枯草芽孢杆菌HAB-8菌株分离筛选鉴定及抑菌机理初步研究
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摘要
对枯草芽孢杆菌(Bacillus subtilis)菌株HAB-8的生物学特性及抑菌机理进行了初步研究。经形态学、生理生化特征及16SrDNA技术鉴定HAB-8菌株,应用对峙法测定了抑菌效果,对其在不同营养条件的适应性、耐盐性等进行了测定,运用光学显微镜和扫描电镜对其抑菌机理进行探究。结果表明:经形态学、生理生化特征及16srDNA技术鉴定HAB-8为B.subtilis;HAB-8菌株在6种培养基上形成的菌落,在形态上存在明显差别,直径从16.25～22.50 mm;HAB-8可在0～20%NaCl浓度的条件下生长;PDA平板对峙培养,发现HAB-8菌株,对芒果炭疽病菌等18株植物病原真菌的抑菌活性有较大差异,平均抑制率为58.52%;光学显微镜观察,抑菌带边缘的菌丝,均出现了不同程度的形态变化,菌丝扭曲变形,中部或顶端膨大变粗;孢子变形等;扫描电镜下,观察到细菌与菌丝直接接触,引起菌丝及孢子的畸形。说明拮抗细菌可能除了通过分泌活性物质来进行拮抗之外,还可能通过直接作用来抑制植物病原真菌的生长。
This research was on the purpose to study the biological characteristics and antimicrobial mechanism of Bacillus subtilis strain HAB-8.First,morphology,physiological and biochemical characteristics and 16 S rDNA gene sequence were used for identification of HAB-8 strains; second,the antibacterial activity was detected by flat confrontation experiments.At the same time,the adaptability of different nutritional conditions,salt tolerance was determination; Last,we used optical microscopy and scanning electron microscopy to explore its inhibitory mechanism.The results showed that the identification of bacterial strains HAB-8 was B.subtilis by morphological,physiological and biochemical characteristics,and phylogenetic analysis of 16 srDNA; The colony morphology of HAB-7 is obviously different in different medium plate,colony diameter ranged from 16.25-22.50 mm;HAB-8 available in 0-20% NaCl growth conditions; HAB-8 confrontation of 18 species of pathogenic fungi,and its average bacteriostatic rate in 58.52%; there were different morphological changes in the mycelia of the treatment group,and the mycelium was distorted,the central part of mycelia was enlarged,and the spore was deformed; under scanning electron microscopy,we found the bacteria directly contact with mycelium and cause deformities of mycelium and spores.It is suggested that antagonistic bacteria may inhibit the growth of plant pathogenic fungi not only by secreting active substances,but also by direct action.
This research was on the purpose to study the biological characteristics and antimicrobial mechanism of Bacillus subtilis strain HAB-8.First,morphology,physiological and biochemical characteristics and 16 S rDNA gene sequence were used for identification of HAB-8 strains; second,the antibacterial activity was detected by flat confrontation experiments.At the same time,the adaptability of different nutritional conditions,salt tolerance was determination; Last,we used optical microscopy and scanning electron microscopy to explore its inhibitory mechanism.The results showed that the identification of bacterial strains HAB-8 was B.subtilis by morphological,physiological and biochemical characteristics,and phylogenetic analysis of 16 srDNA; The colony morphology of HAB-7 is obviously different in different medium plate,colony diameter ranged from 16.25-22.50 mm;HAB-8 available in 0-20% NaCl growth conditions; HAB-8 confrontation of 18 species of pathogenic fungi,and its average bacteriostatic rate in 58.52%; there were different morphological changes in the mycelia of the treatment group,and the mycelium was distorted,the central part of mycelia was enlarged,and the spore was deformed; under scanning electron microscopy,we found the bacteria directly contact with mycelium and cause deformities of mycelium and spores.It is suggested that antagonistic bacteria may inhibit the growth of plant pathogenic fungi not only by secreting active substances,but also by direct action.
引文
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[27]Jin P F,Wang H N,Liu W B,et al.A new cyclic lipopeptide isolated from Bacillus amyloliquefaciens HAB-2and safety evaluation[J].Pesticide Biochemistry and Physiology,2017.
[28]杨廷雅,孙亮,周婷婷,等.短短芽孢杆菌Brevibacillus brevs HAB-5主要抑菌活性成分的分析及其特性研究[J].中国生物防治学报,2014,30(2):222-231.
[29]王皓楠,靳鹏飞,刘文波,等.解淀粉芽胞杆菌HAB-6抑菌活性成分的分析[J].植物保护学报,2016,43(3):452-458.
[30]刘文波,熊燕红,秦春秀,等.解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HAB-7的培养基优化及抑菌活性[J].中国植保导刊,2017,37(6):5-13.
[31]俞志祥,马宏,胡斌,等.常见致食物中毒病原菌在不同培养基上菌落形态分析[J].现代预防医学,2005,32(12):1808-1810.
[32]许曼琳,段永平,吴祖建,等.芽孢杆菌两菌株对香蕉炭疽病菌的抑制作用及其机制[J].云南农业大学学报,2009,24(4):522-527.
[2]Khosro I,Sara K R,Saeed Z et al.Antagonism of Bacillus species against Xanthomonas campestris pv.campestris and Pectobacterium carotovorum subsp.carotovorum[J].African Journal of Microbiology Research,2012,6(7):1615-1620.
