In order to facilitate the correct identification of Phytophthora species, we provide standards of procedures (SOPs) for all steps involved with molecular identification using sequencing analysis of the ITS rDNA and COI barcoding regions. Each SOP is available as a downloadable PDF.
SOP-PID-01.01 Molecular: Equipment, materials, and reagents for DNA extraction and PCR
SOP-PID-02.01 Molecular: DNA extraction for Phytophthora species using Qiagen kit (suggested)
SOP-PID-03.01 Molecular: Primer dilutions for PCR amplifications
SOP-PID-04.01 Molecular: Gels: electrophoresis, and photos
SOP-PID-05.04 Molecular: Identification of Phytophthora species with ITS rDNA and COI barcoding genes
All sequences submitted to NCBI by the Beltsville Lab for seven genes from the ex-types and other well-authenticated specimens can be found here. The seven genes are: internal transcribed spacer (ITS rDNA), elongation factor 1-alpha (EF1a), shock protein 90 (HSP90), beta-tubulin, 60S ribosomal protein L10 (RPL10), Ras-related protein ypt1 (ypt), and cytochrome c oxidase subunit 1 (COI), mitochondrial.
Sequences for new species not yet included in IDphy, as well as sequences for putative new species in progress, can be found here.
Identifying Phytophthora unknowns
For quick reference, here are the steps from SOP-PID-05 for sequence analysis of your unknown. Once you have your FASTA sequence, follow these steps to identify Phytophthora unknown species using vouchers of the ITS rDNA gene of 139 ex-types and 22 well-authenticated species. If this is your first time conducting this analysis, use the example sequences in SOP-PID-05 (page 6) to practice the steps for better understanding. The example below uses example sequence number 1.
Confirm identity using a full clade analysis
The example shown below uses sequence number 2 from SOP-PID-05.
If you follow steps 1-10 with an unknown specimen, and each test matches the unknown with the same result, and that match is from a type or another well-authenticated specimen, then you can confirm that the ITS rDNA sequence for your specimen is a particular species, based on comparison with sequences of the ex-type and other types in the same clade.