Study On Identification & Inhibiting Mechanism And Media Optimization Of Bacillus Subtilis Bs2004

The strain of Bs2004 is a biocontrol microbe which separates from the soil. It can obviously increase the production of potato and lettuce, effectively prevent the plant disease, such as Sclerotinia sclerotiorum,Plasmodiophora brassicae. In order to develop the biocontrol ability of the strain, the variety identification,liquid culture condition,inhibiting mechanism and zymolytic media were studied. The final reports were as follows:The result of the identification demonstrated that the morphological characterization, culturing pattern, physiology and biochemistry characteristics were accordant with those of Bacillus subtilis described by Bergey's Manual of Determinative Bacteriology (version 8). and by common bacterium Manual of Systematic Determinative Bacteriology. Therefore, Bs2004 was initially identified as Bacillus subtilis.The cultural condition of Bs2004, such as medium type, original pH value, culture temperature, oxygenic conditions, inoculation conditions and culture time had been studied. According to the studying results, the best cultural conditions were medium YSP, inoculation age 12 hours, amount of inoculation 3%, original pH 7.5, culture temperature 32℃, amount of load 50mL(in 250mL flask), rotation speed 120r/m, and culture cycle 24 hours.Through studying on the antagonistic mechanism, the results indicated that both Bs2004 strain and its non-volatile metabolites can inhibit the hyphae growth of the five trial plant pathogens, such as Sclerotinia sclerotiorum, Rhizoctonia solani, Botrytis cinerea Pers, Sclerotium rolfsii Sacc. It shows that the hyphae of five pathogenic fungi appeared various malformations, with protoplasm agglomerated after inhibited by Bs2004 strain, according to the results of microscope observated in the dual culture test. Cell-free culture filtrates of Bs2004 had been tested for their inhibitive effects to the hyphae growth of the five trial plant pathogens at different concentrations diluted by medium. The results showed that different concentration filtrates had inhibitory effects to different pathogens, and the higher concentration filtrates, the stronger inhibitory effects. The inhibition effect to Sclerotium rolfsii Sacc was feeble, EC₅₀ value was 7.95%, while it was strong to Sclerotinia sclerotiorum, EC₅₀ value was 31.00%. The stability of Cell-free culture filtrates of Bs2004 was tested after being treated at high temperature and different pH values. the results demonstrated that it is unstable under acid condition, while it is stable after high temperature.About eight fermentation culture media, which are suitable for bacillus sp., were compared on the basis of fermentation yield and economic cost in shake-flask culture. The eighth medium, which was economic and brings the highest fermentation yield, was chosen as the basic medium for fermentation of Bs2004. The basic medium was composed of Sucrose 10g, Corn flour 20g, Bean flour 15g, Yeast extract powder 3g, MnSO₄·H₂O 0.1g, K₂HPO₄ 2g,CaCO₃ 2g,FeSO₄-7H_O 0.02g ,MgSO₄·7H₂O 0.1g,distilled water 1000ml. Plackett-Burman design and Response Surface Analysis provided by SAS software were used to optimize the basic medium for fermentation of Bs2004. FeSO₄·7H₂O, MgSO₄·7H₂O, bean flour and CaCO₃ were chosen as the four major prominence components from nine variables. After regression analysis performed base on RSA experiment, a regression model equation was formed as: y=(157.429—1.625x₅—1.875x₈＋3.4583x₃—1.875x6—5.5967x₅²â€”0.8125x₈x₅—5.9717x₈²â€”2.5625x₃x₅—0.5625x₃x₈—5.2217x₃²â€”1.0625x₆x₅＋0.9375x₆x₈—2.0625x₆x₃—8.7217 x₆²)×10⁷. According to this equation, it were easy to predicted out the optimum concentration of those four major factors which were MgSO₄·7H₂O 0.056g/L, FeSO₄·7H₂O 0.057g/L, CaCO₃ 1.631g/L, Bean flour 19.847g/L, while the fermentation yield of Bs2004 reach to the highest 1.59×10⁹cfu/ml. After experimented on base of the predicted factor medium concentration in the shake-flask, the results showed that the regression equation was validated by the practical fermentation yield of 1.55×10⁹cfu/mL. The fermentation yield was increased by 6.61×10⁸ from 8.92×10⁸cfu/mL in shake-flask condition when Bs2004 was cultivated in the optimized medium instead of the original one.