Isolation And Identification Salvia Miltiorrhiza Endophytic Bacterium DS-1 And The Research Of Antibiotic Activity

In this study, a bio-control bacteria DS-1 that has good antagonist activity was isolated from salvia miltiorrhiza leaves.Conduct a preliminary study to its fermentation broth?the fermentation conditions?antibacterial substance and the field test.1?Isolating endophytic bacteria from salvia miltiorrhiza with tissue isolation method,30 strains of endophytic bacteria were obtained,using plate confrontation method to determine its bacteriostatic activity, the antagonistic effects showed that 3 of them expressed strong antibiotic activity on Alternaria alternata.Through the study of the strain and its fermentation broth,finding that the DS-1 performed high antagonist activity to Alternaria alternata, Alternaria solani?Botrytis cinerea and Fusarium graminearum.2?In the experiment, the antagonistic strain DS-1 was identified by morphology?physiological and biochemical characteristics as well as the 16SrDNA.In the end,the strain was identified as Bacillus amyloliquefaciens.3?In the experiment,single factor test and orthogonal experiments were adopted to optimize the fermentation conditions of the DS-1.Comprehensive consideration, we must consider two factors:the growth of bacteria and the activity of metabolites,we obtained the optimum fermentation conditions:basic medium is YSP medium.the best carbon source is lactose, the best nitrogen source is beef extract and tryptone, pH 9,liquid volume in flask 150mL, bacterial load 5%, fermentation time 48h, and rotational speed 130r/min.4?Extraction of fermentation broth with 5 kinds of organic solvents,we found that all of them had no antimicrobial activity.But it showed bacteriostatic activity by way of fractional salting out,and combination with the characteristics that under the condition of high temperature,the fermented liquid is easy to lose activity.speculating its antimicrobial components is protein.5?Salt fractionation with ammonium sulfate to fermentation broth and detect its activity,it showed that the best salting saturation is 20?40%.Through study of stabilization of the crude protein by different temperature?different pH and ultraviolet light, we can see that the protein was found to be thermostable under 100?, the antifungal activity was best under 9.0,the rough proteins were unsensitive to ultraviolet light.With ammonium sulphate fractional precipitation, a antifungal protein purified by DEAE-52 chromatography and Sephadex G-100 chromatography,the molecular weight was estimated about 40KDa by SDS-PAGE.6?In the article,control efficacy and growth-promoting ability of DS-1 were researched, the growth-promoting effect showed that DS-1 fermentation broth had no significant promoting effect to seed germination rate;In the aspect of buds length, seeds that treated by the dilution of fermentation broth at 100 times are longer than control group and other treatment groups,the growth rate reached 32%.The results of effects of field efficacy trials showed,7 days later when first applying pesticide, the control effect to Fulvia fulva that treated by the dilution of fermentation broth at 10 times?100 times and 75% Chlorothalonil wettable powder at 500 times reached 45.53%?30.14%?53.34%; 7 days later when second applying pesticide, the control effect to Fulvia fulva that treated by the dilution of fermentation broth at 10 times?100 times and 75% Chlorothalonil wettable powder at 500 times reached 54.15%, 45.55%?64.67%, and the control effect in second applying pesticide is much better than that of the first.The experimental results showed that the bio-control strain has good development potential.