The Antagonistic Mechanisms Of B1,B2 To Several Pathogenic Fungi And The Identification And Purification Of The Antifungal Substances

1. The growth and the antagonistic mechanisms of several Bacillus spp. For B1,B2,B3, the time when the OD600 reach the peak is 24h and the optimum time to produce antibiotic substances is 72h,48h,48h; for B4,B6 , the time when the OD600 reach the peak is 48h, the optimum time to produce antibiotic substances is 60h. The antagonistic activity reaches the peak and then keeps stable.B1, B2 and the mixtures of B1+2, B146, and B2346 were tested for their inhibition against pathogenic fungi: Botrytis cinerea Pers., Sclerotium cepivorum Berk., Sclerotinia sclerotiorum(Lib.)de Bary and Rhizoctonia solani Kühn. The results showed that the formation of spores and the growth of colony were inhibited effectively. The inhibition activity of the several antagonistic bacteria against S. cepivorum is the strongest. The R(the ratio of the colony radius of the CK- the growth of the fungi against the bacteria to the colony radius of the CK ) is 0.55,0.54,0.58,0.64,0.67, respectively. And the mixtures of B2346 had high antagonistic activity against the 4 fungi tested, The R is 0.53,0.62,0.66,0.67 , respectively. The microscopic characteristics of antagonistic mechanism were showed in two aspects: 1. The hyphy twisted and deformed, the cytoplasm agglutinated and deformed various structures, then the cells degenerated; 2. The hyphy wall was destroyed and broken, the cytoplasm oozed out of the cell, the hyphy wall disintegrated and the cells collapsed. 2. Identification of B1, B2 To identify the B1, B2 strains, 30 assays of physiological and biochemical determination were done. And according to the previous studies, which established the position of the two strains in bacterial phylogeny through spore stain and 16SrDNA sequence analysis, B1, B2 strains were identified as Bacillus subtilis.3. Identification and purification of the antifungal substances from B1, B2. Liquid cultures of Bacillus subtilis B1, B2 strain were precipitated by acidification to pH 2~3, the active fraction was extracted from the precipitate with ethyl ether and was dried at 40℃ on a rotary evaporator. The compound in the extract separated by thin-layer chromatography using toluene: ethyl acetate: formic acid (90%) (6:3:1) as the developing solvent. Detection of the antifungal compound was done by bioassay with 1×106 spores/ml of Cladosporium sp. Detected 3 and 2 inhibition bands from B1, B2 cultivar respectively: Rf＝0.83,Rf＝0.56,Rf＝0.4 and Rf＝0.89,Rf＝0.40. Molecular weight and structure determination using the IR and GC－MS revealed that the antifungal substances from B1, B2 were acylamino and fatty acid matters which were the degrading products of peptide and amino acid. The antifungal substances from B1, B2 are peptide and amino acid.4. Effect on the control of Sclerotinia sclerotiorum in vitro and field test by seed treatment with B1,B2 The germination of rape seed was inhibited by 5× dilution of bacteria suspension, but the germination rate and the root growth was promoted by 20× dilution. Treated with mixtures of B146 and B2346 (20×dilution), the germination rate reached to 98.5%, 98.0% and the root growth promoted by 54.0% and 52.5% compared with the CK. B1, B2 and the mixtures of antagonistic bacteria exhibited apparent inhibition activity to the growth of mycelium and the formation of sclerotium in vitro. The best obtained in B2346, the mycelium spread just on the surface of the soil, and the sclerotium weight is 0.010g/100g soil, CK is 2.436g/100g soil, the inhibiting rate is 590.97%.The results from field trial demonstrated that the effect of promoting plant growth is apparent in the mixture of B1+ B2 and the mutant's mixture of B1R+B2R. The best results obtained in B1R+B2R: the growth of the root, the height of the plant and the number of the branch, compared with the CK, promoted by 23.78%, 12.15% and 36.45%, respectively. Two mixtures also improved the number of the pods, increased by 22.18% and 37.02%, respectively. The control effect of B1+ B2,B1R+B2R and Shi-le-shi against S. sclerotiorum...