| Summary: | Pinosylvin stilbenes are natural phenolic compounds found in the Pinaceae family and act as phytoalexins. Some pinosylvin stilbenes have strong nematicidal activity against pine wood nematodes (PWNs: <i>Bursaphelenchus xylophilus</i>). Here, we established the efficient production of two pinosylvin stilbenes, dihydropinosylvin monomethylether (DPME) and pinosylvin monomethylether (PME), by cell suspension culture of <i>Pinus koraiensis</i> after fungal elicitation. DPME and PME were found in small amounts (less than 40 µg/g DW) in the stem bark and leaves of <i>P. koraiensis</i> plants. Cell suspension cultures were established from the cultures of calli derived from mature zygotic embryos of <i>P. koraiensis</i> in 1/2 Litvay medium containing 2.2 μM 2,4-D and 2.2 μM BA. Two types of fungal elicitors, fungal cell extract (CE) and fungal medium filtrate (MF), were prepared from three species of fungi (<i>Penicillium chrysogenum, P. pinophilum</i>, and <i>P. roquefortii</i>). CE and MF treatments strongly stimulated the production of PME and DPME in cultured cells. The production of PME in suspension cells of <i>P. chrysogenum, P. pinophilum</i>, and <i>P. roquefortii</i> MF treatments after 3 days was 5734 µg/g DW, 4051 µg/g DW, and 6724 µg/g DW, respectively. Pinosylvin synthase (<i>PkSTS</i>) and pinosylvin <i>O</i>-methyltransferase (<i>PkPMT</i>) are key genes in DPME and PME biosynthesis. qPCR analysis revealed that the expression of the <i>PkSTS</i> and <i>PkPMT</i> in cultured cells was highly enhanced after fungal elicitor treatment. The cell extracts after MF treatment resulted in 92.5 ± 7.8% immobilization of the adult PWNs and 63.7 ± 3.5% immobilization of the juvenile PWNs within 24 h. However, control cell extracts without MF treatment showed 11.3 ± 1.4% nematicidal activity against adult PWNs. Our results suggest that pinosylvin stilbenes can be produced from the cell culture of <i>P. koraiensis</i> after fungal elicitor treatment and can be used as nematicidal compounds against PWNs.
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