烟草根黑腐病拮抗菌的分离鉴定和生防作用特性研究  

Studies on isolation and identification of antagonistic bacteria against tobacco root black rot pathogen,Thielaviopsis basicola and their biocontrol characteristics

作　　者：黄婉媛 李彩斌 彭宇 李章海[3] 黄衍章[1] 丁婷[1] HUANG Wanyuan;LI Caibin;PENG Yu;LI Zhanghai;HUANG Yanzhang;DING Ting(Key Laboratory of Biology and Sustainable Management of Plant Diseases and Pests of Anhui Higher Education Institutes,Anhui Province Key Laboratory of Integrated Pest Management on Crops,College of Plant Protection,Anhui Agricultural University,Heifei 230061,China;Bijie Branch,Guizhou Provincial Tobacco Company,Bijie 551700,Guizhou,China;Tobacco and Health Research Center,University of Science and Technology of China,Hefei 230026,China)

机构地区：[1]安徽农业大学植物保护学院,植物病虫害生物学与绿色防控安徽普通高校重点实验室,作物有害生物综合治理安徽省重点实验室,安徽合肥230061 [2]贵州省烟草公司毕节市公司,贵州毕节551700 [3]中国科学技术大学烟草与健康研究中心,安徽合肥230061

出　　处：《浙江农业学报》2023年第4期873-883,共11页Acta Agriculturae Zhejiangensis

基　　金：贵州省烟草公司科技项目(2020XM09);贵州省烟草公司毕节市公司科技项目(2020520500240016)。

摘　　要：为挖掘对烟草根黑腐病菌有较强拮抗效果,且可在烟株根部稳定定殖的生防菌资源,以烟草根黑腐病菌为靶标,利用稀释涂布法和平板对峙法分离筛选出高效拮抗活性菌株,对其进行系统发育分析以及粗脂肽抑菌活性检测;并运用PCR技术检测拮抗细菌的脂肽编码基因,利用趋化性及生物膜成膜等试验研究烟草根系分泌物及其产生的各种有机酸对拮抗菌根部定殖的影响。结果表明:从烟草根际土壤中分离得到两株Thielaviopsis basicola高效拮抗菌株S2-1和SNSY15-5,分子鉴定表明,S2-1和SNSY15-5均为芽孢杆菌属细菌;拮抗菌S2-1和SNSY15-5脂肽粗提物对T.basicola毒力较强,EC 50值分别为1.93和2.56 mg·mL^(-1),SNSY15-5含有参与脂肽类家族细菌素(bmyB)、生物素操纵子(bioA)和假定蛋白(yngG)合成的基因;S2-1菌株中含有参与脂肽类家族生物素操纵子(bioA)合成的基因;烟草品种K326根系分泌物产生的苹果酸对S2-1和SNSY15-5有较强的吸引作用,S2-1和SNSY15-5在苹果酸中的数量分别达到63.35×10^(4) CFU·mL^(-1)和42.35×10^(4) CFU·mL^(-1),K326烟草根系分泌物及其草酸可促进S2-1生物膜形成,而K326烟草根系分泌物及其柠檬酸则促进SNSY15-5生物膜形成。该研究结果为开发效果稳定的微生物菌剂提供理论基础。In order to explore the biocontrol bacteria resources which had the strong antagonistic effect on tobacco black root rot and could stably colonize the tobacco roots,taking Thielaviopsis basicola as the target,the highly antagonistic strains were isolated and obtained by dilution-plate method and plate confrontation method,and the phylogenetic analysis and antibacterial activity of crude lipopeptide of antagonistic strains were carried out;then the genes coding lipopeptide of antagonistic strains were analyzed by PCR,the effects of K326 tobacco root exudates and various organic acids on root colonization of antagonistic bacteria were studied by chemotaxis and biofilm formation tests.Results indicated that the effective antagonistic strains S2-1 and SNSY15-5 were isolated from tobacco rhizosphere soil,and they were Bacillus microorganisms.The crude lipopeptides of the S2-1 and SNSY15-5 were high virulent to Thielaviopsis basicola,and the EC 50 of them was 1.93 and 2.56 mg·mL^(-1),respectively.SNSY15-5 had the biocontrol marker genes involved in the synthesis of bmyB,bioA and yngG,while S2-1 had the biocontrol marker gene involved in the synthesis of bioA.Malic acid produced by K326 root exudates had better attraction to S2-1 and SNSY15-5,and the amount of S2-1 and SNSY15-5 in malic acid reached 63.35×10^(4) CFU·mL^(-1) and 42.35×10^(4) CFU·mL^(-1),respectively.The biofilm formation of S2-1 could be promoted by the K326 root exudates and oxalic acid,while the biofilm formation of SNSY15-5 could be promoted by the K326 root exudates and citric acid.The research results provide a theoretical basis for developing microbial agent with stable effect.

关 键 词：烟草根黑腐病 根际细菌 脂肽 趋化性 生物膜 

分 类 号：S435.72[农业科学—农业昆虫与害虫防治]