Molecular Detection of Phytophthora ramorum by Real-Time PCR Using Taqman, SYBR®Green and Molecular Beacons with three genes

G. J. Bilodeau & R. C. Hamelin, Natural Resources Canada, Canadian Forest Service, Laurentian Forestry Centre, P.O. Box 3800, 1055 du P.E.P.S., Quebec, Quebec, G1V 4C7, Canada. gbilodeau@cfl.forestry.ca; C. A. Lévesque, Agriculture and Agri-food Canada, National Program on Environmental Health- Biodiversity , 960 Carling avenue, Ottawa, Ontario, K1A 0C6, Canada; A.W.A.M. de Cock, Centraalbureau voor Schimmelcultures, P.O. Box 85167, NL-3508 AD Utrecht, The Netherlands; C. Duchaine, Département de Biochimie-Microbiologie, Université Laval, Quebec, Canada; Centre de Recherche, Hôpital Laval, 2725 chemin Ste-foy, G1V 4G5 Ste-Foy, Quebec, Canada; G. Kristjansson, Pest DNA Diagnostics Laboratory, Centre for Plant Quarantine Pests, CFIA, Ottawa, Ontario, K2H 8P9, Canada.

Sudden oak death, caused by Phytophthora ramorum, is a severe disease that can affect numerous species of trees and shrubs. This pathogen has been spread via nursery stock and quarantine measures are currently in place to prevent further spread. Molecular assays have been developed to rapidly detect and identify P. ramorum but one difficulty encountered with some of these assays is the inability to reliably distinguish between P. ramorum and closely related species.

In order to overcome cross reactions, ß-tubulin and elicitin regions from a collection of Phytophthora species were sequenced and searched for polymorphisms. New assays were designed using existing internal transcribed spacer (ITS) sequences as well as new ß-tubulin and elicitin sequences. Primers specific to P. ramorum were designed to amplify a 171 bp fragment of ß-tubulin and these were used in a real-time PCR assay in conjunction with molecular beacons, Taqman probes and SYBR®Green in order to compare the three reporter systems. The best performing system was also used to compare the three DNA regions.. The real-time PCR assays differentiated P. ramorum from the 65 Phytophthora species tested, including P. lateralis. The assays were also used with DNA extracted from plants infected with P. ramorum. Overall, ITS and elicitin Taqman assays had the best combination of sensitivity and specificity.

With the availability of the Phytophthora ramorum genome, more genes were analysed for polymorphisms. Multiple intraspecific and interspecific polymorphisms were found in most genes assayed. Genotyping assays are currently being developed to allow simultaneously diagnosis at the species level as well as for multilocus fingerprinting of individuals. This should be a useful tool in understanding the origin and migration of P. ramorum.

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