枯草芽胞杆菌8-32对病原真菌的拮抗效应及对植物促生长机制

doi:10.3969/j.issn.1674-7968.2019.05.014

摘要
图/表
参考文献
相关文章 (6)

全文: PDF (4002 KB) HTML (1 KB)
输出: BibTeX | EndNote (RIS)

摘要植物真菌病害是造成各国农业损失的主要原因之一，受该病害影响，全球粮食及其他农作物减产高达20%。前期工作中，本实验室筛选到1株对引起大豆根腐病的尖孢镰刀菌(Fusarium oxysporum)有较强拮抗作用的枯草芽胞杆菌(Bacillus subtilis) 8-32，生防、促生效果显著。为解析枯草芽胞杆菌8-32的拮抗和促生机制，并进一步开发应用该菌种，本实验验证了该菌株对植物病原菌孢子萌发、芽管及菌丝生长的抑制作用；根据已报道基因，采用PCR方法扩增其拮抗基因，高效液相色谱-质谱(high performance liquid chromatography-mass spectrometry, HPLC-MS)法鉴定其产生的拮抗物质，并检测该菌株产生的促生物质。结果表明，菌株8-32可抑制多种病原菌孢子的萌发和芽管的伸长，最大抑制率分别为100%和95.40%，还可以导致病原菌菌丝断裂；PCR扩增得到与拮抗相关的基因4个，分别为枯草杆菌素A合成酶基因(subtilosin A gene, sboA)、枯草杆菌蛋白酶基因(subtilisin gene, qk)、丰原素合成酶基因(fengycin gene, FenB)和假定蛋白合成基因(hypothetical protein yndj gene, yndj)；HPLC-MS结果表明，菌株8-32主要通过伊枯草菌素(iturin)家族发挥抑菌效果，并且其具有较高的吲哚乙酸(indolo acetic acid, IAA)产生能力，发酵72 h后上清液中IAA含量为45.29 mg/L。本研究为进一步在分子水平上研究枯草芽胞杆菌的生物防治提供了科学基础，为菌株8-32的应用提供理论支持。

	服务

	把本文推荐给朋友
	加入我的书架
	加入引用管理器
	E-mail Alert
	RSS
	作者相关文章
	王欢
	贾田惠
	杨可欣
	时向哲
	高同国
	朱宝成

关键词 ：枯草芽胞杆菌, 拮抗机理, 孢子萌发, 芽管生长, 脂肽类化合物

Abstract：Plant diseases caused by pathogenic fungi are one of the main reasons for the decline in crop quality and yield in countries around the world, which result in 20% reduction on the yields of global grain and other crops.Therefore, biological control has become one of the hotspots in plant fungal disease controls.In previous study, a strain Bacillus subtilis 8-32 was screened, which was tested to have strong antagonism to Fusarium oxysporum causing soybean root rot, and 8-32 had significant growth-promoting and biocontrol effect in greenhouse experiments.In this study, the growth-promoting and antagonistic mechanism of B.subtilis 8-32 was investigated for the further development and application of this strain.In detail, the antifungal spectrum of B.subtilis 8-32 was studied by plate confrontation method.The inhibition of B.subtilis 8-32 on spore germination, germ tube elongation and mycelium growth of main plant pathogenic fungi such as Fusarium oxysporum was testified in this study.Besides, molecular level study was carried out to reveal related antagonistic genes according to the reported literature, these genes were directly amplified by PCR using the main antagonistic gene primers of Bacillus such as iturin, surfactin, fengycin.Meanwhile, the lipopeptide antagonists were identified by high performance liquid chromatography-mass spectrometry (HPLC-MS).Plant growth-promoting substances (including indole acetic acid and siderophores) secreted by B.subtilis 8-32 were detected.The results showed that the antifungal spectrum of B.subtilis 8-32 strain was extensive.It could antagonize 7 pathogenic fungi which caused different crop diseases such as soybean root rot and wheat wilt, among which the effects were stronger for Rhizoctonia solani and Pomacea graminis, and the inhibition zone radius was 1.2 cm and 1.1 cm, respectively.The results showed that the strain 8-32 could inhibit spore germination and germ tube elongation of pathogenic fungi, and the maximum inhibition ratio reached to 100% and 95.40%, respectively.And the strain 8-32 also inhibited the growth of pathogenic fungi such as Fusarium oxysporum, Fursarium solani, Colletotrichum gloeosporivides, Alternaria brassicea through mycelial breakage.The results of PCR amplification of the antagonistic genes showed that 4 distinct single bands were amplified from 7 pairs of primers.After sequencing and NCBI Blast, the similarities between sequences and lipopeptide antibiotic genes subtilosin A (sboA), subtilisin gene (qk), fengycin gene (FenB) and hypothetical protein yndj gene (yndj) were 100%, respectively.Finally, the antagonistic substances secreted from B.subtilis 8-32 identified by HPLC-MS, including a family of lipopeptides, which were identified as homologues of iturin, that had antifungal effect.In addition, the quantitative detection on plant growth-promoting substances showed that B.subtilis 8-32 had a weak ability to produce siderophores, but strong on IAA production, which reached to 45.29 mg/L after 72 h fermentation.All the above results will lay a foundation for further study on the biological control of B.subtilis 8-32 at the molecular level, and will provide a theoretical basis for the commercial application of B.subtilis 8-32.

