Pine wilt disease (PWD), which is caused by pine wood nematodes (PWN), is one of the most serious forest diseases worldwide. To clarify the mechanism of resistance to PWD, we compared metabolites from resistant and susceptible Japanese black pine (Pinus thunbergii) families after inoculation with PWN. After 2 weeks to 1 month post inoculation, the number of PWN dramatically increased in susceptible plants, but not in resistant plants. At this PWN-proliferation phase, ethyl acetate soluble fractions extracted from PWN-inoculated plants were analyzed by gas chromatogramphymass spectrometry (GC-MS). Although most compounds were qualitatively and quantitatively similar between resistant and susceptible plants, resistant plants accumulated 2.0-fold more linoleic acid (LA) than susceptible plants. On the other hand, benzoic acid (BA) was barely detected in resistant plants, but it accumulated in susceptible plants as the number of PWN increased. Susceptible plants contained greater levels of the nematicidal compounds pinosylvin and pinosylvin monomethyl ether, compared with resistant plants. These results suggested that LA is involved in the resistance reaction against PWN-proliferation, and that BA could be a good biomarker for PWD.

Understanding resistance mechanisms to pine wilt disease is essential to a successful breeding programme because plant selection cannot always guarantee absolute resistance against every isolate of the pine wood nematode, Bursaphelenchus xylophilus. To examine resistance factors in Japanese black pine, Pinus thwibergii, we devised a novel in vitro bioassay system in which we tested proliferation of pine wood nematodes co-cultured with wood slices or methanol extracts from pines. Proliferation of pine wood nematodes was inhibited in assays with fresh wood slices from resistant Japanese black pines but not with susceptible pine or without wood suces (control). When resistant wood slices were extracted by methanol, the inhibition effect of proliferation of pine wood nematodes was diminished, whereas methanol extraction from susceptible wood slices did not affect the proliferation. To verify whether nematode proliferation was inhibited by pine extracts, methanol extracts were loaded on paper disks and used in the assay. Populations of pine wood nematodes were significantly suppressed when methanol extracts originated from a resistant pine, but not from a susceptible pine. These results strongly suggest that methanol extracts from resistant pine trees contain the inhibitors of pine wood nematode proliferation. This bioassay system is available not only for identifying inhibitors of pine wood nematode proliferation but also for rapid screening of resistant pines.