Identification of Isolates to a Species Level   

There are several gel-based techniques that can be used to identify isolates to a species level when isolates are present in either pure cultures or when amplifying from infected plant tissue.

1. Isolates in Pure Culture
2. From Infected Plant Tissue
3. References

Isolates in Pure Culture 

RFLP analysis of the ITS region

 Ristaino et al. (1998) reported that amplification of the ITS region using the standard primers ITS-4 and ITS-5 described by White et al. (1990) followed by digestion with the restriction enzymes HaeIII, MspI or RsaI generated restriction profiles that could be useful for identification to a species level. Using a larger number of species and substituting ITS-6 for primer ITS-5, Cooke et al. (2000) reported that digestion with AluI, MspI or TaqI was effective in differentiating many species in the genus. Due to the small sizes of the digested fragments restriction digests are run on 3% NuSieve 3:1 gels for accurate size estimations. There is a excellent website that has restriction fragment sizes for many species and tools to assist in the identification of unknown isolates as well as additional background information on individual species (PhytID). More recently 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 but did not yield an amplified product.

RFLP analysis of the mitochondrially encoded cox1 and cox2 gene cluster

 PCR primers spanning the mitochondrially encoded cox 1 and 2 genes have been identified that were capable of amplifying target DNA from all 153 isolates of 31 species in the genus Phytophthora that were tested. Digestion of the amplicons with restriction enzymes generated species-specific RFLP banding profiles that were effective for isolate classification to a species level. Of the 24 species where multiple isolates were examined, intraspecific polymorphisms were not observed for16 species (P. cinnamomi – 4 isolates, P. colocasiae – 6 isolates, P. cryptogea – 2 isolates, P. drechsleri – 3 isolates, P. fragariae var. fragariae – 5 isolates, P. hibernalis- 5 isolates, P. ilicis – 3 isolates, P. infestans- 11 isolates, P. lateralis – 3 isolates, P. mirabilis - 4 isolates, P. nemorosa – 2 isolates, P. nicotianae -12 isolates, P. pseudosyringae – 6 isolates, P. phaseoli- 6 isolates, P. ramorum – 24 isolates, and P. sojae – 3 isolates) while 5 species exhibited limited intraspecific polymorphism that could be explained by the addition/loss of a single restriction site. For example, P. cactorum isolate 385 was identical to the other seven isolates of P. cactorum with the exception of an additional MspI site. Likewise, P. citricola isolate Cr-4 differed from the other two isolates by an additional AluI site, and P. palmivora isolate 329 and Pl-10 differed from isolates Pl-5 and Pl-14 by an additional MspI site. Phytophthora erythroseptica isolate 368 had an identical banding profile as two isolates of P. cryptogea and differed from the other eight isolates of P. erythroseptica by an additional AluI site. For P. capsici the results were more variable; seven isolates recovered from vegetable crops were identical and differed from an additional three isolates from vegetables (Cp-30, Cp-32 and 307) by the absence of an MspI site. One additional isolate, Cp-1 isolated from Theobroma cacao, had identical AluI and RsaI RFLP profiles as the other isolates, but had a different MspI site than Cp-30, Cp-32, and 307 and a different TaqI site than all the other P. capsici isolates. Intraspecific polymorphisms were observed for P. megakarya, P. megasperma, and P. syringae; however, these differences may be a reflection of the variation that exists in these species as reported in the literature. While digestion with AluI alone could differentiate most species tested, single digests with a total of 4 restriction enzymes were used in this investigation to enhance the accuracy of the technique and minimize the effect of intraspecific variability on correct isolate identification. The use of the computer program BioNumerics simplified data analysis and identification of isolates. Successful template amplification was obtained with DNA recovered from hyphae using a boiling miniprep procedure, thereby reducing the time and materials needed for conducting this analysis.  Procedures for RFLP analysis 

Martin FN, Tooley PW. 2004 Identification of phytophthora isolates to species level using restriction fragment length polymorphism analysis of a polymerase chain reaction-amplified region of mitochondrial DNA. Phytopathology 94:983-991 (pdf reprint)

SSCP analysis

Single strand conformational polymorphism (SSCP) analysis of the ITS region of the rDNA has been found to be useful for identification of isolates to a species level (Kong et al. 2003). The ITS1 region is amplified, denatured and separated on a nondenaturing polyacrylamide gel with the resulting banding profiles useful for species identification. A technique for direct colony amplification of the ITS-1 region has been published (Kong et al. 2005). An overview of SSCP migration patterns for the genus Phytophthora, along with morphological keys and photomicrographs of the different species has been recently published (Gallegly and Hong 2008).

