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Real-Time Fluorescent Polymerase Chain Reaction Detection of Phytophthora ramorum and Phytophthora pseudosyringae Using Mitochondrial Gene Regions

April 2006 , Volume 96 , Number  4
Pages  336 - 345

Paul W. Tooley , Frank N. Martin , Marie M. Carras , and Reid D. Frederick

First, third, and fourth authors: U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), Foreign Disease-Weed Science Research Unit, Ft. Detrick, MD 21702; and second author: USDA-ARS, 1636 East Alisal St., Salinas, CA 93905

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Accepted for publication 24 November 2005.

A real-time fluorescent polymerase chain reaction (PCR) detection method for the sudden oak death pathogen Phytophthora ramorum was developed based on mitochondrial DNA sequence with an ABI Prism 7700 (TaqMan) Sequence Detection System. Primers and probes were also developed for detecting P. pseudosyringae, a newly described species that causes symptoms similar to P. ramorum on certain hosts. The species-specific primer-probe systems were combined in a multiplex assay with a plant primer-probe system to allow plant DNA present in extracted samples to serve as a positive control in each reaction. The lower limit of detection of P. ramorum DNA was 1 fg of genomic DNA, lower than for many other described PCR procedures for detecting Phytophthora species. The assay was also used in a three-way multiplex format to simultaneously detect P. ramorum, P. pseudosyringae, and plant DNA in a single tube. P. ramorum was detected down to a 10-5 dilution of extracted tissue of artificially infected rhododendron ‘Cunningham's White’, and the amount of pathogen DNA present in the infected tissue was estimated using a standard curve. The multiplex assay was also used to detect P. ramorum in infected California field samples from several hosts determined to contain the pathogen by other methods. The real-time PCR assay we describe is highly sensitive and specific, and has several advantages over conventional PCR assays used for P. ramorum detection to confirm positive P. ramorum finds in nurseries and elsewhere.

Additional keywords: coxI, coxII.

The American Phytopathological Society, 2006