The virulence of two species (three isolates) of pine wood nematodes in stressed adult Japanese red and black pines was tested using an inoculation experiment. Three thousand nematodes, Bursaphelenchus xylophilus isolate ‘Ka4’ (virulent), isolate ‘C14-5’ (less virulent) or B. mucronatus isolate ‘M’ (less virulent), were inoculated into 15-yr-old naturally shaded and stressed pine trees planted in two separated experimental stands, in which shelterwood cutting had not been carried out after planting. The inoculation was conducted at the beginning of August 2010, and the trees were then visually examined every month for disease symptoms until February 2011. Trees that died during the experimental period were cut and brought back to the laboratory for reisolation attempts of the pathogenic nematodes. The Ka4 isolate killed all inoculated trees within one to 2-mo, whereas C14-5 and M each killed about half. The inoculated nematodes were reisolated from all the dead trees and some of the surviving ones. These results corroborated those of previous research by using small saplings (i.e., that less virulent nematodes can kill shaded (stressed) trees). Further, a feeding preference experiment using their vector beetles, Monochamus alternatus Hope, showed that both healthy and stressed trees are at equal risk of being used by beetles and hence of getting infected by the nematodes. Therefore, pine wilt disease in shading-stressed trees is assumed to occur in susceptible pine trees in natural pine stands. The dead tree is used by insects as an oviposition resource.
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