Study On The Pesticide Active Substance Of Endophytic Fungus Periconia Igniaria Stdif10 Isolated From Medicinal Plant Stephania Dielsiana

In this paper,the pesticide active substance of endophytic fungus Periconia igniaria Stdif10isolated from medicinal plant Stephania dielsiana was studied.The antimicrobial activity and anti-insect activity were determined with the crude extract from fermented product,three different polar solvent extracts and water layer extract of this strain,by using mycelium growth rate method,drug medium coating method and filter paper drug film method.Based on the bioassay-guided fractionation,the extract of petroleum ether layer from the main active part was systematically separated.and the active compounds were isolated by thin layer chromatography and silica gel column chromatography,as well as solvent recrystallization.The structure of the isolated compound was identified by mass spectrometry?MS?,¹H NMR,¹³C NMR and related literature.The bioactivity of the isolated compounds was evaluated.The inhibition effect of P.igniaria Stdif10 of crude extract from fermentation products against Colletotrichum capsici,Cochliobolus miyabeanus,Pestalotiopsis theae,Alternaria oleracea,Ceratocystis paradoxa,Phytophthora parasitica var.nicotianae,Exserohilum turcicum,Diaporthe citri,Alternaria citri was determined by mycelia growth rate method,the results showed that the EC₅₀0 value of crude extract against nine kinds of plant pathogenic fungi was between 0.212 1 mg/mL and 0.507 9 mg/mL,showing the most remarkable inhibition effect against Diaporthe citri,and had lowest inhibitory activity against Exserohilum turcicum.The inhibition effect of crude extract against Enterobacter aerogenes,Escherichia coli,Proteusbacillus vulgaris,Salmonella typhi,Pseudomonas aeruginosa,Staphyloccocus aureus,Bacillus cereus,Bacillus megaterium,Bacillus subtilis,Bacillus anthraci was determined by drug medium coating method,the results showed that the MIC values of crude extract against ten kinds of animal pathogenic bacteria ranged from 0.125 0 mg/mL to 0.500 0 mg/mL,and had best inhibition effect against Bacillus subtilis and Bacillus cereus,the inhibitory effect against gram-positive bacteria was better than that of gram-negative bacteria.The contact toxicity and repelling action against Sitophilus zeamais of crude extract was determined by filter paper medicine film method,the results were as follows:the LC₅₀0 value of crude extract against Sitophilus zeamais was 3.437 8 mg/cm²,the repelling rate against the Sitophilus zeamais was all higher than 50%,after 72 h treatment with the three concentration gradients of 3.932 0,7.863 0and 15.726 0 mg/cm²,respectively,the results showed that the crude extract from fermentation products of P.igniaria Stdif10 had a certain anti-insect activity.The inhibitory effect of three solvent extracts and water layer extract of crude extract from fermentation products against nine kinds of plant pathogenic fungi was further determined,the results showed that the activity of petroleum ether layer extract was better than ethyl acetate layer extract,n-butanol extract and water layer extract,the EC₅₀0 value of petroleum ether layer extract against nine kinds of plant pathogenic fungi was between 0.054 7 mg/mL and 0.319 8mg/mL,and the EC₅₀0 value of ethyl acetate layer extract against nine kinds of plant pathogenic fungi was between 0.524 1 mg/mL and 2.181 4 mg/mL,while the activity of n-butanol extract and water layer extract was low.The inhibitory effect of extracts and water layer extract against ten kinds of animal pathogenic bacteria was further determined,the results showed that the MIC value of petroleum ether layer extract against ten kinds of animal pathogenic bacteria was between 0.125 0 and 0.500 0 mg/mL,and the MIC value of ethyl acetate layer extract against ten kinds of animal pathogens was between 0.250 mg/mL and 1.000 mg/mL,the result indicated that petroleum ether layer extract had the best antibacterial activity,while n-butanol layer extract and water layer extract have little antibacterial activity.The anti-insect activities of extracts and water layer extract were further determined,and the results showed that the petroleum ether layer extract had significant contact toxicity against Sitophilus zeamais,with the LC₅₀0 value against Sitophilus zeamais at 2.295 1 mg/cm² after 72 h treatment,ethyl acetate layer extract was slightly lower,with the LC₅₀0 value against Sitophilus zeamais at 2.582 6 mg/cm² after 72 h treatment.Moreover,petroleum ether layer extract also has good repelling action against Sitophilus zeamais after 48 h of treatment,with the three concentration gradients of 3.932,7.863 and 15.726 mg/cm²,respectively,and the repellent rate against Sitophilus zeamais were all greater than 62.00%.In the same processing conditions,the repellent rate of petroleum ether layer extract was higher than ethyl acetate layer extract?54.80%?,The n-butanol layer extract and water layer extract did not show any activity against Sitophilus zeamais.The methods of chromatographic separation and activity tracing were used for the separation of active substances compounds from the petroleum ether layer extract of crude extract of P.igniaria Stdif10 fermentation product.Ceratocystis paradoxa and Sitophilus zeamais were used as tested pathogen and insect for the determination of activity.Six compounds,namely Xss-1,Xss-2,Xss-3,Xss-4,Xss-5 and Xss-6,were obtained by thin layer chromatography,silica gel column chromatography and recrystallization methods.Based on the antimicrobial experiment,the first five compounds were found to have a certain degree of biological activity,and Xss-6 was determined to be an antimicrobial active substance.The structure of the compound was preliminarily identified by means of mass spectrometry and nuclear magnetism.The antimicrobial activity and toxicity of Xss-1,Xss-5 and Xss-6 were tested,by using a mycelium growth rate method with treatment concentration at 0.1 mg/mL,the inhibition rate of Xss-1,Xss-5 and Xss-6 against nine kinds of plant pathogenic fungi ranged at 4.65 to 23.96%,1.98 to 23.00%,17.83 to 100.00%,respectively.Xss-6 has the best inhibitory effect against Ceratocystis paradoxa and displayed the lowest inhibitory effect against Colletotrichum capsici,the EC₅₀0 value of Xss-6 against nine kinds of plant pathogenic fungi ranged from 0.020 3 to0.557 0 mg/mL.The inhibitory effect of Xss-1,Xss-5 and Xss-6 against ten kinds of animal pathogens was determined by filter paper method,the results showed that when treated with the concentration of 5 mg/mL,Xss-1 had the best inhibitory effect against Salmonella typhi with the diameter of the inhibition zone at 0.87�0.05 cm,and Xss-5 has the best inhibitory effect against candida albicans with the diameter of the inhibition zone at 1.13�0.02 cm,the inhibition zone diameter of Xss-6 against Staphyloccocus aureus,Salmonella typhi,Bacillus anthraci,Escherichia coli,Proteusbacillus vulgaris was all greater than 1.73�0.12 cm,and had some inhibitory effect against Micrococcus lysodeikticus,with the diameter of inhibition zone at0.85�0.12 cm.The toxicity of Xss-1,Xss-5 and Xss-6 against Sitophilus zeamais were determined,the results showed that the mortality of the control group,Xss-1,Xss-5 and Xss-6 on Sitophilus zeamais were 13.33%,13.33%,10.00%and 16.67%,respectively.After 7 d having no significant difference,the three compounds had limited effects on the physiological activities of Sitophilus zeamais combined with the results of the antifeedant activity and oviposition repellent activity.The insect resistant active substance need to be further isolated.