Isolation And Identification Of Active Components Of Endophytic Fungi Of Melia Azedarach Against Bursaphelenchus Xylophilus And Its Nematicidal Mechanism

Bursaphelenchus xylophilus is a plant-parasitic nematode that causes pine wilt disease and has a highly destructive effect on pine trees.It spreads rapidly through the pine sawyer beetle,which acts as a vector for the nematode,leading to an epidemic of the disease.The use of chemical pesticides has been the most effective strategy for controlling pine wilt disease and has been widely used in Asian countries.However,most pesticides are harmful to non-target organisms,and their use can lead to severe environmental and human health problems.Therefore,researchers are endeavoring to find natural compounds from plant or microbial metabolic products that are environmentally friendly and have more efficient insecticidal activity to replace synthetic pesticides in controlling pine wilt disease,while protecting the environment and saving costs.This study isolated and purified endophytic fungi from healthy tissues of Melia azedarach（stem,leaves,and fruits）,and a total of 31 endophytic fungi were obtained.The strains were cultured in shake flasks,and extracellular and intracellular metabolites were prepared by ethyl acetate extraction of fermentation broth and mycelia,respectively.The nematocidal activity was screened by the immersion method,and metabolites from strain J352-03 showed significant nematocidal activity against B.xylophilus with LC₅₀values of 0.46 mg·m L^-1and 0.42 mg·m L^-1after a 48 h immersion treatment.At a drug concentration of 2.00 mg·m L^-1,the inhibition rates of egg hatching were 66.54%and 76.04%,respectively.The fungus was identified as Diaporthe sp.based on morphology and r DNA-ITS sequence analysis.This active strain was scaled up for shake flask cultivation to prepare fermented products.Cytochalasin H,an active compound,was isolated from the ethyl acetate extract of the fermentation broth by combining activity tracing with silica gel column chromatography,Sephadex G-20 column chromatography,and semi-preparative high-performance liquid chromatography.The compound showed significant nematocidal activity against B.xylophilus with an LC₅₀value of 0.37 mg·m L^-1and an inhibition rate of 72.70%at a drug concentration of 1.00 mg·m L^-1after a 48 h immersion treatment.Chemical structure identification was based on nuclear magnetic resonance spectroscopy(¹H-NMR,¹³C-NMR),mass spectrometry（ESI-MS）,and comparison with literature data.To explore the mode of action of Cytochalasin H on B.xylophilus,the study investigated the effects of the active compound on the activities of superoxide dismutase（SOD）,catalase（CAT）,acetylcholinesterase（ACh E）,and adenosine triphosphatase（ATPase）in B.xylophilus.The results showed that Cytochalasin H had different levels of inhibition on the catalytic activity of the four enzymes in B.xylophilus.To gain further insights into the mode of action of Cytochalasin H on B.xylophilus,ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry（UHPLC-Q-TOF MS）was used to investigate the metabolic pathway intervention of Cytochalasin H in B.xylophilus.A total of 905 metabolites were identified in all nematode samples（ESI+484,ESI-421）,and 154 significant differential metabolites（VIP>1,P<0.05）were screened by combining several statistical methods.Cluster and correlation analyses of the significant differential metabolites between the treatment and control groups revealed differences in the expression patterns of different metabolites and their cross-regulation.Based on the nematode metabolic pathway as a reference,91 metabolites were annotated to 60metabolic pathways.Enrichment analysis identified eight pathways（P<0.05）:alanine,aspartate,and glutamate metabolism;ABC transporters;purine metabolism;biotin metabolism;neuroactive ligand-receptor interaction;taurine and hypotaurine metabolism;arginine biosynthesis;and D-glutamine and D-glutamate metabolism,which were identified as the main differential pathways between the treatment group and the control group.The above results provide a foundation for the development and application of new microbial-derived nematocidal agents.