| Summary: | A rich source of bioactive secondary metabolites from microorgannisms are widely used to control plant diseases in an eco-friendly way. To explore ideal candidates for prevention of pine wilt disease (PWD), a bacterial strain from rhizosphere of <i>Pinus thunbergii</i>, <i>Lysinimonas</i> M4, with nematicidal activity against pine wood nematode (PWN), <i>Bursaphelenchus xylophilus</i>, was isolated. Two nematicidal compounds were obtained from the culture of <i>Lysinimonas</i> M4 by silica gel chromatography based on bioactivity-guided fractionation and were subsequently identified as 2-coumaranone and cyclo-(Phe-Pro) by nuclear magnetic resonance (NMR) and mass spectrometry (MS). The 2-coumaranone and cyclo-(Phe-Pro) showed significant nematicidal activity against PWN, with LC<sub>50</sub> values at 24 h of 0.196 mM and 0.425 mM, respectively. Both compounds had significant inhibitory effects on egg hatching, feeding, and reproduction. The study on nematicidal mechanisms revealed that 2-coumaranone and cyclo-(Phe-Pro) caused the accumulation of reactive oxygen species (ROS) in nematodes, along with a notable decrease in CAT and POS activity and an increase in SOD activity in nematodes, which might contribute to the death of pine wood nematodes. Bioassay tests demonstrated that the two compounds could reduce the incidence of wilting in Japanese black pine seedlings. This research offers a new bacterial strain and two metabolites for biocontrol against PWN.
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