Isolation And Analysis Of Antifungal Compounds Produced By Bacillus Subtilis Strain CAB-1

Bacillus Subtilis strain CAB-1 was isolated from rhizosphere soil of cucumber and showed strong inhibition activity against many vegetable diseases such as cucumber powdery mildew, tomato gray mold, tomato leaf mold etc. The objective of this study was to isolate and identify the antifungal protein,lipopeptides and volatile compounds produced by B. subtilis strain CAB-1 and to analyze their inhibitory activities.Antifungal protein was isolated from the cultural supernatant of B. subtilis strain CAB-1 using 40% Ammonium sulfate precipitation. The crude protein effectively restrained the conidia germination and the growth of mycelia of Sphaerotheca fulginea. The crude protein reduced the disease index of cucumber by cotyledon-spraying method, the protective and curative effect were 73.33% and 76.85 respectively. The optimum culture condition for antifungal protein production was 2% inoculums volume to NO.3 medium, 100 mL loading amount in a 250 mL flask, at 30℃for 48 h on a rotary orbital shaker with 180 r/min speed. The crude protein produced by strain CAB-1 was not sensitive to Trypsin and Proteinase K, while the antifungal activity was reduced 14% by Pepsin. The crude protein showed stable activity at 100℃, and retained 72% antifungal activity at 121℃for 30 min. The antifungal activity of crude protein kept stable over a wide pH value. The antifungal activity maintained in organic solvents such as methanol, chloroform, ethyl acetate and acetone. The antifungal protein was purified by DEAE-Sepharose Fast Flow chromatography, and five fractions were harvested. FractionⅢshowed higer antifungal activity and a single band with a molecular mass of 66 kDa was collected following the SDS-PAGE method. According to the analysis of MALDI-TOF-MS, the similarity of amino acid sequence between the the purified protein and putative phage-related pre-neck appendage protein was very high.Lipopeptides were isolated from the cultural supernatant of strain CAB-1 by the methods of hydrochloric acid precipitation. The protective value of lipopeptides-treated plot was 76.17%. According to MALDI-TOF-MS analysis, the lipopeptides revealed two groups [M+H]+ ion peaks at 1447.725,1461.813,1477.109 and 1500.041,1513.839,1527.566 respectively. And the first group was determined as Fengycin A (C15-C17), however, the other group of homologues was undetermined.We also assayed the fumigating effect of the volatiles compounds(VCs) from strain CAB-1 to S. fulginea . VCs fumigation reduced the disease index of cucumber and the protective value was 85.44%. The VCs produced by CAB-1were identified by using GC-MS method, yielded 27 compounds, which included a range of alcohols, phenols, amines, alkanes, amides etc. Comparing with different doses and different times treatments, 2 plates fumigating for 4 days in 0.012 m~3 airtight space was our choice in the controlled greenhouse.