Studies Of Bacillus Subtilis Isolate B6-1 Co-producing Lipopeptides And Poly-γ-glutamic Acid With Antifungal Activity Against Phytopathogens

For the first time,the Bacillus strain B6-1,isolated from the rhizosphere of crops, co-produced the antifungal substances and poly-γ-glutamic acid,and was identified as Bacillus subtilis.The co-productions of antagonistic substances and poly-γ-glutamic acid were obtained when using soybean and sweet potato residues with perlites cultivated with B6-1 in solid-state fermentation.The antifungal compounds were purified and identified as lipopeptides fengycins.Suppression effect of residues cultivated with B6-1 on cucumber wilts and fertilizer synergistic effect were studied.The results were followed as:1.313 distinct presumptive Bacillus isolates were obtained from the rhizosphere of crops.37 of them displayed distinct and stable antagonism against at least one of indicators tested through dual culture assays.B6-1 showed the broadest antifungal spectrum,strongest antagonistic activity.Based on the physiological,biochemical charactcristics and 16S rDNA data,the strain B6-1 can be designated as Bacillus subtilis.2.The results from antagonistic mechanism demonstrated the production of antifungal metabolites.The culture filtrate of Bacillus subtilis B6-1 displayed oil displacement,droplet collapsc,hemolytic activity and aggregational behavior.The antifungal compounds could bc precipitated by acid and remain its activities against heat, pH,UV.All the results above implied the antifungal compounds belong to lipopeptides.3.Using a procedure including ultrafiltration,acid precipitation and solid-phase extraction,the antifungal compounds were purified.The major antifungal compounds showed samc UV spectra in methanol and a homologous[M+H]~+ ion peak at m/z 1450, 1464,1478,1506,respectively.These mass data correspond well to those determined by other authors,indicating the presence of C15,C16,C17,C19 fengycins,corroborating the deduction above.But there are no reports about hemolytic activity of fengycins.So other lipopeptides with hemolytic activity may be present.Further studies would be done in future.4.In the nutrient broth fermentation,B6-1 produced the lipopeptides but noγ-PGA. And in the optimized medium for production ofγ-PGA,the yield ofγ-PGA reached 3.47%but the lipopeptides could not be detected.The averageγ-PGA yield in SSF was 36.3 g/(kg dry substrate) in 250 ml flask.At the same time,the production of lipopeptides was verified.The maximum dilution in SSF that can be detected was 70 folds,remarkably higher compared with 20 folds in NB.5.When the residue mixtures cultivated with B6-1were introduced,the disease occurrence was effectively reduced to 17%.At full nutrition solution,when residue cultures were introduced,the dry weight of shoots and roots and the roots to shoots ratio increased by 33.8%,12.2%,18.3%,respectively.At 1/2 nutrition solution,they increased by 47.4%,26.4%,20.4%respectively,and at 1/3 nutrition solution,increased 69.5%, 17.4%,43.8%,respectively.The addition of residue cultures significantly increased the dry weight of both roots and shoots and the roots to shoots ratio(R/S),at all levels of nutrients.The increases were more significant at lower nutrient levels.6.The survival ratio of lactic acid bacteria was improved by adding more than 1.5% PGA during freezing and frozen-storage and the effect was better as the frozen-storage was prolonged.The acidification activity remained strong and the lag phase of lactic acid bacteria was shorter.