Abstract: [Objective]The aim of this study is to explore the pathogenicity of aseptic Bursaphelenchus xylophilus (pine wood nematode, PWN) and non-aseptic PWN on Pinus densiflora, to better understand the role of PWN in pine wilt disease development, and to provide useful information on pathogenicity mechanism and disease control. [Method] The embryogenic calli of P. densiflora were inoculated with aseptic PWNs under aseptic conditions and cultured for 5 days. The effect of aseptic PWNs on the activity of embryogenic callus cells were evaluated by staining the embryogenic calli with the triphenyltetrazoliumchloride (TTC) solution. Tissue-cultured microshoots and 4-year-old seedlings cultured in green house were inoculated with aseptic PWNs and non-aseptic PWNs, respectively. In 18 and 35 days after inoculation, the pine wilt ratio was recorded and the PWNs were isolated from inoculated seedlings. The pathogenicity of aseptic PWN and non-aseptic PWN on P. densiflora was analyzed. [Result]The inoculation by aseptic PWNs caused severe water stain in embryogenic calli of P. densiflora, and the TTC assay showed beige/light pink of embryogenic calli, revealing that the embryogenic cells lost viability. The embryogenic calli of control treatment remained healthy and showed bright red in TTC assay. Both the aseptic PWN and non-aseptic PWN wilted the tissue-cultured microshoots of P. densiflora. The wilting rates of the microshoots were 70% and 60% in 18 days after inoculation with aseptic PWNs and non-aseptic PWNs, respectively. PWNs were recovered from all wilted microshoots with average number of PWNs 599+567/microshoot for aseptic PWNs inoculation and 365+240/microshoot for non-aseptic PWNs. The number of nematodes recovered from wilted microshoots showed significant difference (P < 0.01) between aseptic PWN and non-aseptic PWN treatments. Fewer PWNs (10-20/microshoot) were also recovered from the remaining healthy-looking microshoots. The aseptic PWN could induce wilting of potted-seedlings of P. densiflora, as the same as non-aseptic PWN, with average of 80% wilting ratio in 35 days after inoculations. PWNs were recovered from wilted seedlings in both treatments. The number of recovered PWNs per seedling was 34 733±34 162 and 25 057±21 410 for aseptic PWNs and non-aseptic PWNs inoculations, respectively. No significant difference was found in number of recovered PWNs between the two treatments (P = 0.508). The number of recovered PWNs in two healthy-looking seedlings of aseptic PWNs treatment was 486 and 22, while only one healthy-looking seedlings of non-aseptic PWNs treatment contained 646 PWNs. [Conclusion] The aseptic and non-aseptic PWN could cause wilting of P. densiflora. The PWN is the main factor causing wilt of Japanese red pine, while the bacteria carried by the PWN are not necessary for the development of PWD.

Key words: pine wilt disease, Bursaphelenchus xylophilus, asepsis, pathogenicity, Pinus densiflora