| Bacillus subtilis Tpb55 was separated from tobacco leaves which showed strong biological control ability. In this paper, the effect of Tpb55 on tobacco black shank was studied firstly, and then the green fluorescent protein gene was transferred into Tpb55 and expressed successfully, and the biological activity and stability of Tpb55-GFP were determined. Greenhouse experiments were made to study the colonization of Tpb55-GFP in soil with different depth and location, different pH, different organic matter and different texture types. Field experiments were made to study the prevention and control ability of Tpb55 to black shank, and the 454 high-throughput technology was used to analyze the strain tpb55 of tobacco rhizosphere micro ecological impact. Specific conclusions are as follows:1. The dual culture assay results showed Tpb55 strains to exert stronger inhibition on mycelial growth of P. nicotianae., and the inhibitory effect was obvious. In scanning electron microscope, Phytophthora nicotianae mycelial morphology was been damaged, a single mycelium ruptured, bended, withered morphology, fold the protoplasm leaked, the mycelium growth disorderly.The fermentation liquid by rotary evaporation and extracted by ethyl acetate, EC50 was 0.33 mg/mL.2. Greenhouse experiments showed that black shank disease index of Tpb55, 58 % metalaxyl manganese zinc(1:400) and control were 21.44, 19.12 and 71.60, respectively. This showed theTpb55 strain to have potential in the prevention and control of tobacco black shank. In 2013 and 2014 field experiment showed that the Tpb55 treatment of the disease index were 21.44 and 14.23. The disease index of 58 % metalaxyl manganese zinc(1:400) were 19.12 and 11.71 and control disease index were 31.55 and 39.51. Although the disease index of Tpb55 treatment higher than 58 % metalaxyl manganese zinc(1:400), data had no significant difference.3. Tpb55-GFP strain was constructed of the fluorescent protein gene of jellyfish. Compared with the original strain Tpb55, the inhabition ability on Phytophthora nicotianae and Alternaria alternata of the labeled strain Tpb55-GFP was basically the same. They can formed obvious antibacterial circle and there was no significant difference between them. The inhabition rate on Phytophthora nicotianae and Alternaria alternata of labeled strain Tpb55-GFP and original strain Tpb55 were higher than 60%, these results suggested that Tpb55-GFP could be used for further experiments.4. Colonization dynamics results showed that Tpb55-GFP could successfully colonize in the tobacco rhizosphere. Compared to the different depth of the soil colonization, Tpb55-GFP in the rhizosphere of tobacco had stronger colonization ability. On the 30 th day, the rhizosphere of colonization density was 3.42? 106 cfu/g. Laser confocal microscopy showed that Tpb55-GFP can form biofilm formation in root, then distribute in tobacco cells and vascular tissu. The different pH soil colonization experiments showed that pH 6-7 were suitable for Tpb55-GFP colonization. Different organic matter experiments showed that rotten peanut shell were suitable for Tpb55-GFP colonization rich organic small molecular substances. Different types of soil colonization experiments showed that clay: sand(2:1) was suitable for Tpb55-GFP colonization. Because this type of soil was similar to the character of loam, which had the ability of keeping certain moisture and nutrients and air exchange.5. 454 high throughput sequencing showed that species and number of bacteria were different between control and treatment in the development of disease process. Chao 1, ACE and Shannon indexes showed that the bacterial diversity and richness of Tpb55 treatment increased and were higher than that of control. In 10 d and 22 d after Tpb55 inoculation, the number of sequences with high homology of V1-V3 regions of Tpb55 16 S rDNA was 31 and 45, respectively. The disease index of tobacco black shank in inoculated tobacco(5.29) was significantly lower than control(38.52). Tpb55 could improve the diversity of soil bacterial community and ecosystem stability,which presented a reason for its biocontrol efficacy on tobacco black shank. |
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