马铃薯早疫病拮抗细菌的筛选、鉴定及抑菌物质研究  

Screening,Identification and Its Antifungal Substances of Antagonistic Bacterium Against Potato Early Blight

作　　者：于水清 杨毅清[1] 张岱[1] 庞民好[1] 杨志辉[1] 朱杰华[1] YU Shui-qing;YANG Yi-qing;ZHANG Dai;PANG Min-hao;YANG Zhi-hui;ZHU Jie-hua(College of Plant Protection,Agricultural University of Hebei,Hebei Baoding 071001,China)

机构地区：[1]河北农业大学植物保护学院,河北保定071000

出　　处：《西南农业学报》2021年第6期1234-1241,共8页Southwest China Journal of Agricultural Sciences

基　　金：河北省薯类产业技术体系创新团队专项基金(HBCT2018080205);国家重点研发计划(2018YFD0200806);现代农业产业技术体系建设专项资金(CARS-09-P18)。

摘　　要：【目的】为获得拮抗马铃薯早疫病菌的优良生防菌株,研究其抑菌活性物质对马铃薯早疫病菌的影响。【方法】利用平板对峙法对生防菌进行筛选,通过形态学特征和分子生物学的方法对生防菌进行鉴定,并检测其分泌生防作用相关的酶的能力。通过扫描电镜观察生防菌次级代谢产物对早疫病菌丝影响,利用高效液相色谱(High Performance Liquid Chromatography, HPLC)技术对生防菌次级代谢产物的活性成分进行鉴定,结合琼脂扩散法进一步验证活性成分。【结果】将筛选得到的生防菌株鉴定为贝莱斯芽胞杆菌(Bacillus velezensis),命名为C16。菌株C16能分泌淀粉酶和表面活性素,具有固氮能力,发酵液和脂肽粗提物能显著抑制马铃薯早疫病菌丝的生长,发酵液的抑菌圈可达31.5 mm, 10 mg/mL脂肽粗提物的抑菌带可达12.67 mm。早疫病菌分别与发酵液和脂肽粗提物共培养后,经扫描电镜观察发现,菌丝扭曲变形,表面产生褶皱,以及出现囊泡化等现象。对脂肽粗提物进行成分分析,发现其含有表面活性素Surfactin和伊枯草素Iturin两种物质,通过琼脂扩散法测定两种活性物质的抑菌活性,发现伊枯草素Iturin为主要抑菌活性成分。【结论】本研究筛选获得拮抗马铃薯早疫病菌的优良生防菌株为贝莱斯芽胞杆菌C16,其主要抑菌物质为伊枯草素Iturin。本研究结果为利用微生物菌剂防治马铃薯早疫病提供了备选菌种资源,同时为菌株C16的开发利用奠定了基础。【Objective】The present paper aimed to obtain antagonistic Bacillus strain which showed strong activity against Alternaria solani in potato,and the effect of the active substances by this strain on potato early blight was evaluated.【Method】Biological bacteria isolated from potato rhizosphere soil was screened using plate confrontation method.By analysis of the morphological characteristics,physiological and bio-chemical indexesand gyrB sequence,the effects of the strain with significant antifungal on pathogen was identified.High performance liquid chromatography(HPLC)was used to determine the active components of fermentation broth of biological strain.【Result】Biological bacteri-um against A.solani was identified as Bacillus velezensis C16.Strain C16 was able to secrete amylase and surfactinand had the ability of ni-trogen fixation,the fermentation broth and lipopeptide extract of strain C16 could significantly inhibit the mycelial growth of A.solani,and the inhibition zone of fermentation broth and 10 mg/mL lipopeptide extract were 31.50 and 12.67 mm,respectively.Using scanning electron microscopy(SEM),the deformed hyphae of A.solani co-cultured with fermentation broth and lipopeptide extract were observed,meanwhile,the treated hyphae also appeared with wrinkles and vesicles in surface.By HPLC,Surfactin and Iturin were identified as the main com-pounds in C16 lipopeptides.Among them,Iturin showed a strong inhibitory effect on A.solani mycelial growth.【Conclusion】In this study,B.velezensis C16 showed great biological ability against A.solani in potato.Iturin was the major antifungal substance in lipopeptides pro-duced by strain C16.This study could provide a candidate strain resource for the development of microbial agents to control potato early blight and lay a foundation for development and utilization of strain C16.

关 键 词：马铃薯早疫病 芽胞杆菌 生防因子 脂肽粗提物 高效液相色谱 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]