中国农业科学2008,4182537-2545.docVIP

柑橘溃疡病生防菌株CQBS03的鉴定及其培养特性研究 陈 力，王中康，黄冠军，曹月青，夏玉先，殷幼平 （重庆大学生物工程学院基因工程研究中心，重庆市基因功能及调控技术重点实验室，重庆 400030） 摘要：【目的】筛选鉴定柑橘溃疡病菌拮抗菌，研究其培养特性及拮抗物质的初步性质，为柑橘溃疡病的生物制剂研制奠定基础。【方法】利用对峙培养法筛选对柑橘溃疡病菌具有良好抑制作用的生防菌，并通过对菌株形态、生理生化特征以及16S rDNA序列分析鉴定其分类地位；以单因素试验和正交设计方法对影响CQBS03菌株抑菌活性物质产生的各种培养条件进行优化；利用硫酸铵沉淀获得抑菌物质粗提物并测定其对温度、蛋白酶及氯仿的敏感性。【结果】经鉴定CQBS03为枯草芽孢杆菌Bacillus subtilis。CQBS03抑菌活性成分主要存在于培养液中，能被80%饱和度硫酸铵沉淀，最高可耐受的温度范围为60～70℃；活性成分在280 nm 处有最大吸收峰，分子量大于10 kD，对蛋白酶K和胰蛋白酶稳定，而对氯仿部分敏感。培养特性研究表明，CQBS03最适培养基为YPG液体培养基，抑菌物质产生的最适培养条件为：pH 8.0左右，28℃培养72 h。【结论】枯草芽孢杆菌CQBS03所产生的抑菌物质主要成分是蛋白质，且属于外泌型蛋白；该抑菌蛋白对多种植物病原菌有抑菌作用，尤其对柑橘溃疡病菌抑菌活性高，稳定性好，是一株极具开发潜力的生防菌株。 关键词：枯草芽孢杆菌；拮抗细菌；生物防治；柑橘溃疡病；抑菌蛋白 Evaluation of Bacillus subtilis Strain CQBS03 Against Xanthomonas axonopodis pv. citri CHEN Li, WANG Zhong-kang, HUANG Guan-jun, CAO Yue-qing, XIA Yu-xian, YIN You-ping (The Key Laboratory of Gene Function and Regulation of Chongqing Municipal Education Commission, The Bioengineering College of Chongqing University, Chongqing 400030) Abstract: 【Objective】 Citrus bacterial canker caused by Xanthomonas axonopodis pv. citri ( Xac.) is one of the most important plant diseases. The aims screening higher activities antibacterial strain to control this disease, and studing the antibacterial substance characteristics.【Method】The bacterial strain CQBS03 was isolated from microorganism flora of citrus leaf surface. The strain was identified based on the bacteriological characteristics and the 16S rDNA sequence. The culture conditions of CQBS03 strain were optimized by single factor and orthogonal experiment. Crude antibacterial substance was obtained by ammonium sulfate from fermented liquid of the CQBS03 strain, and the thermal stable, optimum reactive temperature and pH were tested. 【Result】The strain was identified as Bacillus subtilis. The antibacterial activity substance from CQBS03 could designate as one kind of protein based on the large molecular weight (more than 10 kD), unstable at higher temperature (60℃ to 70℃), partly sensi