Specific Detection of the Dwarf Japanese Pear Pathogen, Fomitiporia torreyae, by the Polymerase Chain Reaction

Abstract

Little is known about Fomitiporia torreyae, a pathogen of Japanese pear dwarf. Therefore, we used the polymerase chain reaction (PCR) to develop a method for the specific detection of the pathogen in decayed wood. In the ribosomal DNA internal transcribed spacer (rDNA ITS) region, the degree of sequence homology between F. torreyae and related species was between 84 and 99%. We used the F. torreyae rDNA ITS sequence to design specific primers (FP-f1 and FP-r1) for detection of the pathogen. When the primers were tested with several isolates of F. torreyae and eight other basidiomycetes isolates, we obtained the expected PCR products (356 bp) only from the F. torreyae isolates. We then developed a method for the extraction of mycelial DNA from decayed tissues of infected trees. Samples were grated by using emery paper with a buffer containing 10% PEG and 0.35 M sorbitol, and the resulting DNA samples were used for PCR reactions. The sensitivity of detection was improved by the use of nested PCR reactions. Thus we were able to specifically detect the Japanese pear dwarf pathogen, F. torreyae, in decayed wood by PCR.