A simple, polymerase chain reaction-restriction fragment length polymorphism-aided diagnosis method for pine wilt disease

For diagnosis of pine wilt disease, a simple PCR-RFLP method was developed to identify and to differentiate two similar nematode species, based on a l iving or preserved single specimen. Pinewood nematodes, Bursapbelenchus xyl ophilus, and Bursaphelenchus mucronatus were examined. A single nematode in 1 mu l of distilled water was put on a glass slide. When the water had alm ost dried the nematode was crushed with a filter raper chip, 1.5 mm x 1.5 m m, with the aid of forceps. The filter paper chip containing nematode remai ns was immediately placed into PCR buffer as the DNA template. The primer s et used was to amplify ribosomal DNA containing; the inter-transcribed spac er (ITS) 1, 5.8S and ITS2 regions. The PCR product was consistently obtaine d from a single nematode, and digesting the produce with restriction endonu clease, Hinf I, enabled discrimination between B. xylophilus and B. mucrona tus. This method was simple, convenient and definitive, and could successfu lly determine the pathogen in the diagnosis of pine wilt disease. This meth od was applicable also to nematode specimens preserved under various condit ions except in the case of those preserved in aldehyde-containing fixatives .