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Evaluation of Molecular Markers for Phytophthora ramorum Detection and Identification: Testing for Specificity Using a Standardized Library of Isolates

First author: United States Department of Agriculture--Agricultural Research Service (USDA-ARS), Salinas, CA; second author: University of California, Riverside; third and tenth authors: USDA Animal and Plant Health Inspection Service (APHIS), PPQ, CPHST, NPGBL, Beltsville, MD; fourth and ninth authors: Natural Resources Canada, Canadian Forest Service, Quebec, QC, Canada; fifth author: USDA-ARS, Ft. Detrick, MD; sixth author: University of California, Berkeley; seventh author: Central Science Lab, York, UK; eighth author: USDA Forest Service, Southern Research Station, Southern Institute of Forest Genetics, Saucier, MS; eleventh author: California Department of Food and Agriculture, Plant Pest Diagnostics Branch, Sacramento; and twelfth author: USDA-APHIS-PPQ-CPHST, Raleigh, NC.

Given the importance of Phytophthora ramorum from a regulatory standpoint, it is imperative that molecular markers for pathogen detection are fully tested to evaluate their specificity in detection of the pathogen. In an effort to evaluate 11 reported diagnostic techniques, we assembled a standardized DNA library using accessions from the World Phytophthora Genetic Resource Collection for 315 isolates representing 60 described Phytophthora spp. as well as 11 taxonomically unclassified isolates. These were sent blind to collaborators in seven laboratories to evaluate published diagnostic procedures using conventional (based on internal transcribed spacer [ITS] and cytochrome oxidase gene [cox]1 and 2 spacer regions) and real-time polymerase chain reaction (based on ITS and cox1 and 2 spacer regions as well as β-tubulin and elicitin genes). Single-strand conformation polymorphism (SSCP) analysis using an automated sequencer for data collection was also evaluated for identification of all species tested. In general, the procedures worked well, with varying levels of specificity observed among the different techniques. With few exceptions, all assays correctly identified all isolates of P. ramorum and low levels of false positives were observed for the mitochondrial cox spacer markers and most of the real-time assays based on nuclear markers (diagnostic specificity between 96.9 and 100%). The highest level of false positives was obtained with the conventional nested ITS procedure; however, this technique is not stand-alone and is used in conjunction with two other assays for diagnostic purposes. The results indicated that using multiple assays improved the accuracy of the results compared with looking at a single assay alone, in particular when the markers represented different genetic loci. The SSCP procedure accurately identified P. ramorum and was helpful in classification of a number of isolates to a species level. With one exception, all procedures accurately identified P. ramorum in blind evaluations of 60 field samples that included examples of plant infection by 11 other Phytophthora spp. The SSCP analysis identified eight of these species, with three identified to a species group.