Antifungial Mechanism And Preliminary Separation Of Antimicrobial Substances Of Actinomycetes JY-22

In 2007,a strain of actinomycetes was named JY-22,screened out from the soil layer of Jinyun Mountain in Chongqing,showing significantly antagonistic action on fungi.Based on JY-22 morphological and molecular level(16S rDNA sequence),the antagonistic actinomycetes strain JY-22 was identified as Streptomyces hygroscopicus.Our laboratory preliminary work optimized the fermentation conditions of antagonistic actinomycetes strain JY-22,and evaluated the stability of JY-22 sterile fermentation broth.The results showed that JY-22 strain sterile fermentation broth has a relatively stable at 80℃ water bath for 3 hours or exposed to UV irradiation for 30 min.Streptomyces hygroscopicus strain JY-22 has antagonistic effects on pathogens of most plant fungal diseases,with inhibition rate to fungal pathogens from 41.9% to 75.7%.Therefore,it is necessary to study the antifugal mechanism and characteristics of metabolic active products of the Streptomyces hygroscopicus strain JY-22.In this paper,taking Alternaria alternata as the target fugi,we investigated the antifungal mechanism of active metabolite of actinomycetes strain JY-22 and the separation and purification process of its active product to provide a basis for further exploitation.The conclusions are following: 1.The antibacterial mechanism of JY-22 on Alternaria alternataThe objective of this study is to understand the antifungal activity and mechanism of production by Streptomyces hygroscopicus strain JY-22 against alternaria alternate so as to provide a scientific basis for further product development and application.10 fungal cakes of Alternaria alternate were cultured in 100 mL of PD medium.After incubation for 36 h,added Actinomycete strain JY-22 sterile fermentation broth.The concentration of sterile fermentation broth in PD medium was 10%,5%,2.5%,1%,0%,respectively.The permeability of the cell membrane was measured by the conductivity method after 24 h.The effects on soluble protein,ergosterol and malondialdehyde were measured by mycelial wet weight method and ultraviolet absorption method after 96 h.The results showed that JY-22 sterile fermentation broth had antagonistic effect on the Alternaria alternata,and the swelling deformity was observed at the tip of the mycelium,protoplast leakage in mycelium,cytoplasmic condensation appearanced cystic swelling.Treated with the 10% JY-22 sterile fermentation broth,the inhibition rate of mycelium reached 75%,the electrolyte leakage increased with conductivity increased,the content of malondialdehyde increased significantly,and ergosterol and the content of soluble protein decreased.The results showed that actinomycete JY-22 had a significant antagonistic effect on the Alternaria alternata,Its bioactivity lies in its doing damage to the pathogen cell membrane and increasing the permeabilitu of plasma membrane through inhibiting the synthesis of ergossterol in the cell membrane and causing lipid peroxidation of the cell membrane,which resulted in the poor and abnormal mycelia growth,suggesting that cell membrane is one of the main action sites of JY-22.2.Separation and purification of JY-22 active substancesThe activity type of JY-22 strain was determined by Doskochulova paper chromatography,Betina solvent system paper chromatography and pH paper chromatography.The separation and purification method of JY-22 active substance was determined by the treatment and separation and purification of JY-22 sterile fermentation filtrate.The results showed that the JY-22 active substance could be a new acid-base amphoteric polyene and the polar alkaloids.The JY-22 fermentation filtrate was progressively subjected to ethanol precipitation,petroleum ether degreasing,n-butanol extraction,Macroporous D101 resin adsorption(water and ethanol as eluent,the ethanol gradients were 0%,30%,95%),silica gel column chromatography(silica: methanol: concentrated ammonia = 8: 2: 0.2),Sephadex LH-20 column chromatography(the mixed solvent of water and methanol as an eluent)and preparation of high performance liquid separation(methanol and water containing 0.05% concentrated aqueous ammonia elution).Through above process,we obtained obtain the main active product of JY-22.3.Efficacy test of JY-22 active productWith Alternaria alternata as target pathogen,the pesticide effect of n-butanol extract from JY-22 was determined by using the bacteriostatic circle method.To determine whether it had synergistic effect with pesticides by the formulation of JY-22 n-butanol extract combined with pyraclostrobin(25%).The results showed that the inhibitory concentration EC50 of JY-22 n-butanol extract was 35.68 mg/L and EC90 was 408.08 mg/L.Formulation combined with pyraclostrobin(25%),which was different modes of action,while V(pyraclostrobin 4 mg/L):V(JY-22 n-butanol crude extract 125 mg/L)= 1:6,The evaluation of EC90 had synergistic effect,when the application amount of pyraclostrobin of 1.21 mg/L,reduced by 20 times compared to the use of pyraclostrobin alone,JY-22 n-butanol crude extract used 227 mg,which reduced 180 mg compared to the use of JY-22 crude extract alone.