This technique has recently been modified by T. Kubisiak using an automated sequencer for data collection, thereby improving the accuracy of data collection and simplifying data analysis and comparison among isolates (see Martin et al. 2009). One microliter of each sample was PCR amplified in replicate using the oomycete specific primers ITS6 and ITS7 as described in Kong et al. (2003). ITS6 was fluorescently 5΄-end labeled with 6-FAM and ITS7 with HEX. A 1:10 dilution of the PCR product was denatured and analyzed using the default criteria specified in the "High Throughput Fluorescent SSCP Analysis User Bulletin" from Applied Biosystems (Foster City, CA) on an ABI3100 (bulletin can be found at Amplified products from all of the samples were separated on an ABI 3100 genetic analyzer using 5% GeneScan polymer containing 10% glycerol. Amplified products from a subset of samples were also separated on an ABI 3130 XL using 9% CAP polymer containing 10% glycerol. All runs were performed using an oven temperature of 20C. All other pertinent information regarding the running conditions can be found in the supplementary data file in the online e-Xtra. For both machines and polymer types ROX500 was used as an internal migration rate standard. To set up a robust migration rate standard for automated scoring, ROX500 peaks that had consistently strong signal strength were identified by examining peak heights across a number of capillaries. Eight consistently strong ROX500 signal peaks were identified using the ABI 3100 and 5% GeneScan polymer, whereas nine were identified using the ABI 3130 XL and 9% CAP polymer (a picture of the ROX500 electropherograms, the peaks labeled, and their assigned scanline values are available in the Supplementary Data file in the online e-Xtra). Adjusted scanline values (observed scanline values divided by 10) were assigned to these peaks using the data from a single capillary. Standardized ROX500 values were then used to estimate adjusted scanline values for all 6-FAM and HEX peaks. Adjusted 6-FAM and HEX scanline values were then converted back to approximate scanline values by multiplying by 10. Using these separation protocols, only two main peaks (one 6-FAM and one HEX) were observed for a number of isolates and species in contrast to the typical four-banded pattern previously reported using slab gel systems (Kong et al. 2003). For those isolates and species where multiple peaks were observed, only the 6-FAM and HEX peaks with the largest peak height/area under the peak were used for data analyses, i.e., mean tests and UPGMA. Mean and standard deviation of scanline estimates for each species were dependent upon the number of isolates analyzed per species, with a minimum of two reads per sample.

Additional details of the modified technique

· List of species examined
· Table of migration types
· Cluster analysis of migration types
· Excel file of migration distances as well as more detailed procedures for running and scoring the samples


Martin FN, Coffey MD, Zeller K, Hamelin RC, Tooley P, Garbelotto M, Hughes KJ, Kubisiak T, Bilodeau GJ, Levy L, Blomquist C, Berger PH. 2009 Evaluation of molecular markers for Phytophthora ramorum detection and identification: testing for specificity using a standardized library of isolates. Phytopathology 99:390-403  (pdf of reprint)

From Infected Plant Tissue 

RFLP analysis of Phytophthora genus-specific amplicon The primers Phy-8b and Phy10b are specific for amplification of Phytophthora spp. (they do not amplify plants or the closely related genus Pythium) and hence, can be used for amplification from infected plant tissue. They are used for the first round amplification of species-specific diagnostic markers for P.  ramorum and several other species. The amplicon spans the spacer region between the mitochondrially encoded cox1 and cox2 genes and is about 450 bp in size (there is some size variation among species). Due to the high AT content of this region there have been only two restriction enzymes that have been found to differentiate some species by RFLP analysis of this region (other restriction enzymes have been identified, but the fragments are too numerous or small to be of use when viewed on a gel). There are several phylogenetically closely related species that cannot be differentiated using this technique. This data is currently being compiled and will be added to the website at a later time. Please contact Frank Martin if you have any questions about this technique.

SSCP analysis This analysis is covered in the above section and the approach is applicable for analysis using DNA extracted form infected plant tissue (T. Kubisiak, unpublished). It should be noted that amplification from a variety of host DNA and Pythium spp. has not been extensively evaluated when using an automated sequencer to collect the data so it may be a good idea to further evaluate the primer specificity.


Cooke, D.E.L., Duncan, J.M., Williams, N.A., Hagenaar-de Weerdt, M., and Bonants, P.J.M. 0 Identification of Phytophthora species on the basis of restriction fragment analysis of the internal transcribed spacer regions of the ribosomal RNA. XXX/EPPO Bulletin 30:519-523

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)

Gallegly, M.E. and Hong, C. 2008. Phytophthora: Indentifying species by morphology and DNA fingerprints. APS Press, St. Paul, MN. 168 pp.

Kong P, Richardson PA, Hong C. 2005 Direct colony PCR-SSCP for detection of multiple pythiaceous oomycetes in environmental samples. J. Microbiological Methods 61:25-32

Kong P, Hong C, Richardson PA, Gallegly ME. 2003 Single-strand-conformation polymorphism of ribosomal DNA for rapid species differentiation in genus Phytophthora. Fungal Genet. Biol. 39:238-249 (pdf of reprint)

Martin FN, Tooley PW. 2004 Identification of phytophthora isolates to species level using restriction fragment length polymorphism analysis of a polymerase chain reaction-amplified region of mitochondrial DNA. Phytopathology 94:983-991 (pdf reprint)

Ristaino JB, Madritch M, Trout CL, Parra G. 1998 PCR amplification of ribosomal DNA for species identification in the plant pathogen genus Phytophthora. Appl. Environ. Microbiol. 64:948-954

White, T. J., Bruns, T., Lee, S., and Taylor, J. 1990. Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. Pages 315-322 in: PCR Protocols: A Guide to Methods and Applications. M. A. Innis, D. H. Gelfand, J. J. Sninsky, and T. J. White, eds. Academic Press, Inc., San Diego, CA.