【作者】 陈亮；

【导师】 封克； 周晓见；

【作者基本信息】 扬州大学 ， 环境科学， 2011， 硕士

【摘要】 本试验以烟草青枯病病原菌为防治对象,对从黔江健康烟区烟草根际土壤中分离出的87株细菌进行直接对峙筛选初步筛选,获得2株对烟草青枯病菌有较好拮抗性的细菌5D15和5B18,通过全细胞脂肪酸分析和16SrDNA分析,鉴定了两株细菌的种属；然后优化了两株生防细菌的培养基组成,制备了2株细菌的发酵产物,通过滤纸片法对粗提物再次筛选,确定了2株细菌的发酵产物都能够抑制烟草青枯病病原菌；最后用薄层层析法确定了发酵产物的柱分离条件,对2株菌的发酵产物进行硅胶柱分离,通过核磁共振波谱法、质谱法和红外光谱法鉴定了活性物质的化学组成,为烟草青枯病生物防治的研究提供了参考。主要结论如下：(1)采用系列稀释法和平板涂布法,从黔江健康烟区土样中分离出87株细菌。直接对峙试验结果表明,细菌5D15和5B18对烟草青枯病病原菌有较好的拮抗作用,拮抗半径分别达到8.4mm和10.2mm。对两株细菌进行发酵,制备其发酵产物,配成25mg/mL的粗提物溶液,进行滤纸片法抑菌试验,结果显示,菌株5D15和5B18提取物的抑菌半径分别为6.2mm和8.0mm。(2)对5D15和5B18进行全细胞脂肪酸分析和16SrDNA分析,结果表明,5D15与铜绿假单孢菌亲缘关系最近,达到99.6%,5B18与芽孢杆菌亲缘关系最近,达到99.4%。(3)通过正交试验,优化了生防细菌5D15和5B18的培养基条件。对5D15的培养基设计了四因素五水平正交试验,发现氯化钠的浓度对5D15的生长影响最为显著,当氯化钠浓度为10.0g/mL时,5D15的生长情况最好。对5B18,首先比较了牛肉膏蛋白胨培养基和马铃薯葡萄糖培养基的影响,发现马铃薯葡萄糖培养基更适合5B18的生长,设计了三因素四水平正交试验,考察了马铃薯、葡萄糖和pH三种因素对5B18的影响。结果表明,马铃薯的浓度对5B18的生长速度影响最为显著,当马铃薯浓度为350g/L时,5B18生长条件最好。(4)将5D15的发酵产物初步分成极性不同的三部分：正己烷相、二氯甲烷相和甲醇相,滤纸片法活性测试结果显示,活性组分集中于二氯甲烷相。采用薄层层析法选择分离5D15活性产物的溶剂体系和溶剂比例,结果表明,二氯甲烷和甲醇体系适于5D15活性产物的分离,分离条件为甲醇含量分别为2%、4%、6%、10%和20%的梯度洗脱。对柱分离产物进行活性追踪,发现16～25号流出物对烟草青枯病病原菌有抑制活性,然后采用结晶法进一步纯化活性产物,高效液相色谱面积归一化法结果显示,活性产物含量为76.11%。采用MIC法测得纯化产物对烟草青枯病的半抑制浓度介于40～160μ g/mL之间。核磁共振波谱分析和红外光谱分析结果表明,活性成分主要为环二肽-(酪氨酸-亮氨酸)。(5)将5B18的发酵产物初步分成极性不同的三部分：正己烷相、二氯甲烷相和甲醇相,滤纸片法活性测试结果显示,活性组分集中于二氯甲烷相和甲醇水相。采用薄层层析法选择分离5B18活性产物的溶剂体系和溶剂比例,结果表明,二氯甲烷和甲醇体系适于5D15二氯甲烷相的分离,分离条件为甲醇含量分别为4%、8%、12%和20%的梯度洗脱。对柱分离产物进行活性追踪,发现27号流出物对烟草青枯病病原菌有抑制活性,高效液相色谱分析结果显示,活性产物中主要含有13种组分。更多还原

【Abstract】 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更多还原