Identification of Phytophthora isolates to species level using RFLP analysis of a PCR amplified region of mitochondrial DNA

PCR primers spanning the mitochondrially encoded cox I and II 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.

• Reference

  • Martin, F. N. and Tooley, P. W.  2004. Identification of Phytophthora isolates to species level using RFLP analysis of a PCR amplified region of mitochondrial DNA.  Phytopathology 94: 983-991(.pdf)

• RFLP analysis

  • Primers and amplification conditions (9/28/04)

    • Modifications to primers (will be added when work is completed)

  • Restriction analysis (9/28/04)

  • Data (9/28/04)