摘要
【背景】腐皮镰孢(Fusarium solani)是陇南花椒根腐病的主要病原物,是危害花椒生产的重要原因之一。【目的】筛选拮抗腐皮镰孢的生防菌株,为开发防治花椒根腐病的微生物农药提供新的生物资源。【方法】采用平板对峙法结合16S rRNA基因序列分析等方法从花椒根围中分离具有较强抑菌效果的细菌并进行筛选鉴定;通过Box-Benhnken中心组合设计试验探究该菌最佳发酵条件;采用液质联用(LC-MS)方法对该菌发酵液的有机溶剂提取相进行非靶向代谢组学分析及KEGG、Lipidmaps数据库注释。【结果】分离筛选出一株拮抗菌株贝莱斯芽胞杆菌(Bacillus velezensis) W-1,该菌株对腐皮镰孢的抑菌率为89%,对藤仓赤霉(Gibberella fujikuroi)、禾谷镰孢(Fusarium graminearum)、梅毒镰孢(Fusarium syphilis)、层生镰孢(Fusarium proliferatum)和松针刺盘孢(Colletotrichum fioriniae)的抑制率为57%-90%。优化后W-1的最佳发酵条件为pH值6.5、温度30℃、接种量5%、转速180r/min;发酵菌液浓度OD600值达到1.93,与LB基础培养基菌浓度相比提高了2.6倍;无菌发酵液在pH 2.0-9.0范围内抑菌活性稳定,紫外线(UVC 200-280 nm,100μW/cm2)照射10 min后抑菌活性逐渐下降,温度超过80℃后抑菌活性显著降低。最佳抑菌活性成分提取的有机溶剂是乙酸乙酯,有机提取相代谢物中含有脂肽类化合物表面活性素、磷酸寡肽抗生素、丁酰苷菌素、聚酮类化合物、溶菌杆素、环二肽、短杆菌肽等多种拮抗物质。【结论】贝莱斯芽胞杆菌W-1能分泌多种抑菌活性物质,对多种植物病原真菌抑制效果较强;其发酵液耐酸碱、耐高温,在花椒根腐病的生物防治中具有较大应用潜力。
[Background]Fusarium solani is the main pathogen of Zanthoxylum bungeanum root rot in Longnan,affecting the production of Z.bungeanum.[Objective]To screen out a strain for the biocontrol of F.solani and provide new bioresources for the development of microbial agents for the control of Z.bungeanum root rot.[Methods]In this study,we employed the plate confrontation method and 16S rRNA gene sequencing to screen and identify the bacterial strain with strong antimicrobial activity from the rhizosphere soil of Z.bungeanum.Box-Benhnken design was employed to optimize the fermentation conditions of the strain.Liquid chromatography-mass spectrometry(LC-MS)was used to study the changes of metabolites in the organic solvent phase of the bacterial fermentation broth.[Results]The antagonistic strain screened out was identified as Bacillus velezensis W-1,which showed the inhibition rates of 89%against F.solani and 57%–90%against Gibberella fujikuroi,Fusarium graminearum,Fusarium syphilis,Fusarium proliferatum,and Colletotrichum fioriniae.The fermentation conditions of W-1 were optimized as pH 6.5,30°C,inoculum amount of 5%,and 180 r/min.The OD600 value of the fermentation broth obtained under the optimal conditions reached 1.93,which was 2.6 times higher than that of the LB basal medium.The antimicrobial activity of the sterile fermentation broth remained stable in the range of pH 2.0−9.0 and gradually decreased after the broth was irradiated with ultraviolet light(UVC 200−280 nm,100μW/cm2)for 10 min.The antimicrobial activity significantly decreased when the temperature exceeded 80°C.Ethyl acetate was the best organic solvent for the extraction of antimicrobial ingredients.The organic phase of the extract contained rich antagonistic substances,such as lipopeptides(surfactants),phosphate oligopeptides(antibiotics),butirosin,polyketone compounds,lysobactin,cyclic dipeptides,and gramicidin.[Conclusion]B.velezensis W-1 can secrete a variety of antimicrobial substances and has strong inhibitory effects on multiple common pathogenic fungi of plants.Moreover,its fermentation broth can tolerate acid,alkali,and high temperature,demonstrating great potential for application in the biocontrol of Z.bungeanum root rot.
作者
田凤鸣
陈强
何九军
张晓娜
王国斌
TIAN Fengming;CHEN Qiang;HE Jiujun;ZHANG Xiaona;WANG Guobin(Technique College of Agriculture and Forestry,Longnan Teachers College,Longnan 742500,Gansu,China;Center for Research and Development of Longnan Characteristic Agro-Bioresources,Longnan 742500,Gansu,China;Prickly Ash Peel Service Center of Wudu District,Longnan 742500,Gansu,China)
出处
《微生物学通报》
CAS
CSCD
北大核心
2023年第7期2950-2969,共20页
Microbiology China
基金
甘肃省青年科技基金(21JR7RK913)
2021年度陇南市科技计划(2021-13)
2021年甘肃省高等学校创新基金(2021B-376)。
关键词
花椒根腐病
腐皮镰孢
拮抗菌
发酵优化
Zanthoxylum bungeanum root rot
Fusarium solani
antagonistic bacteria
optimization of fermentation conditions
