【作者】 陈卫良；

【导师】 李德葆；

【作者基本信息】 浙江大学 ， 植物病理学， 2001， 博士

【摘要】 拮抗细菌菌株Bacillis subtilis 5、Enterobacter cloacae B8分离自水稻 叶片，平皿试验表明它们具有对水稻白叶枯病菌的拮抗作用；Bacillus subtilis 504分离自水稻根部，平皿试验显示具有对水稻白叶枯病菌和水稻 纹枯病菌的拮抗作用。对这三个菌株产生的拮抗物质进行了分离纯化和部 分理化性质、防病效果的测定。研究的主要结果如下：一、拮抗细菌Bacillus subtilis 5菌株 Bacillus subtilis 5菌株分离自杭州郊区的水稻叶片，平皿拮抗活性检 测结果表明该菌株具有对水稻白叶枯病菌较强的拮抗作用。通过菌株生长 曲线的制作和活性测定，结果发现当细菌生长处于对数生长中期时(OD₆₀₀ 吸收值约为2.0)开始检测到拮抗活性，在细菌生长开始衰退前出现了一个 拮抗活性高峰。 B.subtilis 5菌株经KMB液体培养基培养后，其培养液经离心去菌、 硫酸铵沉淀、分子量截流试验，结果发现在截流管的冲出液部分（分子量 小于10KDa）和管内截流部分（分子量大于10KDa）均含有拮抗活性，显 示出多种拮抗物质的存在。这二部分的拮抗物质的活性不受热和有机溶剂 处理的影响，对蛋白酶K部分敏感。拮抗粗提液经FPLC-DEAE Sephacel 色谱柱、FPLC-疏水色谱柱和HPLC-反相C18色谱柱等色谱分离纯化，得 到二种分子量分别约为40KDa和38KDa的二种拮抗蛋白，一种低分子量 拮抗物质。经中国科学院上海生化所和北京大学进行部份氨基酸序列的测 定，结果发现38KDa的拮抗蛋白的N端12个氨基酸序列为： MRINHNIAALNT。经国际联网检测，这十二个氨基酸序列与一种细菌的 鞭毛分泌蛋白有高度同源性，但分子量上有很大差异。另一个40Kda的拮 抗蛋白的N端20个氨基酸序列为：AETVFKQNHAASGFLAGRYD。经国 际联网检测，发现与B.subtilis全序列中的一个基因有高度同源性，推 测该区为含有信号肽的未知蛋白前体，从序列分析该基因紧跟着一个可能 的Serine/threonine protein kinase基因。另外，经质谱初步测定这种 低分子量拮抗物质的分子量为659Da。 细菌菌株培养后所得菌体经超声波破碎、离心，所得菌体提取液也具 有拮抗活性。经FPLC-DEAE Sephacel色谱柱和FPLC-疏水色谱柱部分分 离纯化和SDS-PAGE电泳分析发现，其拮抗物质与38KDa的拮抗蛋白大小 一致。 利用 B SSbtilis 5菌株的去菌培养液进行田间防病试验，结果发现其防 效仅为6．8％，与叶育双农药的防效71．3％有很大差距。二、BSCjllus subtiljs 504菌株 伪d儿ss的u1拈504菌株分离自杭州郊区的水稻根部，平皿抬抗 活性检测结果表明该菌株对水稻白叶枯病菌和水稻纹枯病菌均有较强的桔 抗作用。B SUb tiliSS菌株经删B液体培养基培养后，其培养液经过离 心、硫酸按沉淀，所得的桔抗粗提液具有对热、有机溶剂、蛋白酶K的稳 定性。桔抗粗提液经 FPLC－DEAE Sephacel离子交换色谱柱、FPLC－source 15 phenyl疏水色谱柱和 HPLC－Doill50分子筛色谱柱等色谱分离纯化走 SDS－－ PAGE电泳检测，结果得到种分子量分别约为2．5－SKDa的桔抗物质，该物 质对尤 solani和L 口厂y厂ae pv．o厂y丁ae均有桔抗活性。。、Entezvinsct。cloa。e BS菌株 E cloacae BS菌株分离自浙江天台水稻叶片，平皿桔抗活性检测结果 表明该菌株具有对水稻白叶枯病菌较强的桔抗作用。可用 50mmol／L Tris·HCI …7．5)等缓冲液或二氯甲烷等有机溶剂将该菌株产生的桔抗物质从经菌株 培养后的KMB固体培养基中抽提出来，该桔抗物质可被硫酸铰沉淀。同 时，将 50mmol几 Trs·HCI中H7．0)缓冲液提取所得的桔抗粗提液再用二氯 甲烷宰取后发现，桔抗物质转移到二氯甲烷有机相层。由此可见桔抗物质 虽然可溶解于水溶液，但更易溶于二氯甲烷等有机溶剂。桔抗提取液经 80℃ 或 100℃温度处理 20min后，桔抗活性不受影响，经高压灭菌 20min后活 性部分消失。不同有机溶剂或蛋白酶K处理后桔抗活性保持稳定。但将桔 抗物质在 4℃冰箱中放置 15天后，经 HPLC分析发现其桔抗活性峰型已发 生了变化，即桔抗物质发生了降解。桔抗粗提液经硅胶簿层色谱和 HPLC． PronyC 5川 反相色谱柱分离纯化后，通过质谱分析发现该桔抗物质的分于 量约为力3 Dation。 利用E cloacae BS菌株的桔抗提取液进行田间防病试验，结果发现 其防效可达61％，显示出较好的防治效果。更多还原