Key words： Bacillus subtilis Antagonistic mechanism Spore germination Germ tube growth Peptide lipid compounds

ZTFLH:

S476

基金资助:河北省教育厅拔尖人才项目(No.BJ2016029)、河北自然科学基金青年项目(No.C2015204031)、河北农业大学大学生创新创业训练计划资助项目(No.2018156)、河北农业大学教改项目(No.2015JM04))、河北省教育厅课题“生物技术”品牌特色专业建设研究(No.2012GJJG054)和教育部、河北省教育厅“生物技术”专业综合改革试点项目(No.201202)

通讯作者: gtgrxf@163.com

引用本文:

王欢,贾田惠,杨可欣,时向哲，高同国,朱宝成. 枯草芽胞杆菌8-32对病原真菌的拮抗效应及对植物促生长机制[J]. 农业生物技术学报, 2019, 27(5): 908-918.
WANG Huan,JIA Tian-Hui,YANG Ke-Xin,SHI Xiang-Zhe,GAO Tong-Guo*,ZHU Bao-Cheng. Antagonistic Effect on Pathogenic Fungi and Plant Growth Promoting Mechanism of Bacillus subtilis 8-32. 农业生物技术学报, 2019, 27(5): 908-918.

链接本文:

http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2019.05.014 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2019/V27/I5/908

[1] 陈华, 王丽, 袁成凌, 等. 2008. 高效液相色谱-电喷雾质谱法分离和鉴定枯草芽孢杆菌产生的脂肽类化合物[J]. 色谱, 26(3): 343-347.
(Chen H, Wang L, Yuan C L, et al. 2008. Isolation and identification of lipopeptides produced by Bacillus subtilis using high performance liquid chromatography and electrospray ionization mass spectrometry[J]. Chinese Journal of Chromatography, 26 (3): 343-347.)

[2] 陈莉, 缪卫国, 刘海洋, 等. 2007. 短短芽孢杆菌A57菌株对棉花主要病原真菌的拮抗作用及其最佳液体发酵条件[J]. 中国生物防治学报, (s1): 22-27.
(Chen L, Miao W G, Liu H Y, et al. 2007. Antagonism of Brevibacillus brevs A57 strains of cotton mainly pathogenic fungus and its optimal fermentation conditions[J]. Chinese Journal of Biological Control, (s1): 22-27.)

[3] 陈娜. 2012. 芽孢杆菌AH-E-1抗真菌肽的分离纯化、性质及产生条件研究[D]. 硕士学位论文, 天津大学, 导师: 曲红梅, pp.19.
(Chen N. 2012. Isolation, purification, characterization and production conditions of Bacillus AH-E-1 antifungal peptide[D]. Thesis for M.S., Tianjin University, Supervisor: Qu H M, pp.19.)

