The Isolation, Identification And Metabolite Of Biocontrol Bacteria From Tobacco Field In Qianjiang

In order to control tobacco wilt pathogen Ralatonia solanacearum,87strains were isolated from rhizosphere of healthy tobacco by serial diluting isolation. Two among them,5D15and5B18, were found with strong activity against the growth of R. Solancearum through the dual culture method. The antagonistic activity of the extracts from active bacterium was further confirmed by disc diffusion assay. The16S rDNA analysis and the whole cell fatty acid analysis were then performed, the results of which indicate that the active strain,5D15and5B18, were Pseudomonas aeruginosa and Bacillus amyloliquefaciens, respectively. After that, extracts of the two active strains were separated by silicagel column, and the bioactive chemicals were identified by NMR, IR and HPLC. The main conclusions of the study are as follows:(1)87strains of bacteria were isolated by series dilution method. Two strains were found to be highly effective against Ralatonia solanacearum. They are5D15and5B18. The semi-diameter of the two strains against R. solanacearum are8.4mm and10.2mm, respectively. The disc plate tests were also carried out to check whether the crude extracts is effective. The result showed that the semi-diameter of the two strains5D15and5B18were6.2mm and8.0mm.(2) The16S rDNA analysis and the whole cell fatty acid analysis were performed to identify the active strain, the results of which show that the similarity between5D15and Pseudomonas aeruginosa is99.6%, and the one between5B18and Bacillus amyloliquefaciens is99.4%. Therefore, the active strains are Pseudomonas aeruginosa and Bacillus amyloliquefaciens. (3) Orthogonal tests were carried out to optimize the nutrient medium of the biocontrol strains. The results showed that the concentration of NaCl was the remarkable factor to influence the growth of bacterium5D15with optimal concentration of10g/L. In the case of5B18, the results show that PDA medium is more suitable when compared to NA medium. And in PDA medium,350g/L potato achieved fast growth of5B18.(4) Three fractions in n-hexane, dichloromethane and methonal were obtained from the extract of5D15. Disc plate assays were carried out to test the anti-pathogen activity of the three fractions, the results of which show that the dichloromethane fraction is the most effective one. TLC test showed that dichloromethane and methonal acting as flowing solvents achieve best isolation, and the elution gradient was2%、4%、6%and20%of methonal. The purity of the bioactive chemical was76.11%by HPLC. The structure of the substance was identified to be cyclo-(tyr-leu) by NMR and IR. EC50of the chemical against R. solanacearum was between40μg/mL to160μg/mL.(5) Three fractions in n-hexane, dichloromethane and methonal were obtained from the extract of5B18. Disc plate assays were carried out to test the anti-pathogen activity of the three fractions, the results of which show that the dichloromethane fraction is the most effective one. TLC test showed that dichloromethane and methonal acting as flowing solvents achieve best isolation, and the elution gradient was4%、8%、12%and20%of methonal. The result of HPLC showed that about13chemicals were found in the second part...