Isolation And Identification Of Nematicidal Bacteria From Rootzone Soils Of Pinus Thunbergii And Characterization Of Derived Active Components Against Bursaphelenchus Xylophilus

Pine wilt disease（PWD）is a serious forest disease caused by the pine wood nematode（PWN）,Bursaphelenchus xylophilus.Due to the widespreading,rapid onset,and difficulties in prevention,PWD became a significant threat on pine forests worldwide.Studies have shown that some microorganisms and their metabolites have antagonistic activity against PWN.Finding microbial sources for biological control in nature has become a hot topic of research.Thus,the exploration of microbial-derived nematicides from nature has become a research trend in recent years.In this study,40 bacterial strains were isolated from the rootzone of a healthy Japanese black pine（Pinus thunbergii）tree,located on campus of Qingdao University.Three strains with nematicidal activity were selected by maceration method.The corrected mortality rates of fermentation broth of all three strains were higher than 85%.The M4 strain with the highest nematicidal effect was selected by double dilution of the culture.The strain was identified as Leifsonia sp.M4 by morphological observation,physiological and biochemical characteristics,and 16S r DNA sequencing analysis.Moreover,the ethyl acetate extract of M4 strain culture exhibited strong nematicidal activity.Two compounds with obvious nematicidal effect were isolated from the ethyl acetate extract by repeated silica gel column chromatography and thin-layer chromatography based on the bioactivity-guided fractionation.According to the analysis results of nuclear magnetic resonance（NMR）spectroscopy and mass spectrometry（MS）,two compounds were identified as 2-coumaranone（1）and cyclo-（Phe-Pro）（2）,with LC₅₀ values against PWN of 196μM and425μM at 24 h,respectively.The following studies further demonstrated that compounds 1 and 2 also significantly inhibited the hatching rate,fecundity and food consumption ability of PWNs.To investigate their nematicidal mechanisms,reactive oxygen fluorescence staining was carried out.Results showed that the reactive oxygen species（ROS）were significantly accumulated in PWNs treated with 2-coumaranone or cyclo-（Phe-Pro）.The assay of antioxidant enzyme activities revealed that the treatment with nematicidal compounds resulted in a significant decrease of peroxide（POD）and catalase（CAT）while a significant increase for superoxide（SOD）activities in nematodes.The findings indicated that one of the nematicidal effect of the two compounds might be caused by triggering excessive ROS（H₂O₂）production in PWNs.To further verify the preventive effect of 2-coumaranone（1）and cyclo-（Pro-Phe）（2）against PWN in vivo,100μL of each compound at a concentration of 50 m M was injected into a healthy fresh Japanese black pine branch pre-infected by PWNs.The assay results showed that both nematicidal compounds effectively reduced the wilting symptoms in pine branches.A similar result was got when Japanese black pine seedlings were used instead of branches.The experiments on pine branches and seedlings indicated that 2-coumaranone（1）and cyclo-（Pro-Phe）（2）were effective in PWD control.Overall,potential microorganisms were identified in this study for biocontrol purpose of PWD.Meanwhile,this study offered clues to explore nematicides from microorganisms and provided theoretical references on biological control of PWD.