Physiological Effects Of Plant-derived Nematicidal Coumarins On Pine Wood Nematode,bursaphelenchus Xylophilus And Their Action Mechanism

Pine wilt disease（PWD）is a destructive pine disease,and pine wood nematode（PWN）,Bursaphelenchus xylophilus is the the causal agent of PWD.So far,trunk injection of nematicides against PWN has been considered one of the most immediate and effective strategies for PWD management.However,commonly-used synthetic nematicides has been bringing more and more concerns on drug resistance,threat to non-target organisms and loss of biodiversity.Eco-friendly plant secondary metabolites with various bioactivities and high biocompatibility to host pines have formed a promising resource for screening potential alternatives of nematicides.In this study,the ethanol extracts of Cnidium monnieri seeds,Angelica dahurica roots and Fraxinus rhynchophylla barks with significant nematicidal activity against PWN were selected for exploring the nematicidal constituents based on the nematicidal assays.Through bioactivity-guided assays,eleven nematicidal compounds against PWN were separately isolated from the three botanical extracts and identified to be osthol 1,xanthotoxin 2,cindimine 3,isopimpinellin 4,marmesin 5,isoimperatorin 6,imperatorin 7,bergapten 8,fraxetin 9,esculetin 10 and esculin 11,respectively,based on mass and nuclear magnetic resonance（NMR）spectral data analysis.The eleven compounds all belong to coumarins,and their LC₅₀ values against PWN at 72 h were 64.93μg/m L,54.68μg/m L,24.73μg/m L,92.16μg/m L,122.96μg/m L,43.08μg/m L,35.72μg/m L,52.07μg/m L,30.37μg/m L,72.20μg/m L and 143.31μg/m L,respectively.The effects of the eleven nematicidal coumarins on PWN vitality were further studied,and the results showed that they all had inhibitory effects on the egg hatching,feeding ability,and reproduction of PWN.Especially,cindimine 3,isoimperatorin 6,imperatorin 7,and fraxetin 9 have significant inhibitory effects on PWN vitality.The action mechanism against PWN of the eleven nematicidal coumarins was investicated.All the eleven nematicidal coumarins showed inhibitory effects on the acetylcholinesterase（ACh E）,Ca²⁺-ATPase and Na⁺-K⁺-ATPase of PWN,especially on the ACh E and Ca²⁺-ATPase.Cindimine 3,imperatorin 7 and fraxetin 9 had remarkable inhibitory effect on the ACh E and Ca²⁺-ATPase of PWN.The results indicated that ACh E and Ca²⁺-ATPase may be the main targets of these coumarins against PWN.In addition,the structure-activity relationship was discussed based on the chemical structures and the nematicidal activities of the eleven nematicidal compounds.The core structure 2H-1-benzopyran-2-one of coumarins should be the key effective structure for the nematicidal activity.Furthermore,α,β-unsaturated ester group in cindimine 3,the hydroxyl group on 8′-C of fraxetin 9,and the isopentyloxy existing in both isoimperatorin6 and imperatorin 7 could contribute to the nematotoxicity since these coumarins showed excellent anti-PWN effect.Finally,bioassays on the infected black pine seedlings by PWN demonstrated that the eleven nematicidal coumarins could markedly weaken PWN pathogenicity,which indicated that the coumarins had the potential to be developed as nematicidal agents or related lead compounds.This research provided foundational evidences for the development of new eco-friendly plant-derived coumarin nematicidal agents,and promising theoretical support for the PWD management.