Differentiation of European and North American genotypes of Phytophthora ramorum by real-time polymerase chain reaction primer extension

Phytophthora ramorum, the organism responsible for Sudden oak death, causes severe damage to oak (Quercus spp.) and tanoak (Lithocarpus desilflorus) trees in California and Oregon. There are now >100 host plants that can be affected by this pathogen in nurseries and in the wild in Europe and North America. Molecular diagnosis and genotyping are important tools to monitor and prevent spread of this pathogen, to enforce quarantines, and to support eradication progams. By amplifying and sequencing DNA in a worldwide panel of strains of P. ramorum, we discovered single-nucleotide polymorphisms (SNP) in two genes (beta-tubulin and cellulose binding elicitor lectin (CBEL)) that differ in the European and the North American P. ramorum isolates. Using these SNPs in diagnostic assays and distinguishing among strains of different geographic origins, we developed primer extension assays in real-time polymerase chain reaction using allele-specific oligonucleotides. All European isolates genotyped were homozygous for the SNP at position 279 in the tubulin gene (beta-tub279; G/G) but heterozygous at position 858 (beta-tub858; A/G). By contrast, all the California isolates were heterozygous for beta-tub279 (T/G) but homozygous for beta-tub858 (A/A). For CBEL, all European isolates were heterozygous for the SNPs at position 245 (CBEL245; G/C) and 412 (CBEL412; G/A), but the California samples were homozygous at both SNP loci CBEL245 (G/G) and CBEL412 (G/G). This assay was also used directly on DNA extracted from the ELISA lysates from field samples collected in British Columbia in 2005 by the Canadian Food Inspection Agency. Although most samples had the North American DNA profiles, there were several samples with the European profiles from the nurseries. This finding could provide information on the biology, epidemiology, etiology, host range, and populations of P. ramorum from North America and Europe and provide useful tools in P. ramorum surveys.