Preliminary Study On Antimicrobial Substances Of Marine Bacterium Strain TC-1 From South China Sea Against Fusarium Oxysporum F.sp. Cubense Race 4(foc4) Causing Banana Vascular Wilt

With the research on soil microorganism becoming more and more further, the screen and study on antimicrobial substances of soil microorganism will be difficult. The marine microorganisms with differential inheritance and metabolism produce new substances with novel molecular structure different from soil microorganism.As the result, it is significative to be a world-wide focus to screen marine microorganism and exploit new antimicrobial substances.The strain TC-1 was identified as Bacillus subtilis isolated from the South China Sea sediment collected from the Coast of Donghai Island,Zhanjiang,Guangdong province during 2005.The study on extracellular metabolic substances with inhibitory activity against Fusarium oxysporum f.sp. cubense (E. F. Smith) Snyder et Hansen Race 4 (Foc4) from the strain TC-1, the results show as follows:1. The cell-free fermentation liquid (CFL) of marine bacteria TC-1 has a strong inhibitory activity against Foc4. Culture supernatant diluted of 50 times had 55.2% inhibition rate against Foc4 and 1000 times had 6.7% inhibition rate. When the CFL was heat-processed at 100℃after 60min,the diameter of inhibition halo was 20.0mm, no significant difference from CFL with no heat-processed, which showed that the CFL was of better thermal stability.The inhibition effect of CFL had no significant influence when the value of pH ranged from 2 to 10, which illustrated the change of pH had no influence on the inhibition action of antimicrobial substance in the CFL. The antimicrobial activity was reduced by high concentration of salt. Antimicrobial substances was extracted resultfully by ethyl acetate. The CFL had conspicuous antagonistic effect on Focl, F. oxysporum Schl. f. sp.sesami (Zap.) Cast, Rhizoctonia solani AG-6 syn-20, R.solani AG-4 SDBC-71, Colletotrichum capsici (syd.) Butl, Aspergillus niger, Rhizopus nigricans, Ralstonia solanacearum (E. F. Smith) Yabuuchi, Escherichia coli, Staphylococcus aureus, the diameters of inhibition halos were 22.0 mm,15.0 mm,23.0 mm,16.0 mm, 25.0 mm,21.5 mm,14.2 mm,25.7 mm,16.0 mm,20.0 mm respectively.2. The strain TC-1 could produce antifungal volatiles which strongly inhibited the growth of Foc4. It is speculated that antimicrobial protein and conjugated double bond compound in the CFL by ultraviolet analysis. There were 17 amino acids including Glu,Asp,Leu and so on in the CFL through amino acid analysis. With GC/MS,it's definited that 32 kinds of chemical composition were in the CFL,such as alcohols,acids, halide,esterified esters,et al,of which 27 kinds were new.3. The crude protein extracted by ammonium sulfate that the range from 50% to 60% had obvious inhibitory activity against Foc4. When the crude protein was processed at 120℃after 20min,the diameter of inhibition halo was 20.0 mm, no significant difference from protein without heat-processed,which showed that the crude protein had good thermal stability. The inhibition effect of the crude protein had no significant influence when the value of pH ranged from 2 to 9,which illustrated the change of pH had no influence on the inhibition action of the crude protein.And the crude protein was stable to prolease and ultraviolet radiation. There was one antimicrobial protein by column chromatography, and the diameter of inhibition halo was 16.7mm against Foc4.The result of SDS-PAGE showed that the molecular weight was 50.3KD. The crude protein had conspicuous antagonistic effect on Focl, F. oxysporum Schl. f. sp.sesami (Zap.) Cast, R. solani AG-6 syn-20, R. solani AG-4 SDBC-71, C. capsici (syd.) Butl, A. niger, R. nigricans, R. solanacearum (E. F. Smith) Yabuuchi, E. coli, S. aureus, the diameters of inhibition halos were 22.7 mm, 18.0 mm,31.0 mm,28.0 mm,30.0 mm,27.8 mm,20.7 mm,28.0 mm,23.3 mm,10.0 mm respectively.4. The ethyl acetate extract could inhibit Foc4, so it was explained that there were some antimicrobial substances of nonprotein against Foc4. The result of TLC and functional group analysis showed that the antimicrobial substance was peptide matter. Biosurfactant produced by the strain TC-1 could be evaluated by surface tension, diameter of ejecting oil and hemolysis.The composition lowering surface tension was lipopeptide.5. The antimicrobial substance called lipopeptide biosurfactant was obtained from the CFL, and the molecular weight was 1097.3 Da. The critical micell concentration (CMC) of the biosurfactant was about 35 mg/L. It possessed a high emulsifying activity to hydrophobic organic substance, such as liquid paraffin. And the diameter of ejecting oil was 11.6 cm. The biosurfactant had more endurable to high temperature. It also could keep in a wide pH range from 2 to 12 and its surface activity in high-salinity solution like 15%. The lipopetide was found to inhibit the growth of Foc4 significantly and it had inhibiton effect at 0.05 g/L. The inhibition effect of the lipopetide was stable to temperature and in nature and alkalescence condition. The lipopeptide had conspicuous antagonistic effect on Focl, F. oxysporum Schl. f. sp.sesami (Zap.) Cast, R. solani AG-6 syn-20, R. solani AG-4 SDBC-71, C. capsici (syd.) Butl, A. niger, R. nigricans, P. spp., S. cerevisiae, R. solanacearum (E. F. Smith) Yabuuchi, E. coli, the diameters of inhibition halos were 21.7 mm,14.5 mm,25.0 mm,4.0 mm,28.3 mm,14.8 mm,24.8 mm,19.0 mm,13.8 mm,21.0 mm,12.0 mm respectively.