| In this study,endophytic actinomycetes were isolated from different tissues of the Zanthoxylum bungeanum Maxim,and an endogenous actinomycete HJG-5,which had antibacterial activity against many pathogenic fungi was obtained.The factors such as the taxonomic status,the fermentation conditions,the physical and chemical properties of the fermentation filtrate,the mechanism of antibacterial activity and the biocontrol and antimicrobial active substances were studied in order to provide a theoretical basis for the development and application of microbial pesticides1.Eleven endophytic actinomycetes were isolated from different tissues of Zanthoxylum bungeanum Maxim by tissue homogenate method.Six strains had different antibacterial activity by plate confrontation and mycelial growth method.The strain HJG-5 and its fermentation could effectively inhibit most of tested plant fungal pathogens,especialy the Alternaria solani and Monilinia Laxa,the inhibition rate of the fermentation broth could reach more than 80%.The strain HJG-5 was identified as the strain of Streptomyces cinnamocastaneus by the strain morphology and culture characteristics,physiological and biochemical identification and 16S rDNA sequence analysis2.The single experiment and the orthogonal method are used to optimize fermentation conditions.The results showed that the optimum medium was PD medium,the best carbon source was maltose,and the optimum nitrogen source was tryptone.The optimal culture conditions were as follows:rotational speed 180r·min-1,pH 8,liquid volume 125ml/250ml,inoculation amount 8%,fermentation time 9d.The results showed that the antibacterial activity of A.solani was significantly improved by using the optimized fermentation conditions3.The stability of the strain HJG-5 fermentation filtrate showed that its metabolites had good thermal stability,and the inhibition rate was about 80%within 80 ?,and was stable in neutral and weak alkaline environment.Which was insensitive to ultraviolet light.At 4 ?,the inhibition rate of A.solani could reach 85.15%of the original antibacterial activity.4.The mechanism of antibacterial activity of strain HJG-5 was studied by microscopic observation and conductivity method.The results showed that the mycelium of A.solani was abnormal growth,abnormal swelling,uneven thickness,cytoplasmic aggregation,extravasation and other phenomena.5.The effects of the fermentation of HJG-5 on the seed germination and seedling growth of tomato were showed that the fermentation filtrate had the best effect in 20X dilution,the seed germination index increased by 16.75%compared with the contro,the plant height,root length,fresh weight and dry weight of tomato seedlings were respectively increased by 29.0%,20.4%,24.9%and 35.9%compared with the control group,while the high concentration treatment had different inhibitory effect.6.The in vitro control effect of different concentrations of fermentation filtrate on A.solani was determined by stab inoculation method.The results showed that the antibacterial effect of the undiluted fermentation broth was the best,and the antibacterial rate of the leaves in vitro and fruit in vitro were 80.60%and 82.96%,respectively.With the increase of the dilution factor,the control effect is gradually reduced7.The antimicrobial active substances in the fermentation filtrate of strain HJG-5 were extracted with five different polar organic solvents.The results showed that N-Butyl alcoho was the best extraction solvent,that is,the active substance was a highly soluble liposoluble substance The crude extract was separated by silica gel column elution and TLC,ten components were obtained,the antimicrobial activity of component 8 was the best,and the diameter of the inhibition zone was 18.63mm. |
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