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A nested PCR-based method for detecting the pine wood nematode, Bursaphelenchus xylophilus, from pine wood

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Several methods were examined for extracting DNA from the woody tissues of Japanese black pine, including the general CTAB method which proved to be the most successful. To detect the pine wood nematode, the causal agent of pine wilt disease (PWD) in pine wood tissues, PWN-specific PCR was done using a DNA mixture obtained from 80 mg of pine wood tissues and a single pine wood nematode as the DNA template. The results showed that nested PCR could amplify the ITS region specific to the PWN, even though the amount of nematode DNA was extremely small. Also, the PWN could be detected in diseased tissues of several Pinus spp. which had been inoculated with nematodes. The method described here can also be used as a simple and quick detection method for a wide range of wood-inhabiting pathogens.


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