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Molecular Detection of Phytophthora ramorum by Real-Time Polymerase Chain Reaction Using TaqMan, SYBR Green, and Molecular Beacons

First and eighth authors: Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, P.O. Box 3800, 1055 du P.E.P.S., Sainte-Foy, Quebec, G1V 4C7, Canada; second author: Agriculture and Agri-Food Canada, National Program on Environmental Health-Biodiversity, 960 Carling Avenue, Ottawa, Ontario, K1A 0C6, Canada; third author: Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands; fourth author: Département de biochimie et de Microbiologie, Université Laval, Pavillon Alexandre-Vachon, Quebec, Canada; Centre de Recherche de l'Hôpital Laval, 2725 chemin Sainte-Foy, Sainte-Foy, Quebec, G1V 4G5, Canada; fifth author: Pest DNA Diagnostics Laboratory, Centre for Plant Quarantine Pests, CFIA, Ottawa, Ontario, K2H 8P9, Canada; and sixth and seventh authors: U.S. Department of Agriculture-Agricultural Research Service (USDA-ARS), 1636 East Alisal St., Salinas, CA 93905

Sudden oak death, caused by Phytophthora ramorum, is a severe disease that affects many species of trees and shrubs. This pathogen is spreading rapidly and quarantine measures are currently in place to prevent dissemination to areas that were previously free of the pathogen. Molecular assays that rapidly detect and identify P. ramorum frequently fail to reliably distinguish between P. ramorum and closely related species. To overcome this problem and to provide additional assays to increase confidence, internal transcribed spacer (ITS), β-tubulin, and elicitin gene regions were sequenced and searched for polymorphisms in a collection of Phytophthora spp. Three different reporter technologies were compared: molecular beacons, TaqMan, and SYBR Green. The assays differentiated P. ramorum from the 65 species of Phytophthora tested. The assays developed were also used with DNA extracts from 48 infected and uninfected plant samples. All environmental samples from which P. ramorum was isolated by PARP-V8 were detected using all three real-time PCR assays. However, 24% of the samples yielded positive real-time PCR assays but no P. ramorum cultures, but sequence analysis of the coxI and II spacer region confirmed the presence of the pathogen in most samples. The assays based on detection of the ITS and elicitin regions using TaqMan tended to have lower cycle threshold values than those using β-tubulin and seemed to be more sensitive.