【Abstract】 Antagonistic bacterial strains, Bacillus subtilis 5 and Enterobacter cloacae B8 antagonistic bacterial strains, which were isolated from the rhizosphere of rice, showed strongly antagonistic activities to Xanthomonas oryzae pv.oryzae in vitro test. Antagonistic bacterial strain B. subtilis 504 isolated from the rhizosphere of rice showed strongly antagonistic activities to Xanthomonas oryzae pv.oryzae and Rhizoctonia solani in vitro test. The antagonistic substances produced by these three antagonistic bacterial strains were isolated and purified, the partial properties of the antagonistic substances were studied. The mainly results were as follows:1. Bacillus subtilis 5:B. subtilis 5 was screened out from the phyllosphere of rice in Hangzhou, China. It showed strongly antagonistic activity to rice bacterial blight pathogen in vitro test. The antagonistic substances from the cell free culture could be precipitated by (NH4)2SO4 with 75% saturation. The antagonistic activities were thermostable, resistance to organic solvents and trypsin, but partial sensitive to proteinase K. Two antagonistic proteins with 40Kda and 38Kda were isolated and purified by substantially chromatography of FPLC DEAE sephacel column, and FPLC source 15phenyl column. The sequence of the first 20 amino acids of the N-terminal of the 40 Kda antagonistic protein was determined, so was the first 12 amino acids of the N-terminal of the 38 Kda antagonistic protein. One antagonistic substance with 659 Da, which determined by MS, was also isolated and purified by substantially chromatography of FPLC DEAE sephacel column, FPLC source 15phenyl column and HPLC ProRPC 5/10 column.The antagonistic substance from the bacterial cells was isolated and purified partially by FPLC DEAE sephacel column, and FPLC source 15phenyl column after disruption with ultrasonication, and precipitation with (NH4)2SO4. The result from the SDS-PAGE electrophoresis showed the MW of the antagonistic protein was 38Kda.The control efficiency of antagonistic substance extract to rice bacterial blight in field was 6.8%. It was far less than that of Saikuzuo, which was 71.3%.2. Bacillus subtilis 504B, subtilis 504 was screened out from the rhizosphere of rice in Hangzhou, China. It showed strongly antagonistic activity to X. oryzae pv. oryzae, and Rhizoctonia salani in vitro test. The antagonistic substances from the cell free culture could be precipitated by 1.0 mol/L (NH4)2SO4. The antagonistic activities were thermostable, resistance to organic solvents and proteinase K. One antagonistic peptide with MW of 2.5-5Kda was isolated and purified from the cell free culture after the chromatography of FPLC DEAE sephacel column, and HPLC Diol 150 Column, substantially. It showed antagonistic activity to both X. oryzae pv. oryzae and R. solani.3. Enterobacter cloacae B8E. cloacae B8 was screened out from the phyllosphere of rice in Tiantai, Zhejiang, China. It showed strongly antagonistic activity to rice bacterial blight pathogen in vitro test. The antagonistic substance produced by E. cloacae B8 could be extracted from the cultured solid medium by 50mmol/L Tris-HCl (ph7.5) or CH2C12, and could be precipitated by (NH4)2SO4 with 75% saturation. The antagonistic activities were thermostable, resistance to organic solvents and proteinase K, but the antagonistic substance was degraded gradually during the storage at 4癈. The antagonistic substance was purified by silica gel thin layer chromatography and HPLC ProRPC 5/10 column chromatography, the MW of the antagonistic substance was 453 Da obtained from the results of LC-MS analysis.The control efficiency of antagonistic substance extract to rice bacterial blight in field could reach to 61%, which was potential on the application of plant disease control in future after more studies.更多还原