Screening And Identification Of Bacteria Strains Against Bacterial Wilt And Fusarium Wilt Of Tomato And The Study Of Their Antagonistic Mechanism

Tomatoes,with great nutritional value and economic benefits,was large-scale cultivated in the greenhouse, plastichouses and other protected facilities.vegetables disease outbreaks frequently because of the high temperature and humidity facilities environment provide the various pests and pathogens the suitable growing and breeding conditions.Soil-borne diseases (bacterial wilt,fusarium wilt,etc.) is the main diseases that affect the facilities tomato production. This article includes collecting soil samples,screening and identification of antagonistic bacteria,study of colonization and so on.The results were as follows:(1)Biological control on plant diseases could reduce the remainder of chemical pesticides and protect natural ecological environment.Tomato Fusarium wilt and bacterial wilt are two common diseases in tomato production. The aim of this study was to screen and evaluate the antagonistic bacteria against Fusarium oxysporum f.sp.lycopersici and Ralstonia solanacearum.367strains were isolated and purified from root and soil of tomato,pepper and watermelon in Ningxia, Jiangsu and Fujian province.Antagonistic examination against Fusarium oxysporum f.sp.lycopersici and Ralstonia solanacearum revealed that22strains among all tested strains showed strong and steady antagonism and produced protease. Several strains among the above22strains could produce cellulose and siderophore.On pot experiment, strain PTS-394, H-70, L-1and SJ-280.of22strains could control Fusarium wilt and bacterial wilt effectively with the control effect68.64-77.40%and60.00-80.00%. The molecular identification experiments indicated the strain PTS-394, H-70,L-1and SJ-280were Bacillus subtilis.(2)Bacillus subtilis PTS-394was labeled with green fluorescent protein gene to investigate its colonization in the soil of tomato rhizosphere.The recombinant plasmid pGFP22carrying both the green fluorescent protein gene and chloramphenicol resistance gene was transferred into original strain PTS-394by electroporation to get the marked strain PTS-GFP. Cell growth rate and antibacterial activities of the marked strain were tested.The experimental results showed that the green fluorescent protein gene marker doesn’t bring a significant adverse effects on its cell growth.And antibacterial activities testing proved that there was no obvious difference between wild strain PTS-394and engineered strain PTS-GFP.The bacteria isolation and culture method and Real-Time PCR were used to detect the colonization of PTS-GFP,and experimental results showed that PTS-GFP had a downward trend when irrigating0-15d and then declined slowly after15th day and remained constant at6.5×104cfu/g.And the RT-PCR result was consistent with the result of bacteria isolation method.(3)The Plackett-Burman Design and response surface methodology were adopted to optimize the liquid fermentation medium and conditions of the biocontrol agent of Bacillus sublitis PTS-394. By the statistical analysis of PB design,the significant composition factors affecting the yielding capacity were Corn starch, Soybean powder, Yeast extract.And according to the evaluation by the response surface model established in this study,the optimal composition was11.48g/L,13.35g/L,0.60g/L.Then the production of spores and antibacterial bandwidth4.436×109cfu/mL and6.20mm,which were104.7%and44.8%higher than that of initial medium.The significant condition factors were temperature, pH,Medium volume.The optimal combination of conditions was30.84℃,pH6.93,65.15mL/250mL.Then the bacteria content and antibacterial bandwidth were increased by39.2%and46.0%.