抗烟草黑胫病菌生防细菌筛选、鉴定及体外拮抗机理初探  

Screening,identification and antagonistic mechanism in vitro of biocontrol bacteria against tobacco black shank pathogen

作　　者：付克剑 崔永和 黄智华 张继来 张立猛 邓毅书[3] 张智浩 聂强 周洪印 苏友波[1] FU Ke-jian;CUI Yong-he;HUANG Zhi-hua;ZHANG Ji-lai;ZHANG Li-meng;DENG Yi-shu;ZHANG Zhi-hao;NIE Qiang;ZHOU Hong-yin;SU You-bo(College of Resources and Environment,Yunnan Agricultural University,Kunming,Yunnan 650201,China;Yuxi Company,Yunnan Tobacco Company,Yuxi,Yunnan 653100,China;College of Architectural Engineering,Yunnan Agricultural University,Kunming,Yunnan 650201,China;Honghe Henglin Chemical Co.,Honghe,Yunnan 652300,China;College of Plant Protection,Yunnan Agricultural University,Kunming,Yunnan 650201,China)

机构地区：[1]云南农业大学资源与环境学院,云南昆明650201 [2]云南省烟草公司玉溪市公司,云南玉溪653100 [3]云南农业大学建筑工程学院,云南昆明650201 [4]红河恒林化工有限公司,云南红河652300 [5]云南农业大学植物保护学院,云南昆明650201

出　　处：《南方农业学报》2023年第8期2245-2256,共12页Journal of Southern Agriculture

基　　金：云南省重大科技专项计划项目(202002AE320005);中国烟草总公司云南省公司科技计划一般项目(2020530000242025)。

摘　　要：【目的】从云南烟草黑胫病高发区烟草根际土壤分离对烟草黑胫病菌具有高效拮抗作用的菌株,明确拮抗菌株的分类地位,并进行体外拮抗机理初探,为烟草黑胫病的防治提供生防资源和参考依据。【方法】采用平板对峙法从烟草根际土壤中分离对烟草黑胫病菌具有拮抗作用的菌株,通过生理生化特性及16S rDNA序列分析对具有较强拮抗活性的菌株进行鉴定,并测定生防细菌在体外对烟草疫霉的生物防治活性及作用方式。【结果】从采集的烟草根际土壤样品中共分离出6株对烟草疫霉具有拮抗活性的细菌菌株,其中菌株CD-1表现出最强的拮抗活性,其抑菌带宽度达10.12 mm;根据生理生化特性和16S rDNA序列分析,将该菌株鉴定为地衣芽孢杆菌(Bacillus licheniformis)。菌株CD-1具有广谱抗真菌特性,其胞内和胞外均存在抗真菌物质,其中使用二氯甲烷作为细菌发酵液萃取剂所提取的抗真菌物质的抑制效果最好;在pH 6~8、温度40~80℃时表现出相对稳定的抑制活性;菌株CD-1在NB培养基中培养的上清液提取物和细胞提取物对烟草疫霉均表现出较强的抑制活性,其抑制率分别达76.91%和67.23%;菌株CD-1分泌的抗真菌物质能破坏烟草疫霉的孢子质膜,抑制孢子萌发,其中以抗真菌粗提取物0.4 mg/mL处理烟草疫霉孢子6 h后的孢子失膜率达53.62%,孢子萌发被完全抑制,同时检测到有明显的离子和核酸渗漏。【结论】地衣芽孢杆菌CD-1产生的代谢产物抗菌谱广,抑菌活性强,具有开发成生防菌剂的潜力。【Objective】Strains with high antagonistic activity against the pathogen of tobacco black shank were isolated from tobacco rhizosphere soil in the high incidence area of tobacco black shank disease in Yunnan.The taxonomic status of strains was clarified and the in vitro antagonism mechanism was preliminary studied to provide biocontrol resources and reference basis for the prevention and control of tobacco black shank disease.【Method】Strains antagonistic to tobacco black shank pathogen were isolated from tobacco rhizosphere soil using the plate standoff method.The strains with strong antagonistic activity were identified by physiological and biochemical characteristics and 16S rDNA sequence analysis.The biocontrol activity and mode of action of biocontrol bacteria against Phytophthora nicotianae in vitro were determined.【Result】Six bacterial strains with antagonistic activity against P.nicotianae were isolated from the collected tobacco rhizosphere soil samples,among which strain CD-1 showed the strongest antagonistic activity,with a radius of inhibition zone of 10.12 mm.According to physiological and biochemical characteristics and 16S rDNA sequence analysis,the strain was identified as Bacillus licheniformis.Strain CD-1 had broad-spectrum antifungal properties,and both intracellular and extracellular antifungal substances existed.Among them,the antifungal substance extracted using dichloromethane as bacterial fermentation liquid extractant had the best inhibitory effect.When pH was in the range of 6-8 and the temperature was between 40-80℃,it showed relatively stable inhibitory activity.Strain CD-1 cultured in NB medium showed strong inhibitory activity against P.nicotianae in both culture supernatant extracts and cell extracts,with inhibition rates of 76.91%and 67.23%,respectively.The antifungal substances secreted by strain CD-1 could destroy the spore plasma membrane of P.nicotianae and inhibit spore germination.Among them,the spore membrane loss rate after treating P.nicotianae spores with the

关 键 词：烟草黑胫病 生防细菌 分离鉴定 体外拮抗机理 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]