[3]Mizumoto S,Hirai M,Shoda M.Enhanced iturin A production by Bacillus subtilis and its effect on suppression of the plant pathogen Rhizoctonia solani[J].Applied Microbiology and Biotechnology,2007,75(6):1267-1274.
[4]Schisler D A,Boehm M J,Slininger P J.Greenhouse and field evaluation of biological control of Fusarium head blight on durum wheat[J].Plant Disease,2002,86(11):1350-1356.
[5]黄海婵,裘娟萍.枯草芽孢杆菌防治植物病害的研究进展[J].浙江农业科学,2005(3):213-215.
[6]刘培福.枯草芽孢杆菌D221发酵条件优化及对辣椒根腐病防治效果初探[D].哈尔滨:东北农业大学,2011:2-18.
[7]Kilian U,Steiner B,Krebs,et al.FZB24(r)Bacillus subtilis mode of action of amicrobial agent enhancing plant vitality[J].Pflanzenschutz-Nachrichten Bayer 1/00,2000,1:72-93.
[8]易有,肖浪涛,王若仲,等.内生枯草芽孢杆菌B-001对烟草幼苗的促生作用及其生长动态[J].植物保护学报,2007,12,34(6):619-623.
[9]何红,邱思鑫,蔡学清,等.辣椒内生细菌BS-1和BS-2在植物体内的定殖及鉴定20株生防细菌的鉴定及其拮抗作用测定[J].微生物学报,2004,44(1):13-18.
[10]Chaurasia B,Pandey A,Palni lm,et al.Diffusible and volatile compounds produced by an antagonistic bacillus subtilis strain cause ctructural deformations in pathogenic fungi in vitro[J].Microbiology Research,2005,160(1):75-81.
[11]黄曦,许兰兰,黄荣韶,等.枯草芽孢杆菌在抑制植物病原菌中的研究进展[J].生物技术通报,2010(1):24-29.
[12]梁建根,施跃峰,竺利红,等.植物病害生物防治的研究现状[J].现代农业科技,2008(18):158-159.
[13]方中达.植病研究方法[M].3版.北京:中国农业出版社,1999:189-192.
[14]陈莉,缪卫国,刘海洋,等.短短芽孢杆菌A57对棉花主要病原真菌的拮抗机理[J].西北农业学报,2008,17(4):149-155.
[15]孙力军,陆兆新,别小妹,等.培养基对解淀粉芽孢杆菌ES-2菌株产抗菌脂肽的影响[J].中国农业科学,2008,41(10):3389-3398.
[16]钱存柔,黄仪秀.微生物学实验教程[M].北京:北京大学出版社,1997.
[17]张晓舟,徐剑宏,李顺鹏.植病生防芽胞杆菌的分离筛选与初步鉴定[J].土壤,2005,37(1):85-88.
[18]何红,蔡学清,洪永聪,等.辣椒内生细菌的分离及拮抗菌的筛选[J].中国生物防治,2002,18(4):171-175.
[19]东秀珠,蔡妙英.常见细菌系统鉴定手册[M].北京:科学出版社,2001,
[20]周德庆.微生物学实验手册[M].上海:上海科学技术出版社,1986,
[21]王多兵,任伟,张富春,等.盐穗木根际土壤中耐盐菌B6的分离与鉴定[J].安徽农业科学,2012(3):957-960,969.
[22]许曼琳.芽孢杆菌(Bacillus sp.)的抑菌作用和抗菌蛋白基因TasA的克隆和表达[D].福州:福建农林大学,2005.
[23]郭素枝,季清娥.昆虫扫描电镜样品干燥法[J].福建农业大学学报,2001(2):262-265.
[24]谢联辉.植物病理学[M].北京:科学出版社,2006:379.
[25]Baker K F.Evolving concepts of biocontrol of plant pathogens[J].Annual Review of Phytopathology,1987,25:67-85.
[26]孙亮,刘文波,杨廷雅,等.Bacillus subtilis HAB-1在不同培养基上的形态观察及抑菌活性测定[J].广东农业科学,2013,40(13):76-80.
[27]Jin P F,Wang H N,Liu W B,et al.A new cyclic lipopeptide isolated from Bacillus amyloliquefaciens HAB-2and safety evaluation[J].Pesticide Biochemistry and Physiology,2017.
[28]杨廷雅,孙亮,周婷婷,等.短短芽孢杆菌Brevibacillus brevs HAB-5主要抑菌活性成分的分析及其特性研究[J].中国生物防治学报,2014,30(2):222-231.
[29]王皓楠,靳鹏飞,刘文波,等.解淀粉芽胞杆菌HAB-6抑菌活性成分的分析[J].植物保护学报,2016,43(3):452-458.
[30]刘文波,熊燕红,秦春秀,等.解淀粉芽孢杆菌(Bacillus amyloliquefaciens)HAB-7的培养基优化及抑菌活性[J].中国植保导刊,2017,37(6):5-13.
[31]俞志祥,马宏,胡斌,等.常见致食物中毒病原菌在不同培养基上菌落形态分析[J].现代预防医学,2005,32(12):1808-1810.
[32]许曼琳,段永平,吴祖建,等.芽孢杆菌两菌株对香蕉炭疽病菌的抑制作用及其机制[J].云南农业大学学报,2009,24(4):522-527.