Screening Of An Active Strain Against Bursaphelenchus Xylophilus And Analysis Of Its Active Ingredients

Pine wilt disease(PWD)is a devastating disease caused by Bursaphelenchus xylophilus causing wilting and rapid death,which seriously threatens the development of forestry in China.In the present,the chemical pesticides in market is poor,virulent and high,so we should start from the natural world to develop the biogenic agents with high efficiency and low toxicity.The secondary metabolites of endophytic fungi are abundant and have good insecticidal activity,which can be used in the study of the activity of pine nematodes.In this paper,7 strains of endophytic fungi were used as the test strains,and the bacterial strains were screened.Determination of the stability of fermentation filtrate of active strain;The extraction,isolation,identification and study of pine branch hydroponics were studied in order to determine the high activity compounds in the fermentation broth of the active strains.1.The poisonous activities of three strains of Paecilomyces lilacinus and four strains of Pochonia chlamydospora fermentation broth to B.xylophilus were researched under indoor conditions,and by growth rate method research of active strains fermentation filtrate stability under different conditions.The results showed that the fermentation filtrate of Paecilomyces lilacinus PT1 strains was the most effective in the treatment of B.xylophilus,and the mortality rate of the pine nematode was 88.37%after 72 h.PT1 strains fermentation filtrate stability is better,show the good thermal stability(with the exception of extreme high temperature of 121? environment);Can resist strong acid and strong alkali and ultraviolet radiation;It can remain stable under the influence of oxidation reducer,but in metal ion determination,Zn2+,Fe2+ With Fe3+ The stability of fermentation liquid was greatly affected.The good stability of PT1 fermentation broth can ensure that the secondary metabolites are not damaged,showing good research value and application prospect.2.The most active extracts were obtained by screening different extraction solvents and extraction ratios.The extraction,separation,purification and identification of the active strains were carried out using thin layer chromatography,silica gel column chromatography and GC-MS analysis.The active constituents of the B.xylophilus in the fermentation filtrate of PT1were determined.Test results show that the selected from the 7 kinds of organic solvent ethyl acetate(1:3 proportion)extraction phase is PT1 strains active ingredients more suitable extraction solvent and proportion,impregnation method results show that the PT1 strains ethyl acetate(1:3 proportion)extract processing 24 h and 48 h LC50 were 36.001 ?g/mL and 26.782?g/mL.When the mixture ratio of petroleum ether and n-butanol is 6:4,the thin layer chromatography have 5 bands,and the effect is the best.The active component YZ-4 was obtained by the separation of the high efficient extractant via column chromatography with petroleum ether-n-butanol 6:4 as eluent.A total of 4 compounds were identified by GC-MS analysis of YZ-4 components,respectively:Oleic acid,Pseudopelletierine,n-Hexadecanoic acid and Phthalic acid-butyl hexyl ester.The active strain PT1 fermentation product is expected to develop new microbial source insecticide.3.Inoculation of B.xylophilus on pine needles of Pinus massoniana and put into 4 kinds of compound solution,avermectin solution,"CK + B.xylophilus" and control group(distilled water),periodically observe the pine branch's disease index,transpiration intensity and turpentine content.The test results show that the compound pseudopelletierine and phthalic acid-butyl hexyl ester can effectively control the development of pine wood nematode disease,12 d after treatment,the disease index is 0.00,and abamectin treatment group of the same condition index,slightly lower than that of CK treatment group's condition index(0.26);After 9 days,there was no significant change in the transpiration intensity and turpentine flux of Masson pine branches.However,the transpiration intensity and turpentine content of the "CK+ B.xylophilus" treatment group significantly decreased or even stopped.The results indicate that compound pseudopelletierine and phthalic acid-butyl hexyl ester of forest pest fields can effectively resist development and to maintain normal physiological state,masson pine for Finus plant set up a good protective barrier.