[4] 陈倩倩, 刘波, 王阶平, 等. 2016. 芽胞杆菌FJAT-28592抗真菌脂肽的研究[J]. 农业生物技术学报, 24(02): 261-269.
(Chen Q Q, Liu B, Wang J P, et al. 2016. Anti-fungal lipopetides produced by Bacillus siamensis FJAT-28592[J]. Journal of Agricultural Biotechnology,24(02):261-269.)

[5] 邓建良, 刘红彦, 王鹏涛, 等. 2010. 生防芽胞杆菌脂肽抗生素研究进展[J]. 植物保护, 36(03): 20-25.
(Deng J L, Liu H Y, Wang P T, et al. 2010. Advances in lipopeptides from Bacillus spp.[J]. Plant Protection, 36(03): 20-25.)

[6] 傅晓方, 韩红江, 郝勇锋, 等. 2012.玉米内生固氮菌的分离鉴定及对小麦幼苗的促生效应[J]. 西北农业学报, 21(1): 66-71.
(Fu X F, Han H J, Hao Y F, et al. 2012. Isolation and identification of endophytic diazotrophic bacteria from maize and their effect on wheat seedling growth[J]. Acta Agriculturae Boreali-occidentalis Sinica, 21(1): 66-71.)

[7] 高同国, 贾天瑶, 郭晓军, 等. 2015a. 枯草芽孢杆菌8-32拮抗菌株的紫外诱变选育[J]. 河北大学学报(自然科学版), 35(6): 610-615.
(Gao T G, Jia T Y, Guo X J, et al. 2015. Ultraviolet mutation of antagonistic strain Bacillus subtilis 8-32[J]. Journal of Hebei University (Natural Science Edition), 35(6): 610-615.)

[8] 高同国, 李术娜, 张冬冬, 等. 2015b. 大豆根腐病生防细菌优势菌株的筛选、鉴定及生防效果验证[J]. 大豆科学, 34(04): 661-665.
(Gao T G, Li S N, Zhang D D, et al. 2015b. Screening and identification of biocontrol dominant bacterium against to Soybean Root Rot[J]. Soybean Science, 34(04): 661-665.)

[9] 高同国, 张冬冬, 郭晓军,等 2015c. 芽孢杆菌8-32对大豆根腐病的生防效果[J]. 湖北农业科学, 54(18): 4489-4492.
(Gao T G, Zhang D D, Guo X J, et al. 2015a. Biocontrol effect of Bacillus sp. 8-32 against Soybean Root Rot[J]. Hubei Agricultural Sciences, 54(18): 4489-4492.)

[10] 候美玲, 辛媛媛, 郝志敏, 等. 2012. 玉米内生芽胞杆菌的抗菌活性物质及其拮抗玉米大斑病菌机理的初步研究[J].农业生物技术学报, 20(09): 1018-1027.
(Hou M L, Xin Y Y, Hao Z M, et al. 2012. Antifungal activity substance of endogeny Bacillus in Maize (Zea mays) and mechanisms against Setosphaeria turcica[J]. Journal of Agricultural Biotechnology, 20(09): 1018-1027.)

[11] 胡云云, 高同国, 张冬冬, 等. 2016. 大豆根腐病拮抗菌枯草芽孢杆菌8-32抗菌物质性质的初步研究[J]. 湖北农业科学, 55(4): 917-920.
(Hu Y Y, Gao T G, Zhang D D, et al. 2016. A preliminary study on physicochemical properties of antibacterial substances from Bacillus subtilis 8-32 against soybean root rot[J]. Hubei Agricultural Sciences, 55(4): 917-920.)

[12] 康振生. 2010. 我国植物真菌病害的研究现状及发展策略[J]. 植物保护, 36(03): 9-12.
(Kang Z S. 2010. Current status and development strategy for research on plant fungal diseases in China[J]. Plant Protection, 36(03):9-12.)

