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ARS Home » Northeast Area » Frederick, Maryland » Foreign Disease-Weed Science Research » Research » Publications at this Location » Publication #63007


item Tooley, Paul
item Carras, Marie
item Falkenstein, Kathy

Submitted to: Mycologia
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 4/19/1996
Publication Date: N/A
Citation: N/A

Interpretive Summary: Phytophthora is a group of fungi that cause disease on many species of crop plants. Many species look very similar to one another and are difficult to identify and tell apart. Scientists who wish to determine which species is causing a disease may need to go through a very long procedure to identify the organism, requiring weeks or months before an identification can be made. Sometimes, these identifications are based on the presence of special structures on the fungus, which may require special conditions for production. An alternative method is to use the DNA of the fungus and modern methods for characterization of specific DNA fragments. Using a technique called PCR, or polymerase chain reaction, certain DNA fragments can be produced in large numbers, then compared by their sizes after chemical treatment with enzymes that cut the DNA fragments into smaller fragments. Using this approach, we have found that six different species of Phytophthora fungus can be separated and compared in less time than using other methods of identification

Technical Abstract: The polymerase chain reaction (PCR) was used to amplify the ITS2 region of nuclear ribosomal DNA from six Phytophthora species which comprise taxonomic Group IV. We included six or more isolates for P. infestans, P. mirabilis, and P. colocasiae, three for P. ilicis, and only two for P. phaseoli and P. hibernalis due to scarcity of cultures of these species. Digestion of the ca. 600 bp PCR product with restriction enzymes AluI, DraI, HhaI, HinfI, MspI, and TaqI followed by electrophoresis on agarose gels revealed variation in restriction fragment sizes which allowed relationships among species to be assessed. With AluI and TaqI, P. ilicis, P. colocasiae, and P. hibernalis each showed unique banding patterns different from the common banding pattern shared by P. infestans, P. mirabilis, and P. hibernalis. HinfI allowed differentiation of P. ilicis and P. hibernalis from P. infestans, P. mirabilis, P. phaseoli, and P. colocasiae, all of which shared a common banding pattern. MsPI allowed differentiation of P. hibernalis from the other five species. Species groupings determined by restriction analysis of ITS2 were consistent with those based on morphological criteria. These results show that restriction analysis of PCR-amplified ITS2 regions may be useful as an adjunct to morphological and other characters in Phytophthora species identification