一株植物病原拮抗细菌的分离筛选及拮抗物质  

Isolation and screening of a plant pathogen-antagonistic bacterium and antagonistic substance

作　　者：陈志杰 谢江辉[2] 陈宇丰 周登博[2] 高祝芬[2] 戚春林[1] CHEN Zhi-jie1,2, XIE Jiang-hui2 , CHEN Yu-feng1,2, ZHOU Deng-bo2, GAO Zhu- fen2, QI Chun-lin1(1Institute of Tropical Agriculture and Forestry, Hainan University, Haikou 570228, China; 2Institute of Tropical Bioscience and Biotechnology , China Academy of Tropical Agricultural Sciences, Haikou 571101, China)

机构地区：[1]海南大学热带农林学院,海口570228 [2]中国热带农业科学院热带生物技术研究所,海口571101

出　　处：《生态学杂志》2018年第5期1595-1604,共10页Chinese Journal of Ecology

基　　金：现代农业产业技术体系建设专项资金项目(CARS-32);国家重点研发计划-香蕉化肥农药减施增效技术集成(2017YFD0202105)资助

摘　　要：从黄豆酱中分离筛选对植物病害有防治效果的拮抗菌,提取发酵液中的拮抗物质,研究其抑菌机理及基本性质。采用稀释平板法分离细菌、平板对峙法筛选拮抗菌,通过形态特征、生理生化特征及16S rRNA鉴定菌种,硫酸铵沉淀法提取发酵液中的拮抗物质,考马斯亮蓝法测得蛋白含量,扫描电镜观察拮抗物质抑菌作用,琼脂孔扩散法测定拮抗物质抑菌谱及其活性的稳定性。从黄豆酱中分离出18株细菌,通过筛选,得到一株拮抗效果最好的菌株,菌体抑菌率62.78%,发酵液抑菌率53%,命名为4-z-3,经鉴定为Bacillus siamensis。4-z-3发酵液在硫酸铵饱和度为20%~30%时提取的粗提物抗菌活性最大,为60.8%。最适饱和度下提取的粗提物的蛋白质含量为3.45%。拮抗物质对多种病原菌有良好的拮抗效果。经过4-z-3粗提物处理的病原真菌,菌丝受到破坏,细胞形状改变,细胞完整性缺失。粗提物的稳定性方面,对热不敏感,仅在90和100℃时抑菌活性下降明显;对酸碱不敏感,pH值为4时仍有81.6%的抑菌活性;对蛋白酶不敏感,抑菌活性最低时降低了19.6%;对紫外线不敏感,照射12 h之后抑菌活性仍有92.5%。本研究表明,菌株4-z-3是一株具有发展潜力的农业生防菌,分泌的胞外拮抗物质能够有效防治多种植物病害,且抑菌活性稳定。In this study,the antagonistic bacteria against plant diseases were isolated and screened from soybean paste. The antagonistic substances were extracted from fermentation broth.After that,we examined the inhibition mechanism and basic properties of antagonistic substances.Bacteria were isolated by dilution-plate method. Antagonistic bacteria were screened through plate-confrontation method. The strains were identified by morphological observation,physiological and biochemical experiments, and phylogenetic tree based on the strain ＇s 16 S r DNA sequences. The antagonistic substance of fermentation broth was extracted by ammonium sulfate fractional precipitation. The protein content of the crude extracts was measured by Bradford method. The inhibition effects of antagonistic substances were observed by scanning electron microscope. The antimicrobial spectrum of antagonistic substances and stability of antimicrobial activity of antagonistic substances were determined through agar bore diffusion method. Within the 18 bacterial strains isolated from soybean paste,the best antagonistic bacterium strain was screened and named as 4-z-3,which was identified as Bacillus siamensis. Its inhibitory rate to pathogen was 62.78%,and the inhibitory rate of fermentation broth of 4-z-3 to pathogen was 53%. The crude extracts of 4-z-3 had the highest inhibition rate at 60.8% when the saturation of ammonium sulfate was 20%-30%. At the optimum saturation of ammonium sulfate,the protein content of the crude extracts was 3. 45%. The antagonistic substances had good antagonistic effect against various pathogens. The hyphae of the pathogenic fungi treated with crude extracts of 4-z-3 were destroyed,which caused great absence of cell shape and integrity. In terms of stability,crude extract was insensitive to heat,with the inhibition activity significantly decreased only at 90 and100 ℃. The crude extract was insensitive to acids and alkalis,with 81.6% inhibition activities of crude extracts being remained at p H 4. The crude extract was

关 键 词：芽孢杆菌 香蕉枯萎病 拮抗细菌 抑菌机理 生物防治 

分 类 号：S476.1[农业科学—农业昆虫与害虫防治]