Studies On Identification, Fermentation Conditions And Antifungal Sbustance Of The Antagonistic Bacterium Against Fusarium Graminearum

Wheat scab, caused by Fusarium graminearum, is also called red head wheat. It is a destructive disease that can seriously influence the yield of wheat in the wheat growth area, the yield loss may reach 50% in some area. The prevention against the disease was always through agricultural and chemical mesasures since long before at home and abroad, it is some certain effct that fungicide application of chemical pesticides, but application of chemieal pestieides brought entironment pollution, such as the anti-pest predators, patheogen and pest resistance, serious environmental pollution, pesticide residues harm hunman and animal health. Biongic pesticides is not these problems, biological control by natural antagonistic organisms is a potential measure against plant diseases, so the antagonistic bacterium (named:AF0907) was studied primarily in the test.In terms of the traditional identification way, according to its morphological, cultural, and physiological biochemical characteristics of AF0907, plus the analysis the 16S rDNA sequence, it was identified as Bacillius subtilis. The sequence data for the 16S rDNA gene of the strain AF0907 has been deposited in GenBank with the accession number GU272021. The assay results using plates and hyphal extension showed that antagonist produced by Bacillus subtilis had obvious inhibitive effect on Fusarium graminearum.The medium recipe and fermentation conditions of the antagonistic bacterium were optimized through measuring the effect of different carbon or nitrogen sources on the diameter of inhibition zone. We can get the initial medium:glucose 10g. yeast ectract lg. peptone 5g. beef extract 4g. NaCl 10g.dH2O 1000mL. On the basis of the optimized medium. the effects of a number of factors on the production of Bacillus subtilis were studied, including original pH, incubative temperature and time, inoculation quantity and volume of culture media. Finally, the result showed that the optimum medium was glucose 10g, yeast ectract 1g, peptone 10g, beef extract 1g, NaCl 10g, dH2O 1000mL; the favorable fermentation condition was determned: temperature 30℃, original pH7.0, the optimal load is 250mL flask contains 100mL medium in triangle bottle, inoculation quantity 9%, fermentin time 72h. Under these conditions the strain can grow better and gain the highest antifungus activity with 30.08mm diameter of inhibition zone.Using ammonium sulphate to explore the separation conditions, and some properties of the crude antifungal substance were studied subsequently, and the result showed that the antifungal substance could be separated primarily by ammonium sulphate precipitation. The inhibition of antagonistic substances produced the strongest activity when the saturation of ammonium sulphate was at the range of 80%; The stability experiment result indicated that antagonistic substance was stable against heat, and sensitive to Proteinase K and Chloroform. The antimicrobial substance deposited by ammonium sulphate at 80% saturation was purified by DEAE-52 cellulose column. The fraction contained antifungal activity exhibited one band with SDS-PAGE, The aim of this research is to obtain the purified antimicrobial peptide and analyze the sequence of amino acid:His-Glu-Phe-Pro-Tyr-Lys-His-Met-Tyr-Gln-Val-Met-His-Leu.