[13] 孔少元. 2015. 小麦全蚀病拮抗芽孢杆菌的分离筛选及盆栽生防试验[D]. 硕士学位论文, 河北农业大学, 导师: 王世英. pp: 10.
(Kong S Y. 2015. Separation of screening and potted biocontrol test of antagonistic Bacillus strains against take-all of wheat[D]. Thesis for M.S., Hebei Agricultural University, Supervisor: Wang S Y, pp:10)

[14] 李红亚, 李术娜, 朱宝成. 2011. 棉花黄萎病拮抗细菌B. velezensis 6-61拮抗蛋白发酵条件的优化[J]. 中国农学通报, 27(21): 93-99.
(Li H Y, Li S N, Zhu B C. 2011. Fermentation conditions optimization of antagonistic bacteria B. velezensis 6-61 against Verticillium dahliae Kleb[J]. Chinese Agricultural Science Bulletin, 27 (21): 93-99.)

[15] 李晶,杨谦. 2008.生防枯草芽孢杆菌的研究进展[J].安徽农业科学, 36(01): 106-111; 132.(Li J, Yang Q. 2008. Research progress on biocontrol Bacillus subtilis[J]. Journal of Anhui Agricultural Sciences, 36(01): 106-111; 132.)

[16] 李旭, 马福海, 王秀娟,等. 2012. 大豆根腐病生防菌KJB04-11的鉴定及其产生的脂肽类抗生素[J]. 生态学杂志, 31(6): 1453-1460.
(Li X, Ma H F, Wang X J, et al. 2012. Identification of bio-control bacterial strain KJB04-11 against soybean root rot disease and the produced lipopeptide antibiotics by the strain[J]. Chinese Journal of Ecology, 31(6): 1453-1460.)

[17] 刘雪, 穆常青, 蒋细良, 等. 2006 .枯草芽孢杆菌代谢物质的研究进展及其在植病生防中的应用[J]. 中国生物防治学报, 22(s1): 179-184.
(Liu X, Mu C Q, Jiang X L, et al. 2006 . Research progress of the metabolic substances produced by Bacillus subtillis and their application on biocontrol of plant disease[J]. Chinese Journal of Biological Control, 22(s1): 179-184.)

[18] 马芳芬. 2016. 枯草芽孢杆菌BS-02降解玉米黄曲霉毒素的研究[D]. 硕士学位论文, 河南工业大学, 导师: 殷海成, pp. 41.
(Ma F F. 2016. Study on degradation of Bacillus subtilis BS-02 to aflatoxin in corn[D]. Thesis for M.S., Henan University of Technology, Supervisor: Yin H C, pp. 41.)

[19] 孙冰冰, 李伟, 魏军, 等. 2015. 生防芽孢杆菌的研究进展[J]. 天津农业科学, 21(12): 102-107.
(Sun B B, Li W, Wei J, et al. 2015. Research progress in biocontrol of Bacillus spp[J]. Tianjin Agricultural Sciences, 21(12): 102-107.)

[20] 程水明, 刘仁荣. 2015. 微生物学实验[M]. 武汉：华中科技大学出版社, pp.198.
(Cheng S M, Liu R R. 2015. Microbiology Experiment[M]. Huazhong University of Science & Technology Press, Wuhan, China, pp. 198.)

[21] 石灵芳, 姜明国, 吴家法, 等. 2016. 海洋枯草芽孢杆菌UMBR1027产抑金黄色葡萄球菌活性蛋白分离鉴定及发酵条件优化[J]. 农业生物技术学报, 24(09): 1354-1363.(Shi L F, Jiang G M, Wu J F, et al. 2016. Isolation, identification of the antimicrobial protein on Staphyloccus aureus and optimization of fermentation process from marine Bacillus subtilis UBMR1027[J]. Journal of Agricultural Biotechnology, 24(09): 1354-1363.)

[22] 王超, 郭坚华, 席运官,等. 2017. 拮抗细菌在植物病害生物防治中应用的研究进展[J]. 江苏农业科学, 45(18): 1-6.
(Wang C, Guo J H, Xi Y G, et al. 2017. Research progress on application of antagonistic bacteria in biological control of plant diseases[J]. Jiangsu Agricultural Sciences, 45(18): 1-6.)

