Breeding, Reconstructed Gene And The Control Efficacy Of Ferment Liquid Of The Broad-spectrum Antagonistic Strain To Botrytis Cindered

First,we achieved some kinds of antagonistic bacteria by the preliminary screening and secondary screening from the samples.Then we studied the broad-spectrum antagonistic strain with identification, reconstructed gene, the optimization of fermentation condition and the control efficacy of ferment liquid.A total of 31 strains were islated from samples. The strain of D2 with significant antagonistic activities against tomato Botrytis cinerea was isolated from samples. Acording to its morphological,physiological and biochemical characteristics, D2 was possibly identified as Bacillus.In order to enhance the inhibitory effect of this strain, a 6.4 kb DNA fragment containing Chitinase gene from pUC1965 was inserted into vector pBE2 to construct the recombination plasmids called pBE2-chib. A new strain named D2-chib was formed by transferring the recombination plasmids into Bacillus D2. Chitin plate culture and PCR identification of Chitinase gene confirmed that the Chitinase gene was transferred successfully into the wild strain D2.The control effect of D2-chib and D2 were 91.8% and 86.7% respectively. D2-chib had a significant difference inhibitory effect against Botrytis cinerea on tomato compared to wild strain D2. The original chitinase activity of D2-chib using the vector pBE2 was 4.06 U/ml which was 33.8 times comparing to the D2.Obased on one-way analysis of variance and orthogonal experimental design, we have found the optimization of fermentation condition. An optimum medium came up acordingly,which consisted of 1% glucose,2% sucrose, 1%yeast extract,4% soybean powder,3%bran,0.04% MgSO4,0.4% CaCl2. Carbon sources had significant influence on the effect of antagonistic substance. Then the effects of sucrose, CaCl2, bran, yeast extract, soybean powder, MgSO4 as follow. The optimum fermentation condition was contained:the transfer volume of the seed liquid for 5%(cultivated 24 hours), the volume ration of cultural medium to flask is 30 mL to 250 mol,the pH of the fermentation liquid for 7.2 fermentation time for 48 hours. The main effect the effect of antagonistic substance was pH. Then the effects of the transfer volume of the seed liquid, cell age, the volume ration of cultural medium as follow.At the end of the experiment, we found the effects of fermentation broth of D2-chib strain had abviously antagonistic activities against tomato Botrytis cindered through the greenhouse experiment. The optimum concertration was 100 times Fermentation diluent with 91.34% control effect.Beside, we found that Bacillus could be able to colonize in leaf and rhizosphere of tomato by strong ability of competition.The result indicated that the strains not only have no poison effct, but also had significant effect on control of Botrytis cindered with effect on the growth of seedlings.As a result, D2-chib is a potential applied valuable broad-spectrum antagonistic plant growth-promoting rhizobacteria.