Screening, Identification And Application Of Antagonistic Strains Against Tomato Bacterial Wilt Caused By Ralstonia Solanacearum

Tomata bacterial wilt caused by Ralstonia solanacearum is a serious soil spreading disease which distributes widely in the world. It is a difficult problem to effectively control the disease. The research focuses on the biocontrol of tomato bacterial wilt. The antagonistic strains were isolated and carried on the effect of biocontrol by greenhouse potted trial and field plot trial. It provided the theory basis and the technical support for the preparation of highly effective biocontrol agent.1. Forty-five bacterial strains isolated from the greenhouse soil collected from Shouguang city and Cangshan city, Shandong province, nineteen strains preserved in our laboratory were tested, from which forteen strains inhibiting Ralstonia solanacearum were screened out by inhibitory zone method. After using the aseptic filtrate treatment, six strains showed a wide inhibitory zone among the fourteen strains. Results in greenhouse potted trial showed that the control effect of X10 using liquid microbial agent and solid microbial agent reached 81.8 % and 65.4 %, respectively.2. The morphology, physiological and biochemical characters of strain X10 were studied, which showed that strain X10 was consistant with Brevibacillus laterosporus. The analysis of 16S rDNA sequence from strain X10 suggested that strain X10 was clustered together with B.laterosporus in phylogenetic tree and the sequence identity was 99 %. So strain X10 was identified to B. laterosporus.3. The crude protein extracted from the Brevibacillus laterosporus X10 culture could inhibit the growth of Ralstonia solanacearum. The crude protein is stable against heat, which could tolerate 90 °C. The inhibitory activity is influenced by treatment with trypsin, pepsin and proteinase K. It is sensitive to chloroform. Along with the time of ultraviolet irradiation extending, the inhibitory activity is reduced. The antimicrobial protein could keep its antibacterial activity stable for 28 d at 25 °C. The active pH range of the crude protein to the pathogen is wide, from 5.0 to 11.0.4. According to the results of greenhouse potted trial, we prepared solid biocontrol agent and carried on the field plot trial in Shouguang city and Cangshan city, Shandong province. The results indicated that the control effects of two different inoculation in vegetable shed were 58.42 % and 68.68%, respectively. In the same of time, the solid agent could promoter the growth and yields in vegatable shed.5. High quality genomic DNA of Brevibacillus laterosporus X10 was extracted. The promoter library of X10 was constructed in E. coli DH5a with pUC19-GFP as the promoter probe vector by shotgun-cloning method. 14 positive clones designated as P1~P14 were obtained by screening. The inserting fragments in the recombinant plasmids from P2, P3, P6, P9, P10, P13 and P14 were sequenced and subjected to online promoter prediction.The results show that P3 and P6 have typical prokaryotic promoter elements: -10box and -35box, others haven't. P6 was inserted into the colon site of shuttle vector pNW33N, whice provided a powerful tool to inspect the colony of X10 in the root of tomato.