Analysis The Antagonistic Mechanism Of Bacillus Amyloliquefaciens 262AG6 Against Colletotrichum Coccodes And Optimization Its Fermentation Process

The study was focused on endophytic bacteria 262AG6 from Kobresia humilis on the East Qilian Mountains alpine grassland.The strains were determined through biological functions and physiological and biochemical tests,and it was identified.At the same time,the fermentation process and the characteristics of antagonistic substances were analyzed.The main conclusions were as following:1.Identification and determination of the biological function of 262AG6Endophyticbacteria262AG6wasrod-shapedwithasineof1.63³.65μm×0.38⁰.95μm,Gram-positive.Combinedthemorphological characteristics,physiological and biochemical characteristics,16S rDNA and gyrB DNA sequence analysis,262AG6 was identified as Bacillus amyloliquefaciens.The inhibitory rate of the strain was 81.33%against Colletotrichum coccodes,and the inhibition effect against the Fusarium avenaceum,Phoma foveata,Rhizoctonia solani,Alternaria solani,Bipolaris sorokiniana,Fusarium solani,Fusarium oxysporumwere above 50%,and the antibacterial capability was stable.The concentration of IAA were 8.04mg·L^-1 and 7.98mg·L^-1in the King with 100mg·L^-1 tryptophan and without tryptophan,respectively.And it had the ability of dissolve inorganic phosphorus.2.The Optimization of fermentation medium and conditions of 262AG6Results showed that the optimal medium of 262AG6 were as follows:potatoes200 g,glucose 25g,KNO₃ 16g,NaCl 15g,water 1000mL;The Optimal culture conditions of strains 262AG6 separately were:culture temperature 34℃,liquid medium volume 40mL/150mL,shaking speed 150 r/min,pH8,inoculum size 14%.Study on the growth curve in the 100L fermentation tank was found,0⁴h is in a period of adjustment,and could enter the logarithmic phase after 4h.After 26h,the biomass of the strain tended to be stable and entered a stable phase,so the optimal fermentation time was 26h.262AG6 inhibits the growth of phytopathogenic fungi by secreting extracellular products,the best medium for producing antimicrobial substances was BPY（beef extract 5g,peptone 10g,yeast extract 5g,glucose 5g,NaCl 5g,water 1000mL,pH 8）.And Its sterile fermentation of crude extract had excellent resistance to adverse environmental conditions（high temperature,acid and alkali,ultraviolet）.Antibacterial activity of fermentation crude extracts was significantly decreased after extracting by chloroform and ethyl acetate,the results showed that the bacteria produced some strong antibacterial substances can be extracted by organic solvent.3.Study on the antibacterial mechanism of bio-control of strain 262AG6The antibacterial substance in the fermentation crude extract of 262AG6 can be separated by ethyl acetate and chloroform.The inhibition rate of chloroform extract on Colletotrichum coccodes was significantly higher than that of ethyl acetate extract.The GC-MS analysis of organic solvent extract showed that,262AG6 produced a volatile substance containing at least alkanes,esters,aldehydes,acids,ammonia,benzene and some nitrogen-containing substances were 28,The content of Phthalic acid,mono-（2-ethylhexyl）ester in chloroform extract was the highest,and the content of alkanes was up to 23.835%,and the content of alkane hydrocarbon in the extract of ethyl acetate was up to 82.65%.Determinated that 262AG6 fermentation produced volatileantibacterialsubstancescontaining15 kinds of compounds,Cyclo（leucylprolyl）、Butyl isobutyl phthalate、Eicosane、Heneicosane、Docosane、Tricosane、Tetracosane、Pentacosane、Phthalic acid,mono-（2-ethylhexyl）ester、Hexacosane、Octacosane、Nonacosane、Pyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-、Bis（2-ethylhexyl）adipateandPyrrolo[1,2-a]pyrazine-1,4-dione,hexahydro-3-（phenylmethyl）-.