A rapid detection of the pinewood nematode, Bursaphelenchus xylophilus in stored Monochamus alternatus by rDNA amplification

Pine wilt disease (PWD) caused by pinewood nematode (PWN) Bursaphelenchus xylophilus has originated serious loss to pine forestry around the world. Monochamus alternatus is the most important vector of B. xylophilus in Japan and China. It is necessary to develop an efficient method to store M. alternatus for subsequent PCR detection of B. xylophilus. In the present study, beetle samples carrying nematodes were stored in 75% alcohol. A simple procedure was firstly developed for isolating B. xylophilus DNA in 2 mg stored beetle tissue carrying nematodes for subsequent nematode detection by PCR amplification. The B. xylophilus- specific amplicon of 403 bp (DQ855275) was generated by PCR assay from the stored beetle tissue. To our knowledge, this is the first report of a rapid detection of the pine wood nematode -B. xylophilus in stored M. alternatus by rDNA amplification established without a separate nematode extraction. This result will provide a useful method to detect B. xylophilus in stored M. alternatus.