[23] 杨合同, 王少杰, 王建平, 等. 1994. 荧光假单胞菌噬铁素的性质研究[J]. 山东科学, 7 (1): 53-56.
(Yang H T, Wang S J, Wang J P, et al. 1994. Study on the properties of siderophore produced by Pseudomonas fluorescens[J]. Shandong Science, 7 (1): 53-56.)

[24] 赵翔, 陈绍兴, 谢志雄, 等. 2006a. 高产铁载体荧光假单胞菌Pseudomonas fluorescens sp-f的筛选鉴定及其铁载体特性研究[J]. 微生物学报, 46(5): 691-695.
(Zhao X, Chen S X, Xie Z X, et al. 2006a. Isolation, identification and over- siderophores production of Pseudomonas fluorescens sp-f[J]. Acta Microbiologica Sinica, 46(5): 691-695.)

[25] 赵翔, 谢志雄, 陈绍兴, 等. 2006b. 适合高产铁载体细菌筛选,检测体系的改进与探析[J]. 微生物学通报, 33(6):95-98.
(Zhao X, Xie Z X, Chen S X, et al. 2006b. Improvement and analysis in over-siderophores production bacteria filtrating and detecting[J]. Microbiology China, 33(6): 95-98.)

[26] 郑小亮, 董超, 牛瑞艳, 等. 2018. 枯草芽孢杆菌Zl-2抗菌蛋白特性及对小麦赤霉病菌的抑制作用[J]. 黑龙江大学自然科学学报, 35(02): 206-211.
(Zheng X L, Dong C, Niu R Y, et al. 2018. Characterization and inhibition effects of antifungal protein from Bacillus subtilis ZI-2[J]. Journall of Natural Science of Heilongjiang University, 35(02): 206-211.)

[27] 朱文静, 伍辉军, 高学文. 2011. 芽孢杆菌对大豆根腐病防治效果研究[J]. 大豆科学, 30(4): 621-625.
(Zhu W J, Wu H J, Gao X W. 2011. Control efficacy of Bacillus spp.to soybean root rot[J]. Soybean Science, 30(4): 621-625.)

[28] Fravel D R. 2005. Commercialization and implementation of biocontrol[J]. Annual Review of Phytopathology, 43: 337-359.

[29] Hafeez FY, Yasmin S, Ariani D. 2006. Plant growth promoting bacteria as biofertilizer[J]. Agronomy Sustainable Development, 26: 143-150.

[30] Lopez-Berges M S, Capilla J, Turra D. 2012. HapX-mediated iron homeostasis is essential for rhizosphere competence and virulence of the soilborne pathogen Fusarium oxysporum[J]. Plant Cell, 24: 3805-3822.

[31] Njoroge S M C, Kabir Z, Martin F N. 2009.Comparison of crop rotation for verticillium wilt management and effect on Pythium species in conventional and organic strawberry production[J]. Plant Disease, 93(5): 519-527.

[32] Persmark M, Expert D, Neilands J B. 1989. Isolation, characterization, and synthesis of chrysobactin, a compound with siderophore activity from Erwinia chrysanthemi[J]. The Journal of Biological Chemistry, 264(6): 3187-3193.

[33] Cho S J, Lee S K, Cha B J, et al. 2003. Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03[J]. Fems Microbiology Letters, 223(1): 47-51

[34] Huang X Q, Lu Z X, Zhao H Z, et al. 2006. Antiviral activity of antimicrobial lipopeptide from Bacillus subtilis fmbj against pseudorabies virus, porcine parvovirus, newcastle disease virus and infectious bursal disease virus in vitro[J]. International Journal of Peptide Research and Therapeutics, 12(4): 373-377.

[35] Yun C, Zhi H X, Ning L, et al. 2012. Isolation and identification of lipopeptides produced by B. subtilis SQR 9 for suppressing Fusarium wilt of cucumber[J]. Scientia Horticulturae, 135(24): 32-39.