Isolation And Identification Of Pine Wood Nematode Bacteria And Study Of Its Active Ingredients

Pine wilt disease?PWD?caused by pine wood nematode?PWN?,Bursaphelenchus xylophilus,is a serious forest disease.The main measures of preventing and controlling PWN are utilizing chemical agents,which might cause environmental pollution and drug resistance of PWN.In order to search for environment-friendly natural products,samples were collected from the campus of Qingdao University and used for bacteria isolation,and a total of 120 strains of bacteria were isolated.Twenty strains were found to have nematicidal activity,and two strains of bacteria G5-3 and G8-2 with high nematicidal activity was screened out by immersion method.The results showed that the corrected lethal rate of G5-3and G8-2 after 48 h for PWN culture was 98.26%and 93.10%respectively.On the basis of morphology,biochemical-physiological characters and 16S rDNA sequence analysis,the two stains were identified as Pseudoduganella violaceinigra G5-3 and Novosphingobium pokkalii G8-2.Using single factor analysis method to study the culture conditions of the strains and the optimum culture conditions were determined,the optimal initial pH values of the medium were 30°C and 7.0 for P.violaceinigra G5-3,and 35°C and 6.0 for N.pokkalii G8-2.The optimal inoculum age for these two strains was 15 h and optimal culture time was3 days.The stability of the active substances produced by the two strains were preliminarily analyzed,and the results showed that the active substances produced by the two strains had better thermal stability and higher activity stability against nematodes under weakly alkaline conditions.The active constituents of the two strains were analyzed,and it was found that the volatile components in the fermentation supernatant of the strains had higher nematicidal activity than the fermentation supernatants,which indicated that the main active components were volatiles.The volatiles of the two strains were analyzed by solid-phase microextraction coupled with gas chromatography/mass spectrometry.The main compounds in the volatiles,2,5-dimethyl pyrazine,4-dimethylaminopyridine,benzyl acetate,phenethyl butyrate,phenylethyl alcohol and acetophenone possessed nematicidal activity against B.xylophilus with LC₉₀0 values of 33.72,1.83,0.92,0.20,13.22 and 10.11 mmol/L in 48 h as assayed using the pure commercial compounds,respectively.Subsequently,the nematicidal mechanisms of the five compounds including 4-dimethylaminopyridine,benzyl acetate,phenethyl butyrate,phenylethyl alcohol and acetophenone with high nematicidal activity were studied.Firstly,the effects of the compounds on the morphology of PWN were observed by scanning electron microscopy.The test five compounds were found to have no significant effect on the morphology of the nematode.Subsequently,the effects of the test five compounds on the activities of cellulase,amylase,glutathione S-transferase and acetylcholinesterase in nematodes were determined.The experimental results showed that these five compounds had little effect on amylase activity,but had a greater effect on the activity of cellulase,glutathione S-transferase and acetylcholinesterase.Cellulase showed a significant decrease in activity after 36 h.The five volatiles had a certain activation effect on glutathione S-transferase and acetylcholinesterase in the early stage of treatment for PWN,and then both enzymes gradually decreased.Therefore,it is speculated that volatiles may act by inhibiting the activity of these three enzymes.This study provides a theoretical basis for the utilization of microorganisms and biological control of PWN.