| Cotton Verticillium wilt is a soil-borne vascular bundle disease caused by Verticillium dahliae,and is the most serious fungal disease hazards in production in China.Screening for its control methods for stable and high yield of cotton in China is of great significance.Here we have isolated the antagonistic Bacillus strains against Verticillium dahliae from the health continuously-cropping cotton rhizosphere soil in Xinjiang,and explored the antibacterial properties and antagonistic mechanism of antagonistic bacteria,to provide resources and theoretical guidance for the bio-control of cotton verticillium wilt in Xinjiang.A total of 146 strains were obtained and 29 strains of them have the varying degrees of antagonism to Verticillium dahliae,and 9 strains were highly antagonistic.In which strain J-02 and J-29 belonged to the Bacillus pumilus,strain J-15 and the rest strains are similar to B.subtilis.We found that the inhibition activity of strain J-02 is more stable to heat,and in the pH range of 5-9;The strongest inhibitory effect of strain J-02 on Verticillium dahliae is in the decline phase;Strain J-02 can inhibit the germ tube of Verticillium dahilae elongate to form mycelium;In pot experiment,strain J-02 showed a certain protective effect on Cotton Verticillium wilt.Strain J-15 showed protective effect on Cotton Verticillium wilt in pot experiment.Extracting antibacterial substance of strain J-15 with salting-out method,the results showed that the 80% ammonium sulfate saturation was the best.Strain J-15 is sensitive to temperature,its cell will die after 100? treatment,however,the activity of its antifungal substance decreased only 18.3% after 100? treatment for 1h;The inhibition activity of J-15 treated with pH 7-9 was not significantly different with control group,while its antifungal substance could tolerate pH 2-10,and pH 2-4 can completely precipitated antifungal substance without affect the inhibitory activity;The strongest inhibitory effect of strain J-15 on Verticillium dahliae is in the stable phase,while after culturing 12-24 h,OD600 reach 1.7 above is the best time to extract antifungal substance from strain J-15;In addition,crude antifungal substance of strain J-15 are not sensitive to pepsin,protease K,ultraviolet irradiation and acetone.The antifungal substance of strain J-15 was extracted by acetone after pH 2 acid recipitation,and SDS-PAGE electrophoresis and spectral scan were performed,after purified with DEAE-Sepharose Fast Follow anion exchange chromatography,the activity substance was identified by mass spectrometry.Results show,after inactive protein in crude substance of strain J-15 removed by acetone precipitation,there no specific protein was detected by electrophoresis;there were two absorption peaks at 210 nm and 270 nm,but the light absorption was lower at 280 nm;Pure antifungal substance was obtained using 0.1 mmol/L Tris-HCl buffer(pH=6.5),0-1 mol NaCl linear elution,while it is not absorbable at 280 nm;The result of Mass spectrometry show its molecular weight may be 828 Da.To explore the effects of antifungal substance of strain J-15 on spore germination,mycelium growth and defense enzyme activity of Verticillium dahliae.Results show,antifungal substance of strain J-15 inhibited the germination of spores with low concentration and cause germination loss with high concentration;Plate test found that antifungal substance can cause mycelium be short,rough and deformity,as well as sporulation capacity decreased;Compared with the control group,antifungal substance of strain J-15 could inhibit the activities of SOD,POD and CAT,and the content of MDA was relatively high in Verticillium dahliae.The results show that the mechanism of antifungal substance of strain J-15 is mainly manifested in the inhibition of spore germination and mycelium elongation,as well as the damage of the antioxidant system and membrane system of Verticillium dahliae. |
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