The Screening And Genetic Modification Of Antagonistic Bacteria Against Botrytis Cinerea From Tomato

In order to deal with Tomato Botrytis cinerea, six strains antagonistic to Botrytis cinerea were isolated from the soil growing tomato infected by Botrytis cinerea in Zibo, which were B-01, B-02, B-03, B-04, B-06 and B-07 respectively. The inhibition zone test showed that Bacillus had strong inhibiting activity. According to the size of inhibition zone, B-02, B-04 and B-06's inhibitory founction are better, with the inhibition radius more than14mm, of which B-06 has the strongest inhibitory founction, with inhibition radius of 20.1mm. Morphological and biochemical characteristics as well as sequence analysis of 16S rDNA revealed that they are G+, short rod, with flagella and elliptical spora.According to the criteria of《Bergey's Manual of Determinative Bacteriology》(the ninth edition), it was confirmed that strains B-01, B-02, B-03, B-04 belonged to Bacillus cereus,B-06 belonged to Bacillus subtilis, and B-07 belonged to Bacillus pumilus.The filtrate's inhibitory activity to Botrytis Cinerea was mensurated for determining the inhibition activity. The result suggested that the filtrate of B-02 and B-06 has very strong inhibitory activity. On the control side, the hypha spreaded forward, and behaved normally, while on the tested side dropped with filtrate, the hypha growed slowly, and couldn't grow all around, even with the hypha distortion, with the color deepened. At the same time, the filtrate could inhibit sprouting of Botrytis cinerea. However, the serial dilution of the filtrate would reduce the inhibition activity notably.β-1, 3-glucanase can degradeβ-1, 3-glucan in cell walls of most plant pathogenic fungi and suppress the growth of the fungi. Two enzymes EcoRⅠand SalⅠwere used to cut plasmid pUC1940 into two segments, one was 2.7kb and the other was about 4.1kb, containingβ-1, 3-glucanase gene. Similarly, two shuttling plasmids pBE2 and pHY300PLK were cut with EcoRⅠand SalⅠ. The 4.1kb fragment were ligated to construct the recombinational plasmids, pBE2-glu and pHY300PLK-glu, which were transferred into six Bacillus strains respectively. As the result, successful transformants were obtained in B-04, named B-04-glu, with the highest transfer ratio in 1400V, 50uF, 200?, 2mm. Compared with the wild strain, B-04-glu could expressβ-1, 3-glucanase. There was no significant difference between the growth of the B-04-glu and B-04, which indicated the transfer ofβ-1, 3-glucanase did not influence the growth of the host. The stability determination of the recombinational plasmids revealed a high hereditary stability of more than 90%. Moreover, B-04-glu had increased inhibition effect on Botrytis cinerea in the antagonistic experiment.