DNA-basedmethod for rapid identi¢cationofthepinepathogen, Phytophthora pinifolia

Phytophthora pinifolia causes a needle and shoot disease in Pinus radiata, referred to as ‘Daño Foliar del Pino’. This newly discovered disease requires intensive research efforts that necessitate the processing of large numbers of samples for which accurate identification, often by people not experienced in Phytophthora taxonomy, is required. The aim of this study was, therefore, to develop species-specific primers for P. pinifolia that amplify the internal transcribed spacer region of the ribosomal operon and the nuclear Ypt1 gene, respectively. The primers were tested over several Phytophthora spp., as well as fungi isolated from P. radiata. In all cases, only P. pinifolia was amplified. In addition to the species-specific primers, a PCR-restriction fragment length polymorphism protocol using available Phy-tophthora genus-specific primers was also used to generate a species-specific profile for P. pinifolia. This provided a characteristic profile that allows the identification of P. pinifolia, and it could also discriminate between 27 different species of Phytophthora. Both techniques reported in this study make it possible to identif