Specific polymerase chain reaction identification of Venturia nashicola using internally transcribed spacer region in the ribosomal DNA

A technique based on the. polymerase chain reaction (PCR) was developed for the identification of Venturia nashicola using nucleotide sequence informa tion of the ribosomal DNA region. The complete internal transcribed spacer (ITS) region of V nashicola strains and phylogenetically related species wa s amplified with the two universal ITS1 and ITS4 primers, sequenced, and di gested with five, restriction enzymes. The alignment of nucleotide sequence s and analyses of digestion patterns indicated constant polymorphisms betwe en V nashicola and related species at nucleotides 126 and 127, which overla pped a TaqI restriction site. An oligonucleotide primer named A 126 was des igned for identifying this variable region. A primer set (A126 and ITS4) th at allowed the amplification of a 391-bp DNA fragment within the ITS region by PCR was specific to V nashicola when it was checked against fungal geno mic DNAs of related fungi. This primer set was a good candidate for a speci es-specific reagent in a procedure for identification of V nashicola by PCR .