Submitted to: Phytopathology
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/10/2004
Publication Date: 8/20/2004
Citation: Martin, F.N., Tooley, P.W. 2004. Identification of phytophthora isolates to species level using rflp analysis of a pcr amplified region of mitochondrial dna. Phytopathology. v. 94. p. 983-991. Interpretive Summary: The ability to accurately identify isolates of Phytophthora to a species level is often complicated by similarity in morphological characteristics among some species and intraspecific variation that can some times exist in these characteristics. Furthermore, it can also take a week or longer for the morphological features to fully develop to allow isolate classification. A procedure is described in this submission where a region of the mitochondrial DNA is amplified by polymerase chain reaction and cut with restriction enzymes to generate different sized fragments that can be used to rapidly and accurately identify isolates to a species level.
Technical Abstract: PCR primers spanning a variable region of the mitochondrial genome in the genus Phytophthora have been identified that are capable of amplifying target DNA from all of the 153 isolates of 31 species that were tested. Digestion of the amplicons with restriction enzymes generated 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 for 16 species while 5 species exhibited limited intraspecific polymorphism that could be explained by the addition/loss of a single restriction site. 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 that has been 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 RFLP polymorphisms on correct isolate identification. 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.