A rapid in vitro bioassay system for testing resistance factors of pine trees to Bursaphelenchus xylophilus

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 thunbergii, 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 slices (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.