| With the increasing awareness of plant protection and sustainable agriculture, it is very important to develop pollution- free and nuisance-free biological pesticides. Therefore, the study on bacteria producing antibiotics has been a hot topic. Biocontrol bacteria are diverse and widely distributed, which are vital sources for many kinds of natural products. Pseudomonas diminuta HD13 is an antagonistic bacterial strain isolated from microbial agents by the fungal research team of Biochemical Engineering Center of Guizhou Province. The study preliminarily explored the antifungal stability and mechanism of HD13.HD13 showed broad-spectrum antibacterial activity in confrontation experiment. The best inhibitory effects are on Thanatephorus cucumeris(A.B Frank) Donk and Phytophthora nicotianae Bredade Haan. Through single- factor test and orthogonal experiment, the best medium components for HD13 consist of: sucrose 20.0g/L, beef extracts 7.0g/L, Na Cl 2.0g/L, K2HPO4 4.0 g/L. The optimum absorb wavelength for HD13 is 460 nm. The optimal fermentation condition results indicate that the inhibitory effect can be the best when keeping primary alkalinity acidity neutral, inoculation quantity is 8%, inoculating for 4.5 days with 110r/min under 30?. It is better for the accumulation of antifungal compounds when shaking cultivation for 3 days and then static culture for 15 days.The antifungal compounds of HD13 meanly concentrate in extracellular substances, which fermentation liquid holds high stability, insensitive to hot a nd alkalinity, against UV irradiation, storage time is stable. The inhibitory activity is stronger when precipitating it with 50%-90% ammonium sulfate. The study preliminarily explored the relationship between OD value in fermentation liquid and inhibitory activity. The linear regression equation of OD value and the amount of live bacteria(y) is: y=1.5856x+0.0605,R2=0.9919; O D value and wet weight(y): y=7.2232x+1.0802,R2=0.9919; OD value and dry weight(y): y=0.951x+0.0722,R2=0.9158; OD value and inhibitory rates to fungal pathogen(y): y=84.166x--3.9601,R2 = 0.9482?The linear regression equation of the n-butanol extracts of HD13 fermentation liquid and virulence to Thanatephorus cucumeris(A.B Frank) Donk is: y = 0.66 x + 5.01,R2=0.95, IC50 is 1.25g/L. The antifungal extracts are isolated into two fractions: A and B. Through primary analysis, fraction A is relatively singular, which may be lipopeptide or protein, B contain small molecules.The inhibitory result showed that fraction A had cer tain inhibitory effect to four plant pathogens especially to Thanatephorus cucumeris(A.B Frank) Donk, which IC50 was 124?g/m L, MFC value was 215?g/m L. Fraction A could lead to teratogenic morphology in Thanatephorus cucumeris(A.B Frank) Donk, which destroyed the integrity of fungal pathogen cell wall and increased the permeability of membrane system. The gel shift assay demonstrated that fraction A could combine non-specifically with the DNA of Thanatephorus cucumeris(A.B Frank) Donk, which manifested that fraction A could not only take effect on membrane system but also target other intracellular substances.After 7 generation, the inhibitory rate decreased and target compounds were not stable, which revealed that there are probably many kinds of bioactive compounds produced by the strain HD13. |
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