Direct molecular detection of the pinewood nematode, Bursaphelenchus xylophilus, from pine wood, bark and insect vector

The pinewood nematode (PWN), Bursaphelenchus xylophilus, is the causal agent of pine wilt disease. The international economic impact of the introduction of the PWN into new areas has highlighted the need for the development of accurate and reliable detection methods of B. xylophilus, which are essential to define aspects of its control and management. In the present study, a methodology was developed for the direct detection of PWN by conventional PCR assay, with a species specific set of primers based on PWN satellite DNA, using total DNA extracted directly from maritime pine, Pinus pinaster, wood and bark samples, and from the insect vector, Monochamus galloprovincialis. This methodology involves homogenisation of wood, bark and insects using liquid nitrogen, DNA extraction and one or two PCR amplification steps, which permit the rapid and direct detection of one single nematode present in 100 mg of wood and bark and in one entire insect without the preliminary steps of nematode extraction.