Preliminary Purification And Characterization Of The Antimicrobial Compounds Produced In Lysobacter Capsici X2-3

Lysobacter capsici X2-3 was isolated from rhizosphere of wheat and showed in vitro antagonistic activity to fungi and Oomycetes such as R. cerealis, R. solani, B. sorokiniana and P. myriotylum. To obtained the antimicrobial compounds, the cultural conditions of the strain were optimized firstly and crude compounds were extracted subsequently. Antifungal activity of the crude extracts to R. solani were tested after treated with high temperatures, proteinase K and UV-light. Mycelia morphology of R. solani and germination of B. sorokiniana spores were observed after treated with crude extracts. The main results are as follows:1?Optimization of cultural conditions of X2-3Mycelium growth rate of R. solani were used to test the antifungal activity of cell free culture broth of strain X2-3 at different medium. Cultures from PDB medium showed high antifungal activity against colony growth to R. solani. The fermentation conditions of strain X2-3 were optimized using PDB as the basic medium. The results showed that the optimized condition was as set as follows: pH 5.0, 2% inoculums, 100 mL of solution into a 250 m L flask, 28? incubation at a speed of 180r/min for 120 h. The cell free of X2-3 incubated at this conditions showed high antifungal activity to R. solani.2?Extraction of antimicrobial compounds from cell free culture broth of strain X2-3To extracted the antimicrobial compounds, precipitated complex were obtained from culture broth of X2-3 using ammonium sulfate. As indicated in the result that the antifungal compounds were mainly precipitated from the culture broth but no antifungal activity was found at the supernatant.Five solvents named petroleum ether, dichloromethane, chloroform, ethyl acetate and methanol were used to extract antimicrobial compounds from precipitated complex. Crudes extracted by methanol showed highest activity against R. solani with the inhibition zone about 5mm in radius. Crudes extracted by acetic ether and chloroform showed lower activity against R. solani with the radius only 2mm and 1mm respectively. Ultraviolet spectrophotometer was used to scan the wavelength range of 200-800 nm, and it was found that the antimicrobial substance had the maximum absorption at 244 nm Solvents.Solvents of dichloromethane, chloroform, ethyl acetate and methanol were selected as eluents in extraction of crudes through silica gel column chromatography. The result showed that eluted fractions using methanol as eluent having highest absorption at 220 nm with absorption value as 2.5AU.3?Stability of antimicrobial compoundsIn order to understand the stability of antimicrobial compounds, antifungal activity of the crude extracts was analyzed after treated with temperature, ultraviolet and proteinase K respectively. Results showed that compounds produced by X2-3 showed high antifungal activity after treated with treatments of 40 to 80?. The antifungal activity was significantly decreased after treated at temperature of 90 and 100? respectively, indicating that the compounds was stability in high thermal.The antimicrobial activity of the crude extracts was almost no difference from the control after 2h treatment with proteinase K, indicating that the compounds were no sensitive to proteinase K.After the crude extracts were irradiated by ultraviolet 1h, 2h and 4h, there was no significant difference between the antimicrobial activity and the control. The results showed that the antifungal compounds had better stability in ultraviolet light.4?Analysis of antimicrobial properties of the crude extractsThe crude extracts had obvious inhibitory activity against fungi and Oomycetes such as B. sorokiniana, Colletotrichum truncatum, Fusarium graminearum and P. myriotylum. Bacillus subtilis and Ralstonia solanacearum were used as indicators to analyze antibacterial activity of the crude extracts. It was found that the compounds had obvious antimicrobial activity against gram positive bacteria, and its minimal inhibitory concentration was 1.72ug/ml, but it had no effect on R. solanacearum.5?Preliminary analysis of the mechanism of the crude extracts on pathogenic fungiAs indicators of R. solani and B. sorokiniana, effects of the crude extracts on mycelial morphology and spore germination were determined. It was found that the mycelium of R. solani showed abnormal phenomenon such as local swelling and abnormal branching after the treatment of the crude extracts. It was also found that the treated spore germination rates of B. sorokiniana were about 1% after 8h, while the control were about 85%, which indicated that the compounds could significantly inhibit spore germination.