Genus specific diagnostic markers
There have been several Phytophthora genus-specific primer pairs that have been reported in the literature, however not all of them have been confirmed to be specific to the point where false positives are not obtained with the closely related genus Pythium.
· ITS – Drenth et al. (2006) reported that primer pair A2 and I2 amplified a region from the ITS region ranging from 752 to 832 bp from all Phytophthora spp. that were tested and that RFLP analysis of the amplicon using MspI, RsaI or TaqI could be used to identify isolates to a species level. A total of 9 Pythium spp. were also tested and did not yield an amplified product.
Drenth, A., Wagels, G., Smith, B., Sebdall, B., O'Dwyer, C.O., Irvine, G., and Irwin J.A.G. 2006 Development of a DNA-based method for detection and identification of Phytophthora spp. Aust. Plant Pathology 35:147-159 (pdf of reprint)
· ITS – Kox et al. (2007) reported a TaqMan real time PCR assay using primer pair FITS_15Ph and RITS_279Ph and the probe All-phy. This marker amplified 73 isolates representing 38 Phytophthora spp., but when tested against 13 species from closely related genus Pythium, 5 species were found to give a positive amplification (P. intermedium, P. irregulare, P. oedochum, P. sylvaticum, and P. undulatum).
Kox LF, Brouwershaven IR, Vossenberg BT, Beld HE, Bonants PJ, Gruyter J. 2007 Diagnostic Values and Utility of Immunological, Morphological, and Molecular Methods for In Planta Detection of Phytophthora ramorum. Phytopathology 97:1119-1129 (pdf of reprint)
· Ras-related protein Ypt1 –The ras-related Ypt-1 gene has several conserved exons separated by variable introns that exhibit enough variation to be useful for development of Phytophthora species-specific diagnostic markers. Conserved sequences in exon 3 and 6 were used to develop a Phytophthora genus-specific primer pair (Yph1F and Yph2R) that generated an amplicon ranging in size from 419-478 bp depending on the species (this primer pair did not amplify 9 Pythium spp. that were tested; Schena, L. et al 2008). Nested within this amplicon and based on sequence differences in introns 3, 4, and 5 the species-specific primers were developed. (details)
Schena, L., Duncan, J.M., Cooke, D.E.L. 2008 Development and application of a PCR-based 'molecular tool box' for the identification of Phytophthora species damaging forests and natural ecosystems. Plant Pathology 57:64-75
· Cox spacer – Martin, FN, Tooley, PW, and Blomquist, C (2004) reported that primers Phy-8b and Phy-10b amplified the spacer region between the mitochondrially encoded cox1 and cox2 genes that was specific for Phytophthora and did not amplify the plant or Pythium spp. tested. Sequence or RFLP analysis of this amplicon can also be helpful for identification of species present in infected tissue.
Frank N. Martin 2004 Molecular Detection of Phytophthora ramorum, the Causal Agent of Sudden Oak Death in California, and Two Additional Species Commonly Recovered from Diseased Plant Material. Phytopathology 94:621-631 (pdf of reprint)