Screening Of Mulberry Bacterial Blight Antagonistic Endophytic Bacterium And Research On Antimicrobial Active Substances

Mulberry bacterial blight, as one kind of bacterial disease of mulberry, is caused by Pseudomonas syringae pv.mori. According to the symptoms, the disease can be divided into three types, which are the mulberry broken stipe, shrunken leaf, and black wilt. The disease generally occurs in various mulberry cultivation places throughout the country, which brings great economic losses to silkworm farmers. Now, the control of mulberry bacterial blight mainly depends on chemical bactericide. However, chemical agromedicine not only causes environmental pollution, but puts increasingly side effects on sericulture. Mulberry endophytes can be used to reduce losses caused by diseases, promote the development of sericulture industry and good for the environment. This is a new strategy that biological control mulberry diseases with mulberry endophytes.In the research, one endophyte of mulberry, which has steady and significant inhibitory activity to Pseudomonas syringae pv.mori, was screened out and identified. The research was carried out to optimize fermentation conditions of antagonistic endophyte, and purify the major active component from the supernatant of antagonistic endophytes. This research has made the following results:1. Isolation, screening and identification of endophytic antagonistic bacteria.77endophytic bacteria were obtained from healthy mulberry Tongxiangqing, and the strain SWg2exhibited strong and stable inhibitory activity to the pathogen of mulberry bacterial blight. The active component of strain SWg2was secretory and the generation of active substances with a high genetic stability, which could keep antibacterial activity after passage up to10times. Morphological characteristics of the strain were observed, physiological and biochemical were tested, and the phylogenyetic analysis based on16S rRNA were performed. The results showed that the colony of strain SWg2exhibited milky white on PDA medium with smooth edges, and it’s Gram negative without spores and facultative anaerobic growth. Oxidase test, H2S production test and nitrite gas test were all negative. Both maltose and fructose can be used by the strain SWg2, but lactose and starch cannot be used. Glucose fermentation test was characterized to be fermentation. The strain can oxidize various simple sugars and polysaccharides, and use it as the only carbon source. Thesequence of SWg2strain16S rRNA gene shared more than98%identity with lots of stains of Pantoea sp. Results of phylogenetic analysis demonstrated that SWg2strain and Pantoea agglomerans ATCC27993(FJ611872) were in the same minimum branch. According to above analysis, the antagonistic endophytic strain SWg2to mulberry bacterial blight was identified as P. agglomerans.2. Optimization of fermentation conditions ofpantoea agglomerans SWg2.Single-factor and orthogonal experiment were used to optimize P. agglomerans SWg2fermentation conditions. The result showed that best carbon of P. agglomerans SWg2producing active substances was Glycerin, nitrogen was NH4NO3, metal ions was Mg2+, and phosphate was KH2PO4. The initial pH of fermentation medium was7.5, bottling volume is20%, incubation temperature was28℃, rate of rotation is170r/min, the amount of4.00%is seed liquid inoculum and culture time is5d. Orthogonal test results showed that the optimal fermentation medium was consisted of2.00%glycerol,2.00%(NH4)2SO4,0.10%KH2PO4and0.15%MgSO4·7H2O. Compared with potato dextrose medium and beef extract peptone medium, inhibition zone diameter was increased by186.82%and215.18%by optimized fermentation medium. Optimization fermentation conditions laid a good foundation for the production of active substance.3. The exploration of the properties of antibacterial substance and purification of the active ingredient.Physical and chemical properties of antibacterial substances indicated that the main active ingredient was soluble in ethanol, methanol and acetonitrile and insoluble in n-butyl alcohol, methylene chloride, chloroform, ethyl acetate, petroleum ether. The ability of withstand heat was tested by incubation at temperatures of100℃for30min that they could survive at high temperatures. And pH tested by incubation at pH3-12, the results showed that they could survive at acid environment (pH3-6) but not alkali-resistant (pH>9). Based on the above test results, the following protocol were used to isolate the active component. Ethanol precipitation was used to remove contaminate proteins at the first step. Then, it was extracted by acetonitrile. The extract was dried and dissolved in0.02mol/L pH5.8Na2HPO4-citric acid buffer, applied to anion exchange chromatography of DEAE sepharose FF. The main active component was eluted at0.13mol/L to0.14mol/L NaCl. MIC test results of different steps, which were fermentation supernatant, organic extract and after ion-exchange column chromatogr-aphy, were3.30×104μg/mL,6.20×103μg/mL and2.37×102μg/mL respectively. The antibiotic component had been purified nearly70times after IEC. Final purification was done by HPLC on C1g column. The conditions were as follows:0.1%acetic acid for channel "A" and100%acetonitrile for channel "B" and acetonitrile was ramped from0to100%after30min. the active component was collected at the retention time of6.27min. The following research will be carried out to explore the antimicrobial mechanism.