A Study On The Active Substances From Biocontrol Strain B10-26

Objective: Screening to better prevention medicinal plant diseases Bio-control bacteria,researching physicochemical properties and structural information of active substances fromB10-26fermentation broth.Methods: Comparing the inhibitory effect of five strains of Bio-control bacteria onRehmannia glutinosa and Dioscorea opposite diseases by the method of plate toxic medium,filter out the strain which has the best control efficacy, and observe the changes of mycelialmorphology; Comparing the extract efficiency by organic extraction, ammonium sulfateprecipitation and hydrochloric acid precipitation, the physicochemical properties of activesubstances, such as temperature、pH、protease、UV stability and ninhydrin reaction weretested. The structure of active substances were identified by the method of identification ofprotein glue and LC-MS/MS.Results:1. Screening of Bio-control bacteria5strains of Bio-control bacteria all have certain inhibitory effect to4pathogenic fungis,but there is a big difference in the inhibitory effect. The strain YCB9and B10-26toAscochyta molleriana wint were the best, colony diameter16.0mm and17.0mm respectively,and the inhibitory effect of strain B10-26is the best to the other three pathogenic fungis.Through the observation of morphological changes in hyphae, the active substances ofB10-26secretion can make mycelium expansion occurs vesicles, so can inhibite the mycelialgrowth.2. The study of crude extract and physicochemical propertiesOrganic extraction, ammonium sulfate precipitation and hydrochloric acid precipitationall can extract active substances from the fermentation broth of B10-26, but ammoniumsulfate precipitation is best, the extract efficiency is100%. The results showed that the activesubstances are insensitivity to temperature、pH、protease and UV, ninhydrin reaction waspositive, it can produce blue-violet, so the active substances may contain proteins or peptides.3. Isolation、purification and structure identification of active substancesAt last obtain a mixture composed of a plurality of active substance, the structure ofactive substances were identified by the method of identification of protein glue andLC-MS/MS, considered the active substances include lipopeptide antibiotics:C14BacillomycinD、 C15BacillomycinD、 C16BacillomycinD、 C17BacillomycinD，C14FengycinA、 C15FengycinA、 C16FengycinA、 C17FengycinA， C16FengycinB、C17FengycinB、C18FengycinB，C14Mycosubtilin、C14Mycosubtilin、C14Mycosubtilin、Surfactin，specific types of surfactin not clear，and polyene antibiotic Dihydrobacillaene.Conclusion: The strain B10-26was chosed, the physical and chemical properties of activesubstances is stability. After isolation and purification, found that contains a variety of activesubstances in the fermentation broth. There are BacillomycinD、FengycinA、fengycinB、Mycosubtilin、Surfactin and Dihydrobacillaene.