PCR amplification and a second primer pair specific for amplification of sequences from all Phytophthora spp. The plant primers amplified the desired amplicon size in the 29 plant species tested and did not interfere with amplification by the Phytophthora genus-specific primer pair. The Phytophthora genus-specific primer pair amplified a diagnostic fragment from all 28 species evaluated, but not from 30 Pythium spp. or from 29 plant species, although occasional faint bands were observed for three additional plant species. The products of the first round amplification were amplified with nested primers that were specific for P. ramorum or a P. ilicis-like species. These species-specific primers amplified the target sequence from all isolates of the pathogen under evaluation. The limit of detection using this marker system was approximately 0.02 pg of total DNA. This marker system was validated with samples collected from naturally infected plants from the field by sequence analysis, morphological identification of cultures, and for P. ramorum, amplification with a previously published rDNA ITS species-specific primer pair. The specificity of the Phytophthora genus-specific primers suggests they will have utility for pathogen detection in other Phytophthora pathosystems.