| 254 strains were isolated and purified from the soil samples, which were collected from rhizosphere of the crops in the suburban districts of Shanghai. One strain of P13 was screened out from 254 isolates, which expressed stronger antagonistic effect against Sclerotinia sclerotiorum through dual culture method. Besides, it could also inhibit some other pathogens of agricultural importance with various extents. Through EM (Electron Microscope) observation, colony analysis and a series of physiological and biochemical experiments, this strain was identified as Pseudomonas fluorescent.In order to strengthen its antagonistic effect, the experiment studied the optimum culture medium components and conditions of flask fermentation of P13. The results showed (1000ml medium) peptone 25g, glycerol 5ml,K2HPO4 1.5g,MgSO4.7H2O 2g; 28℃, 48h, pH8, media amount 100ml/500ml and inoculation amount 5%.Do not know characteristics of antifungal substances P13 produced and there is no invariable method of being purified. According to the method that had been reported, we extracted antibiotic substances from liquid containing such substances with organic solvent. It was found that ethyl acetate and n-butanol were better choice. In order to ensure bioactivity, the effects of heat, pH and UV on the stability of antifungal substances in the fermented broth were studied. The ion character of the antibiotics was analysized by pH paper chromatography, and the result showed antifungal substances were probably alkaline antibiotics. The most intensive absorption wavelength by antifungal substances was 265nm.To make sure why the hyphal growth was inhibited, we researched and found antibiotic substances P13 produced had great influence on hyphal growth by means of optics and electron microscopy techniques, including twist of hyphae, necrosis of the hyphal wall, concentration and leakage of the cytoplasma. This is the preliminary study about the antagonistic mechanism by the way of microscopically observation.
|
PDF Full Text Request