Submitted to: Phytopathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: November 24, 2005
Publication Date: April 1, 2006
Citation: Tooley, P.W., Martin, F.N., Carras, M.M., Frederick, R.D. 2006. Real-time fluorescent pcr detection of the sudden oak death pathogen, phytophthora ramorum, using mitochondrial gene regions. Phytopathology. 95:336-345. Interpretive Summary: We describe in this paper the application of a novel methodology to the detection of an important plant pathogenic fungus. The fungus, Phytophythora ramorum, is related to the fungus that caused the Irish potato famine in the mid 1800's. It has killed thousands of oak trees on the Calfornia coast since 1995 and is a threat to the Eastern oak forests of the U.S. Detection tests are needed that can reliably detect fungus at very low levels, allow many samples to be processed at once, and reduce contamination. The test described in this paper uses a DNA detection methodology known as "real-time PCR" to detect minute amounts of the pathogen's DNA from infectd leaf tissue. The test is rapid and sensitve, and detects only the sudden oak death pathogen with high reliability. We used the test to detect the pathogen in infected plant samples from the field in California, proving its utility with actual field material. We also modified the test so it can detect two different pathogens in a single test tube, making the test more efficient. The detection assay described here will provide a needed tool for detecting the sudden oak death pathogen in news areas.
Technical Abstract: A real-time PCR detection method for the sudden oak death pathogen, P. ramorum, was developed based on mitochondrial DNA sequence and using an ABI Prism 7700 (Taqman) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species which causes symptoms similiar to those of P. ramorum on certain hosts. The Phytophthora-specific primer-probe system was used in a multiplex assay with a plant primer-probe systems utilizing FAM and CAL Orange dyes to allow plant DNA to serve as a positive control in each reaction. With the FAM dye system, the lower limit of detection of P. ramorum DNA was 1 fg genomic DNA. P. ramorum was detected in artificially inoculated Rhododendron 'Cunningham's White' (12 mg fresh weight tissue) to a lower limit of a 10 -5 dilution. We also detected P. ramorum from infected California field samples from various hosts which had been determined to contain the pathogen via convential PCR and isolation on selective agar medium. The assay we describe will prove useful in allowing rapid, sensitive and specific detection of the sudden oak death pathogen on ornamentals and other host species with greater ease of use compared with conventional PCR procedurs.