| Objective: This study isolated and screened endophytic bacteria inhibited on Fusarium Wilt of cottons(Fusarium oxysporium)from Glycyrrhiza uralensis, Sophora alopecuroides and Resina Ferulae. Separated the antibacterial substance of antagonistic bacteria A preliminarily, explored the antibacterial mechanism, and observed its effect on cottons.Methods: The isolation and screening of antagonistic bacteria by plate culture method. The basic properties of antagonistic bacteria including growth curve, the optimum p H, sugar fermentation, starch hydrolysis test and Gram staining; identified the 16 Sr DNA molecular and established the phylogenetic tree. Cell disruption by the ultrasonic method. Separated the intracellular or extracellular secretion by centrifugal. The separation of antibacterial intracellular protein by ammonium sulfate precipitation dialysis method. The detection of protein concentration by Coomassie brilliant blue G-250 method. The purification of antibacterial protein by Sephadex G-75 column chromatography preliminarily. Determinated the molecular weight of inhibitory protein by SDS-PAGE method. The influence in antibacterial substances deal with the Fusarium oxysporium on the mycelial growth, spore production and spore germination by plate culture method, microscopic counting method and the concave slide culture method respectively. Calculated the germination rate and survival rate. Detected the activity of POD, PPO, PAL in different culture time of cottons, which deal with endophytic bacteria or pathogenic fungi.Results: 1, The study separated and screened out 4 strains inhibited on Fusarium oxysporium from Glycyrrhiza uralensis, Sophora alopecuroides and Resina Ferulae. The antagonistic bacteria A had the best Inhibite effect, the circlediameter of bacteriostatic ring up to 26.96 mm in the first test. After identification, antagonistic bacteria A, B,C, D belong to Bacillus amyloliquefaciens, Bacillus tequilensis, Bacillus sp. and Klebsiella pneumoniae respectively.2, The Inhibitory rate which intracellular secretion deal with the Fusarium oxysporium was larger than the rate in extracellular secretion on the mycelial growth, spore production and spore germination. Among them, the inhibition effect of pathogen spore production was best, and the average inhibition rate was 40.14%, the antagonistic of maximum inhibition rate from the strain in logarithmic phase, and the rate up to 88.81%.3, The biggest inhibition rate against Fusarium wilt was 19.55%, which deal with the precipitated protein by 80% concentration of ammonium sulphateon. To maintain the antibacterial of the crude protein activity, the highest tolerable temperature was 60℃, the maximum tolerance of p H was 8 and the lowest tolerance of p H was 4.4, The testing of Inhibite crude protein by Sephadex G-75 column chromatography and SDS-PAGE showed that, the peak appeared between 200 s and 400 s. The protein molecular size between 25 k Da and 100 k Da. In this time, the inhibited rate by collected eluent in the spore production of Fusarium wilt pathogen up to 34.79%, larger than the inhibited rate of mycelial growth and spore germination.5, The antagonistic bacteria A could improve the adult plant rate of cottons. In the different stages of cotton seedlings, the determination results of POD, PPO, PAL enzyme activity showed that, there was no significant difference in the same period and different processing of three kinds of enzyme activity in cotton seedlings. Except in the 40 days after sowing the cottons, the PAL enzyme activity of cottons,which antibacterial A and Fusarium oxysporum treated, were increased significantly.Conclusion: the study separated and screened out 4 strains inhibited on Fusarium oxysporium from Glycyrrhiza uralensis, Sophora alopecuroides and Resina Ferulae. The test of antagonistic bacteria A extracellular or intracellular secretion inhibiting mycelium growth, spore production and spore germination showed that, antibacterial substance produced in the logarithmic phase, and intracellular secretion had better inhibited effection. The main way of inhibited Fusarium oxysporum was inhibited the spore. The stability of antibacterial protein in different temperature and p H can meet the need of production practice. The molecular sizes of protein were between 25 k Da and 100 k Da, and still have some antibacterial activities. The antagonistic bacteria A could improve the adult plant rate of cottons, but the active oxygen system of cottons was not enhanced significantly. |
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